Enhancing Pulmonary Host Defence via Administration of Granulocyte- Colony stimulating factor.

All patent and non-patent references cited in the application, or in the present application, are also hereby incorporated by reference in their entirety.

Field of invention

The present invention provides a method for enhancing pulmonary host defense in a subject suffering from, for example, but not limited to lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization by administering to the subject an effective amount of an agent capable of binding to and stimulating the G-CSF receptor such as granulocyte colony stimulating factor (G- CSF) via pulmonary airway administration.

Background of invention

Respiratory infections are a major cause of human morbidity and still a leading cause of mortality. The lower respiratory tract is normally a sterile environment and the pulmonary host defense is normally well developed to clear bacteria, fungi and virus. The cellular host defense response includes both humeral factors as well as resident and recruited cells. In the cellular host defense the alveolar macrophage is an integral component and its long-lifespan aids function. After a modest dose of antigen, i.e. challenge with infectious agents, alveolar macrophages has the main clearing function in the lung. After a massive exposure of infectious agents, however, the phagocytic capacity of alveolar macrophages is overwhelmed and the alveolar macrophages orchestrate the inflammatory response, provided that sufficient cytokine concentrations for macrophage stimulation are present. However, in severe infection and in high alveolar oxygen content, the most important cytokines for the activation and proliferation of alveolar macrophages are reduced or even lacking. Furthermore, the

process of apoptosis of alveolar immunocompetent cells, for example macrophages, is enhanced in severe pulmonary inflammation, leaving the lung with a severely reduced cellular host defense in severe infections.

The G-CSF receptor (G-CSFR) is expressed on the progenitor cells of neutrophlic granulocytes and on mature committed cells. The G-CSF receptor belongs to the superfamily of cytokine/hematopoietic receptors. Although the majority of family members, including the receptors for the interleukins from interleukin-2 (IL-2) to IL-7 and granulocyte-macrophage colony-stimulating factor (GM-CSF), are activated through the formation of heteromeric complexes, G-CSF receptor protein, consisting of a single chain polypeptide, is believed to form a homodimeric complex upon ligand binding (Fukunaga, R., et al., J. Bio. Chem., 1990,265: 14008).

Granulocyte colony stimulating factor (G-CSF) is a hematopoietic growth factor and glycoprotein which stimulates the survival, proliferation, differentiation and function of neutrophil granulocyte progenitor cells and mature neutrophils.

Intravenous G-CSF is used to treat neutropenia associated with cancer chemotherapy, myodysplasia and aplastic anemia and is used to decrease frequency of infection in patients with congential, idiopathic and cell neutropenia.

WO 08/017126 has recently disclosed pulmonary administration of inhibitors of G-CSF or the G-CSF receptor for the treatment of pulmonary diseases characterized by or associated with infiltrations of neutrophils and complications arising there from.

Summary of invention

A first aspect of the invention is to provide a composition comprising an agent capable of binding to and stimulating the G-CSF receptor for use as a medicament for pulmonary administration in a subject, for the enhancement of the pulmonary host defense. In a particularly preferred embodiment said agent is G-CSF or a functional homologue thereof.

Another aspect, the present invention relates to a method for enhancing pulmonary host defense in a subject in need thereof which comprises administering to the subject via pulmonary administration an effective amount of granulocyte colony stimulating factor (G-CSF) or a functional homologue thereof.

Yet another aspect, the present invention is to provide a composition comprising an agent capable of binding to and stimulating the G-CSF receptor for use as a medicament for intratracheal, intrabronchial, intraalveolar or bronchio-alveolar administration in a subject, for alleviating symptoms or treating a subject suffering from a disease selected from the group consisting of lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization. In a particularly preferred embodiment said agent is G-CSF or a functional homologue thereof.

Another aspect of the present invention relates to the use of G-CSF for the manufacture of a medicament or for use as a medicament for pulmonary administration to enhance pulmonary host defense in a subject in need thereof. G-CSF via pulmonary administration is particularly useful in treating pulmonary diseases, alleviating symptoms and/or treating conditions including, but not limited to lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia

(HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization.

An aspect of the invention relates to the use of an agent capable of binding to and stimulating the G-CSF receptor for the preparation of a medicament for pulmonary administration in a subject, for the enhancement of the pulmonary host defense. In a particularly preferred embodiment said agent is G-CSF or a functional homologue thereof.

A further aspect of the invention relates to the use of an agent capable of binding to and stimulating the G-CSF receptor in the preparation of a medicament for pulmonary administration in a subject, for alleviating symptoms or treating a subject suffering from a disease selected from the group consisting of lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization. In a particularly preferred embodiment said agent is G-CSF or a functional homologue thereof.

A further aspect of the invention relates to the use of an agent capable of binding to and stimulating the G-CSF receptor in the preparation of a medicament for pulmonary administration in a subject, for alleviating symptoms or treating a subject suffering from a disease selected from the group consisting of lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization. In a particularly preferred embodiment said agent is G-CSF or a functional homologue thereof.

Yet another aspect of the present invention relates to a method of enhancing pulmonary host defense in a subject comprising administering to the subject via pulmonary administration an effective amount of an capable of binding to and stimulating the G-CSF receptor. In a particularly preferred embodiment said agent is G- CSF or a functional homologue thereof.

Yet another aspect of the present invention relates to a method for alleviating symptoms or treating a subject suffering from a disease selected from the group consisting of lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization comprising administering to the subject via pulmonary administration

of an effective amount of an agent capable of binding to and stimulating the G-CSF receptor. In a particularly preferred embodiment said agent is G-CSF or a functional homologue thereof.

The method of the present invention is particularly useful in alleviating symptoms and/or treating conditions including, but not limited to lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization.

For purposes of the present invention by "enhancing pulmonary host defense" it is meant any detectable change in the host which increase defense to infection in the pulmonary system of the host. Enhancement of the pulmonary host defense can be determined, for example, by monitoring local pulmonary host defense parameters such as changes in white cell count and/or cytokine release from tracheal aspirate or obtained from bronchoalveolar lavage. Alternatively or in addition, enhancement of the pulmonary host defense can be determined by flow cytometric analysis of cells from the lung such as alveolar macrophages with or without selected and/or specified surface receptors and/or subgroups recruited by, for example, tracheal aspirate and/or from bronchoalveolar lavage fluid. Methods for flow cytometric analysis of cells from the lung are described Garn et al. in Experimental and Toxicologic Pathology 2006 57:S2:21 -24.

For purposes of the present invention by "an agent capable of binding to and stimulating the G-CSF receptor" may be any type of agent, for example the agent may be selected from the group comprising proteins, peptides, polypeptides, antibodies or an antigen-binding fragment thereof, antisense-RNA, antisense-DNA, siRNA, other polynucleotides, or organic molecules that are capable of binding to and stimulating the G-CSF receptor thereby stimulating the activities of the G-CSF receptor, including but not limited to stimulation of the survival, proliferation, differentiation and function of neutrophil granulocyte progenitor cells and mature neutrophils or preventing infectious complications of some neutropenic states

Detailed description of the invention

The present invention relates to the pulmonary administration, by any appropriate method including, but not limited to, intratracheal, intrabronchial, bronchio-alveolar or intraalveolar administration, to a human subject inclusive of both adults and children, of an agent capable of binding to and stimulating the G-CSF receptor such as purified or concentrated natural human of granulocyte colony stimulating factor (G-CSF), or a functional homologue thereof, however prepared, to enhance the pulmonary host defense.

Administration of an effective amount of an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF or a functional homologue of thereof via intratracheal, intrabronchial, intraalveolar or bronchio-alveolar administration is particularly useful in alleviating symptoms and/or treating subjects suffering from pulmonary conditions including, but not limited to lung cancer, both small cell lung cancer and squamous or non-small cell lung cancer, to lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal but not limited to, Candidiasis species and Aspergillus species and/or viral infection and/or bacterial, fungal and/or viral colonization of the airways and/or lung parenchyma.

The pulmonary host defence

The respiratory system is the first line of defense against inhaled substances. The system comprises a complex and multilayered defense system involving mechanical, reflex, cellular mechanisms as well as producing local and systemically derived defense molecules.

The upper airways and the major bronchi are protecting the lungs with the anatomic barriers they represent associated with the cough reflex, the mucociliary apparatus and the secretory immunoglobulin A (IgA). Below, the superficial layers of the mucosa, a tight network of dendritic cells will sense and catch any invading organisms and bring

them to the lymph nodes around the airways or in the hilum. In the respiratory units beyond the respiratory bronchioles particles will be caught by alveolar macrophages in a milieu rich in elements such as IgG, complement, surfactant and fibronectin. In these units when needed various amounts of neutrophils and lymphocytes will be recruited.

The pulmonary epithelium in particular protects the airspace and preserves normal respiratory functions by providing a barrier function and by secreting substances which targets environmental challenges. The pulmonary epithelium can be injured by infection, such as infections by viruses, which permits bacterial attachment.

The cellular part of the pulmonary defense involves mucins, antibiotic substances and/or antioxidants such as surfactants, immunoglobulins and complement proteins and antiproteases. Collectins have also been implicated in the cellular pulmonary defense.

Inflammatory cells are recruited to the airways via chemoattractants secreted by the pulmonary epithelium, which in turn leads to the migration of inflammatory cells across the epithelium. The epithelium further releases cytokines to regulate the inflammatory cell activities. The epithelial cells release factors such as IL-8 and G-CSF, whereas macrophages release IL-1 and TNF, which further stimulates the epithelial cells.

As described herein above, under severe infection and in high alveolar oxygen content, the most important cytokines for the activation and proliferation of alveolar macrophages are reduced or even lacking. Furthermore, the process of apoptosis of alveolar immunocompetent cells, for example macrophages, is enhanced in severe pulmonary inflammation, leaving the lung with a severely reduced cellular host defense in severe infections. By supplementing the insufficient or lacking cytokines for proliferation and enhancing the immunocompetence of the alveolar cells like alveolar macrophages, as a therapeutic action, the down-regulated cellular host defense may be enhanced or even normalized. Such a therapeutic intervention may be brought about by a local pulmonary deposition, e.g. via inhalation, bronchoalveolar lavage (BAL) using bronchoscopic delivery of the cytokines for the maintenance and upregulation of pulmonary host defense. Reduced macrophage function leads to reduced activation and recruitment of otyher immunocompetent cells like T-cells, e.g. CD4+ and CD8+ T cells.

For purposes of the present invention by "enhancing pulmonary host defense" it is meant any detectable change in the host which increase defense to infection in the pulmonary system of the host. Enhancement of the pulmonary host defense can be determined, for example, by monitoring local pulmonary host defense parameters such as changes in white cell count and/or cytokine release from tracheal spirate or obtained from bronchoalveolar lavage. Alternatively or in addition, enhancement of the pulmonary host defense can be determined by flow cytometric analysis of cells from the lung such as alveolar macrophages with or without selected and/or specified surface receptors and/or subgroups recruited by, for example, tracheal aspirate and/or from bronchoalveolar lavage fluid. Methods for flow cytometric analysis of cells from the lung are described Garn et al. in Experimental and Toxicologic Pathology 2006 57:S2:21 -24.

G-CSF receptor

The G-CSF receptor (G-CSFR) is expressed on the progenitor cells of neutrophlic granulocytes and on mature committed cells. The G-CSF receptor belongs to the superfamily of cytokine/hematopoietic receptors. Although the majority of family members, including the receptors for the interleukins from interleukin-2 (IL-2) to IL-7 and granulocyte-macrophage colony-stimulating factor (GM-CSF), are activated through the formation of heteromeric complexes, G-CSF receptor protein, consisting of a single chain polypeptide, is believed to form a homodimeric complex upon ligand binding (Fukunaga, R., et al., J. Bio. Chem., 1990,265: 14008).

Homodimerization of the G-CSF receptor has been shown to be essential for signal transduction (Wells, J. A., and Vos, A. M., Annu. Rev. Biochem., 1996, 65: 609). The G-CSF receptor does not contain an intrinsic protein kinase domain although tyrosine kinase activity seems to be essential to transduction of the G-CSF signal. The signal from G-CSF receptor activation through G-CSF induced receptor homodimerization is mediated by noncovalent binding of various tyrosine kinases, e. g. JAK1 and JAK2 (Barge, R. M. Y., et al, Blood, 1996,87: 2148-2153), and thereafter the phosphorylation of transcription factors Stats such as Stat3 and Statδ (Tian, S-S., et al, Blood, 1994,84: 1760-1764; Watowich, S. S., et al., Annu. Rev Cell Dev.Biol., 1996,12: 91 ; Dong F., et

al, J. Immunol., 1998, 161 : 6503-6509). These tyrosine kinases play an essential role for G-CSF receptor phosphorylation and Stat activation in response to G-CSF (Tian S- S. et al blood, 1996,88: 4435-4444; Shimoda, K.,et al., Blood, 1997,90: 597-604).

According to the present invention an agent capable of binding G-CSFR may be capable of binding any G-CSFR or variant thereof including but not limited to the three splice variants of G-CSFR identified bye SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 5.

Agents capable of binding G-CSFR

In one preferred embodiment of the present invention, an agent is administered to the subject, said agent being capable of binding to and stimulating the G-CSF receptor thereby interfering with the activity of G-CSF, either directly or indirectly.

The agent capable of exhibiting the mentioned effect may be any type of agent, for example the agent may be selected from the group comprising proteins, peptides, polypeptides, antibodies or an antigen-binding fragment thereof, antisense-RNA, antisense-DNA, siRNA, other polynucleotides, or organic molecules. The antibody or functional equivalent thereof may be any type of antibody known in the art, for example a polyclonal or a monoclonal antibody derived from a mammal or a synthetic antibody, such as a single chain antibody or hybrids comprising antibody fragments. In addition functional equivalents of antibodies may be antibody fragments, in particular epitope binding fragments. Furthermore, antibodies or functional equivalent thereof may be small molecule mimicking an antibody. Naturally occurring antibodies are immunoglobulin molecules consisting of heavy and light chains. Functional equivalents of antibodies may be a fragment of an antibody, preferably an antigen binding fragment or a variable region. Examples of antibody fragments useful with the present invention include Fab, Fab', F(ab') ₂ and Fv fragments. The antibody can also be a single chain antibody ("SCA"), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. Such single chain antibodies are also refered to as "single-chain Fv" or "sFv" antibody fragments.

In a preferred embodiment the agent is an antibody or a polypeptide, and the agent is more preferably a polypeptide. The agent may any of the G-CSF peptides described herein below; most preferably the agent capable of binding to and stimulating the G- CSF receptor is G-CSF.

Most preferably the agent capable of binding to and stimulating the G-CSF receptor does not act as an antagonist on the receptor, rather as an agonist.

The agent may bind to any part of the receptor relevant for the stimulation of the receptor.

G-CSF

Colony-stimulating factors are glycoproteins that stimulate the growth of hematopoietic progenitors and enhance the functional activity of mature effector cells. In brief, at the level of immature cells, CSFs assure the self-renewal of the staminal pool and activate the first stage of hematopoietic differentiation; in the middle stage, when cell proliferation is associated to a progressive acquisition of 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.

Granulocyte colony stimulating factor (G-CSF) is a glycoprotein which stimulates the survival, proliferation, differentiation and function of neutrophil granulocyte progenitor cells and mature neutrophils. The amino acid sequence of human granulocyte colony stimulating factor has been reported by Nagata et al. (Nature, 319, 415- 418, 1986). The native protein functions as a monomer. There are two known splice variants of G- CSF of 174 and 180 amino acids respectively. The human form of G-CSF was cloned by groups from Japan and the U.S.A. in 1986 (see e.g., Nagata et al. Nature 319: 415- 418, 1986).

Intravenous G-CSF is used to treat neutropenia associated with cancer chemotherapy, myodysplasia and aplastic anemia and is used to decrease frequency of infection in patients with congential, idiopathic and cell neutropenia.

G-CSF is capable of forming a complex with its receptor thereof in a ratio of 2:2 upon the dimerization of the receptor (Horan et al., Biochemistry, 35, 4886-96, 1996). It is reported by Aritomi et al. that amino acids of human G-CSF, which exist in contact region or adjacent region thereof when the receptor is bound to human G-CSF, include G4, P5, A6, S7, S8, L9, P10, Q1 1 , S12, L15, K16, E19, Q20, L108, D109, D1 12, T1 15, T1 16, Q1 19, E122, E123 and L124 (Aritomi et al., (1999) Nature 401 :713-717).

The more-abundant and more-active 174-amino acid form has been used in the recombinant production of G-CSF. Recombinant human G-CSF in clinical use is a potent stimulant of neutrophil granulopoiesis and has demonstrated efficacy in preventing infectious complications of some neutropenic states. Recombinant human G-CSF can be used to accelerate neutrophil recovery from myelosuppressive treatments. The recombinant human G-CSF synthesised in an E. coli expression system is called filgrastim. The structure of filgrastim differs slightly from the natural glycoprotein. When recombinant human G-CSF is synthesised in Chinese hamster ovary (CHO) cells it is called lenograstim.

G-CSF has been reported to decrease the morbidity of cancer chemotherapy by reducing the incidence of febrile neutropenia, the morbidity of high-dose chemotherapy supported by marrow transplantation, and the incidence and duration of infection in patients with severe chronic neutropenia. Further, G-CSF has recently been shown to have therapeutic effect when administered after the onset of myocardial infarction.

Granulocyte colony stimulating factor (G-CSF) is a hematopoietic growth factor that promotes neutrophil proliferation and function. Intravenous G-CSF is used to treat neutropenia associated with cancer chemotherapy, myodysplasia and aplastic anemia and is used to decrease frequency of infection in patients with congential, idiopathic and cell neutropenia.

Prendiville et al. describe the development of painful neutrophilic skin lesions in a child with cystic fibrosis and cyclic neutropenia receiving G-CSF for idiopathic neutropenia (Pediatric dermatology (United States) 2001 , 18: 417-21 )

G-CSF for use in the present invention may be natural or recombinant G-CSF as described herein below. G-CSF for use in the present invention may be produced by

any methods including but not limited to the methods described herein below or G-CSF may be purchased by any commercial vendor.

Functional homologues

A functional homologue of G-CSF is a polypeptide having at least 70 % sequence identity with SEQ ID NO. 1 or SEQ ID NO. 2 and has one or more G-CSF functions, such as binding to and stimulating G-CSFR, the stimulation of the survival, proliferation, differentiation and function of neutrophil granulocyte progenitor cells and mature neutrophils or preventing infectious complications of some neutropenic states.

Preferably said homologue of any of the predetermined sequences herein, such as SEQ ID NO: 1 or SEQ ID NO 2 may be defined as:

i) homologues comprising an amino acid sequence capable of binding selectively to the G-CSF receptor and stimulating the receptor

ii) homologues capable of stimulation of the survival, proliferation, differentiation or function of neutrophil granulocyte progenitor cells and mature neutrophils

iii) homologues capable of preventing infectious complications of some neutropenic states

iv) homologues exhibiting pulmonary host defense enhancing activities

Methods for assaying the functional activity of the functional homologue for use in the present invention include those described by US 20031 18612, which is hereby incorporated by reference.

Methods for assaying the binding of the functional homologue to the G-CSF receptor may be determined using standard binding assays such as the assay described by Yamasaki et al. (Drugs. Exptl. Clin. Res. 24:191 -196 (1998)

Preferably, evolutionary conservation between G-CSF of different closely related species, e.g. assessed by sequence alignment, can be used to pinpoint the degree of evolutionary pressure on individual residues. Preferably, G-CSF sequences are compared between species where G-CSF function is conserved, including but not limited to mammals including rodents, monkeys and apes. Residues 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 modifications or alterations of the human G-CSF sequence does not interfere with the activity of the G-CSF molecule according to the invention. Such G-CSF molecules are herein referred to as functional equivalents of human G-CSF, and may be such as variants and fragments of native human G-CSF as described here below.

As used herein the expression "variant" refers to polypeptides or proteins which are homologous to the basic protein, which is suitably human G-CSF, but which differs from the base sequence from which they are derived in that one or more amino acids within the sequence are substituted for 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.

A person skilled in the art will know how to make and assess 'conservative' amino acid substitutions, by which one amino acid is substituted for another with one or more shared chemical and/or physical characteristics. Conservative amino acid substitutions are less likely to affect the functionality of the protein. 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. Within the meaning of the term "conservative amino acid substitution" as applied herein, one amino acid may be substituted for another within groups of amino acids characterised by having

Within the meaning of the term "conservative amino acid substitution" as applied herein, one amino acid may be substituted for another within the groups of amino acids indicated herein below:

Lower levels of similarity:

Polarity: i) Amino acids having polar side chains (Asp, GIu, Lys, Arg, His, Asn, GIn, Ser,

Thr, Tyr, and Cys,)

ii) Amino acids having non-polar side chains (GIy, Ala, VaI, Leu, lie, Phe, Trp, Pro, and Met)

Hydrophilic or hydrophobic: iii) Hydrophobic amino acids (Ala, Cys, GIy, lie, Leu, Met, Phe, Pro, Trp, Tyr, VaI)

iv) Hydrophilic amino acids (Arg, Ser, Thr, Asn, Asp, GIn, GIu, His, Lys)

Charges: v) Neutral amino acids ( Ala, Asn, Cys, GIn, GIy, lie, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, VaI)

vi) Basic amino acids (Arg, His, Lys)

vii) Acidic amino acids ((asp, GIu)

High level of similarity:

viii) Acidic amino acids and their amides (GIn, Asn, GIu, Asp)

ix) Amino acids having aliphatic side chains (GIy, Ala VaI, Leu, lie)

x) Amino acids having aromatic side chains (Phe, Tyr, Trp)

xi) Amino acids having basic side chains (Lys, Arg, His)

xii) Amino acids having hydroxy side chains (Ser, Thr)

xiii) Amino acids having sulphor-containing side chains (Cys, Met),

Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine.

A functional homologue within the scope of the present invention is a polypeptide that exhibits at least 70% sequence identity with human G-CSF as identified by SEQ ID NO. 1 or SEQ ID NO. 2, 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 or SEQ ID NO. 2.

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 may be used for searching 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. Moreover, sequence alignments may be performed using a range of penalties for gap opening and extension. For example, 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.

Accordingly, 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.

It is clear from the above outline that 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.

Aside from the twenty standard amino acids and two special amino acids, selenocysteine and pyrrolysine, there are a vast number of "nonstandard amino acids" which are not incorporated into protein in vivo. Examples of nonstandard amino acids include the sulfur-containing taurine and the neurotransmitters GABA and dopamine. Other examples are lanthionine, 2-Aminoisobutyric acid, and dehydroalanine. Further non standard amino are ornithine and citrulline.

Non-standard amino acids are usually formed through modifications to standard amino acids. For example, taurine can be formed by the decarboxylation of cysteine, while dopamine is synthesized from tyrosine and hydroxyproline is made by a posttranslational modification of proline (common in collagen). Examples of 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.

Both standard and non standard amino acid residues described herein can be in the "D" or or "L" isomeric form.

It is contemplated that a functional equivalent according to the invention may comprise any amino acid including non-standard amino acids. In preferred embodiments a functional equivalent comprises only standard amino acids.

The standard and/or non-standard 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.

Suitably variants will be at least 70% identical and accordingly, variants 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 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 the predetermined sequence of human G-CSF of or SEQ ID NO. 1 or or SEQ ID NO. 2..

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.

In addition to the peptidyl compounds described herein, 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 modelling and chemical designing known to those of skill in the art. For example, esterification and other alkylations may be em- ployed to modify the amino terminus of, e.g., a di-arginine peptide backbone, to mimic a tetra peptide structure. It will be understood that all such sterically similar constructs fall within the scope of the present invention.

Peptides with N-terminal alkylations and C-terminal esterifications are also encom- passed within the present invention. Functional equivalents also comprise glycosylated and covalent or aggregative conjugates formed with the same molecules, including dimers or 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.

The term "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 addition mutants capable of binding to and stimulating the G-CSF receptor. 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 mutants capable of binding to and stimulating the G-CSF receptor. The deletion of at least one amino acid may be an addition or deletion of from preferably 2 to 180 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.

Preferably, the number of substitutions, deletions, or additions is 20 amino acids or less, such as 15 amino acids or less, for example 10 amino acids or less, such as 9 amino acids or less, for example 8 amino acids or less, such as 7 amino acids or less, for example 6 amino acids or less, such as 5 amino acids or less, for example 4 amino acids or less, such as 3 amino acids or less, for example 2 amino acids or less (such as 1 ), or any integer in between these amounts. In one aspect of the invention, the substitutions include one or more conservative substitutions, such as 20 or fewer conservative substitutions, for example 18 or fewer, such as 16 or fewer, for example 14 or fewer, such as 12 or fewer, for example 10 or fewer, such as 8 or fewer, for example 6 or fewer, such as 4 or fewer, for example 3 or fewer, such as 2 or fewer conservative substitutions.

Deletion mutants suitably comprise at least 20 or 40 consecutive amino acid and more preferably at least 80 or 100 consecutive amino acids in length. Accordingly such a fragment may be a shorter sequence of the sequence as identified by SEQ ID NO: 1 or SEQ ID NO: 2 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 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 or SEQ ID NO: 2.

It is preferred that functional homologues of G-CSF comprises at the most 500, more preferably at the most 400, even more preferably at the most 300, yet more preferably at the most 200 amino acids. In one preferred embodiment a functional homologues of G-CSF comprises at the most 180 such as at the most 175, for example at the most 160, such as at the most 150 amino acids, for example at the most 144 amino acids. In one preferred embodiment a functional homologues of G-CSF comprises at the most 174 or 180 amino acids.

The term "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.

There are two known splice variants of G-CSF of 174 - and 180 amino acids respectively. There are two known sequence variations of human G-CSF; a L157M substitution in variant 1 and a A174T substitution in variant 2. Accordingly, in one embodiment of the invention functional homologues of G-CSF comprise a sequence with high sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2 or any of the splice variants. The variants described are functional equivalents within the present invention.

Amino acids thought to be in the contact or adjacent region when G-CSF is bound to its receptor include G4, P5, A6, S7, S8, L9, P10, Q1 1 , S12, L15, K16, E19, Q20, L108,

D109, D1 12, T115, T1 16, Q1 19, E122, E123, and L124. Preferably a G-CSF fragment comprises at least some of the above mentioned residues, more preferably most of the indicated residues.

Different isoforms of G-CSF including but not limited to those described in for example WO 06/135176 are also functional homologues within the scope of the present invention. Further analogues of G-CSF which are within the scope of this invention are described in for example WO 02/028896, WO 02/077034, WO 01/51510 and WO 03/06501 which are hereby incorporated by references.

Recombinant production

The present invention relates to the pulmonary administration, of an agent capable of binding to and stimulating the G-CSF receptor, preferably granulocyte colony stimulating factor (G-CSF), or a functional homologue of thereof, however prepared, to enhance the pulmonary host defense. G-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.

In one embodiment of the invention, G-CSF is produced recombinantly by host cells.

Thus, in one aspect of the present invention, G-CSF is produced by host cells comprising a first nucleic acid sequence encoding the G-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 G-CSF in general comprises the steps of:

-providing a host cell

-preparing a gene expression construct comprising a first nucleic acid encoding G-CSF operably linked to a second nucleic acid capble 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 G-CSF.

The recombinant G-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 G-CSF.

In one embodiment of the invention, the recombinantly produced G-CSF is excreted by the host cells. When G-CSF is excreted 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 G-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 G-CSF and secretion of G- CSF into the culture medium,

-thereby obtaining culture medium comprising G-CSF.

The composition comprising G-CSF and nucleic acids may thus in this embodiment of the invention be the culture medium or a composition prepared from the culture medium.

In another embodiment of the invention said composition is an extract prepared from animals, parts thereof or cells or an isolated fraction of such an extract.

In an embodiment of the invention, G-CSF is recombinantly produced in vitro in host cells and is isolated from cell lysate, cell extract or from tissue culture supernatant. In a

more preferred embodiment G-CSF is produced by host cells that are modified in such a way that they express G-CSF. In an even more preferred embodiment of the invention said host cells are transformed to produce and excrete G-CSF.

The synthesis of the protein of interest may be by use of in vitro or in vivo cultures. One process for producing recombinant G-CSF according to the invention is characterised in that the host cell culture is may be eukaryotic, and for example a mammalian cell culture or a yeast cell culture.

Useful mammalian cells may for example be human embryonal kidney cells (HEK cells), such as the cell lines deposited at the American Type Culture Collection with the numbers CRL-1573 and CRL-10852, chick embryo fibroblast, hamster ovary cells, baby hamster kidney cells, human cervical carcinoma cells, human melanoma cells, human kidney cells, human umbilical vascular endothelium cells, human brain endothelium cells, human oral cavity tumor cells, monkey kidney cells, mouse fibroblast, mouse kidney cells, mouse connective tissue cells, mouse oligodendritic cells, mouse macrophage, mouse fibroblast, mouse neuroblastoma cells, mouse pre-B cell, mouse B lymphoma cells, mouse plasmacytoma cells, mouse teratocacinoma cells, rat astrocytoma cells, rat mammary epithelium cells, COS, CHO, BHK, 293, VERO, HeLa, MDCK, WI38, and NIH 3T3 cells.

In another embodiment of the invention the host cells are either prokaryotic cells or yeast cells. Prokaryotic cells may for example be E. coli. Yeast cells may for example be Saccharomyces, Pichia or Hansenula.

Medical indications

Administration of an effective amount of an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF or a functional homologue thereof via intratracheal, intrabronchial, intraalveolar or bronchio-alveolar administration is particularly useful in alleviating symptoms and/or treating subjects suffering from conditions including, but not limited to lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis

carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization.

Infections may for example be an infection by bacteria, fungi, viruses, parasites. For example infection by parasites such as Plasmodium falciparum. For example infection by one or more bacteria selected from the group consisting of Achromobacter xylosoxidans, Acinetobacter calcoaceticus, preferably A. anitratus, A. haemolyticus, A. alcaligenes, and A. Iwoffii, Actinomyces israelii, Aeromonas hydrophilia, Alcaligenes species, preferably A. faecalis, A. odorans and A. denitrificans, Arizona hinshawii, Bacillus anthracis, Bacillus cereus, Bacteroides fragilis, Bacteroides melaninogenicus, Bordetella pertussis, Borrelia burgdorferi, Borrelia recurrentis, Brucella species, preferably B. abortus, B. suis, B. melitensis and B. canis, Calymmatobacterium granulomatis, Campylobacter fetus ssp. intestinalis, Campylobacter fetus ssp. jejuni, Chlamydia species, preferably C. psittaci and C. trachomatis, Chromobacterium violaceum, Citrobacter species, preferably C. freundii and C. diversus, Clostridium botulinum, Clostridium perfringens, Clostridium difficile, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium, preferably C. ulcerans, C. haemolyticum and C. pseudotuberculosis, Coxiella burnetii, Edwardsiella tarda, Eikenella corrodens, Enterobacter, preferably E. cloacae, E. aerogenes, E. hafniae (also named Hafnia alvei) and E. agglomerans, Erysipelothrix rhusiopathiae, Escherichia coli, Flavobacterium meningosepticum, Francisella tularensis, Fusobacterium nucleatum, Gardnerella vaginalis, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter species, Klebsiella species, preferably K. pneumoniae, K. ozaenae og K. rhinoscleromatis, Legionella species, Leptospira interrogans, Listeria monocytogenes, Moraxella species, preferably M. lacunata and M. osloensis, Mycobacterioum bovis, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma species, preferably M. pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia species, preferably N. asteroides and N. brasiliensis, Pasterurella haemolytica, Pasteurella multocida, Peptococcus magnus, Plesiomonas shigelloides, Pneumococci, Proteus species, preferably P. mirabilis, P. vulgaris, P. rettgeri and P. morganii (also named Providencia rettgeri and Morganella morganii respectively), Providencia species, preferably P. alcalifaciens, P. stuartii and P. rettgeri (also named Proteus rettgeri), Pseudomonas aeruginosa, Pseudomonas mallei, Pseudomonas pseudomallei, Rickettsia, Rochalimaia henselae, Salmonella species, preferably S. enteridis, S. typhi and S. derby, and most preferably Salmonella species

of the type Salmonella DT104, Serratia species, preferably S. marcescens, Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei, Spirillum minor, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptobacillus moniliformis, Streptococcus, preferably S. faecalis, S. faecium and S. durans, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema carateum, Treponeam pallidum, Treponema pertenue, preferably T. pallidum, Ureaplasma urealyticum, Vibrio cholerae, Vibrio parahaemolyticus, Yersinia enterocolitica, and Yersinia pestis.

Infections also comprise protozoan infections such as, but not limited to, Trichomonas infections, such as Pentatrichomonas infections. For example T. buccalis, T. tenax, T. foetus, T. galli ^' nae, T. gallina ^' rum, T. ho ^' minis, T. intestinalis, T. te ^' nax, T. vaginalis.

In other embodiments of the invention G-CSF may be used for the treatment of any condition caused by fungal infections including, but not limited to : Aspergillosis, Blastomycosis, Candidiasis, Coccidioidomycosis, Cryptococcosis, Histoplasmosis, Paracoccidiomycosis, Sporotrichosis, Zygomycosis. The composition may also be used to treat fungal infections in conditions such as Chromoblastomycosis, Mycotic keratitis, Endogenous oculomycosis, Extension oculomycosis, Lobomycosis, Mycetoma, Nail, Hair, and Skin diseases (for example Onychomycosis (Tinea unguium), Piedra, Pityriasis versicolor, Tinea barbae, Tinea capitis, Tinea corporis, Tinea cruris, Tinea favosa, Tinea nigra, Tinea pedis), Otomycosis, Phaeohyphomycosis, Rhinosporidiosis.

In preferred embodiments the subject is suffering from a disease selected from the group consisting of lung cancer, Pneumocystis carinii, panbronchiolitis, bronchietasis, pneumonia with or without bacterial, fungal or viral infection or colonization, community acquired pneumonia with or without bacterial, fungal or viral infection or colonization, nosocomial pneumonia with or without bacterial, fungal or viral infection or colonization, ventilatory associated pneumonia with or without bacterial, fungal or viral infection or colonization and cystic fibrosis with or without bacterial, fungal or viral infection or colonization. More preferred the subject is suffering from a disease selected from the group consisting of lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis,

bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization.

Administration

Methods of intratracheal, intrabronchial, intraalveolar or bronchio-alveolar administration include, but are not limited to, spraying, lavage, inhalation, flushing or instillation, using as fluid a physiologically acceptable composition in which an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF have been dissolved. When used herein the terms "intratracheal, intrabronchial or intraalveolar or bronchio-alveolar administration" include all forms of such administration whereby an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF is applied into the trachea, the bronchi or the alveoli, respectively, whether by the instillation of a solution of an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF, by applying an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF in a powder form, or by allowing an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF to reach the relevant part of the airway by inhalation of an agent capable of binding to and stimulating the G- CSF receptor, such as G-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/alveolar 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 the an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF been dissolved or indeed by any other effective form of intrabronchial administration including the use of inhaled powders containing an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF in dry form, with or without excipients, or the direct application of an agent capable of binding to and stimulating the G-CSF receptor, such as G-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 or a suspension of an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF, or the inhalation of nebulized fluid droplets containing dissolved an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF, a suspension of an agent

capable of binding to and stimulating the G-CSF receptor, such as G-CSF, obtained by use of any nebulizing apparatus adequate for this purpose.

In another embodiment, intratracheal, intrabronchial or intraalveolar administration does not include inhalation of G-CSF but the instillation or application of a solution of G-CSF or a powder or a gel containing G-CSF into the trachea or lower airways.

Other preferred methods of administration may include using the following devices:

1 . Pressurized nebulizers using compressed air/oxygen mixture 2. Ultrasonic nebulizers

3. Electronic micropump nebulizers (e.g. Aeroneb Professional Nebulizer)

4. Metered dose inhaler (MDI)

5. Dry powder inhaler systems (DPI),

The aerosol may be delivered by via a) facemasks or b) via 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.

Thus, in one embodiment the effective amount of an agent capable of binding to and stimulating the G-CSF receptor is administered by intratracheal, intrabronchial, intraalveolar or bronchio-alveolar administration.

In another embodiment the subject is administered a solution of an agent capable of binding to and stimulating the G-CSF receptor via bronchoalveolar lavage.

In another embodiment the subject is administered a solution of an agent capable of binding to and stimulating the G-CSF receptor via blind tracheal washing.

In another embodiment the subject is administered a nebulized solution or a suspension of an agent capable of binding to and stimulating the G-CSF receptor.

In another embodiment the subject is administered a nebulized aerosol or inhaled powder form of an agent capable of binding to and stimulating the G-CSF receptor.

In another embodiment the subject is administered a pegylated, liposomal or nanoparticle prepared form of G-CSF or a functional homologue thereof.

In another embodiment the subject is administered G an agent capable of binding to and stimulating the G-CSF receptor by direct application of an agent capable of binding to and stimulating the G-CSF receptor during bronchoscopy.

In another embodiment the subject is a mammal.

In another embodiment the mammal is a human.

In another embodiment the human is a child younger than 12 years of age.

In another embodiment the human is an adult older than 12 years of age.

Preferred concentrations for a solution comprising an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF or a functional homologue thereof are in the range of 0.1 μg to 10000 μg active ingredient per ml solution. The suitable concentrations are often in the range of from 0.1 μg to 5000 μg per ml solution, such as in the range of from about 0.1 μg to 3000 μg per ml solution, and especially in the range of from about 0.1 μg to 1000 μg per ml solution, such as in the range of from about 0.1 μg to 250 μg 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 0.2 to about 2.5 mg and especially in the range from 0.2 to 1 .0 mg per ml solution.

Pharmaceutical composition

Pharmaceutical compositions or formulations for use in the present invention include an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF in combination with or preferably dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent, or as a pegylated preparation carried to the lower airways or as a liposomal or nanoparticle preparation administered as an aerosol via inhalation, or as a lavage fluid administered via a bronchoscope as a bronchoalveloar lavage or as a blind intratracheal wash or lavage. A variety of aqueous

carriers may be used, including, but not limited to 0.9% saline, buffered saline, physiologically compatible buffers and the like. The 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

In one embodiment a freeze-dried an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF preparation may be pre-packaged for example in single dose units. In an even more preferred embodiment 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. Conventional 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. The most frequently used lipid for liposome formation are: 1 ^-Dilauroyl-sn-Glycero-S-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-Dimyristoyl-sn-Glycero-3-Phosphate

(Monosodium Salt) (DMPA), 1 ,2-Dipalmitoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DPPA), 1 ,2-Dioleoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DOPA), 1 ,2- Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1 -glycerol)] (Sodium Salt) (DMPG), 1 ,2- Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1 -glycerol)] (Sodium Salt) (DPPG), 1 ,2- Dioleoyl-sn-Glycero-3-[Phospho-rac-(1 -glycerol)] (Sodium Salt) (DOPG), 1 ,2-

Dimyristoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt) (DMPS), 1 ,2-Dipalmitoyl- sn-Glycero-3-[Phospho-L-Serine) (Sodium Salt) (DPPS), 1 ,2-Dioleoyl-sn-Glycero-3- [Phospho-L-Serine] (Sodium Salt) (DOPS), 1 ,2-Dioleoyl-sn-Glycero-3- Phosphoethanolamine-N-(glutaryl) (Sodium Salt) and 1 ,1 ',2,2'-Tetramyristoyl 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. 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).

Possible lipids applicable for liposomes are supplied by Avanti, Polar Lipids, Inc,

Alabaster, AL. Additionally, 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 ferrioxianine, are preferred.

A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), 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 multilamellar vesicles of heterogeneous sizes. In this method, 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. If desired, the film may be redissolved 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. 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 multilamellar 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.

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 pharmaceutical administration including but not limited to IV injection such as, TWEEN-80, PLURONIC F-68, n-octyl-beta-D-glucopyranoside, and the like. In addition, phospholipids, such as those described for use in the production of liposomes, may also be used for micelle formation.

In some cases, it will be advantageous to include a compound, which promotes delivery of the active substance to its target.

Dose

According to the present invention a pharmaceutically effective amount or a therapeutically effective amount is to be understood as an amount sufficient to induce a desired biological result. The result can be alleviation of the signs, symptoms, or causes of a disease, for example of lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization, preferably, the result is a significant alleviation of signs, symptoms or causes of lung cancer, pneumonia, ventilator associated pneumonia (VAP), hospital acquired pneumonia (HAP), community acquired pneumonia (CAP), severe community acquired pneumonia (sCAP), Pneumocystis carinii, panbronchiolitis, bronciectasis and cystic fibrosis with bacterial, fungal and/or viral infection and/or bacterial, fungal and/or viral colonization

,. For example, an effective amount is generally that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer, preferably such a relief of symptoms is a significant relief. Thus, by "effective amount" of an agent capable of binding to and stimulating the G- CSF receptor it is meant a dose, which, when administered via pulmonary administration, achieves a concentration of an agent capable of binding to and stimulating the G-CSF receptor in the subject's airways and/or lung parenchyma which enhances pulmonary host defense.

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 kilo body weight normally of the order of several hundred μg active ingredient per administration with a preferred range of from about 0.1 μg to 10000 μg per kilo body weight. Doses expected to provide an effective amount of an agent capable of binding to and stimulating the G-CSF receptor comprise an agent capable of binding to and stimulating the G-CSF receptor are often in the range of from 0.1 μg to 5000 μg per kilo body weight, such as in the range of from about 0.1 μg to 3000 μg per kilo body weight, and especially in the range of from about 0.1 μg to 1000 μg per kilo body weight, preferably in the range of 1 μg to 700 μg, even more preferably about 50 μg to about 500 μg administered via inhalation once or twice daily.

Suitable daily dosage ranges are per kilo body weight per day normally of the order of several hundred μg active ingredient per day with a preferred range of from about 0.1 μg to 10000 μg per kilo body weight per day. Using monomeric forms of G-CSF for example, the suitable dosages are often in the range of from 0.1 μg to 5000 μg per kilo body weight per day, such as in the range of from about 0.1 μg to 3000 μg per kilo body weight per day, and especially in the range of from about 0.1 μg to 1000 μg per kilo body weight per day, when based on monomeric forms having a sequence identical to sequence ID NO: 1 or SEQ ID NO: 2, for functional homologues and fragments the dose is calculated based on the molecular weight of the monomeric form to the molecular weight of the homologues or fragments.

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 -2 days to 2 months, such as in the range of 1 -2 days to 1 month.

Medical packaging

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.

It is preferred that 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.

Thus, it is preferred that the medical packaging comprises an amount of dosage units corresponding to the relevant dosage regimen. Accordingly, in one embodiment, 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, bronchio-alveolar or intraalveolar administration. In a preferred embodiment the packaging is in the form of a vial, ampule, tube, blister pack, cartridge or capsule.

When the medical packaging comprises more than one dosage unit, it is preferred that the medical packaging is provided with a mechanism to adjust each administration to one dosage unit only.

Preferably, 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. Accordingly, in one embodiment the medical packaging comprises instructions for administering the pharmaceutical composition .

Even more preferably a freeze-dried preparation of an agent capable of binding to and stimulating the G-CSF receptor, such as G-CSF may be pre-packaged for example in single dose units. In an even more preferred embodiment the single dose unit is adjusted to the patient.

Examples

Example 1

Protocol for local pulmonary treatment with G-CSF through Bronchoalveolar lavage (BAD

/. Patient group to be treated:

Patients with disease selected from the group consisting of lung cancer, Pneumocystis carinii, panbronchiolitis, bronchietasis, pneumonia with or without bacterial, fungal or viral infection or colonization, community acquired pneumonia with or without bacterial, fungal or viral infection or colonization, nosocomial pneumonia with or without bacterial, fungal or viral infection or colonization, ventilatory associated pneumonia with or without bacterial, fungal or viral infection or colonization and cystic fibrosis with or without bacterial, fungal or viral infection or colonization with reduced oxygenation capacity as revealed by a reduced PaCyFiO ₂ ratio, i.e. < 200 mmHg (arterial oxygen tension in mmHg over inspired oxygen fraction) in spite of treatment with full antibiotic coverage towards the isolated microbiological agent or treatment of underlying disease.

//. Treatment regime: Local administration of 5 mg G-CSF dissolved in 20 ml of normal saline via Bronchoalveolar lavage (BAL).

///. Analysis of results:

a) Monitoring of oxygenation capacity as by monitoring the PaO2/FiO2 ratio (arterial oxygen tension in mmHg over inspired oxygen fraction). A successful treatment results in an increase in oxygen capacity with a PaO ₂ /FiO ₂ ratio, i.e. > 200 mmHg. b) Radiography of the lung field before and after treatment. As the patients have infiltrations in the lung a successful treatment leads to reduction of these infiltrations as monitored by radiography.

Example 2

Protocol for local pulmonary treatment with G-CSF through inhalation

/. Patient group to be treated:

Patients with disease selected from the group consisting of lung cancer, Pneumocystis carinii, panbronchiolitis, bronchietasis, pneumonia with or without bacterial, fungal or viral infection or colonization, community acquired pneumonia with or without bacterial, fungal or viral infection or colonization, nosocomial pneumonia with or without bacterial, fungal or viral infection or colonization, ventilatory associated pneumonia with or without bacterial, fungal or viral infection or colonization and cystic fibrosis with or without bacterial, fungal or viral infection or colonization with reduced oxygenation capacity as revealed by a reduced PaO ₂ /FiO ₂ ratio, i.e. < 200 mmHg (arterial oxygen tension in mmHg over inspired oxygen fraction) in spite of treatment with full antibiotic coverage towards the isolated microbiological agent or treatment of underlying disease.

//. Treatment regime: Local administration of 3 x 5 mg G-CSF via a micropump nebulizer (e.g. Aeroneb® nebulizer).

///. Analysis of results: a) Monitoring of oxygenation capacity as by monitoring the PaO ₂ /FiO ₂ ratio (arterial oxygen tension in mmHg over inspired oxygen fraction). A successful treatment results in an increase in oxygen capacity with a PaO ₂ /FiO ₂ ratio, i.e. > 200 mmHg. b) Radiography of the lung field before and after treatment. As the patients have infiltrations in the lung a successful treatment leads to reduction of these infiltrations as monitored by radiography.