EP4683647A1 - Use of low molecular weight hyaluronic acid for promoting ciliated cell differentiation - Google Patents

Abstract

Mucociliary clearance is accomplished by the coordinated efforts of ciliated and goblet cells, two major airway epithelial cell types. It is important to have a sufficient number of ciliated cells to be able to propel the mucus out of the airways. Too few ciliated cells and/or too much mucus production can lead to toxin-laden mucus accumulation in the airways that can lead to respiratory diseases. Many lung diseases are associated with a diminished number of ciliated cells. Therefore, there is a need for identifying drugs that promote ciliated cell differentiation. The inventors show that hyaluronic acid (HA) with a low molecular weight, from 15,000 to 45,000 Daltons promotes ciliated cell differentiation in different physiopathological conditions that include cystic fibrosis and chronic obstructive pulmonary disease.

Description

USE OF LOW MOLECULAR WEIGHT HYALURONIC ACID FOR PROMOTING CILIATED CELL DIFFERENTIATION

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular pneumology.

BACKGROUND OF THE INVENTION:

The epithelium lining the conducting airways acts as the front-line defense to protect the lungs from inhaled pathogens and noxious agents by trapping and expelling them from the airway by the process of mucociliary clearance. Mucociliary clearance is accomplished by the coordinated efforts of ciliated and goblet cells, two major airway epithelial cell types. Goblet cells secrete mucus, which forms a viscoelastic gel that covers the epithelial surface and traps inhaled harmful particles and pathogens. The coordinated beating of cilia on the apical surface of the ciliated cells propels this mucus with entrapped noxious particles out of the airways before they can damage the lung. Efficient mucociliary clearance depends upon a proper balance between ciliated and goblet cells. It is important to have a sufficient number of ciliated cells to be able to propel the mucus out of the airways. Too few ciliated cells and/or too much mucus production can lead to toxin-laden mucus accumulation in the airways that can lead to respiratory diseases. In the respiratory tract, ciliated cells have -200 cilia per cell, each with a diameter of 250 nm and a length of -6 pm and there are an estimated 10⁹ ciliated cells/cm² of upper and large airway surfaces (Livraghi, Alessandra, and Scott H. Randell. "Cystic fibrosis and other respiratory diseases of impaired mucus clearance. " Toxicologic pathology 35.1 (2007): 116-129). Many lung diseases are associated with a diminished number of ciliated cells. For instance, the COPD bronchial epithelium is imprinted by an altered programming of lineage differentiation that includes a defect in the generation of ciliated cells that is seen in large conducting airways (Gohy, S., Carlier, F.M., Fregimilicka, C. et al. Altered generation of ciliated cells in chronic obstructive pulmonary disease. Sci Rep 9, 17963 (2019)) and small airways (Luczka-Majerus, E, Bonnomet, A, Germain, A. et al. Ciliogenesis is intrinsically altered in COPD small airways. Eur Respir J 60 (6), 2200791 (2022)). In a murine experimental asthma model, it has been reported that the number of ciliated cells tended to decrease (Reader, J. Rachel, et al. "Pathogenesis of mucous cell metaplasia in a murine asthma model. " The American journal of pathology 162.6 (2003): 2069-2078) while the loss of cilia has been reported following smoke exposure (Sisson, Joseph H., et al. "Smoke and viral infection cause cilia loss detectable by bronchoalveolar lavage cytology and dynein ELISA. " American journal of respiratory and critical care medicine 149.1 (1994): 205-213) as well as in cystic fibrosis (Bedrossian, Carlos WM, et al. "The lung in cystic fibrosis: a quantitative study including prevalence of pathologic findings among different age groups. "Human pathology 7.2 (1976): 195-204). Therefore, there is a need for identifying drugs that promote ciliated cell differentiation.

SUMMARY OF THE INVENTION:

The present invention is defined by the claims. In particular, the present invention relates to the use of low molecular weight hyaluronic acid for promoting ciliated cell differentiation.

DETAILED DESCRIPTION OF THE INVENTION:

The first object of the present invention relates to a method of promoting differentiation of ciliated cells in the airway epithelium of a patient suffering from a chronic airway disease comprising administering to the patient a therapeutically effective amount of hyaluronic acid having a low molecular weight from 15,000 to 45,000 Daltons.

As used herein, the term “patient” is interchangeable with the term “individual” or “subject”, and may refer to a subject to be treated by the methods disclosed herein. In some embodiments, the patient is a mammal. Non-limiting examples of mammals include rodents (e.g., mice and rats), primates (e.g., lemurs, bushbabies, monkeys, apes, and humans), rabbits, dogs (e.g., companion dogs, service dogs, or work dogs such as police dogs, military dogs, race dogs, or show dogs), horses (such as race horses and work horses), cats (e.g., domesticated cats), livestock (such as pigs, bovines, donkeys, mules, bison, goats, camels, and sheep), and deer. In some embodiments, the mammal is a human. In some embodiments, the patient is a human infant. In some embodiments, the patient is a human child. In some embodiments, the patient is a human adult.

As used herein, the term “airway epithelium” has its general meaning in the art and refers to the type of mucociliated columnar epithelium found lining most of the respiratory tract as respiratory mucosa, where it serves to moisten and protect the airways.

As used herein, the term “ciliated cell” has its general meaning in the art and refers to the ciliated epithelial cells that are adapted in the respiratory tract to form an epithelial lining with coordinated metachronal ciliary activity that provides the propelling force for the transport of mucus along the airways.

As used herein, the term “differentiation” refers to a phenomenon in which the structure or function of cells is specialized during the division, proliferation and growth thereof, that is, the morphology or function of cells or tissues of organisms changes to perform their tasks. Cell differentiation that is a final stage resulting in the specialization of cells in the developmental or regeneration processes is a phenomenon in which genes in cells are expressed in different manners because their different activities, and as a result, the cells have structurally and functionally completely different characteristics.

In particular, the method of the present invention is particularly suitable for increasing the number of ciliated cells in the airway epithelium of the patient. The method of the present invention is also particularly suitable for restoring airway and pulmonary homeostasis.

In particular, the method of the present invention is particularly suitable for preventing extensive airway mucus plugging in a patient suffering from a chronic airway disease.

As used herein, the term “mucus” has its general meaning in the art and refers to a usually clear viscous fluid that is secreted by mucous cells and glands of the respiratory tract. Mucus moistens, lubricates and protects the tissues from which it is secreted. It comprises mucin macromolecules, which are the gel forming constituents of mucus.

As used herein, the term “extensive airway mucus plugging” refers to a large number of occluded airways caused by mucus plugs (e.g. completely occluded airways) in one or more segments of the lungs. Identification of extensive airway mucus plugging may be determined by assessing the quantity of mucus in an airway within the lung of the subject. In some embodiments, extensive airway mucus plugging can indicate complete occlusion of about 5- 10%, about 10-20%, about 20-30%, about 30-40%, about 40-50%, about 50-60%, about 60- 70%, about 70-80%, about 80-90%, about 90-100%, or about 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% of segments.

In particular, the method of the present invention is particularly suitable for preventing mucus occlusion of an airway lumen. As used herein, the term “mucus occlusion of an airway lumen” indicates a complete opacification of an airway by mucus with or without bronchial dilatation as indicated by, e.g., lung imaging. In some embodiments, mucus plugs can be detected (e.g. seen) in sections (such as longitudinal sections) as tubular structures with or without branching or in cross-section as rounded opacities.

In some embodiments, the method of the present invention is particularly suitable for improving mucociliary clearance in patient suffering from a chronic airway disease.

As used herein, the term “mucociliary clearance” refers to the ability of the mucus to be cleared from the respiratory tract of the patient. Thus the expression “improving mucociliary clearance” is any improvement of mucus clearance from a starting level. This would be determined by the ability of a patient to eject mucus from the respiratory tract. The terms would be understood by a person of ordinary skill in the art.

According to the present invention the chronic airway disease thus features aberrant mucus production and loss of ciliated cells. Examples of chronic airway diseases include, but are not limited to cystic fibrosis; chronic or acute bronchitis; bronchiectasis (non-CF and CF bronchiectasis); acute tracheitis (bacterial, viral, mycoplasmal or caused by other organisms); acute or chronic sinusitis; atelectasis (lung or lobar collapse) resulting from acute or chronic mucus plugging of the airways (sometimes seen in a variety of diseases such as asthma); and bronchiolitis (viral or other). In some embodiments, the patient has a chronic airway disease selected from, cystic fibrosis (CF), chronic obstructive pulmonary disease, bronchiectasis and asthma.

As used herein, the term "asthma" refers to diseases that present as reversible airflow obstruction and/or bronchial hyper-responsiveness that may or may not be associated with underlying inflammation. Examples of asthma include allergic asthma, atopic asthma, corticosteroid naive asthma, chronic asthma, corticosteroid resistant asthma, corticosteroid refractory asthma, asthma due to smoking, asthma uncontrolled on corticosteroids and other asthmas as mentioned, e.g., in the Expert Panel Report 3: Guidelines for the Diagnosis and Management of Asthma, National Asthma Education and Prevention Program (2007) ("NAEPP Guidelines"), incorporated herein by reference in its entirety. As used herein, the term “severe asthma” has its general meaning in the art and refers to asthma which requires treatment with high doses of corticosteroid and P2-adrenergic receptor agonist to prevent it from becoming uncontrolled or which remains uncontrolled despite therapy.

As used herein, the term "COPD" refers to chronic obstructive pulmonary disease. The term "COPD" includes two main conditions: emphysema and chronic obstructive bronchitis.

As used herein the term "cystic fibrosis" has its general meaning in the art and refers to an inherited autosomal disease associated with mutations in the gene encoding the cystic fibrosis transmembrane conductor regulator (CFTR). The method of the invention may be performed for any type of cystic fibrosis such as revised in the World Health Organisation Classification of cystic fibrosis and selected from the E84 group: mucoviscidosis, Cystic fibrosis with pulmonary manifestations, Cystic fibrosis with intestinal manifestations and Cystic fibrosis with other manifestations. In some embodiments, the subject harbours at least one mutation in the CFTR gene, including, but not limited to F508del-CFTR, R117H-CFTR, and G55 ID CFTR (see, e.g., http://www.genet.sickkids.on.ca/cftr, for CFTR mutations).

As used herein, the term “hyaluronic acid” or “HA” refers to the polymer having the formula: where n is the number of repeating units. All sources of hyaluronic acid are useful in this invention, including bacterial and avian sources. However, hyaluronic acid of bacterial origin is preferable. Hyaluronic acids useful in this invention have a molecular weight from 15,000 to 45,000 Daltons (“low molecular weight”). Preferably, the hyaluronic acid of the present invention has a molecular weight of 25,000 Daltons. In some embodiments, the hyaluronic acid of the present invention is administered to the subject in the form of a salt. In some embodiments, a salt of sodium, potassium, lithium, calcium, barium, strontium, magnesium, aluminum, or ammonium is used. In particular, the hyaluronic acid of the present invention is used in the form of a sodium salt. Commercial sources of hyaluronic acid typically include those from Sigma-Aldrich (e.g. CAS Number: 9067-32-7 or CAS Number: 9067-32-7).

By a "therapeutically effective amount" is meant a sufficient amount of the HA of the present invention for providing a therapeutic effect (for promoting the differentiation of ciliated cells) at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compound will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Preferably, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Typically, the active ingredient of the present invention (i.e. the HA of the present invention) is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. The term "Pharmaceutically" or "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. In the pharmaceutical compositions of the present invention, the active ingredients of the invention can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. In some embodiments, the pharmaceutical composition of the invention is administered topically (i.e. in the respiratory tract of the subject). Therefore, the compositions can be formulated in the form of a spray, aerosol, solution, emulsion, or other form well-known to one of skill in the art. If the method of the invention comprises intranasal administration of a composition, the composition can be formulated in an aerosol form, spray, mist or in the form of drops. In particular, the active ingredients for use according to the present invention can be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebuliser, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (composed of, e.g., gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES: Figure 1: Quantification of the ciliated cell number in the air-liquid interface (ALI) cultures of human F508del / F508del cystic fibrosis (CF) primary airway epithelial cells. Airway epithelial cells were seeded in bi-compartmental chambers and cultured in liquid-liquid condition until confluence was reached. At confluence, culture medium from the upper chamber was removed to create an air-liquid interface (ALI) that will favor epithelial cell differentiation. From the ALI creation, the different treatments (15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa; ARD - Pomade - France - as prepared according to W02004050187), 15-30 kDa sodium-hyaluronic acid (HA-Na 15-30 kDa) (Sigma Aldrich) or 1400 kDa sodium-hyaluronic acid (HA-Na 1400 kDa) (Contipro) were added at 1 mg/mL to the culture medium in the basal chamber. At ALI day 25, immunodetection of a ciliated cell marker (Ari 13b protein) was realized on the different cultures and the number of stained cells was quantified by computer- assisted imaging. n = 10 different F508del / F508del CF patients for HA-Na 15-45 kDa. n = 5 different F508del / F508del CF patients for HA-Na 15-30 kDa. n = 7 different F508del / F508del CF patients for HA-Na 1400 kDa. p < 0.001 (***); p < 0.01 (**); p < 0.05 (*). ns: non-significant.

Figure 2: Quantification of the ciliated cell number in the air-liquid interface (ALI) cultures of human F508del / F508del cystic fibrosis (CF) primary airway epithelial cells. Airway epithelial cells were seeded in bi-compartmental chambers and cultured in liquid-liquid condition until confluence was reached. At confluence, culture medium from the upper chamber was removed to create an air-liquid interface (ALI) that will favor epithelial cell differentiation. From the ALI creation, the different treatments (15-45 kDa sodium-hyaluronic acid: HA-Na 15-45 kDa; ARD - Pomade - France - as prepared according to W02004050187) or 15-45 kDa calcium-hyaluronic acid (HA-Ca 15-45 kDa; ARD), or 15-45 kDa potassium -hyaluronic acid (HA-K 15-45 kDa; ARD)), were added at 1 mg/mL to the culture medium in the basal chamber. At ALI day 25, immunodetection of a ciliated cell marker (Ari 13b protein) was realized on the different cultures and the number of stained cells was quantified by computer-assisted imaging, n = 10 different F508del / F508del CF patients for HA-Na 15-45 kDa. n = 9 different F508del / F508del CF patients for HA-Ca 15-45 kDa. n = 8 different F508del / F508del CF patients for HA-K 15-45 kDa. p < 0.001 (***); p < 0.01 (**); p < 0.05 (*). ns: non-significant.

Figure 3: Quantification of the ciliated cell number in the air-liquid interface (ALI) cultures of human F508del / F508del cystic fibrosis (CF) primary airway epithelial cells. Airway epithelial cells were seeded in bi-compartmental chambers and cultured in liquid-liquid condition until confluence was reached. At confluence, culture medium from the upper chamber was removed to create an air-liquid interface (ALI) that will favor epithelial cell differentiation. From the ALI creation, the treatment (15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa; ARD - Pomade - France - as prepared according to W02004050187) was added at 2, 1, 0.5, 0.1 and 0.01 mg/mL to the culture medium in the basal chamber. At ALI day 25, immunodetection of a ciliated cell marker (Ari 13b protein) was realized on the different cultures and the number of stained cells was quantified by computer-assisted imaging. n = 2 different F508del / F508del CF patients, p < 0.001 (***); p < 0.01 (**); p < 0.05 (*). ns: non-significant.

Figure 4: Quantification of the ciliated cell number in the air-liquid interface (ALI) cultures of human non-cystic fibrosis (CF) primary airway epithelial cells. Airway epithelial cells were seeded in bi -compartmental chambers and cultured in liquid-liquid condition until confluence was reached. At confluence, culture medium from the upper chamber was removed to create an air-liquid interface (ALI) that will favor epithelial cell differentiation. From the ALI creation, the different treatments (15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa) (ARD - Pomade - France - as prepared according to W02004050187), 15-30 kDa sodium-hyaluronic acid (HA-Na 15-30 kDa) (Sigma Aldrich) or 1400 kDa sodium-hyaluronic acid (HA-Na 1400 kDa) (Contipro)) were added at 1 mg/mL to the culture medium in the basal chamber. At ALI day 25, immunodetection of a ciliated cell marker (Ari 13b) was realized on the different cultures and the number of stained wells was quantified by computer-assisted imaging, n = 7 different non-CF patients for HA-Na 15-45 kDa and HA-Na 15-30 kDa. n = 6 different non-CF patients for HA-Na 1400 kDa. p < 0.001 (***) ; p < 0.01 (**); p < 0.05 (*). ns: non-significant.

Figure 5: Quantification of the ciliated cell number in the air-liquid interface (ALI) cultures of human non-cystic fibrosis (CF) primary airway epithelial cells. Airway epithelial cells were seeded in bi -compartmental chambers and cultured in liquid-liquid condition until confluence was reached. At confluence, culture medium from the upper chamber was removed to create an air-liquid interface (ALI) that will favor epithelial cell differentiation. From the ALI creation, the different treatments (15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa) (ARD - Pomade - France - as prepared according to W02004050187), 15-45 kDa calcium-hyaluronic acid (HA-Ca 15-45 kDa) (ARD) or 15-45 potassium-hyaluronic acid (HA- K 15-45 kDa) (ARD)) were added at 1 mg/mL to the culture medium in the basal chamber. At ALI day 25, immunodetection of a ciliated cell marker (Ari 13b) was realized on the different cultures and the number of stained wells was quantified by computer-assisted imaging, n = 7 different non-CF patients, p < 0.001 (***) ; p < 0.01 (**); p < 0.05 (*). ns: non-significant.

Figure 6: Quantification of the ciliated cell number in the air-liquid interface (ALI) cultures of human non-cystic fibrosis (CF) primary airway epithelial cells. Airway epithelial cells were seeded in bi -compartmental chambers and cultured in liquid-liquid condition until confluence was reached. At confluence, culture medium from the upper chamber was removed to create an air-liquid interface (ALI) that will favor epithelial cell differentiation. From the ALI creation, the treatment (15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa; ARD - Pomade - France - as prepared according to W02004050187) was added at 2, 1, 0.5, 0.1 and 0.01 mg/mL to the culture medium in the basal chamber. At ALI day 25, immunodetection of a ciliated cell marker (Ari 13b protein) was realized on the different cultures and the number of stained cells was quantified by computer-assisted imaging. n = 4 different non-CF patients for HA-Na 15-45 kDa at 1, 0.1 and 0.01 mg/mL. n = 3 different non-CF patients for HA-Na 15-45 kDa at 2 and 0.5 mg/mL. p < 0.001 (***); p < 0.01 (**); p < 0.05 (*). ns: non-significant.

Figure 7: Quantification of the ciliated cell number in the air-liquid interface (ALI) cultures of human COPD primary airway epithelial cells. Airway epithelial cells were seeded in bi-compartmental chambers and cultured in liquid-liquid condition until confluence was reached. At confluence, culture medium from the upper chamber was removed to create an air-liquid interface (ALI) that will favor epithelial cell differentiation. From the ALI creation, the different treatments (15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa) (ARD - Pomade - France - as prepared according to W02004050187), 15-30 kDa sodium-hyaluronic acid (HA-Na 15-30 kDa) (Sigma Aldrich) or 1400 kDa sodium-hyaluronic acid (HA-Na 1400 kDa) (Contipro)) were added at 1 mg/mL to the culture medium in the basal chamber. At ALI day 25, immunodetection of a ciliated cell marker (Ari 13b) was realized on the different cultures and the number of stained wells was quantified by computer-assisted imaging, n = 4 different COPD patients, p < 0.001 (***) ; p < 0.01 (**); p < 0.05 (*). ns: non-significant. Figure 8: Quantification of the ciliated cell number in the air-liquid interface (ALI) cultures of human COPD primary airway epithelial cells. Airway epithelial cells were seeded in bi-compartmental chambers and cultured in liquid-liquid condition until confluence was reached. At confluence, culture medium from the upper chamber was removed to create an air-liquid interface (ALI) that will favor epithelial cell differentiation. From the ALI creation, the different treatments (15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa) (ARD - Pomade - France - as prepared according to W02004050187), 15-45 kDa calcium -hyaluronic acid (HA-Ca 15-45 kDa) (ARD) or 15-45 potassium-hyaluronic acid (HA-K 15-45 kDa) (ARD)) were added at 1 mg/mL to the culture medium in the basal chamber. At ALI day 25, immunodetection of a ciliated cell marker (Ari 13b) was realized on the different cultures and the number of stained wells was quantified by computer-assisted imaging, n = 4 different COPD patients, p < 0.001 (***) ; p < 0.01 (**); p < 0.05 (*). ns: non-significant.

EXAMPLE 1:

Sodium form of Hyaluronic Acid of 15-45 kDa exhibits a stimulatory effect of the ciliated cell differentiation during the regeneration of the human Cystic Fibrosis (CF) airway epithelium carrying the F508del / F508del class II mutation (Figure 1). In particular, treatment with 15-45 kDa sodium -hyaluronic acid (HA-Na 15-45 kDa) leads to a significant increase in the number of ciliated cells (8.85 fold) in the cultures of human CF airway epithelial cells, in comparison to control cultures, whereas treatment with 15-30 kDa sodium-hyaluronic acid (HA-Na 15-30 kDa) leads to a 9.99 fold non-significant increase in the number of ciliated cells.

Hyaluronic Acid of 15-45 kDa in its sodium and potassium forms exhibits a stimulatory effect of the ciliated cell differentiation during the regeneration of the human Cystic Fibrosis (CF) airway epithelium carrying the F508del / F508del class II mutation (Figure 2). In particular, treatment with 15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa) leads to a significant increase in the number of ciliated cells (8.85 fold) in the cultures of human CF airway epithelial cells, in comparison to control cultures whereas treatment with 15-45 kDa potassiumhyaluronic acid (HA-K 15-45 kDa) leads to a 4.38 fold non-significant increase in the number of ciliated cells in the cultures of human CF airway epithelial cells, in comparison to control cultures. Sodium form of Hyaluronic Acid of 15-45 kDa exhibits a dose-response stimulatory effect of the ciliated cell differentiation during the regeneration and differentiation of the human Cystic Fibrosis (CF) airway epithelium carrying the F508del / F508del class II mutation (Figure 3). In particular, treatment with 15-45 kDa sodium-hyaluronic acid (HA-Na) leads to an increase in the number of ciliated cells in the cultures of human CF airway epithelial cells at 2 mg/mL (2.45 fold), 1 mg/mL (2.11 fold), 0.5 mg/mL (1.71 fold), 0.1 mg/mL (1.45 fold) and 0.01 mg/mL (1.28 fold), in comparison to control cultures.

EXAMPLE 2:

Sodium form of Hyaluronic Acid of 15-45 kDa exhibits a stimulatory effect of the ciliated cell differentiation during the regeneration of the human airway epithelium of non-Cystic Fibrosis (non-CF) patients (Figure 4). In particular, treatment with 15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa) and 15-30 kDa sodium-hyaluronic acid (HA-Na 15-30 kDa) lead to an increase in the number of ciliated cells (HA-Na 15-45 kDa: 1.66 fold; HA-Na 15-30 kDa: 1.34 fold) in the cultures of human non-CF airway epithelial cells, in comparison to control cultures.

Hyaluronic Acid of 15-45 kDa exhibits a stimulatory effect of the ciliated cell differentiation in its sodium, calcium and potassium forms during the regeneration of the human airway epithelium of non-Cystic Fibrosis (non-CF) patients (Figure 5). In particular, treatment with 15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa) leads to an increase in the number of ciliated cells (1.66 fold) in the cultures of human non-CF airway epithelial cells, in comparison to control cultures, as well as HA-Ca 15-45 kDa (2.15 fold) and HA-K 15-45 kDa (2.21 fold).

Sodium form of Hyaluronic Acid of 15-45 kDa does not exhibit any stimulatory effect of the ciliated cell differentiation during the regeneration and differentiation of the human non-Cystic Fibrosis (non-CF) airway epithelium (Figure 6).

EXAMPLE 3:

Sodium form of Hyaluronic Acid of 15-45 kDa exhibits a stimulatory effect of the ciliated cell differentiation during the regeneration of the human airway epithelium of COPD patients (Figure 7). In particular, treatment with 15-45 kDa sodium-hyaluronic acid (HA-Na 15-45 kDa) and 15-30 kDa sodium-hyaluronic acid (HA-Na 15-30 kDa) lead to an increase in the number of ciliated cells (HA-Na 15-45 kDa: 4.3 fold; HA-Na 15-30 kDa: 3.93 fold) in the cultures of human COPD airway epithelial cells, in comparison to control cultures.

Hyaluronic Acid of 15-45 kDa exhibits a stimulatory effect of the ciliated cell differentiation in its sodium and potassium forms during the regeneration of the human airway epithelium of COPD patients (Figure 8). In particular, treatment with 15-45 kDa sodium -hyaluronic acid (HA-Na 15-45 kDa) leads to an increase in the number of ciliated cells (4.3 fold) in the cultures of human COPD airway epithelial cells, in comparison to control cultures, as well as HA-K 15- 45 kDa (5.1 fold).

REFERENCES:

Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

Claims

CLAIMS:

1. A method of promoting differentiation of ciliated cells in the airway epithelium of a patient suffering from a chronic airway disease comprising administering to the patient a therapeutically effective amount of hyaluronic acid having a low molecular weight from 15,000 to 45,000 Daltons.

2. The method of claim 1 for restoring airway and pulmonary homeostasis.

3. The method of claim 1 for preventing extensive airway mucus plugging in a patient suffering from a chronic airway disease.

4. The method of claim 1 for improving mucociliary clearance in patient suffering from a chronic airway disease.

5. The method according to any one of claims 1 to 4 wherein the patient suffers from asthma, cystic fibrosis or COPD.