WO2015110536A1 - Facteur de stimulation des colonies de granulocytes et macrophages pour le traitement et la prévention de formes graves de l'asthme bronchique - Google Patents

Facteur de stimulation des colonies de granulocytes et macrophages pour le traitement et la prévention de formes graves de l'asthme bronchique Download PDF

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WO2015110536A1
WO2015110536A1 PCT/EP2015/051266 EP2015051266W WO2015110536A1 WO 2015110536 A1 WO2015110536 A1 WO 2015110536A1 EP 2015051266 W EP2015051266 W EP 2015051266W WO 2015110536 A1 WO2015110536 A1 WO 2015110536A1
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csf
composition
treatment
bronchial asthma
administered
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PCT/EP2015/051266
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Lars Heslet
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Reponex Pharmaceuticals Aps
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    • 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
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/193—Colony stimulating factors [CSF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics

Definitions

  • Granulocyte-macrophage colony-stimulating factor for the treatment and prevention of severe forms of bronchial asthma
  • the present invention relates to granulocyte-macrophage colony-stimulating factor (GM-CSF) for use in the treatment of severe persistent and acute severe bronchial asthma, the latter formerly known as status asthmaticus.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the invention further relates to the administration of GM-CSF to a patient with said forms of bronchial asthma in need thereof.
  • Granulocyte-macrophage colony-stimulating factor was originally identified as a hemopoietic growth factor.
  • Human GM-CSF stimulates the growth of myeloid and erythroid progenitors in vitro and activates monocytes, macrophages and granulocytes in several immune and inflammatory processes (Gasson et al., 1990b; Gasson et al.,
  • GM- CSF GM- CSF also was discovered as having a variety of effects on cells of the immune system expressing the GM-CSF receptor (for review, see Hamilton, 2002; de Groot et al., 1998).
  • Granulocyte-macrophage colony-stimulating factor inhalation therapy has been disclosed for treating patients suffering from idiopathic pulmonary alveolar proteinosis, a rare lung disease characterized by the accumulation of surfactant that fills the terminal airways and alveoli, thereby impairing respiratory function (Tazawa et al., 2006). Summary of invention
  • the present invention relates to GM-CSF for use in the treatment of severe persistent and acute severe bronchial asthma.
  • the GM-CSF may be
  • Figure 1 A theory of the consequence of the stimulation of the GM-CSF receptor on the surface of cells in the airways (the lumen of the small airways) and in the blood compartments, such as the blood or tissue.
  • A) denotes inhalation of GM-CSF into the airways and B) systemic administration, e.g. in an asthmatic patient.
  • C) denotes the air- blood barrier, which may act as a "sealed lining", with lack of penetration of proteins.
  • the air-blood barrier (C) ensures that inhaled GM-CSF mainly or only enhances the macrophage transformation to dendritic cells on the "airside” and the recruitment of T - Cells (CD 8+ and CD 16+) to the airways by IL-2.
  • the cytokine IL-2 is expressed by macrophages located at the bronchioli and alveoles.
  • ET denotes eosinophilic toxins secreted as a result of the TH2 response.
  • MF denotes macrophages.
  • Figure 2 A theory of the consequence of the stimulation of the GM-CSF receptor on the surface of cells in the airways (the lumen of the small airways) and in the blood compartments, such as the blood or tissue It is hypothesised that systemic
  • GM-CSF inhaled GM-CSF
  • inhaled GM-CSF may counteract the already existing immunoinflammatory condition, enhancing the formation of the TH1 subset. Inhaled GM-CSF does not bring about a negative reaction - due to the fact that it does not penetrate the air-blood barrier and enter the systemic compartment as shown in figure 1 .
  • Figure 3 Bar graph showing the effect of 2 times daily inhalation of 300 micrograms of GM-CSF to a patient suffering from bronchiectasis.
  • the X axis is days, first dose was given the day before the day represented by the first column and the last dose was given in the morning of day 7.
  • the present invention relates to GM-CSF for use in the treatment of severe forms of bronchial asthma.
  • the GM-CSF may be administered in any suitable way known to a person of skill, particularly by inhalation of a nebulized solution of GM-CSF or of GM- CSF in powder form, or by any other appropriate means of intratracheal, intrabronchial or bronchoalveolar administration.
  • the GM-CSF may be administered to human subjects including both adults and children.
  • the GM-CSF may be purified or concentrated natural human GM-CSF or a functional homologue thereof, however prepared.
  • the GM-CSF may further be a recombinantly produced human GM-CSF.
  • the GM-CSF may be purified or concentrated natural human GM-CSF, or recombinantly produced human GM-CSF, or a functional homologue thereof, however prepared.
  • Bronchial asthma is caused by a combination of genetic and environmental factors. Thus may also be classified as atopic (extrinsic) or non-atopic (intrinsic).
  • Symptoms include wheezing, coughing, chest tightness, and shortness of breath.
  • Mild bronchial asthma is clinically characterized, by a reduced forced expiratory volume in 1 second (FEV1 ) and/or in peak expiratory flow rate (PFR).
  • FEV1 forced expiratory volume in 1 second
  • PFR peak expiratory flow rate
  • Treatment of acute symptoms in its initial condition with 100 % reversibility is usually an inhaled short-acting 2 -adrenergic receptor agonist (such as salbutamol). Symptoms can be prevented by avoiding triggers, such as allergens and irritants,.
  • the BA become severe BA or acute severe BA 1 , an acute chronic inflammatory condition, only responsive to anti-inflammatory treatment, firstly to increasing doses of inhaled, preferably steroids; and finally to increased doses of systemically administered steroids, as e.g . peroral glucocorticoids.
  • bronchial asthma Acute severe BA.
  • diagnosis of bronchial asthma is usually made on the basis of the pattern of symptoms and signs and/or the response to therapy over time.
  • the prevalence of bronchial asthma has increased significantly since the 1970s. As of 2010, 300 million people were affected worldwide. In 2009 bronchial asthma caused 250,000 deaths globally. Despite this, with proper control of bronchial asthma with step down therapy, prognosis is generally good.
  • Bronchial asthma is defined by the Global Initiative for Asthma as "a chronic inflammatory disorder of the airways in which many cells and cellular elements play a role.
  • the chronic inflammation is associated with airway hyper-responsiveness that leads to recurrent episodes of wheezing, breathlessness and as objective dyspnea, combined with the subjective sensation of chest tightness. Further, coughing particularly at night or in the early morning becomes more and more frequent. '
  • BA asthma is a chronic obstructive condition
  • chronic obstructive pulmonary disease refers specifically to combinations of disease that are irreversible such as bronchiectasis, chronic bronchitis, and emphysema.
  • the airway obstruction in bronchial asthma is usually reversible; however, if left untreated, the chronic inflammation from bronchial asthma can lead the lungs to become irreversibly obstructed because of airways remodeling.
  • bronchial asthma affects the bronchi, not the alveoli.
  • bronchial asthma The persistent forms of bronchial asthma may be classified as Mild, Moderate and Severe BA.
  • GM-CSF may be used to treat all types of clinical classifications of bronchial asthma mentioned herein above, but particularly persistent forms of bronchial asthma, such as severe persistent bronchial asthma, which display a limited clinical response, e.g. no reversibility to currently used therapeutics (clinically and or related to measurements of lung function FEV1 and or PEFR ⁇ 15% after intervention with ⁇ 2 agonists).
  • GM-CSF is used to treat acute severe bronchial asthma
  • Severe BA Irreversible condition fie to conventional treatment therapy including systemic steroids) also its acute form of BA either prophylactically, preemptively or therapeutically. Patients suffering from severe bronchial asthma and those at risk of developing acute severe bronchial asthma may particularly benefit from treatment with GM-CSF. Please note, that the maximal conventional intervention toward the antiinflammatory component is not tappered off or even modified - due to the effect of iGM-CSF.
  • dendritic cells may also clear the rejected muciosa.
  • Prophylaxis may be equivalent to reducing risk of the development of symptoms associated with bronchial asthma, such as airway hyperresponsiveness, recurrent episodes of wheezing, breathiessness, chest tightness, coughing particularly at night or in the early morning and widespread, but variable airflow obstruction within the lung.
  • GM-CSF is believed to exert its effect on macrophages in the small airways, which are thus stimulated to transform into dendritic cells which are able to stop the rejection process, and the formation of dendritic cells will enhance the clearing of the rejected material of the airways associated with severe forms of bronchial asthma.
  • a prophylactically intervention i a predefined group of BA, such as falling response to maximal anti-asthmatic intervention including treatment with systemic steroid Comparison of the effect and AE S of Inflammatory and immuno-inflammatory processes in severe asthma
  • Bronchiolitis is already present in BA, thought to be induced by eosinohilic toxin (ET) released by activated eosiophilic cells, being peribonchial cuffed around the bronchioli.
  • E eosinohilic toxin
  • the GM-CSF should only be administerred as inhaled therapy not as systemical intervention.
  • Colony-stimulating factors are glycoproteins that stimulate the growth of hematopoietic progenitors and enhance the functional activity of mature effector cells.
  • CSFs ensure the self-renewal of the staminal pool and activate the first stage of hematopoietic differentiation; in the middle stage, when cell proliferation is associated with a progressive acquisition of the characteristics of mature cells, they enormously enhance the number of differentiating cells; in the terminal stage they control the circulation and the activation of mature cells.
  • Mature GM-CSF is a monomeric protein of 127 amino acids (SEQ ID NO: 1 ) with several potential glycosylation sites. The variable degree of glycosylation results in a molecular mass or weight range between 14 kDa and 35 kDa. Non-glycosylated and glycosylated GM-CSF show similar activity in vitro (Cebon et al., 1990). The crystallographic analysis of GM-CSF revealed a barrel-shaped structure composed of four short alpha helices (Diederichs et al., 1991 ). There are two known sequence variants of GM-CSF. The active form of the GM-CSF protein is found extracellularly as a homodimer in vivo. The protein sequence of GM-CSF of Homo Sapiens (SEQ ID NO:1 ):
  • GM-CSF-R GM-CSF receptor
  • the native receptor is composed of at least two subunits, alpha and beta.
  • the alpha subunit imparts ligand specificity and binds GM-CSF with nanomolar affinity (Gearing et al., 1989; Gasson et al., 1986).
  • the beta subunit is also part of the interleukin-3 and interleukin-5 receptor complexes and, in association with the GM-CSF receptor alpha subunit and GM-CSF, leads to the formation of a complex with picomolar binding affinity (Hayashida et al., 1990).
  • GM-CSF interacts with the beta subunit of its receptor via a very restricted region in the first alpha helix of GM-CSF (Shanafelt et al., 1991 a; Shanafelt et al., 1991 b; Lopez et al., 1991 ). Binding to the alpha subunit could be mapped to the third alpha helix, helix C, the initial residues of the loop joining helices C and D, and to the carboxy-terminal tail of GM-CSF (Brown et al., 1994).
  • GM-CSF trimeric receptor complex leads to the activation of complex signaling cascades involving molecules of the JAK/STAT families, She, Ras, Raf, the MAP kinases, phosphatidylinositol-3 -kinase and N FKB, finally leading to transcription of c-myc, c-fos and c-jun.
  • Activation is mainly induced by the beta subunit of the receptor (Hayashida et al., 1990; Kitamura et al., 1991 ; Sato et al., 1993).
  • the shared beta subunit is also responsible for the overlapping functions exerted by IL-3, IL-5 and GM-CSF (for review see: de Groot et al., 1998).
  • GM-CSF functions especially as a pro-inflammatory cytokine.
  • Macrophages e.g. alveolar macrophages types I & II and monocytes as well as neutrophils and eosinophils are activated by GM-CSF, resulting in the release of other cytokines and chemokines, matrix-degrading proteases, increased HLA expression and increased expression of cell adhesion molecules or receptors for CC-chemokines which in turn leads to increased chemotaxis of inflammatory cells into inflamed tissue.
  • macrophages, eosinophils and basophils express GM-CSF receptors on their cell surface.
  • the macrophages are on the air side of the air-blood barrier and the eosinophils are on the other side in the blood compartment at the level of the peripheral airways.
  • the present invention may decrease the T h 2 immune response that is mediated by IgE antibodies, decrease activation of eosinophilic cells, and decrease eosinophilic toxin (ET).
  • the present invention may further increase a T h 1 immune response by increasing cellular immunity and transforming the resting macrophages into dendritic cells. The increase in the T h 1 immune response may occur simultaneously with, or before or after the decrease in the T h 2 immune response.
  • the switch of the derailed T h 2 response to promotion of the T h 1 subset is caused by the sole stimulation of the GM- CSF receptors on cells on air side (for example the cells in or bordering the lumen) of the small airways. Subsequently dendritic cells orchestrate the immune defense in concert with a T helper cell inflammation.
  • GM-CSF is a protein of a considerable size, and therefore the transport of the protein across the air-blood barrier is minor and in some cases non-existent.
  • Administration of GM-CSF into the airways may thus ensure that the majority of the GM-CSF reaches the cells in the airways (in the lumen of the bronchioles or alveoli), and not the cells in the blood or tissue compartments.
  • GM-CSF only enhances the macrophage transformation into dendritic cells on the "air side".
  • the macrophages located in the bronchioles and alveoli express the cytokine IL-2, which is responsible for the recruitment of T cells (CD8+ and CD16+) to the airways. This means that the initially derailed hyperinflammatory state is modified by recruiting T cells from the circulation.
  • a functional homologue of GM-CSF is a polypeptide having at least 50% sequence identity with SEQ ID NO. 1 and has one or more GM-CSF functions, such as the stimulation of the growth and differentiation of hematopoietic precursor cells from various lineages, including granulocytes, macrophages, eosinophils and erythrocytes.
  • GM-CSF regulates multiple functions of alveolar macrophages (AMs).
  • AMs alveolar macrophages
  • GM-CSF stimulation of AMs has been documented to enhance their selective response to noxious ingestants, i.e., stimulation of inflammation during bacterial phagocytosis, while the responses to non-noxious ingestants are generally mollified, i.e., anti-inflammatory responses during phagocytosis of apoptotic cells.
  • Further AM functions are enhanced by GM-CSF stimulation with subsequent proliferation, differentiation, accumulation and activation.
  • these GM-CSF effects also encompass cell adhesion, improved chemotaxis, Fc-receptor expression, complement- and antibody-mediated
  • GM-CSF also enhances AM cell adhesion, pathogen-associated molecular-pattern receptors, like Toll-like receptors and TLR trans-membranous signaling, surfactant protein and lipid uptake and degradation (Trapnell and Whitsett, 2002).
  • GM-CSF interacts with the AM's recognition receptors, the so-called Toll-like receptors (TLR).
  • TLR Toll-like receptors
  • GM-CSF is important for the pulmonary host defense in pneumonia because of its interaction with the TLRs' participation in host defense, resulting in enhanced clearance of the causative microorganism (Chen et al., 2007).
  • the lung has its own innate GM-CSF production, which is reduced in pneumonia and hyperoxia in relation to high 0 2 exposure, as seen in e.g. ventilator-associated pneumonia (VAP), contributing to the impairment of host defense secondary to apoptosis with poor response to infections.
  • VAP ventilator-associated pneumonia
  • the hyperoxic injury seems to be counteracted by activation of AMs by GM-CSF (Altemeier et al., 2007; Baleeiro et al., 2006) with subsequent clearance of P. aeruginosa via expression of the TLR signaling pathway (Baleeiro et al., 2006).
  • GM -CSF stimulates the in-vitro conversion of AMs into immature dendritic cells (DCs), which may be further matured by means of specific agents with respect to activating the homing of matured DCs to a specific receptor or target (Zobywalski et al., 2007).
  • the evolutionary conservation of amino-acid residues in GM-CSFs of different closely related species can be used to pinpoint the degree of evolutionary pressure on individual amino-acid residues.
  • GM-CSF amino-acid sequences are compared between species where GM-CSF function is conserved, for example, but not limited to, mammals including rodents and non-human primates (monkeys and apes). Residues that remain constant between species or are under high selective pressure are more likely to represent essential amino acids that cannot easily be substituted than residues that change between species. It is evident from the above that a reasonable number of
  • GM-CSF molecules are herein referred to as functional equivalents of human GM-CSF, and may be such as variants and fragments of native human GM-CSF as described here below.
  • the expression “variant” refers to polypeptides or proteins which are homologous to the index protein, which is suitably human GM-CSF, but which differ from the index sequence from which they are derived in that one or more amino acids within the sequence are substituted by other amino acids.
  • Amino acid substitutions may be regarded as "conservative" where an amino acid is replaced with a different amino acid with broadly similar properties. Non-conservative substitutions are where amino acids are replaced with amino acids of a different type. Broadly speaking, fewer non-conservative substitutions will be possible without altering the biological activity of the polypeptide.
  • amino acids may be grouped according to shared characteristics.
  • a conservative amino acid substitution is a substitution of one amino acid within a predetermined group of amino acids for another amino acid within the same group, wherein the amino acids within a predetermined groups exhibit similar or substantially similar characteristics.
  • one amino acid may be substituted for another within groups of amino acids characterized by having i) polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,) ii) non-polar side chains (Gly, Ala, Val, Leu, lie, Phe, Trp, Pro, and Met) iii) aliphatic side chains (Gly, Ala Val, Leu, lie) iv) cyclic side chains (Phe, Tyr, Trp, His, Pro) v) aromatic side chains (Phe, Tyr, Trp) vi) acidic side chains (Asp, Glu) vii) basic side chains (Lys, Arg, His) viii) amide side chains (Asn, Gin ix) hydroxy side chains (Ser, Thr) x) sulfur-containing side chains (Cys, Met), and xi) amino acids being monoa
  • a functional homologue within the scope of the present invention is a polypeptide that exhibits at least 50% sequence identity with human GM-CSF as identified by SEQ ID NO. 1 , preferably at least 60% sequence identity, for example 70% sequence identity, and preferably functional homologues have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90 % sequence identity, such as at least 91 % sequence identity, for example at least 91 % sequence identity, such as at least 92% sequence identity, for example at least 93% sequence identity, such as at least 94% sequence identity, for example at least 95% sequence identity, such as at least 96% sequence identity, for example at least 97% sequence identity, such as at least 98% sequence identity, for example 99% sequence identity with SEQ ID NO: 1 .
  • Sequence identity can be calculated using a number of well-known algorithms and applying a number of different gap penalties. Any sequence alignment algorithm, such as but not limited to FASTA, BLAST, or GETSEQ can be used for searching for homologues and calculating sequence identity. Moreover, when appropriate, any commonly known substitution matrix, such as but not limited to PAM, BLOSSUM or PSSM matrices, may be applied with the search algorithm. For example, a PSSM (position-specific scoring matrix) may be applied via the PSI-BLAST program.
  • Any sequence alignment algorithm such as but not limited to FASTA, BLAST, or GETSEQ can be used for searching for homologues and calculating sequence identity.
  • any commonly known substitution matrix such as but not limited to PAM, BLOSSUM or PSSM matrices, may be applied with the search algorithm. For example, a PSSM (position-specific scoring matrix) may be applied via the PSI-BLAST program.
  • sequence alignments may be performed using a range of penalties for gap opening and extension.
  • the BLAST algorithm may be used with a gap opening penalty in the range 5-12, and a gap extension penalty in the range 1 -2.
  • a variant or a fragment thereof according to the invention may comprise, within the same variant of the sequence or fragments thereof or among different variants of the sequence or fragments thereof, at least one substitution, such as a plurality of substitutions introduced independently of one another.
  • the same variant or fragment thereof may comprise more than one conservative amino-acid substitution from more than one group of conservative amino acids as defined herein above.
  • nonstandard amino acids include the sulfur-containing taurine, the neurotransmitter gamma-amino butyric acid (GABA) and the neurotransmitter precursor dihydroxyphenylalanine (DOPA).
  • GABA neurotransmitter gamma-amino butyric acid
  • DOPA neurotransmitter precursor dihydroxyphenylalanine
  • Other examples are lanthionine, 2-aminoisobutyric acid, and dehydroalanine.
  • Further nonstandard amino acids are ornithine and citrulline.
  • nonstandard amino acids include hydroxylated amino acids such as hydroxyproline and hydroxylysine.
  • Non-standard amino acids are usually formed through modifications to standard amino acids.
  • taurine can be formed by the decarboxylation of cysteine
  • DOPA is synthesized from tyrosine
  • hydroxyproline is made by a posttranslational modification of proline (common in collagen).
  • non-natural amino acids are those listed e.g. in 37 C.F.R. section 1 .822(b)(4), all of which are incorporated herein by reference.
  • a functional equivalent according to the invention may comprise the inclusion or substitution in the sequence of any amino acid including nonstandard amino acids.
  • a functional equivalent comprises only standard amino acids.
  • the standard and/or nonstandard amino acids may be linked by peptide bonds or by non-peptide bonds.
  • the term peptide also embraces post-translational modifications introduced by chemical or enzyme-catalyzed reactions, as are known in the art. Such post-translational modifications can be introduced prior to partitioning, if desired.
  • Amino acids as specified herein will preferentially be in the L-stereoisomeric form.
  • Amino-acid analogs can be employed instead of the 20 naturally-occurring amino acids. Several such analogs are known, including fluorophenylalanine, norleucine, azetidine-2- carboxylic acid, S-aminoethyl cysteine, 4-methyl tryptophan and the like.
  • Suitable variants will have at least 60% sequence identity, for example 70% sequence identity, and variants will preferably have at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 91 % sequence identity, for example at least 92% sequence identity, such as at least 93% sequence identity, for example at least 94% sequence identity, such as at least 95% sequence identity, for example at least 96% sequence identity, such as at least 97% sequence identity, for example at least 98% sequence identity, such as at least 99 % sequence identity with the predetermined sequence of human GM-CSF (SEQ ID NO: 1 ).
  • Functional equivalents may further comprise chemical modifications such as ubiquitination, labeling (e.g., with radionuclides, various enzymes, etc.), pegylation (derivatization with polyethylene glycol), or by insertion (or substitution by chemical synthesis) of amino acids (amino acids) such as ornithine, which do not normally occur in human proteins.
  • chemical modifications such as ubiquitination, labeling (e.g., with radionuclides, various enzymes, etc.), pegylation (derivatization with polyethylene glycol), or by insertion (or substitution by chemical synthesis) of amino acids (amino acids) such as ornithine, which do not normally occur in human proteins.
  • sterically similar compounds may be formulated to mimic the key portions of the peptide structure and that such compounds may also be used in the same manner as the peptides of the invention. This may be achieved by techniques of molecular modeling and chemical designing known to those of skill in the art. For example, esterification and other alkylations may be employed to modify the amino terminus of, e.g., a di-arginine peptide backbone, to mimic a tetrapeptide structure. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
  • Functional equivalents also comprise glycosylated and covalent or aggregative conjugates formed with the same molecules, including dimers or complexes with unrelated chemical moieties. Such functional equivalents are prepared by linkage of functionalities to groups which are found in fragment including at any one or both of the N- and C-termini, by means known in the art.
  • Functional homologues may further include peptides with N-terminal carboxylation and/or C-terminal amidation.
  • a functional homologue may comprise a peptide having one or more N-terminal modifications selected from the list including alkylations and carboxylations, and/or a peptide having one or more C-terminal modifications selected from the list including esterifications and amidations.
  • functional homologues also comprise glycosylated and covalent or aggregative conjugates which are homodimers.
  • fragment thereof may refer to any portion of the given amino-acid sequence. Fragments may comprise more than one portion from within the full-length protein, joined together. Suitable fragments may be deletion or addition mutants.
  • the addition of at least one amino acid may be an addition of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids. Fragments may include small regions from the protein or combinations of these. Suitable fragments may be deletion or addition mutants.
  • the addition or deletion of at least one amino acid may be an addition or deletion of from preferably 2 to 250 amino acids, such as from 10 to 20 amino acids, for example from 20 to 30 amino acids, such as from 40 to 50 amino acids.
  • the deletion and/or the addition may, independently of one another, be a deletion and/or an addition within a sequence and/or at the end of a sequence.
  • Deletion mutants suitably comprise at least 20 or 40 consecutive amino acids and more preferably at least 80 or 100 consecutive amino acids in length.
  • such a fragment may be a shorter sequence of the sequence as identified by SEQ ID NO: 1 comprising at least 20 consecutive amino acids, for example at least 30 consecutive amino acids, such as at least 40 consecutive amino acids, for example at least 50 consecutive amino acids, such as at least 60 consecutive amino acids, for example at least 70 consecutive amino acids, such as at least 80 consecutive amino acids, for example at least 90 consecutive amino acids, such as at least 95 consecutive amino acids, such as at least 100 consecutive amino acids, such as at least 105 amino acids, for example at least 1 10 consecutive amino acids, such as at least 1 15 consecutive amino acids, for example at least 120 consecutive amino acids, wherein said deletion mutants preferably has at least 75% sequence identity, for example at least 80% sequence identity, such as at least 85% sequence identity, for example at least 90% sequence identity, such as at least 91 % sequence identity, for example at least 92% sequence identity, such as at least 93% sequence identity, for example at least 94% sequence identity, such as at least 95% sequence identity, for example at least 96 % sequence identity, such as at least
  • functional homologues of GM-CSF comprises at most 500, more preferably at most 400, even more preferably at most 300, yet more preferably at most 200, such as at most 175, for example at most 160, such as at most 150 amino acids, for example at most 144 amino acids.
  • fragment thereof may refer to any portion of the given amino acid sequence. Fragments may comprise more than one portion from within the full-length protein, joined together. Portions will suitably comprise at least 5 and preferably at least 10 consecutive amino acids from the basic sequence. They may include small regions from the protein or combinations of these.
  • GM-CSF There are two known variants of human GM-CSF; a T1 15I substitution in variant 1 and an 11 17T substitution in variant 2. Accordingly, in one embodiment of the invention functional homologues of GM-CSF comprises a sequence with high sequence identity to SEQ ID NO: 1 or any of the splice variants. Analogs of GM-CSF are for example described in U.S. Pat. Nos. 5,229,496, 5,393,870, and 5,391 ,485 by Deeley et al. Such analogues are also functional equivalents comprised within the present invention.
  • GM-CSF is used according to the present invention in homo- or heteromeric form.
  • Homo- and heteromeric forms of GM-CSF may comprise one or more GM-CSF monomers or functional homologous of GM-CSF as defined herein above.
  • Homo- and heteromers include dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, nonamers and decamers.
  • a homodimer, trimer or tetramer of GM-CSF is used.
  • the present invention relates to the pulmonary administration of GM-CSF, or a functional homologue thereof, however prepared, to treat bronchial asthma in a patient in need thereof.
  • GM-CSF can be produced in various ways, such as isolation from for example human or animal serum or from expression in cells, such as prokaryotic cells, yeast cells, insect cells, mammalian cells or in cell-free systems.
  • GM-CSF is produced recombinantly by host cells.
  • GM-CSF is produced by host cells comprising a first nucleic acid sequence encoding the GM-CSF operably associated with a second nucleic acid capable of directing expression in said host cells.
  • the second nucleic acid sequence may thus comprise or even consist of a promoter that will direct the expression of protein of interest in said cells.
  • a skilled person will be readily capable of identifying useful second nucleic acid sequence for use in a given host cell.
  • the process of producing recombinant GM-CSF in general comprises the steps of: providing a host cell, preparing a gene expression construct comprising a first nucleic acid encoding GM-CSF operably linked to a second nucleic acid capable of directing expression of said protein of interest in the host cell, - transforming the host cell with the construct, cultivating the host cell, thereby obtaining expression of GM-CSF.
  • the recombinant GM-CSF thus produced may be isolated by any conventional method, such as any of the methods for protein isolation described herein below.
  • the skilled person will be able to identify a suitable protein isolation steps for purifying GM-CSF.
  • the recombinantly produced GM-CSF is excreted by the host cells.
  • the process of producing a recombinant protein of interest may comprise the following steps: providing a host cell, preparing a gene expression construct comprising a first nucleic acid encoding GM-CSF operably linked to a second nucleic acid capable of directing expression of said protein of interest in said host cell, transforming said host cell with the construct, - cultivating the host cell, thereby obtaining expression of GM-CSF and secretion of GM-CSF into the culture medium, hereby obtaining culture medium comprising GM-CSF.
  • the composition comprising GM-CSF and nucleic acids may thus in this embodiment of the invention be the culture medium or a composition prepared from the culture medium.
  • said composition is an extract prepared from animals, parts thereof or cells or an isolated fraction of such an extract.
  • GM-CSF is recombinantly produced in vitro in host cells and is isolated from cell lysate, cell extract or from tissue culture supernatant.
  • GM-CSF is produced by host cells that are modified in such a way that they express GM-CSF.
  • said host cells are transformed to produce and excrete GM-CSF.
  • Administration of an effective amount of GM-CSF or a functional homologue thereof via e.g. intratracheal, intrabronchial or bronchoalveolar administration is particularly useful in alleviating symptoms and/or treating subjects suffering from bronchial asthma, particularly severe bronchial asthma.
  • the administration or treatment may either be prophylactic, preemptive or therapeutic.
  • an effective amount of GM-CSF or a functional homologue thereof is administered via pulmonary administration, such as by inhalation or intratracheal, intrabronchial or intraalveolar administration.
  • Inhalation of an effective amount of GM-CSF or a functional homologue thereof, such as for example via a nebulized solution, or via a powder form, is useful in alleviating symptoms or signs of severe forms of bronchial asthma and/or treating subjects suffering from such forms of bronchial asthma.
  • GM-CSF is administered systemically, preferably subcutaneously.
  • GM-CSF Intravenous administration of GM-CSF is not recommended as GM-CSF is rapidly metabolized and cleared from the circulation.
  • GM-CSF according to the present invention may be administered in any suitable way or form to achieve an effect on bronchial asthma, preferably by pulmonary
  • the GM-CSF or a functional equivalent thereof is administered via inhalation such as by inhalation of a nebulized solution or powder comprising GM-CSF or a functional homologue thereof.
  • GM-CSF GM-CSF
  • Methods of intratracheal, intrabronchial or bronchoalveolar administration include, but are not limited to, spraying, lavage, inhalation, flushing or installation, using as fluid a physiologically acceptable composition in which GM-CSF have been dissolved.
  • intratracheal, intrabronchial or intraalveolar administration include all forms of such administration whereby GM-CSF is applied into the trachea, the bronchi or the alveoli, respectively, whether by the instillation of a solution of GM- CSF, by applying GM-CSF in a powder form, or by allowing GM-CSF to reach the relevant part of the airway by inhalation of GM-CSF as an aerosolized or nebulized solution or suspension or inhaled powder or gel, with or without added stabilizers or other excipients.
  • Methods of intrabronchial or intraalveolar administration include, but are not limited to, bronchoalveolar lavage (BAL) according to methods well known to those skilled in the art, using as a lavage fluid a physiologically acceptable composition in which GM-CSF has been dissolved or indeed by any other effective form of intrabronchial
  • BAL bronchoalveolar lavage
  • administration including the use of inhaled powders containing GM-CSF in dry form, with or without excipients, or the direct application of GM-CSF, in solution or suspension or powder form during bronchoscopy.
  • Methods for intratracheal administration include, but are not limited to, blind tracheal washing with a similar solution of dissolved GM-CSF or a GM-CSF suspension, or the inhalation of nebulized fluid droplets containing dissolved GM-CSF or a GM-CSF suspension obtained by use of any nebulizing apparatus adequate for this purpose.
  • intratracheal, intrabronchial or intraalveolar administration does not include inhalation of the product but the instillation or application of a solution of GM-CSF or a powder or a gel containing GM-CSF into the trachea or lower airways.
  • Other preferred methods of administration may include using the following devices:
  • Electronic micropump nebulizers e.g. Aeroneb Professional Nebulizer
  • the aerosol may be delivered by a) facemasks or b) endotracheal tubes in intubated patients during mechanical ventilation (device 1 , 2 and 3).
  • the devices 4 and 5 can also be used by the patient without assistance, provided that the patient is able to self- activate the aerosol device.
  • the treatment with GM-CSF or a functional homologue thereof is particularly effective when GM-CSF or a functional homologue thereof reaches the parts of the airways where the macrophages are protecting the airways against incoming (inhaled) antigens such as the small airways.
  • the small airways at the level of the bronchiole are specifically vulnerable with respect to the allergic response, because of the air-blood barrier, which separates the air compartment in the airway lumen from the blood compartment.
  • the lumen of these airways may be completely blocked by edema fluid, edema of the airway wall, constriction of the airway smooth muscle, rejected mucosa cells and cellular debris, all consequences of the inflammatory process involving the activation of eosinophilic granulocytes.
  • the bronchioles may be blocked so as to hinder the penetration of an inhaled GM-CSF aerosol. Adequate alleviation of the condition will require delivery of GM-CSF to the target site both above and preferably below the site of blockage to the passage of aerosol.
  • the small airways (bronchioles and alveoli) may be obtained by:
  • Previously intervention e.g. already in the early phase of acute severe asthma, i.e. when the patient no longer respond to 2 -agonists, or when the asthmatic condition becomes unresponsive to maximal anti-asthmatic therapy including short- and long-acting bronchodilators and corticosteroids administered both orally and by inhalation.
  • PEEP positive end-expiratory pressure
  • CPAP and PEEP may increase the collateral ventilation (CV) (Menkes et al 1979), so to say "from behind” via the ventilation pores between the terminal units of peripheral airways.
  • CV collateral ventilation
  • the phenomenon of CV can be particularly useful in pulmonary disease with anatomical partial or total block of the airways, since it can increase delivery of drugs to the site of interest which is the peripheral airways.
  • CPAP and PEEP may cause air to bypass obstructed airways through collateral channels including interalveolar pores, bronchiole-alveolar communications, and interbronchiolar pathways. Resistance through these channels located at the small airways increases with decreasing lung volume.
  • inhaled GM-CSF or a functional homologue thereof is administered via inhalation combined with collateral ventilation, such as CPAP and/or PEEP.
  • concentrations for a solutionaccording to the invention, comprising GM-CSF and/or functional homologues or variants of GM-CSF are in the range of 0.1 ⁇ g to 10000 ⁇ g active ingredient per mL of solution.
  • the suitable concentrations are often in the range of from 0.1 ⁇ g to 5000 ⁇ g per mL of solution, such as in the range of from about 0.1 ⁇ g to 3000 ⁇ g per mL of solution, and especially in the range of from about 0.1 ⁇ g to 1000 ⁇ g per mL of solution, such as in the range of from about 0.1 ⁇ g to 250 ⁇ g per mL solution.
  • a preferred concentration would be from about 0.1 to about 5.0 mg, preferably from about 0.3 mg to about 3.0 mg, such as from about 0.5 to about 1 .5 mg and especially in the range from 0.8 to 1 .0 mg per mL of solution.
  • GM-CSF is administered systemically, e.g. by subcutaneous injection.
  • the GM-CSF is used to treat a mammal, such as a human subject.
  • the human subject may be a child of less than 12 years or an adult older than 12 years.
  • compositions or formulations for use in the present invention include GM-CSF or functional homologue thereof combination with, preferably dissolved in, a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent, or carried to the lower airways as a pegylated preparation or as a liposomal or nanoparticle preparation administered as an aerosol via inhalation, or as a lavage fluid administered via a bronchoscope as a bronchoalveolar lavage or as a blind intratracheal wash or lavage.
  • aqueous carriers may be used, including, but not limited to 0.9% saline, buffered saline, physiologically compatible buffers and the like.
  • compositions may be sterilized by conventional techniques well known to those skilled in the art.
  • the resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and freeze-dried, the freeze-dried preparation being dissolved in a sterile aqueous solution prior to administration
  • a freeze-dried GM-CSF preparation may be pre-packaged for example in single dose units.
  • the single dose unit is adjusted to the patient.
  • the compositions may contain pharmaceutically acceptable auxiliary substances or adjuvants, including, without limitation, pH adjusting and buffering agents and/or tonicity adjusting agents, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.
  • the formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like.
  • liposomes are typically composed of phospholipids (neutral or negatively charged) and/or cholesterol.
  • the liposomes are vesicular structures based on lipid bilayers surrounding aqueous compartments. They can vary in their physiochemical properties such as size, lipid composition, surface charge and number and fluidity of the phospholipids bilayers.
  • lipid for liposome formation 1 ,2-Dilauroyl-sn-Glycero-3-Phosphocholine (DLPC), 1 ,2-Dimyristoyl-sn-Glycero-3- Phosphocholine (DMPC), 1 ,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC), 1 ,2- Distearoyl-sn-Glycero-3-Phosphocholine (DSPC), 1 ,2-Dioleoyl-sn-Glycero-3- Phosphocholine (DOPC), 1 ,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine (DMPE), 1 ,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE), 1 ,2-Dioleoyl-sn-Glycero- 3-Phosphoethanolamine (DOPE), 1 ,2-Dimyristo
  • Cardiolipin (Ammonium Salt). Formulations composed of DPPC in combination with other lipids or modifiers of liposomes are preferred e.g. in combination with cholesterol and/or phosphatidylcholine.
  • Long-circulating liposomes are characterized by their ability to extravasate at body sites where the permeability of the vascular wall is increased.
  • the most popular way of producing long-circulating liposomes is to attach hydrophilic polymer polyethylene glycol (PEG) covalently to the outer surface of the liposome.
  • PEG polyethylene glycol
  • Some of the preferred lipids are: 1 ,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethylene glycol)-2000] (Ammonium Salt), 1 ,2-Dipalmitoyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-5000] (Ammonium Salt), 1 ,2- Dioleoyl-3-Trimethylammonium-Propane (Chloride Salt) (DOTAP).
  • the liposome suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damage on storage.
  • Lipophilic free-radical quenchers such as alpha-tocopherol and water-soluble iron-specific chelators, such as ferrioxamine, are preferred.
  • a variety of methods are available for preparing liposomes, as described in e.g. Szoka et al. (1980), and in U.S. Pat. Nos. 4,235,871 , 4,501 ,728 and 4,837,028, all of which are incorporated herein by reference.
  • Another method produces multi-lamellar vesicles of heterogeneous sizes.
  • the vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film.
  • the film may be re-dissolved in a suitable solvent, such as tertiary butanol, and then lyophilized to form a more homogeneous lipid mixture which is in a more easily hydrated powder-like form.
  • a suitable solvent such as tertiary butanol
  • This film is covered with an aqueous solution of the targeted drug and the targeting component and allowed to hydrate, typically over a 15-60 minute period with agitation.
  • the size distribution of the resulting multi-lamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents such as deoxycholate.
  • Micelles are formed by surfactants (molecules that contain a hydrophobic portion and one or more ionic or otherwise strongly hydrophilic groups) in aqueous solution.
  • surfactants molecules that contain a hydrophobic portion and one or more ionic or otherwise strongly hydrophilic groups
  • Common surfactants well known to one of skill in the art can be used in the micelles of the present invention.
  • Suitable surfactants include sodium laureate, sodium oleate, sodium lauryl sulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 and PLURONIC F-127 (Wyandotte Chemicals Corp.).
  • Preferred surfactants are nonionic polyoxyethylene and polyoxypropylene detergents compatible with IV injection such as, TWEEN-80, PLURONIC F-68, n-octyl-beta-D-glucopyranoside, and the like.
  • phospholipids such as those described for use in the production of liposomes, may also be used for micelle formation.
  • GM-CSF GM-CSF
  • a dose which, when administered to a patient in need thereof, e.g. by pulmonary administration, achieves a concentration in the subject's airways and/or lung parenchyma which has a beneficial effect on bronchial asthma, i.e. by alleviating and/or preventing asthma symptoms.
  • the preparations are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically effective.
  • the quantity to be administered depends on the subject to be treated, including, e.g. the weight and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are per kilogram of body weight normally of the order of several hundred ⁇ g active ingredient per administration with a preferred range of from about 0.1 ⁇ g to 10000 ⁇ g per kilogram of body weight.
  • Doses expected to provide an effective amount of GM-CSF comprise GM-CSF are often in the range of from 0.1 ⁇ g to 5000 ⁇ g per kilogram of body weight, such as in the range of from about 0.1 ⁇ g to 3000 ⁇ g per kilogram of body weight, and especially in the range of from about 0.1 ⁇ g to 1000 ⁇ g per kilogram of body weight, preferably in the range of 5 ⁇ g to 1000 ⁇ g, even more preferred about 100 ⁇ g to about 800 ⁇ g administered via inhalation once, twice or three times daily.
  • the patient is administered a total daily dose of GM-CSF of from 200 ⁇ g to 1000 ⁇ g, such as from 300 ⁇ g to 900 ⁇ g, such as from 400 ⁇ g to 800 ⁇ g.
  • Suitable daily dosage ranges are per kilogram of body weight per day normally of the order of several hundred ⁇ g active ingredient per day with a preferred range of from about 0.1 ⁇ g to 10000 ⁇ g per kilogram of body weight per day.
  • the suitable dosages are often in the range of from 0.1 ⁇ g to 5000 ⁇ g per kilogram of body weight per day, such as in the range of from about 0.1 ⁇ g to 3000 ⁇ g per kilogram of body weight per day, and especially in the range of from about 0.1 ⁇ g to 1000 ⁇ g per kilogram of body weight per day, when based on monomeric forms having a sequence identical to sequence ID NO: 1 , for functional homologues and fragments the dose is calculated according to the molecular weight of the monomeric form to the molecular weight of the homologues or fragments.
  • the dose of functional homologues and fragments is further calculated according to the ratio of their biological activity to that of the parent compound.
  • GM-CSF may e.g. be administered by inhalation to a patient suffering from severe asthma in a dose ranging from about 300 ⁇ g administered once a day to about 600 ⁇ g administered three times a day.
  • Patients suffering from severe forms of bronchial asthma characterized by clogging of the airways may particularly benefit from GM-CSF administered via inhalation and systemic treatment with GM-CSF, such as by administration of about 300 ⁇ g GM-CSF subcutaneously.
  • GM-CSF is administered to a patient suffering from severe bronchial asthma and in risk of developing acute severe bronchial asthma.
  • Duration of dosing will typically range from 1 day to about 4 months, such as 2 days to about 3 months, for example in the range of 1 or 2 days to 2 months, such as in the range of 1 or 2 days to 1 month .
  • a duration of dosing may be in the range of 1 to 14 days, such as 2 to 3 days, or 3 days to 4 days, or 4 to 5 days, such as in the range of 5 to 14 days, such as 5 to 6 days, or 6 to 7 days, or 7 to 14 days, such as one week to two weeks, for example two to four weeks, such as one month to two months, for example 2 to 4 months, or for as long as symptoms and signs of disease are detectable.
  • the duration of dosing has the length to allow for said transformation.
  • the duration can be in the range of 5 to 14 days, for example 5 days, or for example 6 days, or for example 13 days, or for example 14 days, even more preferably in the range of 7 to 12 days such as for example 7 days, or for example 8 days, or for example 9 days, or for example 10 days, or for example 1 1 days, or for example 12 days.
  • a dosage regimen may alternate between periods of administration of the
  • a pause in treatment in such a dosage regimen may last 5 to 10 days, for example 5 days, or for example 6 days, or for example 7 days, or for example 8 days, or for example 9 days, or for example 10 days or more, for example 10 days to 4 months or until symptoms or signs of bronchial asthma are observed.
  • inhaled GM-CSF produces an enhanced survival in animal models.
  • an increased dose rate may further counteract the deleterious effect of the pre-existing severe asthmatic condition.
  • Examples of dosage regimens may include a cycle of 10 days of treatment with the pharmaceutical composition according to the present invention and 7 days of pause in treatment.
  • the GM-CSF according to the present invention is administered to a patient in need thereof whenever there is a need to alleviate or prevent symptoms of bronchial asthma.
  • the compounds used in the invention may be administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses.
  • the formulations may conveniently be presented in unit dosage form by methods known to those skilled in the art.
  • the compounds according to the invention are provided in a kit.
  • Such a kit typically contains an active compound in dosage forms for administration.
  • a dosage form contains a sufficient amount of active compound such that a desirable effect can be obtained when administered to a subject.
  • the medical packaging comprises an amount of dosage units corresponding to the relevant dosage regimen.
  • the medical packaging comprises a pharmaceutical composition comprising a compound as defined above or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable carriers, vehicles and/or excipients, said packaging comprising from 1 to 7 dosage units, thereby having dosage units for one or more days, or from 7 to 21 dosage units, or multiples thereof, thereby having dosage units for one week of administration or several weeks of administration.
  • the dosage units can be as defined above.
  • the medical packaging may be in any suitable form for intratracheal, intrabronchial or intraalveolar administration.
  • the packaging is in the form of a vial, ampule, tube, blister pack, cartridge or capsule.
  • the medical packaging comprises more than one dosage unit
  • the medical packaging is provided with a mechanism to adjust each administration to one dosage unit only.
  • a kit contains instructions indicating the use of the dosage form to achieve a desirable affect and the amount of dosage form to be taken over a specified time period.
  • the medical packaging comprises instructions for administering the pharmaceutical composition.
  • a freeze-dried GM-CSF preparation may be pre-packaged for example in single dose units.
  • the single dose unit is adjusted to the patient.
  • a composition comprising as the sole active ingredient an effective amount of granulocyte-macrophage colony-stimulating factor (GM-CSF) or a functional homologue thereof for use in the treatment of severe bronchial asthma, acute severe bronchial asthma, severe bronchial asthma in risk of developing acute severe bronchial asthma in a subject.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the composition of embodiment 1 wherein the subject does not respond or has significantly lowered response to existing treatment, such as treatment with any one of a beta2 agonist or prostaglandin or steroid treatment
  • composition of embodiments 1 or 2 wherein the composition is treatment of airway inflammation in severe bronchial asthma or acute severe bronchial asthma in a subject.
  • composition of embodiment 3, wherein the treatment of airway immuno- inflammation is by changing from a T H 2 helper immunoinflammatory reaction the airway mucosa into a T H 1 subset.
  • composition according to any one of the preceding embodiments, wherein the composition is made for inhalation as a nebulized solution, a suspension or as a powder form
  • composition according to any one of the preceding embodiments, wherein the composition is made for administration by inhalation combined with collateral ventilation, such as continuous positive airway pressure (CPAP) and/or positive end-expiratory pressure (PEEP) ) in order to enhance the deposition of iGM-CSF into small airways where the airways are blocked by rejected mucosa cells, or to enhance the deposition into of the effective iGM-
  • collateral ventilation such as continuous positive airway pressure (CPAP) and/or positive end-expiratory pressure (PEEP)
  • CPAP continuous positive airway pressure
  • PEEP positive end-expiratory pressure
  • composition is made for administration by bronchoalveolar lavage or blind tracheal washing.
  • composition of any of embodiments 1 to 10, wherein the subject is
  • GM-CSF or a functional homologue thereof by direct application of GM-CSF or a functional homologue thereof during bronchoscopy.
  • an effective amount of GM-CSF or a functional homologue thereof is administered in doses of 5 ⁇ g to 1000 ⁇ g, such as about 100 ⁇ g to about 800 ⁇ g administered.
  • Collateral ventilatoin ie ventilation via th3 preformed holes between neighboring alveoles.
  • Collateral ventilatoin is enhanced by PEEP and or CPAP 17.
  • composition of embodiment 18, wherein the human subject is an adult older than 12 years of age.
  • composition according to any one of the preceeding embodiments, wherein the composition either comprises another active substance, or is for use in combination with another active substance, which is a substance that does not activate cAMP in a cell.
  • composition according to any one of the preceeding embodiments, wherein the composition either comprises another active substance, or is for use in combination with another active substance which is a substance that does activate cAMP in a cell.
  • composition according to embodiment 22, wherein the composition is for use in a patient suffering from acute severe asthma.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • the medicament comprises a composition as defined in embodiments 1 to 24.
  • a method of treatment wherein the compositions of any one of embodiments 1 - 24 is used for treatment, preemptive treatment or prophylactic treatment in a patient suffering from severe bronchial asthma, or acute severe bronchial asthma.
  • a composition comprising an effective amount of granulocyte-macrophage colony-stimulating factor (GM-CSF) or a functional homologue thereof for use in preventing airway mucosa rejection, such as small airway mucosa rejection.
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • composition according to embodiment 28 or 29, wherein the composition is the composition according to any one of embodiments 1 -24.
  • composition according to any one of the preceeding embodiments, wherein the condition is atopic asthma.
  • composition according to any one of embodiments 1 -32, wherein the condition is non-atopic asthma.
  • composition or method of any one of the previous embodiments, wherein the condition is not mild or moderate bronchial asthma.
  • composition or method of any one of the previous embodiments wherein the composition is for use in reducing eosinophilic toxin in a mammal such as a human.
  • a mammal such as a human.
  • 37. The composition or method of embodiment 36, wherein the eosinophilic toxin is present in an airway mucosa, or in a tissue connected with an airway mucosa.
  • composition or method according to anyone of the previous embodiments wherein the composition is made for use in treating a patient suffering from decrease in lung function as measured by FEV1 and or PEFR, wherein said decrease in lung function is not reversible by intervention with ⁇ 2 agonists.
  • material of the airways is in a patient suffering from severe bronchial asthma, acute severe bronchial asthma, severe bronchial asthma in risk of developing acute severe bronchial asthma.
  • Example 1 Treatment of severe bronchial asthma
  • a patient suffering from severe bronchial asthma will be treated by pulmonary administration of a composition comprising GM-CSF as the sole active ingredient.
  • the patient may be administered a dosage of_300 ⁇ g per day.
  • the patient may be administered one dosage / day for 10 days, followed by a non-treatment period.
  • Clinical case In example, a middle aged man with a history of bronchial asthma since childhood may have experienced increasing dyspnea over 4 days. Day 0 is the day of hospitalization.
  • Day 16 The patient will experience increasing dyspnea and wheezing, with decreasing effect of even large doses of prednisolone. Over the next days he will experience constant severe dyspnea, even when sitting in an upright position in order to sleep.
  • Day 19 The patient start inhalation of GM-CSF at a dose of 300 ⁇ g a day via a micropump nebulizer. At about day 25: The condition will stabilize with a clear decrease in shortness of breath.
  • Example 3 Treatment of Bronchiectasis (BE) with GM-CSF BE is characterized by chronic cough, production of copius amounts of sputum and abnormal airway dilation of the small airways (diameter > 2 mm) as revealed by a HR, CT or MR scan.
  • the disease is characterized i.e. by coughing and by a local inflammation of the mucosa in the small airways. This local reaction causes a rejection of the mucosa of the small airways.
  • GM-CSF administered using a NebulizerA micro pump nebulizer, to the smaller airways of the lungs.
  • Administration was planned to be in cycles, wherein the first cycle was with a start dose of 300 x 2 microgram GM-CSF per day, in total 600 microgram GM-CSF per day for 10 days. Subsequently, one week without treatment, followed by about a 1 week pause in the treatment.
  • the next cycle to follow the pause was similar to the first cycle with a start dose of 300 x 2 microgram GM-CSF per day, which in total was 600 microgram GM-CSF per day.
  • the second cycle was also planned to last for 10 days.
  • the first period of treatment was initiated the day before day 1 in Figure 1 , and it was terminated in the morning of the 7 th day of Figure 3. It was observed that during the first period of treatment, the sum of duration of coughing per day was reduced significantly to about one third of the initial level, please see the levels on days 5, 6 and 7 in figure 3. Coughing increased again when treatment was stopped, see days 8 and 9 in Figure
  • Non-patent literature Altemeier WA, Zhu X, Berrington WR, Harlan JM, Liles WC (2007) Fas (CD95) induces macrophage proinflammatory chemokine production via a MyD88-dependent, caspase- independent pathway. J Leukoc Biol 82:721 -728.
  • Clark SC Kamen R (1987) The human hematopoietic colony-stimulating factors.
  • GM-CSF granulocyte/macrophage colony- stimulating factor
  • Rapoport AP Abboud CN, DiPersio JF (1992) Granulocyte-macrophage colony- stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF):

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Abstract

La présente invention concerne le facteur de stimulation des colonies de granulocytes et macrophages (GM-CSF) et son utilisation pour traiter les formes graves de l'asthme bronchique, par administration par les voies respiratoires d'une quantité efficace de GM-CSF ou d'un homologue fonctionnel de celui-ci.
PCT/EP2015/051266 2014-01-22 2015-01-22 Facteur de stimulation des colonies de granulocytes et macrophages pour le traitement et la prévention de formes graves de l'asthme bronchique WO2015110536A1 (fr)

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