WO2014096032A1

WO2014096032A1 - Gm-csf for treatment of chronic sinusitis

Info

Publication number: WO2014096032A1
Application number: PCT/EP2013/077137
Authority: WO
Inventors: Lasse HESLET
Original assignee: Trifoilium Aps
Priority date: 2012-12-18
Filing date: 2013-12-18
Publication date: 2014-06-26

Abstract

Compositions comprising one or more cytokines and methods for their use in inhibiting and/or alleviating chronic inflammation in the nasal and/or paranasal sinus tissue in a subject in need thereof are provided.

Description

GM-CSF FOR TREATMENT OF CHRONIC SINUSITIS

Field of invention

Background of invention

Nasal polyposis is a chronic inflammatory condition of the upper airways leading to the formation of nasal polyps, which can be broadly defined as abnormal polypoidal masses arising from the mucous membranes of the nose and paranasal sinuses, i.e. as overgrowth of the mucosa in the nasal or paranasal tissue. The condition is often associated with symptoms such as nasal blockage, congestion, hyposmia or anosmia and if associated with chronic sinusitis a purulent nasal discharge. Secondary symptoms comprise post nasal drip, rhinorrhea, facial pain, headache, sleep disturbance and lowered quality of life. The mechanisms involved in the formation of the condition are presently not determined, however, the condition is often associated with other diseases such as chronic rhinosinusitis or sinusitis, allergic fungal sinusitis (AFS) , asthma, aspirin intolerance/salicylate sensitivity, primary ciliary dyskinesia, cystic fibrosis (CF), Kartagener's syndrome, Young's syndrome, Churg-Strauss syndrome and nasal mastocytosis.

Steroids or corticoids such as beclomethasone, dipropionate or flunisolide are presently used for treatment of nasal polyps, however symptoms often return when the treatment is stopped. The disadvantages of therapies based upon the above mentioned compounds is development of local hypersensibility and that the

compounds cannot be used for prolonged treatment because of adverse effects.

Nasal polyps may also be removed by surgery, (such as for example functional endoscopic sinus surgery (FESS) or polypectomy) but are found to recur in a majority of cases. Sinus surgery requires great amount of precision as this involves risk of damage to orbit matter. Thus, patients may be subjected to multiple surgeries with their attendant costs and risks of complications and/or may experience a long-term reduction in quality of life due to continued symptoms (Bachert et al. Drugs 2005:

65(1 1 ):1537-1552). Thus, new non-invasive treatment methods which can reduce the formation of nasal polyps and the occurrence of related symptoms, as well as reduce the risks of damages caused by the presently used treatment methods are needed.

Summary of invention

The present invention relates to the use of GM-CSF for treatment of chronic nasal and/or paranasal sinus inflammation which can lead to nasal and/or paranasal sinus polyposis, including methods for treatment of said conditions, pharmaceutical compositions and kits of parts comprising GM-CSF for treatment of said conditions. The new treatment method targets the underlying chronic inflammation of the nasal tissue and thereby has an effect on the condition leading to formation of nasal polyps.

Thus the present invention relates to a composition comprising granulocyte- macrophage colony-stimulating factor (GM-CSF), or a functional variant, homologue or derivative thereof, for use in the treatment, prevention or alleviation of chronic nasal and/or paranasal sinus inflammation, preferably nasal and/or paranasal sinus polyposis.

Compositions comprising (GM-CSF), or a functional variant, derivative or homologue thereof may be administered locally for example by intranasal and/or intrasinus administration, and may be administered as a solution, a suspension, an aerosol, a nebulized solution, a nebulized suspension, as a pegylated, liposomal or nanoparticle prepared form or as a powder. Compositions according to the present invention may further be administered via nasal drops, spraying, lavage, flushing, inhalation or exhalation, injection or instillation, insertion of a depot, preferably by injection or via CPAP.

Chronic nasal and/or paranasal sinus inflammation according to the present invention may be associated with a condition selected from the group consisting of chronic rhinosinusitis, chronic sinusitis, allergic fungal sinusitis (AFS) , asthma, aspirin intolerance/salicylate sensitivity, primary ciliary dyskinesia, cystic fibrosis (CF), Kartagener's syndrome, Young's syndrome, Churg-Strauss syndrome, nasal mastocytosis, exposure to chromium and allergic rhinitis. The compositions comprising GM-CSF, or a functional variant, derivative or homologue thereof, may be administered at an effective amount, such as from between 100 to 1000 microgram per dose, for example 200-800 microgram per dose, such as 300 microgram per dose, once, twice, three times or four times a day in a period lasting for 1 day or for between 1 to 14 days, such as 1 to 3 days, 3 to 5 days, 5 to 7 days, 7 to 10 days, 10 to 14 days.

Another aspect of the present invention provides a method for treating, preventing, reducing risk of, or alleviating of chronic nasal and/or paranasal sinus inflammation in a subject in need thereof, said method comprising administering to the subject an effective amount of a composition comprising granulocyte-macrophage colony- stimulating factor (GM-CSF), or a functional variant, derivative or homologue thereof. In a preferred embodiment, said chronic nasal and/or paranasal sinus inflammation leads tonasal and/or paranasal sinus polyposis. Description of Drawings

Figure 1 : An example of an airtight mask used for continuous positive airway pressure. The in- and expiratory sides are separated via T-piece equipped with valves (not shown). A medicament can be applied via the inspiratory side by use of a nebulizer.

Detailed description of the invention

Nasal and paranasal sinus polyposis

It is an aspect of the present invention to provide a composition comprising

granulocyte-macrophage colony-stimulating factor (GM-CSF), or a functional variant, derivative or homologue thereof, for use in the treatment, prevention or alleviation of chronic nasal and/or paranasal sinus inflammation, preferably nasal and/or paranasal sinus polyposis and associated symptoms. Prevention may be equivalent to reducing risk of acquiring said condition and symptoms. It is also an aspect of the present invention to provide use of a composition comprising granulocyte-macrophage colony-stimulating factor (GM-CSF), or a functional variant, derivative or homologue thereof, for manufacture of a medicament for the treatment, prevention or alleviation of chronic nasal and/or paranasal sinus inflammation, preferably nasal and/or paranasal sinus polyposis and associated symptoms.

Nasal polyposis is a chronic inflammatory condition of the upper airways (e.g. the nasal or paranasal sinus tissue) leading to the formation of polyps in the nasal or paranasal sinus tissue. Broadly defined, nasal polyposis is can be characterized by abnormal polypoidal masses arising from the mucous membranes of the nose and paranasal sinuses, i.e as overgrowth of the mucosa. Nasal polyps are often found at the outflow tract of the sinuses. Nasal polyps are covered with respiratory epithelium and are typically filled with edematous stroma and activated inflammatory cells, particularly eosinophils, but also mast cells, neutrophils, lymphocytes and plasma cells can be present.

Nasal and/or paranasal sinus polyps can be categorized as edematous, cystic, or fibrous. Edematous polyps are characterized by massive tissue edema, resulting from a leakage of plasma through widened endothelial junctions in the blood vessels. The typical histological characteristics of nasal polyps include edematous fluid with sparse fibrous cells, and few mucous glands with no innervation, squamous metaplasia of the surface epithelium, proliferation of stromal and epithelial elements and a thickening of the basement membrane. Eosinophils are the dominant cell type of nasal polyps. Activated eosinophils release toxic granule proteins and pro-inflammatory mediators, which may cause tissue damage and dysfunction. Recent evidence suggests that eosinophils may also be involved in tissue remodeling and immunoregulation. Eosinophils can synthesize, store and secrete at least 35 inflammatory and immunoregulatory cytokines, chemokines, and growth factors, including GM-CSF. These may play roles in the regulatory functions of eosinophils. Eosinophils often localize at sites of Th2-type chronic inflammation.

The underlying mechanisms causing nasal and/or paranasal sinus polyposis are still largely unknown, but the condition can be associated with a number of diseases including but not limited to chronic rhinosinusitis or sinusitis, allergic fungal sinusitis (AFS) , asthma, aspirin intolerance/salicylate sensitivity, primary ciliary dyskinesia, cystic fibrosis (CF), Kartagener's syndrome, Young's syndrome, Churg-Strauss syndrome and nasal mastocytosis. Exposure to some forms of chromium can also cause nasal polyps and associated diseases. Nasal and/or paranasal sinus polyps can also be accompanied by allergic rhinitis. In various embodiments of the present invention, the nasal and/or paranasal sinus polyposis may not or may be associated with the above mentioned diseases. Primary symptoms of nasal and/or paranasal sinus polyposis are nasal blockage, congestion, hyposmia or anosmia and if associated with chronic sinusitis a purulent nasal discharge. Secondary symptoms comprise post nasal drip, rhinorrhea, facial pain, head ache, pain referred to upper teeth, sense of pressure over the forehead and/or the face, snoring, a sensation of itching around the eyes, sleep disturbance and lowered quality of life. Symptoms of nasal and/or paranasal sinus polyposis may also include other signs of inflammation of the nasal and/or paranasal sinus tissue such as for example fever. The present invention may help to reduce, alleviate or treat said symptoms of nasal and/or paranasal sinus polyposis. Diagnosis of nasal and/or paranasal sinus polyposis can be made by history, clinical examination, radiography, nasal endoscopy and additional tests for allergy, aspirin sensitivity, bacteriology, and pulmonary function tests.

Nasal and/or paranasal sinus polyps are usually classified into antrochoanal polyps and ethmoidal polyps. Antrochoanal polyps arise from the maxillary sinuses and are usually single and unilateral, and often found in children. Ethmoidal polyps arise from the ethmoidal sinuses which are often multiple and bilateral, and often found in adults. An individual polyp can be an antral-choanal polyp, a benign massive polyp, or any benign or malignant tumor (eg, encephaloceles, gliomas, hemangiomas, papillomas, juvenile nasopharyngeal angiofibromas, rhabdomyosarcoma, lymphoma,

neuroblastoma, sarcoma, chordoma, nasopharyngeal carcinoma, inverting papilloma).

A variety of objective measures suitable for assessing, e.g., quantifying, the severity of nasal and/or paranasal sinus polyposis are available. For example, olfactory function can be assessed using the Smell Identification Test (Doty RL. The Smell Identification Test(TM) Administration Manual. 3rd ed. Sensonics, Inc.; Haddon Heights, New Jersey, pp. 1 -17, 1995). The Lund-Mackay CT score (Lund VJ, Mackay IS. Staging in rhinosinusitis. Rhinology. 107: 183-184, 1993) and/or Lund-Kennedy endoscopy score (Lund VJ, Kennedy DW. Quantification for staging sinusitis. International Conference on Sinus Disease: terminology, staging, therapy. Ann Oto Rhinol Laryngol. 104(Suppl): 17-21 , 1995). The Lund-Mackay system assigns a score of 0-2 dependent on the absence, partial, or complete pacification of each sinus system and of the ostiomeatal complex, deriving a maximum score of 12 per side. Nasal polyps can be graded based on size and/or number. A variety of grading methods, e.g., based on endoscopy and/or CT, are available for assessment of nasal polyposis. For example, these include: (1 ) lateral imaging projecting the extension of the polyps by drawing on a schematic picture of the lateral wall of each nasal cavity; (2) assessment of polyp obstruction estimating the proportion of the total nasal cavity volume occupied by polyps; (3) nasal airway patency-determining the relationship between the patient's patent airway lumen and an imaginary maximal nasal airway lumen; (4) a four point scoring system of

Lildholdt et al, involving determining their relationship to fixed anatomical landmarks; and (5) a three point scoring system of Lund and Mackay (see, e.g., Johansson L, Acta Otolaryngol., 120(l):72-6, 2000, and references therein). For example, polyps can be scored as 1 , 2, or 3 (on a scale of 0 to 3) in each of the right and left nasal cavities, where 0 indicates no polyps; 1 , polyps within the middle meatus; 2, polyps not confined to the middle meatus; and 3, completely obstructive polyps; or where 0 indicates no polyps, 1 , polyps restricted to middle meatus; 2, polyps below middle turbinate; and 3, massive polyposis. Validated, disease-specific quality of life (QOL) instruments, such as the Rhinosinusitis Disability Index (RSDI), the Chronic Sinusitis Survey (CSS), or the Sinonasal Outcomes Test (SNOT) can be used. The RSDI is a 30-question survey (range: 0-120) developed to predict rhinosinusitis specific health outcomes in three domains (physical, functional, and emotional) (Benninger MS, Senior BA. The development of the rhinosinusitis disability index. Arch Otolaryngol Head Neck Surg. 123 : 1 175-1 179, 1997). Higher scores on the RSDI represent a higher level of disease impact and worse QOL status. The CSS is a 6 item disease-specific questionnaire developed for assessing health status and treatment effectiveness in CRS. Higher total and subscale scores (range: 0-100) represent a lower impact of disease and better QOL status (Gliklich RE, Metson R. Techniques for outcomes research in chronic rhinosinusitis. Laryngoscope. 105:387-390, 1995). The 20-ltem Sino- Nasal Outcome Test (SNOT-20) is a validated, self-administered, quality of life instrument specific for patients with symptoms of rhinosinusitis (Piccirillo JF,et al. Psychometric and clinimetric validity of the 20-ltem Sino-Nasal Outcome Test (SNOT- 20). Otolaryngol Head Neck Surg. 126(l):41 -47, 2002). The instrument measures physical problems, functional limitations, and emotional consequences of sinusitis by asking subjects to score 20 items, including the need to blow the nose, sneezing, runny nose, cough, postnasal discharge, thick nasal discharge, ear fullness, dizziness, ear pain, facial pain/pressure, difficulty falling asleep, waking up at night, lack of a good night's sleep, waking up tired, fatigue, reduced productivity, reduced concentration, frustrated/restless/irritable, and being sad and embarrassed. The more recently developed 22 item Sinonasal Outcome Test (SNOT-22), a modification of the SNOT- 20, can be used (see, e.g., Hopkins C, et al. Psychometric validity of the 22-item Sinonasal Outcome Test. Clin Otolaryngol., 34(5):447-54, 2009). The Medical Short Form-36 is a general health-related QOL instrument. The SF-36 contains measures overall QOL status in eight individual domains: general health, physical functioning, physical role, bodily pain, vitality, social functioning, emotional role, and mental health. Higher subscale scores (range: 0-100) represent a lower impact of disease severity and better overall health.

According to the present invention, the beneficial effects on nasal and/or paranasal sinus polyposis of treatment with GM-CSF can be assessed by one or more of the abovementioned assessment methods.

In one embodiment, the subject is a mammal. In one embodiment, the mammal is a human. In one embodiment, the human is a child younger than 15 years of age. In one embodiment, the human is an adult 15 years of age or older.

GM-CSF

Colony-stimulating factors (CFS) 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, CSF's 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. The cytokine according to the present invention is GM-CSF. Mature GM-CSF is a monomeric protein of 127 amino acids with several potential glycosylation sites. The variable degree of glycosylation results in a molecular weight range between 14kDa and 35kDa. 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. GM-CSF exerts its biological activity by binding to its receptor. The most important sites of GM-CSF receptor (GM-CSF-R) expression are on the cell surface of myeloid cells, like macrophages (type I & II), eosinophils and granulocytes, epithelial cells and endothelial cells, whereas lymphocytes are GM-CSF-R negative. 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) . The binding domains on GM-CSF for the receptor have been mapped: 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 b; Shanafelt et al., 1991 a; 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 carboxyterminal tail of GM-CSF (Brown et al., 1994).

Formation of the 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 NFkB, 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).

Apart from its hemopoietic growth and differentiation stimulating activity, GM-CSF functions especially as a proinflammatory cytokine. Macrophages, e.g. macrophages type I & II and monocytes as well as neutrophils and eosinophils become 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-chemokinesm which in turn, leads to increased chemotaxis of inflammatory cells into inflamed tissue.

GM-CSF a drug with seemingly "many faces"

Without being bound by a specific theory, the inventors hypothesize that GM-CSF mediates different immune responses depending on the cells being stimulated by GM- CSF. This may explain the seemingly paradoxical effect of GM-CSF, that the protein has seemingly different effects on each side of the mucous barrier.

It appears that on the air side of the mucous barrier (the surface of the nasal or paranasal sinus epithelium) GM-CSF has an inhibitory effect on the chronic

inflammation - the so-called T helper cell inflammatory pattern, type two (T_H2) mediated inflammation and augmenting effect on the T helper cell inflammatory pattern, type one (T_H1 ) response. This effect is different from the autocrine effect of endogenous GM-CSF produced inside the inflamed tissue (for example eosinophils, neutrophils and macrophages can be stimulated in respect to endogenous GM-CSF production). In a preferred embodiment, GM-CSF is administered to the epithelial side of the nasal cavity and/or paranasal sinuses.

Without being bound by theory, the inventors believe that GM-CSF administered on the surface of the epithelium in the airways has a positive effect in that it helps to transform a chronic inflammation regulated by Th2 cells into a Th1 regulated inflammation. The transformation can also lead to decreased production of the cytokines IL-10 and IL-20. Because of the large molecular size and the polarity of the protein, the penetration of GM-CSF in the inflammatory tissue is minimal, if not absent, and thus the effects of GM-CSF on eosinophil-associated chronic inflammation are avoided.

However, systemically administered GM-CSF, i.e. S.C., I.M. and or IV may further aggravate the chronic inflammation. Provided there is a major contribution of eosinophilic element in the pathogenesis of the condition, the consequence of such systemically administered GM-CSF can be an unwarranted maintenance of the chronic inflammatory condition - in as much as the targets of the GM-CSF is not only the macrophage (MF) and granulocytes (PMNL) but also the corresponding GM-CSF receptor on the surface of the eosinophilic cells as a part of the local autocrine effect.

Below is a table summarizing the hypothesis of the two faces of GM-CSF and the seemingly opposite effects, i.e. the T_H1 or T_H2 inflammatory effect (Table 1 The autocrine effect of GM-CSF)

1 ) Surface of polyps which is generally impermeable of GM-CSF, where only macrophages are positioned.

2) The DC orchestrate and communicate with other cells in the body either as a direct cell-to-cell contact-based interaction of cell-surface proteins but may also interact at a distance via cytokines, or as the complex interplay as a combined effect of the direct and indirect distant effect, DC will- in contact microbial extracts - induce the dendritic cells to rapidly produce IL-12, which in turn induce the combined DC and the CD4 T cells to produce the T_H1 phenotype. The ultimate consequence is priming and activation of the immune system to attack the antigens with the dendritic cell present on its surface. The fully GM- CSF activated DC subsequently become the "antigen presenting cell". However, there are differences in the cytokines produced depending on the type of dendritic cell. The plasmacytoid DC has the ability to produce huge amounts of type-1 IFN's, which recruit more activated macrophages to allow phagocytosis. Dendritic cells and T cells involves different signal molecules containing peptide fragments on the DC and the T-cell receptor interaction between co-stimulation of CD molecules on the surface of the DC and on the T- cell surface. Finally IL-12 secretion from the DC. In total these signals promote the T helper 1 (T_H1 ) response, (P. Toby H. Coates, Bridget L. Colvin, Holger Hackstein and Angus W. Thomson. The interaction between dendritic cells (DCs) and T cells involves three signals.

In the macrophage, the primary signal for activation is IFN-γ secreted from T_H1 type CD4 T cells. The secondary signal is CD40L (CD154) on the T_H1 T cell which binds CD40 on the macrophage cell surface. As a result, the macrophage expresses more CD40 and TNF receptors on its surface which helps increase the level of activation. The increase in activation results in the induction of potent microbicidal substances in the macrophage, including reactive oxygen species and nitric oxide, leading to the destruction of ingested microbe. The T_H1 T cell also produces IL-4, which directly influences B cells. As a result of this net stimulation, the B cell can undergo division, antibody isotype switching, and differentiation to plasma cells. The end-result is a B cell that is able to mass-produce specific antibodies against an antigenic target. Early evidence for these effects were that in CD40 or CD154 deficient mice, there is little class switching or germinal centre formation, and immune responses are severely inhibited.

The beneficial effects of GM-CSF on the surface of the epithelium can thus be assessed by use of conventional methods in the art for example to determine the levels of IFN-γ secreted from T_H1 type CD4 T cells, CD40L (CD154) expressed on the surface of the T_H1 cell, augmentation in macrophages expressing CD40 and TNF receptors on the surface, induction of potent microbicidal substances in the macrophages on the surface of the epithelium, for example reactive oxygen species and nitric oxide, and increased production of IL-4 from the T_H1 -cells. Wong et al., Science Vol. 228, pp. 810-815 (1985) and Kaushansky et al., Proc. Natl. Acad. Sci. USA, Vol. 83, pp. 3101 -3105 (1986) have described the production of recombinant GM-CSF in mammalian cells. Burgess et al., Blood, Vol. 69, pp. 43-51 (1987) describes the purification of GM-CSF produced in Escherichia coli.

Functional homologues of GM-CSF

A functional homologue of GM-CSF is a polypeptide having at least 50 % sequence identity with the known and naturally occurring sequence of GM-CSF 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.

Granulocyte macrophage colony-stimulating factor (GM-CSF) is a cytokine important for the stimulation of proliferation, differentiation and survival of many hemopoietic cells including mature neutrophils, macrophages and dendritic cells. GM-CSF is produced by many cell types within the body (e.g. fibroblasts, endothelial cells) when stimulated by microbial products or inflammatory cytokines and its activity is important to the innate immune response. T lymphocytes also produce it when stimulated with a specific antigen. An important issue here is that the so-called 'autocrine effect' of the GM-CSF is always an up regulation of both the number of the cells and the quality of the cells. Thus the outcome of the autocrine effect activating the GM-CSF receptors depend entirely on the cell types like macrophage, granulocyte and the eosinophilic cell. GM-CSF regulates multiple functions of macrophages. GM-CSF stimulation of macrophages has been documented to enhance macrophages selective response to noxious ingestants, i.e., stimulation of inflammation during bacterial phagocytosis, nonnoxious ingestants are generally mollified, i.e., anti-inflammatory responses during phagocytosis of apoptotic cells. Further the multiple functions of the macrophage are enhanced by GM-CSF stimulation with subsequent proliferation, differentiation, accumulation and activation. Further these GM-CSF effects also encompasses cell adhesion, improved chemotaxis, Fc-receptor expression, complement- and antibody- mediated phagocytosis, oxidative metabolism, intracellular killing of bacteria, fungi, protozoa, and viruses, cytokine signaling, and antigen presentation. Further GM-CSF enhances defects in macrophages cell adhesion, pathogen associated molecular pattern receptors, like Toll-like receptors and TLR trans-membranous signaling, surfactant protein and lipid uptake and degradation (Trapnell BC and Whitsett JA. GM- CSF regulates pulmonary surfactant homeostasis and alveolar macrophage-mediated innate host defense. Annu. Rev. Physiol. 2002.64:775-802).

Further GM-CSF interacts with the macrophages recognition receptors, the so-called toll like receptors (TLR). GM-CSF is important in respect to the macrophages due to its interaction with the Toll like receptor's (TLR's) participation in the host defense resulting in enhanced clearance of the causative microorganism (Chen GH, Olszewski MA, McDonald RA, Wells JC, Paine R 3rd, Huffnagle GB, Toews GB. Role of granulocyte macrophage colony-stimulating factor in host defense against pulmonary Cryptococcus neoformans infection during murine allergic bronchopulmonary mycosis. Am J Pathol. 2007 Mar;170(3):1028-40). Lung has its own innate GM-CSF production, which is reduced in pneumonia and hyperoxia, in relation to high 0₂ exposure as seen in, e.g. ventilator associated pneumonia (VAP) contributing impairment of host defense secondary to apoptosis with poor response to infections. The hyperoxic injury seems to be counteracted by activation of alveolar macrophages with GM-CSF (Altemeier WA, Sinclair SE. Hyperoxia in the intensive care unit: why more is not always better. Curr Opin Crit Care. 2007 Feb;13(1 ):73-8. & Baleeiro CE, Christensen PJ, Morris SB, Mendez MP, Wilcoxen SE, Paine R. GM-CSF and the impaired pulmonary innate immune response following hyperoxic stress. Am J Physiol Lung Cell Mol Physiol. 2006 Dec;291 (6):L1246-55. Epub 2006 Aug 4) with subsequent clearance of P. aeruginosa via expression of the TLR signaling pathway (Baleeiro CE, Christensen PJ, Morris SB, Mendez MP, Wilcoxen SE, Paine R. GM-CSF and the impaired pulmonary innate immune response following hyperoxic stress. Am J Physiol Lung Cell Mol Physiol. 2006 Dec;291 (6):L1246-55. Epub 2006 Aug 4).

Finally GM -CSF produces in-vitro conversion of the resting macrophages into immature dendritic cells (DC), which may further be matured with specific agents in respect to activate the homing of matured DCs to a specified receptor or target.

(Zobywalski A, Javorovic M, Frankenberger B, Pohla H, Kremmer E, Bigalke I, Schendel DJ. Generation of clinical grade dendritic cells with capacity to produce biologically active IL-12p70. J Transl Med. 2007 Apr 12;5:18). Evolutionary conservation between GM-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, GM-CSF sequences are compared between species where GM-CSF function is conserved, for example 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 GM-CSF sequence does not interfere with the activity of the GM-CSF molecule according to the invention. Such 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.

As used herein the expression "variant" refers to polypeptides or proteins, which are homologous to the basic protein, which is suitably human GM-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 i) polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser, Thr, Tyr, and Cys,) ϋ) 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) sulphor-containing side chains (Cys, Met), and/or

xi) amino acids being monoamino-dicarboxylic acids or monoamino- monocarboxylic-monoamidocarboxylic acids (Asp, Glu, Asn, Gin).

A functional homologue within the scope of the present invention is a polypeptide that exhibits at least 50% sequence identity with human GM-CSF, such as at least 60% sequence identity, for example at least 70% sequence identity, such as 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 human GM-CSF.

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 60% identical, preferably at least 70% 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 GM-CSF. 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 employed 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 encompassed 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 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 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 of human GM-CSF 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 human GM-CSF. It is preferred that functional homologues of GM-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, 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.

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 variants of human GM-CSF; a T1 15I substitution in variant 1 and a 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 human GM-CSF 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 to Deeley, et al. Such analogues are also functional equivalents comprised within the present invention.

In one embodiment 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, septamers, heptamers, octamers, nonamers and decamers.

In one embodiment, a homodimer, trimer or tetramer of GM-CSF is used.

The protein sequence of GM-CSF of Homo Sapiens (SEQ ID NO:1 ):

MWLQSLLLLG TVACSISAPA RSPSPSTQPW EHVNAIQEAR RLLNLSRDTA AEMNETVEVI SEMFDLQEPT CLQTRLELYK QGLRGSLTKL KGPLTMMASH YKQHCPPTPE TSCATQIITF ESFKENLKDF LLVIPFDCWE PVQE GM-CSF according to the present invention may be commercially available, e.g.

sargramostim (GM-CSF [Leukine^®; Immunex, Seattle, WA])

Recombinant production

Granulocyte-macrophage colony-stimulating factor (GM-CSF), or functional variants or homologues thereof, 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 (for example E. Coli), yeast cells, insect cells, mammalian cells or in cell-free systems. In one embodiment of the invention, GM-CSF is produced recombinantly by host cells. Thus, in one aspect of the present invention, GM-CSF is produced by host

cellscomprising 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 a recombinant GM-CSF in general comprises the steps of:

-providing a host cell

-preparing a gene expression construct comprising a first nucleic acid encoding the 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 the 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 the GM- CSF.

In one embodiment of the invention, the recombinantly produced GM-CSF is excreted by the host cells. When the GM-CSF is excreted the process of producing a

recombinant protein of interest may comprise the steps of

-providing a host cell

-preparing a gene expression construct comprising a first nucleic acid encoding the 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 the GM-CSF and secretion of the GM-CSF into the culture medium,

-thereby obtaining culture medium comprising the 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.

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

Pharmaceutical composition

Pharmaceutical compositions or formulations for use in the present invention include granulocyte-macrophage colony-stimulating factor (GM-CSF), or functional variants or homologues thereof, preferably dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent, or carried to the relevant site as a pegylated preparation or as a liposomal or nanoparticle preparation. 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 GM-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, biodegradable polymers 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 ,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-Dimyristoyl-sn- Glycero-3-Phosphate (Monosodium Salt) (DMPA), 1 ,2-Dipalmitoyl-s/ 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. Liposomes may also be modified for example by attaching 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 e.g. 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 multi-lamellar 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 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.

Exemplary synthetic polymers which can be used to form a biodegradable delivery system include: polyamides, polycarbonates, polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkylene terepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, poly-vinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes, polyurethanes and co-polymers thereof, alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, polymers of acrylic and methacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, cellulose sulphate sodium salt, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate), polyethylene, polypropylene, poly(ethylene glycol), poly(ethylene oxide), poly(ethylene terephthalate), polyvinyl alcohols), polyvinyl acetate, poly vinyl chloride, polystyrene and polyvinylpyrrolidone. Examples of biodegradable polymers include synthetic polymers such as polymers of lactic acid and glycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes, poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone), and natural polymers such as alginate and other polysaccharides including dextran and cellulose, collagen, chemical derivatives thereof (substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art), albumin and other hydrophilic proteins, zein and other prolamines and hydrophobic proteins, copolymers and mixtures thereof. In general, these materials degrade either by enzymatic hydrolysis or exposure to water in vivo, by surface or bulk erosion. In some embodiments, a bioadhesive polymer is used. Dose

By "effective amount" of GM-CSF according to the present invention it is meant a dose, which, when administered to a patient in need thereof, achieves a concentration which has a beneficial biological effect, i.e. by alleviating and/or preventing inflammation of the nasal tissue or formation of nasal polyposis or symptoms associated with nasal polyposis.

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 the relevant cytokines 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 5 μg to 1000 μg, even more preferred about 100 μg to about 800 μg. The preparations may be administered once, twice or three times 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. 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.

GM-CSF may e.g. be administered by inhalation to a patient suffering from nasal polyposis in a dose ranging from about 10 to 1000 μg per dose, such as 50-100,

100-200, 200-300, 300-400, 400-500, 500-600, 600-700, 700-800, 800-900, 900-1000 μg per dose. Preferably the dosage per administration is 100 to 1000 μg, for example 200 to 800 μg per dosage, such as 200 to 400 μg per dosage, preferably about 300 μg per dosage. Each dose can be administered once a day, twice a day, three times a day, four times a day, five times a day or six times a day.

Duration of dosing will typically range from 1 day to about 4 months, such as in the range of 1 day to 2 days, for example 2 days to 3 days, such as in the range of 3 days to 4 days, for example 4-5 days, such as 5-6 days, for example 6-7 days, for example 7-14 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, as long as symptoms and disease is detectable.

The transformation of a resting macrophage into a fully immuno-competent dendritic cell after in vitro incubation of macrophages with GM-CSF takes approximately 10 days. In one embodiment, a duration of a dose has the length allowing for said a transformation, thus the duration can be 7-14 days, such as 8-12 days, 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 dose regime may alternate between periods of GM-CSF administration and periods with no administration (a pause in treatment). A period with a pause of treatment in such a dose regime may last 5-10 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 1 to 4 months.

Examples of dosage regimes may include a cycle of 10 days treatment with GM-CSF or functional variants or homologues thereof and 7 days pause of treatment.

The conversion of resting macrophages (MF) into dendritic (DC) - cells may be boosted by repeating a dosage regime. Thus dosage regimes can be repeated one, two, three, four, five or more times in order to obtain an effective treatment.

In one embodiment, a dosage regime is repeated once, two times, three times or for example for the rest of the lifespan of a subject in need.

In another embodiment, patients are treated with a dosage regime of 10 days treatment with GM-CSF or functional variants or homologues thereof, followed by 7 days pause in said treatment and subsequently repeating the dosage regime 2-3 or more times.

In one embodiment, the administration of GM-CSF or functional variants or

homologues thereof is administered in combination with CPAP for about 7 days per month or every second month or actuated by pre-specified symptoms e.g. fever.

Additional Therapy

The invention encompasses administration of granulocyte-macrophage colony- stimulating factor (GM-CSF), or functional variants or homologues thereof in

combination with additional therapy for nasal polyposis. Such additional therapy may include, in certain embodiments, any appropriate therapy for the condition known in the art. Additional therapy can include co-administration of one or more therapeutic agents such as antibiotics , a corticosteroid, leukotriene antagonist, anti-lgE agent, antihistamine, decongestant, beta- agonist, an anti-infective agent or other cytokines.

In some embodiments granulocyte-macrophage colony-stimulating factor (GM-CSF), or functional variants or homologues thereof is administered in combination with one or more cytokines known in the art, for example selected from the group consisting of granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M-CSF), stem cell factor (SCF), and/or an interleukin series (IL-1 to IL-16) and combinations thereof, or functional variants or homologues thereof.

Examples of anti-infective agents include antibacterial agents, antifungal agents, antiviral agents, and antiseptics. Examples of antibacterial agents include

aminoglycosides, amphenicols, ansamycins, lactams, lincosamides, macrolides, nitrofurans, quinolones, sulfonamides, sulfones, tetracyclines, and any of their derivatives. Exemplary antifungal agents include polyenes, allylamines, azoles (e.g., imidazoles, triazoles, and thiazoles), and echinocandins. Exemplary compounds include amphotericin B, nystatin, miconazole, or ketoconazole.

Exemplary corticosteroids include 21 -acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamino-acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, rofleponide palmitate, tixocortol, triamcinolone, (e.g., triamcinolone acetonide, triamcinolone benetonide, triamcinolone hexacetonide). In some embodiments, a corticosteroid is selected from mometasone furoate, fluticasone propionate, fluticasone furoate, rofleponide palmitate, budesonide, triamcinolone acetonide, prednisolone, beclomethasone dipropionate, ciclesonide, and flunisolide.

Exemplary decongestants include 1 -desoxyephedrine, ephedrine, ephedrine hydrochloride, ephedrine sulfate, naphazoline, naphazoline hydrochloride,

oxymetazoline and pharmaceutically acceptable salts thereof, oxymetazoline hydrochloride, phenylephrine, phenylpropanolamine, menazoline, phenylephrine hydrochloride, propylhexedrine, xylometazoline and xylometazoline hydrochloride.

Exemplary anti-histamines include anti-histamine, such as azelastine, loratidine, brompheniramine, chlorpheniramine, mizolastine, promethazine, doxylamine, des loratidine, triprolidine, clemastine, fexofenadine, cetirizine and levocetirizine, and the pharmaceutically - acceptable salts and derivatives thereof.

As used herein, the term "leukotriene antagonist" encompasses leukotriene receptor antagonists (e.g., zafirlukast) and leukotriene synthesis inhibitors (e.g., zileuton). An "anti-lgE agent" is an agent that inhibits or antagonizes IgE, e.g., by binding to IgE and blocking its interaction with its receptor(s). Exemplary anti-lgE agents include antibodies (e.g., omalizumab) or IgE receptor antagonists.

Administration

An effective amount of GM-CSF according to the present invention, or functional variants or homologues thereof, are preferably administered by intranasal and/or intrasinus administration allowing GM-CSF to reach the nasal cavity and/or paranasal sinuses in either one side of the head of an individual in need or both sides of the head of an individual in need. Such local administration (i.e. intranasal or intrasinus administration) allowing GM-CSF, or functional variants or homologues thereof, to reach the "air-side" of the mucous membrane (i.e. the surface of the epithelium of the nasal cavity and/or paranasal sinuses) in the nasal cavity and/or paranasal sinus(es), by is a convenient route of administering GM-CSF or functional variants or homologues thereof.

CSF according to the present invention, or functional variants or homologues thereof can be administered by the intravenous, subcutaneous, pulmonary, intramuscular, or oral route in various embodiments of the invention.

Methods of intranasal and/or intrasinus administration include but are not limited to, nasal drops, spraying, lavage, inhalation (including administration of GM-CSF using CPAP), flushing, injection or instillation, using as fluid a physiologically acceptable composition in which the cytokine have been dissolved. When used herein the term "intranasal administration" includes all forms of such administration whereby the cytokine is applied into the nasal cavity or the paranasal sinuses, respectively, whether by the injection or instillation of a solution of the cytokine, by the cytokine in a powder form, or by allowing GM-CSF to reach the relevant part by inhalation of the cytokine as an aerosolized or nebulized solution or suspension or inhaled powder or gel, or as a deposited composition comprising the cytokine, with or without added stabilizers or other excipients.

Continuous positive airway pressure (CPAP) can be used in combination with administration of GM-CSF, or functional variants or homologues thereof in order to keep the sinus orifices open using a CPAP mask during the GM-CSF administration

Continuous positive pressure (CPAP) is conventionally used to keep the orifices of the sinuses open by applying a constant mild (5 - 10 cm H₂0) to moderate airway pressure (10 -15 cm H₂0 or even higher) into the nasal airways via a standard medical apparatus. Such a medical apparatus can consist of an airtight facial mask having. a valve separating the inspiratory side (where the GM-CSF or functional variants or homologues thereof is being delivered via a nebulizer) In such a medical apparatus, resistance is applied on the expiratory side of the valve in the form of a different boreholes calibrated to give an expiratory wanted pressure or by applying a standard expiratory valve (figure 1 ). GM-CSF or functional variants or homologues thereof can be applied breath wise via CPAP by a relatively shot inspiratory breath of for example 50-200 ml such as about 100 ml containing GM-CSF aerosol, and immediately after the inspiration to initiate expiration. In this way the amount of GM-CSF, or functional variants or homologues thereof, being wasted is keept to a minimum.

A variety of devices can be used to administer a nasal spray. Suitable devices are known in the art and include, e.g., squeeze bottles, pump sprays, and airless sprays. In some embodiments, a nasal spray contains GM-CSF or functional variants or homologues thereof, dissolved or suspended in a solution or mixture of excipients in a nonpressurized dispenser that delivers a spray containing a metered dose GM-CSF or functional variants or homologues thereof. The dose can be metered by the spray pump or may have been premetered during manufacture. In some embodiments, a nasal spray device is designed to be capable of discharging up to several hundred metered sprays of formulation containing GM-CSF or functional variants or

homologues thereof. In some embodiments, a nasal spray device or dry powder delivery device is designed for unit dosing. In some embodiments, the device is disposable, e.g., it contains a single dose (or two doses, one to each nostril) and is not designed to be refilled.

Mechanical pumps or actuators are often employed to deliver nasal formulations as sprays. A variety of devices are available. In some embodiments, a nasal spray is delivered using a Becton-Dickinson Accuspray (TM) Nasal Delivery System or similar technology. It creates a spray by forcing liquid through a pressure swirl atomizer when the user depresses the plunger on the device. A thin intact sheet of liquid is formed in the shape of a cone at the exit orifice, and breaks up into droplets of an appropriate size for delivery of drugs to the nasal mucosa. A variety of nasal administration systems are available from the Pharma Division of Erich Pfeiffer GmbH (now Aptar Pharma). Available systems include ones suitable for administering liquids and others suitable for powders. In some embodiments, a nasal spray delivery device with the capability to prevent the entrance of microorganisms is used. For example, pumps may employ sterile filtration in conjunction with a venting system in order to prevent microorganisms from entering. Another common approach involves a mechanical tip seal that closes off the orifice at all times except during spraying of the formulation. In some embodiments an airless spray is used, which prevents entry of air into the dispensing device after use. Such approaches may be of particular use if a composition does not contain an antimicrobial agent. Pressurised metered dose inhalers can also be used, e.g., containing a hydrofluoroalkane as a propellant. In some embodiments, GM-CSF or functional variants or homologues thereof, is administered locally using a nebulizer. A nebulizer device may produce a dispersion of droplets in a gas streams by various methods.

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

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

2. Pressurized nebulizers using compressed air/oxygen mixture

3. Metered dose inhaler (MDI)

4. Dry powder inhaler systems (DPI),

Jet nebulizers can, for example, use a compressed air or other compressed gas supply to draw liquid up a tube and through an orifice and introduce it into a flowing gas stream as droplets suspended therein, after which the fluid is caused to impact one or more stationary baffles to remove excessively large droplets. Ultrasonic nebulizers use an electrically driven transducer to subject a fluid to high-frequency oscillations, producing a cloud of droplets which can be entrained in a moving gas stream. Handheld nebulizers may atomize a liquid with a squeeze bulb air supply. A variety of nebulizers are available, e.g., from PARI Respiratory Equipment, Inc. For example, PARI SinuStar(TM) Nasal Aerosol Delivery System delivers aerosols to the upper airway including the sinuses. The PARI Sinus Therapy System is described as combining efficient nebulization with a vibrating pulse to efficiently deliver aerosol into the paranasal sinuses. The SinusAero(TM) Nasal Nebulizer (Sinus Dynamics) is another nebulizer system of use. In some embodiments, controlled particle dispersion technology (CPD) is used. CPD employs the principle of vertical flow, by which inherent airflows of the nasal cavity are disrupted. CPD allows delivery of formulations to the entire nasal cavity, olfactory region, and paranasal sinuses. See, e.g., PCT/US2004/029001 (WO/2005/023335). For example, ViaNase ID (Kurve Technology, Bothell, WA) is a CPD-powered electronic atomizer that can be used to deliver GM-CSF or functional variants or homologues thereof, is for treatment of CRS and/or nasal polyposis.

In some embodiments, an approach that utilizes the exhalation breath of a user as the driving force to deliver a metered dose of a liquid substance is employed. For example, bidirectional intranasal drug delivery can be used, which delivers a drug while the subject exhales and is reported to reduce lung deposition. It uses the concept that exhalation against resistance leads to closure of the soft palate, thus separating the nasal cavity from the mouth and cutting off communication between the cranial surface of the soft palate and the posterior margin of the nasal septum. Under such conditions, air can enter through one nostril through the sealing nozzle, turn -180 degrees, and exit through the other nostril in the reverse direction. A single-use or multidose liquid reservoir or powder delivery device can be used. For example, OptiNose (Oslo, Norway) has developed devices embodying this approach. See, e.g., Djupesland PG, Breath actuated device improves delivery to target sites beyond the nasal valve.

Laryngoscope, 1 16(3):466-72, 2006. See also PCT/IB2007/004353

(WO/2008/081326).

In some embodiments, a Direct-Haler Nasal device is used (Direct-Haler, Copenhagen, Denmark, now owned by Trimel BioPharma, Etobicoke, Ontario). In this device, the subject blows air out of the mouth and into the device, upon which a nasal dry powder dose is delivered into the nostril. See, e.g., Keldmann, T., Advanced Simplification of Nasal Delivery Technology: Anatomy + Innovative Device = Added Value Opportunity. ON drugdelivery, 3rd issue, pp. 4-7 (2005). In some embodiments, a device or delivery method is selected such that a significant fraction of the administered material is deposited in the nasal cavity posterior to the nasal vestibule. In some embodiments a significant fraction of the administered material is deposited in the region lined with respiratory epithelium (a ciliated pseudo- stratified columnar epithelium). In some embodiments, a significant fraction of the administered material is deposited in the region above the inferior meatus

encompassing the middle turbinate, the middle meatus, the sinus ostia of the maxillary, frontal and ethmoidal sinuses, and the olfactory region. Parameters such as the spray- cone angle, configuration of the delivery device, particle size range, tap density, etc. can be selected to direct the composition to a desired location, e.g., to achieve a more posterior deposition, increase delivery to the middle meatus or sinus ostia, etc. In some embodiments, a delivery device includes a nosepiece which is inserted into the one nostril of a subject and a nozzle through which a substance is delivered to the nasal cavity. Particles having a desired range or distribution of aerodynamic and/or physical particle sizes (e.g., diameters) can be used. In some embodiments, a size range is selected to reduce the likelihood that an inhaled particle would reach or be retained in the lung and/or to reduce the likelihood that a particle would be exhaled.

In some embodiments, GM-CSF or functional variants or homologues thereof, is administered to the nose or to one or more paranasal sinuses as a depot or in a composition that forms a depot upon administration. In some embodiments, the depot forms upon contact with nasal or sinus secretions. In certain embodiments the depot decreases in size and/or density over time (e.g., by degradation and/or disintegration), releasing GM-CSF or functional variants or homologues thereof. The depot may be in the form of a gel or a material having physical properties (e.g., viscosity, elasticity, hardness, and/or compressibility) characteristic of a gel, wherein a "gel" may be defined as a colloidal system in which a porous network of interconnected particles (typically of nanometer scale) spans the volume of a liquid medium. In some embodiments, the composition comprises GM-CSF or functional variants or homologues thereof, and an excipient that modulates the rate of deposit

degradation/disintegration and/or modulates a physical characteristic of the depot. In some embodiments, a composition comprises a bioadhesive, mucoadhesive, and/or viscosity- modifying substance. In some embodiments, the substance reduces the clearance of GM-CSF or functional variants or homologues thereof, in the nasal cavity or sinuses. For example, the composition may adhere to the nasal or sinus mucosa. In some embodiments, the composition comprises GM-CSF or functional variants or homologues thereof, and a gel-forming substance. In some embodiments, the composition comprises GM-CSF or functional variants or homologues thereof, and an excipient that modulates the rate of degradation/disintegration and/or modulates a physical characteristic of the depot. In some embodiments, the excipient is a sugar alcohol or amino acid.

In some embodiments, GM-CSF or functional variants or homologues thereof, is administered as a gel or ointment. A gel, ointment, or other pharmaceutical composition of the invention can contain one or more thickening agents, soothing substances, humectants, or emollients such as glycerin, aloe, propylene glycol, etc. In some embodiments, a composition contains a substance that enhances absorption through mucus and/or into nasal or sinus mucosa.

In some embodiments, GM-CSF or functional variants or homologues thereof, is delivered to the nasal cavity and/or paranasal sinuses by use of an implant. Often, an implant comprises a polymeric material. In some embodiments, an implant is biodegradable, e.g., by way of diffusion or by degradation of the matrix. Such degradation may release GM-CSF or functional variants or homologues thereof. An implant, e.g., a biodegradable implant, could have any of a variety of shapes, e.g., rods, pellets, beads, strips, or microparticles, and may be delivered into a sinus in various pharmaceutically acceptable carriers. Such implants may be designed to have a size, shape, density, viscosity, and/or mucoadhesiveness that prevents them from being substantially cleared by the mucociliary lining of the sinuses during a desired treatment period. See, e.g., PCT/US2004/007828 (WO/2004/082525) for a description of certain biodegradable implants and devices and methods for their deployment. In some instances, an instrument for visualizing the sinus ostium or sinus wall is used. Examples of such instruments include endoscopes and computed tomography (CT) scanners. Preferred concentrations for a solution comprising a cytokine according to the present invention and/or functional homologues or variants 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 μg, preferably from about 0.3 μg to about 3.0 μg, such as from about 0.5 to about 1.5 μg and especially in the range from 0.8 to 1.0 μg per ml solution. In one embodiment the intranasal and/or intrasinus administered GM-CSF, or a functional variant, derivative or homologue thereof, is to be administered in

combination with systemic and/or subcutaneous administration of GM-CSF. Medical packaging

The GM-CSF, or a functional variant, derivative or homologue thereof, 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 GM-CSF, or a functional variant or homologue thereof, 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 GM- CSF, or a functional variant or homologue thereof, 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 GM-CSF, or a functional variant or homologue thereof, 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 intranasal and/or intrasinus 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 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.

Embodiments

1 . A composition comprising granulocyte-macrophage colony-stimulating factor (GM-CSF), or a functional variant or homologue thereof, for use in the treatment, prevention or alleviation of chronic nasal and/or paranasal sinus inflammation.

2. The composition for use according to the embodiment 1 , wherein said

composition is for use in the treatment, prevention or alleviation of nasal and/or paranasal sinus polyposis.

The composition for use according to the preceding embodiments, wherein said composition is administered locally.

The composition for use according to the preceding embodiments, wherein said GM-CSF, or a functional variant or homologue thereof, is to be administered by intranasal and/or intrasinus administration.

The composition for use according to the preceding embodiments, wherein said GM-CSF, or a functional variant or homologue thereof, is to be administered by intranasal and/or intrasinus administration to the surface of the epithelium of the nasal cavity and/or paranasal sinuses.

The composition for use according to the preceding embodiments, wherein said GM-CSF, or a functional variant or homologue thereof, is to be administered as a solution, a suspension, an aerosol, a nebulized solution, or a nebulized suspension.

The composition for use according to the preceding embodiments, wherein said GM-CSF, or a functional variant or homologue thereof, is to be administered as a powder. The composition for use according to the preceding embodiments, wherein said GM-CSF, or a functional variant or homologue thereof, is to be administered via nasal drops, spraying, lavage, flushing, inhalation, injection or instillation. The composition for use according to the preceding embodiments, wherein said

GM-CSF, or a functional variant or homologue thereof, is to be administered by injection. The composition for use according to the preceding embodiments, wherein said GM-CSF, or a functional variant or homologue thereof, is to be administered via

CPAP. The composition for use according to the preceding embodiments, wherein said GM-CSF, or a functional variant or homologue thereof, is to be administered by inhalation and/or exhalation. The composition for use according to the preceding embodiments, wherein said GM-CSF, or a functional variant or homologue thereof, is to be administered as an inserted depot. The composition for use according to the preceding embodiments, wherein said GM-CSF, or a functional variant or homologue thereof, is to be administered in a pegylated, liposomal or nanoparticle prepared form. The composition for use according to the preceding embodiments, wherein said composition is for use in combination with one or more other pharmaceutically active components.

The composition for use according to the preceding embodiments, wherein said composition further comprises one or more additional pharmaceutically active component. The composition according to embodiment 14 or 15, wherein the additional pharmaceutically active component is selected from the list of antibiotics , a corticosteroid, leukotriene antagonist, anti-lgE agent, anti-histamine, decongestant, beta- agonist, an anti-infective agent or other cytokines The composition according to embodiment 16, wherein the additional pharmaceutically active component is selected from the list of granulocyte colony-stimulating factor (G-CSF), macrophage colony-stimulating factor (M- CSF), stem cell factor (SCF), and/or an interleukin series (IL-1 to IL-16) and combinations thereof, or functional variants or homologues thereof. The composition for use according to the preceding embodiments, wherein said chronic nasal and/or paranasal sinus inflammation is associated with a condition selected from the group consisting of chronic rhinosinusitis, chronic sinusitis, allergic fungal sinusitis (AFS) , asthma, aspirin intolerance/salicylate sensitivity, primary ciliary dyskinesia, cystic fibrosis (CF), Kartagener's syndrome, Young's syndrome, Churg-Strauss syndrome, nasal mastocytosis, exposure to chromium and allergic rhinitis. The composition for use according to the preceding embodiments, wherein said GM-CSF, or a functional variant or homologue thereof, is to be administered at an effective amount, such as from between 100 to 1000 microgram per dose, for example 200-800 microgram per dose. The composition for use according to embodiment 20, wherein said dose is administered once, twice, three times or four times daily. The composition for use according to any one of embodiments 19-20, wherein said dose is selected from the list of 75, 150, 300, 600, 1200, 2400, and 4800 microgram GM-CSF or a functional variant thereof.

The composition for use according to embodiments 20 and 21 , wherein said one or more doses are administered for 1 day or for between 1 to 14 days, such as 1 to 3 days, 3 to 5 days, 5 to 7 days, 7 to 10 days, 10 to 14 days, or for 7-14 days, such as 8-12 days, such as 8 days, 9 days, 10 days, 1 1 days or 12 days, or for one week to two weeks, for two to four weeks, for one month to two months, for 2 to 4 months, or for as long as symptoms and disease is detectable.

23. The composition for use according to embodiments 15 to 17, wherein said one or more doses are administered in a dosage regime with pauses of treatment having a duration of 5 to10 days, such as 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more such as 1 to 4 months.

24. The composition for use according to embodiments 15 to 18, wherein said one or more doses are administered in a dosage regime that is repeated once, two times, three times or for the rest of the life time of the subject in need thereof, 25. A method for treating, preventing, reducing risk of, or alleviating of chronic nasal and/or paranasal sinus inflammation in a subject in need thereof, said method comprising administering to the subject an effective amount of a composition comprising granulocyte-macrophage colony-stimulating factor (GM-CSF), or a functional variant or homologue thereof.

26. The method according to embodiment 20 for treatment for use in the treatment, prevention or alleviation of nasal and/or paranasal sinus polyposis.

Examples Example 1

Treatment in a hyperacute phase:

Group 1

Treated subjects: Patients having symptoms of fever and pain originating from a paranasal sinus, tenderness over the surface of the involved sinus and with significant liquid as a sign of closed orificii.

Treatment: Intrasinus injection of GM-CSF in a dose of 300 microgram diluted in a total volume of 25 - 50 ml . After treatment with GM-CSF, the symptoms of nasal/paranasal polyposis are alleviated or relieved. Group 2

Treated subjects: Patients having symptoms of pain originating from a paranasal sinus and tenderness over the surface of the involved sinus.

Treatment: CPAP with an aerosol containing GM-CSF in a total dose of 300 microgram once per day in order to ensure intrasinus cavity application.

Treatment in the chronic phase:

Treated subjects: Patients one or more of the common signs and symptoms of chronic sinusitis with nasal polyps including: Runny nose, persistent stuffiness, postnasal drip decreased or absent sense of smell, loss of sense of taste, facial pain or headache, pain referred to upper teeth, sense of pressure over your forehead and face, snoring and finally a sensation of Itching around the eyes.

Treatment: GM-CSF in a total dose of 300 microgram per day delivered via CPAP until the symptoms are relieved.

After treatment with GM-CSF, the symptoms of nasal/paranasal polyposis are alleviated or relieved. Example 2

Treatment in a chronic and or subacute phase:

Group 1

Treated subjects: Patients having signs and symptoms consisting of low grade fever and pain originating from a paranasal sinus, tenderness over the surface of the involved sinus and with significant liquid as a sign of closed orificii.

Treatment: Intrasinus injection of GM-CSF, administered either as injection and or via a drain placed into the sinus, a dose escalation study in dosages ranging from of 0 (control), 75, 150, 300, 600, 1200, 2400, and 4800 microgram diluted in saline per day to a total volume of 25 - 100 ml. The recombinant GM- CSF growth factor is administered bilaterally as a combination of aerosol in each nostril together with an increased continuous intranasal pressure (nasal CPAP), in order to penetrate the "sinus-nasal orificii"(opening)).

The study can be done in one patient, where the effect of increasing dosages can be observed, or the study can be done in groups, each group comprising one or more patients, wherein each group of patients receive either one of 0, 75, 150, 300, 600, 1200, 2400, and 4800 microgram GM-CSF. It is expected that all the exemplified dosages 75, 150, 300, 600, 1200, 2400, and 4800 microgram GM-CSF will have an effect on disease variables. However, this experiment is designed to identify an optimal dosage for treatment.

Care will be taken to make sure that the composition is not injected indo the edema fluid, but rather injected into the Intrasinus lumen. This is in order to avoid activation of eosinofilic cells.

The composition is administered once daily for 10 days, followed by 10 days without treatment. After each period without treatment, the status of the disease is evaluated, in order to decide whether or not to initiate a new round of treatment.

In one part of the experiment, the length of the period without treatment will be varied in order to identify the optimal length of this. This will be tested using an effective dosage selected from the group of either one of 75, 150, 300, 600, 1200, 2400, and 4800 microgram GM-CSF and compared to 0 microgram (negative control). The length of non treatment periods that will be tested in this part of the experiment will be a 2 day period without treatment, until initiation of the next administration period, other groups will have a non treatment period selected from 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 days without treatment before the next 10 day period with daily administrations of GM-CSF. A more narrow selection of non treatment intervals may be chosen, such as 3, 5, 10, 30 and 60 days of non treatment.

In one part of this experiment, 150, 300 or 1200 microgram GM-CSF is administered daily for 10 days with 5, 10 or 30 days of non treatment interval followed by 10 days of daily administration and another period of 5, 10 or 30 days of without treatment. After the end of each period without treatment, status of disease is evaluated.

Evaluation of the status and possible alleviation or relieve of the disease signs and symptoms is done by both visual observation of swelling of the epithelium, or by observation of the level of fever and level of pain and tenderness relief.

After treatment with GM-CSF, the symptoms of nasal/paranasal polyposis are alleviated or relieved. Group 2

Treated subjects: Patients having chronic signs and symptoms of pain originating from bilareaklnasal stenosis paranasal sinus and or tenderness over the surface of the involved sinus.

Treatment: Binasal - CPAP with an aerosol containing GM-CSF in a in a dose escalation study in dosages ranging from of 0 (control), 75, 150, 300, 600, 1200, 2400, and 4800 microgram as a total dose for binasal administration per day in order to ensure intrasinus cavity application. It is expected that all the exemplified dosages 75, 150, 300, 600, 1200, 2400, and 4800 microgram GM-CSF will have an effect on disease parameters. However, this experiment is designed to identify an optimal dosage for treatment. The study can be done in one patient, where the effect of increasing dosages can be observed, or the study can be done by testing two different dosages in different nostrils. If groups of patients comprising one or more patients are included, the study is designed to provide an amount of either one of 0, 75, 150, 300, 600, 1200, 2400, and 4800 microgram GM-CSF per day to each group. The treatment period will be set to 10 days, followed by 10 days without treatment..

In one part of this experiment, 150, 300 or 1200 microgram GM-CSF is administered daily for 10 days with 5, 10 or 30 days of non treatment interval followed by 10 days of daily administration and another period of 5, 10 or 30 days of without treatment. After the end of each period without treatment, status of disease is evaluated. Treatment in the chronic phase:

Evaluation of the status and possible alleviation or relieve of the disease is done by visual observation (inspection) of swelling of the nasal epithelium, or by observation of signs and symptoms of infection and or sign of systemic inflammation (fever and blood se- Procalcitonin test (PCT) and se- c-reactive protein (CRP)) state and level of pain and tenderness relief, or improvement of some of the symptoms of chronic sinusitis with nasal polyps including: Runny nose, persistent stuffiness, postnasal drip decreased or absent sense of smell, loss of sense of taste, facial pain or headache, pain referred to upper teeth, sense of pressure over your forehead and face, snuffling/twang, and a constant feeling of stuffiness and the dysfunctional speech dominated by nasal speech, sneezing, troubles of sleeping due to snoring and finally a sensation of itching around the nostrils and eyes.

After treatment with GM-CSF, the signs and symptoms of nasal/paranasal polyposis are relieved, (defervesced over some weeks finally to be alleviated or to completely disappear)

Surrogate markers like biopsy verified disapperance of eosinophils and e.g,. verified via FACS analysis, and immunohistochemistry (biopsy verified) will be used to evaluate the status of the disease. Finally in order to monitor the most important surrogate marker, the transition of the T Helper type of inflammation from the eosinophilic dominant immuno-inflammatory TH2 state to the TH 1 type of inflammation¹ - the main effect of the GM-CSF therapy. T_H1 Cellular immunity T-cells: CD4+, CD8 & CD16 accompanied by the typical cytokines: IFN gamma, IL-2, TNF, IL-2 & IL-3.

Claims

A composition comprising granulocyte-macrophage colony-stimulating factor (GM-CSF), or a functional variant or homologue thereof, for use in the treatment, prevention or alleviation of chronic nasal and/or paranasal sinus inflammation.

The composition for use according to the preceding claims, wherein said composition is for use in the treatment, prevention or alleviation of nasal and/or paranasal sinus polyposis.

The composition for use according to the preceding claims, wherein said composition is administered locally.

The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered by intranasal and/or intrasinus administration.

The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered by intranasal and/or intrasinus administration to the surface of the epithelium of the nasal cavity and/or paranasal sinuses.

The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered as a solution, a suspension, an aerosol, a nebulized solution, or a nebulized suspension.

7. The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered as a powder.

8. The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered via nasal drops, spraying, lavage, flushing, inhalation, injection or instillation.

The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered by injection.

10. The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered via

CPAP.

1 1 . The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered by inhalation and/or exhalation.

12. The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered as an inserted depot.

13. The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered in a pegylated, liposomal or nanoparticle prepared form.

14. The composition for use according to the preceding claims, wherein said

chronic nasal and/or paranasal sinus inflammation is associated with a condition selected from the group consisting of chronic rhinosinusitis, chronic sinusitis, allergic fungal sinusitis (AFS) , asthma, aspirin intolerance/salicylate sensitivity, primary ciliary dyskinesia, cystic fibrosis (CF), Kartagener's syndrome, Young's syndrome, Churg-Strauss syndrome, nasal mastocytosis, exposure to chromium and allergic rhinitis.

15. The composition for use according to the preceding claims, wherein said GM- CSF, or a functional variant or homologue thereof, is to be administered at an effective amount, such as from between 100 to 1000 microgram per dose, for example 200-800 microgram per dose.

16. The composition for use according to claim 15, wherein said dose is

administered once, twice, three times or four times daily.

17. The composition for use according to claims 15 and 16, wherein said one or more doses are administered for 1 day or for between 1 to 14 days, such as 1 to 3 days, 3 to 5 days, 5 to 7 days, 7 to 10 days, 10 to 14 days, or for 7-14 days, such as 8-12 days, such as 8 days, 9 days, 10 days, 1 1 days or 12 days, or for one week to two weeks, for two to four weeks, for one month to two months, for 2 to 4 months, or for as long as symptoms and disease is detectable.

18. The composition for use according to claims 15 to 17, wherein said one or more doses are administered in a dosage regime with pauses of treatment having a duration of 5 to10 days, such as 5 days, 6 days, 7 days, 8 days, 9 days, 10 days or more such as 1 to 4 months.

19. The composition for use according to claims 15 to 18, wherein said one or more doses are administered in a dosage regime that is repeated once, two times, three times or for the rest of the life time of the subject in need thereof,

20. A method for treating, preventing, reducing risk of, or alleviating of chronic nasal and/or paranasal sinus inflammation in a subject in need thereof, said method comprising administering to the subject an effective amount of a composition comprising granulocyte-macrophage colony-stimulating factor (GM-CSF), or a functional variant or homologue thereof.

21 . The method according to claim 20 for treatment for use in the treatment,

prevention or alleviation of nasal and/or paranasal sinus polyposis.