WO2012073025A1

WO2012073025A1 - Glucosaminoglucans such as heparin for use in the treatment of pulmonary inflammation such as copd

Info

Publication number: WO2012073025A1
Application number: PCT/GB2011/052366
Authority: WO
Inventors: Frazer Giles Morgan
Original assignee: Vectura Ltd
Current assignee: Vectura Ltd
Priority date: 2010-11-30
Filing date: 2011-11-30
Publication date: 2012-06-07
Anticipated expiration: 2013-05-30
Also published as: TW201306847A

Abstract

According to the invention there is provided heparin for use in the treatment of COPD, wherein, after administration to a subject, the heparin reduces inflammation in the lungs of the subject.

Description

GLUCOSAMINOGLUCANS SUCH AS HEPARIN FOR USE IN THE TREATMENT OF PULMONARY INFLAMMATION SUCH AS COPD

The present invention relates to compositions for providing treatment of diseases and disorders of lung disease, including asthma (e.g. steroid resistant asthma), cystic fibrosis, idiopathic pulmonary fibrosis, non-cystic fibrosis bronchiectasis and, particularly, chronic obstructive pulmonary disease (COPD).

COPD

Worldwide, COPD ranks as the sixth leading cause of death. It is projected to be the fourth leading cause of death worldwide by 2030 due to an increase in smoking rates and demographic changes in many countries. COPD is the 4th leading cause of death in the U.S., and the economic burden of COPD in the U.S. in 2007 was $42.6 billion in health care costs and lost productivity. The largest portion of total expenditure (over 70%) is for inpatient hospitalization for exacerbations (Sullivan S D, et al., Chest 1 17:5-9, 2000; Ramsey S D, et al., Eur Respir J 21 :29 S-35S, 2003). This is explained by the high cost of hospitalization for medical care. In the recent medical literature, the average length of hospital stay for patients with COPD exacerbations ranges from 5.9 days to 12 days. Any therapy effective at improving the dysfunction of patients' airways would allow physicians to discharge patients from the hospital sooner, or avoid the need for them to be admitted to hospital entirely, thereby reducing the overall economic burden of COPD.

COPD is generally recognized as one of the most serious and disabling conditions in middle-aged and elderly patients. In the US, 80 to 90% of COPD cases are due to cigarette smoking (medicinenet.com - COPD causes, Young et al. Eur. Respir. J. 34(2), 2009, 380-386). COPD can also be caused by prolonged exposure to certain dusty environments, for example mining and powder associated manufacturing industries.

COPD is an incurable disease wherein chronic inflammation of the bronchial passageways plays a prominent role. Cigarette smoking and other inhaled irritants, perpetuate the cycle of inflammatory response that leads to further airway narrowing and hypersensitivity. With disease progression, patients have increasing difficulty clearing secretions, due in part, to poor ciliary function. Consequently, they develop a chronic productive cough, wheezing, and dyspnea. Further inflammation is compounded by opportunistic bacterial colonisation of the airways which can result in the formation of obstructions in the pulmonary airways.

Acute COPD exacerbations occur more often during the winter months. An acute exacerbation of COPD can be defined as a sustained worsening of the patient's symptoms from their stable state that is in excess of their normal day-to-day variations, and is acute in its onset. Acute exacerbations manifest in a number of ways, in particular, increased wheezing and/or coughing, chest tightness, increased sputum production, reduced ability to tolerate exercise, increased fluid retention, increased fatigue and worsening of dyspnea. Although infectious agents account for most exacerbations, exposure to allergens, pollutants, or inhaled irritants may also play a role. Infectious agents known to cause acute exacerbations of COPD include: rhinoviruses, influenza, parainfluenza, coronavirus, adenovirus, respiratory syncytial virus, Chlamydia pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae, Moraxella catarrhalis, Staphylococcus aureus, Mycoplasma pneumoniae, and Pseudomonas aeruginosa. Pollutants known to cause acute exacerbations include nitrogen dioxide, particulates, sulfur dioxide, and ozone.

The airway inflammatory response consists of macrophage and CD8 T lymphocytes in the airway wall and neutrophils in the airways.

During acute COPD exacerbations the cellular infiltrate changes dramatically. The concentration of cellular elements in bronchoalveolar lavage (BAL) rises substantially compared to patients with stable COPD, resulting in neutrophil levels that make them the dominant inflammatory cell in the airways.

Neutrophilic airway inflammation is dramatically induced in all COPD exacerbations. In the BAL from patients with acute exacerbations, neutrophils constitute over half of all cellular elements as a percentage of BAL differential counts, compared to only 18% (Papi A, et al., Am J Respir Crit Care Med 173: 1 1 14-1 121 , 2006) in the BAL fluid of patients with stable COPD and 5% (Papi A, et al., Am J Respir Crit Care Med 173: 1 1 14-1 121 , 2006) in the BAL from non-smokers and healthy smokers. The cause of this neutrophilic inflammatory influx is a dramatic increase in neutrophilstimulating cytokines within BAL and cytokine-secreting cells within the airway epithelium.

Cytokine upregulation results in prominent increase in neutrophil protease human leukocyte elastase (HLE) within the airway epithelium. This is significant because of its potential for producing proteolytic airway injury and also because HLE and other neutrophil proteases stimulate bronchial mucus hypersecretion and possibly activate airway epithelial epidermal growth factor, thereby stimulating pro-inflammatory signaling cascades.

When cytokines signal for neutrophil influx from the vascular space into the lung, the neutrophils change velocity along the vascular wall. After finding an intercellular junction between endothelial cells, the neutrophil then migrates from the vascular space to the airways where it adheres to cells via Intracellular Adhesion Molecules (ICAM) expressed on the surface of the airway. As neutrophils move along the airway wall they activate and firmly adhere to the airway epithelium. The neutrophil can engulf invading microbes and particulates in addition to causing indiscriminate injury to inflamed tissues.

Acute exacerbations of COPD are accompanied by evidence not only of increased airway inflammation but also of increased systemic inflammation. The most commonly used biomarker of inflammation is C-reactive protein (CRP). CRP bind bacteria, oxidized lipids, and apoptotic cells and facilitates their clearance from the immune system. CRP has been shown to be associated with increased lung inflammation in stable patients with COPD.

An effective method for reducing neutrophil injury in the lung would be to retard neutrophil migration into the lung from the bloodstream, before neutrophils become activated and release proteolytic enzymes and oxidants into the lung environment. One way to achieve this might be by an intravenous drug that retards neutrophil migration into the lung, thereby decreasing overall lung and systemic inflammation from COPD exacerbations. Currently, corticosteroids are used for this purpose, but have disadvantages, including the induction of muscle weakness, an increased catabolic state, the induction of osteoporosis, induction of elevated blood pressure, and the induction of glucose resistance leading in some cases to the diabetic state. The anti-inflammatory effect of corticosteroids in COPD exacerbations is also modest, and leads to only moderate reductions in systemic inflammation measured by CRP.

Cigarette smoking is implicated in diagnosed cases of COPD. Cigarette smoking causes an influx of activated leukocytes into the lungs with subsequent degranulation and release of proteases. Cigarette-derived oxidants inactivate α-1 -anti-proteinase by oxidizing an important methionine near the active site. Elastase delivered to the alveolar lung unit as a result of the influx due to cigarette smoking, concurrent with oxidative inactivation of α-1 -anti-proteinase activity, produces an imbalance of protease/anti-proteinase activity that is thought to be a major cause of human emphysema from cigarette smoking. Similarly, murine models have recently been developed with analogous inflammatory mechanisms.

Induction of emphysema requires chronic exposure to tobacco smoke (TS) over a period of 6 months, which also induces lung tissue inflammation. Whilst chronic exposure TS mouse model is the current gold standard of COPD, these studies are inherently time consuming, labour intensive and expensive

Mouse models used to study TS-induced pulmonary inflammation allow short term evaluation of anti-inflammatory compounds for the treatment of COPD (J.C. Fox et al., ATS, 2007). In this study the effect of exposing C57/B16 mice to tobacco smoke for 4 or 1 1 days (6 cigarettes/day) was investigated. The study demonstrated that TS exposure for 4 or 1 1 days induced significant BAL cell influx which mainly comprised of macrophages and neutrophils. The degree of BAL cell influx did not differ significantly when examined 2, 4 or 24 hrs post exposure and was generally similar following 4 or 1 1 days exposure (J.C. Fox et al., ATS, 2007). In this model BAL inflammatory cell influx can be inhibited by PDE4 inhibitors and p38 MAP kinase inhibitors. Inflammatory mediators in both BAL and lung tissue homogenate provide useful endpoints.

Periodic exacerbations of COPD are a major cause of morbidity, mortality, and health care costs in patients with COPD. Patients who suffer exacerbations have a worse quality of life and a more rapid decline in both health status and lung function as measured by forced expired volume 1 (FEV1 ).

Because no curative therapy is available, management of severe exacerbations of COPD are generally directed at relieving symptoms and restoring functional capacity. Pharmacological management includes the use of bronchodilators, anticholinergics, corticosteroids, antibiotics, and methylxanthines, as well as oxygen therapy and noninvasive ventilation.

Bronchodilators are used to treat the increased breathlessness that occurs during exacerbations of COPD. Inhaled β2 agonists are typically administered during an acute exacerbation using nebulizers, hand-held metered dose, or dry powder inhalers. Specific examples of β2 agonists include albuterol, salbutamol, fomoterol, and terbutaline. Inhaled anticholingergics (such as ipratropium and tiotroprium) may also be used for bronchodilation and can also be administered by a nebulizer, metered- dose inhalers, or dry powdered inhaler. Combination products, such as ipratropium- albuterol, are used to simplify the medication regimen.

In the absence of significant contraindications, oral corticosteroids are typically recommended, often in conjunction with other therapies, in all patients suffering from acute exacerbation of COPD. For severe exacerbations requiring inpatient therapy, prednisolone or methylprednisolone is commonly used.

Corticosteroids are the mainstay of anti-inflammatory therapy, but the use thereof in the treatment of acute exacerbations of COPD is complicated by side effects. Betaadrenergic agonists, acting by stimulation of β2 adrenergic receptors on airway smooth muscle, are used as bronchodilators to directly reverse constricted airways. Nonselective anti-cholinergic drugs, such as ipratropium bromide, are available for use as bronchodilators.

Asthma

Asthma is an inflammatory disease of lung airways in which the airways are prone to narrowing in response to provoking stimuli. Although airflow obstruction is a feature of asthma it is not considered to be COPD because pulmonary function is recoverable in asthma patients.

Cystic fibrosis

Cystic fibrosis (also known as CF or mucoviscidosis) is a common disease which affects the entire body, causing progressive disability and often early death. Approximately 30,000 Americans have CF, making it one of the most common life shortening inherited diseases. The name cystic fibrosis refers to the characteristic scarring (fibrosis) and cyst formation within the pancreas, first recognized in the 1930s. Difficulty breathing is the most serious symptom and results from frequent lung infections that is treated with, though not cured by, antibiotics and other medications, which ultimately leads to need for lung transplantation. A multitude of other symptoms, including sinus infections, poor growth, diarrhea, and infertility result from the effects of CF on other parts of the body.

There is currently no cure for CF. There are already a number of products approved for the treatment of the disease but the unmet medical need remains high in CF patients. Treatment of the pulmonary aspects of disease in CF patients includes physiotherapy to help clear mucus from lungs, antibiotics to help control infection and prevent progressive lung damage, Deoxyribonuclease (DNase) I (Pulmozyme®) to clear mucus from the lungs.

Cystic Fibrosis patients exhibit a complex respiratory pathlogy that involves the interplay of four main factors, these include: bacterial infections, broncho-constriction and airway obstruction, inflammation and bronchiectasis and, finally, increased mucus viscoeleasticity and impaired mucociliary clearance. Each of these pathlogy factors have the potential to influence the extent of the other three factors. For example, increased bacterial infection leads to increased inflammation and bronchiectasis with a concomitant increase in broncho-constrictions and airway obstruction. An increase in inflammation and bronchiectasis results in an increase in mucus viscoeleasticity and impaired mucociliary clearance. An airway with an impaired mucociliary clearance mechanism lends itself to bacterial infections thereby perpetuating the cycle. Mucolytic and antibiotic therapies used in the treatment of CF are typically delivered via nebulisation of liquids. It is recognised that such nebulisation delivery systems are considered sub-optimal for the following reasons: dosing is typically restricted to the clinic or at-home environment; inadequate cleaning and repeated use of nebuliser systems can lead to the unwanted aerosol isation of microbes as well as drug. This is a significant treatment issue in view of the increased susceptibility of CF patients to lung infection; and poor delivery efficiency of nebulised drug (maximum 10% of delivered dose reaching target organ) results in extended dosing periods and the delivery of drug to the environment rather than into the patient. Such environmental contamination may contribute to increased levels of antibiotic resistance in clinical settings.

Idiopathic pulmonary fibrosis (IPF)

Idiopathic pulmonary fibrosis (IPF) (or cryptogenic fibrosing alveolitis (CFA)) is a chronic, progressive form of lung disease characterized by fibrosis of the supporting framework (interstitium) of the lungs.

Non-cystic fibrosis bronchiectasis

Non-cystic fibrosis bronchiectasis (Bronchiectasis) is dilation and destruction of larger bronchi caused by chronic infection and inflammation. Common causes are immune defects, and recurrent infections, though some cases appear to be idiopathic. Symptoms are chronic cough and purulent sputum expectoration; some patients may also have fever and dyspnea.

There is still a need for improved treatments for lung diseases. Summary of the Invention

In a first aspect of the present invention, there is provided a glycosanninoglycan (e.g. heparin) for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject.

Thus according to further aspects of the present invention, there is provided:

(i) a method of treatment and/or prevention of an inflammatory lung disease, comprising the administration of a glycosaminoglycan (e.g. heparin) to a subject, optionally wherein, after administration, the glycosaminoglycan reduces inflammation in the lungs of the subject;

(ii) use of a glycosaminoglycan (e.g. heparin) in the preparation of a medicament for the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject; and

(iii) a kit comprising a glycosaminoglycan (e.g. heparin) for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject.

In a yet further aspect of the present invention, there is provided a glycosaminoglycan delivered by an inhalation device for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject.

Thus according to further aspects of the present invention, there is provided: (i) a method of treatment and/or prevention of an inflammatory lung disease, comprising the delivery of a glycosaminoglycan (e.g. heparin) by an inhalation device to a subject, optionally wherein, after administration, the glycosaminoglycan reduces inflammation in the lungs of the subject;

(ii) use of a glycosaminoglycan (e.g. heparin) and an inhalation device in the preparation of a medicament for the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject; and

(iii) a kit comprising a glycosaminoglycan (e.g. heparin) and an inhalation device for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject.

In a further aspect of the present invention, there is provided a glycosaminoglycan (e.g. heparin) in combination with another active agent for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, inflammation in the lungs of the subject is reduced.

Thus according to further aspects of the present invention, there is provided:

(i) a method of treatment and/or prevention for an inflammatory lung disease, comprising the administration of a glycosaminoglycan (e.g. heparin) and another active agent to a subject, optionally wherein, after administration to a subject, inflammation in the lungs of the subject is reduced; and

(ii) use of a glycosaminoglycan (e.g. heparin) in the preparation of a medicament for the treatment and/or prevention of an inflammatory lung disease in combination with another active agent, optionally wherein, after administration to a subject, inflammation in the lungs of the subject is reduced.

In a further aspect of the present invention, there is provided a glycosaminoglycan (e.g. heparin) for use in reducing inflammation.

Thus according to further aspects of the present invention, there is provided:

(i) a method of reducing inflammation, comprising the administration of a glycosaminoglycan (e.g. heparin) to a subject;

(ii) use of a glycosaminoglycan (e.g. heparin) in the preparation of a medicament for the reduction of inflammation; and

(iii) a kit comprising a glycosaminoglycan (e.g. heparin) for use in the reduction of inflammation.

In a further aspect of the present invention, there is provided a glycosaminoglycan (e.g. heparin) delivered by an inhalation device for use in reducing inflammation.

Thus according to further aspects of the present invention, there is provided:

(i) a method of reducing inflammation, comprising the delivery of a glycosaminoglycan (e.g. heparin) by an inhalation device to a subject;

(ii) use of a glycosaminoglycan (e.g. heparin) and an inhalation device in the preparation of a medicament for reducing inflammation; and

(iii) a kit comprising a glycosaminoglycan (e.g. heparin) and an inhalation device for use in reducing inflammation. In a further aspect of the invention subjects to be treated are COPD subjects who are undergoing an acute episode of COPD exacerbation.

In a further aspect of the invention subjects to be treated are COPD subjects who are undergoing a chronic episode of COPD exacerbation.

In a yet further aspect of the invention subjects to be treated are CF subjects.

In yet a further aspect of the invention, there is provided a pharmaceutical formulation of a glycosaminoglycan (e.g. heparin) for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject.

In yet a still further embodiment of the invention, there is provided a device for the administration of a glycosaminoglycan (e.g. heparin) for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject.

Brief description of the Drawings

Figure 1 shows the reduction in total cell numbers obtained from BAL following administration of heparin doses of 100 pg/kg, 300 pg/kg, 1000 pg/kg and 2000 pg/kg compared with sham control and the positive control ADS1 15398. Figure 2 shows the reduction in total macrophage numbers obtained from BAL following administration of heparin doses of 100 pg/kg, 300 pg/kg, 1000 pg/kg and 2000 pg/kg compared with sham control and the positive control ADS1 15398.

Figure 3 shows the reduction in total epithelial cell numbers obtained from BAL following administration of heparin doses of 100 pg/kg, 300 pg/kg, 1000 pg/kg and 2000 pg/kg compared with sham control and the positive control ADS1 15398.

Figure 4 shows the reduction in total neutrophil numbers obtained from BAL following administration of heparin doses of 100 pg/kg, 300 pg/kg, 1000 pg/kg and 2000 pg/kg compared with sham control and the positive control ADS1 15398.

Figure 5 shows the reduction in total eosinophil numbers obtained from BAL following administration of heparin doses of 100 pg/kg, 300 pg/kg, 1000 pg/kg and 2000 pg/kg compared with sham control and the positive control ADS1 15398.

Figure 6 shows the reduction in total lymphocyte numbers obtained from BAL following administration of heparin doses of 100 pg/kg, 300 pg/kg, 1000 pg/kg and 2000 pg/kg compared with sham control and the positive control ADS1 15398.

Figure 7 shows a change in mucus viscoelasticity following administration of heparin at 0.16 mg/ml, 0.5 mg/ml, 1 .6 mg/ml, and 5 mg/ml, compared with control, saline solution and the positive control Nacystelyn. Figure 8 shows a change in mucus viscoelasticity (Relative velocity (% of Ringer control)) following administration of unfractionated heparin at 0.5 mg/ml, 1 .6 mg/ml, and 5 mg/ml (Column 1 -3), compared with low molecular weight heparin at 0.5 mg/ml, 1 .6 mg/ml, and 5 mg/ml (Column 4-6), compared with the positive control Nacystelyn (Column 7) demonstrating a dose dependent increase in mucociliary velocity and comparability with clinically relevant Nacystelyn dose.

Figure 9 shows the mean platelet counts following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared to lactose placebo. Data demonstrate no systemic exposure is present following heparin administration throughout the course of the study.

Figure 10 shows mean activated partial thromboplastin time (aPTT) following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate no systemic exposure is present following heparin administration throughout the course of the study.

Figure 1 1 shows induced sputum neutrophil elastate summary following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate reduction in neutrophil elastate levels following 100 mg dose.

Figure 12 shows induced sputum total cell count summary following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate reduction in total cell count levels following administration of unfractionated heparin compared lactose placebo.

Figure 13 shows induced sputum IL-8 summary following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate reduction IL-8 levels following administration of 100 mg dose heparin compared with lactose placebo.

Figure 14 shows Plasma neutrophil elastate summary following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate the absence of plasma inflammatory markers levels.

Figure 15 shows plasma neutrophil counts summary following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate the absence of plasma inflammatory markers levels.

Figure 16 shows plasma NE/AAT complex summary following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate the absence of plasma inflammatory markers levels.

Figure 17 shows Plasma CRP summary following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate the absence of plasma inflammatory markers levels. Figure 18 shows plasma IL-8 summary following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate the absence of plasma inflammatory markers levels.

Figure 19 shows plasma IL-6 summary following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate the absence of plasma inflammatory markers levels.

Figure 20 shows plasma inflammatory mediator summary following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo. Data demonstrate the absence of plasma inflammatory markers levels for total neutrophils, neutrophil elastate neutrophil elastate/alpha-1 antitrypsin complex, IL-8, IL-6 and CRP. In Figure 20, the first bar in the hiostogram represents Low dose, the second bar represents Mild dose and the third bar represents High dose.

Figure 21 shows mucus observations following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo as a change from baseline to week 4 of the study. Data demonstrate that the patients' mucus becomes thinner following heparin treatment. The sputum volume increases versus placebo. The mucus colour becomes clearer indicating a reduction in bacterial infection. The sputum become less sticky and therefore easier to clear from the lungs.

Figure 22 shows Staphylococcus aureus and Pseudomonas aeruginosa levels in the sputum following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo as a change from baseline to week 4 of the study. Data demonstrate the absence of a pro-microbial effect.

In each histogram in Figures 21 and 22, where present, the first bar represents Low dose, the second bar represents Mild dose, the third bar represents High dose, and the fourth bar represents Placebo.

Detailed Description of Invention

We have found that the anticoagulant drug heparin reduces inflammation (e.g. pulmonary neutrophil levels) to the level achieved using the p38 MAP kinase inhibitor, ADS1 15398. Additionally, we have found that heparin is capable of reducing inflammation in a clinical setting too (e.g. for CF patients).

The present invention provides methods for treating and preventing symptoms of an inflammatory lung disease, including asthma, cystic fibrosis, idiopathic pulmonary fibrosis, non-cystic fibrosis bronchiectasis and, particularly, chronic obstructive pulmonary disease (COPD). The methods comprise administration of heparin to a patient with an inflammatory lung disease such as asthma, cystic fibrosis, idiopathic pulmonary fibrosis, non-cystic fibrosis bronchiectasis, COPD or any combination thereof. The administration of heparin is particularly beneficial for reducing lung inflammation, and as shown herein by reducing pulmonary neutrophil levels to those achieved by the p38 MAP kinase inhibitor, ADS1 15398. A drug that is capable of reducing inflammation (e.g. neutrophil levels), bacterial infections and pulmonary mucus viscosity in the pulmonary airways would be of tremendous benefit in the treatment of CF or, particularly, COPD exacerbations. Heparin combines these desired effects and may avoid the need for CF patients or, particularly, COPD patients to administer multiple medications.

The term "inflammatory lung disease" is a disease which can be associated with inflammation of the respiratory system (e.g. the lungs).

The present invention demonstrates that the administration of a glycosaminoglycans (e.g. heparin) is particularly beneficial for reducing lung inflammation (e.g. by reducing pulmonary neutrophil levels to those achieved by the p38 MAP kinase inhibitor, ADS1 15398).

Embodiments of the invention, which may used alone or be in any combination, include those wherein:

(a) the glycosaminoglycan is selected from chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate, hyaluronan and, particularly, heparin (e.g. the glycosaminoglycan is selected from danaparoid sodium, dermatan sulphate and, particularly, heparin);

(b) the administration of glycosaminoglycan (e.g. heparin) results in a reduction of inflammation (e.g. as measured by inflammatory markers, as described herein (e.g. total sputum cell count, neutrophil elastase levels etc)) of at least 10% (e.g. at least 20%, 30%, 40%, 50% or, particularly, 60%); (c) the glycosaminoglycan (e.g. heparin) is administered intranasally or by pulmonary inhalation (e.g. the heparin is administered by pulmonary inhalation);

(d) the glycosaminoglycan (e.g. heparin) is in the form of a dry powder formulation;

(e) the glycosaminoglycan is heparin;

(f) the heparin is unfractionated heparin (e.g. unfractionated heparin sodium, such as oxidised or unoxidised unfractionated heparin sodium (e.g. unoxidised unfractionated heparin sodium));

(g) the heparin is low molecular weight heparin (e.g. enoxaparin);

(h) the total daily dose (measured as the FPD) of glycosaminoglycan (e.g. heparin) is between 0.5 mg and 6000 mg (e.g. the total daily dose of heparin is between 1 mg and 2000 mg, such as between 2 mg and 1500 mg, between 6 mg and 600 mg, between 12 mg and 300 mg, such as between 18 mg and 240 mg, between 24 mg and 180 mg, such as between 36 mg and 120 mg (e.g. between 50 and 75 mg));

(i) the total daily dose (measured as the FPD) of heparin is between 0.5 mg and 6000 mg (e.g. the total daily dose of heparin is between 1 mg and 2000 mg, such as between 2 mg and 1500 mg, between 6 mg and 600 mg, between 12 mg and 300 mg, such as between 18 mg and 240 mg, between 24 mg and 180 mg, such as between 36 mg and 120 mg (e.g. between 50 and 75 mg));

(j) the inflammation is as measured by total cell count obtained from induced sputum or, particularly, bronchoalveolar lavage;

(k) the inflammation is as measured by an inflammation marker (e.g induced sputum neutrophil elastase, induced sputum IL-8 or, particularly, pulmonary eosinophil levels, pulmonary lymphocyte levels, pulmonary macrophage levels or, more particularly, pulmonary neutrophil levels or any combination thereof (e.g. pulmonary macrophage levels or, particularly, pulmonary neutrophil levels)); (I) the reduction in inflammation achieved is equivalent to that achieved by the p38 MAP kinase inhibitor, ADS1 15398 (e.g. wherein the ADS1 15398 is given in a dose of 0.1 mg/kg to a subject);

(m) the COPD is an episode of acute COPD exacerbation;

(n) the COPD is an episode of chronic COPD exacerbation;

(o) heparin is delivered in a nominal dose of between 0.8 mg and 2100 mg (e.g. between 1 .5 mg and 1 100 mg, between 3 mg and 530 mg, between 8 mg and 300 mg, between 25 mg and 200 mg, between 35 mg and 150 mg, between 70 mg and

140 mg);

(p) heparin is delivered in a fine particle dose of between 25 mg and 200 mg, between 50 mg and 125 mg, such as between 75 and 100 mg or, particularly, between 0.5 mg and 2000 mg (e.g. between 1 mg and 1000 mg, between 2 mg and 500 mg, between 6 mg and 250 mg, between 18 mg and 240 mg, between 30 mg and 130 mg, between 60 mg and 120 mg) for example when measured by a New Generation Impactor (Ph Eur Apparatus at 60 L/min);

(q) the inflammatory lung disease is a disease characterised by neutrophilia;

(r) the inflammatory lung disease may be a disease or condition selected from asthma, cystic fibrosis, idiopathic pulmonary fibrosis, non-cystic fibrosis bronchiectasis and, particularly, chronic obstructive pulmonary disease (e.g. moderate persistent asthma, severe persistent asthma, cystic fibrosis, idiopathic pulmonary fibrosis, non-cystic fibrosis bronchiectasis and, particularly, chronic obstructive pulmonary disease, such as severe persistent asthma, cystic fibrosis and, particularly, chronic obstructive pulmonary disease (e.g. cystic fibrosis and, particularly, chronic obstructive pulmonary disease)). (s) the use further comprises another active agent (e.g. the further active agent is selected from mucolytic agents (e.g. N -acetylcysteine, ambroxol, amiloride, dextrans, heparin, desulphated heparin, low molecular weight heparin and recombinant human DNase);

Bronchodilators (e.g. the 2-agonists bambuterol, bitolterol, broxaterol, carmoterol, clenbuterol, fenoterol, formoterol, indacaterol, levalbuterol, metaproterenol, orciprenaline, picumeterol, pirbuterol, procaterol, reproterol, rimiterol, salbutamol, salmeterol, terbutaline and the like);

Anti-muscarinics (e.g. ipratropium, ipratropium, bromide, oxitropium, tiotropium and glycopyrrolate);

Antibiotic and antibacterial agents (e.g. including the beta-lactams, fluoroquinolones, ketolides, macrolides, sulphonamides and tetracyclines, aclarubicin, amoxicillin, amphotericin, azithromycin, aztreonam chlorhexidine, clarithromycin, clindamycin, colistimethate, dactinomycin, dirithromycin, doripenem, erythromycin, fusafungine, gentamycin, metronidazole, mupirocin, natamycin, neomycin, nystatin, oleandomycin, pentamidine, pimaricin, probenecid, roxithromycin, sulphadiazine and triclosan);

Anti-infective agents (e.g. antivirals (including nucleoside and non-nucleoside reverse transcriptase inhibitors and protease inhibitors) including aciclovir, adefovir, amantadine, cidofovir, efavirenz, famiciclovir, foscarnet, ganciclovir, idoxuridine, indinavir, inosine pranobex, lamivudine, nelfinavir, nevirapine, oseltamivir, palivizumab, penciclovir, pleconaril, ribavirin, rimantadine, ritonavir, ruprintrivir, saquinavir, stavudine, valaciclovir, zalcitabine, zanamivir, zidovudine and interferons);, aminoglycosides (e.g. tobramycin; antifungals for example amphotericin, caspofungin, clotrimazole, econazole nitrate, fluconazole, itraconazole, ketoconazole, miconazole, nystatin, terbinafine and voriconazole; antituberculosis agents for example capreomycin, ciprofloxacin, ethambutol, meropenem, piperacillin, rifampicin and vancomycin; beta-lactams including cefazolin, cefmetazole, cefoperazone, cefoxitin, cephacetrile, cephalexin, cephaloglycin and cephaloridine; cephalosporins, including cephalosporin C and cephalothin; cephamycins such as cephamycin A, cephamycin B, cephamycin C, cephapirin and cephradine);

leprostatics (e.g. clofazimine; penicillins including amoxicillin, ampicillin, amylpenicillin, azidocillin, benzylpenicillin, carbenicillin, carfecillin, carindacillin, clometocillin, cloxacillin, cyclacillin, dicloxacillin, diphenicillin, heptylpenicillin, hetacillin, metampicillin, methicillin, nafcillin, 2-pentenylpenicillin, penicillin N, penicillin O, penicillin S and penicillin V; quinolones including ciprofloxacin, clinafloxacin, difloxacin, grepafloxacin, norfloxacin, ofloxacine and temafloxacin);

tetracyclines including doxycycline and oxytetracycline;

miscellaneous anti-infectives for example linezolide, trimethoprim and sulfamethoxazole.

Nonsteroidal anti-inflammatory agents (e.g. aceclofenac, acetaminophen, alminoprofen, amfenac, aminopropylon, amixetrine, aspirin, benoxaprofen, bromfenac, bufexamac, carprofen, celecoxib, choline, cinchophen, cinmetacin, clometacin, clopriac, diclofenac, diclofenac sodium, diflunisal, ethenzamide, etodolac, etoricoxib, fenoprofen, flurbiprofen, ibuprofen, indomethacin, indoprofen, ketoprofen, ketorolac, loxoprofen, mazipredone, meclofenamate, mefenamic acid, meloxicam, nabumetone, naproxen, nimesulide, parecoxib, phenylbutazone, piroxicam, pirprofen, rofecoxib, salicylate, sulindac, tiaprofenic acid, tolfenamate, tolmetin and valdecoxib);

Other anti-inflammatory agents (e.g. B-cell inhibitors, p38 MAP kinase inhibitors, particularly, ADS1 15398 and TNF inhibitors); PDE4 inhibitors (e.g. cilomilast, etazolate, rolipram, oglemilast, roflumilast, ONO 6126, tolafentrine and zardaverine);

quinazolinediones (e.g. nitraquazone and nitraquazone analogs; xanthine derivatives such as denbufylline and arofylline; tetrahydropynmidones such as atizoram; and oxime carbamates such as filaminast);

Steroids (e.g. alcometasone, beclomethasone, beclomethasone dipropionate, betamethasone, budesonide, butixocort, ciclesonide, clobetasol, deflazacort, diflucortolone, desoxymethasone, dexamethasone, fludrocortisone, flunisolide, fluocinolone, fluometholone, fluticasone, fluticasone proprionate, hydrocortisone, methylprednisolone, mometasone, nandrolone decanoate, neomycin sulphate, prednisolone, rimexolone, rofleponide, triamcinolone and triamcinolone acetonide); Matrix metalloprotease inhibitors (e.g. adamalysins, serralysins, and astacins); Epithelial sodium channel (ENaC) inhibitors (e.g. P-680 and Denufosol;

CFTR Potentiators for example VX-809);

Methylxanthines (e.g. caffeine, theobromine and theophylline);

Drugs for cystic fibrosis management (e.g. Pseudomonas aeruginosa infection vaccines (eg Aerugen™), alpha 1 -antitripsin, amikacin, cefadroxil, denufosol, duramycin, glutathione, mannitol, and tobramycin).

Further embodiments of the invention, which may used alone or be in any combination with those described herein, include those wherein:

(i) the total daily dose (measured as the FPD) of heparin is between 0.1 and 6000 mg, such as between 0.2 and 3000 mg (e.g. between 0.3 and 250 mg, such as between 0.32 and 200 mg, such as between 1 and 175mg, 5 and 150 mg, such as between 10 and 100 mg, such as between 20 and 80 mg (e.g. between 30 and 70 mg, such as between 35 and 50 mg (e.g. between 40 and 45mg)));

(ii) the total daily dose (measured as the nominal dose) of heparin is between 1 and 8000 mg, such as between 2 and 5000 mg (e.g. between 15 and 300 mg (e.g. between 20 and 300 mg), such as between 25 and 250 mg (e.g. between 31 and 250 mg or 30 and 200 mg), such as between 48 and 248 mg, such as between 62 and 150 mg (e.g. between 70 and 130 mg, such as between 80 and 125 mg, e.g. between 90 and 124 mg));

(iii) the total daily dose (measured as the delivered dose) of heparin is between 0.5 and 6000 mg, such as between 0.8 mg and 5000, such as between 10 and 1000 mg (e.g. between 15 and 500 mg, between 25 and 400 mg, between 50 and 300 mg, between 75 and 200 mg (e.g. between 100 and 150 mg));

(iv) the inflammation is as measured by an inflammation marker selected from neutrophil elastase and total cell count;

(vi) heparin is delivered in a nominal dose of between 0.8 mg and 500 mg (e.g. between 1 .5 mg and 400 mg, between 3 mg and 200 mg, between 8 mg and 150 mg, between 10 mg and 100 mg, between 15 mg and 75 mg, between 20 mg and 31 mg) for example when measured by a New Generation Impactor (Ph Eur Apparatus at 60 L/min);

(vii) heparin is delivered in a fine particle dose of between 0.3 and 400 mg, between 0.5 and 200 mg, such as between 1 and 150 mg or, particularly, between 1 .5 mg and 100 mg (e.g. between 5 mg and 80 mg, between 10 mg and 60 mg, between 15 mg and 40 mg, between 20 mg and 30 mg) for example when measured by a New Generation Impactor (Ph Eur Apparatus at 60 L/min); (viii) the total daily dose (measured as the FPD) of heparin is between 0.1 and 6000 mg, such as between 0.2 and 3000 mg (e.g. between 0.3 and 250 mg, such as between 0.32 and 200 mg, such as between 1 and 175mg, 5 and 150 mg, such as between 10 and 100 mg, such as between 20 and 80 mg (e.g. between 30 and 60 mg, such as between 35 and 50 mg (e.g. between 40 and 45mg))) wherein a patient with CF does not suffer from an increased level of haemoptysis following administration of said dose;

(ix) the total daily dose (measured as the nominal dose) of heparin is between 1 and 8000 mg, such as between 2 and 5000 mg (e.g. between 15 and 300 mg, such as between 31 and 250 mg, such as between 48 and 248 mg, such as between 62 and 150 mg (e.g. between 70 and 130 mg, such as between 80 and 125 mg, e.g. between 90 and 124 mg)) wherein a patient with CF does not suffer from an increased level of haemoptysis following administration of said dose;

(x) the total daily dose (measured as the delivered dose) of heparin is between 0.5 and 6000 mg, such as between 0.8 mg and 5000, such as between 10 and 1000 mg (e.g. between 15 and 500 mg, between 25 and 400 mg, between 50 and 300 mg, between 75 and 200 mg (e.g. between 100 and 150 mg)) wherein a patient with CF does not suffer from an increased level of haemoptysis following administration of said dose;

(xi) one or more (e.g. two to eight (such as one to four) nominal doses may be delivered sequentially).

Yet further embodiments of the invention, which may used alone or be in any combination with those described herein, include those wherein: (i) the total daily dose (measured as the FPD) of heparin is between 30 and 50 mg (e.g. 40 mg) for a patient with COPD;

(ii) the total daily dose (measured as the nominal dose) of heparin is between 90 and 1 10 mg (e.g. 100 mg) for a patient with COPD;

(iii) the total daily dose (measured as the delivered dose) of heparin is between 1 15 and 135 mg (e.g. 124 mg) for a patient with COPD.

Yet further embodiments of the invention, which may used alone or be in any combination with those described herein, include those wherein:

(i) the total daily dose (measured as the FPD) of heparin is between 30 and 50 mg (e.g. 40 mg) for a patient with CF;

(ii) the total daily dose (measured as the nominal dose) of heparin is between 90 and 1 10 mg (e.g. 100 mg) for a patient with CF;

(iii) the total daily dose (measured as the delivered dose) of heparin is between 1 15 and 135 mg (e.g. 124 mg) for a patient with CF;

(iv) the total daily dose (measured as the FPD) of heparin is between 30 and 50 mg (e.g. 40 mg) for a patient with CF wherein the patient does not suffer from a cough and/or does not suffer from an increased level of haemoptysis following administration of said dose;

(v) the total daily dose (measured as the nominal dose) of heparin is between 90 and 1 10 mg (e.g. 100 mg) for a patient with CF wherein the patient does not suffer from a cough and/or does not suffer from an increased level of haemoptysis following administration of said dose;

(vi) the total daily dose (measured as the delivered dose) of heparin is between 1 15 and 135 mg (e.g. 124 mg) for a patient with CF wherein the patient does not suffer from a cough and/or does not suffer from an increased level of haemoptysis following administration of said dose.

(a) the glycosaminoglycan formulated as a dry powder is delivered by a dry powder inhaler;

(b) the dry powder inhaler is selected from a passive (Rotahaler and Diskhaler, the the GyroHaler, the Turbohaler, Novolizer or particularly the Monohaler and the device produced by Vectura (as covered by international patent application number WO 2010/086285) or active device (Aspirair (Trade Mark) and the active inhaler device produced by Nektar Therapeutics (as covered by US Patent No. 6,257,233)

(e.g. the dry powder inhaler is a passive device selected from GyroHaler or, particularly, Monohaler and the device produced by Vectura (as covered by international patent application number WO 2010/086285));

(c) the dry powder inhaler is an inhaler comprising a housing having a mouthpiece through which a user may inhale a dose of medicament and a blister support member having a slot to receive a dose containing blister, the housing and the blister support member being pivotable relative to each other between a first position for insertion of a blister into said slot and, a second, pierced position, in which a blister piercing element carried by the housing pierces an inserted blister so that when a user inhales on the mouthpiece, the dose is entrained in an airflow and flows out of the blister through the mouthpiece and into the user's airway

(e.g. said device wherein the housing comprises a substantially cylindrical chamber having an inlet at one end for the flow of drug laden air into the chamber from a pierced blister and an outlet at its opposite end for the flow of drug laden air out of the mouthpiece and into a patient's airway (e.g. such that the substantially cylindrical chamber has at least one bypass air inlet for the flow of clean air into the cyclone chamber to interact with the drug laden air flowing between the inlet and the outlet)).

Human doses of 25 mg, 50 mg and 100 mg correspond to approximately 417 pg/kg, 833 pg/kg & 1667 pg/kg, based on a 60 kg subject (as recommended by FDA).

Consequently, murine heparin doses of 100 pg/kg, 300 pg/kg, 1000 pg/kg and 2000 g/kg correspond to human doses of 6 mg, 18 mg, 60mg and 120 mg, based on a 60 kg subject (as recommended by FDA).

Definitions

The term Chronic Obstructive Pulmonary Disease ("COPD") is used to describe respiratory tract diseases generally characterized by partial or complete airway obstruction that is not completely reversible. This condition may also be referred to as chronic obstructive respiratory disease (CORD), chronic obstructive airways disease (COAD), chronic obstructive lung disease (COLD), or chronic airway limitation (CAL). The term COPD is intended to encompass all such references including, but not limited to, the symptoms described above.

The American Thoracic Society (ATS) defines COPD as a disease involving progressive chronic airflow obstruction. COPD is defined as a disease state characterized by airflow limitation that is not completely reversible. The limitation in airflow is usually degenerative in its progression and is associated with abnormal inflammatory response in the lungs. Underlying conditions that lead to COPD include chronic bronchitis, emphysema and bronchiectasis. Chronic bronchitis is clinically defined as excessive cough and sputum production on most days for at least three months during at least two consecutive years. Emphysema is characterized by chronic dyspnea (shortness of breath) resulting from the destruction of lung tissue. Bronchiectasis is the atypical stretching of the respiratory passages. The weakened passages become scarred allowing for more mucus and bacteria to accumulate, and in turn, results in a recurring cycle of blocked airways and infection.

Any reference herein to the treatment and/or prevention of COPD includes reference to the treatment and/or prevention of any one of the underlying conditions identified above (i.e. chronic bronchitis, emphysema and bronchiectasis), whether alone or in combination with each other.

For the avoidance of doubt, in the context of the present invention, the term "treatment" includes references to therapeutic or palliative treatment of patients in need of such treatment, as well as to the prophylactic treatment and/or diagnosis of patients which are susceptible to the relevant disease states.

The terms "patient" and "patients" include references to mammalian (e.g. human) patients.

The terms "moderate persistent asthma" and "severe persistent asthma" are terms used to classify the severity of asthma (along with intermittent and mild persistent asthma). The classification of these terms can be found in Yawn, BP (September 2008). "Factors accounting for asthma variability: achieving optimal symptom control for individual patients". Primary Care Respiratory Journal 17(3): 138-147.

The term "effective amount" refers to an amount of a compound, which confers a therapeutic effect on the treated patient (e.g. sufficient to treat or prevent the disease). The effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).

When used herein, the term "Nominal Dose" (ND) is the amount of drug metered in the receptacle (also known as the Metered Dose). This is different to the amount of drug that is delivered to the patient which is referred to a Delivered Dose.

The fine particle fraction (FPF) is normally defined as the "fine particle dose" (FPD; the dose that is <5 μιτι) divided by the Emitted Dose (ED) which is the dose that leaves the device. The FPF is expressed as a percentage. Herein, the FPF of ED is referred to as FPF (ED) and is calculated as FPF (ED) = (FPD/ED) x 100%.

FPD may be measured by a Multistage Liquid Impinger, United States Pharmacopoeia 26, Chapter 601 , Apparatus 4 (2003), an Andersen Cascade Impactor or a New Generation Impactor.

When used herein, the term "fine particle fraction" (FPF) may also be defined as the FPD divided by the Metered Dose (MD) which is the dose in the blister or capsule, and expressed as a percentage. Herein, the FPF of MD is referred to as FPF (MD), and is calculated as FPF (MD) = (FPD/MD) x 100%. The term "ultrafine particle dose" (UFPD) is used herein to mean the total mass of active material delivered by a device which has a diameter of not more than 3 μιτι. The term "ultrafine particle fraction" is used herein to mean the percentage of the total amount of active material delivered by a device which has a diameter of not more than 3 μιτι. The term percent ultrafine particle dose (%UFPD) is used herein to mean the percentage of the total metered dose which is delivered with a diameter of not more than 3 μηη (i.e., %UFPD = 100 x UFPD/total metered dose).

The terms "delivered dose" and "emitted dose" or "ED" are used interchangeably herein. These are measured as set out in the current European Pharmacopeia (EP) monograph for inhalation products.

"Actuation of an inhaler" refers to the process during which a dose of the powder is removed from its rest position in the inhaler. That step takes place after the powder has been loaded into the inhaler ready for use.

Particle sizes are geometric unless otherwise stated.

(a) the glycosaminoglycan (e.g. a compound selected from chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate, hyaluronan and, particularly, heparin) may be in the same composition as another inhaled therapeutic or, particularly, the heparin is in a separate composition to a composition comprising and inhaled therapeutic; (b) the glycosaminoglycan (e.g. a compound selected from chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate, hyaluronan and, particularly, heparin) is delivered by pulmonary inhalation;

(c) the glycosaminoglycan (e.g. a compound selected from chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate, hyaluronan and, particularly, heparin) is in the form of a composition such as a dry powder composition;

(d) a composition comprising glycosaminoglycan (e.g. a compound selected from chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate, hyaluronan and, particularly, heparin) comprises at least 5% (e.g. at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%) of heparin by weight, for example at least about 75%, 85%, 95%, 96%, 97%, 98% or 99% (by weight) glycosaminoglycans (e.g. a compound selected from chondroitin sulphate, dermatan sulphate, keratin sulphate, heparan sulphate, hyaluronan and, particularly, heparin).

(e) a composition comprising glycosaminoglycan (e.g. a compound selected from chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate, hyaluronan and, particularly, heparin) further comprises an additive material (e.g. magnesium stearate or leucine, preferably L-leucine);

(f) the glycosaminoglycan (e.g. a compound selected from chondroitin sulphate, dermatan sulphate, keratan sulphate, heparan sulphate, hyaluronan and, particularly, heparin) composition further comprises carrier particles made from one or more excipient materials (e.g. inorganic salts, organic salts, other organic compounds sugar or, more particularly, alcohols, polyols and crystalline sugars (such as mannitol, trehalose, melezitose, dextrose or, particularly, lactose); (g) carrier particles, when present, may have an average particle size between 5 to 1000 pm (e.g. 4 to 500 μητι, such as 20 to 200 μητι, 30 to 150 μπτι, 40 to 70 μπτι, or 60 pm);

(h) the glycosaminoglycan is heparin;

(i) the maximum daily dose of heparin is less than 6000 mg (e.g. less than 2000 mg, such as less than 1500 mg, 600 mg, 300 mg, 240 mg, 180 mg or, particularly, 120 mg);

(j) a dose of heparin is provided as a fine particle dose of heparin of between 0.5 mg and 2000 mg (e.g. between 1 mg and 1000 mg, between 2 mg and 500 mg, between 6 mg and 250 mg, between 18 mg and 240 mg, between 30 mg and 130 mg, between 60 mg and 120 mg), for example when measured by a New Generation Impactor (Ph Eur Apparatus at 60 L/min);

(k) the antiinflammarory action of heparin is dose dependent (e.g. the action of heparin on the measured level of neutrophils and macrophages after BAL following treatment with heparin is dose dependent);

(I) the heparin provides a therapeutic effect within 120 minutes of administration (e.g. between 30 and 60 minutes);

(m) the heparin is administered sequentially, simultaneously or concomitantly with another active agent.

(a) the maximum daily delivered dose of heparin is less than or equal to 2000 mg (e.g. less than or equal to 500 mg, less than or equal to 300 mg, 200 mg, 150 mg, 100 mg, 80 mg, 40 mg, 20 mg, 10 mg, 5 mg or 1 mg); (b) a dose of heparin is provided as a fine particle dose of heparin of between 0.3 and 400 mg, between 0.5 and 200 mg, such as between 1 and 150 mg or, particularly, between 1 .5 mg and 100 mg (e.g. between 5 mg and 80 mg, between 10 mg and 60 mg, between 15 mg and 40 mg, between 20 mg and 30 mg), for example when measured by a New Generation Impactor (Ph Eur Apparatus at 60 L/min);

(c) a heparin dry powder formulation containing between 1 and 20% by weight (e.g. between 2 and 15%, between 5 and 10%, between 7 and 9% by weight) of an additive (e.g. leucine between 3 and 15%, between 5 and 10%, between 7 and 9%, such as L-leucine)).

In one embodiment, a nominal dose of heparin administered to a patient is up to 2100 mg, 1 100 mg, 530 mg, 300 mg or, particularly, up to 200 mg, in particular, the nominal dose is at least 100mg or, particularly, 0.8 mg, 1 .5 mg, 3 mg, 8 mg, 25 mg, 35 mg, 70 mg, and 140 mg).

In a further embodiment, a nominal dose of heparin administered to a patient is up to 2100 mg, 1 100 mg, 530 mg, 300 mg or, particularly, up to 200 mg, in particular, the nominal dose is at least 0.8 mg, 1 .5 mg, 3 mg, 8 mg, 25 mg, 35 mg, 70 mg, 100 mg, 140 mg and 200 mg).

Mechanism of action

Without wishing to be bound by theory, it is thought that glycosaminoglycans (e.g heparin, such as unfractionated heparin, such as unfractionated heparin sodium) possesses anti-inflammatory activity. This activity is thought to arise by heparin, which is a large, negatively charged molecule, binding to numerous physiological substances that are involved in the inflammatory process, such as L- and P-selectins (which reduces the attachment of leukocytes to the wall of the blood vessel at inflammatory sites), cytokines, growth factors, cytotoxic peptides and tissue destructive enzymes. This binding is believed to result from electrostatic forces between the polyanionic forces of the heparin glycosaminoglycan (GAG) chain and the positively charged amino acids of the inflammatory protein in question.

It is also disclosed that heparin can reduce the total amount of activated neutrophil cells, which may help to reduce inflammation. Neutrophils are a potent source of oxygen-derived free radicals involving the enzymatic complex nicotinamide adenine dinucleotide phosphate (NADPH) oxidase.

In addition, heparin has mucolytic, anti-bronchocontriction and anti-infective properties which it is thought may help to reduce overall inflammation by reducing symptoms or a cause of inflammation. For example, the efficient removal of mucus may prevent bacterial build-up in the lungs, which in turn may lead to a reduction in inflammation by reducing the immunological response.

Heparin Compositions for Pulmonary Inhalation

In the past, many of the commercially available dry powder inhalers exhibited very poor dosing efficiency, with sometimes as little as 10% of the active agent present in the dose actually being properly delivered to the user so that it can have a therapeutic effect. This low efficiency is simply not acceptable where a high dose of active agent is required for the desired therapeutic effect. The reason for the lack of dosing efficiency is that a proportion of the active agent in the dose of dry powder tends to be effectively lost at every stage the powder goes through from expulsion from the delivery device to deposition in the lung. For example, substantial amounts of material may remain in the blister/capsule or device. Material may be lost in the throat of the subject due to excessive plume velocity. However, it is frequently the case that a high percentage of the dose delivered exists in particulate forms of aerodynamic diameter in excess of that required.

It is well known that particle impaction in the upper airways of a subject is predicted by the so-called impaction parameter. The impaction parameter is defined as the velocity of the particle multiplied by the square of its aerodynamic diameter. Consequently, the probability associated with delivery of a particle through the upper airways region to the target site of action, is related to the square of its aerodynamic diameter. Therefore, delivery to the lower airways, or the deep lung is dependant on the square of its aerodynamic diameter, and smaller aerosol particles are very much more likely to reach the target site of administration in the user and therefore able to have the desired therapeutic effect.

Particles having aerodynamic diameters of less than 10 μιτι tend to be deposited in the lung. Particles with an aerodynamic diameter in the range of 2 μιτι to 5 μιτι will generally be deposited in the respiratory bronchioles whereas smaller particles having aerodynamic diameters in the range of 0.05 to 3 μιτι are likely to be deposited in the alveoli. So, for example, high dose efficiency for particles targeted at the alveoli is predicted by the dose of particles below 3 μητι, with the smaller particles being most likely to reach that target site.

In one embodiment of the present invention, the composition comprises active particles comprising heparin, at least 50%, at least 70% or at least 90% of the active particles having a Mass Median Aerodynamic Diameter (MMAD) of no more than about 15 μιτι. In another embodiment, at least 50%, at least 70% or at least 90% of the active particles have an MMAD of from about 10 μιτι to about 5 μιτι. In yet another embodiment, at least 50%, at least 70% or at least 90% of the active particles have aerodynamic diameters in the range of about 0.05 μιτι to about 3 μιτι. In one embodiment of the invention, at least about 90% of the heparin containing particles have a particle size (MMAD) of 5 μιτι or less.

Particles having a diameter of less than about 10 μιτι are, however, thermodynamically unstable due to their high surface area to volume ratio, which provides significant excess surface free energy and encourages particles to agglomerate. In a dry powder inhaler, agglomeration of small particles and adherence of particles to the walls of the inhaler are problems that result in the active particles leaving the inhaler as large agglomerates or being unable to leave the inhaler and remaining adhered to the interior of the device, or even clogging or blocking the inhaler.

The uncertainty as to the extent of formation of stable agglomerates of the particles between each actuation of the inhaler, and also between different inhalers and different batches of particles, leads to poor dose reproducibility. Furthermore, the formation of agglomerates means that the MMAD of the active particles can be vastly increased, with agglomerates of the active particles not reaching the required part of the lung. Consequently, it is essential for the present invention to provide a powder formulation which provides good dosing efficiency and reproducibility, delivering an accurate and predictable dose.

Much work has been done to improve the dosing efficiency of dry powder systems comprising active particles having a size of less than 15 μιτι MMAD, reducing the loss of the pharmaceutically active agent at each stage of the delivery. In the past, efforts to increase dosing efficiency and to obtain greater dosing reproducibility have tended to focus on preventing the formation of agglomerates of fine particles of active agent. Such agglomerates increase the effective size of these particles and therefore prevent them from reaching the lower respiratory tract or deep lung, where the active particles should be deposited in order to have their desired therapeutic effect. Proposed measures have included the use of relatively large carrier particles. The fine particles of active agent tend to become attached to the surfaces of the carrier particles as a result of interparticle forces such as Van der Waals forces. Upon actuation of the inhaler device, the active particles are supposed to detach from the carrier particles and are then present in the aerosol cloud in inhalable form. In addition or as an alternative, the inclusion of additive materials that act as force control agents that modify the cohesion and adhesion between particles has been proposed.

However, where the dose of drug to be delivered is very high, the options for adding materials to the powder composition are limited, especially where at least 90% of the composition is made up of the heparin as is particularly disclosed in the present invention. Nevertheless, it is imperative that the dry powder composition exhibit good flow and dispersion properties, to ensure good dosing efficiency.

Heparin may be obtained from a variety of sources, including Bioiberica (such as unfractionated Heparin Sodium (Bioiberica, EM/09/140, F0001 )).

Additional Therapeutic Agents

In accordance with the invention, heparin may be administered alone (i.e. as a monotherapy). In alternative embodiments of the invention, however, heparin may be administered in combination with another therapeutic agent (e.g. another therapeutic agent for the treatment of COPD), as listed hereinbefore.

References herein (in any aspect or embodiment of the invention) to therapeutic agents (which may also be referred to as active agents or active ingredients), such as heparin or ADS1 15398, includes references to such therapeutic agents per se, to tautomers of such compounds, as well as to pharmaceutically acceptable salts or solvates, or pharmaceutically functional derivatives of such therapeutic agents.

Pharmaceutically acceptable salts that may be mentioned include acid addition salts and base addition salts. Such salts may be formed by conventional means, for example by reaction of a free acid or a free base form of an active ingredient (e.g. heparin, ADS1 15398 etc) with one or more equivalents of an appropriate acid or base, optionally in a solvent, or in a medium in which the salt is insoluble, followed by removal of said solvent, or said medium, using standard techniques (e.g. in vacuo, by freeze-drying or by filtration). Salts may also be prepared by exchanging a counterion of an active ingredient (e.g. heparin, ADS1 15398 etc) in the form of a salt with another counter-ion, for example using a suitable ion exchange resin.

Examples of pharmaceutically acceptable salts include acid addition salts derived from mineral acids and organic acids, and salts derived from metals such as sodium, magnesium, or particularly, potassium and calcium.

Examples of acid addition salts include acid addition salts formed with acetic, 2,2- dichloroacetic, adipic, alginic, aryl sulphonic acids (e.g. benzenesulphonic, naphthalene-2-sulphonic, naphthalene-1 ,5-disulphonic and p-toluenesulphonic), ascorbic (e.g. L-ascorbic), L-aspartic, benzoic, 4-acetamidobenzoic, butanoic, (+) camphoric, camphor-sulphonic, (+)-(1 S)-camphor-10-sulphonic, capric, caproic, caprylic, cinnamic, citric, cyclamic, dodecylsulphuric, ethane-1 ,2-disulphonic, ethanesulphonic, 2-hydroxyethanesulphonic, formic, fumaric, galactaric, gentisic, glucoheptonic, gluconic (e.g. D-gluconic), glucuronic (e.g. D-glucuronic), glutamic (e.g. L-glutamic), a-oxoglutaric, glycolic, hippuric, hydrobromic, hydrochloric, hydriodic, isethionic, lactic (e.g. (+)-L-lactic and (±)-DL-lactic), lactobionic, maleic, malic (e.g. (-)- L-malic), malonic, (±)-DL-mandelic, metaphosphoric, methanesulphonic, 1 -hydroxys- naphthoic, nicotinic, nitric, oleic, orotic, oxalic, palmitic, pamoic, phosphoric, propionic, L-pyroglutamic, salicylic, 4-amino-salicylic, sebacic, stearic, succinic, sulphuric, tannic, tartaric (e.g.(+)-L-tartaric), thiocyanic, undecylenic and valeric acids.

Particular examples of salts are salts derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulphuric acids; from organic acids, such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic, arylsulphonic acids; and from metals such as sodium, magnesium, or particularly, potassium and calcium. As mentioned above, the active agents discussed herein also includes any solvates of the active ingredients and their salts. Particular solvates that may be mentioned herein are solvates formed by the incorporation into the solid state structure (e.g. crystal structure) of the active agents described herein of molecules of a non-toxic pharmaceutically acceptable solvent (referred to below as the solvating solvent). Examples of such solvents include water, alcohols (such as ethanol, isopropanol and butanol) and dimethylsulphoxide. Solvates can be prepared by recrystallising the active ingredient with a solvent or mixture of solvents containing the solvating solvent. Whether or not a solvate has been formed in any given instance can be determined by subjecting crystals of the active ingredient to analysis using well known and standard techniques such as thermogravimetric analysis (TGE), differential scanning calorimetry (DSC) and X-ray crystallography.

The solvates can be stoichiometric or non-stoichiometric solvates. Particular solvates that may be mentioned herein are hydrates, and examples of hydrates include hemihydrates, monohydrates and dihydrates.

For a more detailed discussion of solvates and the methods used to make and characterise them, see Bryn et al., Solid-State Chemistry of Drugs, Second Edition, published by SSCI, Inc of West Lafayette, IN, USA, 1999, ISBN 0-967-06710-3. "Pharmaceutically functional derivatives" of the active ingredients as defined herein includes ester derivatives and/or derivatives that have, or provide for, the same biological function and/or activity as any relevant compound of the invention. Thus, for the purposes of this invention, the term also includes prodrugs of the active ingredients described herein. The term "prodrug" of a relevant active ingredient includes any compound that, following oral or parenteral administration, is metabolised in vivo to form that compound in an experimentally-detectable amount, and within a predetermined time (e.g. within a dosing interval of between 6 and 24 hours (i.e. once to four times daily)). Prodrugs of the active ingredients described herein may be prepared by modifying functional groups present on the compound in such a way that the modifications are cleaved, in vivo when such prodrug is administered to a mammalian subject. The modifications typically are achieved by synthesizing the parent compound with a prodrug substituent. Prodrugs include active ingredients wherein a hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group in a compound of formula I is bonded to any group that may be cleaved in vivo to regenerate the free hydroxyl, amino, sulfhydryl, carboxyl or carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters and carbamates of hydroxyl functional groups, esters groups of carboxyl functional groups, N-acyl derivatives and N-Mannich bases. General information on prodrugs may be found e.g. in Bundegaard, H. "Design of Prodrugs" p. 1-92, Elsevier, New York-Oxford (1985). When used herein, the term "another therapeutic agent" includes references to one or more (e.g. one) therapeutic agents that are known to be useful for (e.g. that are known to be effective in) the treatment of COPD.

When used herein, the term "administered sequentially, simultaneously or concomitantly" includes references to:

(i) administration of separate pharmaceutical formulations (one containing the heparin and one or more others containing the one or more other therapeutic agents); and (ii) administration of a single pharmaceutical formulation containing the heparin and the other therapeutic agent(s).

In a particular embodiment, when heparin is to be administered by pulmonary inhalation, the heparin and other active agent(s) as defined above are delivered from different receptacles.

The other active agents described herein (i.e. those not heparin) may be administered by any suitable route, but may particularly be administered orally, intravenously, intramuscularly, cutaneously, subcutaneously, transmucosally (e.g. sublingually or buccally), rectally, transdermally, nasally, pulmonarily (e.g. tracheally or bronchially), topically, by any other parenteral route, in the form of a pharmaceutical preparation comprising the compound in a pharmaceutically acceptable dosage form. Particular modes of administration that may be mentioned include oral, transdermal, intravenous, cutaneous, subcutaneous, nasal, intramuscular or intraperitoneal administration. Yet more particular modes of administration that may be mentioned include oral and transdermal administration.

The other active agents described herein will generally be administered as a pharmaceutical formulation in admixture with a pharmaceutically acceptable adjuvant, diluent or carrier, which may be selected with due regard to the intended route of administration and standard pharmaceutical practice. Such pharmaceutically acceptable carriers may be chemically inert to the active compounds and may have no detrimental side effects or toxicity under the conditions of use. Suitable pharmaceutical formulations may be found in, for example, Remington The Science and Practice of Pharmacy, 19th ed., Mack Printing Company, Easton, Pennsylvania (1995). For parenteral administration, a parenterally acceptable aqueous solution may be employed, which is pyrogen free and has requisite pH, isotonicity, and stability. Suitable solutions will be well known to the skilled person, with numerous methods being described in the literature. A brief review of methods of drug delivery may also be found in e.g. Langer, Science (1990) 249, 1527.

Otherwise, the preparation of suitable formulations may be achieved routinely by the skilled person using routine techniques and/or in accordance with standard and/or accepted pharmaceutical practice.

The amount of the other active agents described herein in any pharmaceutical formulation used in accordance with the present invention will depend on various factors, such as the severity of the condition to be treated, the particular patient to be treated, as well as the compound(s) which is/are employed. In any event, the amount the other active agents described herein in the formulation may be determined routinely by the skilled person.

For example, a solid oral composition such as a tablet or capsule may contain from 1 to 99 % (w/w) active ingredient; from 0 to 99% (w/w) diluent or filler; from 0 to 20% (w/w) of a disintegrant; from 0 to 5% (w/w) of a lubricant; from 0 to 5% (w/w) of a flow aid; from 0 to 50% (w/w) of a granulating agent or binder; from 0 to 5% (w/w) of an antioxidant; and from 0 to 5% (w/w) of a pigment. A controlled release tablet may in addition contain from 0 to 90 % (w/w) of a release-controlling polymer. A parenteral formulation (such as a solution or suspension for injection or a solution for infusion) may contain from 1 to 50 % (w/w) active ingredient; and from 50% (w/w) to 99% (w/w) of a liquid or semisolid carrier or vehicle (e.g. a solvent such as water); and 0-20% (w/w) of one or more other excipients such as buffering agents, antioxidants, suspension stabilisers, tonicity adjusting agents and preservatives.

Depending on the disorder, and the patient, to be treated, as well as the route of administration, the other active agents described herein may be administered at varying therapeutically effective doses to a patient in need thereof.

However, the dose administered to a mammal, particularly a human, in the context of the present invention should be sufficient to effect a therapeutic response in the mammal over a reasonable timeframe. One skilled in the art will recognize that the selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by inter alia the pharmacological properties of the formulation, the nature and severity of the condition being treated, and the physical condition and mental acuity of the recipient, as well as the potency of the specific compound, the age, condition, body weight, sex and response of the patient to be treated, and the stage/severity of the disease.

Administration may be continuous or intermittent (e.g. by bolus injection). The dosage may also be determined by the timing and frequency of administration. In the case of oral or parenteral administration the dosage can vary from about 0.01 mg to about 2000 mg per day (e.g. 0.5 mg, 3 mg, 6 mg or 10 mg per day of ADS1 15398) of the other active agents described herein. In any event, the medical practitioner, or other skilled person, will be able to determine routinely the actual dosage, which will be most suitable for an individual patient. The above-mentioned dosages are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this invention.

The aspects of the invention described herein (e.g. the above-mentioned compounds, combinations, methods and uses) may have the advantage that, in the treatment of the conditions described herein, they may be more convenient for the physician and/or patient than, be more efficacious than, be less toxic than, have better selectivity over, have a broader range of activity than, be more potent than, produce fewer side effects than, or may have other useful pharmacological properties over, similar compounds, combinations, methods (treatments) or uses known in the prior art for use in the treatment of those conditions or otherwise.

Additives

The tendency of fine particles to agglomerate means that the FPF of a given dose can be highly unpredictable and a variable proportion of the fine particles will be administered to the lung, or to the correct part of the lung, as a result. This is observed, for example, in formulations comprising pure drug in fine particle form. Such formulations exhibit poor flow properties and poor FPF. In an attempt to improve this situation and to provide a consistent FPF and FPD, dry powder compositions according to the present invention may include additive material which is an anti-adherent material and reduces cohesion between the particles in the composition.

The additive material is selected to reduce the cohesion between particles in the dry powder composition. It is thought that the additive material interferes with the weak bonding forces between the small particles, helping to keep the particles separated and reducing the adhesion of such particles to one another, to other particles in the formulation if present and to the internal surfaces of the inhaler device. Where agglomerates of particles are formed, the addition of particles of additive material decreases the stability of those agglomerates so that they are more likely to break up in the turbulent air stream created on actuation of the inhaler device, whereupon the particles are expelled from the device and inhaled. As the agglomerates break up, the active particles may return to the form of small individual particles or agglomerates of small numbers of particles which are capable of reaching the lower lung.

The additive material may be in the form of particles which tend to adhere to the surfaces of the active particles, as disclosed in WO 1997/03649. Alternatively, the additive material may be coated on the surface of the active particles by, for example a co-milling method as disclosed in WO 2002/43701 .

Particularly, the additive material is an anti-adherent material and it will tend to reduce the cohesion between particles and will also prevent fine particles becoming attached to surfaces within the inhaler device. Advantageously, the additive material is an anti- friction agent or glidant and will give the powder formulation better flow properties in the inhaler. The additive materials used in this way may not necessarily be usually referred to as anti-adherents or anti-friction agents, but they will have the effect of decreasing the cohesion between the particles or improving the flow of the powder. The additive materials are sometimes referred to as force control agents (FCAs) and they usually lead to better dose reproducibility and higher FPFs.

Therefore, an additive material or FCA, as used herein, is a material whose presence on the surface of a particle can modify the adhesive and cohesive surface forces experienced by that particle, in the presence of other particles and in relation to the surfaces that the particles are exposed to. In general, its function is to reduce both the adhesive and cohesive forces.

The reduced tendency of the particles to bond strongly, either to each other or to the device itself, not only reduces powder cohesion and adhesion, but can also promote better flow characteristics. This leads to improvements in the dose reproducibility because it reduces the variation in the amount of powder metered out for each dose and improves the release of the powder from the device. It also increases the likelihood that the active material, which does leave the device, will reach the lower lung of the patient.

It is favourable for unstable agglomerates of particles to be present in the powder when it is in the inhaler device. As indicated above, for a powder to leave an inhaler device efficiently and reproducibly, the agglomerated particles of such a powder should be large, particularly larger than about 30 μιτι, preferably larger than 50 μιτι and more preferably larger than 70 μιτι as measured optically using for example a scanning electron microscope with a suitable reference sample of known size. Such a powder may be in the form of either individual particles having a size of about 40 μιτι or larger and/or agglomerates of finer particles, the agglomerates having a size of about 40 μιτι or larger. The agglomerates formed can have a size of 100 μιτι or 200 μιτι and, depending on the type of device used to dispense the formulation, the agglomerates may be as much as about 1000 μιτι. With the addition of the additive material, those agglomerates are more likely to be broken down efficiently in the turbulent airstream created on inhalation. Therefore, the formation of unstable or "soft" agglomerates of particles in the powder may be favoured compared with a powder in which there is substantially no agglomeration. Such unstable agglomerates are stable whilst the powder is inside the device but are then disrupted and broken up upon inhalation.

It is particularly advantageous for the additive material to comprise an amino acid. Amino acids have been found to give, when present as additive material, high respirable fraction of the active material and also good flow properties of the powder. A particular amino acid that may be mentioned is leucine, in particular L-leucine, dileucine and tri-leucine. Although the L-form of the amino acids is generally used, the D- and DL-forms may also be used. The additive material may comprise one or more of any of the following amino acids: aspartame, leucine, isoleucine, lysine, valine, methionine, cysteine, and phenylalanine. Additive materials may also include, for example, metal stearates such as magnesium stearate, phospholipids, lecithin, colloidal silicon dioxide and sodium stearyl fumarate, and are described more fully in WO 1996/23485, which is hereby incorporated by reference. Advantageously, the powder includes at least 80%, at least 90%, or particularly 95% (e.g. at least 96%, 97% 98% or 99%) by weight of glycosaminoglycan (e.g. heparin), optionally comprising other active ingredients, such as those discussed herein based on the weight of the powder. The optimum amount of additive material will depend upon the precise nature of the additive and the manner in which it is incorporated into the composition. In some embodiments, the powder advantageously includes not more than 8%, more advantageously not more than 6%, preferably 5% by weight of additive material based on the weight of the powder. As indicated above, in some cases it will be advantageous for the powder to contain about 1 % by weight of additive material. In other embodiments, the additive material or FCA may be provided in an amount from about 0.1 % to about 10% by weight, and particularly from about 0.15% to 7%, most particularly from about 0.5% to about 6%.

When the additive material is micronised leucine or lecithin, it is particularly provided in an amount from about 0.1 % to about 10% by weight (e.g. 0.5%, 1 %, 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9%). Particularly, the additive material comprises from about 3% to about 7%, particularly about 5%, of micronised leucine. Particularly, at least 95% (e.g. at least 96%, 97%, 98% or 99%) by weight of the micronised leucine has a particle diameter of less than 150 μιτι, particularly less than 100 μιτι, and most particularly less than 50 μιτι. Particularly, the mass median diameter of the micronised leucine is less than 10 μιτι.

If magnesium stearate or sodium stearyl fumarate is used as the additive material, it is particularly provided in an amount from about 0.05% to about 10%, from about 0.15% to about 5%, from about 0.25% to about 3%, or from about 0.5% to about 2.0% depending on the required final dose.

In a further attempt to improve extraction of the dry powder from the dispensing device and to provide a consistent FPF and FPD, dry powder compositions according to the present invention may include particles of an inert excipient material, which act as carrier particles. These carrier particles are mixed with fine particles of active material and any additive material which is present. Rather than sticking to one another, the fine active particles tend to adhere to the surfaces of the carrier particles whilst in the inhaler device, but are supposed to release and become dispersed upon actuation of the dispensing device and inhalation into the respiratory tract, to give a fine suspension.

The inclusion of carrier particles is less attractive where very large doses of active agent are to be delivered, as they tend to significantly increase the volume of the powder composition. Nevertheless, in some embodiments of the present invention, the compositions include carrier particles. In such an embodiment, the composition comprises at least about 10% (by weight) of the active ingredient(s) (e.g. heparin alone, or optionally in combination with one or more active ingredients), or at least about 15%, 17%, or 18% or 18.5% (by weight) of the active ingredient(s) (e.g. heparin alone, or optionally in combination with one or more active ingredients). More particularly, the carrier particles are present in small amount, such as no more than 90% (e.g. 85%, 83% or, more particularly 80%) by weight of the total composition, in which the total active ingredient (e.g. heparin) and magnesium stearate content would be about 18.5 and 1 .5% by weight, respectively. Carrier particles may be of any acceptable inert excipient material or combination of materials. For example, the carrier particles may be composed of one or more materials selected from sugar alcohols, polyols and crystalline sugars. Other suitable carriers include inorganic salts such as sodium chloride and calcium carbonate, organic salts such as sodium lactate and other organic compounds such as polysaccharides and oligosaccharides. Advantageously, the carrier particles comprise a polyol. In particular, the carrier particles may be particles of crystalline sugar, for example mannitol, trehalose, melezitose, dextrose or lactose. Most particularly, the carrier particles are composed of lactose.

Thus, in one embodiment of the present invention, the composition comprises active particles comprising heparin and carrier particles. The carrier particles may have an average particle size of from about 5 to about 1000 μιτι, from about 4 to about 40 μιτι, from about 60 to about 200 μιτι, or from 150 to about 1000 μιτι. Other useful average particle sizes for carrier particles are about 20 to about 30 μιτι or from about 40 to about 70 μιτι.

In an alternate embodiment, the carrier particles are present in small amount, such as no more than 50% (e.g. 60%, 70% or, more particularly, 80%) by weight of the total composition, in which the total active ingredient (e.g. heparin) and magnesium stearate content, by weight, would be 18 and 2% respectively. As the amount of carrier in these formulations changes, the amounts of additive and heparin will also change, but the ratio of these constituents particularly remains approximately 1 :9 to about 1 :13. In an alternate embodiment, the formulation does not contain carrier particles and comprises an active ingredient (e.g. heparin) and additive, such as at least 30% (e.g. 60%, 80%, 90%, 95% or, more particularly, 97%) by weight of the total composition comprises of pharmaceutically active agent. The active agent may be a glycosaminoglycan (e.g heparin) alone or it may be a combination of the glycosaminoglycan (e.g. heparin) and another drug which would benefit patients (e.g. COPD patients). The remaining components may comprise one or more additive materials, such as those discussed above.

In a further embodiment the formulation may contain carrier particles and comprises of the active ingredient(s) (e.g. heparin alone, or optionally in combination with one or more active ingredients) and additive, such as at least 30% (e.g. 60%, 80%, 90%, 95% or, more particularly, 97%) by weight of the total composition comprises the pharmaceutically active agent and wherein the remaining components comprise additive material and larger particles. The larger particles provide the dual action of acting as a carrier and facilitating powder flow.

In a particular embodiment, the composition comprises heparin (30% w/w) and lactose having an average particles size of 45-65 μιτι. In a particular embodiment, the composition comprises heparin (90% w/w, e.g. 95%, such as 96%, 97%, 98% or 99%) and lactose having an average particles size of 45-65 μιτι.

The compositions comprising active ingredient(s) (e.g. heparin alone, or optionally in combination with one or more active ingredients) and carrier particles may further include one or more additive materials. The additive material may be in the form of particles which tend to adhere to the surfaces of the active particles, as disclosed in WO 1997/03649. Alternatively, the additive material may be coated on the surface of the active particles by, for example a co-milling method as disclosed in WO2002/43701 or on the surfaces of the carrier particles, as disclosed in WO2002/00197.

In one embodiment, the additive is coated onto the surface of the carrier particles. This coating may be in the form of particles of additive material adhering to the surfaces of the carrier particles (by virtue of interparticle forces such as Van der Waals forces), as a result of a blending of the carrier and additive. Alternatively, the additive material may be smeared over and fused to the surfaces of the carrier particles, thereby forming composite particles with a core of inert carrier material and additive material on the surface. For example, such fusion of the additive material to the carrier particles may be achieved by co-jet milling particles of additive material and carrier particles. In some embodiments, all three components of the powder (active, carrier and additive) are processed together so that the additive becomes attached to or fused to both the carrier particles and the active particles. In one illustrative embodiment, the compositions include an additive material, such as magnesium stearate (up to 10% w/w) or leucine, said additive being jet-milled with the particles of heparin and/or with the lactose.

In a particular embodiment described herein, the formulation comprises one or more of:

(a) an additive material (e.g. magnesium stearate); and (b) a carrier (e.g. lactose fines).

In certain embodiments of the present invention, the heparin formulation is a "carrier free" formulation, which includes only the active ingredient (e.g. heparin) or its pharmaceutically acceptable salts or esters and one or more additive materials.

In a preferred embodiment described herein, the formulation comprises an additive material, preferably wherein the additive material is an amino acid, more preferably wherein the amino acid is L-leucine.

Advantageously, in these "carrier free" formulations, at least 90% by weight of the particles of the powder have a particle size less than 63 μιτι, particularly less than 30 μιτι and more particularly less than 10 μιτι. As indicated above, the size of the heparin (or its pharmaceutically acceptable salts) particles of the powder should be within the range of about from 0.1 μιτι to 5 μιτι for effective delivery to the lower lung. Where the additive material is in particulate form, it may be advantageous for these additive particles to have a size outside the preferred range for delivery to the lower lung.

The powder includes at least 60% by weight of the glycosaminoglycan (e.g. heparin) or a pharmaceutically acceptable salt or ester thereof based on the weight of the powder. Advantageously, the powder comprises at least 70%, or at least 80% by weight of glycosaminoglycan (e.g. heparin) or a pharmaceutically acceptable salt or ester thereof based on the weight of the powder. Most advantageously, the powder comprises at least 90%, at least 95% (e.g. at least 96%), or at least 97% (e.g. at least 98% or 99%) by weight of heparin or a pharmaceutically acceptable salt or ester thereof based on the weight of the powder. It is believed that there are physiological benefits in introducing as little powder as possible to the lungs, in particular material other than the active ingredient to be administered to the patient. Therefore, the quantities in which the additive material is added are particularly as small as possible. Most particularly the powder, therefore, would comprise more than 99% by weight of heparin or a pharmaceutically acceptable salt or ester thereof.

In a particular embodiment, at least some of the heparin is in amorphous form as determined by X-Ray Powder Diffraction (XRPD) analysis. A formulation containing amorphous heparin will possess particular dissolution characteristics. A stable form of amorphous heparin may be prepared using suitable sugars such as trehalose and melezitose.

Preparing Dry Powder Inhaler Formulations

Where the compositions of the present invention include an additive material, the manner in which this is incorporated will have a significant impact on the effect that the additive material has on the powder performance, including the FPF and FPD.

In one embodiment, the compositions according to the present invention are prepared by simply blending particles of the active ingredient(s) (e.g. heparin alone, or optionally in combination with one or more active ingredients) of a selected appropriate size with particles of additive material and/or carrier particles. The powder components may be blended by a gentle mixing process, for example in a tumble mixer such as a Turbula (trade mark). In such a gentle mixing process, there is generally substantially no reduction in the size of the particles being mixed. In addition, the powder particles do not tend to become fused to one another, but they rather agglomerate as a result of cohesive forces such as Van der Waals forces. These loose or unstable agglomerates readily break up upon actuation of the inhaler device used to dispense the composition the compositions of the present invention

Compressive Milling Processes

In an alternative process for preparing the compositions according to the present invention, the powder components undergo a compressive milling process, such as processes termed mechanofusion (also known as 'Mechanical Chemical Bonding') and cyclomixing.

As the name suggests, mechanofusion is a dry coating process designed to mechanically fuse a first material onto a second material. It should be noted that the use of the terms "mechanofusion" and "mechanofused" are supposed to be interpreted as a reference to a particular type of milling process, but not a milling process performed in a particular apparatus. The compressive milling processes work according to a different principle to other milling techniques, relying on a particular interaction between an inner element and a vessel wall, and they are based on providing energy by a controlled and substantial compressive force. The process works particularly well where one of the materials is generally smaller and/or softer than the other.

The fine active particles and additive particles are fed into the vessel of a mechanofusion apparatus (such as a Mechano-Fusion system (Hosokawa Micron Ltd) or the Nobilta or Nanocular apparatus, where they are subject to a centrifugal force and are pressed against the vessel inner wall. The powder is compressed between the fixed clearance of the drum wall and a curved inner element with high relative speed between drum and element. The inner wall and the curved element together form a gap or nip in which the particles are pressed together. As a result, the particles experience very high shear forces and very strong compressive stresses as they are trapped between the inner drum wall and the inner element (which has a greater curvature than the inner drum wall). The particles are pressed against each other with enough energy to locally heat and soften, break, distort, flatten and wrap the additive particles around the core particle to form a coating. The energy is generally sufficient to break up agglomerates and some degree of size reduction of both components may occur.

These mechanofusion and cyclomixing processes apply a high enough degree of force to separate the individual particles of active material and to break up tightly bound agglomerates of the active particles such that effective mixing and effective application of the additive material to the surfaces of those particles is achieved. An especially desirable aspect of the processes is that the additive material becomes deformed in the milling and may be smeared over or fused to the surfaces of the active particles.

However, in practice, these compression milling processes produce little or no size reduction of the drug particles, especially where they are already in a micronised form (i.e. <10 μιτι). The only physical change which may be observed is a plastic deformation of the particles to a rounder shape. Other Milling Procedures

The process of nnilling may also be used to formulate the dry powder compositions according to the present invention. The manufacture of fine particles by milling can be achieved using conventional techniques. In the conventional use of the word, "milling" means the use of any mechanical process which applies sufficient force to the particles of active material that it is capable of breaking coarse particles (for example, particles with a MMAD greater than 100 μιτι) down to fine particles (for example, having a MMAD not more than 50 μιτι). In the present invention, the term "milling" also refers to deagglomeration of particles in a formulation, with or without particle size reduction. The particles being milled may be large or fine prior to the milling step. A wide range of milling devices and conditions are suitable for use in the production of the compositions of the inventions. The selection of appropriate milling conditions, for example, intensity of milling and duration, to provide the required degree of force will be within the ability of the skilled person.

Impact milling processes may be used to prepare compositions comprising heparin according to the present invention, with or without additive material. Such processes include ball milling and the use of a homogenizer.

Ball milling is a suitable milling method for use in the prior art co-milling processes. Centrifugal and planetary ball milling are especially particular methods.

Alternatively, a high pressure homogeniser may be used in which a fluid containing the particles is forced through a valve at high pressure producing conditions of high shear and turbulence. Shear forces on the particles, impacts between the particles and machine surfaces or other particles, and cavitation due to acceleration of the fluid may all contribute to the fracture of the particles. Suitable homogenisers include EmulsiFlex high pressure homogenisers which are capable of pressures up to 4000 bar, Niro Soavi high pressure homogenisers (capable of pressures up to 2000 bar), and Microfluidics Microfluidisers (maximum pressure 2750 bar). The milling process can be used to provide the microparticles with mass median aerodynamic diameters as specified above. Homogenisers may be more suitable than ball mills for use in large scale preparations of the composite active particles.

The milling step may, alternatively, involve a high energy media mill or an agitator bead mill, for example, the Netzsch high energy media mill, or the DYNO-mill (Willy A. Bachofen AG, Switzerland).

If a significant reduction in particle size is also required, co-jet milling is used particularly, as disclosed in the earlier patent application published as WO2005/025536. The co-jet milling process can result in composite active particles with low micron or sub-micron diameter, and these particles exhibit particularly good FPF and FPD, even when dispensed using a passive DPI.

The milling processes apply a high enough degree of force to break up tightly bound agglomerates of fine or ultra-fine particles, such that effective mixing and effective application of the additive material to the surfaces of those particles is achieved.

These impact processes create high-energy impacts between media and particles or between particles. In practice, while these processes are good at making very small particles, it has been found that neither the ball mill nor the homogenizer was particularly effective in producing dispersion improvements in resultant drug powders in the way observed for the compressive process. It is believed that the second impact processes are not as effective in producing a coating of additive material on each particle.

Conventional methods comprising co-milling active material with additive materials (as described in WO 2002/43701 ) result in composite active particles which are fine particles of active material with an amount of the additive material on their surfaces. The additive material is particularly in the form of a coating on the surfaces of the particles of active material. The coating may be a discontinuous coating. The additive material may be in the form of particles adhering to the surfaces of the particles of active material. Co-milling or co-micronising particles of active agent and particles of additive (FCA) or excipient will result in the additive or excipient becoming deformed and being smeared over or fused to the surfaces of fine active particles, producing composite particles made up of both materials. These resultant composite active particles comprising an additive have been found to be less cohesive after the milling treatment.

At least some of the composite active particles may be in the form of agglomerates. However, when the composite active particles are included in a pharmaceutical composition, the additive material promotes the dispersal of the composite active particles on administration of that composition to a patient, via actuation of an inhaler. Milling may also be carried out in the presence of a material which can delay or control the release of the active agent.

The co-milling or co-micronising of active and additive particles may involve compressive type processes, such as mechanofusion, cyclomixing and related methods such as those involving the use of a Hybridiser or the Nobilta. The principles behind these processes are distinct from those of alternative milling techniques in that they involve a particular interaction between an inner element and a vessel wall, and in that they are based on providing energy by a controlled and substantial compressive force, particularly compression within a gap of predetermined width.

In one embodiment, if required, the microparticles produced by the milling step can then be formulated with an additional excipient. This may be achieved by a spray drying process, e.g. co-spray drying with excipients. In this embodiment, the particles are suspended in a solvent and co-spray dried with a solution or suspension of the additional excipient. Particular additional excipients include trehalose, melezitose and other polysaccharides. Additional pharmaceutical effective excipients may also be used.

In another embodiment, the powder compositions are produced using a multi-step process. Firstly, the materials are milled or blended. Next, the particles may be sieved, prior to undergoing mechanofusion. A further optional step involves the addition of carrier particles. The mechanofusion step is thought to "polish" the composite active particles, further rubbing the additive material into the active particles. This allows one to enjoy the beneficial properties afforded to particles by mechanofusion, in combination with the very small particles sizes made possible by the jet milling.

The reduction in the cohesion and adhesion between the active particles can lead to equivalent performance with reduced agglomerate size, or even with individual particles.

High shear blending

Scaling Scaling up of pharmaceutical product manufacture often requires the use one piece of equipment to perform more than one function. An example of this is the use of a mixer-granulator which can both mix and granulate a product thereby removing the need to transfer the product between pieces of equipment. In so doing, the opportunity for powder segregation is minimised. High shear blending often uses a high-shear rotor/stator mixer (HSM), which has become used in mixing applications. Homogenizers or "high shear material processors" develop a high pressure on the material whereby the mixture is subsequently transported through a very fine orifice or comes into contact with acute angles. The flow through the chambers can be reverse flow or parallel flow depending on the material being processed. The number of chambers can be increased to achieve better performance. The orifice size or impact angle may also be changed for optimizing the particle size generated. Particle size reduction occurs due to the high shear generated by the high shear material processors while it passes through the orifice and the chambers. The ability to apply intense shear and shorten mixing cycles gives these mixers broad appeal for applications that require agglomerated powders to be evenly blended. Furthermore conventional HSMs may also be widely used for high intensity mixing, dispersion, disintegration, emulsification and homogenization.

It is well known to those skilled in the production of powder formulations that small particles, even with high-power, high-shear, mixers a relatively long period of "aging" is required to obtain complete dispersion, and this period is not shortened appreciably by increases in mixing power, or by increasing the speed of rotation of the stirrer so as to increase the shear velocity. High shear mixers can also be used if the auto- adhesive properties of the drug particles are so that high shear forces are required together with use of a force-controlling agent for forming a surface-energyreducing particulate coating or film.

Spray Drying and Ultrasonic Nebulisers

Spray Spray drying may be used to produce particles of inhalable size comprising the heparin. The spray drying process may be adapted to produce spray-dried particles that include the active agent and an additive material which controls the agglomeration of particles and powder performance. The spray drying process may also be adapted to produce spray-dried particles that include the active agent dispersed or suspended within a material that provides the controlled release properties. Furthermore the dispersal or suspension of the active material within an excipient material may impart further stability to the active compounds. In a particular embodiment the heparin may reside primarily in the amorphous state. A formulation containing amorphous heparin will possess particular dissolution characteristics. This would be possible in that particles are suspended in a sugar glass which could be either a solid solution or a solid dispersion. Particular additional excipients include trehalose, melezitose and other polysaccharides.

Spray drying is a well-known and widely used technique for producing particles of active material of inhalable size. Conventional spray drying techniques may be improved so as to produce active particles with enhanced chemical and physical properties so that they perform better when dispensed from a DPI than particles formed using conventional spray drying techniques. Such improvements are described in detail in the earlier patent application published as WO 2005/025535.

In particular, it is disclosed that co-spray drying an active agent with an FCA under specific conditions can result in particles with excellent properties which perform extremely well when administered by a DPI for inhalation into the lung.

It has been found that manipulating or adjusting the spray drying process can result in the FCA being largely present on the surface of the particles. That is, the FCA is concentrated, but not exclusively located at the surface of the particles as distinct crystals, rather than being homogeneously distributed throughout the particles. This clearly means that the FCA will be able to reduce the tendency of the particles to agglomerate. This will assist the formation of unstable agglomerates that are easily and consistently broken up upon actuation of a DPI.

Advantageously, the particles comprise at least 2%, at least 3%, or particularly 4% (by weight the particles) of additive on the surface of the particles as determined by X-Ray Powder Diffraction (XRPD) analysis or atomic force microscopy or combination thereof, optionally comprising active ingredients based on the weight of the powder. The optimum amount of additive material will depend upon the precise nature of the additive and the manner in which it is incorporated into the composition.

It has been found that it may be advantageous to control the formation of the droplets in the spray drying process, so that droplets of a given size and of a narrow size distribution are formed. Furthermore, controlling the formation of the droplets can allow control of the air flow around the droplets which, in turn, can be used to control the drying of the droplets and, in particular, the rate of drying. Controlling the formation of the droplets may be achieved by using alternatives to the conventional 2-fluid nozzles, especially avoiding the use of high velocity air flows.

In particular, it is preferred to use a spray drier comprising a means for producing droplets moving at a controlled velocity and of a predetermined droplet size. The velocity of the droplets is particularly controlled relative to the body of gas into which they are sprayed. This can be achieved by controlling the droplets' initial velocity and/or the velocity of the body of gas into which they are sprayed, for example by using an ultrasonic nebuliser (USN) to produce the droplets. Alternative nozzles such as electrospray nozzles or vibrating orifice nozzles may be used.

In one embodiment, a USN is used to form the droplets in the spray mist. USNs use an ultrasonic transducer which is submerged in a liquid. The ultrasonic transducer (a piezoelectric crystal) vibrates at ultrasonic frequencies to produce the short wavelengths required for liquid atomisation. In one common form of USN, the base of the crystal is held such that the vibrations are transmitted from its surface to the nebuliser liquid, either directly or via a coupling liquid, which is usually water. When the ultrasonic vibrations are sufficiently intense, a fountain of liquid is formed at the surface of the liquid in the nebuliser chamber. Droplets are emitted from the apex and a "fog" emitted.

Whilst USNs are known, these are conventionally used in inhaler devices, for the direct inhalation of solutions containing drug, and they have not previously been widely used in a spray drying apparatus. It has been discovered that the use of such a nebuliser in spray drying has a number of important advantages and these have not previously been recognised. The particular USNs control the velocity of the particles and therefore the rate at which the particles are dried, which in turn affects the shape and density of the resultant particles. The use of USNs also provides an opportunity to perform spray drying on a larger scale than is possible using conventional spray drying apparatus with conventional types of nozzles used to create the droplets, such as 2-fluid nozzles.

The attractive characteristics of USNs for producing fine particle dry powders include: low spray velocity; the small amount of carrier gas required to operate the nebulisers; the comparatively small droplet size and narrow droplet size distribution produced; the simple nature of the USNs (the absence of moving parts which can wear, contamination, etc.); the ability to accurately control the gas flow around the droplets, thereby controlling the rate of drying; and the high output rate which makes the production of dry powders using USNs commercially viable in a way that is difficult and expensive when using a conventional two-fluid nozzle arrangement. USNs do not separate the liquid into droplets by increasing the velocity of the liquid. Rather, the necessary energy is provided by the vibration caused by the ultrasonic nebuliser.

Further embodiments, may employ the use of ultrasonic nebuliser (USN), rotary atomisers or electrohydrodynamic (EHD) atomizers to generate the particles.

Delivery Devices

The inhalable compositions in accordance with the present invention are particularly administered via a dry powder inhaler (DPI), but can also be administered via a pressurized metered dose inhaler (pMDI), via a liquid instillate, or even via a nebulised system. In an embodiment of the current invention, pulmonary inhalation incorporates inhalation by way of an inhaler (e.g. DPI or pMDI) or via a nebulised system.

In a dry powder inhaler, the dose to be administered is stored in the form of a nonpressurized dry powder and, on actuation of the inhaler, the particles of the powder are expelled from the device in the form of a cloud of finely dispersed particles that may be inhaled by the patient.

Dry powder inhalers can be "passive" devices in which the patient's breath is the only source of gas which provides a motive force in the device. Examples of "passive" dry powder inhaler devices include the device produced by Vectura (as covered by international patent application number WO 2010/086285 or, particularly, the Rotahaler and Diskhaler (GlaxoSmithKline), the Monohaler (MIAT), the GyroHaler (Trade Mark) (Vectura) the Turbohaler (Astra-Draco) and Novolizer (Trade Mark) (Viatris GmbH). Alternatively, "active" devices may be used, in which a source of compressed gas or alternative energy source is used. Examples of suitable active devices include Aspirair (Trade Mark) (Vectura) and the active inhaler device produced by Nektar Therapeutics (as covered by US Patent No. 6,257,233).

It is generally considered that different compositions perform differently when dispensed using passive and active type inhalers. Passive devices create less turbulence within the device and the powder particles are moving more slowly when they leave the device. This leads to some of the metered dose remaining in the device and, depending on the nature of the composition, less deagglomeration upon actuation. However, when the slow moving cloud is inhaled, less deposition in the throat is often observed. In contrast, active devices create more turbulence when they are activated. This results in more of the metered dose being extracted from the blister or capsule and better deagglomeration as the powder is subjected to greater shear forces. However, the particles leave the device moving faster than with passive devices and this can lead to an increase in throat deposition.

It has been surprisingly found that the compositions of the present invention with their high proportion of heparin perform well when dispensed using both active and passive devices. Whilst there tends to be some loss along the lines predicted above with the different types of inhaler devices, this loss is minimal and still allows a substantial proportion of the metered dose of heparin to be deposited in the lung. Once it reaches the lung, the heparin is rapidly absorbed and exhibits excellent bioavailability. Particularly, "active" dry powder inhalers that may be mentioned herein are referred to as Aspirair® inhalers and are described in more detail in WO 2001/00262, WO2002/07805, WO 2002/89880 and WO 2002/89881 , the contents of which are hereby incorporated by reference. Particular "passive" dry powder inhalers that may be mentioned herein are "passive" dry powder inhalation devices that are described in WO 2010/086285. It should be appreciated, however, that the compositions of the present invention can be administered with either passive or active inhaler devices.

In an alternative embodiment, the composition is a solution or suspension, which is dispensed using a pressurised metered dose inhaler (pMDI). The composition according to this embodiment can comprise the dry powder composition discussed above, mixed with or dissolved in a liquid propellant such as HFA 134a or HFA 227. In a yet further embodiment, the composition is a solution or suspension and is administered using a pressurised metered dose inhaler (pMDI), a nebuliser or a soft mist inhaler. Examples of suitable devices include pMDIs such as Modulite® (Chiesi), SkyeFineTM and SkyeDryTM (SkyePharma). Nebulisers such as Porta-Neb®, InquanebTM (Pari) and AquilonTM, and soft mist inhalers such as eFlowTM (Pari), AerodoseTM (Aerogen), Respimat® Inhaler (Boehringer Ingelheim GmbH), AERx® Inhaler (Aradigm) and MysticTM (Ventaira Pharmaceuticals, Inc.).

Where the composition is to be dispensed using a pMDI, the composition comprising heparin optionally further comprises a propellant (i.e. further comprises a propellant). In embodiments of the present invention, the propellant is CFC-12 or an ozonefriendly, non-CFC propellant, such as 1 ,1 ,1 ,2-tetrafluoroethane (HFC-134a), 1 ,1 ,1 ,2,3,3,3- heptafluoropropane (HFC-227), HCFC-22 (difluororchloromethane), HFA-152 (difluoroethane and isobutene) or combinations thereof. Such formulations may require the inclusion of a polar surfactant such as polyethylene glycol, diethylene glycol monoethyl ether, polyoxyethylene sorbitan monolaurate, polyoxyethylene sorbitan monooleate, propoxylated polyethylene glycol, and polyoxyethylene lauryl ether for suspending, solubilizing, wetting and emulsifying the active agent and/or other components, and for lubricating the valve components of the MDI.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.

As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The term "or combinations thereof as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of particular embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Examples Example 1

Unfractionated heparin sodium (100 pg/kg, 300 pg/kg, 1000 pg/kg & 2000 pg/kg) was administered intranasally (i.n.) to evaluate the efficacy of the on pulmonary inflammation induced by 4 days of tobacco smoke (TS) exposure in C57BL/6J mice. The inhaled p38 MAP kinase inhibitor, ADS1 15398, administered at 100pg/kg i.n. 1 h prior to each TS exposure was used as a reference agent.

Experimental Procedure

The total study period was approximately 5 days from the start of the study to harvesting of samples from the last subject. Evaluable subjects were randomly assigned to a treatment group.

Vehicle for intra-nasal dosing

0.2% Tween 80 (Product number: C-4888, Lot no 82K0153). Test Substance

Unfractionated Heparin Sodium (Bioiberica, EM/09/140, F0001 ) pre-weighed in vials (number to agreed) ready to make up fresh for each day of dosing.

Reference control

ADS1 15398 (p38 MAP kinase inhibitor) Animals

70 (10 per group) Female Mice 16-20g (C57BL/6J), full barrier-bred and certified free of specific micro organisms on receipt. Individual Pentel markings on tails were used as identification method.

Treatment Groups

No. Groups: 7; Group Size: 10; Dose Volume: 50 μΙ_ per mouse (25 μΙ_ per nares); Treatment times: intra-nasal, once daily

Table 1 : Experimental Design

O_{h 1 h dil tt}o sam a_{r pr}a_ynce

s ex_pos_i

Group Compound

Sham* Code n Dosing** Mg/kg Frequency

No. Number

Exposure

Ϊ Sham Vehicle A 10 i.n. — CO

I—

2 TS Vehicle B 10 i.n. — o

3 TS Heparin C 10 i.n. 100

4 TS Heparin D 10 i.n. 300 O i-

5 TS Heparin E 10 i.n. 1000

6 TS Heparin F 10 i.n. 2000

TS ADS115398 G 10 i.n. 100

* The sham group of mice was exposed to air for an equivalent length of time on each exposure day (Sham controls); ** For intra-nasal dosing mice assumed to weigh 20g. Cell and Tissue Harvesting

A bronchoalveolar lavage (BAL) was performed using 0.4 mL of phosphate buffered saline (PBS). Cells recovered from the BAL were used for the total cell and differential cell counts carried out using cytospin prepared slides. The BAL supernatants were stored at -40°C.

Data Analysis

A test for normality was carried out on the cell data. Then a preliminary analysis was carried out using a one-way analysis of variance test (one-way ANOVA) followed by a Bonferroni's multiple comparison post-test to compare control and treatment groups andlor a Mann Whitney test. Data that was not normally distributed, was assessed using a Kruskal-Wallis test followed by Dunn's multiple comparisons test. Data p<0.05. was considered statistically significant.

Results

There is a statistically significant (P= <0.01 ) reduction in total cell count in all of the heparin treated groups. This appears to be dose related with the top two dose levels (1000 pg/kg and 2000 pg/kg) demonstrating maximal activity. In addition the level of inhibition (-50%) at 1000 and 2000 pg/kg is equivalent to that of the positive control (Inhaled P38MAP Kinase inhibitor). Table 2: Summary of cell data followinq intranasal administration of unfractionated heparin sodium (100

- 2000 uq/kq) or ADS1 15398 (100uq/kq)

Compound Heparin Sodium ( Mg/kg) ADS115398

Treatment 100 300 1000 2000 100Mg/kg

Inhibition % % p value % p value % p value % p value p value

Total cells 23 <0.01 26 <0.01 50 <0.001 46 <0.001 47 <0.001

Macrophages 26 <0.05 1 1 ns 38 <0.001 34 <0.01 34 <0.01

Epithelial cells 18 ns 43 <0.05 64 <0.001 57 <0.001 50 <0.01

Neutrophils 19 ns 39 <0.001 64 <0.001 61 <0.001 67 <0.001

Eosinophils 39 ns 85 <0.05 71 ns 83 <0.05 72 ns

Lymphocytes 26 ns 55 <0.001 68 <0.001 62 <0.001 72 <0.001

Analysis of the doses in this study on the basis of lung surface area (based on a standard calculation used and presuming 30% of an intranasal dose is delivered to the lungs) gives human equivalent doses of 2.43 mg, 7.3 mg, 24.3 mg and 48.6 mg from murine doses of 100 pg/kg, 300 pg/kg, 1000 pg/kg and 2000 pg/kg. Analysis of the total cell data from our study indicates that we have identified a clear dose response relationship. The lowest dose of 100 pg/kg significantly reduces the total cell count, however looking at the distribution in terms of individual animals it is clear that some animal respond very well and other less so. At 300 pg/kg, the overall mean effect is of a similar magnitude (26% vs 23% reduction for 100 pg/kg) there is also considerably less variation between animals. This may suggest that the 100 pg/kg dose represent the very beginning of the dose response curve that by 300 pg/kg there is a clearly established effect. At 1000 pg/kg & 2000 pg/kg similar response are observed both in magnitude (50 and 46% at 1000 pg/kg & 2000 pg/kg) and tightness of data, suggesting that a maximum effect is achieved by 1000 pg/kg.

This application discloses a good understanding of the dose effect relationship for unfractionated heparin in this model. This provides the rationale to select doses for further pre-clinical or clinical studies.

Example 2 (Sputum samples from adult cystic fibrosis patients and Frog palate model)

The experiments were carried out using rheometry and frog palate protocols similar to that described by Sun et al. (2002) Rancourt et al. (2004) and O'Brien et al. (2004). Concentrations of heparin were investigated, and the results compared with vehicle control (normal 0.15M saline) and a historical control (nacystelyn 1 mM).

Materials and Methods

Given the target concentration for in vitro mucolytic testing in cystic fibrosis (CF) sputum - 5 mg/mL final cone, our basic protocol (Sun 2002) called for the measurement of viscoelasticity of a 10-15 mg aliquot of sputum by magnetic rheometry (King 1988), incubation of the sputum sample in 10% vol/wt of mucolytic solution or vehicle at 10 times the final desired concentration (37 °C / 30 min), and re- measurement of the viscoelasticity. The fall in viscoelastic modulus (expressed as a logarithm) is considered the magnitude of viscoelastic effect for the particular concentration of mucolytic agent. Because there is a small dilution effect associated with vehicle incubation, the results are corrected for vehicle control. 0.15M (0.9%) NaCI was employed as vehicle, and nacystelyn 1 mM (N-acetylcysteine L-lysinate, 309 Mg/mL) as a positive control.

For experiments involving direct application of heparins to frog palate, a modified Ringer solution consisting of 2/3 standard non-lactated Ringer solution (Baxter) and 1/3 de-ionized water was used as vehicle. This modified Ringer solution is termed "frog Ringer" solution.

Sputum samples from adult cystic fibrosis patients

Sputum samples collected from adult patients with cystic fibrosis attending the University of Alberta Hospital outpatient clinic were used. Small quantities (75-100 mg per sample) of suitable material were available in our deep freezer for initial testing. The patients met the general profile of mild to moderate CF lung disease, regular sputum producers, not taking Pulmozyme or other mucolytic, and chronically infected with Pseudomonas aeruginosa. The sputum samples were obtained under a protocol approved by the University of Alberta Health Research Ethics Board.

Sputum rheology by magnetic microrheometry (King 1988)

The magnetic microrheometer is used to measure the bulk viscosity and elasticity of microliter quantities of mucus. A 100 m steel ball is carefully positioned in a 1 -10 μί sample of mucus and oscillated by means of an electromagnetic field gradient. The motion of this sphere is tracked with the aid of a photocell. Plots of ball displacement versus magnetic force are used to determine the viscosity and elasticity of the mucus as a function of applied frequency (1 -100 rad/s). These rheological properties can be used to predict the effectiveness of mucus in clearance, both by ciliary action and for clearance by airflow interaction (King 1987). This instrument is particularly suited to the proposed studies involving multiple treatments of sputum because of the minimal sample requirement.

Frog palate studies of mucociliary clearance

The frog palate epithelium is lined with cilia and secretes and clears mucus much the same as the mammalian trachea. Mucociliary clearance continues at a steady rate for several hours after sacrifice and excision of the palate (King 1998). During this period, the rate of palatal mucociliary clearance can be modulated by agents that alter the ciliary activity or that change the properties of the superficial fluid layer (mucus and periciliary fluid). By waiting longer (1 -2 days in the bullfrog), mucus secretion ceases while ciliary activity continues for at least 5-6 days (Rubin 2002). During this extended period, mucus from endogenous or exogenous (e.g. cystic fibrosis) sources or mucus simulants are transported at rates that are reflective of their viscoelastic properties (King 1998). Studies during the initial and extended periods may be described as using "non-depleted" and "depleted" frog palate respectively, referring to the presence or absence of a mucus layer covering the cilia.

Mucociliary velocity (MCV) is measured by observing the rate of movement of endogenous mucus, using a calibrated macroscope and a stopwatch. MCV is computed as the distance of marker particle travel divided by elapsed time (mm/s). A mean of five consecutive runs for each test solution was used to compute each value of MCV. Rheometry

From each of 10 CF sputum samples, 6 aliquots of ca. 10-15 mg each were incubated for 30 minutes at 37°C with either 0.9% NaCI or with one of the 4 concentrations of unfractionated heparin outlined above, or with N-acetylcysteine L-lysinate (309 g/mL). The incubation solutions are gently layered on top of the sputum to avoid mechanical degradation due to stirring. To minimize dilution effects, 1 part of mucolytic solution at 10 times the final desired concentration was added to 9 parts of sputum (vol/wt). Prior to and following incubation, sputum viscoelasticity at 10 rad/s was determined by magnetic microrheometry. The mucolytic effect of each solution was defined by the mean fractional decrease in G^* (vector sum of viscosity and elasticity) over the 10 samples tested (Sun 2002).

Depleted frog palate (heparin dose-response)

The effect of the mucolytic treatment on mucociliary clearability was tested using the depleted frog palate model. The transport velocity of heparin-treated sputum samples on frog palate was measured and compared with the transport velocity of control samples treated with either saline vehicle or nacystelyn (Sun 2002).

Rheometry (heparin molecular weight effect)

The basic design was similar to above except for the nacystelyn control, which was not necessary to repeat. Two heparin fractions differering in molecular weight were obtained from Neoparin, Inc (Alameda, CA, U.S.A.); these were heparin decasaccharide (m.w. 3000 Da) and heparin polysaccharide IV (m.w. 12,000 Da). Depleted frog palate (heparin molecular weight effect)

For the CF sputum samples described above, the transport velocity of the heparin- treated sputum samples on frog palate was measured and compared with the transport velocity of untreated control sputum (Sun 2002).

Non-depleted frog palate

This model, using endogenous mucus, represents an open system in which material exchange across the epithelium could modify the mucolytic activity. The protocol was designed to compare the mucokinetic effects of heparin in the isolated cilioactive frog palate at two concentrations previously shown to suggest biological activity in CF sputum, in terms of viscoelasticity and transportability testing, with the same concentrations of a heparin fraction of lower molecular weight, namely the Neoparin decasaccharide. The doses for these two heparin preparations were 16 mg/nnL and 50 mg/nnL, which correspond to nominal final mucus layer concentrations of 1 .6 and 5 img/mL. In order to control for possible variations in ciliary activity over the course of the experiment, the order of application of unfractionated heparin and Neoparin decasaccharide was varied. Each experiment was initiated with a frog Ringer solution control, and a second Ringer solution was applied between the two sets of heparin treatments. After the heparin treatments were completed, a final positive control, namely Nacystelyn (NAL) solution at 1 mM concentration was applied, resulting in a nominal mucus concentration of 100 μΜ. The NAL treatment was placed as the last active treatment to avoid any possible interference with the comparison between the two heparin preparations. The solution application procedure was as follows: A 5 μΙ_ volume of mucolytic solution was gently delivered to the nasal end of the palate (upstream in terms of mucociliary flow), allowing approx. 2 minutes for the fluid to disperse, and for clearance to stabilize. Then the movement of charcoal marker particles over a fixed displacement of 9 mm was observed under a calibrated macroscope, from which velocity is calculated as displacement/time (mm/min). Five readings per solution over ca. 10-15 minutes were taken in order to calculate an average velocity. The velocity for the Ringer treatment was taken as 100% for any particular palate, and the velocity for each mucolytic treatment was expressed as a percentage of the preceding Ringer control.

Estimation of the final concentrations of heparins in the frog palate epithelial surface fluid is subject to a number of assumptions. The volume of mucus lining the palatal surface varies with the resting metabolic state and the time after excision. Based on the work of Festa et al. (1997) for freshly excised palates from large bullfrogs, this volume has been estimated to be of the order of 50 μΙ_. Thus, with the addition of 5 μΙ_ of mucolytic solution, it is assumed that the final concentration in the epithelial fluid is tenfold lower.

Statistical treatment of data

Data measurements were expressed as mean ± standard error. Overall significance of the viscoelasticity and clearability results was tested using a one-way analysis of variance. For comparison between two groups, a paired Student T-test was used. The level of significance was set at p<0.05. Interpretation

The primary variable of interest is the decrease in mucus viscoelasticity, expressed as delta log G^* (corrected for vehicle treatment). A statistically significant decrease in log G^* at a given concentration of heparin is taken as evidence supporting mucolytic activity. Mucociliary clearability (rate of clearance of mucolytic-treated sputum relative to vehicle-treated sputum) is a second variable of interest. Mucolytic treatments that reduce the degree of crosslinking without destroying the basic mucous gel structure should result in an improvement in in vitro clearability (King 1994). Differences between means were tested by analysis of variance, and significance between groups was tested by means of paired t-tests. Based on previous experience, a biologically significant decrease in log G^* is achieved with a change of about 0.2 log units, and a biologically significant increase in clearability would be achieved with a 10% increase in MCV (King & Rubin 1994).

A dose-dependent, continuous increase in mucolytic activity of heparin was seen (Figure 7) over the concentration range 0.16 mg/mL to 5.0 mg/mL (final cone, in sputum). There was a corresponding increase in in vitro mucociliary clearability of the heparin-treated sputum over this same dose range (Data not shown). For comparison, treatment of aliquots of the same samples with Nacystelyn (NAL) at a concentration of 309 pg/nriL (100 μΜ final cone, in sputum) was statistically indistinguishable from heparin at 5.0 mg/mL, both for the change in logG^* and the change in MCV.

Heparin solutions increased frog palate mucociliary velocity (MCV) compared with frog Ringer solution in a dose-dependent fashion (Figure 8): At 1 .6 mg/mL nominal final cone, the velocity increased to 1 16.2% ± 3.6% of Ringer control; at 5.0 mg/mL, MCV further increased to 127.9 ± 3.6%. The effects of heparin decasaccharide were similar: At 1 .6 mg/mL, MCV increased to 1 14.7% ± 2.1 % of Ringer control; at 5.0 mg/mL, MCV attained a mean value of 126.3 ± 3.1 %. At the lowest concentrations of heparins, MCV was very near 100% of Ringer control. The positive reference solution was NAL 100 μΜ, for which MCV attained a mean value of 130.2% ± 4.8% of control. This value of MCV was not significantly different from the MCV for unfractionated heparin or heparin decasaccharide at 5.0 mg/mL. The second Ringer velocity was 98.9% ± 1 .0% of the first, a nonsignificant difference, indicating the stability of the control over the course of the experiment.

Both unfractionated heparin and Neoparin decasaccharide, stimulated clearance in this animal mucociliary model in a dose-dependent manner. The differences between the mucokinetic activity of unfractionated heparin and the heparin decasaccharide fraction were not statistically significant. The lack of any significant effect of heparin molecular weight on mucokinetic capacity over the investigated range suggests that there is little diffusion of heparin out of the mucus layer over the time frame of observation (ca. 15 min). At the highest dose, the mucokinetic effects of unfractionated heparin and heparin decasaccharide were similar to and statistically indistinguishable from the positive reference compound Nacystelyn at 100 μΜ.

Example 3

A Phase l/ll randomised, placebo-controlled, double blind trial with four parallel groups with a 1 :1 :1 :1 ratio was conducted to assess the safety, tolerability, pharmacodynamics and exploratory efficacy of unfractionated heparin sodium presented as 25 mg premetered doses (i.e. a 31 mg nominal dose) in hard capsules (hydroxypropyl-methyl cellulose) to be delivered directly to the lungs of patients with Cystic Fibrosis (CF) using the Monohaler^® dry powder inhalation (DPI) device.

Experimental Procedure

The study period was approximately 22 months. Each patient took part in: Screening period of 4 weeks prior to Baseline (Day 1 ); Treatment period of 4 weeks; Follow-up period of 2 weeks. The duration of participation in the trial was approximately 10 weeks for each patient. Evaluable subjects were randomly assigned to a treatment group.

Formulation for dosing

Delivered daily doses of unfractionated Heparin Sodium (from - Bioiberica as described in Example 1 ) 50 mg, 100 and 200 mg containing L-leucine (Ajinomoto) were administered by oral inhalation (based upon nominal daily doses of 62 mg, 124 mg and 248 mg, respectively). Each individual placebo capsule contained lactose (Respitose) and L-leucine (Ajinomoto) dry powder blend.

Study Subjects (Inclusion criteria)

Male or female >16 years; Non-smoker; Written informed consent obtained prior to any trial specific procedures; confirmed diagnosis of CF lung disease (i.e., respiratory clinical symptoms and positive sweat test or disease inducing mutations) by CF expert / Investigator; patient considered, in the Investigator's opinion, to be clinically stable and has at Screening and Baseline an FEV1 1 40 - 90% of predicted value for age, sex and height; FEV1 value at Baseline is within +/-15% of FEV1 value 4 weeks earlier at Screening; regular mucus production due to CF; ease of sputum expectoration (i.e., clearability) VAS score of < 80 mm; Neutrophil elastase and / or IL-8 levels above detectable levels and/or upper limit of normal range for specified laboratory; Adequate contraceptive measures (the subject [and his/her partner] should use adequate contraceptive measures, consisting of two forms of contraception, at least one of which must be a barrier method); Able to comply with all the requirements of the protocol; Able to use inhaler satisfactorily.

Treatment Groups

A total of 77 randomised evaluable patients with moderate CF were included in the study. These patients had to meet all of the inclusion criteria and provide written informed consent prior to any trial specific procedures

Dose Administration and Dosing Regimen:

Patients were randomised to receive one of three daily delivered dose levels 50 mg, 100 mg, 200 mg heparin inhalation powder or matching placebo; 4 capsules to be self- administered by inhalation by the patient twice daily (i.e. 8 capsules per day in total) for 4 consecutive weeks. The treatment was double blind. Dosing scheduled for 09h00 am (±2 hours) (to be taken post morning physiotherapy and any bronchodilator treatment) and 21 h00 pm (±2 hours). Note: The patient was advised that on the morning of each centre visit (i.e., Baseline [Day 1 ], Week 2 and Week 4) his / her morning dose was taken at the trial centre after all pre-treatment assessments have been completed. Of the four groups, one group received 8 placebo capsules (4 to be taken am and 4 to be taken pm) and no active capsules, one group received 2 active capsules (1 to be taken am and 1 to be taken pm) along with 6 placebo capsules (3 to be taken am and 3 to be taken pm), another group received 4 active capsules (2 to be taken am and 2 to be taken pm) and 4 placebo capsules (2 to be taken am and 2 to be taken pm) and the final group received 8 active capsules (4 to be taken am and 4 to be taken pm).

Five visits to the trial centre were included: 1 . Screening (Days -28 [± 4 days]); 2. Baseline (Day 1 ); 3. Week 2 (Day 15 [± 2 days]); 4. Week 4 (Day 29 [± 2 days]); 5. Week 6 Follow-up (Day 43 [± 4 days]).

Safety and Tolerability Parameters:

Monitoring and assessment of: AEs / serious adverse events (SAEs); Sitting vital sign parameters (blood pressure, heart rate, respiratory rate, temperature), weight and physical examination; Clinical laboratory parameters, including haematology, clinical chemistry and urinalysis; Platelet counts were measured at screening, baseline, Week 2 and Week 4.

Pharmacodynamic (PD) Parameters:

Expectorated sputum measurement parameters such as (i.e., rheological viscoelasticity / physicochemical measurement parameters); Induced sputum markers neutrophil elastase, interleukins (IL-6 and IL-8) and cell counts (i.e., total cell count, % neutrophil count, % macrophages); EBC pH levels may be measured at a few selected sites with the relevant experience; Blood plasma markers, neutrophil elastase, neutrophil elastase / AAT complex, neutrophil count, IL-6, IL-8 and CRP levels; aPTT and platelet count.

Primary Trial Objectives:

To investigate the safety and tolerability of heparin inhalation powder in patients with CF.

Secondary Trial Objectives:

To investigate the pharmacodynamics (PD) of heparin inhalation powder by means of: Expectorated mucus properties (i.e., rheological properties including viscoelasticity / physicochemical measurements);

The effect on inflammatory markers in induced sputum (i.e., neutrophil elastase, interleukins [IL-6 and IL-8]) and cell counts (i.e., total cell count, % neutrophil count, % macrophages);

The effect on pH levels in exhaled breath condensate (EBC) (measured at selected centres);

The effect on inflammatory markers in blood plasma (i.e., neutrophil elastase, neutrophil elastase / alpha-1 antitrypsin [AAT] complex, IL-6, IL-8, neutrophil count and C-reactive protein [CRP] level); and

The effect on blood coagulation (i.e., activated partial thromboplastin time [aPTT] and platelet count). To evaluate efficacy by:

Visual analogue scales (VAS) to be completed based on change in symptoms, including: cough resolution; expectorated sputum clearability, thickness, volume, colour, viscoelasticity (stickiness); breathlessness; general well being (including feeling, energy, physical activity, appetite and sleep);

Microbiological analysis of expectorated sputum for effects on bacterial growth, density, antibiotic sensitivity (The identification, culture, density and antibiotic sensitivity of the following organisms are included: Pseudomonas aeruginosa [mucoid and other types], Burkholderia cepacia, Haemophilus influenza and Staphylococcus aureus);

Volume and weight of 24 hour cumulative expectorated sputum sample;

Pulmonary function measured by forced expiratory volume in one second (FEV1 ), forced vital capacity (FVC), forced mid-expiratory flow (FEF25-75), and arterial oxygen saturation (SaO2); and

Response to the Cystic Fibrosis Questionnaire (CFQ);

Data Analysis

Patients randomised to receive placebo were pooled together. Summary statistics were produced by delivered dose level i.e., 0 mg (placebo), 50 mg, 100 mg and 200 mg Frequency counts and percentages for treatment-emergent adverse, events (TEAEs), related TEAEs and for SAEs coded using the Medical Dictionary for Regulatory Activities (MedDRA) were summarised by system organ class and preferred term. Appropriate summary statistics for clinical laboratory parameters, vital signs, weight and physical examination were presented by visit. Change from Baseline (Day 1 ) to Week 4 in VAS (cough resolution; expectorated sputum clearability, thickness, volume, colour, viscoelasticity [stickiness]; breathlessness; weight and volume of 24 hour cumulative expectorated sputum sample, general well being [including feeling, energy, physical activity, appetite and sleep]), inflammatory markers, mucus rheological measurements; microbiological measurements (bacterial growth, density and antibiotic sensitivity of identified organisms) and pulmonary function tests (FEV1 , FVC and FEF25-75) were analysed using an analysis of covariance (ANCOVA) model with Baseline level as covariate. Estimates of the difference between dose levels were presented with the 95% confidence intervals for the comparison of each active dose versus placebo.

Results

Safety (local and systemic) and tolerability

Mean platelet counts following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo demonstrated no systemic exposure following heparin administration throughout the course of the study (see Figure 9).

Likewise mean activated partial thromboplastin time (aPTT) following administration of a delivered dose of 50 mg, 100 mg, 200 mg unfractionated heparin compared lactose placebo demonstrate demonstrated no systemic exposure following heparin administration throughout the course of the study (see Figure 10). Induced sputum neutrophil elastase, total cell count and IL-8 levels

These data demonstrate that key study objective and a priori acceptance criteria achieved with a neutrophil elastase mean reduction > 0.3 log (compared to placebo) and a total cell count mean reduction > 0.3 log (compared to placebo), see Figures 1 1 and 12. A > 0.3 log reduction is clinically relevant and is correlated with a preservation of lung function in CF patients.

As shown by Figure 13, IL-8 levels are reduced relative to placebo by the administration of heparin and demonstrate a trend of reduction in inflammation.

Plasma inflammatory mediator summary

These data demonstrate that no systemic effect is observed following administration with orally inhaled heparin, as measured by plasma marker levels (e.g. plasma neutrophil count, neutrophil elastase/alpha-1 antitrypsin complex, CRP, IL-8 and IL-6 - see Figures 14 to 20).

Sputum clearability and Suputum colour (VAS score)

As demonstrated by the data in Figure 21 , each of the doses provided to the patients showed an improved efficacy when compared to the group on placebo. The effects reported by the patients on sputum colour (less sputum colour suggests less infection) and sputum clearability shows that heparin is having a perceived effect in reducing inflammation and easing the symptoms of CF. Haemoptysis

Heparin is an anti-coagulant and may cause bleeding in certain groups of patients, which includes CF patients, who have a tendency to suffer from haemoptysis (lung bleeding).

While previous studies using nebulised heparin have shown no increase in haemoptysis, it is noted that such nebulised solutions administer heparin in droplets that impact throughout the upper airway, but do not reach the lower reaches of the lung (lung extremities). As the lung extremities contain the vast bulk of the vasculature and hence the potential for haemoptysis, a nebulised formulation would not be expected to cause significant haemoptysis. However, based upon the information currently available, if heparin were to reach the lower reaches of the lung, it would be expected to cause haemoptysis. We have surprisingly found that this is not the case.

In the current study, a significant proportion of the heparin delivered to the patient was capable of reaching the lung bronchi extremities (e.g. each 25 mg of heparin delivered dose contained a fine particle dose of 10 mg). According to the results obtained in this study, 7% of the heparin receiving patients reported haemoptysis compared to 5% of the patients receiving placebo. As described above, this result is surprising given the anti-coagulant nature of heparin and the fact that CF patients are prone to haemoptysis. Cough

Cough was reported at the low (1 1 % of patients) and high (10% of patients) doses, as well as placebo (15% of patients), but no cough was reported in patients taking the medium dose. In previous clinical studies, incidences exceeding 20% of patients reported cough due to the formulations used.

Claims

1 . A glycosanninoglycan (e.g. heparin) for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan (e.g. heparin) reduces inflammation in the lungs of the subject.

2. A method of treatment and/or prevention of an inflammatory lung disease, comprising the administration of a glycosaminoglycan (e.g. heparin) to a subject, optionally wherein, after administration, the glycosaminoglycan (e.g. heparin) reduces inflammation in the lungs of the subject.

3. The use of a glycosaminoglycan (e.g. heparin) in the preparation of a medicament for the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan (e.g. heparin) reduces inflammation in the lungs of the subject.

4. A kit comprising a glycosaminoglycan for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject.

5. The glycosaminoglycan, method, use or kit according to Claims 1 to 4 respectively, wherein the glycosaminoglycan is in the form of a dry powder.

6. The glycosaminoglycan, method, use or kit according to any preceding claim, wherein the administration of the glycosaminoglycan (e.g. heparin) results in a reduction of inflammation of at least 10%.

7. The glycosanninoglycan, method, use or kit according to Claim 6, wherein the administration of the glycosaminoglycan (e.g. heparin) results in a reduction of inflammation of at least 20%.

8. The glycosaminoglycan, method, use or kit of Claim 7, wherein the administration of the glycosaminoglycan (e.g. heparin) results in a reduction of inflammation of at least 30%.

9. The glycosaminoglycan, method, use or kit of Claim 8 wherein the administration of the glycosaminoglycan (e.g. heparin) results in a reduction of inflammation of at least 50%.

10. The glycosaminoglycan, method, use or kit of Claim 9 wherein the administration of the glycosaminoglycan (e.g. heparin) results in a reduction of inflammation of at least 60%.

1 1 . The glycosaminoglycan, method, use or kit according to any preceding claims, wherein the glycosaminoglycan (e.g. heparin) is administered intranasally or by pulmonary inhalation.

12. The glycosaminoglycan, method, use or kit according to any preceding claims, wherein the glycosaminoglycan is heparin.

13. The glycosaminoglycan, method, use or kit according to Claim 1 1 , wherein the heparin is unfractionated heparin.

14. The glycosaminoglycan, method, use or kit according to Claim 13,, wherein the heparin is unfractionated heparin sodium.

15. The glycosaminoglycan, method, use or kit according to any one of Claims 1 to 13, wherein the heparin is low molecular weight heparin.

16. The glycosaminoglycan, method, use or kit according to any preceding claim, wherein the total daily dose (measured as the FPD) of heparin is between 0.1 mg and 6000 mg.

17. The glycosaminoglycan, method, use or kit according to Claim 16,, wherein the total daily dose (measured as the FPD) of heparin is between 0.5 mg and 6000 mg.

18. The glycosaminoglycan, method, use or kit according to Claim 17, wherein the total daily dose of heparin is between 2 mg and 240 mg.

19. The glycosaminoglycan, method, use or kit according to Claim 18, wherein the total daily dose of heparin is between 5 mg and 80 mg.

20. The glycosaminoglycan, method, use or kit according to Claim 19, wherein the total daily dose of heparin is between 10 mg and 40 mg.

21 . The glycosaminoglycan, method, use or kit according to Claim 19, wherein the total daily dose of heparin is between 12 mg and 50 mg.

22. The glycosaminoglycan, method, use or kit according to any preceding claim, wherein the total daily delivered dose of heparin is between 0.8 and 200 mg.

23. The glycosaminoglycan, method, use or kit according to any preceding claim, wherein heparin is delivered in a fine particle dose of between 0.3 mg and 2000 mg.

24. The glycosaminoglycan, method, use or kit according to Claim 23, wherein heparin is delivered in a fine particle dose of between 0.5 mg and 2000 mg.

25. The glycosaminoglycan, method, use or kit according to Claim 24, wherein heparin is delivered in a fine particle dose of between 2 mg and 240 mg.

26. The glycosaminoglycan, method, use or kit according to Claim 25, wherein heparin is delivered in a fine particle dose of between 6 mg and 35 mg.

27. The glycosaminoglycan, method, use or kit according to Claim 23, wherein heparin is delivered in a fine particle dose of between 0.5 mg and 20 mg.

28. The glycosaminoglycan, method, use or kit according to any preceding claim, wherein the inflammation is as measured by total cell count obtained from bronchoalveolar lavage.

29. The glycosanninoglycan, method, use or kit according to any preceding claim, wherein the inflammation is as measured by pulmonary neutrophil levels.

30. The glycosaminoglycan, method, use or kit according to any preceding claim, wherein the reduction achieved is equivalent to that achieved by the p38 MAP kinase inhibitor, ADS1 15398.

31 . The glycosaminoglycan, method, use or kit according to Claim 30, wherein the ADS1 15398 is administered by pulmonary inhalation or intranasally to a subject in a dose of 0.1 mg/kg.

32. The glycosaminoglycan, method, use or kit according to any preceding claim, further comprising another active agent.

33. The glycosaminoglycan, method, use or kit according to Claim 32, wherein the further active agent is selected from mucolytic agents; bronchodilators; anti- muscarinics; antibiotic and antibacterial agents; anti-infective agents; aminoglycosides; leprostatics; miscellaneous antiinfectives; nonsteroidal antiinflammatory agents; other anti-inflammatory agents;

PDE4 inhibitors; quinazolinediones; steroids; matrix metalloprotease inhibitors; epithelial sodium channel (ENaC) inhibitors; methylxanthines; and drugs for cystic fibrosis management.

34. The glycosaminoglycan, method, use or kit according to Claim 33, wherein the further active agent is selected from mucolytic agents; bronchodilators; anti- muscarinics; antibiotic and antibacterial agents; anti-infective agents; aminoglycosides; leprostatics; miscellaneous antiinfectives; nonsteroidal antiinflammatory agents; other anti-inflammatory agents; PDE4 inhibitors; quinazolinediones; steroids; and drugs for cystic fibrosis management.

35. The glycosaminoglycan, method, use or kit according to Claim 34, wherein the further active agent is ADS1 15398.

36. The glycosaminoglycan, method, use or kit according to any preceding claim wherein the inflammatory lung disease is a disease characterised by neutrophilia.

37. The glycosaminoglycan, method, use or kit according to any preceding claim wherein the inflammatory lung disease is a disease or condition selected from asthma, cystic fibrosis, idiopathic pulmonary fibrosis, non-cystic fibrosis bronchiectasis and, chronic obstructive pulmonary disease.

38. The glycosaminoglycan, method, use or kit according to Claim 37, wherein the inflammatory lung disease is chronic obstructive pulmonary disease (COPD).

39. The glycosaminoglycan, method, use or kit according to Claim 38, wherein the COPD is an episode of acute COPD exacerbation.

40. The glycosaminoglycan, method or use according to Claim 37, wherein the the inflammatory lung disease is cystic fibrosis.

41 . The glycosanninoglycan, method or use according to any preceding claim, wherein the glycosaminoglycan further comprises between 1 and 20%, preferably 2 to 15% by weight of an additive (e.g. leucine).

42. A passive or active inhaler device in combination with a glycosaminoglycan.

43. The device of Claim 42, wherein the device is a passive inhaler device.

44. The device of Claim 43, wherein the device is selected from Monohaler and the device produced by Vectura (as covered by international patent application number WO 2010/086285.

45. The device of any one of Claims 42 to 44, wherein the glycosaminoglycan is in the form of a dry powder.

46. The device of any one of Claims 42 to 44, wherein the glycosaminoglycan is heparin.

47. A device for the administration of a glycosaminoglycan (e.g. heparin) for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject.

48. The device of Claim 47, wherein the device is a passive inhaler device.

49. The device of Claim 48, wherein the device is selected from Monohaler and the device produced by Vectura (as covered by international patent application number WO 2010/086285.

50. The device of any one of Claims 47 to 49, wherein the glycosaminoglycan is in the form of a dry powder.

51 . The device of any one of Claims 47 to 50, wherein the glycosaminoglycan is heparin.

52. Use of a drug delivery device to deliver a glycosaminoglycan a pharmaceutical formulation of a glycosaminoglycan (e.g. heparin) for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject.

53. The device of Claim 52, wherein the device is a passive inhaler device.

54. The device of Claim 53, wherein the device is selected from Monohaler and the device produced by Vectura (as covered by international patent application number WO 2010/086285.

55. The device of any one of Claims 52 to 54, wherein the glycosaminoglycan is in the form of a dry powder.

56. The device of any one of Claims 52 to 55, wherein the glycosaminoglycan is heparin.

57. A pharmaceutical formulation of a glycosaminoglycan.

58. A pharmaceutical formulation of a glycosaminoglycan (e.g. heparin) for use in the treatment and/or prevention of an inflammatory lung disease, optionally wherein, after administration to a subject, the glycosaminoglycan reduces inflammation in the lungs of the subject.

59. The pharmaceutical formulation according to Claim 57 or Claim 58, wherein the glycosaminoglycan is a dry powder.

60. The pharmaceutical formulation according to any one of Claims 57 to 59, wherein the glycosaminoglycan is heparin.

61 . The pharmaceutical formulation according to any one of Claims 57 to 60, wherein the formulation further comprises between 1 and 20% or preferably 2 to 15% by weight of an additive (e.g. leucine).

62. The pharmaceutical formulation according to any one of Claims 57 to 61 , wherein the formulation further comprises a carrier.

63. The pharmaceutical formulation according to any one of Claims 57 to 62, wherein the glycosoaminoglycan comprises 80 to 99% by weight of the formulation.