MX2008005174A - Treatment of hypersensitivity - Google Patents

Abstract

The present invention relates to a method for inhibiting a hypersensitivity reaction in a subject, wherein said method comprises administering an effective amount of chaperonin (10).

Description

HIPERSENSITIVITY TREATMENT FIELD OF THE INVENTION The present invention relates to the hypersensitivity treatment by administering a therapeutically effective amount of eukaryotic chaperonin (CpnlO) and in particular, the invention relates to the use of eukaryotic CpnlO for the treatment of allergic conditions associated with hypersensitivity, including fasma.

BACKGROUND OF THE INVENTION The sensitization of an individual to a particular antigen or combination of antigens, after which .. the subsequent exposure causes extreme allergic reactions, can be harmful and even fatal. When an immune response is presented, of adaptation in an exaggerated or inappropriate manner, it is said that the individual who experiences the reaction is hypersensitive. Hypersensitivity reactions are the result of immune responses that act inappropriately and can be triggered by many antigens. A form of hypersensitivity occurs when an IgE response is directed against harmless environmental antigens, such as pollen or dust mites. The resulting release of pharmacological mediators by IgE-sensitized mast cells produces an acute inflammatory reaction with symptoms such as asthma or rhinitis. The present invention reflects the surprising discovery that eukaryotic CpnlO can inhibit hypersensitivity reactions, including asthma.

SUMMARY OF THE INVENTION According to a first aspect of the present invention, there is provided a method for inhibiting a hypersensitivity reaction in an individual, wherein said method comprises administering an effective amount of eukaryotic chaperonin. According to a second aspect of the present invention, there is provided a method for treating or preventing a disorder associated with hypersensitivity reaction in an individual, the method comprising administering to the individual an effective amount of eukaryotic chaperonin, in which the chaperonin 10 modulates the signaling coming from a Toll-type receiver. In accordance with a third aspect of the present invention, a composition is provided for treating or To prevent a disorder associated with hypersensitivity reaction in an individual, the composition comprises an effective amount of eukaryotic chaperonin, together with at least one concomitant therapy. According to a fourth aspect of the present invention, there is provided a method for treating or preventing a disorder associated with hypersensitivity reaction in an individual, the method comprising administering an effective amount of the composition according to the third aspect of '. the invention. The hypersensitivity reaction may involve the activation of basophils, eosinophils, mast cells, neutrophils and lymphocytes, and may involve signaling activation of the Toll-like receptor (TLR). The hypersensitivity reaction may reflect elevated levels of eosinophils and immunoglobulin E. An example of a hypersensitivity reaction is an inflammatory reaction. More specifically, examples of a hypersensitivity reaction include food allergy, dermatitis, allergic conjunctivitis, rhinitis, eczema, anaphylaxis, and respiratory diseases associated with airway inflammation. Examples of said respiratory diseases include asthma, such as allergic asthma, intrinsic asthma and occupational asthma.

The activation of TLR plays an important role in the etiology of inflammatory lung diseases ranging from acute respiratory distress syndrome (ARDS) to asthma and chronic obstructive pulmonary disease (COPD). Toll-like receptors can be selected from the group consisting of: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 and TLR10. The eukaryotic chaperonin 10 can be one. chaperonin 10 obtained from natural sources, produced in recombinant form or produced by synthesis. The eukaryotic chaperonin 10 can be of mammalian origin. The chaperonin 10 can be chaperonin 10 of human. Chaperonin 10 may comprise the polypeptide sequence as indicated in SEQ ID NO: 1, 2, 3, 4, 7, 9, 11, 13, 15 or 17. The chaperonin 10 can be acetylated or non-acetylated. Chaperonin 10 may be lacking or substantially lacking in protein folding activity. Eukaryotic chaperonin 10 can be administered in the form of a polynucleotide that codes for chaperonin 10. The polynucleotide encoding chaperonin 10 can be located in a genetic construct, operably linked to a promoter. The eukaryotic chaperonin 10 can be encoded by a polynucleotide which may comprise the sequence as indicated in SEQ ID NO: 5, 6, 8, 10, 12, 14, 16, or 18. The methods may also comprise the administration of at least one additional agent. The agent can be an immunomodulator. The immunomodulator may be a type I interferon such as IFNa or IFN (¾.) Concomitant therapy may include the administration of agents, such as anti-inflammatory compounds, bronchodilator compounds or immunosuppressive agents, or a combination thereof. it can be an immunosuppressant drug or a specific antibody directed against B or T lymphocytes, an antibody against a cytokine for example IL-3, IL-5, IL-13, GM-CSF, or surface receptors that mediate its activation, including IgE and the FcEpsilon receptor.The immunosuppressant drug can be cromoglycolates, theophylline, leukotriene antagonist, antihistamines or a combination thereof.The composition according to the third aspect can also comprise a corticosteroid.The above aspects and modalities contemplate the use of wild-type and modified forms of eucaryotic chaperonin 10 including chaperonin 10 mammal, and for example human chaperonin 10, as well as full-length chaperonin 10 polypeptides and fragments or derivatives thereof that retain immunomodulatory activity.

Definitions In the context of this description, the term "comprising" means "mainly including, but not necessarily only." Also, the variations of the word "comprising", such as "comprises" and "comprise", have meanings that vary accordingly. As used in the present invention, the terms "treatment", "treating" and variations thereof, refer to any and all uses that correct a disease state or symptoms, prevent the establishment of the disease, or otherwise prevent, hinder, retard, or reverse the progression of the disease or other undesirable symptoms in any way at all. As used in the present invention, the term "effective amount" includes within its meaning a non-toxic but sufficient amount of an agent or compound to provide the desired therapeutic or prophylactic effect. The exact amount required varies from individual to individual depending on factors such as the species being treated, the age and general condition of the individual, the severity of the condition being treated, the particular agent being administered and the mode of administration, etc. Therefore, it is not possible to specify an exact "effective amount". However, for any given case, an appropriate "effective amount" can be determined by one skilled in the art using only routine experimentation. The term "polypeptide" means a polymer consisting of amino acids linked together by peptide bonds. The terms "polypeptide" and "protein" are used interchangeably in the present description, although for the purposes of the present invention a "polypeptide" may constitute a portion of a full-length protein. The term "polynucleotide" as used in the present invention refers to a single-chain or double-stranded polymer of deoxyribonucleotide, ribonucleotide or known analogs or natural nucleotides bases, or mixtures thereof. As used in the present invention the terms "modular", "modulates" and variations thereof refer to increasing or decreasing the level of activity, production, secretion or functioning of a molecule in the presence of a particular molecule or modulating agent of the invention compared to the level of activity, production, secretion or other functioning thereof in the absence of the molecule or modulating agent. These terms do not imply the quantification of the increase or decrease. The modulation can be of any magnitude sufficient to produce the desired result and can be direct or indirect. The term "immunomodulator" as used in the present invention refers to a molecular mediator secreted by one or more cell types and which plays a role in the activation, maintenance, maturation, inhibition, suppression or increase of an immune response.

BRIEF DESCRIPTION OF THE FIGURES AND THE LIST OF SEQUENCES The present invention is described below, by way of example only, with reference to the appended figures. Figure 1. Percent of eosinophils of the total number of cells counted in the bronchoalveolar lavage (BAL) of sheep (n = 5 per group) treated with vehicle, 0.5, 4, or 16 mg1 of CpnlO delivered via intravenous (A) or vehicle, 0.5, or 4 mg of CpnlO delivered directly into the left lobe of the lung, supplying the vehicle in the right lobe (B) followed by exposure > to house dust mites (HDM for its acronym in English). Wash samples are taken at 48 hours after exposure to HDM. Figure 2A. HDM specific IgE levels previous (dO) and later (d7) to HDM exposure in group i.v. after administration of 4 mg of CpnlO. Figure 2B. Previous serum IgE (dO) and subsequent serum levels (d7) at exposure to HDM in the intrapulmonary group after administration of 4 mg of CpnlO. Figure 3. Respiratory tract of representative bronchioles of two sheep 48 hours after exposure to domestic dust mites (HDM). Ewes are administered either an intra-pulmonary infusion of vehicle (1), or a single IV injection of 4 mg of CpnlO before exposure to HDM (2). Notice the infiltrating inflammatory cells which completely surround the airway in the control sheep with vehicle (arrow) which are mostly absent in the respiratory tract of the sheep to which iv CpnlO is administered before exposure to HDM ( xlOO H and E). Figure . Terminal bronchioles representative of two sheep 48 hours after exposure to mites of household dust (HDM). Ewes are administered either an intra-pulmonary infusion of vehicle (1), or a single IV injection of 4 mg of CpnlO before exposure to HDM (2). Notice the infiltrating inflammatory cells which completely surround the airway in the control sheep with vehicle (arrow) which are mostly absent in the respiratory tract of the sheep to which iv CpnlO is administered before exposure to HDM ( xlOO H and E). Figure 5. High magnification micrograph of a bronchial respiratory tract in sheep # 6 48 hours after exposure to HDM and administration of an intrapulmonary vehicle (control). Notice the red mucosa stained with PAS which completely obstructs the lumen of the bronchial gland (arrow). Note also the epithelial cell and mucus secretory cell hyperplasia and mucosal lining of the airway lumen. (x400 dyed with PAS Azul Alciano). Figure 6. Micrograph of a bronchial respiratory tract of sheep # 44 48 hours after exposure to HDM and an iv injection of 4 mg of CpnlO. Note that the lumens of the bronchial gland are mostly devoid of mucosa except for a small region of blue mucosa located on the left side of the gland (arrow). Also note that the cells Secretory mucus and epithelial cells lining the airway lumen s.on more typical of an unexposed animal. (x250 stained with PAS Blue Alciano). Sequence Listing: 'The amino acid sequence of human wild type CpnlO (Accession No. of GenBank X75821) is provided in SEQ ID NO: 1. The amino acid sequences of two modified forms of CpnlO, with additional amino acid residues at the N-terminal end are provided in SEQ ID NOs: 2, 3 and 4. The nucleotide sequence coding therefor is provided in SEQ ID NOs: 5 and 6. Another modified form of wild type CpnlO of human comprises deletion of the mobile vessel as indicated in SEQ ID NO: 7. The nucleotide sequence encoding the same is provided in SEQ ID NO: 8. In addition to these, other modified forms of wild-type CpnlO from human include deletions within of the mobile ship as provided in SEQ ID NOs: 13, 15 and 17. The nucleotide sequences encoding them are provided in SEQ ID NOs: 14, 16 and 18. Additional modified forms of wild-type CpnlO from human comprise the deletion of the beta-hairpin cover loop (SEQ ID. NO: 9) or the deletion of both the mobile vessel and the beta fork cover loop (SEQ ID NO: 11). The nucleotide sequences coding for them are provided in SEQ ID NOs: 10 and 12.

DETAILED DESCRIPTION OF THE NTION Hypersensitivity reactions are the result of immune responses that act inappropriately and can be triggered by many antigens. A form of hypersensitivity occurs when an IgE response is directed against harmless environmental antigens, such as pollen or dust mites. The resulting release of pharmacological mediators by mast cells sensitized to IgE produces an acute inflammatory reaction with symptoms such as asthma or rhinitis. Inflammation of the airways is crucial for the pathogenesis of asthma and lves the recruitment and activation of eosinophils, mast cells, neutrophils and lymphocytes into lung tissue and bronchioalveolar spaces (Busse et al., 2001). Mite antigen from house dust has been shown to be the most common allergen that causes allergic asthma in humans, and sensitized individuals develop specific IgE and IgG humoral responses (Roche et al., 1997). The appropriate animal-asthma model could allow definite and controlled stigations to be carried out with direct relevance to the human disease. The present ntion is exemplified by evaluating the modulating effects of CpnlO in an asthma model in sheep However, it will be appreciated that the concept can be applied to other disorders of the hypersensitivity reaction type. Previous models of asthma in sheep are based on animals sensitized to nematode allergens (Ascaris) and the results are extrapolated to the evaluation of physiological and pharmacological effects of potential antiasthmatic drugs. Recently a model of allergic lung inflammation has been established in sheep using the house dust mite as an allergen with direct relevance to allergic disease in humans. In this model, sensitized sheep develop allergen-specific IgE responses with recruitment of neutrophils, eosinophils and lymphocytes activated into lung tissue and BAL with kinetics similar to that of humans exposed to the HDM allergen (Bischof et al., 2003) . The present ntion provides a method for inhibiting a hypersensitivity reaction in an individual, wherein said method comprises administering an effective amount of eukaryotic chaperonin.

In accordance with aspects and embodiments of the present ntion, it is administered to an individual in the need for treatment is an effective amount of eukaryotic CpnlO, for example, human CpnlO, also known as a 10 kDa heat shock protein (HsplO). For example, the individual to be treated is a human, and accordingly, the CpnlO polypeptide is the human CpnlO polypeptide. Those skilled in the art will appreciate that the precise identity of the CpnlO used in accordance with the present ntion may vary depending on a number of factors, for example, the species to be treated, so that the CpnlO may be selected to be obtained from the species to be treated. Typically, CpnlO is recombinant CpnlO. The methods described in Orton et al., 2000 (Immunol Cell Biol 78: 603-607), Ryan et al, 1995 (J Biol Chem 270: 22037-22043) and Johnson et al., 2005 (J Biol Chem 280: 4037 -4047) are examples of suitable production methods for the recombinant CpnlO protein, although one skilled in the art will appreciate that the present ntion is not limited by the purification or production method used and that any other method for producing CpnlO can be used. to be used in accordance with the methods and compositions of the present ntion. Polypeptides and peptide fragments of eukaryotic CpnlO for use in accordance with the present ntion can be obtained using standard acid techniques or recombinant nucleic acid can be synthesized, for example using conventional liquid or solid phase synthesis techniques. CpnlO peptides can be produced by digestion of a polypeptide with one or more proteinases such as endoLys-C, endoArg-C, endoGlu-C and V8 protease of Staphylococcus. The digested peptide fragments can be purified by, for example, high performance liquid chromatography (HPLC) techniques. The embodiments of the invention also contemplate the administration of a polynucleotide encoding eukaryotic CpnlO. In such situations the polynucleotide is typically operably linked to a promoter such that the appropriate polypeptide sequence is produced after the polynucleotide is administered to the individual. The polynucleotide can be administered to individuals in a vector. The vector can be a plasmid vector, a viral vector, or any other suitable vehicle adapted for the insertion of exogenous sequences, their introduction into eukaryotic cells and the expression of the introduced sequences. Typically, the vector is a eukaryotic expression vector and can include expression and processing control sequences such as a promoter, an enhancer, ribosome binding sites, polyadenylation signals, and transcription termination. The nucleic acid construct to be administered may comprise naked DNA or may be in the form of a composition, together with one or more pharmaceutically acceptable carriers. The eukaryotic CpnlO polypeptide may have the amino acid sequence as indicated in SEQ ID NO: 1, 2, 3, 4, 7, 9, 11, 13, 15 or 17. The nucleotide sequence of the polynucleotide encoding CpnlO may be be as that indicated in SEQ ID NO: 5, 6, 8, 10, 12, 14, 16 or 18 or display sufficient sequence identity therewith so that it hybridizes to the sequences of SEQ ID NO: 5, 6, 8 , 10, 12, 14, 16 or 18. In alternative embodiments, the nucleotide sequence of the polynucleotide may share at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 96%, 97%, 98% or 99% identity with the sequences indicated in SEQ ID NO: 5, 6, 8, 10, 12, 14, 16 or 18. Within the scope of the terms "polypeptide" and "polynucleotide" such as used in the present invention are fragments and variants thereof. By way of example only, the peptide fragments of CpnlO as described in WO 95/15338 and PCT / AU2006 / 001278, the descriptions of which are incorporated in the present invention for reference, can be used in accordance with aspects and modalities of the present invention.

The term "fragment" refers to a nucleic acid or polypeptide sequence that encodes a constituent or that is a constituent of the CpnlO protein of incomplete length. In terms of the polypeptide the fragment possesses qualitative biological activity in common with the full-length protein. A biologically active fragment of CpnlO used according to the present invention typically can possess at least about 50% of the immunomodulatory activity of the corresponding full-length protein, more typically at least about 60% of. said activity, more typically at least about 70% of said activity, more typically at least about 80% of said activity, more typically at least about 90% of said activity, and more typically still at least 95% of said activity. As described later in the present description, the inventors of the present invention have also shown that the addition of a glycine residue to the N-terminal end of CpnlO enhances the immunomodulatory activity. It is contemplated that the presence of an acetyl group or an amino acid that shares structural homology with an acetyl group such as an alanine residue or a glycine residue increases the immunomodulatory activity of CpnlO.

The term "variant" as used in the present invention refers to substantially similar molecules. In general terms, nucleic acid sequence variants encode polypeptides possessing qualitative biological activity in common. In general terms, the polypeptide sequence variants also possess qualitative biological activity in common. In addition, these polypeptide sequence variants can share at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 82%, 85%, 90%, 95%, 96 %, 97%, 98% or 99% sequence identity and 70%, 75%, 80%, 82%, 85%, 90%, 95%, 96%, 97%, 98% or 99% similarity of sequence. In addition, a variant polypeptide may include analogs, in which the term "analog" means a polypeptide that is a derivative of CpnlO, which derivative comprises the addition, deletion, substitution of one or more amino acids, such that the polypeptide substantially retains the same function as the original CpnlO. It is well known in the art that some amino acids can be changed within a polypeptide without altering the activity of the polypeptide (conservative substitutions). The term "conservative amino acid substitution" refers to a substitution or replacement of an amino acid by another amino acid with similar properties within a polypeptide chain (primary sequence of a protein). By example, the substitution of the glutamic acid-charged amino acid (Glu) for the amino acid with similar charge aspartic acid (Asp) could be a conservative amino acid substitution. The amino acid additions can result from the fusion of a CpnlO polypeptide or fragment thereof with a second polypeptide or peptide, such as a polyhistidine tag, maltose binding protein fusion, glutathione S transferase fusion, fluorescent protein fusion green, or the addition of an epitope tag such as FLAG or c-myc. For example, the wild-type CpnlO polypeptide of human can comprise an additional portion of the GSM tripeptide at the N-terminus (SEQ ID NO: 2, see for example WO 95/15338, the disclosure of which is incorporated herein). invention for reference) or an additional alanine residue (A) at the N-terminus. (SEQ ID NO: 3, WO 2004/041300, the descriptions of which are incorporated in the present invention for reference), or an additional glycine (G) at the N-terminal end, SEQ ID NO: 4 (see for example PCT / AU2006 / 001278). The wild-type CpnlO polypeptide may or may not include the N-terminal initiation methionine. The wild-type CpnlO polypeptide can be modified at the N-terminus or at the C-terminus by the addition, deletion, or substitution of one or more amino acid residues.

In another example, the human wild type CpnlO polypeptide may lack the movable loop (SEQ ID NO: 7), the Beta hairpin loop loop (SEQ ID NO: 9) or both (SEQ ID NO: 11) which can be seen, for example, in PCT / AU2006 / 001278, the description of which is incorporated in the present invention for reference. In addition, the wild-type CpnlO polypeptide of human can contain tripeptide deletions in the mobile loop as indicated in SEQ ID NO: 13, 15 or 17 (see for example PCT / AU2006 / 001278, the description of which is incorporated in the present invention for reference The present invention also contemplates the use of polynucleotides encoding said modified forms of CpnlO The CpnlO variants can be generated by mutagenesis of a CpnlO protein or mutagenesis of a coding nucleic acid, such as by mutagenesis. randomization or site-directed mutagenesis using methods well known to those skilled in the art, Such methods can be found for example in Current Protocols In Molecular Biology (Chapter 9), Ausubel et al., 1994, John Wiley &Sons, Inc. , New York, the disclosure of which is incorporated herein by reference. "Variants and analogs also encompass polypeptides complexed with Other chemical portions, proteins merged or modified in some other way after the transition. Examples of suitable modifications are described in International Patent Application No. PCT / AU2005 / 000041, the disclosure of which is incorporated herein by reference. In addition, the CpnlO polypeptide or fragment thereof may possess other post-translational modifications, including side chain modifications such as for example modifications with acetyl, amidine, carbamoyl, reductive alkylation and other modifications as known to those skilled in the art. . A further example of a variant of chaperonin 10 is one that lacks or substantially lacks protein folding activity, and examples of such modifications are described in International Patent Application (PCT) No. PCT / AU2006 / 001278, whose Description is incorporated in the present invention for reference.

Production of CpnlO In accordance with the present invention CpnlO polypeptides can be produced using standard techniques of recombinant DNA and Molecular Biology which are well known to those skilled in the art. The guidelines can be obtained, for example, from standard texts such as Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, New York, 1989 and Ausubel et al., Current Protocols in Molecular Biology, Greene Publ. Assoc. and iley-Intersciences, 1992. The methods described in Morton et al., 2000. { Immunol Cell Biol 78: 603-607), Ryan et al, 1995 (J Biol Chem 270: 22037-22043) and Johnson et al., 2005 (J Biol Chem 280: 4037-4047) are examples of suitable purification methods for CpnlO polypeptides, although one skilled in the art will appreciate that the present invention is not limited by the purification or production method used and that any other method for producing CpnlO can be used to be used in accordance with the methods and compositions of the invention. present invention. CpnlO peptides can be produced by digestion of a polypeptide with one or more proteinases such as endoLys-C, endoArg-C, endoGlu-C and V8 protease from Staphylococcus. The digested peptide fragments can be purified by, for example, high performance liquid chromatography (HPLC) techniques. The purification of the CpnlO polypeptides of the invention can be scaled for large scale production purposes. For example, as described in the present description, the inventors of the present invention have developed a biological process for the production of large quantities (grams) of highly pure CpnlO polypeptides, of clinical grade by batch fermentation in E. coli. The CpnlO polypeptides of the present invention, as well as fragments and variants thereof, can also be synthesized using standard methods of liquid or solid phase chemistry well known to those skilled in the art. For example, such molecules can be synthesized following the solid phase chemistry methods of Steward and Young (Steward, JM and Young, JD, Solid Phase Peptide Synthesis. (2nd edition) Pierce Chemical Co., Illinois, USA (1984). In general, said method of synthesis comprises sequentially adding one or more amino acids or amino acids suitably protected to a growing peptide chain Typically, any of the amino or carboxyl group of the first amino acid is protected using an appropriate protecting group. The protected group can then be bound to an inert solid support or used in solution by adding the next amino acid in the sequence having the complementary group (amino or carboxyl) suitably protected and under appropriate conditions to form the amide bond. this newly added amino acid residue and the next amino acid (protected) is added, and so on. 4 After all the desired amino acids have been ligated, any remaining protecting groups are removed, and if necessary any solid support, sequentially or concurrently to produce the final polypeptide. The amino acid changes in the CpnlO polypeptides can be made using techniques well known to those skilled in the relevant art. For example, amino acid changes can be made by nucleotide replacement techniques which include the addition, deletion or substitution of nucleotides (conservative and / or non-conservative), provided that the appropriate reading frame is maintained. Exemplary techniques include random mutagenesis, site-directed mutagenesis, oligonucleotide-mediated or polynucleotide-mediated mutagenesis, deletion of selected region or regions by the use of existing or genetically engineered restriction enzyme sites, and the polymerase chain reaction. The generation of immunomodulatory activity by the CpnlO polypeptides of the invention may involve the formation of heptamers of the CpnlO polypeptides. The evaluation of the immunomodulatory activity for purposes of the present invention may be by any of a number of techniques known to the art. experts in the field. As exemplified in the present invention, the immunomodulatory activity of the CpnlO polypeptides can be determined by measuring the ability of the polypeptide to modulate TLR4 Toll-like receptor signaling, using for example a luciferase bioassay, and typically in the presence of an agonist. TLR4 such as lipopolysaccharide. Alternatively or in addition to, the immunomodulatory activity can be determined using other in vitro, ex vivo or in vivo tests, for example by measuring the production of NF- ?? Or the production of cytokines in cells such as peripheral blood mononuclear cells.

Compositions and routes of administration In general, compositions suitable for use in accordance with the methods of the present invention can be prepared in accordance with methods and procedures that are known to those skilled in the art and can therefore include a carrier, diluent and / or pharmaceutically acceptable adjuvant. In accordance with the present invention, a CpnlO composition comprising CpnlO alone or as a pharmaceutical composition comprising a pharmaceutically acceptable carrier, adjuvant and / or diluent can be prepared. Alternatively, the composition of CpnlO it may also comprise an immunosuppressive agent. The immunosuppressive agent can be an immunosuppressant drug or a specific antibody directed against B or T lymphocytes, or surface receptors that mediate its activation. For example, the immunosuppressant drug may be cyclosporine, tacrolimus, sirolimus, mycophenolate mofetil, methotrexate, cromoglycolates, theophylline, leukotriene antagonist or antihistamine, or a combination thereof. In addition, the pharmaceutical composition for use according to the invention may also comprise a spheroid, such as a corticosteroid. The compositions can be administered using normal routes. In general, the compositions can be administered parenterally (eg, intravenous, intraspinal, subcutaneous or intramuscular), oral or topically. The administration can be systemic, by regions or local. The particular route of administration used in any given circumstance will depend on a number of factors, including the nature of the condition to be treated, the severity and degree of the condition, the required dose of the particular compound to be delivered and the effects potential side effects of the compound. In general, compositions can be prepared suitable in accordance with methods that are known to those skilled in the art and may include a pharmaceutically acceptable diluent, adjuvant and / or excipient. The diluents, adjuvants and excipients must be "acceptable" in terms of being compatible with the other ingredients of the composition, and not deleterious to the recipient thereof. Examples of pharmaceutically acceptable carriers or diluents are demineralized or distilled water; Saline solution; vegetable oils such as peanut oil, safflower oil, olive oil, cottonseed oil, corn oil, sesame oil such as peanut oil, safflower oil, olive oil, cottonseed oil, oil of corn, sesame oil, peanut oil or coconut oil; silicone oils, including polysiloxanes, such as methyl polysiloxane, phenyl polysiloxane and methylphenyl polysiloxane; volatile silicones; mineral oils such as liquid paraffin, soft paraffin or squalane; cellulose derivatives such as methyl cellulose, ethyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose or hydroxypropyl methyl cellulose; lower alkanols, for example ethanol or iso-propanol; lower aralkanols; lower polyalkylene glycols or lower alkylene glycols, for example polyethylene glycol, polypropylene glycol, ethylene glycol, propylene glycol, 1/3-butylene glycol or glycerin; fatty acid esters such as isopropyl palmitate, isopropyl myristate or ethyl oleate; polyvinyl pyrrolidone; agar; carrageenan; gum tragacanth or gum acacia, and petrolatum. Typically, the vehicle or vehicles constitute from 10% to 99.9% by weight of the compositions. The compositions of the invention may be in a form suitable for administration by injection, in the form of a formulation suitable for oral ingestion (such as capsules, tablets, capsule-shaped tablets, elixirs, for example), in the form of an ointment, cream or lotion suitable for topical administration, in a form suitable for delivery as an ophthalmic drop, in the form of an aerosol (such as liquid or powder) suitable for administration by inhalation, such as by intranasal inhalation or oral inhalation, in an appropriate form for parenteral administration, i.e., subcutaneous, intramuscular or intravenous injection. For administration as an injectable solution or suspension, non-toxic parenterally acceptable diluents or vehicles may include Ringer's solution, isotonic saline, phosphate-buffered saline, ethanol, and 1,2-propylene glycol. Some examples of vehicles, diluents, excipients and adjuvants suitable for oral use include peanut oil, liquid paraffin, sodium carboxymethylcellulose, methylcellulose, sodium alginate, acacia gum, tragacanth gum, dextrose, sucrose, sorbitol, mannitol, gelatin and lecithin. In addition, these oral formulations may contain suitable flavoring and coloring agents. When used in the form of a capsule, the capsules may be coated with compounds such as glyceryl monostearate or glyceryl distearate which retard disintegration. The adjuvants typically include emollients, emulsifiers, thickening agents, preservatives, bactericides and pH regulating agents. The solid forms for oral administration may contain binders acceptable in human and veterinary pharmaceutical practice, sweeteners, disintegrating agents, diluents, flavors, coating agents, preservatives, lubricants and / or time delay agents. Suitable binders include acacia gum, gelatin, corn starch, tragacanth gum, sodium alginate, carboxymethylcellulose or polyethylene glycol. Suitable sweeteners include sucrose, lactose, glucose, aspartame or saccharin. Suitable disintegrating agents include corn starch, methylcellulose, polyvinylpyrrolidone, guar gum, xanthan gum, bentonite, alginic acid or agar. Suitable diluents include lactose, sorbitol, mannitol, dextrose, kaolin, cellulose, calcium carbonate, calcium silicate or dicalcium phosphate. Suitable flavoring agents include peppermint oil, oil of wintergreen, cherry, orange or raspberry flavors. Suitable coating agents include polymers or copolymers of acrylic acid and / or methacrylic acid and / or their esters, waxes, fatty acid alcohols, zein, shellac or gluten. The appropriate conservatives. they include sodium benzoate, vitamin E, alpha-tocopherol, ascorbic acid, methyl-paraben, propyl-paraben or sodium bisulfite. Suitable lubricants include magnesium stearate, stearic acid, sodium oleate, sodium chloride or talc. Suitable time delay agents include glyceryl monostearate or glyceryl distearate. Liquid forms for oral administration may contain, in addition to the above agents, a liquid carrier. Suitable liquid carriers include water, oils such as olive oil, peanut oil, sesame oil, sunflower oil, safflower oil, peanut oil, coconut oil, liquid paraffin, ethylene glycol, propylene glycol, polyethylene glycol, ethanol, propanol , isopropanol, glycerol, acid alcohols fat, triglycerides or mixtures thereof. Suspensions for oral administration may also comprise dispersing agents and / or suspending agents. Suitable suspending agents include sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, sodium alginate or acetyl alcohol. Suitable dispersing agents include lecithin, polyoxyethylene esters of fatty acids such as stearic acid, mono-oleate or di-oleate, di-stearate or polyoxyethylene sorbitol di-laurate, mono-oleate or di-oleate, di-stearate or di- polyoxyethylene sorbitan laurate and the like. Emulsions for oral administration may also comprise one or more emulsifying agents. Suitable emulsifying agents include dispersing agents as exemplified above or natural gums such as guar gum, acacia gum or tragacanth gum. Methods for preparing compositions that can be administered parenterally are apparent to those skilled in the art, and are described in greater detail in, for example, Remington's Pharmaceutical Science, 15th edition, Mack Publishing Company. Easton, Pa. , incorporated in the present invention for reference. Topical formulations of this invention, comprise an active ingredient together with one or more acceptable carriers, and optionally any other therapeutic ingredients. Formulations suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin to the site where the treatment is required, such as balms, lotions, creams, ointments or pastes, and drops suitable for administration to the patient. eyes, ears or nose The drops according to the present invention may comprise sterile aqueous or oily solutions or suspensions. These can be prepared by dissolving the active ingredient in an aqueous solution of a bactericidal and / or fungicidal agent and / or any other suitable preservative, and optionally including a surfactant. The resulting solution can then be clarified by filtration, transferred to an appropriate container and subjected to sterilization. Sterilization can be achieved by autoclaving or maintaining at a temperature between 90 ° C-100 ° C for half an hour, or by filtration, followed by transfer to a container using an aseptic technique. Examples of suitable bactericidal and fungicidal agents for inclusion in the drops are nitrate or phenylmercuric acetate (0.002%), benzalkonium chloride (0.01%) and chlorhexidine acetate. (0.01%). Suitable solvents for the preparation of an oily solution include glycerol, dilute alcohol and propylene glycol. Lotions in accordance with the present invention include those suitable for application to the skin or eye. An ocular lotion may comprise a sterile aqueous solution optionally containing a bactericide and may be prepared using methods similar to those described above in relation to the preparation of drops. Lotions or balsams for application to the skin may also include an agent for accelerating drying and for refreshing the skin such as an alcohol or acetone, and / or a humectant such as glycerol, or oil such as castor oil or peanut oil. . The creams, ointments or pastes according to the present invention are semi-solid formulations of the active ingredient for external application. These can be made by mixing the active ingredient in finely divided or powdered form, alone or in solution or suspension in an aqueous or non-aqueous fluid, with a greasy or non-greasy base. The base may comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol, beeswax, a metal soap, a mucilage; an oil of natural origin such as sweet almond, corn, peanut, castor oil or olive oil; wool fat or its derivatives, or such a fatty acid as stearic or oleic acid together with an alcohol such as propylene glycol or macrogols. The composition may incorporate any suitable surfactant such as an anionic, cationic or nonionic surfactant such as sorbitan esters or polyoxyethylene derivatives thereof. Suspending agents such as natural gums, cellulose derivatives or inorganic materials such as siliceous silicas, and other ingredients such as lanolin may also be included. The compositions can also be administered in the form of liposomes. Liposomes are generally obtained from phospholipids or other lipid substances, and are formed by monolamellar or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid that can form liposomes can be used. The compositions in liposome form may contain stabilizers, preservatives, excipients and the like. The preferred lipids are phospholipids and phosphatidylcholines (lecithins), both natural and synthetic. Methods for forming liposomes are known in the art and specific reference is made to this in: Prescott, Ed., Methods in Cell Biology, Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq. , whose contents are incorporated in the present invention for reference.

Dosage For the purposes of the present invention, molecules and agents can be administered to individuals as compositions either in therapeutic or preventative form. In a therapeutic application, the compositions are administered to a patient already suffering from a disease, in an amount sufficient to cure or at least partially arrest the disease and its complications. The composition must provide an amount of the molecule or agent sufficient to effectively treat the patient. The therapeutically effective dose level for any particular patient depends on a variety of factors including: the disorder being treated and the severity of the disorder; the activity of the molecule or agent used; the composition used; age, body weight, general health, gender and diet of the patient; the time of administration; the route of administration; the speed of sequestration of the molecule or agent; the duration of the treatment; the drugs used in combination or coincident with the treatment, together with other related factors well known in medicine.

The person skilled in the art can, by routine experimentation, determine an effective, non-toxic amount of the agent or compound that may be required to treat applicable diseases. In general terms, an effective dose is expected to be in the range of about 0.0001 mg to about 1000 mg per kg of body weight per 24 hours; typically, about 0.001 mg to about 750 mg per kg of body weight per 24 hours; approximately 0.01 mg to approximately 500 mg per kg of body weight per 24 hours; about 0.1 mg to about 500 mg per kg of body weight per 24 hours; about 0.1 mg to about 250 mg per kg of body weight per 24 hours; about 1.0 mg to about 250 mg per kg of body weight per 24 hours. More typically, an effective dose range is expected to be in the range of about 1.0 mg to about 200 mg per kg of body weight per 24 hours; about 1.0 mg to about 100 mg per kg of body weight per 24 hours; about 1.0 mg to about 50 mg per kg of body weight per 24 hours; about 1.0 mg to about 25 mg per kg of body weight per 24 hours; approximately 5.0 mg to approximately 50 mg per kg of body weight per 24 hours; about 5.0 mg to about 20 mg per kg of body weight per 24 hours; about 5.0 mg to about 15 mg per kg of body weight per 24 hours. Alternatively, an effective dose may be up to about 500 mg / m2. In general terms, an effective dose is expected to be in the range of about 25 to about 500 mg / m2, preferably about 25 to about 350 mg / m2, more preferred about 25 to about 300 mg / m2, even more preferred about 25 to about 250 mg / m2, still more preferred about 50 to about 250 mg / m2, and even more preferably still about 75 to about 150 mg / m2. Typically, in therapeutic applications, the treatment may be for the entire duration of the disease state. Furthermore, it will be apparent to the person skilled in the art that the optimum amount and separation of the individual doses are determined by the nature and degree of the disease state being treated, the form, route and site of administration, and the nature of the particular individual. who is being treated. Further, said optimum conditions can be determined using conventional techniques. It will also be apparent to the person skilled in the art that those skilled in the art can establish the optimum course of treatment, such as, the number of doses of the composition administered per day for a defined number of days, using tests of course determination of conventional treatment.

CpnlO agonists and antagonists The present invention also contemplates the use of CpnlO agonists and antagonists and methods of screening and production of said agonists and antagonists. The CpnlO agonists and antagonists can be designed or specifically selected in accordance with their effect on TLR signaling and immunomodulatory secretion. The antibodies can act as agonists or antagonists of CpnlO, or fragments or analogs thereof. Preferably, appropriate antibodies are prepared from discrete regions or fragments of the polypeptide. of CpnlO, in particular; those involved in conferring protease activity and / or binding to partner or substrate. An antigenic CpnlO polypeptide contains at least about 5, and preferably at least amino acids approximately. Methods for the generation of appropriate antibodies will be readily appreciated by those skilled in the art. For example, an anti-CpnlO monoclonal antibody, typically containing Fab portions, can be prepared using the hybridoma technology described in Antibodies-A Laboratory Manual, Harlow and Lane, eds. , Cold Spring Harbor Laboratory, N.Y. (1988). In essence, in the preparation of monoclonal antibodies directed against CpnlO, or fragment or analogue thereof, any technique that provides for the production of antibody molecules using continuous cell lines in culture can be used. These include the hybridoma technique originally developed by Kohler et al., 1975, Nature, 256: 495-497, as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today , 4, -72), and the hybridoma-EBV technique to produce human monoclonal antibodies (Colé et al., In Monoclonal Antibodies and Cancer Therapy, pp. 77-96, Alan R, Liss, Inc., (1985) Immortal antibody producing cell lines can be created using techniques other than fusion, such as direct transformation of B lymphocytes with oncogenic DNA, or transfection with Epstein-Barr virus See, for example, M. Schreier et al., " Hybridoma Techniques "(1980); Hammerling et al., "Monoclonal Antibodies and T-cell Hybridomas" (1981); Kennett et al. , "Monoclonal Antibodies" (1980). In summary, the means to produce a hybridoma from which the monoclonal antibody is produced, a myeloma or other self-perpetuating cell line are fused with lymphocytes obtained from the spleen of a hyperimmunized mammal with a recognition factor binding portion thereof, or recognition factor, or a DNA binding portion. specific origin of the same. Hybridomas that produce a monoclonal antibody useful in the practice of this invention are identified by their ability to exhibit immunoreaction with the recognition factor of the present invention and their ability to inhibit the specified transcription activity in the target cells. A monoclonal antibody useful in the practice of the present invention can be produced by initiating a monoclonal hybridoma culture comprising a nutrient medium containing a hybridoma that secretes antibody molecules of appropriate antigenic specificity. The culture is maintained under conditions and for a sufficient time for the hybridoma to secrete the antibody molecules in the medium. The antibody-containing medium is then collected. The antibody molecules are then They can isolate using well-known techniques. Similarly, there are several methods known in the art that can be used for the production of polyclonal antibodies. For the production of anti-CpnlO polyclonal antibody, several host animals can be immunized by injection with CpnlO, or a fragment or analogue thereof, including but not limited to rabbits, chickens, mice, rats, sheep, goats, and the like. In addition, the CpnlO polypeptide or fragment or analogue thereof can be conjugated to an immunogenic carrier, for example bovine serum albumin (BSA) or limpet hemocyanin (KLH). In addition, various adjuvants can be used to increase the immune response, including but not limited to Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surface-active substances such as lysolecithin, "pluronic" polyols, polyanions, peptides, oily emulsions, limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. Screening for the desired antibody can also be achieved using a variety of techniques known in the art. Tests for immunospecific binding of antibodies may include, but are not limit to, radioimmunoassays, ELISAs (enzyme-linked immunosorbent assay), sandwich immunoassays, immuno-radiometric tests, gel diffusion precipitation reactions, immunodiffusion tests, in situ immunological tests, Western blot analysis, precipitation reactions , agglutination tests, complement fixation tests, immunofluorescence tests, tests with protein A, and immunoelectrophoresis tests, and the like (see, for example, Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley &Sons, Inc., New York). The antibody binding can be detected by virtue of a detectable label in the primary antibody. Alternatively, the antibody can be detected due to its binding to a secondary antibody or reagent that is appropriately labeled. A variety of methods are known in the art to detect binding in an immunoassay and are within the scope of the present invention. The antibody (or fragment thereof) created against CpnlO or a fragment or analogue thereof has binding affinity for CpnlO. Preferably, the antibody (or fragment thereof) has binding affinity or an avidity of greater than about 10 5 M 1, more preferred greater than about 10 6 M 1, even more preferred greater than about 10 7 M 1 and more preferably greater than approximately 108 M_1. In terms of obtaining an appropriate amount of an antibody according to the present invention, the antibody (s) can be made using batch fermentation with serum-free medium. After fermentation, the antibody can be purified through a multi-step procedure that incorporates the steps of chromatography and inactivation / viral clearance. For example, the antibody can be first separated using affinity chromatography with protein A and then treated with solvent / detergent to inactivate any lipid-coated viruses. Additional purification can be used, typically by anionic and cation exchange chromatography to remove residual proteins, solvents / detergents and nucleic acids. The purified antibody can be further purified and formulated in 0.9% saline using columns for gel filtration. The formulated bulk preparation can then be sterilized and filtered to remove virus and dispensed. Agonists and antagonists other than antibodies are also contemplated. A candidate agonist or antagonist can be identified by an ability to form a molecular complex with one or more of the TLRs, and / or its adapter molecules as an agonist. can identify a candidate antagonist by an ability to prevent or alter the formation of a molecular complex comprising CpnlO, and one or more of the TLRs and / or their adapter molecules to act as an antagonist. Techniques and methods for identifying and producing agonists and antagonists are well known to those skilled in the art, including screening of groups of molecules such as groups of synthetic chemical compounds such as combinatorial groups, computer-aided screening of structural databases, modeling and / or computer-aided design, or more traditional biophysical techniques that detect molecular binding interactions. The present invention is described below with reference to specific examples, which should not be considered in any way as limiting the scope of the invention.

EXAMPLES EXAMPLE 1 Evaluation of efficacy of CpnlO in an asthma model by house dust mite in sheep Inflammation of the airway is crucial 4 for the pathogenesis of asthma and involves the recruitment and activation of eosinophils, mast cells, neutrophils and lymphocytes into lung tissue and bronchioalveolar spaces (Busse et al., 2001). The house dust mite antigen has been shown to be the most common allergen that causes allergic asthma in humans, and sensitized individuals develop specific IgE and IgG humoral responses (Roche et al., 1997). The appropriate animal-asthma model could allow definite and controlled investigations to be carried out with direct relevance to the human disease. Previous models of asthma in sheep are based on animals sensitized to nematode allergens (Ascaris) and the results are extrapolated to the evaluation of physiological and pharmacological effects of potential antiasthmatic drugs. Recently a model of allergic lung inflammation has been established in sheep using the house dust mite as an allergen with direct relevance to allergic disease in humans. In this model, sensitized sheep develop allergen-specific IgE responses with recruitment of neutrophils, eosinophils and lymphocytes activated into lung tissue and BAL with kinetics similar to that of humans exposed to the HDM allergen (Bischof et al., 2003) .

The experiments described in the present invention consider whether CpnlO administered by intravenous injection or instilled directly into the lung may or may not affect the clinical and immunological manifestations of the allergic response to exposure to domestic dust mites in sensitized sheep.

Materials and methods Sheep (n = 10) were immunized with solubilized extract of house dust mite and selected for high allergen-specific IgE responses and bronchoalveolar lavage (BAL) eosinophilia. The animals are then treated with CpnlO (0.5, 4, or 16 mg) or vehicle delivered by intravenous injection (n = 5), or direct delivery on one side of the lung and vehicle on the other side of the lung before exposure to HDM using bronchoscope with optical fiber. The bronchoalveolar lavage fluid and blood samples are collected at 6 and 48 hours and 7 days after exposure to HDM to enumerate differential cell counts, BAL cytokine quantification, and serum IgE quantitation. BAL cells are also stored in RNA isolation medium (Trizol) and frozen for subsequent RT-PCR analysis.

Results In general, the results presented in the present invention show that CpnlO has a dramatic and dose-responsive effect on allergic inflammation in an asthma model in sheep.

Intravenous delivery of CpnlO Intravenous administration of CpnlO results in a neutrophil reduction of the BAL at the 6-hour time point in a dose-dependent manner. The number of neutrophils present in BAL 6 hours after the second exposure to HD alone, without treatment with CpnlO, is an average of 34% among all 6 sheep in the intravenous group. The percentage of neutrophils, expressed as an average of the group, declines as the dose of CpnlO increases. At the 6-hour time point, doses of 0.5 mg, 4 mg and 16 mg of CpnlO result in 15%, 12% and 8% of neutrophils, in BAL fluid, respectively. Intravenous administration of CpnlO results in a reduction of BAL eosinophils at the time point of 48 hours in a dose-dependent manner. The number of eosinophils present 48 hours after the second exposure to HDM alone, without treatment with CpnlO, is an average of 31% among all 6 sheep in the group intravenous. The percentage of eosinophils, expressed as a group average, generally declines as the dose of CpnlO increases. At the 48-hour time point, doses of 0.5 mg, 4 mg and 16 mg of CpnlO result in 15%, 16% and 11% eosinophils, in BAL fluid, respectively.

Intrapulmonary supply of CpnlO The percentages of BAL neutrophils at the 6 and 48 hour time points differ between the right and left lungs of individual sheep through most of the treatments. However, there is no consistent trend between the left (treated with CpnlO) and the right (treated with vehicle) lungs. The percentages of BAL eosinophils at the 6 and 48 hour time points also differ between the right and left lungs of individual sheep through most treatments. In general, there is no consistent trend between the left lung (treated with CpnlO) and the right lung (treated with vehicle) with the exception of the 4 mg dose of CpnlO at 48 hours after exposure. The percentage of BAL eosinophils in each sheep is lower in the left lung 48 hours after exposure to HDM and the administration of 4 mg (total dose) of CpnlO to the left lung compared to the right lung control. The data averaged from all the sheep in the intra-pulmonary group show that the local infusion of 4 mg of CpnlO to the left lung reduces the level of eosinophilia in that lung to 50% of the level of the right lung (control, treated with vehicle).

Baseline IgE responses Serum IgE levels are evaluated before (dO) and seven days after (d7) each of the HDM exposures, as shown in Figure 5. In the absence of CpnlO (Ia and 2nd exposures to HDM), serum IgE levels are clearly elevated after exposing the airway to HDM. Sheep # 23 is the only sheep that does not show a high IgE response after initial exposures to HDM. Compared to baseline IgE levels before the first exposure to HDM, slightly elevated IgE levels were observed before the second and third exposures to HDM; in each case this may reflect a slow return to baseline values after exposure to HDM 2 weeks earlier.

Responses of IgE to exposure to HDM in the presence of CpnlO CpnlO, regardless of the dose or mode of administration, has a marked effect on specific serum IgE responses of HDM as assessed 7 days after exposure to HDM . The administration of CpnlO results in the blocking of serum IgE responses, with maintenance close to baseline levels particularly evident with the fourth and fifth exposures to HDM.

Eosinophils of the bronchoalveolar lavage BAL is sampled in the baseline (0 hours), 6 hours and 48 hours after exposing the airways to HDM and drug treatment and the numbers of eosinophils are counted. The results to date are plotted in figure 1 as percent of eosinophils of the total cell population in the wash fluid sampled at 48 hours after exposure to HDM. Intravenous injection of CpnlO administered before exposure to HDM results in a reduction of up to 15-fold in BAL eosinophilia at maximum eosinophil recruitment at 48 hours after exposure. When CpnlO is administered unilaterally in the left lung, and the vehicle administered only to the right lung (control) using a fiberoptic bronchoscope, shows a 4-fold reduction in the percentage of eosinophils in the BAL coming from the lung treated with CpnlO against the control lung.

Serum immunoglobulin responses to HDM exposure Serum is collected on day 0 and day 7 after administration of either vehicle or CpnlO and exposure to HDM. The sera are then analyzed and HDM specific IgE levels are evaluated using ELISA as described in more detail in Bischof et al, 2003, and the results are presented in the present invention as Figure 2. The data indicate serum levels of HDM specific IgE after each exposure to HDM with vehicle control administration, and after 4 mg of CpnlO and exposure to HDM.

Changes in airway epithelium, mucus secreting cell and bronchial gland Mucus-secreting cells Mucus-secreting cell hyperplasia is a characteristic feature of the pathology of asthma that is induced by allergic inflammation of the respiratory tract. It is known that after allergen challenge, the number of mucus-secreting cells per unit length of the airway epithelium increases. Concomitant with an increase in number, the mucus-secreting cells also increase in size and show large multicolored cytoplasm when stained with histological PAS / Alcian blue stains. The analysis of airway epithelium, mucus secreting cells and bronchial glands is carried out on histological slides stained with PAS / alciano blue. This analysis is performed in glandular bronchial cartilaginous airways which have a diameter between 1800 and 3300 μ.

Intrapulmonary administration of CpnlO to sheep exposed to HDM The average number of mucus-secreting cells per mm of basement membrane of the respiratory tract is 33 in both the left and in the lungs of sheep administered with intrapulmonary CpnlO (n = 4 sheep). The mucus-secreting cells are large and filled with granules stained red and blue (Figure 5).

Intravenous administration of CpnlO to sheep exposed to HDM Compared to the intrapulmonary delivery groups, there are fewer mucus secreting cells in the bronchial epithelium of sheep treated with 4 mg of CpnlO by the intravenous route of administration. The average number of mucus-secreting cells per mm of airway basement membrane is 16 in sheep given intravenous CpnlO (n = 6 sheep). This is less than half the number of mucus-secreting cells of the intrapulmonary group treated with vehicle (33 mucus-secreting cells per mm of basement membrane of the airway). Mucus-secreting cells in the intravenous CpnlO group are generally less mature in the sense that they are smaller, more idal, and have less cytoplasmic staining.

Epithelium It is known that the epithelium that lines the airways is an important tissue component in asthma conditions because it forms a first line barrier against exogenous materials transported by 4 Intrapulmonary administration of CpnlO to sheep exposed to HDM In the intra-pulmonary groups of both vehicle and CpnlO, the airway epithelium is more columnar and in some airways it is hyperplastic (Figure 5).

Intravenous administration of CpnlO to sheep exposed to HDM In contrast to the intra-pulmonary group, the airway epithelium is more id (Figure 6).

Bronchial glands The glands associated with the bronchial airways can be stimulated to secrete mucus into the glandular lumens after allergen challenge. When stained with histological PAS / Alcian blue dyes, the mucus can be stained red (indicating neutral mucins) or blue (indicating acid mucins).

Intra-pulmonary administration of CpnlO to sheep exposed to HDM Analysis of histological slides stained with PAS / Alciano blue reveals that the bronchial glands of both groups of sheep treated intra-pulmonally secrete mucus 48 hours after exposure to HDM and treatment with CpnlO / vehicle (figure 5). The mucus in two sheep is stained red with PAS (figure 5) while in the other two sheep the mucus is predominantly dyed blue. A systematic analysis of all available bronchial glands to be graded in each sheep indicates that on average the bronchial gland lumen is occluded by 23% with mucus in vehicle-treated lungs and 27% occluded in lungs treated with CpnlO (n = 4 sheep).

Intravenous administration of CpnlO to sheep exposed to HDM There is markedly less mucus present in the glandular lumens of sheep treated intravenously compared with those observed in intra-pulmonary groups (Figure 6). The analysis of histological slides stained with PAS / Alcian blue reveals that the bronchial glands of sheep treated intravenously have small amounts of mucus in the lumen 48 hours after exposure to HDM and treatment with intravenous CpnlO. The mucus is predominantly blue in color (for example figure 6). A systematic analysis of all available bronchial glands to be graded in sheep treated intravenously it indicates that on average the lumen of the bronchial gland is occluded in 4.8% with mucus (n = 6 sheep).

Discussion The data from this test clearly show that CpnlO delivered systemically has the ability to alleviate allergic inflammation which is underlying the pathophysiology of asthma. It is known that neutrophils, eosinophils and IgE antibodies are all important components of the inflammation associated with asthma. This test shows that CpnlO has significant buffering effects on eosinophilia and neutrophilia in BAL and specific IgE level of serum HDM after exposure to HDM. The evaluation of the administration of CpnlO to sheep sensitized to an asthma-inducing allergen relevant to asthma in humans has shown a dose-related reduction in eosinophils that accumulate in the bronchoalveolar space at 48 hours after exposure to antigen The eosinophilia in BAL of sheep exposed to HDM is similar to the level of eosinophils found in human asthmatics (Metzger et al, 1987). Another feature of the model used in this study is that sheep with elevated IgE serum titers Specific (ie, allergic sheep) are correlated with those individuals that generate elevated and prolonged BAL eosinophilia when exposed locally to HDM, compared to non-allergic and control sheep (Bischof, 2003). The intravenous administration of CpnlO has clear effects dependent on the attenuation dose of allergic inflammation. The high dose (16 mg) of CpnlO is the most effective to reduce the percentage of neutrophils and eosinophils in BAL. The 16 mg dose of CpnlO also has a significant buffering effect on the blood eosinophil level 24 and 48 hours after exposure compared to eosinophils counted at the same time points for the 4 mg dose tests and the second exposure to HDM alone. CpnlO seems to have no detrimental effects on the sheep throughout the entire trial because their main clinical signs remain within the normal physiological range throughout the entire experimental period. This indicates that CpnlO is well tolerated in sheep at all doses used, regardless of whether the supply is through the intravenous or intrapulmonary route. The post mortem histological analysis of the animal results in some interesting observations. The Intravenous administration of 4 mg of CpnlO results in a marked reduction of a number of pathological parameters associated with allergic inflammation. In general, intravenously treated sheep have less infiltration of inflammatory cells into the airway walls, have fewer mucus-secreting cells and are less mature, and have a lower mucus content in bronchial glandular lumens. There seems to be little difference between the degree of pathology between lungs infused with vehicle (control) and with CpnlO. In general, sheep exposed by intrapulmonary route have a pathology similar to that observed in animals exposed to HDM alone. In summary, this test clearly demonstrates that the systemic delivery of CpnlO has the ability to attenuate the underlying inflammatory components of asthma.

EXAMPLE 2 Compositions for treatment In accordance with the best mode of carrying out the invention provided in the present description, specific preferred compositions are indicated below. The following should be considered as examples merely illustrative of compositions and in no way as a limitation to the scope of the present invention.

EXAMPLE 2 (A) Composition for parenteral administration A composition for parenteral injection can be prepared to contain from 0.05 mg to 5 g of an appropriate agent or compound as described herein in 10 ml to 2 liters of 1% carboxymethylcellulose. Similarly, an intravenous infusion composition can comprise 250 ml of sterile Ringer's solution, and 0.05 mg to 5 g of an appropriate agent or compound as described in the present invention.

EXAMPLE 2 (B) Composition for oral administration A composition of an appropriate agent or compound can be prepared in the form of a capsule by filling a standard two-piece hard gelatin capsule with 500 mg of the agent or compound, in powder form, 100 mg of lactose, 35 mg of talc and 10 mg. mg of magnesium stearate.

References Busse WW, Lemanske RF Jr. Asthma. N Engl J Med 2001; 344: 350-62. Roche N, Chinet TC, Huchon GJ. Allergic and nonallergic interactions bet in house dust mite allergens and airway mucosa. Eur Respir J 1997; 10: 719-26. Bischof RJ, Snibson K, Shaw R, eeusen ENT. Induction of allergic inflammation in the lungs of sensitized sheep after local challenge with house dust limit. Clin Exp Allergy 2003; 33: 367-75. Metzger WJ, Zavala D, Richerson HB et al. Local allergen challenge and bronchoalveolar lavabe of allergic asthmatic lungs. Description of the model and local airway inflammation. Am Rev Respir Dis 1987; 135: 433-40.

Claims (25)

NOVELTY OF THE INVENTION Having described the present invention, it is considered as a novelty and therefore the content of the following is claimed as property: CLAIMS

1. - A method for inhibiting a hypersensitivity reaction in an individual, characterized in that said method comprises administering an effective amount of eukaryotic chaperonin.

2. - The method according to claim 1, characterized in that the hypersensitivity reaction involves activation. of cells that are selected from the group comprising: basophils, eosinophils, mast cells, neutrophils and lymphocytes.

3. The method according to claim 1, characterized in that the hypersensitivity reaction involves the activation of Toll-like receptor (TLR) signaling.

4. - The method according to any of claims 1 to 3, characterized in that the hypersensitivity reaction involves high levels of eosinophils and immunoglobulin E.

5. - The method according to any of claims 1 to 4, characterized in that the hypersensitivity reaction is an inflammatory reaction.

6. - The method according to claim 5, characterized in that the hypersensitivity reaction is selected from the group comprising: food allergy, dermatitis, allergic conjunctivitis, rhinitis, eczema, anaphylaxis and respiratory diseases associated with inflammation of the respiratory tract .

7. - The method according to claim 6, characterized in that the respiratory disease is selected from the group comprising: asthma, allergic asthma, intrinsic asthma, occupational asthma, Acute Respiratory Distress Syndrome (ARDS) and Chronic Obstructive Pulmonary Disease (COPD).

8. - The method according to any of claims 1 to 7, characterized in that said eukaryotic chaperonin comprises a polypeptide sequence that is selected from the group comprising SEQ ID NO: 1, 2, 3, 4, 7, 9, 11, 13, 15 or 17.

9. The method according to claim 8, characterized in that said polypeptide sequence is encoded by a polynucleotide sequence that is selected from the group that comprises SEQ ID NO: 5, 6, 8, 10, 12, 14, 16 or 18.

10. A method for treating or preventing a disorder associated with a hypersensitivity reaction in an individual, the method comprising administering to the individual an amount effective of chaperonin 10, characterized in that the chaperonin 10 modulates the signaling of a Toll-like receiver.

11. - The method according to claim 10, characterized in that the Toll-like receptor is selected from the group comprising: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, and TLR10.

12. - The method according to claim 10 or 11, characterized in that said eukaryotic chaperonin comprises a polypeptide sequence that is selected from the group comprising SEQ ID NO: 1, 2, 3, 4, 7, 9, 11, 13, 15 or 17.

13. The method according to claim 12, characterized in that said polypeptide sequence is encoded by a polynucleotide sequence which are selected from the group comprising SEQ ID NO: 5, 6, 8, 10, 12, 14, 16 or 18. 14. The method according to any of claims 1 to 13, characterized in that the method also comprises the administration of at least one additional agent.

14. - The method according to claim 13, characterized in that the agent is an immunomodulator.

15. - The method according to claim 14, characterized in that the immunomodulator is a type I interferon.

16. The method according to claim 15, characterized in that the interferon is IFNa or? G? ß.

17. A composition for treating or preventing a disorder associated with a hypersensitivity reaction in an individual, the composition comprising an effective amount of chaperonin., together with an immunosuppressive agent.

18. The composition according to claim 17, characterized in that the immunosuppressive agent is selected from the group comprising anti-inflammatory compounds and bronchodilator compounds.

19. The composition according to claim 17, characterized in that the immunosuppressant agent is selected from the group comprising: cyclosporin, tacrolimus, sirolimus, mycophenolate mofetil, methotrexate, cromoglycolates, theophylline, leukotriene antagonist, and antihistamine, or a combination of them.

20. The composition according to any of claims 17 to 19, characterized in that the immunosuppressive agent is a specific antibody directed against B or T lymphocytes.

21. The composition according to any of claims 17 to 20, characterized in that The immunosuppressive agent is a specific antibody directed against B or T lymphocyte surface receptors that mediate its activation.

22. The composition according to any of claims 17 to 21, characterized in that the composition also comprises a spheroid.

23. - A method for treating or preventing a disorder associated with a hypersensitivity reaction in an individual, the method comprising administering an effective amount of the composition according to any of claims 17 to 22.

24. - The composition in accordance with any of claims 17 to 23, characterized in that the eukaryotic chaperonin 10 comprises a polypeptide sequence that is selected from the group comprising SEQ ID NO: 1, 2, 3, 4, 7, 9, 11, 13, 15 or 17.

25. - The composition according to claim 24, characterized in that the sequence of The polypeptide is encoded by a polynucleotide sequence that is selected from the group comprising SEQ ID NO: 5, 6, 8, 10, 12, 14, 16 or 18.