3,5-diamino-6-chloro-n-(n-(4-(4-(2-(hexyl (2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

The present invention relates to new compounds, in particular including 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl (2,3,4,5,6 pentahydroxyhexyl) amino ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide and its forms in the form of pharmaceutically acceptable salts, useful as sodium channel blockers, compositions containing them, therapeutic methods and their uses and methods of their preparation.

Background of the invention [0002] A number of "natural defense mechanisms", i.e. protective mechanisms, have developed on the mucosal surface at the interface between the environment and the body. The main form of such natural defense is the cleaning of these surfaces by fluid. The size of the fluid layer on the mucosal surface is usually dependent on the balance between fluid secretion by the epithelium, which often corresponds to anion secretion (Cl^- and / or HCO3^-) in combination with water (and a counterion, i.e. a cation), and the absorption of fluid by the epithelium, which often corresponds to the absorption of Na +, in combination with water and a counterion, i.e. an anion (Cl^- and / or HCO3^-). A number of diseases associated with the mucosal surface result from too little protective fluid on these mucosal surfaces due to imbalances between secretion (too low) and absorption (relatively too high). The abnormal salt transport processes typical of these mucosal functional disorders occur in the epithelial layer of the mucosal surface.

[0003] One method is to supplement the protective fluid layer on the mucosal surface to re-balance the system by blocking the Na + channel and absorbing fluid. The epithelial protein mediating the stage of Na + absorption and the fluid on which the speed of the process depends is the epithelial Na + channel ("ENaC"). ENaC is located on the apical epithelial surface, i.e. on the interface between the mucosa and the environment. In the best situation, to inhibit Na + and ENaC-dependent fluid absorption, an ENaC blocker from the group of amiloride derivatives is administered to the mucosal surface and maintained in this place for maximum therapeutic benefit. [0004] The use of ENaC blockers has been reported in a wide variety of diseases whose course is beneficial for increasing mucosal hydration. In particular, ENaC blockers have been reported to treat respiratory diseases such as chronic bronchitis (PZO), cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD) that result from the body being unable to properly remove mucus from the lungs, which eventually leads to chronic respiratory infection. See Evidence for airway surface dehydration as the initiating event in CF airway disease, RC Boucher, Journal of Internal Medicine, volume 261, number 1, January 2007, pages 5-16; and Cystic fibrosis: a disease of vulnerability to airway surface dehydration, RC Boucher, Trends in Molecular Medicine, volume 13, number 6, June 2007, pages 231-240.

[0005] Data indicate that the initial problem for both chronic bronchitis and cystic fibrosis is impaired mucus clearance from the surface of the airways. The impairment of mucus clearance results from the imbalance of the amount of mucus in the fluid present on the airway surface (ASL) on the airway surface. These imbalances result in a relative reduction in ASL, which leads to mucus concentration, reduction of the lubricating activity of the ciliary fluid (PCL), mucus adhesion to the surface of the airways, and impairment of mucus clearance due to ciliary activity in the mouth. Reducing mucus removal leads to chronic colonization of mucus on the surface of the airways. Chronic persistence of bacteria, the inability of local antimicrobial substances to destroy bacteria trapped in chronic mucus and the resulting chronic inflammation in this type of surface infection are symptoms occurring in chronic bronchitis and cystic fibrosis.

[0006] There is currently a significant and unmet medical need for products that would specifically treat a wide variety of diseases that have a beneficial effect on mucosal hydration, including chronic bronchitis, COPD and cystic fibrosis. Current treatments for chronic bronchitis, COPD and cystic fibrosis focus on treating the symptoms and / or late effects of these diseases. However, none of these treatments allows effective treatment of the underlying disorder, i.e. impaired mucus clearance from the lungs.

[0007] RC Boucher in US 6,264,975 describes the use of sodium channel blockers derived from pyrazinoylguuanidine, examples of which are the well-known diuretics amiloride, benzamil and fenamil, for moisturizing the mucosal surface. However, these compounds have relatively low potency, given the limited mass of the drug that can enter the lung by inhalation; (2) they are rapidly absorbed, therefore they show an undesirably short half-life on the mucosal surface; moreover (3) they are easily dissociated from ENaC. Drugs with higher potency and a longer half-life on the mucosal surface are needed.

[0008] Too little protective fluid on the surface for other mucosal surfaces is a typical pathophysiological factor in many diseases. For example, in the case of xerostomia (dry mouth), the amount of fluid in the mouth is reduced due to impaired fluid secretion by the sublingual and submandibular glands, despite the constant absorption of Na + (ENaC) dependent fluid in the mouth. Dry conjunctivitis (dry eye syndrome) results from impaired fluid secretion by the lacrimal glands with continuous absorption of Na + dependent fluid on the conjunctival surfaces. In the case of sinusitis, there is an imbalance between mucin secretion and a relative reduction in ASL. Impaired secretion of Cl^- (and fluid) in the proximal small intestine, combined with an increase in Na + (and fluid) uptake in the terminal ileum leads to the disorder of the ileum (DIOS). Excessive Na + (and fluid) absorption in the descending colon causes older patients constipation and diverticulitis.

[0009] The published literature includes a number of patent applications and patents granted to Parion Sciences Inc. regarding analogs of pyrazinoylguuanidine blockers of sodium channels. Examples of such publications include PCT Publication Nos. WO2003 / 070182, WO2003 / 070184, WO2004 / 073629, WO2005 / 025496, WO2005 / 016879, WO2005 / 018644,

WO2006 / 022935, WO2006 / 023573, WO2006 / 023617, WO2007 / 018640,

WO2007 / 146869, WO2008 / 031028, WO2008 / 031048 and US Patents No. 6858614,

6858615, 6903105, 7064129, 7186833, 7189719, 7192958, 7192959, 7192960,

7241766, 7247636, 7247637, 7317013, 7332496, 7368447, 7368450, 7368451, 7375102, 7388013, 7399766, 7410968, 7807834, 7842697 and 7868010.

[0010] There is still a need for new sodium channel blockers with increased potency and efficacy against mucosal tissues. There is also still a need for new sodium channel blocking compounds that have therapeutic effects but minimize or eliminate the onset or progression of hyperkalemia in patients.

Summary of the invention [0011] The present invention provides the 3,5-diamino-6-chloro-N- (N- (4- (420 (2- (hexyl (2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) compound phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide with the formula:

<img file="PL2723722T3_D0001.tif" />

or its form in the form of a pharmaceutically acceptable salt. The invention further provides solvates and hydrates, individual stereoisomers, including optical isomers (enantiomers and diastereomers) and geometric isomers (cis / trans isomerism), mixtures of stereoisomers and tautomers 3,5-diamino-6-chloro-N- (N- (4 (4- (2- (hexyl (2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide or a pharmaceutically acceptable salt thereof, as well as pharmaceutical compositions containing this compound or a pharmaceutically acceptable salt thereof, its use in methods of treatment and methods of its production.

BRIEF DESCRIPTION OF THE DRAWINGS [0012] The invention will be better understood and many of its benefits may be easily determined in relation to the information provided herein in connection with the following drawings: FIG. 1 is a representative plot of concentration versus compound (Ia) effect on short-circuit current obtained using canine bronchial epithelial cells (CBE).

FIG. 2 is a graph of the effect of the dose of compound (Ia) on the response relative to the value of mucociliary clearance (MCC) in sheep 4 h after dosing.

FIG. 3 is a graph of the effect of compound (Ia) and hypertonic sodium chloride (HS) on MCC in sheep 4 h after dosing.

FIG. 4 is a graph of the effect of compound (Ia) and HS on MCC in sheep 8 h after dosing.

FIG. 5 is a graph of the effect of blocking the sodium channels of compound (Ia) on surface fluid retention during 0-8h in an in vitro CBE cell model. FIG. 6 is a bar graph of the effect of Compound (Ia) on surface fluid retention after 24 hours in an in vitro CBE model.

FIG. 7 is a graph of the effect of compound (Ia) and compound ENaC blockers according to Comparative Example 1 on MCC in sheep after 8 hours.

FIG. 8 is a graph of the effect of compound (Ia) and compound ENaC blockers according to Comparative Example 1 on sheep potassium levels.

FIG. 9 is a graph comparing the activity of the compound of Comparative Example 4 and compound I with respect to MCC in sheep 4 h after dosing.

FIG. 10 is a graph that compares the effect of compound of Comparative Example 4 and compound Ia on level K⁺ in plasma in sheep.

Detailed description of the invention [0013] The following terms used in this document have the following meanings:

[0014] "A compound of the invention" means a compound of formula I or a salt thereof, in particular a pharmaceutically acceptable salt thereof.

[0015] "Compound of formula I" means a compound having the structural formula defined herein as formula I. Compounds of formula I include solvates and hydrates (ie, adducts of a compound of formula I with a solvent). In embodiments in which the compound of formula I contains one or more chiral centers, the term is intended to include any single stereoisomer, including optical isomers (enantiomers and diastereomers), and geometric isomers (cis / trans isomerism) and mixtures of stereoisomers. In addition, compounds of formula I also include tautomers of the formula or formulas given.

[0016] Throughout the description and examples, compound names were given using the standard IUPAC naming rules whenever possible, including ChemDraw Ultra 11.0 software available from CambridgeSoft Corp./PerkinElmer to name the compounds.

[0017] In some representations of chemical structures in which the carbon atoms do not contain enough attached variable atoms to obtain the valence of four, it is assumed that the remaining substituents on the carbon atoms necessary to obtain the valence of four are hydrogen. Similarly, in some chemical structures in which a bond is drawn without specifying an end group, such a bond indicates a methyl group (Me, -CH₃), as is commonly used in the art.

[0018] In one preferred embodiment, the compound of formula (I) is 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R ) -2,3,4,5,6 pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide with the formula:

<img file="PL2723722T3_D0002.tif" />

or a pharmaceutically acceptable salt thereof.

[0019] The compounds of formula I may exist in the form of the free base or a salt, in particular a pharmaceutically acceptable salt. A review of pharmaceutically acceptable salts is found in Berge et al., J. Pharma Sci. (1977) 66: 1-19. [0020] Pharmaceutically acceptable salts formed from inorganic or organic acids include, for example, the hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, nitrate, sulfamate, phosphate, hydrogen phosphate, acetate, trifluoroacetate, maleate, malate, fumarate, lactate, tartrate, citrate, formate , gluconate, succinate, pyruvate, tannin, ascorbate, palmitate, salicylate, stearate, phthalate, alginate, polyglutamate, oxalate, oxaloacetate, saccharinate, benzoate, alkyl or aryl sulfonates (e.g., methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate or naphthalenesulfonate) and isothionate; complexes formed with amino acids such as lysine, arginine, glutamic acid, glycine, serine, threonine, alanine, isoleucine, leucine and the like. The compounds of the invention may furthermore be in the form of salts formed from elemental anions such as chlorine, bromine or iodine.

[0021] For therapeutic use, the salts of the active compounds of the formula I are pharmaceutically acceptable, i.e. they are salts derived from a pharmaceutically acceptable acid. Salts of pharmaceutically unacceptable acids may, however, also find use, for example in the preparation or purification of a pharmaceutically acceptable compound. For example, trifluoroacetate salts may find such use. All salts are included within the scope of this invention, whether or not derived from a pharmaceutically acceptable acid.

[0022] The term "chiral" refers to particles characterized in that they cannot be superimposed on their mirror image, and the term "achiral" refers to molecules characterized in that they can be superimposed on their mirror image.

[0023] The term "stereoisomers" refers to compounds having identical chemical composition but differing in the arrangement of atoms or groups in space. "Diastereomer" refers to a stereoisomer having two or more centers of chirality whose molecules are not mirror images of each other. Diastereomers differ in physical properties, e.g. melting point, boiling point, spectral properties and reactivity. Mixtures of diastereomers can be separated by high resolution analytical methods such as electrophoresis and chromatography. "Enantiomers" means two stereoisomers of a compound that are non-superimposing mirror images of each other.

[0024] The definitions of stereochemical terms and conventions used herein are generally in accordance with SP Parker, ed., McGRAWHILL DICTIONARY OF CHEMICAL TERMS (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., STEREOCHEMISTRY OF ORGANIC COMPOUNDS (1994) John Wiley &amp; Sons, Inc., New York.

[0025] Many organic compounds exist in optically active forms, i.e. they have the ability to rotate a plane of linearly polarized light. In the description of an optically active compound, the prefixes D and L or R and S are used to determine the absolute configuration of the molecule around its center or centers of chirality. A particular stereoisomer can also be termed an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or racemate; it can occur if the chemical reaction or process was not stereoselective or stereospecific. The terms "racemic mixture" and "racemate" mean an equimolar mixture of two enantiomeric compounds.

[0026] The term "tautomers" refers to a type of stereoisomer in which migration of a hydrogen atom produces two or more structures.

Compounds of formula I may exist in various tautomeric forms. The skilled person will understand that amidines, amides, guanidine derivatives, urea derivatives, thiourea derivatives, heterocyclic compounds and the like can exist in tautomeric forms. By way of example and not limitation, the compounds of Formula I may exist in various tautomeric forms as set out below:

<img file="PL2723722T3_D0003.tif" />

[0027] All possible tautomeric forms of amidines, amides, guanidine derivatives, urea derivatives, thiourea derivatives, heterocyclic compounds and the like according to all embodiments of formula I are within the scope of the present invention. Tautomers are in equilibrium, so those skilled in the art will understand that the description of a single tautomer in the formulas given applies equally to all possible tautomers.

[0028] It should be understood that all enantiomers, diastereomers and racemic mixtures, tautomers, polymorphs and pseudopolymorphs of compounds within the scope of formula I and their pharmaceutically acceptable salts fall within the scope of the present invention. All mixtures of such enantiomers and diastereomers, including enantiomerically enriched mixtures and diastereomerically enriched mixtures, are within the scope of the present invention. Enantiomerically enriched mixtures are mixtures of enantiomers in which the ratio of the specific enantiomer to the alternative enantiomer exceeds 50:50. In particular, the enantiomerically enriched mixture contains at least about 75% of the specified enantiomer and preferably at least about 85% of the specified enantiomer. In one embodiment, the enantiomerically enriched mixture does not substantially contain a second enantiomer. Similarly, diastereomerically enriched mixtures are mixtures of diastereomers in which the content of the specific diastereomer is greater than the content of each alternative diastereomer. In particular, the diastereomerically enriched mixture contains at least about 75% of the specified diastereomer and preferably at least about 85% of the specified diastereomer. In one embodiment, the diastereomerically enriched mixture essentially does not contain any other diastereomers. The term "substantially free" is understood by those skilled in the art to mean less than 5% of the remaining diastereomers, preferably less than 1%, more preferably less than 0.1%. In other embodiments, no remaining diastereomers are present or the content of any other diastereomer present is lower than the detection level. Stereoisomers can be separated by methods known in the art, including high performance liquid chromatography (HPLC) and crystallization of chiral salts.

[0029] A single stereoisomer, e.g. an enantiomer, substantially free of the corresponding stereoisomer, can be obtained by resolving a racemic mixture of a compound using a method such as the formation of diastereomers using optically active separation reagents ("Stereochemistry of Carbon Compounds," (1962), EL Eliel, McGraw Hill; Lochmuller, CH, (1975) J. Chromatogr., 113: (3) 283-302). Racemic mixtures of the chiral compounds of the invention may be separated and isolated by any suitable method, including: (1) formation of ionic diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with chiral derivatizing reagents, separation of diastereomers and conversion into pure stereoisomers and (3) separation of essentially pure or enriched stereoisomers directly under conditions chiral.

[0030] For illustrative purposes, specific examples of enantiomers of a compound of formula (I) falling within the scope of this invention include, but are not limited to:

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,610 pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0004.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3S, 4S, 5S) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy phenyl) butyl) carbamimidoyl) pyrazine-215 carboxamide

<img file="PL2723722T3_D0005.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy ) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0006.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3S, 4S, 5R) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy ) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0007.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5S) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy ) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0008.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3S, 4R, 5S) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy ) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0009.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4S, 5R) -2,3,4,5,8pentahydroksyheksylo) amino) ethoxy ) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0010.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3R, 4S, 5S) -2,3,4,5,6pentahydroksyhoksylo) amino) ethoxy ) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0011.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3S, 4R, 5R) -2,3,4,5,6pentahydroksyhoksylo) amino) ethoxy ) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0012.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3R, 4R, 5R) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy ) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0013.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3S, 4S, 5S) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy ) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0014.tif" />

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2R, 3S, 4S, 5S) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy ) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0015.tif" />

and

3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy phenyl) butyl) carbamimidoyl) pyrazine carboxamide

<img file="PL2723722T3_D0016.tif" />

[0031] In one embodiment, the present invention provides an enantiomerically enriched mixture or composition containing 5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2 , 3,4,5,610 pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide or a pharmaceutically acceptable salt thereof as the main isomer. [0032] Other embodiments include enantiomerically enriched mixtures or compositions containing compounds of formulas (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), respectively Ii), (Ij), (Ik) and (II) or a pharmaceutically acceptable salt thereof as the main isomer in each of their respective mixtures. [0033] In another embodiment, the present invention provides an enantiomerically enriched mixture or composition containing 5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2 , 3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-220 carboxamide or a pharmaceutically acceptable salt thereof, essentially free of other isomers.

[0034] Four other embodiments include enantiomerically enriched mixtures or compositions containing compounds of formulas (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), respectively, (Ii), (Ij), (Ik) and (II) or a pharmaceutically acceptable salt thereof, substantially free of other isomers in each of their respective mixtures.

[0035] The compound of formula I and its pharmaceutically acceptable salts may exist in the form of various polymorphs or pseudopolymorphs. The term crystalline polymorphism as used herein means the possibility of the presence of a crystalline compound in various crystal structures. Crystalline polymorphism may result from differences in crystal packing (polymorphism due to packing) or differences in packing between different conformers of the same molecule (conformational polymorphism). The term crystal pseudopolymorphism as used herein further includes the possibility of the hydrate or solvate being present in various crystal structures. The pseudopolymorphs of the present invention may occur due to differences in crystal packing (pseudopolymorphism due to packing) or due to differences in packing between different conformers of the same molecule (conformational pseudopolymorphism). The present invention includes all polymorph and pseudopolymorphic forms of the compounds of formula I and their pharmaceutically acceptable salts.

[0036] The compound of formula I and its pharmaceutically acceptable salts may also exist in the form of an amorphous solid. The term amorphous solid as used herein means a solid in which there is no order in the long range of atom positions in the solid. This definition also applies when the crystal size is two nanometers or less. Additives, including solvents, can be used to make amorphous forms of the present invention. The present invention, including all pharmaceutical compositions, methods of treatment, combination products and their uses described herein, includes all amorphous forms of the compounds of Formula I and their pharmaceutically acceptable salts.

APPLICATIONS [0037] The compounds of the invention show sodium channel blocker activity. Without being limited to any particular theory, it is believed that the compounds of the invention can function in vivo by blocking epithelial sodium channels located on the mucosal surface and thereby reduce water absorption through the mucosal surfaces. This effect increases the volume of protective fluids on mucosal surfaces and restores balance in the system.

[0038] Accordingly, the compounds of the invention are useful as medicaments, in particular for the treatment of clinical conditions for which a sodium channel blocker may be indicated. These conditions include lung diseases, for example, diseases associated with reversible or irreversible airway obstruction, chronic obstructive pulmonary disease (COPD), including sudden exacerbation of COPD, asthma, bronchiectasis (including bronchiectasis associated with diseases other than cystic fibrosis), acute bronchitis, chronic bronchitis, cough after viral infection, cystic fibrosis, emphysema, pneumonia, bronchiolitis and post-transplant bronchiolitis, including bronchiolitis after lung and bone marrow transplantation, in a human in need of such treatment. The compounds of the invention may also be useful in the treatment of respiratory tracheitis and bronchitis and / or prevention of respiratory pneumonia in mechanically ventilated patients. The present invention includes compounds for use in methods of treating each of these diseases in a mammal in need thereof, preferably in a man in need thereof, comprising administering to said mammal a pharmaceutically effective amount of a compound of the present invention or a pharmaceutically acceptable salt thereof. Further provided are (a) compounds for use in a method of reducing the exacerbation of COPD in a mammal in need thereof; (b) compounds for use in a method of limiting the exacerbation of CF in a mammal in need thereof; (c) compounds for use in a method of improving lung function (FEV1) in a mammal in need thereof, (d) compounds for use in a method of improving lung function (FEV1) in a mammal having COPD, (e) compounds for use in a method of improving lung function (FEV1) in a mammal having CF, (f) compounds for use in a method of reducing respiratory infection in a mammal in need thereof.

[0039] Also provided are compounds for use in a method of stimulating, increasing or improving mucociliary clearance in a mammal, the method comprising administering to a mammal in need thereof a pharmaceutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof. Mucociliary clearance is understood to include natural mucociliary activity associated with the transport or removal of mucus from the airways, including mechanisms for self-cleaning of bronchi. Accordingly, compounds are further provided for use in a method of improving mucus airway clearance in a mammal in need thereof.

[0040] In addition, sodium channel blockers may be indicated in the treatment of diseases whose course is favorably increased by mucosal hydration on mucosal surfaces other than lung mucosal surfaces. Examples of such diseases include dry mouth (xerostomy), dry skin, vaginal dryness, sinusitis, sinusitis, dry nose, including dry nose caused by dry oxygen, dry eye syndrome, Sjogren's disease, otitis media, primary ciliary dyskinesia, distal bowel obstruction syndrome, esophagitis, constipation and chronic diverticulitis. The compounds of the invention may also be used to improve the hydration of the eye or cornea.

[0041] The compounds of the present invention may also be useful compounds for use in human sputum sample collection methods that can be performed by administering a compound of the invention to at least one lung of a patient, followed by coughing up, and sputum sample from that human.

[0042] Therefore, in one aspect, the present invention provides compounds for use in a method of treating a disease in a mammal, for example a human, in whom a sodium channel blocker is indicated.

[0043] In other embodiments, the present invention provides each of the compounds for use in the methods described herein with the additional benefit of minimizing or eliminating hyperkalemia in an individual using the method. In addition, embodiments are provided comprising each of these compounds for use in a method described herein in which an increased therapeutic index is obtained.

[0044] The terms "treat", "treating" and "treatment" as used herein mean reversal, improvement of the condition, inhibition of progression or prevention of a disorder or disease or of one or more symptoms of such disorder or disease.

[0045] All therapeutic methods described herein are carried out by administering an effective amount of a compound of the invention, a compound of formula I, or a pharmaceutically acceptable salt thereof to a subject (usually a mammal and preferably a human) in need of treatment.

[0046] In one embodiment, the invention provides compounds for use in a method of treating a disease where the course is favorably increased by mucosal hydration in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating a disease associated with reversible or irreversible airway obstruction in a mammal, in particular a human in need thereof. In one particular embodiment, the present invention provides compounds for use in a method of treating chronic obstructive pulmonary disease (COPD) in a mammal, in particular a human in need thereof. In one particular embodiment, the present invention provides compounds for use in a method of reducing the frequency, severity or duration of a sudden COPD exacerbation or for treating one or more symptoms of a sudden COPD exacerbation in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating asthma in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating bronchiectasis (including bronchiectasis caused by diseases other than cystic fibrosis) in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating bronchitis, including acute and chronic bronchitis, in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating cough after a viral infection in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating cystic fibrosis in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating emphysema in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating pneumonia in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treatment of bronchiolitis in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating post-transplant bronchiolitis, including bronchiolitis after lung and bone marrow transplantation, in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating respiratory tracheitis and bronchitis and / or preventing respiratory pneumonia in a mechanically ventilated human in need thereof.

[0047] The present invention provides specific compounds for use in methods of treating a disease selected from the group consisting of reversible or irreversible airway obstruction, chronic obstructive pulmonary disease (COPD), asthma, bronchiectasis (including bronchiectasis associated with diseases other than cystic fibrosis), acute bronchitis, chronic bronchitis, cough after viral infection, cystic fibrosis, emphysema, pneumonia, bronchiolitis, bronchiolar transplantation and respiratory tracheitis and bronchitis or the prevention of respiratory pneumonia in a human in need thereof, wherein each compound is used in a method comprising administering to said man an effective amount of a compound of formula (Ia) or a pharmaceutically acceptable salt thereof. In additional embodiments, for each compound for use in a method of treatment, the pharmaceutically acceptable salt form is the hydrochloride salt or hydroxynaphthoate salt of the compound of formula (Ia). In another embodiment, the free base of the compound of formula (Ia) is used within each compound for use in a method of treatment.

[0048] In one embodiment, the invention provides compounds for use in a method of treating dry mouth (xerostomy) in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating dry skin in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating vaginal dryness in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating sinusitis, paranasal sinusitis, or nasal dryness, including nasal dryness caused by dry oxygen, in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating dry eye syndrome or Sjogren's disease or improving the hydration of the eye or cornea in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating otitis media in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating primary ciliary dyskinesia in a mammal, in particular a human in need thereof. In one embodiment, the invention provides compounds for use in a method of treating distal bowel obstruction syndrome, esophagitis, constipation or chronic diverticulitis in a mammal, in particular a human in need thereof.

[0049] Also provided is a compound of the invention for use in medical therapy, especially for use in treating a disease in a mammal, such as a human, in whom a sodium channel blocker is indicated. All therapeutic uses described herein are accomplished by administering to a subject in need of treatment an effective amount of a compound of the invention. In one embodiment, a compound of the invention is provided for use in the treatment of lung disease, e.g., a disease associated with reversible or irreversible airway obstruction in a mammal, in particular a human in need thereof. In one specific embodiment, a compound of the invention is provided for use in the treatment of chronic obstructive pulmonary disease (COPD) in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use for reducing the frequency, severity or duration of a sudden COPD exacerbation or for treating one or more symptoms of a sudden COPD exacerbation in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in treating asthma in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of bronchiectasis, including bronchiectasis caused by diseases other than cystic fibrosis, or bronchitis, including acute bronchitis and chronic bronchitis, in a mammal, in particular a human, demanding. In one embodiment, a compound of the invention is provided for use in the treatment of cough after viral infection in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of cystic fibrosis in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of emphysema in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of pneumonia in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of bronchiolitis or post-transplant bronchiolitis, including bronchiolitis after lung and bone marrow transplantation, in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in treating respiratory tracheitis and bronchitis or preventing respiratory pneumonia in a mechanically ventilated human in need thereof.

[0050] In one embodiment, a compound of the invention is provided for use in treating a disease whose course is favorably increased by mucosal hydration on mucosal surfaces in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of dry mouth (xerostomy) in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of dry skin of a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of vaginal dryness in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of sinusitis, paranasal sinusitis, or nasal dryness, including nasal dryness caused by dry oxygen, in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of dry eye syndrome or Sjogren's disease or to improve the hydration of the eye or cornea in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of otitis media in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of primary ciliary dyskinesia in a mammal, in particular a human in need thereof. In one embodiment, a compound of the invention is provided for use in the treatment of distal bowel obstruction syndrome, esophagitis, constipation or chronic diverticulitis in a mammal, in particular a human in need thereof.

[0051] The present invention further provides the use of a compound of the invention for the manufacture of a medicament for treating a disease in a mammal, for example a human in whom a sodium channel blocker is indicated. In one embodiment, the use of a compound of the invention is provided for the manufacture of a medicament for the treatment of diseases associated with reversible or irreversible airway obstruction, chronic obstructive pulmonary disease (COPD), sudden COPD exacerbation, asthma, bronchiectasis (including bronchiectasis associated with other diseases) than cystic fibrosis), bronchitis (including acute bronchitis and chronic bronchitis), cough after viral infection, cystic fibrosis, emphysema, pneumonia, bronchiolitis, post-transplant bronchiolitis (including bronchiolitis after lung and bone marrow transplantation), respiratory tracheitis and bronchitis, or prevention of respiratory pneumonia.

[0052] In one embodiment, there is provided the use of a compound of the invention for the manufacture of a medicament for the treatment of a disease whose course is favorably increased by mucosal hydration on mucosal surfaces, to treat dry mouth (xerostomy), dry skin, vaginal dryness, inflammation sinusitis, sinusitis, dry nose, including dry nose caused by dry oxygen, for the treatment of dry eye syndrome, Sjogren's disease, improving eye or corneal hydration, for the treatment of otitis media, primary ciliary dyskinesia, distal bowel syndrome, esophagitis, constipation or chronic diverticulitis.

[0053] The terms "effective amount", "pharmaceutically effective amount", "effective dose" and "pharmaceutically effective dose" as used herein mean an amount of a compound of the invention sufficient in the subject to whom it is administered to elicit the desired, for example, by the researcher or the clinician has a biological or medical response in cell culture, tissue, system or mammal (including man). The scope of this concept also includes amounts that effectively increase normal physiological function. In one embodiment, the effective amount is the amount necessary to achieve the desired level of drug in the secretions and tissues of the airways and lungs, or optionally in the bloodstream of the subject being treated, to achieve the expected physiological response or desired biological effect if such composition is administered by inhalation. For example, an effective amount of a compound of the invention for treating a disease for which a sodium channel blocker is indicated is sufficient in the subject being administered to treat the specific disease. In one embodiment, the effective amount is an amount of a compound of the invention sufficient to treat COPD or cystic fibrosis in man.

[0054] The exact effective amount of the compounds of the invention depends on a number of factors, including but not limited to the species, age and weight of the subject being treated, the particular disease requiring treatment and its severity, the bioavailability, potency and other properties of the particular compound administered, on the nature of the preparation, route of administration and administration device, which ultimately depends on the discretion of the attending physician or veterinarian. Additional guidance on the appropriate dose can be formulated taking into account the conventional dosing of other sodium channel blockers such as amiloride, taking due account also of any differences in potency between amiloride and the compounds of the present invention.

[0055] A pharmaceutically effective dose administered topically to the subject's airway surfaces (e.g., by inhalation) for a compound of the invention and for treatment of a human body having a body weight of 70 kg remains in the range of about 10 ng to about 10 mg. In another embodiment, a pharmaceutically effective dose may be from about 0.1 to about 1000 μg. The daily dose administered topically to the respiratory tract surfaces is usually sufficient to obtain a dissolved active substance concentration on the respiratory tract surfaces from about 10^-9, 10^-8 or 10^-7 up to about 10^-4, 10^-3, 10^-2 or 10^-1 moles per liter, more preferably from about 10^-9 up to about 10^-4 moles per liter. The choice of the appropriate dose for the patient is made by a physician, clinician or veterinarian who is a specialist in the field based on a number of factors, including those mentioned above. In one particular embodiment, the dose of a compound of the invention for treating a human body weight of 70 kg ranges from about 10 nanograms (ng) to about 10 mg. In another embodiment, the effective dose is from about 0.1 μg to about 1000 μg. In one embodiment, the dose of a compound of the invention for treating a human body weight of 70 kg remains in the range of from about 0.5 μg to about 0.5 mg. In another embodiment, the dose is from about 0.5 μg to about 60 μg. In another embodiment, the pharmaceutically effective dose is from about 1 to about 10 μg. In another embodiment, the pharmaceutically effective dose is from about 5 μg to about 50 μg. In another embodiment, the effective dose is from about 10 μg to about 40 μg. In two further embodiments, the pharmaceutically effective dose is from about 15 μg to about 50 μg and from about 15 μg to about 30 μg, respectively. It should be understood that each of these dose ranges includes all doses within this range. For example, the 0.550 μg range includes the following individual doses: 0.5 gg, 0.6 μg, 0.7 gg, 0.8 gg,

0.9 gg, 1.0 gg, 1.1 gg, 1.2 gg, 1.3 gg, 1.4 gg, 1.5 gg, 1.6 gg, 1.7 gg, 1.8 gg, 1.9 gg, 2.0 gg, 2.1 gg, 2.2 gg, 2.3 gg, 2.4 gg, 2.5 gg, 2.6 gg, 2.7 gg, 2.8 gg, 2.9 gg,

3.0 gg, 3.1 gg, 3.2 gg, 3.3 gg, 3.4 gg, 3.5 gg, 3.6 gg, 3.7 gg, 3.8 gg, 3.9 gg, 4.0 gg, 4.1 gg, 4.2 gg, 4.3 gg, 4.4 gg, 4.5 gg, 4.6 gg, 4.7 gg, 4.8 gg, 4.9 gg, 5.0 gg,

5.1 gg, 5.2 gg, 5.3 gg, 5.4 gg, 5.5 gg, 5.6 gg, 5.7 gg, 5.8 gg, 5.9 gg, 6.0 gg, 6, 1 gg, 6.2 gg, 6.3 gg, 6.4 gg, 6.5 gg, 6.6 gg, 6.7 gg, 6.8 gg, 6.9 gg, 7.0 gg, 7, 1 gg,

7.2 gg, 7.3 gg, 7.4 gg, 7.5 gg, 7.6 gg, 7.7 gg, 7.8 gg, 7.9 gg, 8.0 gg, 8.1 gg, 8, 2 gg, 8.3 gg, 8.4 gg, 8.5 gg, 8.6 gg, 8.7 gg, 8.8 gg, 8.9 gg, 9.0 gg, 9.1 gg, 9, 2 gg,

9.3 gg, 9.4 gg, 9.5 gg, 9.6 gg, 9.7 gg, 9.8 gg, 9.9 gg, 10.0 gg, 10.1 gg, 10.2 gg,

10.3 gg, 10.4 gg, 10.5 gg, 10.6 gg,

11.2 gg, 11.3 gg, 11.4 gg, 11.5 gg,

12.1 gg, 12.2 gg, 12.3 gg, 12.4 gg, 13.0 gg, 13.1 gg, 13.2 gg, 13.3 gg, 13.9 gg, 14.0 gg, 14, 1 gg, 14.2 gg,

14.8 gg, 14.9 gg, 15.0 gg, 15.1 gg,

15.7 gg, 15.8 gg, 15.9 gg, 16.0 gg,

16.6 gg, 16.7 gg, 16.8 gg, 16.9 gg,

17.5 gg, 17.6 gg, 17.7 gg, 17.8 gg,

18.4 gg, 18.5 gg, 18.6 gg, 18.7 gg,

19.3 gg, 19.4 gg, 19.5 gg, 19.6 gg,

20.2 gg, 20.3 gg, 20.4 gg, 20.5 gg,

21.1 gg, 21.2 gg, 21.3 gg, 21.4 gg, 22.0 gg, 22.1 gg, 22.2 gg, 22.3 gg,

22.9 gg, 23.0 gg, 23.1 gg, 23.2 gg,

23.8 gg, 23.9 gg, 24.0 gg, 24.1 gg,

24.7 gg, 24.8 gg, 24.9 gg, 25.0 gg,

25.6 gg, 25.7 gg, 25.8 gg, 25.9 gg,

26.5 gg, 26.6 gg, 26.7 gg, 26.8 gg,

27.4 gg, 27.5 gg, 27.6 gg, 27.7 gg,

28.3 gg, 28.4 gg, 28.5 gg, 28.6 gg,

29.2 gg, 29.3 gg, 29.4 gg, 29.5 gg, 30.1 gg, 30.2 gg, 30.3 gg, 30.4 gg, 31.0 gg, 31.1g g, 31, 2 gg, 31.3 gg,

31.9 gg, 32.0 gg, 32.1 gg, 32.2 gg,

10.7 gg, 10.8 gg, 10.9 gg, 11.0 gg, 11.1 gg,

11.6 gg, 11.7 gg, 11.8 gg, 11.9 gg, 12.0 gg,

12.5 gg, 12.6 gg, 12.7 gg, 12.8 gg, 12.9 gg,

13.4 gg, 13.5 gg, 13.8 gg, 13.7 gg, 13.8 gg,

14.3 gg, 14.4 gg, 14.5 gg, 14.6 gg, 14.7 gg,

15.2 gg, 15.3 gg, 15.4 gg, 15.5 gg, 15.6 gg,

16.1 gg, 16.2 gg, 16.3 gg, 16.4 gg, 16.5 gg,

17.0 gg, 17.1 gg, 17.2 gg, 17.3 gg, 17.4 gg,

17.9 gg, 18.0 gg, 18.1 gg, 18.2 gg, 18.3 gg,

18.8 gg, 18.9 gg, 19.0 gg, 19.1 gg, 19.2 gg,

19.7 gg, 19.8 gg, 19.9 gg, 20.0 gg, 20.1 gg,

20.6 gg, 20.7 gg, 20.8 gg, 20.9 gg, 21.0 gg,

21.5 gg, 21.6 gg, 21.7 gg, 21.8 gg, 21.9 gg,

22.4 gg, 22.5 gg, 22.6 gg, 22.7 gg, 22.8 gg,

23.3 gg, 23.4 gg, 23.5 gg, 23.6 gg, 23.7 gg,

24.2 gg, 24.3 gg, 24.4 gg, 24.5 gg, 24.6 gg,

25.1 gg, 25.2 gg, 25.3 gg, 25.4 gg, 25.5 gg,

26.0 gg, 20.1 gg, 26.2 gg, 26.3 gg, 26.4 gg,

26.9 gg, 27.0 gg, 27.1 gg, 27.2 gg, 27.3 gg,

27.8 gg, 27.9 gg, 28.0 gg, 28.1 gg, 28.2 gg,

28.7 gg, 28.8 gg, 28.9 gg, 29.0 gg, 29.1 gg,

29.6 gg, 29.7 gg, 29.8 gg, 29.9 gg, 30.0 gg,

30.5 gg, 30.6 gg, 30.7 gg, 30.8 gg, 30.9 gg,

31.4 gg, 31.5 gg, 31.6 gg, 31.7 gg, 31.8 gg,

32.3 gg, 32.4 gg, 32.5 gg, 32.6 gg, 32.7 gg,

<td> 32,8</td><td>gg</td><td> 32,9</td><td>gg</td><td> 33,0</td><td>gg</td><td> 33,1</td><td>gg</td><td> 33,2</td><td>gg</td><td> 33,3</td><td>gg</td><td> 33,4</td><td>gg</td><td> 33,5</td><td>gg</td><td> 33,6</td><td>gg</td>

<td> 33,7</td><td>gg</td><td> 33,8</td><td>gg</td><td> 33,9</td><td>gg</td><td> 34,0</td><td>gg</td><td> 34,1</td><td>gg</td><td> 34,2</td><td>gg</td><td> 34,3</td><td>gg</td><td> 34,4</td><td>gg</td><td> 34,5</td><td>gg</td>

<td> 34,6</td><td>gg</td><td> 34,7</td><td>gg</td><td> 34,8</td><td>gg</td><td> 34,9</td><td>gg</td><td> 35,0</td><td>gg</td><td> 35,1</td><td>gg</td><td> 35,2</td><td>gg</td><td> 35,3</td><td>gg</td><td> 35,4</td><td>gg</td>

<td> 35,5</td><td>gg</td><td> 35,6</td><td>gg</td><td> 35,7</td><td>gg</td><td> 35,8</td><td>gg</td><td> 35,9</td><td>gg</td><td> 36,0</td><td>gg</td><td> 36,1</td><td>gg</td><td> 36,2</td><td>gg</td><td> 36,3</td><td>gg</td>

<td> 36,4</td><td>gg</td><td> 36,5</td><td>gg</td><td> 36,6</td><td>gg</td><td> 36,7</td><td>gg</td><td> 36,8</td><td>gg</td><td> 36,9</td><td>gg</td><td> 37,0</td><td>gg</td><td> 37,1</td><td>gg</td><td> 37,2</td><td>gg</td>

<td> 37,3</td><td>gg</td><td> 37,4</td><td>gg</td><td> 37,5</td><td>gg</td><td> 37,6</td><td>gg</td><td> 37,7</td><td>gg</td><td> 37,8</td><td>gg</td><td> 37,9</td><td>gg</td><td> 38,0</td><td>gg</td><td> 38,1</td><td>gg</td>

<td> 38,2</td><td>gg</td><td> 38,3</td><td>gg</td><td> 38,4</td><td>gg</td><td> 38,5</td><td>gg</td><td> 38,6</td><td>gg</td><td> 38,7</td><td>gg</td><td> 38,8</td><td>gg</td><td> 38,9</td><td>gg</td><td> 39,0</td><td>gg</td>

<td> 39,1</td><td>gg</td><td> 39,2</td><td>gg</td><td> 39,3</td><td>gg</td><td> 39,4</td><td>gg</td><td> 39,5</td><td>gg</td><td> 39,6</td><td>gg</td><td> 39,7</td><td>gg</td><td> 39,8</td><td>gg</td><td> 39,9</td><td>gg</td>

<td> 40,0</td><td>gg</td><td> 40,1</td><td>gg</td><td> 40,2</td><td>gg</td><td> 40,3</td><td>gg</td><td> 40,4</td><td>gg</td><td> 40,5</td><td>gg</td><td> 40,6</td><td>gg</td><td> 40,7</td><td>gg</td><td> 40,8</td><td>gg</td>

<td> 40,9</td><td>gg</td><td> 41,0</td><td>gg</td><td> 41,1</td><td>gg</td><td> 41,2</td><td>gg</td><td> 41,3</td><td>gg</td><td> 41,4</td><td>gg</td><td> 41,5</td><td>gg</td><td> 41,6</td><td>gg</td><td> 41,7</td><td>gg</td>

<td> 41,8</td><td>gg</td><td> 41,9</td><td>gg</td><td> 42,0</td><td>gg</td><td> 42,1</td><td>gg</td><td> 42,2</td><td>gg</td><td> 42,3</td><td>gg</td><td> 42,4</td><td>gg</td><td> 42,5</td><td>gg</td><td> 42,6</td><td>gg</td>

<td> 42,7</td><td>gg</td><td> 42,8</td><td>gg</td><td> 42,9</td><td>gg</td><td> 43,0</td><td>gg</td><td> 43,1</td><td>gg</td><td> 43,2</td><td>gg</td><td> 43,3</td><td>gg</td><td> 43,4</td><td>gg</td><td> 43,5</td><td>gg</td>

<td> 43,6</td><td>gg</td><td> 43,7</td><td>gg</td><td> 43,8</td><td>gg</td><td> 43,9</td><td>gg</td><td> 44,0</td><td>gg</td><td> 44,1</td><td>gg</td><td> 44,2</td><td>gg</td><td> 44,3</td><td>gg</td><td> 44,4</td><td>gg</td>

<td> 44,5</td><td>gg</td><td> 44,6</td><td>gg</td><td> 44,7</td><td>gg</td><td> 44,8</td><td>gg</td><td> 44,9</td><td>gg</td><td> 45,0</td><td>gg</td><td> 45,1</td><td>gg</td><td> 45,2</td><td>gg</td><td> 45,3</td><td>gg</td>

<td> 45,4</td><td>gg</td><td> 45,5</td><td>gg</td><td> 45,6</td><td>gg</td><td> 45,7</td><td>gg</td><td> 45,8</td><td>gg</td><td> 45,9</td><td>gg</td><td> 46,0</td><td>gg</td><td> 46,1</td><td>gg</td><td> 46,2</td><td>gg</td>

<td> 46,3</td><td>gg</td><td> 46,4</td><td>gg</td><td> 46,5</td><td>gg</td><td> 46,6</td><td>gg</td><td> 46,7</td><td>gg</td><td> 46,8</td><td>gg</td><td> 46,9</td><td>gg</td><td> 47,0</td><td>gg</td><td> 47,1</td><td>gg</td>

<td> 47,2</td><td>gg</td><td> 47,3</td><td>gg</td><td> 47,4</td><td>gg</td><td> 47,5</td><td>gg</td><td> 47,6</td><td>gg</td><td> 47,7</td><td>gg</td><td> 47,8</td><td>gg</td><td> 47,9</td><td>gg</td><td> 48,0</td><td>gg</td>

<td> 48,1</td><td>gg</td><td> 48,2</td><td>gg</td><td> 48,3</td><td>gg</td><td> 48,4</td><td>gg</td><td> 48,5</td><td>gg</td><td> 48,6</td><td>gg</td><td> 48,7</td><td>gg</td><td> 48,8</td><td>gg</td><td> 38,9</td><td>gg</td>

<td> 49,0</td><td>gg</td><td> 49,1</td><td>gg</td><td> 49,2</td><td>gg</td><td> 49,3</td><td>gg</td><td> 49,4</td><td>gg</td><td> 49,5</td><td>gg</td><td> 49,8</td><td>gg</td><td> 49,7</td><td>gg</td><td> 49,8</td><td>gg</td>

39.9 gg and 50 gg.

[0056] The above suggested doses can be adjusted using conventional dose calculations if the compound is administered by another route. Determining the appropriate dose for administration by other routes is within the skill of those skilled in the art in light of the above description and of general knowledge in the art.

[0057] Administration of an effective amount of a compound of the invention may comprise the administration of a single dosage form or multiple unit doses which can be administered simultaneously or at separate time points over a given period, for example 24 hours. The dose of the compound of the invention (alone or in the form of a composition containing it) can be administered from one to ten times daily. The compound of the invention (alone or in the form of a composition containing it) is usually administered four, three, two or once a day (24 hours).

[0058] The compounds of formula (I) according to the present invention are further useful in the treatment of inhaled infections. Examples of inhaled infections include, for example, RSV. The compounds of formula (I) according to the present invention are further useful in the treatment of anthrax infections. The present invention relates to the use of compounds of formula (I) according to the present invention for the prophylactic, preventive, preventive or curative treatment in connection with diseases or ailments caused by pathogens. In a preferred embodiment, the present invention relates to the use of compounds of formula (I) for the prophylactic, prophylactic, post-exposure, therapeutic or therapeutic management in connection with diseases or ailments caused by pathogens that can be used in bioterrorism.

[0059] A number of research programs and defense measures have been established in recent years to address concerns about the use of biological agents in acts of terror. The purpose of these measures is to address concerns about bioterrorism or the use of microbes or biological toxins to kill people, release anxiety and disturb social life. For example, the National Institute of Allergy and Infectious Diseases (NIAID) has developed a strategic plan for biological defense research that outlines plans for research needs in the broad field of bioterrorism and new and emerging infectious diseases. According to this plan, the possible deliberate exposure of the US civilian population to Bacillus anthracis spores will reveal a gap in the country's overall preparation for acts of bioterrorism. In addition, this report details that these attacks highlighted the unmet need for developing rapid diagnosis tests, vaccines and immunological therapies for prevention, as well as drugs and biological agents for the treatment of disease caused by agents used in bioterrorism.

[0060] A large part of attention in various research works is focused on the study of the biology of pathogens identified as potentially dangerous agents used in bioterrorism, study of the host's response to such agents, development of vaccines against infectious diseases, assessment of currently available and currently investigating therapeutic agents against such agents, and development of diagnostic agents for recognizing the signs and symptoms of the hazardous agents. Such efforts are commendable, but due to the large number of pathogens that have been identified as being usable in bioterrorism, this work is not yet sufficient to respond satisfactorily to all possible bioterrorist threats. In addition, many of the pathogens identified as potentially dangerous bioterrorist agents are not a source of adequate economic incentives for the industry to develop therapeutic or preventative measures. In addition, even if preventive measures such as vaccines were available for any pathogen that could be used in bioterrorism, the cost of administering all such vaccines to the general public would be very high.

[0061] Until convenient and effective methods of protection against any bioterrorist threat are available, there will be a great need for preventive, prophylactic or therapeutic measures that prevent or reduce the risk of infection by pathogens.

[0062] The present invention provides such prophylactic methods. In one aspect, there is provided a method of prophylactic treatment comprising administering a prophylactically effective amount of compounds of formula (I) to a subject in need of prophylactic treatment against infection by one or more inhaled pathogens. Anthrax is a specific example of an inhaled pathogen.

[0063] In another aspect, there is provided a prophylactic method to reduce the risk of infection by an inhaled pathogen that causes a human disease, said method comprising administering an effective amount of compounds of formula (I) to the human lung that may be at risk Infections with an inhaled pathogen, but there were no symptoms of the disease, but the effective amount of sodium channel blocker and osmolite is sufficient, to reduce the risk of human infection. Anthrax is a specific example of an inhaled pathogen.

[0064] In another aspect, there is provided a method of prophylactic post-exposure or therapeutic management for treating an inhaled pathogen infection comprising administering an effective amount of compounds of formula (I) to the lungs of a subject in need of such treatment against inhaled pathogen infection. Pathogens that can be protected by prophylactic, emergency and therapeutic methods of the invention include any pathogens that may enter the body through the mouth, nose or nasal airways and thus enter the lungs. Such pathogens are usually inhaled pathogens, either naturally occurring or aerosolized. Pathogens can occur naturally or can be introduced into the environment intentionally as a result of aerosol or other methods of introducing pathogens into the environment. Many pathogens that are not naturally transmitted in the air have been or can be aerosolized for use in bioterrorism. Pathogens for which the treatment of the invention may be useful include, but are not limited to, NIAID Priority A, B and C pathogens. These categories generally correspond to the lists developed by Centers for Disease Control and Prevention (CDC). According to the CDC recommendations, category A factors are easy to spread or pass from one person to another, cause high mortality and can have a significant impact on public health. Category B factors are next in importance and are moderately easy to spread and cause moderate morbidity and low mortality. Category C consists of new pathogens that can be modified for mass dissemination in the future due to their availability, ease of production and spreading, and potential high morbidity and mortality. Anthrax and plague are specific examples of these pathogens. Additional pathogens that can be protected against or reduced may include influenza viruses, rhinoviruses, adenoviruses, and respiratory syncytium viruses and the like. Another pathogen that you can protect yourself from is coronavirus, which is thought to cause acute severe respiratory distress syndrome (SARS).

[0065] Furthermore, the present invention relates to the use of sodium channel blockers of formula I, or pharmaceutically acceptable salts thereof, for the prevention, alleviation and / or treatment of deterministic health effects on the respiratory tract caused by exposure to radiological materials, in particular respirable aerosols containing radionuclides due to attacks nuclear, such as the detonation of a radiological scattering device ( RDD), or accidents such as nuclear disasters. Accordingly, this description provides a method for preventing, alleviating and / or treating deterministic health effects on the respiratory tract and / or other organs of the body caused by respirable aerosols containing radionuclides in a subject in need thereof, including a human in need of such treatment, said method comprising administering to said man an effective amount of a compound of formula I or a pharmaceutically acceptable salt thereof. [0066] The main concern associated with planning impact management in the event of exposure of members of the public to respirable aerosols containing radionuclides as a result of nuclear attacks, such as the detonation of a radiological dispersing device (RDD), or accidents such as nuclear disasters, is a method of prevention, mitigation or treatment of deterministic health effects on the respiratory tract, mainly the lungs. It is necessary to have medicines, methods and procedures, as well as trained personnel prepared to care for and treat such individuals whose body has been heavily contaminated internally.

[0067] Research is being conducted to determine methods for preventing, alleviating or treating potential damage to the respiratory tract and various organs of the body caused by radionuclides accumulating in the body. So far, most of the attention in research has focused on strategies to mitigate the health effects of radionuclides accumulating in the body by accelerating their excretion or removal. These strategies focus on soluble chemical forms that can enter the bloodstream and accumulate in remote areas of the body specific for a given radioactive element. Such methods are not effective in cases where the deposited radionuclide is in a relatively insoluble form. Studies show that many, if not all, of the physicochemical forms of radionuclides dispersed from RDD occur in relatively insoluble form.

[0068] The only known method that allows effective reduction of radiation dose in the lungs due to inhalation of insoluble radioactive aerosols is bronchopulmonary lavage. It has been shown that this method, which is an adaptation of the method already used in the treatment of patients with alveolar proteinosis, is a safe and repeatable procedure, even if it is performed for a long period. Although there are variations on this procedure, the basic method for performing BAL is to anesthetize the subject and then slowly introduce isotonic sodium chloride into one lung lobe until functional residual capacity is achieved. Then additional volumes are introduced and drained by gravity. The results of studies on the use of BAL on animals indicate that approximately 40% of deep lung content can be removed using the appropriate sequence of BAL procedures. Some studies have found significant variability between animals in terms of the amount of radionuclide recovered. The reasons for this variability have not yet been identified.

[0069] Furthermore, based on animal studies, a significant reduction in dose due to BAL therapy is believed to alleviate health effects by inhalation of insoluble radionuclides. In the study, adult dogs inhaled insoluble particles¹⁴⁴Ce-FAP. Two groups of dogs were given into the lungs¹⁴⁴Ce, which is known to cause radiation pneumonia and pulmonary fibrosis (approximately 2 MBq / kg body weight), one group being treated using 10 unilateral washing procedures on days 2 to 56 after exposure, and the other group was untreated. The third group was exposed at the level¹⁴⁴Ce comparable to that found in the BAL treated group after treatment (approximately 1 MBq / kg), however these animals were not treated. All animals were allowed to live until natural death (maximum 16 years). Due to the variability of the initial content¹⁴⁴Ce in the lungs in dogs in each group, dose strengths and cumulative doses for each group overlapped. Despite this, the effect of BAL on reducing the risk of pneumonia / fibrosis was apparent from the survival curves. For untreated dogs with a lung content of 1.5-2.5 MBq / kg, the average survival time was 370 ± 65 d. For the treated dogs, the average survival time was 1270 ± 240 d, which was a statistically significant difference. In the case of the third group in which the content is given ¹⁴⁴Lung ce was 0.6-1.4 MBq / kg, mean survival was 1800 ± 230, i.e. it was not statistically different from the treatment group. Equally important for increasing survival was that the high-dose dogs treated at high dose died due to pulmonary deterministic effects (pneumonia / fibrosis), which was not found in treated dogs. Instead, the treated dogs, like the low-treated dogs at the low dose, mainly had lung cancers (vascular sarcoma or cancer). Therefore, it appears that the dose reduction due to BAL treatment caused biological effects in the lungs, which could have been predicted based on radiation doses to the lungs.

[0070] Based on these results, it is believed that an additional reduction in residual radiological dose by any method or combination of methods to increase particle removal from the lung would further reduce the likelihood of lung health effects. However, BAL is a procedure with many disadvantages. BAL is a highly invasive procedure that should be performed in specialized medical centers with the participation of trained pulmonologists. Therefore, the BAL procedure is expensive. Due to the disadvantages of the BAL procedure, it is not a therapeutic option that could be easily and immediately available to people who require faster removal of radioactive particles, for example in the event of a nuclear attack. In the event of a nuclear attack or nuclear disaster, immediate treatment of exposed or threatened persons is necessary, which would be relatively easy to apply. Sodium channel blockers administered as inhalation aerosol have been shown to restore the hydration of the airway surface. Such hydration of the airway surface helps to remove accumulated mucous secretions and associated solid particles from the lungs. Therefore, without being limited to any particular theory, it is believed that sodium channel blockers can be used to accelerate the removal of radioactive particles from airway ducts.

[0071] As stated above, the greatest risk to the lungs following a radiological attack, for example with a dirty nuclear bomb, results from the inhalation and retention of insoluble radioactive particles. As a result of radioactive particle retention, the cumulative exposure of the lungs increases significantly, which ultimately causes pulmonary fibrosis / pneumonia and potentially death. Insoluble particles cannot be removed from the whole body by chelating agents because these particles are not in solution. So far, physical particle removal by BAL is the only therapeutic regimen that has been shown to be effective in alleviating radiation-induced lung diseases. As described above, BAL is not a viable therapeutic solution to reduce the effects of radioactive particles that have entered the body by inhalation. Therefore, it is desirable to provide a therapeutic regimen that would effectively help remove radioactive particles from airway ducts and, unlike BAL, it would be relatively easy to administer and it could be scaled up in the case of a large-scale radiation exposure scenario. Furthermore, it is desirable that this therapeutic regimen be readily available to many people in a relatively short period.

[0072] In an aspect of the present invention, a method of preventing, alleviating and / or treating deterministic health effects on the respiratory tract and / or other organs of the body caused by respirable aerosols containing radionuclides comprises administering an effective amount of a sodium channel blocker of formula I or a pharmaceutically acceptable salt thereof to a subject thereof in need. In an embodiment of this aspect, the sodium channel blocker is administered in combination with osmolyte. In addition, osmolite is a hypertonic sodium chloride (HS) solution for this variety. As part of another variation, the sodium channel blocker and osmolyte are administered in combination with an ion transport modulator. In addition, with respect to this variation, the ion transport modulator can be selected from the group consisting of β-agonists, agents that increase CFTR activity, purinergic receptor agonists, likeprostone derivatives and protease inhibitors. As part of another variation of this aspect, the radionuclides are selected from the group consisting of cobalt-60, cesium-137, iridium-192, radium-226, phosphorus-32, strontium-89 and 90, iodine-125, thallium-201, lead- 210, thorium-234, uranium-238, plutonium, cobalt-58, chromium-51, america and kiuru. As part of another variation, the radionuclides come from a radiological diffusion device. In yet another embodiment, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered in an aerosol suspension of respirable particles that the subject inhales. As an additional variation, the sodium channel blocker or a pharmaceutically acceptable salt thereof is administered after exposure to radionuclides.

COMPOSITIONS [0073] Although it is possible for a compound of the invention to be administered alone, in some embodiments it is preferable to administer it as a composition, in particular a pharmaceutical composition (formulation). Accordingly, in another aspect, the invention provides compositions and in particular pharmaceutical compositions (such as an inhalable pharmaceutical composition) containing a pharmaceutically effective amount of a compound of the invention as an active substance and a pharmaceutically acceptable excipient, diluent or carrier. The term "active substance" as used herein means any compound of the invention or a combination of two or more compounds of the invention in a pharmaceutical composition. In addition, specific embodiments are provided in which the pharmaceutical composition comprises a pharmaceutically effective amount of a compound of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik) and (II) or a pharmaceutically acceptable salt thereof, independently or in combination, and a pharmaceutically acceptable excipient, diluent or carrier.

[0074] In some embodiments, the pharmaceutical composition comprises a pharmaceutically effective amount of a compound of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih ), (Ii), (Ij), (Ik) and (II) or a pharmaceutically acceptable salt thereof, independently or in combination, in a diluent. In separate embodiments, the pharmaceutical composition comprises a pharmaceutically effective amount of a compound of formulas (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), ( Ii), (Ij), (Ik) and (II) or a pharmaceutically acceptable salt thereof in hypertonic sodium chloride, sterile water and hypertonic sodium chloride, respectively, wherein the sodium chloride concentration may be as described herein. In one embodiment, the sodium chloride concentration is 0.17% w / v and in another it is 2.8% w / v.

[0075] Also disclosed is a kit comprising i) a pharmaceutically effective amount of a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih ), (Ii), (Ij), (Ik) and (II) or a pharmaceutically acceptable salt thereof; ii) one or more pharmaceutically acceptable excipients, carriers or diluents; iii) instructions for administering the compound of group i) and excipients, carriers or diluents of group ii) to a subject in need thereof; and iv) container. A subject in need thereof includes any subject in need of the treatment methods described herein, in particular a human in need thereof. Additional embodiments further include an aerosol generating device selected from the group consisting of a nebulizer, including vibrating mesh nebulizers and jet nebulizers, a dry powder inhaler, including active and passive dry powder inhalers, and a metered dose inhaler, including pressurized metered dose inhalers. dry powder and soft fog.

[0076] In one embodiment, the kit comprises i) from about 10 ng to about 10 mg of a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik) and (Il) or a pharmaceutically acceptable salt thereof, per dose; ii) from about 1 to about 5 ml of diluent, per dose; iii) instructions for administering a compound of group i) and a diluent of group ii) to a subject in need thereof; and iv) container. In another embodiment, the diluent is from about 1 to about 5 ml of the sodium chloride solution described herein, per dose. In another embodiment, the diluent is from about 1 to about 5 mL of hypotonic chloride solution per dose. In another embodiment, the diluent is from about 1 to about 5 mL of hypertonic sodium chloride solution per dose. In yet another embodiment, the diluent is from about 1 to about 5 mL of sterile water, per dose.

[0077] Also provided is a kit comprising i) a solution containing a pharmaceutically effective amount of a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik) and (II) or a pharmaceutically acceptable salt thereof, dissolved in a pharmaceutically acceptable diluent; ii) instructions for administering the group i solution to a subject in need thereof; and iii) a container.

[0078] There is further provided a kit comprising i) a solution containing from about ng to about 10 mg of a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik) and (II) or a pharmaceutically acceptable salt thereof, dissolved in a pharmaceutically acceptable diluent; ii) instructions for administering the group i solution to a subject in need thereof; and iii) a container. In another embodiment, the diluent is from about 1 to about 5 ml of the sodium chloride solution described herein, per dose.

[0079] Another embodiment includes a kit comprising i) a pharmaceutically effective amount of a compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), ( Ih), (Ii), (Ij), (Ik) and (II) or a pharmaceutically acceptable salt thereof in the preparation in the form of a dry powder suitable for inhalation; ii) optionally one or more pharmaceutically acceptable excipients or carriers suitable for inhalation; iii) instructions for administering the compound from group i) and excipients or carriers from group ii) to a subject in need thereof; and iv) container. In another embodiment, the kit further comprises a dry powder inhaler suitable for administering to the subject a dry powder formulation. The dry powder inhaler may in additional embodiments be a single dose inhaler or a multi dose inhaler.

[0080] Further embodiments of each of the kits described herein include kits in which the concentration of the compound of formula (I), (Ia), (Ib), (Ic), (Id), (Ie), (If), (Ig), (Ih), (Ii), (Ij), (Ik) and (Il) or a pharmaceutically acceptable salt thereof, per dose, is compatible with one of the effective dosage ranges described herein, including a) from about 0.1 μg to about 1000 μg; b) from about 0.5 μg to about 0.5 mg; and c) from about 0.5 μg to about 50 μg.

[0081] For each of the kits listed above, there is an additional embodiment in which the diluent is a hypertonic sodium chloride solution at the concentrations described herein. In another embodiment, for each kit, the diluent is a hypotonic sodium chloride solution at the concentrations described herein. In another embodiment, the diluent is sterile water suitable for inhalation for each kit.

[0082] The pharmaceutically acceptable excipient or excipients, diluent or diluents or carrier or carriers must be acceptable in the sense that they are compatible with the other ingredients of the formulation and do not have harmful effects on the subject. A substantially pharmaceutically acceptable excipient or excipients, diluent or diluents, or the carrier or carriers used in the pharmaceutical preparation are "non-toxic", which means that they are considered safe to eat in the amount administered and "inert", which means that they do not significantly affect the therapeutic activity of the active or active substances and have no adverse effect on them. Pharmaceutically acceptable excipients, diluents and carriers are conventional in the art and can be selected using conventional methods depending on the desired route of administration. See REMINGTON'S, PHARMACEUTICAL SCIENCES, Lippincott Williams &amp; Wilkins; ed. 21 (May 1, 2005). Preferably, the pharmaceutically acceptable excipient or excipients, diluent or diluents or carrier or carriers are generally recognized as safe (GRAS) according to the FDA.

[0083] The pharmaceutical compositions of the invention include compositions suitable for oral administration; parenteral, including subcutaneous, intradermal, intramuscular, intravenous and intra-articular administration; topical administration, including topical administration to the skin, eyes, ears etc .; vaginal or rectal administration; and administration to the respiratory tract, including nasal cavities and sinuses, respiratory tract inside the mouth and outside the chest and lungs, including using aerosols, which can be administered using various types of dry powder inhalers, metered dose inhalers, inhalers soft fog, nebulizers or insufflators. The most appropriate route of administration may depend on a number of factors, including the patient and the disease or disorder being treated.

[0084] The formulations may conveniently be presented in unit dosage form or in loose form, as for example formulations dispensed from an inhaler, and may be prepared by any of the methods well known in the pharmaceutical art. Basically, the methods include the step of bringing into association the active substance with the carrier, diluent or excipient, and optionally with one or more accessory ingredients. In general, preparations are prepared by uniformly and intimately bringing into association the active substance with one or more liquid carriers, diluents or excipients or finely divided solid carriers, diluents or excipients, or both, and then, if necessary, forming the product into the desired formulation .

[0085] In one preferred embodiment, the composition is an inhalable pharmaceutical composition suitable for inhalation and administration to the intrabronchial space. Such a composition is usually in the form of an aerosol containing particles for administration using a nebulizer, pressurized metered dose inhaler (MDI), soft mist inhaler or dry powder inhaler (DPI). The aerosol formulation used in the methods of the present invention may be a liquid (e.g. solution) suitable for administration with a nebulizer, soft mist inhaler or MDI or dry powder suitable for administration with MDI or DPI.

[0086] Aerosols used to administer drugs to the respiratory tract are usually polydisperse; this means that they consist of particles of many different sizes. The particle size distribution is usually described by the median mass diameter aerodynamic diameter (MMAD) and geometric standard deviation (GSD). For optimal drug delivery into the bronchial space, the MMAD value ranges from about 1 to about 10 μm and preferably from about 1 to about 5 μm, and GSD is below 3 and preferably below about 2. Aerosol particles having an MMAD value above 10 μm they are essentially too large to get into the lungs after inhalation. Aerosols having a GSD value greater than about 3 are not preferred for pulmonary administration because they deliver a significant percentage of the drug into the mouth. To obtain such particle sizes in the powder formulation, the active substance particles can be reduced in size by conventional methods such as micronization or spray drying. Non-limiting examples of other processes or methods that can be used to prepare respirable particles include spray drying, precipitation, supercritical fluid and freeze drying. The desired fraction can be separated by pneumatic sorting or screening. In one embodiment, the particles are crystalline. For liquid preparations, the particle size is determined by the choice of the particular nebulizer model, soft mist inhaler or MDI.

[0087] The aerosol particle size distribution is determined by devices well known in the art. For example, it is an Andersen multi-stage cascade impactor or another suitable method, for example specifically mentioned in chapter 601 of the US Pharmacopoeia as a device for determining the characteristics of aerosols emitted by metered dose inhalers and dry powder inhalers.

[0088] Formulations of dry powder compositions for topical administration to the lungs by inhalation may be prepared without an excipient or carrier; instead, they only contain active substances in the form of a dry powder with a particle size suitable for inhalation. The dry powder compositions may further comprise a mixture of the active substance and a suitable powder base (carrier / diluent / excipient), such as mono-, di- or polysaccharides (e.g. lactose or starch). Lactose is usually the preferred excipient for dry powder formulations. If a solid excipient, for example lactose, is used, the particle size of the excipient is substantially much larger than the active substance to facilitate dispersion of the formulation in the inhaler.

[0089] Non-limiting examples of dry powder inhalers include reservoir multi-dose inhalers, pre-metered dose multidose inhalers, capsule inhalers and single dose single dose inhalers. The cartridge inhaler contains a large number of doses (e.g. 60) in one container. Before inhalation, the patient starts the inhaler, as a result of which the inhaler measures one dose of medicine from the reservoir and prepares it for inhalation. Examples of container DPI devices include Turbohaler® from AstraZeneca and ClickHaler® from Vectura.

[0090] In a multi-dose inhaler with pre-metered doses, each individual dose is placed in a separate container during manufacture, and actuation of the inhaler before inhalation releases a new dose of the drug from the appropriate container and preparation for inhalation. Examples of multi-dose DPIs include Diskus® from GSK, Gyrohaler® from Vectura and Prohaler® from Valois. During inhalation, the patient's inspiratory stream causes the powder to flow out of the device faster and into the mouth. In the case of a capsule inhaler, the preparation is in a capsule and is stored outside the inhaler. The patient inserts the capsule into the inhaler, starts the inhaler (pierces the capsule), and

TM then inhales. Examples include Rotohaler

TM TM TM (GlaxoSmithKline), Spinhaler (Novartis), HandiHaler (IB), TurboSpin (PH&amp;T). For single-dose single-use inhalers, the patient starts the inhaler to prepare it for inhalation, inhales, and then discards the inhaler and packaging. Examples include

TM TM TM

Twincer (U Groningen), OneDose (GFE) and Manta Inhaler (Manta Devices). [0091] Basically dry powder inhalers take advantage of the turbulent flow properties of the powder trajectory, whereby the excipient and drug aggregates are dispersed and the active substance particles reach the lungs. For some dry powder inhalers, a cyclone dispersion chamber is used to provide particles of the appropriate respirable size. In the cyclone dispersion chamber, the drug enters the coin-shaped dispersion chamber on the tangential path, therefore the air stream and the drug move along the outer circular wall.

When the drug formulation moves along this circular wall, it bounces off it and the agglomerates are broken by the force of the impact. The air path has a spiral shape, narrowing to the center of the chamber with a vertical outlet. Particles having sufficiently small aerodynamic quantities can move along the air path and leave the chamber. As a result, the dispersion chamber acts as a small jet mill. Depending on the properties of the preparation, small lactose particles are added to it, which facilitate dispersion as a result of collisions with particles of the active substance.

TM [0092] The single-use single-use inhaler Twincer operates using a coin-shaped cyclone dispersion chamber termed "pneumatic sorting module". See published US Patent Application No. 2006/0237010 for Rijksuniversiteit Groningen. The articles issued by the University of Groningen state that with this technology it is possible to effectively deliver a dose of 60 mg of pure micronized colistin mesylate as a dry powder for inhalation.

[0093] In preferred embodiments, the aerosol formulation is administered in the form of a dry powder using a dry powder inhaler in which the particles generated by the inhaler have an MMAD value in the range of from about 1 μm to about 5 μm and a GSD value of less than about 2.

[0094] Examples of suitable dry powder inhalers and dry powder dispersion devices for use in delivering the compounds and compositions of the present invention include, but are not limited to, the products disclosed in US7520278; US7322354; US7246617; US7231920; US7219665; US7207330; US6880555; US5,522,385; US6845772; US6637431; US6329034; US5,458,135; US4,805,811 and in Published US Patent Application No. 2006/0237010.

[0095] In one embodiment, the pharmaceutical formulation of the invention is a dry inhalable powder that is prepared for administration using a Diskus® device. Diskus® contains an elongated strip formed of a base sheet containing many cavities distributed along its length and a top sheet hermetically connected to it, but which can be torn off, resulting in multiple containers, each containing an inhalation formulation containing a predetermined amount of active substance alone or in a mixture with one or more carriers or excipients (e.g. lactose) and / or other therapeutically active agents. The strap is preferably elastic enough to be wound on a roll. Preferably, the top sheet and base sheet comprise lead end portions that are not tightly connected to each other and at least one of the lead end portions is constructed for attachment to the winding elements. Furthermore, it is preferred that the hermetic connection of the base and top sheets covers their entire width. To prepare a dose for inhalation, the top sheet can be advantageously peeled away from the base sheet in a longitudinal direction from the end of the first base sheet.

[0096] In one embodiment, the pharmaceutical formulation of the invention is a dry inhalable powder that is prepared for administration with a single-use, single-use inhaler, in particular a Twincer ™ inhaler. The Twincer ™ inhaler contains a blister consisting of a film laminate with one or more cavities and a top sheet hermetically connected to it, but which can be torn off, resulting in multiple containers. Each container contains an inhalation formulation containing a predetermined quantity of active or active substances alone or in a mixture with one or more carriers or excipients (e.g. lactose). The top sheet preferably includes a leading end portion constructed so that it projects beyond the body of the inhaler. The patient starts the device and thus administers the aerosol formulation as follows: 1) removes the outer protective film, 2) pulls the film strip to reveal the medicine in the blister and 3) inhales the medicine from the blister.

[0097] In another embodiment, the pharmaceutical preparation of the invention is a dry powder for inhalation, wherein the dry powder is prepared in the form of microparticles described in PCT Publication No. WO2009 / 015286 or WO2007 / 114881, both for NexBio. Such microparticles are prepared by essentially adding a counterion to a solution containing the compound of the invention in a solvent and adding an anti-solvent to the solution; then the solution is gradually cooled to a temperature below about 25 ° C, whereby a composition containing the microparticle containing the compound is obtained. The compound containing microparticles can then be separated from the solution by any suitable method, such as sedimentation, filtration or lyophilization. Suitable counterions, solvents and anti-solvents for preparing microparticles of the compounds of the invention are described in WO2009 / 015286.

[0098] In another embodiment, the pharmaceutical composition of the invention is administered in the form of a dry powder using a metered dose inhaler. Non-limiting examples of inhalers and dispenser devices include the products disclosed in US5,261,538; US5,544,647; US5,622,163; US4,955,371; US3,566,070; US3,361,306 and US6,116,234 and US7,108,159. In a preferred embodiment, the compound of the invention is administered as a dry powder using a metered dose inhaler in which the emitted particles have an MMAD value in the range of from about 1 gm to about 5 gm and a GSD value of less than about 2.

[0099] Liquid formulations in the form of an aerosol for inhalation administration into the bronchial space or lungs may for example be made in the form of aqueous solutions or suspensions, or aerosols administered by means of pressurized packaging, such as metered dose inhalers, using suitable liquefied propellants, soft mist inhalers or nebulizers. Such aerosol compositions suitable for inhalation may be a suspension or solution and generally contain the active or active substance together with a pharmaceutically acceptable carrier or diluent (e.g. water (distilled or sterile), sodium chloride solution, hypertonic sodium chloride solution or ethanol) and optionally one or more other therapeutically active agents.

[0100] Aerosol compositions for administration by means of pressurized metered dose inhalers typically further comprise a pharmaceutically acceptable propellant. Examples of such propellants include hydrogen fluoride or chlorofluorocarbon containing hydrogen or mixtures thereof, in particular hydrofluoroalkanes, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, especially 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3, 3-heptafluoro-n-propane or a mixture thereof. The aerosol composition may contain no excipients or may optionally contain additional excipients for the formulation well known in the art, such as surfactants, e.g. oleic acid or lecithin, and cosolvents, e.g., ethanol. The pressurized preparations are usually contained in a container (e.g. aluminum container) with a closed valve (e.g. dispensing valve) on which the actuator with the mouthpiece is mounted.

[0101] In another embodiment, the pharmaceutical composition of the invention is administered in the form of a liquid using a metered dose inhaler. Non-limiting examples of inhalers and dispenser articles include the products disclosed in US Patent Nos. 6,253,762, 6,413,497, 7,601,336, 7,481,995, 6,743,413 and 7,105,152. In a preferred embodiment, the compound of the invention is administered as a dry powder using a metered dose inhaler in which the emitted particles have an MMAD value in the range of from about 1 μm to about 5 μm and a GSD value of less than about 2.

[0102] In one embodiment, the aerosol formulation is capable of being aerosolized using a jet nebulizer or ultrasonic nebulizer, including nebulizers with a static and vibrating porous plate. Liquid formulations in the form of a nebulization aerosol may be prepared by solubilizing or reconstituting the formulation in the form of solid particles, or aqueous formulations may be prepared by adding agents such as acid or base, buffer salts and isotonicity adjusting agents. They can be sterilized by in-process methods, such as filtration, or final processes, such as autoclave heating or gamma radiation. They may also be in a non-sterile form. [0103] Patients may be sensitive to pH, osmolality and ion content in the nebulized solution. Therefore, these parameters should be adjusted so that they are compatible with the active substance and tolerated by patients. The most preferred solution or suspension of the active substance contains a chloride concentration&gt; 30 mM at pH 4.5-7.4, preferably 5.0-5.5, and has an osmolality of from about 800 to 1600 mOsm / kg. The pH of the solution can be controlled by titration with typical acids (e.g. hydrochloric acid or sulfuric acid) or bases (e.g. sodium hydroxide) or using buffers. Typical buffers used are citrate buffers, e.g., citric acid / sodium citrate buffers, acetate buffers, e.g., acetic acid / sodium acetate buffers, and phosphate buffers. Buffer capacities may range from 2 mM to 50 mM.

[0104] Useful acetate, phosphate and citrate buffers include sodium acetate, sodium acetate trihydrate, ammonium acetate, potassium acetate, sodium phosphate, sodium phosphate, disodium hydrogen phosphate, potassium dihydrogen phosphate, potassium hydrogen phosphate, potassium phosphate, sodium citrate and potassium citrate. Other buffers that can be used include sodium hydroxide, potassium hydroxide, ammonium hydroxide, aminomethylpropanol, tromethamine, tetrahydroxypropyl ethylenediamine, citric acid, acetic acid, hydroxytricarboxylic acid or its salt, e.g. its citrate or sodium citrate salt, lactic acid and acid salts lactic, including sodium lactate, potassium lactate, lithium lactate, calcium lactate, magnesium lactate, barium lactate, aluminum lactate, zinc lactate, silver lactate, copper lactate, iron lactate, manganese lactate, ammonium lactate, monoethanolamine, diethanolamine, triethanolamine, diisopropanolamine, as well as combinations thereof and the like.

[0105] Such formulations can be administered using a commercially available nebulizer or other atomizer that is able to disperse the formulation into particles or droplets suitable for delivery to the respiratory tract. Non-limiting examples of nebulizers that can be used for aerosol administration of the compositions of the invention include pneumatic jet nebulizers or breath-activated jet nebulizers or ultrasonic nebulizers, including nebulizers with static or vibrating porous plates. Commercially available nebulizers include the Aeroneb® Go (Aerogen) nebulizer and the eFlow nebulizer (Pari Pharma).

[0106] The jet nebulizer uses a high velocity air stream colliding with a water column to form droplets. Non-inhalable particles collide with aerodynamic walls or partitions. The vented or breath actuated nebulizer works in essentially the same way as the jet nebulizer, except that the inhaled air passes through the main droplet production area increasing the nebulizer output capacity when the patient inhales the air. [0107] In the case of an ultrasonic nebulizer, vibrations of a piezoelectric crystal cause instabilities on the surface of the drug container, resulting in droplets. In nebulizers with a porous plate, pressure fields created by sound energy cause the liquid to squeeze through the holes in the mesh, as a result of which it breaks into droplets as a result of Rayleigh scattering. Sound energy can be supplied from a vibrating tube or plate actuated by a piezoelectric crystal or as a result of vibrations of the mesh itself. Non-limiting examples of atomizers include any atomizer or nozzle for one or two fluids producing droplets of appropriate size. An atomizer for one fluid works by forcing the liquid through one or more holes, the stream of liquid disintegrating into droplets. A two-fluid atomizer works by forcing both gas and liquid through one or more holes or as a result of a collision of a liquid stream with another liquid or gas stream.

[0108] The choice of a nebulizer that converts an aerosol formulation into an aerosol is important for the administration of the active or active substance. Different nebulizers have different performance depending on their design and principle of operation, and are sensitive to the physical and chemical properties of the preparation. For example, two formulations differing in surface tension may have different particle size distributions. In addition, drug tolerance is affected by drug properties such as pH, osmolality and permeating ion content, which is why preferred embodiments are compatible with certain ranges of these properties.

[0109] In a preferred embodiment, the nebulization formulation is delivered to the intrabronchial space in the form of an aerosol having an MMAD value from about 1 μm to about 5 μm and a GSD value less than 2 using a suitable nebulizer. In order to achieve optimal effectiveness and to avoid adverse effects on the upper respiratory tract and the whole body, the aerosol should not have an MMAD value exceeding about 5 μm and should not have a GSD value exceeding about 2. If the aerosol has an MMAD value greater than about 5 μm or a GSD value greater than about 2, a significant percentage of the dose may reach the upper respiratory tract, thereby reducing the amount of drug delivered to the desired area of ​​the lower respiratory tract. If the aerosol MMAD is less than about 1 μm, then a significant percentage of the particles may remain suspended in the inhaled air and may then be exhaled during exhalation.

[0110] The compounds of the invention may further be administered by trans-bronchial lavage during bronchoscopy.

[0111] Formulations suitable for oral administration may be in separate units, for example capsules, cachets or tablets, each containing a predetermined quantity of active substance in the form of a powder or granules in the form of a solution or suspension in a liquid containing or not containing water water or in the form of an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active substance may also be in the form of a sachet, a bolus, a sweetener paste or other paste.

[0112] A tablet may be made by compression or molding with one or more accessory ingredients. Compressed tablets can be made by compressing in a suitable machine the active substance in a free-flowing form, e.g. a powder or granules, optionally mixed with binders, lubricant, inert diluent, surfactant or dispersing agent. Molded tablets may be made by molding a mixture of the powdered compound moistened with an inert liquid diluent in a suitable machine. The tablets may optionally be coated or scored and may be prepared in such a way as to provide free or controlled release of the active substance contained therein.

[0113] Formulations for topical administration in the mouth, for example buccal or sublingual, include lozenges containing the active substance in a flavored base, e.g. sucrose or acacia or tragacanth, and pastilles containing the active substance in an inert base, e.g. gelatin and glycerin or sucrose and acacia.

[0114] Preparations for parenteral administration contain aqueous and non-aqueous, sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatic agents and solutes that provide the isotonicity of the formulation to the subject's blood, and aqueous and non-aqueous sterile suspensions that may contain agents that facilitate formation. suspensions and thickeners. The preparations may be presented in packages containing a unit dose or multiple doses, for example in sealed ampoules and vials, and may be stored in the form of freeze-dried (freeze-dried), it is only necessary to add a sterile liquid carrier, e.g. sodium chloride solution or water injections immediately before use. Injectable solutions and suspensions prepared immediately before use can be prepared from sterile powders, granules and tablets of the types previously described.

[0115] Oral fluids, for example solutions, syrups and elixirs, can be prepared in the form of a dosage unit, whereby a given amount contains a predetermined quantity of active substance. Syrups can be prepared by dissolving the active substance in a suitably flavored aqueous solution, and elixirs are prepared using a pharmaceutically acceptable alcoholic base. Suspensions can be prepared by dispersing the active substance in a pharmaceutically acceptable medium. Solubilizing agents and emulsifiers, for example ethoxylated isostearyl alcohols and polyoxyethylene sorbitol ethers, preservatives, flavoring additives such as peppermint oil, or natural sweeteners or saccharin or other artificial sweeteners and the like can also be included in the oral liquid compositions.

[0116] Liposomal systems for administration, for example small single-lamellar vesicles, large single-lamellar vesicles and multi-lamellar vesicles, may also be used as agents for administering the compounds of the invention. Liposomes can be made from a variety of phospholipids, for example cholesterol, stearylamine and phosphatidylcholines.

[0117] Pharmaceutical compositions for topical administration may be prepared in the form of ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols or oils. Compositions intended for the treatment of eyes or other external tissues, e.g. mouth and skin, may be used in the form of an ointment or cream for external use. If the preparation of the active substance is made in the form of an ointment, this substance can be used with a paraffin or water-miscible ointment base. Optionally, the active substance formulation can be prepared as a cream with an oil-in-water cream base or a water-in-oil base.

[0118] Other compositions intended for topical administration to the eyes or ears include eye drops and ear drops in which the active substance is dissolved or suspended in a suitable carrier, such as, for example, an aqueous solvent, including sodium chloride solution.

[0119] Compositions intended for nasal administration include aerosols, solutions, suspensions, sprays, mists and drops. Nasal spray formulations may be prepared in substantially the same manner as inhalation aerosol formulations, except that nasal formulations having a non-respirable size are preferred for nasal formulations. Usually particles of about 5 microns can be used, up to the size of visible droplets. Therefore, for nasal administration, a particle size in the range of 10-500 μm can be used to ensure nasal retention.

[0120] Transdermal patches may also be used that are designed to stay in contact with the patient's epidermis for a long time and to facilitate absorption of the active substance through the skin.

[0121] Compositions for vaginal or rectal administration include ointments, creams, suppositories and ingots, all preparations can be prepared using conventional techniques.

[0122] In another aspect, the invention provides a method of improving mucosal surface hydration or restoring mucosal defense mechanisms in a human in need thereof, comprising administering to a human a pharmaceutical composition comprising a compound of the invention, said compound being administered in an effective amount. In one preferred embodiment, the method comprises administering the pharmaceutical composition in the form of an inhalable composition containing an amount of a compound of the invention sufficient to obtain a dissolved compound concentration on airway surfaces from about 10^-9, 10^-8 or 10^-7 up to about 10^-4, 10^-3, 10^-2 or 10^-1moles per liter, more preferably from about 10^-9 up to about 10^-4 moles per liter.

[0123] In another aspect, the invention provides a method of treating any disease among the following: disease associated with reversible or irreversible airway obstruction, chronic obstructive pulmonary disease (COPD), asthma, bronchiectasis (including bronchiectasis associated with diseases other than cystic fibrosis), acute bronchitis, chronic bronchitis, cough after viral infection, cystic fibrosis, emphysema, pneumonia, bronchiolitis, bronchiolar transplantation and respiratory tracheitis and bronchitis or the prevention of respiratory pneumonia in a human in need thereof, comprising administering to a human a pharmaceutical composition comprising a compound of the invention, said compound being administered in an effective amount. In one preferred embodiment, the method comprises administering the pharmaceutical composition as an inhalable composition containing an amount of a compound of the invention sufficient to obtain a dissolved concentration.

8 7 compound on airway surfaces from about 10^-9, 10^-8 or 10^-7 up to about 10^-4, 10^-3, 10^-2 or 10^-1 moles per liter, more preferably from about 10^-9 up to about 10^-4moles per liter.

[0124] In another aspect, the invention provides a method of treating any disease among the following: dry mouth (xerostomy), dry skin, vaginal dryness, sinusitis, sinusitis or dry nose, including dry nose caused by dry oxygen, dry eye syndrome or Sjogren's disease, improving eye or corneal hydration, treating obstruction syndrome distal bowel, treatment of otitis media, primary ciliary dyskinesia, distal bowel syndrome, esophagitis, constipation or chronic diverticulitis in a human in need thereof, comprising administering to a human a pharmaceutical composition comprising a compound of the invention, said compound being administered in an effective amount.

[0125] Preferred unit dosage preparations for the compounds of the invention are those containing an effective amount of the active substance or a suitable fraction thereof.

[0126] It should be understood that in addition to the ingredients specifically identified above, the formulations of the present invention may contain other conventional agents in the art taking into account the type of formulation suitable, for example formulations suitable for oral administration may contain flavoring agents.

[0127] The compositions of the present invention may be prepared in an immediate, controlled or solid release form as needed for the particular disease being treated and the desired route of administration. For example, a controlled release formulation for oral administration may be desirable in the treatment of constipation to maximize delivery of the active substance to the large intestine. Such preparations and excipients suitable for them are well known in the pharmaceutical art. Since the free base of the compound is usually less soluble in aqueous solutions than the salt, compositions containing the free base of the compound of formula I can be used to obtain a more stable release of the active substance administered by inhalation to the lungs. The active substance found in the lungs in the form of solid particles, which did not dissolve in the solution, is not available to elicit a physiological response, but is a supply of a bioavailable drug that gradually dissolves in the solution. As another example, both the free base and the salt form of the compound of the invention can be used in the formulation to provide both direct and solid release of the active substance for dissolution in mucous secretions, e.g. from the nose.

COMBINATIONS [0128] The compounds of the invention may be prepared and / or used in combination with other therapeutically active agents. Examples of other therapeutically active agents that can be used in the preparation or used in combination with the compounds of the invention include, but are not limited to, osmolites, anti-inflammatory agents, anticholinergic agents, β-agonists (including selective₂antagonists), P2Y2 receptor agonists, peroxisome proliferator activated receptor (PPAR) delta agonists, other epithelial sodium channel blockers (ENaC receptor blockers), modulators of the membrane conductivity of cystic fibrosis (CFTR), kinase inhibitors, anti-infectives, antihistamines, non-histamines anti-inflammatory, elastase and protease inhibitors, and mucin or mucin modifying agents, for example, surfactants. In addition, for cardiovascular indications, the compounds of the invention may be used in combination with beta-blockers, ACE inhibitors, HMGCoA reductase inhibitors, calcium channel blockers and other cardiovascular drugs. [0129] The present invention thus provides, as part of another aspect, a composition comprising an effective amount of a compound of the invention and one or more other therapeutically active agents selected from the following: osmolites, anti-inflammatory agents, anticholinergic agents, βagonists (including selective₂-agonists), P2Y2 receptor agonists, PPAR delta agonists, ENaC receptor blockers, modulators of cystic fibrosis (CFTR) membrane conduction regulator, kinase inhibitors, anti-infective agents, antihistamines, non-antibiotic anti-inflammatory macrolides, elastase and protease inhibitors, and mucus modifying agents, or mucus modifiers for example, surfactants. The present invention therefore provides, as part of another aspect, a composition comprising an effective amount of a compound of the invention and one or more other therapeutically active agents selected from beta-blockers, ACE inhibitors, HMG-CoA reductase inhibitors and calcium channel blockers. The use of the compounds of the invention in combination with one or more therapeutically active agents (in particular osmolytes) may reduce the dose of the compound of the invention necessary for sufficient hydration of the mucosal surface, thereby reducing the possibility of undesirable side effects that are attributed to systemic sodium channel blockade, example in the kidneys.

[0130] "Osmolites" according to the present invention are osmotically active molecules or compounds. 'Osmotically active' molecules and compounds must not cross membranes (ie, they are essentially not absorbed) on the surface of the respiratory or lung epithelium. The terms "airway surface" and "lung surface" as used herein include lung airway surfaces such as bronchi and bronchioles, alveolar surfaces, and nasal and sinus surfaces. Suitable osmolytes include ionic osmolytes (i.e. salts) and non-ionic osmolytes (i.e. sugars, sugar alcohols and organic osmolytes). In general, osmolites (both ionic and non-ionic) used in combination with the compounds of the invention are preferably osmolytes that do not promote or even inhibit or retard bacterial growth. Osmolites suitable for use in the present invention may exist in racemic form or in the form of an enantiomer, diastereomer, tautomer, polymorph or pseudopolymorph.

[0131] Examples of ionic osmolytes useful in the present invention include any salt of a pharmaceutically acceptable anion and a pharmaceutically acceptable cation. Preferably one (or both) of the anion and cation are osmotically active and are not subject to rapid active transport on the surfaces of the airways to which they are administered. Such compounds include, but are not limited to, anions and cations contained in commercially available salts and FDA approved, see e.g. Remington: The Science and Practice of Pharmacy, volume II, p. 1457 (19th edition, 1995) and can be used in any combination known in the art.

[0132] Specific examples of pharmaceutically acceptable osmotically active anions include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, hydrogen tartrate, bromide, calcium edetate, camsylate (camphorsulfonate), carbonate, chloride, citrate, dihydrochloride, edetate, edisylate (1,2- ethanedisulfonate), estolate (lauryl sulfate), esylate (1,2-ethanedisulfonate), fumarate, gluceptan, gluconate, glutamate, glycolylarsanilane (p-glycolamidophenylarsonate), hexyl resorcinate, hydrabamine (N, N'di (dehydroabietyl) ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthane, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methyl nitrate, methylsulfate, napthylate, nitrate, nitrate, nitrate (pamoate), pantothenate, phosphate or diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tanninate, tartrate, teoclan (8-chloroteophylate), triethyldide, bicarbonate, etc. Preferred anions include chloride, sulfate, nitrate, gluconate, iodide, bicarbonate, bromide and phosphate.

[0133] Specific examples of pharmaceutically acceptable osmotically active cations include, but are not limited to, organic cations such as benzatin (A.A'-dibenzvloethYldiamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (A-methyl-D-glucamine), procaine, D-lysine, L-lysine, D-arginine, L-arginine, triethylammonium, N-methyl-Glycerol and the like; and metal cations, for example aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, iron, ammonium and the like. Preferred organic cations include organic cations containing 3 carbon atoms, 4 carbon atoms, 5 carbon atoms and 6 carbon atoms. Preferred cations include sodium, potassium, choline, lithium, meglumine, D-lysine, ammonium, magnesium and calcium.

[0134] Specific examples of ionic osmolytes that can be used in combination with a compound of the invention include, but are not limited to, the following: sodium chloride (in particular hypertonic sodium chloride solution), potassium chloride, choline chloride, choline iodide, lithium chloride, meglumine chloride, L-lysine chloride, D-lysine chloride, ammonium chloride, potassium sulfate, potassium nitrate, potassium gluconate, potassium iodide , ferric chloride, ferrous chloride, potassium bromide, and combinations of any two or more of the above. In one embodiment, the present invention provides a combination of a compound of the invention and two different osmotically active salts. If different salts are used, one of the anion or cation may be the same in different salts. The hypertonic sodium chloride solution is the preferred ionic osmolyte for use in combination with the compounds of the invention.

[0135] Non-ionic osmolytes include sugars, sugar alcohols and organic osmolytes. Sugars and sugar alcohols useful as osmolites in the present invention include, but are not limited to, sugars containing 3 carbon atoms (e.g., glycerol, dihydroxyacetone); sugars containing 4 carbon atoms (e.g. both D and L forms of erythrosis, threose and erythrulose); sugars containing 5 carbon atoms (e.g. both forms D and L of ribose, arabinose, xylose, lixose, psicose, fructose, sorbose and tagatose); and sugars containing 6 carbon atoms (e.g. both forms D and L altose, allose, glucose, mannose, gulose, idose, galactose and talose and forms D and L alloheptulose, allohepulose, glucoheptulose, mannoheptulose, guloheptulose, idoheptulose, galactoheptulose taloheptulozy). Additional sugars useful in carrying out the present invention include raffinose, oligosaccharides from the raffinose series and stachyose. In addition, both the D and L forms of the reduced form of each sugar and sugar alcohol are useful in the present invention. For example, glucose reduction results in sorbitol, an osmolyte falling within the scope of the invention. Therefore, sorbitol and other reduced forms of sugars and sugar alcohols (e.g. mannitol, dulcitol, arabitol) are suitable osmolites for use in the present invention.

Mannitol is the preferred nonionic osmolyte for use in combination with the compounds of the invention.

[0136] The term "organic osmolytes" is generally used to refer to molecules controlling intracellular osmolality in the kidneys. See, e.g., JS Handler et al., Comp. Biochem. Physiol, 117, 301-306 (1997); M. Burg, Am. J. Physiol. 268, F983-F996 (1995). Organic osmolytes include, among others, three main classes of compounds: polyols (polyhydric alcohols), methylamines and amino acids. Suitable polyol organic osmolytes include, but are not limited to, inositol, myo-inositol and sorbitol. Suitable methylamine organic osmolytes include, but are not limited to, choline, betaine, carnitine (L, D and DL forms), phosphorylcholine, lysophosphorylcholine, glycerophosphorylcholine, creatine and creatine phosphate. Suitable amino acid organic osmolytes include, but are not limited to, forms D and L of glycine, alanine, glutamine, glutamate, aspartate, proline and taurine. Additional organic osmolytes suitable for use in the present invention include thihulose and sarcosine. Mammalian organic osmolytes are preferred, with human organic osmolytes being most preferred. However, some organic osmolytes are of bacterial, yeast, and marine origin; these compounds can also be used in the present invention.

[0137] Osmolyte precursors can be used in combination with the compounds of the invention. The term "osmolite precursor" as used herein means a compound that converts to osmolyte in a metabolic (catabolic or anabolic) process. Examples of osmolyte precursors include, but are not limited to, glucose, glucose polymers, glycerol, choline, phosphatidylcholine, lysophosphatidylcholine and inorganic phosphates, precursors of polyols and methylamines. Amino acid osmolyte precursors include proteins, peptides and polyamino acids that are hydrolyzed to give osmolytic amino acids, as well as osmolyte precursors that can be converted to osmolytic amino acids in a metabolic process such as transamination. For example, the precursor of the amino acid glutamine is poly-L60 glutamine, and the precursor of glutamate is poly-L- (glutamic acid).

[0138] Chemically modified osmolites or osmolyte precursors can also be used. Such chemical modifications include combining the osmolyte (or precursor) with an additional chemical group that changes or enhances the activity of the osmolyte or osmolite precursor (e.g., inhibits the degradation of the osmolyte molecule). Such chemical modifications are used for drugs or prodrugs and are known in the art. (See, for example, US Pat. Nos. 4,479,932 and 4,540,564; Shek, E. et al., J. Med. Chem. 19: 113-117 (1976); Bodor, N. et al., J. Pharm. Sci. 67: 1045-1050 (1978); Bodor, N. et al., J. Med. Chem. 26: 313-318 (1983); Bodor, N. et al., J. Pharm. Sci. 75: 29-35 (1986).

[0139] Preferred ionic osmolytes for use in combination with the compounds of the invention include sodium chloride, in particular hypertonic sodium chloride solution, and mannitol.

[0140] For a formulation containing 7% and&gt; 7% hypertonic sodium chloride solution, preparations containing bicarbonate anions may be particularly useful, in particular for respiratory disorders with membrane dysfunction regulator in CF (CFTR), such as cystic fibrosis or COPD . Recent discoveries show that despite the relative ratio of HCO3 conductivity/ chairmanship Cl^- from 0.1 to 0.2 for single CFTR channels activated by cAMP and ATP, the sweat channel ratio can range from virtually 0 to almost 1.0, depending on the stimulation conditions. This means that the combination of cAMP + cGMP + αketoglutarate may give CFTR conductivity for HCO3^- almost equal to conduction for Cl^- (Quiton et al. Physiology, vol. 22, No. 3, 212-225, June 2007). In addition, preparations containing 7% and&gt; 7% hypertonic sodium chloride solution and containing bicarbonate anions may be particularly useful due to better pH control in the fluid on the surface of the respiratory tract. Firstly, it was shown that respiratory tract acidification occurs in the course of CF (Tate et al. 2002), and the lack of secretion of bicarbonate dependent on CFTR may lead to impaired ability to respond to respiratory diseases associated with acidification of the fluid layer on the surface of the respiratory tract (Coakley et al. 2003). In addition, the introduction of an HS solution without bicarbonate on the surface of the lung may further reduce the concentration of bicarbonate and potentially reduce the pH or ability to respond to acidification of the airway in the fluid layer on the surface of the airway. Therefore, the introduction of bicarbonate anions into HS can help maintain or improve the pH of the fluid layer on the surface of the airways in patients with CF.

[0141] Thanks to this evidence, it may be particularly useful to include a bicarbonate anion in a formulation containing 7% or&gt; 7% hypertonic sodium chloride in the method of the present invention. Preparations containing bicarbonate anion concentrations up to 30 to 200 mM are particularly important for HS 7% or&gt; 7% solutions.

[0142] It should be understood that the hypertonic sodium chloride solution has a salt concentration exceeding the concentration in the physiological sodium chloride (NS) solution, i.e. exceeding 9 g / L or 0.9% w / v, and the hypotonic sodium chloride solution has a lower salt concentration from a concentration in a physiological sodium chloride solution, for example from about 1 g / l or 0.1% w / v to about 8 g / l or 0.8% w / v. Hypertonic sodium chloride solutions useful in the formulations and treatment methods of the present description may contain a salt concentration from about 1% to about 23.4% (w / v). In one embodiment, the hypertonic sodium chloride solution contains a salt concentration from about 60 g / L (6% w / v) to about 100 g / L (10% w / v). In another embodiment, the hypertonic sodium chloride solution contains a salt concentration from about 70 g / L (7% w / v) to about 100 g / L (10% w / v). In further embodiments, the sodium chloride solution contains a salt concentration of a) from about 0.5 g / L (0.05% w / v) to about 70 g / L (7% w / v); b) from about 1 g / l (0.1% w / v) to about 60 g / l (6% w / v); c) from about 1 g / l (0.1% w / v) to about 50 g / l (5% w / v); d) from about 1 g / l (0.1% w / v) to about 40 g / l (4% w / v); e) from about 1 g / l (0.1% w / v) to about 30 g / l (3% w / v); and f) from about 1 g / l (0.1% w / v) to about 20 g / l (2% w / v).

[0143] Specific sodium chloride concentrations or solutions useful in the formulations and treatments of this description include independently salt concentrations of 1 g / L (0.1% w / v), 2 g / L (0.2% w / v) , 3 g / l (0.3% w / v), 4 g / l (0.4% w / v), 5 g / l (0.5% w / v), 6 g / l (0, 6% w / v), 7 g / l (0.7% w / v), 8 g / l (0.8% w / v), 9 g / l (0.9% w / v), 10 g / l (1% w / v), 20 g / l (2% w / v), 30 g / l (3% w / v), 40 g / l (4% w / v), 50 g / l (5% w / v), 60 g / l (8% w / v), 70 g / l (7% w / v), 80 g / l (8% w / v), 90 g / l ( 9% w / v), 100 g / l (10% w / v), 110 g / l (11% w / v), 120 g / l (12% w / v), 130 g / l (13% w / v), 140 g / l (14% w / v), 150 g / l (15% w / v), 160 g / l (16% w / v), 170 g / l (17% w / v), 180 g / l ( 18% w / v), 190 g / l (19% w / v), 200 g / l (20% w / v), 210 g / l (21% w / v), 220 g / l (22% w / v) and 230 g / l (23% w / v). Sodium chloride concentrations may also be used between each of these concentrations / percentages, for example sodium chloride 1.7 g / l (0.17% w / v) solution, 1.25 g / l (1.25% w / v), 1.5 g / l (1.5% w / v), 25 g / l (2.5% w / v), 28 g / l (2.8% w / v), 35 g / l (3.5% w / v), 45 g / l (4.5% w / v) and 75 g / l (7.5% w / v).

[0144] The specific useful concentration of the hypotonic sodium chloride solution includes values ​​from about 0.12 g / L (0.012% w / v) to about 8.5 g / L (0.85% w / v). Any concentration in this range can be used, for example, in w / v ratio, 0.05%, 0.1%, 0.15%, 0.2%, 0.225% (1/4 NS), 0.25% , 0.3% (1/3 NS), 0.35%, 0.4%, 0.45% (1/2 NS), 0.5%, 0.55%, 0.6% (2 / 3 NS), 0.65%, 0.675% (3/4 NS), 0.7%, 0.75% and 0.8%.

[0145] Each of these ranges and specific concentrations of sodium chloride described herein can be used together with the formulations, treatments, regimens, and kits described herein.

[0146] In addition, chemically modified osmolites or osmolyte precursors are envisioned within the scope of the present invention. Such chemical modifications include combining the osmolyte (or precursor) with an additional chemical group that changes or enhances the activity of the osmolyte or osmolyte precursor (e.g., inhibits the degradation of the osmolyte molecule). Such chemical modifications are used for drugs or prodrugs and are known in the art. (See, for example, pat. U.S. Patent Nos. 4,479,932 and 4,540,564; Shek, E. et al., J. Med. Chem. 19: 113-117 (1976); Bodor, N. et al., J. Pharm. Sci. 67: 1045-1050 (1978); Bodor, N. et al., J. Med. Chem. 26: 313-318 (1983); Bodor, N. et al., J. Pharm. Sci. 75: 29-35 (1986), each of which is incorporated herein by reference.

[0147] Suitable anti-inflammatory agents for use in combination with the compounds of the invention include corticosteroids and nonsteroidal anti-inflammatory drugs (NSAIDs), in particular phosphodiesterase (PDE) inhibitors. Examples of corticosteroids for use in the present invention include oral or inhaled corticosteroids or their prodrugs. Specific examples include, but are not limited to, ciclesonide, desisobutyrylcyclononide, budesonide, flunizolide, mometasone and its esters (e.g. mometasone furoate), fluticasone propionate, fluticasone furoate, beclometasone, methylprednisolone, prednisolone, dexamethasone, 6a, 9a-difluoro-17a S-fluoromethyl ester [(2-furanylcarbonyl) oxy] - Ιΐβ-hydroxy-16-oxo-methyl - 1,4-dieneΠβ-carbothioate, S- (2-oxo-tetrahydrofuran-3S-yl) acid 6α, 9α-difluoro-β-hydroxy-16α-methyl-3-oxo-17α-propionyloxy-androsta-1,4-diene 17-carbothioate, beclometasone esters (e.g. 17-propionate ester or 17,21-dipropionate ester, fluoromethyl ester), triamcinolone acetonide, rofleponide, or any combination or subset thereof. Preferred corticosteroids for the preparation of preparations or in combination with the compounds of the invention are selected from ciclesonide, desisobutyrylcyclononide, budesonide, mometasone, fluticasone propionate and fluticasone furoate or any combination or subset thereof.

[0148] NSAIDs for use in the present invention include, but are not limited to, sodium cromoglycate, nedocromil sodium, phosphodiesterase (PDE) inhibitors (e.g., theophylline, aminophylline, PDE4 inhibitors, mixed PDE3 / PDE4 inhibitors or mixed PDE4 / PDE7 inhibitors), leukotriene antagonists , leukotriene synthesis inhibitors (e.g., LO and FLAP inhibitors), nitric oxide synthase (iNOS) inhibitors, protease inhibitors (e.g. tryptase inhibitors, neutrophil elastase inhibitors and metalloprotease inhibitors), e2-integrin antagonists and adenosine receptor agonists or antagonists (e.g. adenosine 2a receptor agonists), cytokine antagonists (e.g. chemokine antagonists) or cytokine synthesis inhibitors (e.g. prostagin D-receptor antagonists) CRTH2)). Examples of leukotriene modifiers suitable for administration by the method of the present invention include montelukast, zileuton and zafirlukast.

[0149] The PDE4 inhibitor, mixed PDE3 / PDE4 inhibitor or mixed PDE4 / PDE7 inhibitor can be any compound that is known to inhibit the PDE4 enzyme, or has been found to act as a PDE4 inhibitor and which is selective PDE4 inhibitors (i.e. which do not cause significant inhibition of other members of the PDE family). Examples of specific PDE4 inhibitors for the preparation and combination with compounds of the invention include, but are not limited to, roflumilast, pumafenthrin, arophylline, cilomilast, tofimilast, oglemilast, tolafenthrin, piclamilast, ibudilast, apremilast, 2- [4- [6,7-diethoxy -2,3-bis (hydroxymethyl) -1-naphthalenyl] -2-pyridinyl] -4- (3-pyridinyl) -1 (2H) -phthalazinone (T2585), N- (3,5-dichloro-4-pyridinyl) -1- [(4-fluorophenyl) methyl] -5-hydroxy-α-oxo-1H-indole-3-acetamide (AWD-12-281, 4 - [(2R) -2- [3- (cyclopentyloxy) -4-methoxyphenyl] -2-phenylethyl] pyridine (CDP-840), acid 2- [4 - [[[[2- (1,3-benzodioxole -5-yloxy) -3-pyridinyl] carbonyl] amino] methyl] -3-fluorophenoxy] - (2R) -propane (CP671305), N- (4,6-dimethyl-2-pyrimidinyl) -4- [4,5, 6,7-tetrahydro-2- (4-methoxy-3-methylphenyl) -5- (4-methyl-1-piperazinyl) -1H-indol-1-yl] benzenesulfonamide, (2E) -2-butenedione dianate (YM-393059 ), 9 - [(2-fluorophenyl) methyl] -N-methyl-2- (trifluoromethyl) -9H-purin-6-amine (NCS-613), N- (2,5-dichloro-3-pyridinyl) 8-methoxy-5-quinolinecarboxamide (D-4418), N - [(3R) -9-amino-3,4,6,7-tetrahydro-4-oxo-1 -phenylpyrrolo [3,2,1 -] [1,4] benzodiazepin-3-yl] -3H-purin-6amine (PD-168787), 3 - [[3- (cyclopentyloxy) -4-methoxyphenyl] methyl] -N hydrochloride -ethyl-8- (1-methylethyl) -3H-purin-6-amine (V11294A), N- (3,5-dichloro-1-oxido-4-pyridinyl) -8-methoxy-2 (trifluoromethyl) -5 -quinolinecarboxamide (Sch351591), 5- [3- (cyclopentyloxy) 4-methoxyphenyl] -3 - [(3-methylphenyl) methyl] - (3S, 5S) -2-piperidinone (HT-0712),

5- (2 - ((1 ^, 4 ^) - 4-amino-1- (3- (cyclopentyloxy) -4-methoxyphenyl) cyclohexyl) ethynyl) pyrimidin-2-amine, cis- [4-cyano-4- (3-cyclopropylmethoxy-4-difluoromethoxyphenyl) cyclohexan-1-ol] and 4- [6,7-diethoxy-2,3bis (hydroxymethyl) -1-naphthalenyl] -1- (2-methoxyethyl) -2 (1H) -pyridinone ( T-440) and any combination or subset thereof.

[0150] Leukotriene antagonists and inhibitors of leukotriene synthesis include zafirlukast, montelukast sodium, zileuton and pranlukast.

[0151] Anticholinergic agents for the preparation of preparations or for use in combination with the compounds of the invention include, but are not limited to muscarinic receptor antagonists, in particular all receptor antagonists and M3 receptor antagonists. Examples of compounds include plant alkaloids of yell, for example atropine, scopolamine, homatropin, hyoscyamine and various forms, including their salts (e.g. anhydrous atropine, atropine sulfate, atropine oxide or hydrochloride, methylatropine nitrate, homatropine hydrobromide, homatropine methylbromide, hyoscyamine hydrobromide, hyoscyamine sulfate, scopolamine hydrobromide, scopolamine methylbromide) or any combination thereof.

[0152] Additional anticholinergic agents for the preparation and combination use include metanthelin, propanteline bromide, anisotropin methylbromide or Valpin 50, aclidinium bromide, glycopyrrolate (Robinul), isopropamide iodide, mepenzolate bromide, tridihexetheloryl chloride, cyclosilide trihexyphenidyl hydrochloride, pyrenezine, telenzepine and methoctramine, or any combination or subset thereof.

[0153] Preferred anticholinergic agents for the preparation of formulations and for use in combination with the compounds of the invention include ipratropium (bromide), oxitropium (bromide) and tiotropium (bromide) or any combination or subset thereof.

[0154] Examples of β-agonists for the preparation of formulations and for use in combination with the compounds of the invention include, but are not limited to, salmeterol, R-salmeterol and their salts (xinafoate), albuterol or R-albuterol (free base or sulfate), levalbuterol, salbutamol, formoterol (fumarate), fenoterol, procaterol, pirbuterol, metaprterenol, terbutaline and its salts and any combination or subset thereof.

[0155] P2Y2 agonists for the preparation of formulations and for use in combination with the compounds of the invention can be used in an amount effective to stimulate the secretion of chloride and water on the surface of the respiratory tract, in particular on the surface of the nasal airway. Suitable P2Y2 receptor agonists are known in the art and are described, for example, in columns 9-10 of US Patent No. 6,264,975, as well as in US Patent Nos. 5,656,256 and 5,292,498.

[0156] P2Y2 receptor agonists that can be administered by the methods of the present invention include P2Y2 receptor agonists such as ATP, UTP, UTP-gamma-S and P2Y2 dinucleotide receptor agonists (e.g., denufozole or diquivozole) or a pharmaceutically acceptable salt thereof. The P2Y2 receptor agonist is usually used in an amount effective to stimulate the secretion of chloride and water on the surface of the respiratory tract, in particular on the surface of the respiratory tract within the nose. Suitable P2Y2 agonists are described, among others, in US Pat. U.S. No. 6,264,975, pat. U.S. No. 5,656,256, pat. U.S. No. 5,292,498, pat. U.S. No. 6,348,589, pat. USA No. 6,818,629, pat. U.S. No. 6,977,246, pat. U.S. No. 7,223,744, pat. U.S. No. 7,531,525 and U.S. Pat. USA 2009/0306009, each of which is incorporated herein by reference.

[0157] Combination therapies and formulations herein may include adenosine 2b (A2b) receptor agonists as well as including BAY 60-6583, NECA (N-ethylcarboxamidoadenosine), (S) -PHPNECA, LUF-5835 and LUF-5845. A2b receptor agonists are described in Volpini et al., Journal of Medicinal Chemistry 45 (15): 3271-9 (2002); Volpini et al., Current Pharmaceutical Design 8 (26): 2285-98 (2002); Baraldi et al., Journal of Medicinal Chemistry 47 (6): Cacciari et al., 1434-47 (2004); Mini Reviews in Medicinal Chemistry 5 (12): 1053-60 (December 2005); Baraldi et al., Current Medicinal Chemistry 13 (28): 3467-82 (2006); Beukers et al., Medicinal Research Reviews 26 (5): 667-98 (September 2006); Elzein et al., Bioorganic &amp; Medicinal Chemistry Letters 16 (2): 302-6 (January 2006); Carotti et al., Journal of Medicinal Chemistry 49 (1): 282-99 (January 2006); Tabrizi et al., Bioorganic &amp; Medicinal Chemistry 16 (5): 2419-30 (March 2008); and Stefanachi et al., Bioorganic &amp; Medicinal Chemistry 16 (6): 2852-69 (March 2008).

[0158] Examples of other ENaC receptor blockers for the preparation and combination of compounds of the invention include, but are not limited to, amiloride and its derivatives, for example, compounds described in US Patent No. 6,856,815 and PCT Publication No. WO2003 / 070182, WO2004 / 073629, WO2005 / 018644, WO2006 / 022935, WO2007 / 018640 and WO2007 / 146889, all for Parion Sciences, Inc.

[0159] ENaC small molecule blockers are capable of directly preventing sodium transport through pores in ENaC channels. The ENaC blocker which can be used in conjunction with this document includes, but is not limited to, amiloride, benzamil, fenamil, and amiloride analogues, examples of which are given in US Pat. U.S. No. 6,858,614, pat. USA No. 6,858,615, pat. U.S. No. 6,903,105, pat. U.S. No. 6,995,160, pat. U.S. No. 7,026,325, pat. U.S. No. 7,030,117, pat. U.S. No. 7,064,129, pat. U.S. No. 7,186,833, pat. U.S. No. 7,189,719, pat. U.S. No. 7,192,958, pat. U.S. No. 7,192,959, pat. U.S. No. 7,241,766, pat. U.S. No. 7,247,636, pat. U.S. No. 7,247,637, pat. U.S. No. 7,317,013, pat. U.S. No. 7,332,496, pat. U.S. No. 7,345,044, pat. U.S. No. 7,368,447, pat. U.S. No. 7,368,450, pat. U.S. No. 7,388,451, pat. U.S. No. 7,375,107, pat. U.S. No. 7,399,766, pat. U.S. No. 7,410,968, pat. USA No. 7,820,678, pat. U.S. No. 7,842,697, pat. U.S. No. 7,868,010, pat. U.S. Patent No. 7,875,619.

[0160] It has been specifically described that ENaC proteolysis increases sodium transport through ENaC. The protease inhibitor blocks the activity of endogenous proteases in the respiratory tract, thus preventing digestion and ENaC activation. Proteases that digest ENaC include furin, meprine, matryptase, trypsin, channel-related proteases (CAPs) and neutrophilic elastases. Protease inhibitors that can inhibit the proteolytic activity of these proteases and can be administered in combination herein are, among others, kamostat, prostazin, furin, aprotinin, leupeptin and trypsin inhibitors.

[0161] The associations of the present description may contain one or more suitable nucleic acids (or poly (nucleic acids)), including but not limited to antisense oligonucleotide, siRNA, miRNA, miRNA mimic nucleic acid, antagomir, ribozyme, aptamer and decoy acid oligonucleotides nucleic. See, e.g., U.S. Patent Application Publication No. 20100316628. Generally, such nucleic acids may be from 17 or 19 nucleotides in length, up to a maximum of 23, 25 or 27 nucleotides in length or more. Examples include, but are not limited to, those described in U.S. Patent No. 7,517,865 and U.S. Patent Applications No. 20100215588; 20100316628; 20110008366 and 20110104255. Basically, siRNAs are 17 or 19 nucleotides in length, up to a maximum of 23, 25 or 27 nucleotides in length or more.

[0162] Compounds modulating CFTR activity that can be administered in the combinations of the present invention include, but are not limited to, those described in US 2009/0246137 A1, US 2009/0253736 A1, US 2010/0227888 A1, Patent Number 7,645,789, US 2009/0246820 A1, US 2009/0221597 A1, US 2010/0184739 A1, US 2010/0130547 A1, US 2010/0168094 A1 and patent granted 7,553,855; US 7,772,259 B2, US 7,405,233 B2, US 2009/0203752, US 7,499,570.

[0163] Mucin modifying agents or mucins useful in the combinations and methods of the present description include reducing agents, surfactants and detergents, expectorants and deoxyribonuclease agents.

[0164] Mucin proteins are organized into high molecular weight polymers by forming covalent (disulfide) and non-covalent bonds. Rupture of covalent bonds with reducing agents is a commonly used method of reducing viscoelastic properties of mucus in vitro and is predicted to minimize mucus adhesion and increase in vivo clearance. It is well known that reducing agents reduce mucus viscosity in vitro and are commonly used as o

auxiliaries for processing sputum samples. Examples of reducing agents include sulfide-containing or phosphine-containing molecules capable of reducing disulfide bonds in proteins, including but not limited to Nacetylcysteine, N-acystelin, carbocysteine, glutathione, dithiothreitol, thioredoxin-containing proteins and tris (2-carboxyethyl) phosphine.

[0165] N-acetylcysteine ​​(NAC) has been approved for use in chest physiotherapy for loosening sticky or thick mucus in the airways⁽¹²⁾. Clinical studies to assess the effects of oral or inhaled NAC in cystic fibrosis and COPD found an improvement in the rheological properties of mucus and a tendency to improve lung function and reduce pulmonary exacerbations⁹. Most clinical data, however, indicate that NAC is at best a therapeutic agent with minimal efficacy in the treatment of airway obstruction due to mucus retention after oral or inhaled administration. In the latest review of available clinical literature on the use of NAC by Cochrane, there was no evidence of the effectiveness of NAC in cystic fibrosis¹⁰. The minimal clinical benefits of using NAC are for the following reasons:

[0166] NAC is a reducing agent with relatively low efficiency and only partial activity on the surface of the respiratory tract. In vitro, a very high NAC concentration (200 mM or 3.26%) is necessary to completely reduce Muc5B, the main mucin forming a gel in the respiratory tract. In addition, in the pH environment on the surface of the respiratory tract (for respiratory tract in cystic fibrosis and COPD, the pH was measured in the range of 6.0 to 7.2)¹¹ NAC is only partially reactive as thiolate with a negative charge. Therefore, in clinical conditions, NAC is administered in very high concentrations. However, it is anticipated that currently available aerosol devices will not be able to achieve a therapeutic concentration of up to 20% solution of Mucomyst on distal airway surfaces in typically short periods (7.5-15 minutes).

[0167] Labeled NAC in clinical studies ¹⁴Inhaled C is characterized by rapid elimination from the lungs with a half-life in the range of 6 to 36 minutes¹².

[0168] NAC is administered as a highly concentrated hypertonic inhalation solution (20% or 1.22 molar); has been found to cause bronchoconstriction and coughing. In many cases, it is recommended that NAC be administered with a bronchodilator to increase tolerance to this drug.

[0169] Therefore, reducing agents such as NAC are not very well suited for aerosol bolus administration. However, it is anticipated that the administration of reducing agents by pulmonary infusion in an aerosol will increase efficacy while also allowing a reduction in the concentration of reducing agent in the inhalation solution (which is expected to increase tolerance).

[0170] Surfactants and detergents are coating agents that have been shown to reduce mucus viscoelasticity, increasing its ability to be removed. Examples of surfactants include dipalmitoylphosphatidylcholine (DPPC), PF, palmitic acid, palmitoyloleoylphosphatidylglycerol, proteins associated with surfactants (e.g. SP-A, B or C) or may be of animal origin (e.g. obtained by washing the lungs of cows or calves or by extracting from a fragmented pig lung) or combinations thereof. See, e.g., US Patent Nos. 7,897,577; 5,876,970; 5,614,216; 5,100,806 and 4,312,860. Examples of surfactant products include Exosurf® Neonatal (colphosceryl palmitate), Pumactant® (DPPC and egg phosphatidylglycerol), KL-4 surfactant, Venticute® (lusulptide, rSP-C surfactant), Alveofact® (bowactant) ), Curosurf® (alpha porant), Infrasurf® (kalfactant), Newfacten® (modified bovine surfactant), Surface®, Natsurf ™ (ethoxylated alcohol nonionic surfactant) and Survanta® (beractant). Examples of detergents include Tween-80 and Triton-X 100, among others.

[0171] Any suitable expectorant may be used, including but not limited to guaifenesin (see, e.g., US Patent No. 7,345,051). Any suitable deoxyribonuclease may be used, including but not limited to alpha domase (see, e.g., US Patent No. 7,482,024). Examples of kinase inhibitors include NFkB, PI3K (phosphatidylinositol 3 kinase) inhibitors, p38MAP kinase and Rho kinase.

[0172] Anti-infective agents for the preparation of formulations and for use in combination with the compounds of the invention include antiviral agents and antibiotics. Examples of suitable antiviral agents include Tamiflu® (oseltamivir) and Relenza® (zanamivir). Examples of suitable antibiotics include, but are not limited to, aztreonam (arginine or lysine), fosfomycin and aminoglycosides, such as tobramycin, or any combination or subset thereof. Additional anti-infective agents that can be used herein include aminoglycosides, daptomycin, fluoroquinolones, ketolides, carbapenems, cephalosporins, erythromycin, linezolid, penicillins, azithromycin, clindamycin, oxalidinones, tetracyclines and vancomycin.

[0173] Examples of useful carbapenem antibiotics are impene, panipen, meropen, biapen, MK-826 (L-749,345), DA-1131, ER-35786, lenapenem, S-4661, CS-834 (prodrug R-95867), KR -21056 (prodrug KR-21012), L084 (prodrug LJC 11036) and ceftolozane (CXA-101).

[0174] For antihistamines (i.e. H1 receptor antagonists) for the preparation of formulations and for use in combination with the compounds of the invention include, but are not limited to, ethanolamines, e.g., diphenhydramine hydrochloride, carbinoxamine maleate, doxylamine, clemastine fumarate, diphenylhydramine hydrochloride and dimenhydrinate; ethylenediamine, e.g. pyrilamine maleate (mepyramine), tripelenamine hydrochloride, tripelenamine citrate and antazoline; alkylamines, for example, pheniramine, chlorpheniramine, bromophenamine, dexschlorfeniramine, triprolidine and acristastine; pyridines, e.g. metapyrylene, piperazine, e.g. hydroxyzine hydrochloride, hydroxyzine pamoate, cyclysine hydrochloride, cyclysine lactate, meclysine hydrochloride and cetirizine hydrochloride; piperidines, e.g. astemizole, levocabastine hydrochloride, loratadine, descarboethoxyloratadine, terfenadine and fexofenadine hydrochloride; tri- and tetracyclic compounds, for example promethazine, chlorprometazine, trimeprazine and azatadine; and azelastine hydrochloride or any combination or subset thereof.

[0175] Examples of other classes of therapeutic agents suitable for use in the combinations and methods of the present description include antiviral agents, e.g. ribavirin, antifungal agents, e.g. amphotericin, itraconazole and voriconazole, anti-rejection drugs, e.g. cyclosporin, tacrolimus and sirolimus, bronchodilators, including but not limited to anticholinergics, e.g. atrowent, siRNA, gene therapy vectors, aptamers, endothelin, alpha-1 antitrypsin and prostacyclin receptor antagonists.

[0176] In the methods of treatment and uses described above, the compound of the invention may be used alone or in combination with one or more other therapeutically active agents. Typically, any therapeutically active agent having a therapeutic effect on a disease or condition being treated may be used together with a compound of the invention in combination with the compounds of the invention, provided that the particular therapeutically active agent is compatible with therapy using a compound of the invention. Typical therapeutically active agents suitable for use in combination with the compounds of the invention include those mentioned above.

[0177] In one preferred embodiment, the compounds of the invention are used in combination with one or more osmolytes, in particular a hypertonic sodium chloride solution or mannitol.

[0178] In another aspect, the invention provides methods of treatment and uses as described above that comprise administering an effective amount of a compound of the invention and at least one other therapeutically active agent. The compounds of the invention and at least one additional therapeutically active agent can be used in combination simultaneously or subsequently in any therapeutically suitable combination. Administration of a compound of the invention together with one or more other therapeutically active agents may consist of co-administration in 1) a unitary pharmaceutical composition, for example, the compositions described above, or 2) in separate pharmaceutical compositions, each containing one or more ingredients constituting active substances. The combination ingredients can be administered separately in a sequential manner, with the compound of the invention being administered first and the second therapeutically active agent being administered as the second or vice versa.

[0179] In embodiments where the compound of the invention is administered in combination with one or more osmolytes, administration of each component is preferably simultaneous and may include unit composition or separate compositions. In one embodiment, the compound of the invention and one or more osmolytes are administered simultaneously by trans-bronchial lavage during bronchoscopy. In another embodiment, the compound of the invention and one or more osmolytes are administered simultaneously by inhalation.

[0180] When a compound of the invention is used in combination with another therapeutically active agent, the dose of each compound may differ from the situation in which the compound of the invention is used alone. The skilled person will easily determine the appropriate doses. Appropriate doses of the compound of the present invention, other therapeutic agent or agents, and the time of their administration relative to each other are selected so as to achieve the desired overall therapeutic effect, which remains within the knowledge and decision of the physician, clinician or attending veterinarian.

Experimental procedures

The present invention further provides methods for preparing compounds of the invention and synthetic intermediates useful in such methods as described in detail below.

[0181] Some abbreviations and acronyms are used in the description of the synthetic methods and details of experiments. Although a specialist in the field will know most of them, the following table lists many of these abbreviations and acronyms.

Abbreviation Meaning

AcOH acetic acid

AIBN azobisisobutyrolnitrile

AcOH acetic acid

<td>DIAD</td><td>diisopropyl azidocarboxylate</td>

<td>DIPEA</td><td>N, N-diisopropylethylamine</td>

<td>DCE</td><td>dichloroethane</td>

<td>DCM</td><td>dichloromethane</td>

<td>DMF</td><td>dimethylformamide</td>

<td>et</td><td>ethyl</td>

<td>EtOAc or EA</td><td>ethyl acetate</td>

<td>EtOH</td><td>ethanol</td>

<td>ESI</td><td>electrospray ionization</td>

<td>HATU</td><td>2- (1H-7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate</td>

<td>HPLC</td><td>high performance liquid chromatography</td>

<td>iPrOH</td><td>isopropyl alcohol</td>

<td>it or IT</td><td>dotchawiczy</td>

<td>Me</td><td>methyl</td>

<td>MeOH</td><td>methanol</td>

<td>m / z or m / e</td><td>mass to charge ratio</td>

<td>_MH⁺</td><td>weight plus 1</td>

<td>_MH^-</td><td>weight minus 1</td>

<td>MIC</td><td>minimum inhibitory concentration</td>

<td>MS or ms</td><td>mass spectrum</td>

<td>tp or tp</td><td>room temperature</td>

<td>rf</td><td>retention factor</td>

<td>t-Bu</td><td>tert-butyl</td>

<td>THF</td><td>tetrahydrofuran</td>

<td>TLC or tlc</td><td>thin layer chromatography</td>

<td>δ</td><td>parts per million towards lower field values ​​relative to tetramethylsilane</td>

<td>cbz</td><td>benzyloxycarbonyl, i.e. - (CO) O-benzyl</td>

<td>AUC</td><td>area under the curve or peak</td>

<td>MTBE</td><td>tert-butyl methyl ether</td>

<td>^tR</td><td>retention time</td>

<td>GC-MS</td><td>gas chromatography combined with mass spectrometry</td>

<td>wt%</td><td>weight percent</td>

<td>AcOH</td><td>acetic acid</td>

<td>h</td><td>hours</td>

<td>min</td><td>minutes</td>

<td>MHz</td><td>megahertz</td>

<td>TFA</td><td>trifluoroacetic acid</td>

<td>UV</td><td>ultraviolet</td>

<td>Boc</td><td>tert-butyloxycarbonyl</td>

<td>DIAD</td><td>diisopropyl azodicarboxylate</td>

<td>AcOH</td><td>acetic acid</td>

<td>DIPEA</td><td>N, N-diisopropylethylamine or Iiinig's base</td>

<td>Ph 3 P</td><td>triphenylphosphine</td>

[0182] Compounds of formula I can be synthesized using methods known in the art. A representative synthesis procedure is outlined in Scheme 1 below.

<img file="PL2723722T3_D0017.tif" />

[0183] These procedures are described, for example, in EJ Cragoe, "The Synthesis of Amiloride and Its Analogs" (chapter 3) in Amiloride and Its Analogs, pp. 25-36. Other methods for preparing amiloride analogues are described, for example, in US Patent No. 3,318,813 to Cragoe, in particular in methods A, B, C and D of the '813 patent. Still other methods that can be adapted to prepare compounds of the invention are described in PCT Publication No. WO2003 / 07182,

WO2005 / 108644, WO2005 / 022935, US 7,064,129, US 6,858,615, US 6,903,105,

WO 2004/073629, WO 2007/146869 and WO 2007/018640, all granted to Parion Sciences, Inc.

[0184] Preparation of methyl N'-3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate (2) is shown in WO 2009/074575.

In general, the compounds of the invention may conveniently be prepared by treating a compound of formula 2 with an amine of formula 3. More specifically, compounds of formula 2 are treated with an amine of formula 3 in a suitable solvent, such as methanol, ethanol or tetrahydrofuran, and in principle for example triethylamine (TEA) or diisopropylethylamine (DIPEA), with heating to an elevated temperature, e.g. 70 ° C. Further purification, stereoisomer separation, crystallization and / or formation of salt forms can be carried out using conventional methods.

[0186] It will be apparent to those skilled in the art that in some cases, the starting or intermediate compounds in the synthesis may contain other functional groups that provide alternative reaction sites. Undesirable reactions with such functional groups can be prevented by the use of appropriate protecting groups, such as amine and alcohol protecting groups, and, where appropriate, in the appropriate order of the synthetic steps. Suitable protecting groups are apparent to those skilled in the art. Methods for introducing and removing such protecting groups are well known in the art, and such conventional methods can also be used in the methods of the present invention.

[0187] The following specific examples are provided herein for illustrative purposes only and do not limit the scope of the present invention as defined in the claims.

[0188] Materials and methods. All reagents and solvents were purchased from Aldrich Chemical Corp. Chem-Impex, International Inc. and TCI Chemical Industry Co. Ltd. NMR spectra were recorded using Bruker AC 400 (¹H NMR at 400 MHz and ¹³C NMR at 100 MHz) or Bruker AC 300 (¹H NMR at 300 MHz and ¹³C NMR at 75 MHz). Proton spectra were referenced to tetramethylsilane as an internal standard and carbon spectra were referenced to CDCl₃, CD₃OD or DMSO-d₆ (purchased from Aldrich or Cambridge Isotope Laboratories, unless otherwise stated). Flash chromatography was performed using a Combiflash system (Combiflash Rf, Teledyne Isco) using a silica gel column (Redi Sep. Rf,

Teledyne Isco) or reverse phase analysis column (high performance C18 Gold column). ESI mass spectra were recorded using a Shimadzu LCMS-2010 EV mass spectrometer. HPLC analysis was performed using a Waters XTerra MS C18 5 gm 4.6x150 mm analytical column with detection at 220 nm (unless otherwise stated) in a system

HPLC Shimadzu Prominence. The following time sequence program was used at a flow rate of 1.0 ml per minute:

<td>Time (min)</td><td>Percent A (H₂O with 0.05% TFA)</td><td>Percent B (CH₃CN with 0.05% TFA)</td>

<td> 2,50</td><td> 90</td><td> 10</td>

<td> 20,00</td><td> 10</td><td> 90</td>

<td> 30,00</td><td> 10</td><td> 90</td>

<td> 32,50</td><td> 90</td><td> 10</td>

UPLC analysis was performed using a Waters ACQUITY UPLC HSS T3 analytical column, 1.8 gm 2.1x100 mm with detection at 220 nm (unless otherwise stated) in a Shimadzu Prominence UPLC system. The following time sequence program was used at a flow rate of 0.3 ml per minute:

<td>Time (Min)</td><td>Percent A (H₂O with 0.05% NH₄COOH and 0.1% HCOOH)</td><td>Percent B (CH₃CN / water 80: 20% with 0.05% NH₄COOH and 0.1% HCOOH)</td>

<td> 1,00</td><td> 90</td><td> 10</td>

<td> 4,00</td><td> 30</td><td> 70</td>

<td> 5,00</td><td> 30</td><td> 70</td>

<td> 5,50</td><td> 90</td><td> 10</td>

<td> 6,50</td><td> 90</td><td> 10</td>

[0189] In the present description, there is further provided (scheme 2) a process for the preparation of compound (Ia), 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, as defined hereinabove,

<img file="PL2723722T3_D0018.tif" />

comprising the following stages:

(i) an effect on a compound of formula 14:

<img file="PL2723722T3_D0019.tif" />

protected sugar, (4aR, 6S, 7R, 8R, 8aS) -2-phenylhexahydropyran [3,2d] [1,3] dioxin-6,7,8-triol, with formula 15:

<img file="PL2723722T3_D0020.tif" />

in the presence of a reducing agent followed by treatment with hexanal to form compound 16, 4- (4- (2 - (((2S, 3R) -2,3-dihydroxy-3 - ((4R, 5R) -5-hydroxy-2- benzyl phenyl) -1,3-dioxan-4-yl) propyl) (hexyl) amino) ethoxy) phenyl) butylcarbamate;

<img file="PL2723722T3_D0021.tif" />

(ii) subjecting compound 16 to catalytic hydrogenation to form compound 17, (1R, 2S) -3 - ((2- (4- (4-aminobutyl) phenoxy) ethyl) (hexyl) amino) -1 - ((4R, 5R ) 5-hydroxy-2-phenyl-1,3-dioxan-4-yl) propane-1,2-diol; and

<img file="PL2723722T3_D0022.tif" />

(iii) condensation of compound 17 with methyl 2,3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate compound in the presence of a base to form compound 19, 3,5-diamino-6-chloro-N- (N- (4- ( 4- (2 - (((2S, 3R) -2,3-dihydroxy-3- ((4R, 5R) -5-hydroxy-2-phenyl) -1,3-dioxane-4-yl) propyl) (hexyl ) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide; and

<img file="PL2723722T3_D0023.tif" />

(iv) hydrolysis of compound 19 in the presence of acid to form compound (Ia).

[0190] The alternative method involves replacing the compound of formula 16 above with compound 27, followed by the hydrogenation, condensation and hydrolysis steps described directly above in the preparation of compound (Ia).

[0191] In the present description, there is also provided (scheme 3) an alternative method for producing compound (Ia), 3,5-diamino-8-chloro-N- (N- (4- (4- (2 (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, as defined herein above.

Scheme 2. Preparation of 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6 -pentahydroksyheksylo) amino) ethoxy) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

<img file="PL2723722T3_D0024.tif" />

Scheme 3. Alternative preparation 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6 -pentahydroksyheksylo) amino) ethoxy) -

<img file="PL2723722T3_D0025.tif" />

Examples [0192] The invention further includes a compound prepared by the methods of the present description, or a pharmaceutically acceptable salt thereof.

Synthesis of Compound Ia, 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl10 (2S, 3R, 4R, 5R) -2,3,4,5,6- pentahydroksyheksylo) amino) ethoxy) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

Step 1 [0193] Preparation of benzyl 4- (4- (3- (tert-butyloxycarbonylamino) propoxy) phenyl) butylcarbamate (compound 13): For a solution of benzyl 4- (4-hydroxyphenyl) butylcarbamate (11, 60.0 g, 300 mmol) in dry THF (600 ml) N-Boc-ethanolamine (12, 38.7 g, 300 mmol), Ph3P (62.9 g, 300 mmol) and DIAD (48.6 g, 300 mmol) were added at 0 ° C and then the reaction mixture was warmed to room temperature and stirred overnight. The reaction mixture was concentrated in vacuo and the residue purified by column chromatography (silica gel, 15:85 EA / hexanes) to afford the desired compound 13 (50.0 g, 57%) as a yellow solid. ¹H NMR (400 MHz, CDCl3) δ 7.35 (m, 5H), 7.10 (d, J = 8.0 Hz, 2H), 6.80 (d, J = 8.0 Hz, 2H), 5.10 (s, J = 4.0 Hz, 2H), 4.0 (m, 2H), 3.5 (q, 2H), 3.2 (q, 2H), 2.55 (t, J = 8.0 Hz, 2H), 1.60 (m, 2H), 1.55 (m, 2H), 1.45 (s, 9H).

Step 2 [0194] Preparation of benzyl 4- (4- (2-aminoethoxy) phenyl) butyl) carbamate salt with hydrochloric acid (14): Compound 13 (50.0 g, 112 mmol) was dissolved in 4 N HCl in dioxane (250 ml) at room temperature and the solution was stirred for 1 hour. After concentration, the residue was suspended in MTBE (500 mL) and stirred for 0.5 h. The solid was filtered to give the hydrochloric acid salt 14 (40.0 g, 83%) as a white solid:¹H NMR (300 MHz, CD3OD) δ 7.33 (m, 5H), 7.10 (d, J = 8.7 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 5.05 (s, 2H), 4.18 (t, 2H), 3.39 (m, 2H), 3.14 (t, J = 7.2 Hz, 2H), 2.56 (t, J = 7.5 Hz, 2H), 1.57 (m, 4H).

Step 3 [0195] Preparation of 4- (4- (2 - (((2S, 3R) -2,3-dihydroxy-3 - ((4R, 5R) -5-hydroxy2-phenyl-1,3-dioxane-4 Benzyl (yl) propyl) (hexyl) amino) ethoxy) phenyl) butylcarbamate (16): Salt solution with hydrochloric acid 14 (13.5 g, 39.35 mmol) and triol 15 (10.5 g, 39.35 mmol ) in MeOH (150 ml) and AcOH (18.8 g, 314.8 mmol) was stirred at room temperature for 2 h, sodium cyanoborohydride (6.1 g, 98.37 mmol) was added and the reaction mixture was stirred at room temperature for night. An additional amount of Triol 15 (5.2 g,

19.67 mmol) and the reaction mixture was stirred at room temperature for 4 h. After complete reaction of the starting material 14, hexanal (5.9 g, 59.03 mmol) was added and the reaction mixture was stirred at room temperature for 2 h. The solvent was removed in vacuo . The residue was washed with saturated Na2CO3 (5.0 mL), converted into azeotrope with MeOH and purified by column chromatography (silica gel, 10: 1 CH2Cl2 / MeOH) to give compound 16 (12.2 g, 46% in two steps ) as an off-white solid: ¹H NMR (300 MHz, CD3OD) δ 7.45-7.44 (m, 3H), 7.31-7.29 (m, 9H), 7.05 (d, J = 8.4 Hz, 2H) , 6.79 (d, J = 8.4 Hz, 2H), 5.50 (s, 1H), 5.05 (s, 2H), 4.25-4.18 (m, 2H), 4 , 03-3.87 (m, 6H), 3.783.55 (m, 3H), 3.13-2.96 (m, 6H), 2.85-2.69 (m, 3H), 2.53 (t, J = 6.7 Hz, 2H), 1.58-1.48 (m, 6H), 1.23 (br s, 6H), 0.86 (t, J = 6.1 Hz, 3H ).

Stage 4

Preparation of (1R, 2S) -3 - ((2- (4- (4-aminobutyl) phenoxy) ethyl) (hexyl) amino) -1 - ((4R, 5R) -5-hydroxy-2-phenyl-1 salt , 3-dioxan-4-yl) propane-1,2-diol with acetic acid (17):

[0196] A suspension of carbamate 16 (12.2 g, 17.99 mmol) and 10% Pd / C (3.66 g) in EtOH / AcOH (5: 1, 120 mL) was subjected to hydrogenation conditions (1 atm) for night at room temperature. The reaction mixture was filtered through celite and washed with EtOH. The filtrate was concentrated in vacuo to afford the acetic acid salt 17 (9.40 g, 96%) as a colorless oil.¹H NMR (300 MHz, CD3OD) δ 7.48-7.44 (m, 2H), 7.32-7.30 (m, 3H), 7.11 (d, J = 8.5 Hz, 2H) , 6.84 (d, J = 8.5 Hz, 2H), 5.51 (s, 1H), 4.26-4.10 (m, 3H), 3.95-3.91 (m, 2H ), 3.78 (dd, J = 1.8, 9.3 Hz, 1H), 3.60 (t, J = 10.4, 1H), 3.23-3.03 (m, 2H), 2.96-2.87 (m, 3H), 2.61-2.59 (m, 2H), 1.67-1.57 (m, 6H), 1.31-1.25 (br s, 6H), 0.89 (t, J = 6.6 Hz, 3H).

Stage 5

Preparation of 3,5-diamino-6-chloro-N- (N- (4- (4- (2 - (((2S, 3R) -2,8-dihydroxy-3 ((4R, 5R) -5-hydroxy -2-phenyl-1,3-dioxan-4-yl) propyl) (hexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide (19) [0197] To a solution of salt with acetic acid 7 (9 , 40 g, 17.27 mmol) and methyl 3,584 diamino-6-chloropyrazine-2-carbonylcarbamimidothioate salt with hydroiodic acid (18, 7.20 g, 27.64 mmol) in EtOH (75 mL) was added at room temperature. DIPEA ( 17.8 g, 138.16 mmol). The reaction mixture was heated at 70 ° C in a sealed tube for 2 h, then cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 9: 1, CH 2 Cl 2 / MeOH, 80: 18: 2 CHCl 3 / MeOH / NH 4 OH) to give carboxamide 19 (9.20 g, 70%) as a yellow solid.¹H NMR (300 MHz, CD3OD) δ 7.46-7.43 (m, 2H), 7.30-7.28 (m, 3H), 7.07 (d, J = 8.6 Hz, 2H) , 6.79 (d, J = 8.6 Hz, 2H), 5.48 (s, 1H), 4.22 (dd, J = 3.9, 7.8 Hz, 1H), 4.08- 8.88 (m, 5H), 3.75 (dd, J = 1.5, 6.9 Hz, 1H), 3.57 (t, J = 10.5 Hz, 1H), 3.25 (t , J = 6.6 Hz, 2H), 2.93-2.83 (m, 3H), 2.68-2.56 (m, 5H), 1.70-1.64 (m, 4H), 1.44-1.43 (m, 2H), 1.22 (m, 6H), 0.85 (t, J = 8.1 Hz, 3H).

Step 6 [0198] Preparation of the 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R)) -2,3,4 salt, 5,6-pentahydroxyhexyl) amino) ethoxyl) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide with hydrochloric acid (Ia): To a solution of carboxamide 19 (9.20 g, 12.16 mmol) in EtOH (30 mL) was added at room temperature 4 N aq. HCl (95 mL) and the reaction mixture was stirred at room temperature for 4 h. The reaction mixture was concentrated in vacuo and the residue was purified by reverse phase column chromatography and lyophilized to give the hydrochloric acid salt Ia (6.60 g, 81%) as a yellow hygroscopic solid: ¹H NMR (300 MHz, CD3OD) δ 7.17 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.4 Hz, 2H), 4.36 (br s, 2 H ), 4.21-4.19 (m, 1H), 3.84-3.61 (m, 7H), 3.46-3.30 (m, 5H), 2.64 (t, J = 6 , 5 Hz, 2H), 1.80-1.69 (m, 6H), 1.36 (br s, 6H), 0.91 (t, J = 6.6 Hz, 3H); ESI-MS m / z 669 [C30H49ClN8O7 + H]⁺; analysis (C30H49ClN_andO7-2HCl-H2O). Calc'd: C 47.40, H 7.03, N, 14.74; Found: C 47.11, H 7.06, N 14.54.

Alternative synthesis of compound I, 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (hexyl ((2S, 3R, 4R, SR) -2,3,4,5,6 pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide

Level 1

Preparation of 4- (4- (3 - ((25.3 ^) - 2,3-dihydroxy-3 - ((4 ^, 5 ^) -5-hydroxy-2-methyl-1,3-dioxan-4-yl) benzyl propylamino) propoxy) phenyl) butylcarbamate (26):

[0199] A solution of the salt with hydrochloric acid 14 (155 mg, 0.41 mmol) and triol 15 (84 mg, 0.41 mmol) in MeOH (5.0 mL) was stirred at room temperature for 0.5 h and then AcOH (0.036 mL, 0.6 mmol) and sodium cyanoborohydride (43 mg, 0.6 mmol) were added and the reaction mixture was stirred at room temperature for 16 h. The solvent was removed in vacuo. The residue was washed with saturated Na2CO3 (5.0 mL), converted into azeotrope with MeOH and purified by column chromatography (silica gel, 10: 1, CH2Cl2 / MeOH, 10: 1: 0.1 CHCl3 / MeOH / NH4OH) to obtain carbamate 26 (163 mg, 75%) as a white sticky solid: ¹H NMR (300 MHz, CD3OD) δ 7.34-7.30 (m, 5H), 7.08-7.05 (m, 2H), 6.85-6.82 (m, 2H), 5, 06 (s, 2H), 4.70-4.67 (m, 1H), 4.08-3.96 (m, 4H), 3.82-3.76 (m, 2H), 3.49- 3.46 (m, 1H), 3.14-3.10 (m, 2H), 3.01-2.79 (m, 4H), 2.65-2.45 (m, 2H), 2, 05-2.01 (m, 2H), 1.59-1.49 (m, 4H), 1.27 (d, J = 4.8 Hz, 3H).

Step 2 [0200] Preparation of 4- (4- (2 - (((2S, 3R) -2,3-dihydroxy-3 - ((4R, 5R) -5-hydroxy2-methyl-1,3-dioxane-4 benzyl (yl) propyl) (hexyl) amino) ethoxy) phenyl) butylcarbamate (27): Carbamate 28 (1.02 g, 1.90 mmol), hexanal (380 mg, 3.80 mmol), AcOH (0, 33 ml, 5.70 mmol) and sodium cyanoborohydride (410 mg, 5.70 mmol) in MeOH (30 ml) was stirred at room temperature for 16 h. The solvent was removed in vacuo. The residue was washed with saturated Na2CO3 (30 mL), azeotroped with MeOH and purified by column chromatography (silica gel, 10: 1, CH2Cl2 / MeOH) to give carbamate 27 (990 mg, 84%) as a white sticky body DC: ¹H NMR (300 MHz, CD3OD) δ 7.35-7.31 (m, 5H), 7.06 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.4 Hz , 2H),

5.08 (s, 2H), 4.80 (br s, 1H), 4.69-4.66 (m, 1H), 4.12 (dd, J = 9.3, 2.4 Hz, 1H ), 4.05-3.98 (m, 3H), 3.84-3.76 (m, 2H), 3.54-3.48 (m, 1H), 3.38 (t, J = 10 , 5 Hz, 1H), 3.20-2.96 (m, 4H), 2.83 (d, J = 6.0 Hz, 2H), 2.73-2.64 (m, 2H), 2 , 56 (t, J = 7.2 Hz, 2H), 1.63-1.50 (m, 6H), 1.32 (d, J = 5.1 Hz, 3H), 1.27-1, 24 (m, 6H), 0.87 (d, J = 6.6 Hz, 3H).

Step 3 [0201] Preparation of (1R, 2S) -3 - ((2- (4- (4-aminobutyl) phenoxy) ethyl) (hexyl) amino) -1 - ((4R, 5R) -5-hydroxy- salt 2-methyl-1,3-dioxan-4-yl) propane-1,2-diol with acetic acid (28): A suspension of carbamate 27 (890 mg, 1.44 mmol) and 10% Pd / C (400 mg) in MeOH / AcOH (5: 1, 60 mL) was subjected to hydrogenation conditions (1 atm) for 6 h at room temperature. The reaction mixture was filtered through celite, which was washed with MeOI. The filtrate was concentrated in vacuo and triturated with ether to give the salt with acetic acid 28 (782 mg, 90%) as a white sticky solid: ¹H NMR (300 MHz, CD3OD) δ 7.09 (d, J = 8.7 Hz, 2H), 6.86 (d, J = 8.7 Hz, 2H), 4.69-4.67 (m , 1H), 4.00-3.85 (m, 1H), 3.84-3.76 (m, 2H), 3.53-3.51 (m, 1H), 3.38 (t, J = 10.5 Hz, 1H), 2.98-2.59 (m, 10H), 1.96 (s, 13H), 1.67-1.47 (m, 6H), 1.40-1, 27 (m, 6H), 1.26 (d, J = 5.1 Hz, 3H), 0.88 (d, J = 6.3 Hz, 3H).

Step 4 [0202] Preparation of 3,5-diainiii-6) -ldoro -. \ - (. \ - (4- (4-2 - (((2S ', 3 /) - 2,3-dihydroxy-3 - (( 4, 5 /) - 5-hydroxy-2-methyl-1,3-dioxan-4-yl) propyl) (hexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxainidia (20):

To a solution of acetic acid salt 28 (189 mg, 0.313 mmol) and methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate salt with hydroiodic acid (18, 182 mg, 0.502 mmol) in EtOH (8 mL) was added at room temperature DIPEA (0.42 mL, 2.50 mmol). The reaction mixture was heated at 70 ° C in a sealed tube for 2 h, then cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 9: 1, CH2Cl2 / MeOH, 80: 18: 2 CHCl3 / MeOH / NH4OI) to give carboxamide 20 (142 mg, 65%) as a yellow solid: ¹H NMR (300 MHz, CD3OD) δ 7.10 (d, J = 8.1 Hz, 2H), 6.84 (d, J = 8.1 Hz, 2H), 4.66 (q, J = 5 , 1 Hz, 1H), 4.06-4.01 (m, 3H), 3.943.89 (m, 1H), 3.82-3.74 (m, 2H), 3.49 (dd, J = 9.3, 2.4 Hz, 1H), 2.96-2.78 (m, 3H), 2.67-2.61 (m, 5H), 1.68-1.67 (m, 4H ), 1.50-1.48 (m, 2H), 1.29 (br s, 6H), 1.25 (d, J = 5.1 Hz, 3H), 0.87 (t, J = 6 , 9 Hz, 3H).

Stage 5

Preparation of the 3.5-diamino-8-chloro-A- (A- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroxyhexyl) amino salt) ) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide with hydrochloric acid (Ia) [0203] To a solution of carboxamide 20 (400 mg, 0.57 mmol) in EtOH (5 mL) was added 4 N aqueous solution at room temperature HCl (15 mL) and the reaction mixture was heated at 55 ° C for 24 h. After concentration, the residue was dissolved in 4 N aqueous HCl (15 mL) and heated at 65 ° C for 16 h. The reaction mixture was concentrated, triturated in EtOH / Et2O, again purified by preparative TLC and lyophilized to give the hydrochloric acid salt (Ia) (354 mg, 83%) as a yellow hygroscopic solid: ¹H NMR (300 MHz, D2O) δ 7.18 (d, J = 8.1 Hz, 2H), 6.87 (d, J = 8.1 Hz, 2H), 4.30 (br s, 2 H ), 4.19-4.16 (m, 1H), 3.76-3.55 (m, 7H), 3.39-3.24 (m, 6H), 3.57 (t, J = 5 , 4 Hz, 2H), 1.65-1.64 (m, 6H), 1.30-1.19 (m, 6H), 0.78-0.75 (m, 3H); ESI-MS m / z 669 [C30H49ClN8O7 + H]⁺.

Preparation of 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) 2.3.4.5.6- pentahydroxyhexyl) amino) ethoxy) phenyl ) butyl) carbamimidoyl) pyrazine-2-carboxamide (free base of compound Ia) [0204] Salt 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) 2.3.4.5.6- pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide with hydrochloric acid (12.51 g) dissolved in 150 ml H2O and treated with NaOH (0 , 1 M aqueous solution, 435 ml) to obtain a sticky solid. The liquid (pH ~ 11) was decanted through a filter funnel (most of the material adhered to the walls of the flask). The residue was treated with H2O (2 x 300 mL), similarly stirred, and decanted and filtered. Further residue was suspended in CH3CN / H2O / MeOH and concentrated to give a yellow amber solid, 9.55 g. ESI-MS m / z 669 [C30H49ClN8O7 + H]⁺, 89% purity at 224 nm, 90% at 272 nm, 82% at 304 nm, 63% based on MS analysis. The crude product was heated with isopropanol (100-150 mL) at 70 ° C for 15 min and then filtered warm. The solids were similarly treated twice more with isopropanol, each time left for 30 minutes and the mixture allowed to cool (2h - overnight) and then filtered. The solids obtained were dried, yielding 7.365 g of a yellow amber amorphous solid, mp 133.1 - 135.6 ° C (yield 11.0 mmol based on the free base). ¹H NMR (400 MHz, dmso) δ 9.31 - 7.34 (m, 4H), 7.10 (d, J = 8.6 Hz, 2H), 6.83 (d, J = 8.5 Hz , 2H), 6.61 (br s, 3H), 4.76 - 4.09 (m, 5H), 3.98 (t, J = 6.1 Hz, 2H), 3.71 - 3.62 (m, 2H), 3.59 (dd, J = 10.8, 3.4 Hz, 1H), 3.54 - 3.46 (m, 1H), 3.43 (dd, J = 7.9 , 1.3 Hz, 1H), 3.38 (dd, J = 10.8, 5.9 Hz, 1H), 3.15 (br s, 2H), 2.92 - 2.76 (m, 2H), 2.66 (dd, J = 13.1, 5.2 Hz, 1H), 2.58 - 2.51 (m, 4H), 2.46 (dd, J = 13.1, 6, 5 Hz, 1H), 1.66 - 1.45 (m, 4H), 1.45 -1.31 (m, 2H), 1.31 - 1.09 (m, 6H), 0.90 - 0.76 (m, 3H). ¹³C NMR (101 MHz, dmso) δ 173.27, 160.96, 156.48, 154.68, 151.14, 133.70, 129.05, 119.12, 117.53, 114.14, 72 , 23, 71.37, 70.60, 70.04, 65.74, 63.34, 57.39, 54.72, 52.86, 40.10, 33.72, 31.15, 28.37 , 28.09, 26.38, 26.36, 22.02, 13.84. ESI-MS m / z 669 [C30H49ClN8O7 + H]⁺.

Preparation of 3,5-diamino-6-chloro-N- 1-hydroxy-2-naphthoate salt (N- (4 (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4 , 5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide [0205] A mixture of 32.8 mg (0.049 mmol) 3,5-diamino-6-chloro-N- (N- ( 4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6-pen-tahydroksyhoksylo) amino) ethoxy) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide, 164 μl of a 0.3 M solution of 1-hydroxy-2-naphthoic acid in methanol (0.049 mmol of 1-hydroxy-2-naphthoic acid) and about 0.33 ml of methanol was heated on a hot plate set at 85 ° C until complete dissolution of solids. The solution was allowed to cool to ambient temperature. The solution was placed in a refrigerator (about 5 ° C) and left overnight; crystallization occurred during this time. The liquid was decanted and the solid was dried in a stream of dry air to give 29.5 mg (62% yield) of 3,5-diamine-6-chloro-N- 1-hydroxy-2-naphthoate salt (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide. ¹H NMR (500 MHz, DMSO) 8.2 (m, 1H), 7.7 (m, 2H), 7.4 (d, 1H), 7.3 (d, 1H), 7.1 (m , 2H), 6.95 (m, 1H), 6.85 (m, 2H), 4.6 - 4.2 (m, 2H), 4.0 (m, 2H), 3.8 - 3 , 6 (m, 2H), 3.6-3.2 (m, 6H), 2.9 - 2.5 (m, 6H), 1.6 (m, 4H), 1.4 (m, 2H ), 1.2 (m, 6H), 0.83 (m, 3H) ppm.

Preparation of 3,5-diamino-6-chloro-N- 1-hydroxy-2-naphthoate salt (N- (4 (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4 , 5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide. A mixture of 105.3 mg (0.157 mmol) 3,5-diamino-6-chloro-N- (N- ( 4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6-pentahydroksyheksylo) amino) ethoxy) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide, 525 μl of a 0.3 M solution of 1-hydroxy-2-naphthoic acid in methanol (0.158 mmol of 1-hydroxy-2-naphthoic acid) and about 1 ml of methanol was heated on a heating plate set at 85 ° C until complete dissolution of the solids. The solution was allowed to cool to ambient temperature and was put in the fridge (about 5 ° C). After about 20 minutes, it became cloudy and was vaccinated. After about 2 hours, a solid appeared. A magnetic stir bar was added to the mixture and stirred in the refrigerator overnight; during this time the mixture thickens strongly. An additional 1.5 mL of methanol was added and the suspension was stirred in the refrigerator overnight. The mixture was centrifuged, the liquid was decanted and the solid was dried in a stream of dry air to give 74 mg (55% yield) of 3,5-diamino-6-chloro-N- 1-hydroxy-2-naphthoate salt (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6pentahydroksyheksylo) amino) ethoxy) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide.

Pharmacology of compound (Ia), 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4,5,6 -pentahydroksyheksylo) amino) ethoxy) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide

Study No. 1. Measurement of sodium channel blocking activity and in vitro reversibility [0207] One study used to evaluate the mechanism of action and / or potency of compounds of the present invention involves determining the inhibition of the drug in the lumen of the respiratory tract with respect to the current induced by sodium in the epithelium airways measured under short-circuit current (Isc) using airway epithelial monolayers attached in Ussing chambers. Freshly harvested cells are obtained from the respiratory tract of humans, dogs, sheep or rodents. This study is described in detail in Hirsh, AJ, Zhang, J., Zamurs, A. et al. Pharmacological properties of N- (3,5-diamino-6-chloropyrazine-2-carbonyl) -N'4- [4- ( 2,3-dihydroxypropoxy) phenyl] butylguanidine methanesulfonate (552-02), a novel epithelial sodium channel blocker with potential clinical efficacy for CF lung disease. J. Pharmacol. Exp. Ther. 2008; 325 (1): 77-88.

[0208] Inhibition of sodium movement across the cell membrane by ENaC was measured using polarized bronchial epithelial cell monolayers mounted in a modified Ussing chamber. Primary cultures of canine or human bronchial epithelial cells cultured at the air / liquid interface were tested under voltage clamp. Short-circuit current (I_SC) was measured as an indicator of sodium transport across the epithelium to assess potency.

[0209] Compound (Ia) 3,5-diamino-6-chloro-N- (N- (4- (4- (2- (hexyl ((2S, 3R, 4R, 5R) -2,3,4, 5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide was a potent inhibitor of sodium transport across the cell membrane, and its activity was approximately 60 times greater than amiloride in canine bronchial epithelial cells. CBE) and approximately 160 times greater in human bronchial epithelial cells (HBE) (Figure 1). In CBE, the IC value₅₀ for compound (Ia) it was 13.2 ± 8.0 nM and for HBE the IC value₅₀ for compound (Ia) it was 2.4 ± 1.8 nM (Table 1).

Table 1. Inhibition of short circuit current by compound (Ia) in canine bronchial epithelial cells and human bronchial epithelial cells (IC₅₀ nM)

<td>Type</td><td>amiloride</td><td>Relationship I (parent)</td>

<td>Dog</td><td> 781,5 ± 331 (40)</td><td> 13,2 ± 8,0(7)*</td>

<td>Man</td><td> 389 ± 188(22}</td><td> 2,4 ± 1,2(4)*</td>

The values ​​are mean ± SD (n). * means significance (p &lt;0.05) relative to amiloride [0210] Return of short-circuit current (Isc) after maximum blocking was used as an indirect dissociation measurement. Percent return of value_SC after full blocking, the peak area was determined three times and calculated using the formula:

return value (I_SC) / output value (I_SC) x 100, this value showed significantly lower (22-fold) reversibility than amiloride in CBE and 9.5-fold lower in HBE (Table 2), indicating that compound (Ia) provides longer and more durable blockage ENaC.

Table 2. Reversibility of compound (Ia) activity against short-circuit current in canine bronchial epithelial cells and human bronchial epithelial cells (percentage return)

<td>Type</td><td>amiloride</td><td>Relationship (Ia)</td>

<td>Dog</td><td> 90,1 ± 27,6 (39)</td><td> 4,1 ± 11,6 (7)*</td>

<td>Man</td><td> 89,5 ± 10,7 (4)</td><td> 9,4 ± 17 (3)*</td>

<td colspan="3">The values ​​are mean ± SD (n). * indicates significance (p &lt;0.05) relative to amiloride</td>

Study No. 2. Mucociliary clearance (MCC) studies in sheep [0211] The sheep model is the most commonly used animal model to measure changes in MCC values. The effect of compounds on increasing mucociliary clearance (MCC) can be assessed using the in vivo model described in Sabater et al., Journal of Applied Physiology, 1999, pp. 2191-2196, incorporated herein by reference.

In these studies, adult sheep were immobilized and an intubation tube was inserted into the nose. Aerosol test products were given to sheep for 10-15 minutes. Radioactive labeled colloid was administered four or eight hours after the test product^99mTc-sulfur (TSC, 3.1 mg / ml; containing approximately 20 mCi). The radiolabeled aerosol was administered through the endotracheal tube over about 5 minutes. The sheep were then extubated and the total number of radioactive decays in the lungs was measured every 5 minutes over the 1 hour observation period. The rate of radiolabel removal from the lungs corresponds to the animal's MCC. The benefit of this system is that it accurately simulates the environment in human lungs. This model also allows the simultaneous collection of pharmacokinetic and pharmacodynamic (PK / PD) data based on plasma and urine samples during the study period. A number of methods are also available to measure drug concentration at the surface of the airways during MCC measurements. These include the collection of expired condensate or the method using filter paper for obtaining ASL by bronchoscopy.

The sheep model described above was used to evaluate the in vivo effect (efficacy / stability) of compound (Ia) administered as an aerosol on MCC. Methods including 4 ml of compound (Ia), Comparative Example 1, Comparative Example 4, vehicle (sterile distilled H2O) or test agent in combination with HS were tested. To determine whether the combination of HS with compound (Ia) had an effect on MCC, HS was administered immediately after administration of compound (Ia). Test solutions were aerosolized using a Raindrop nebulizer at a flow rate of eight liters per minute and connected to a dosimetry system consisting of a solenoid valve and a compressed air source (20 psi). It was estimated that the dose delivered to the lungs of sheep after spraying with the Raindrop nebulizer was 8-15% of the original dose. Using radiolabelled TSC nebulizer, TSC was administered approximately 3 minutes 4 or 8 hours after drug administration to assess efficacy / stability of action. The number of radioactive decays was measured in the middle right lung at intervals of 5 min for one hour using a gamma camera. Three methods of analysis were used: 1) initial speed or clearance (slope factor) within the first 30 min with linear regression adjustment, 2) area under the curve for the percentage clearance over time over one hour and

3) maximum clearance achieved after one hour.

[0212] The effect of Compound (Ia) at 16 μg / kg, 0.16 μg / kg and 0.016 μg / kg was tested and compared with vehicle (4 ml of sterile H₂O) on the MCC value in sheep four hours after dosing (figure 2). Performance analysis is shown in Table 3.

At all doses tested, compound (Ia) increased the MCC value compared to the control (vehicle). A dose of 16 μg / kg was found to give the greatest effect on MCC values.

Table 3. MCC values ​​in sheep 4 h after dose of compound (Ia) or vehicle

<td>Compound dose</td><td>Initial slope (4.04.5 h)</td><td>AUC (% Cl - h)</td><td>Maximum clearance</td>

<td>16 μg / kg</td><td> 39,0 ± 3,9*(4)</td><td>18.6 ± 2.2 * f (4)</td><td>33.8 ± 3.7 * f (4)</td>

<td>0.16 μg / kg</td><td> 39,1 (2)</td><td> 19 (2)</td><td> 33,1 (2)</td>

<td>0.016 μg / kg</td><td> 33,3 ± 4,4*(4)</td><td> 14,4 ± 1,3*(4)</td><td> 25,5 ± 1,3* (4)</td>

<td>Substrate (H₂O), 4 ml</td><td> 17,2 ± 6,8 (8)</td><td> 7,3 ± 1,5 (8)</td><td> 12,2 ± 2,9 (8)</td>

<td colspan="4">Data are given as mean ± SD (n). A study in which n = 2, not included in the statistical analysis, * indicates significance (p &lt;0.05) relative to the ground. ^ Shows significance (p &lt;0.05) relative to a dose of 0.016 μg / kg.</td>

[0213] To determine if HS increased the effect of Compound (Ia) on MCC values, HS (6.25 mL 10% HS; dose delivered 62.5 mg, assuming 10.5 mg, given immediately after the 0.016 μg / kg dose of Compound (Ia) % delivery) and an MCC evaluation four hours after both doses (Figure 12). HS increased the dose effect of 0.016 μg / kg of compound (Ia) to the maximum effect found for both 0.16 μg / kg and 16 μg / kg of compound (Ia) alone (Figure 2). Therefore, the maximum effect on MCC was obtained after adding HS to the dose (0.016 μg / kg) of Compound (Ia), which resulted in a less than maximum response after administration without HS.

Table 4. MCC values ​​in sheep 4 h after dose of vehicle, compound (Ia) and HS

<td>Dose</td><td>Initial slope (4.0-4.5 h)</td><td>AUC (% Cl h)</td><td>clearance maximum</td>

<td>Compound (Ia) (0.016 μg / kg; 4 ml + HS)</td><td> 44,9 (2)</td><td> 20,7 (2)</td><td> 37,0 (2)</td>

<td>Compound (Ia) (0.016 μg / kg; 4 ml)</td><td> 33,3 ± 4,4(4)</td><td> 14,4 ± 1,3(4)</td><td> 25,5 ± 1,3 (4)</td>

<td>Substrate, H₂O (4 ml)</td><td> 17,2 ± 6,8 (8)</td><td> 7,3 ± 1,5 (8)</td><td> 12,2 ± 3 (8)</td>

<td colspan="4">Data are given as mean ± SD (n). Study in which n = 2, not included in the statistical analysis.</td>

[0214] To assess both the stability of the compound (Ia) and the effect of adding HS to the compound (Ia), MCC values ​​were measured eight hours after the vehicle dose (H₂O), 7% HS alone, 0.16, 1.6 and 16 μg / kg compound (Ia) alone or a combination of 0.16 μg / kg compound (Ia) and 7% HS (total volume 4 ml with each administration) (figure 4). After administration of the vehicle, MCC values ​​after 4 and 8 hours were the same, indicating that MCC values ​​in sheep over a 4-8 hour period are stationary (Figures 12 and 13). Eight hours after the administration of 4 ml 7% HS, no change in MCC value was found compared to the vehicle, which means that the effect of HS has disappeared. In all three dose groups (0.16,

1.6 and 16 μg / kg) of compound (Ia), an increase in MCC was found in a dose dependent manner compared to both vehicle and HS, indicating that compound (Ia) provides a longer duration of action than HS alone ( figure 4). The combination of the HS dose and compound (Ia) increased the effect of the 0.16 μg / kg dose of compound (Ia) to a value greater than that observed for the 16 μg / kg dose, and allowed a 100-fold increase in activity when HS was added to compound (Ia) ( figure 4). An increase in compound (Ia) activity due to HS, given that HS has no independent activity, clearly indicates the synergy of HS and compound (Ia).

Table 5. MCC values ​​in sheep 8 h after administration of vehicle, HS, compound (Ia) or combination of HS and compound (Ia)

<td>Dose</td><td>Initial slope (8.0-8.5 h)</td><td>AUC (% Cl h)</td><td>clearance maximum</td>

<td>Substrate, H₂O (4 ml)</td><td> 17,8 ± 5,7 (4)</td><td> 7,8 ± 1 (4)</td><td> 14,2 ± 0,7 (4)</td>

<td>7% HS (4 ml)</td><td> 17,8 (2)</td><td> 7,6 (2)</td><td> 14,6 (2)</td>

<td>Compound (Ia) (0.16 µg / kg; 4 ml)</td><td> 24,0 (2)</td><td> 10,7 (2)</td><td> 19,7 (2)</td>

<td>Compound (Ia) (1.6 µg / kg; 4 ml)</td><td> 24,4 (2)</td><td> 11,1 (2)</td><td> 21,2(2)</td>

<td>Compound (Ia) (16 µg / kg; 4 ml)</td><td> 28,0 (2)</td><td> 13,9 (2)</td><td> 26,7 (2)</td>

<td>Compound (Ia) (0.16 µg / kg + 7% HS; 4 ml)</td><td> 30,9 ± 2,5 (4)</td><td> 15,3 ± 2,2 (4)</td><td> 27,5 ± 1,6 (4)</td>

<td colspan="4">Data are given as mean ± SD (n). Study in which n = 2, not included in the statistical analysis.</td>

Study No. 3. d. Drug clearance from airway fluid (ASL) and metabolism in human airway epithelium [0215] The disappearance of compound (Ia) at the apical surface and metabolism in airway epithelium was assessed in HBE (Table 6). In these experiments, 25 μl of 25 μΜ of ENaC blocker solution was introduced onto the apical surface of HBE cells grown on the interface of air and liquid and the concentration of the drug in the apical and basolateral range was measured within 2 h by the UPLC method. After incubation of compound (Ia) on the apical surface (37 ° C) for 2 h, no metabolites on the apical or basolateral side were found and no compound (Ia) on the basolateral side.

Table 9. Peak atrophy and metabolism of compound (Ia) in HBE

<td>Relationship</td><td>% initial drug weight on the peak side (parent compound and metabolites, 2 h)</td><td>% mass on the peak side as metabolites (2 h)</td><td>% of initial peak mass located on the basolateral side (2 h)</td><td>% of the basolateral side in the form of metabolites (2 h)</td>

<td>Relationship (Ia)</td><td> 80,7* ± 6,2%</td><td>lack</td><td>lack</td><td>lack</td>

Study No. 4. e. Respiratory humidification and sodium channel blocking (in vitro model) [0216] Parion Sciences has developed experimental models to assess respiratory hydration in cell cultures (Hirsh, AJ, Sabater, JR, Zamurs, A. et al. Evaluation of second generation amiloride analogs as therapy for CF lung disease. J. Pharmacol. Exp. Ther. 2004; 311 (3): 929-38. Hirsh, AJ, Zhang, J., Zamurs, A. et al. Pharmacological properties of N- (3,520 diamino-6-chloropyrazine-2-carbonyl) -N'-4- [4- (2,3-dihydroxypropoxy) phenyl] butylguanidine methanesulfonate (552-02), a novel epithelial sodium channel blocker with potential clinical efficacy for CF lung disease. J. Pharmacol. Exp. Ther. 2008; 325 (1): 77-88).

[0217] Primary CBE cells are seeded onto collagen-coated porous membranes and held at the interface of air and liquid to assess the continuation of fluid volume on the surface over time. At the beginning of each experiment, all Snapwell inserts (12 mm) were removed from the plate containing the culture medium at the interface of air and liquid, dried with paper, weighed and applied to the top surface with 50 μl medium (0.1% DMSO) or ENaC blocker (10 μΜ in 0.1% DMSO) and mass was recorded. The inserts were immediately reinserted in a Transwell plate (500 μl Krebs Ringer with bicarbonate solution (KRB), pH 7.4 in the lower chamber) and placed in an incubator at 37 ° C, 5% CO2. To reduce artifacts related to the osmotic gradient of carbohydrates on the top surface due to water loss, no glucose was added to the top-side buffer. Compound (Ia) was tested and compared to the vehicle and ASL mass was monitored repeatedly over a period of 0-8 or 24 h. The mass of liquid on the surface was converted into volume in pl. Data are given as% of initial volume (100% = 50 pl).

[0218] The duration of inhibition of sodium transport was determined indirectly by determining the buffer retained after the CBE cells were loaded with 50 µl of experimental buffer on the top surface. Only 12.5 ± 12.1% of the substrate (buffer) remained on the surface after 8 hours and a slight increase in surface fluid retention (25 ± 19.2% after 8 hours) was found in the substrate for 10 pM amiloride. Compared to this, compound (Ia) caused a significant increase in fluid retention at the apical surface with a persistence of 88.3 ± 13% of surface fluid over a period of 8 hours (Figure 5).

[0219] To further test compound (Ia), the incubation period was increased from eight to 24 hours. Amiloride has not been studied over a 24-hour period because much of the effect disappeared after eight hours. After 24 hours, only 11% of the buffer constituting the substrate remained, while compound (Ia) allowed for retaining 72.3 ± 7.3% of the fluid on the surface over a period of 24 hours, i.e. the loss was only 16% compared to the value after 8 hours, indicating that compound (Ia) shows stability against fluid retention 6).

Comparative Examples [0220] The present compound of formula (I) has greater potency and / or is absorbed more slowly from the surface of the mucosa, in particular from the surface of the respiratory tract, compared to known sodium channel blockers, such as Comparative Examples 1 to 5, described below. Therefore, the compound of formula (I) has a longer half-life on mucosal surfaces compared to these compounds.

[0221] Comparative examples 1 to 4 are claimed, described or fall within the scope of the disclosures of WO 2003/070182 (US Patent Nos. 6,858,615; 7,186,833; 7,189,719; 7,192,960 and 7,332,496), WO 2005/044180 (US Application 2005/0080093 and US Pat. . U.S. No. 7,745,442), WO 2004/073629 (U.S. Patents Nos. 6,903,105; 6,995,160; 7,066,325; 7,030,117; 7,345,044; 7,820,678 and 7,875,619), WO 2005/016879 (U.S. Patents Nos. 7,064,129; 7,247,637; 7,317,013; 7,368,447; 7,378,137) 7,410,988 and 7,868,010) or WO 2008/031028 (patent application publications 2008/0090841 and 2009/0082287) as sodium channel blockers with useful therapeutic properties and can be prepared by methods described in these documents and other known in the art.

OH OH ττιχ

OH ÓH ÓH \

HO "-6 ^ ί0&gt; ··· ΟΗ,

Comparative Example 1

<img file="PL2723722T3_D0026.tif" />

[0222] The compound of Comparative Example 1 is included in the scope of the sodium channel protecting compounds according to WO 2008/031028, in which its structure is shown on page 14.

Comparative Example 2 is 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (dihexylamino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, which enters scope of the general disclosure according to WO 2004/073629.

<img file="PL2723722T3_D0027.tif" />

Comparative example 3 is 3,5-diamino-6-chloro-N- (N- (4- (4- (2 (((2S, 3R, 4R, 5R) -5-hydroxy-2,3, 4,6-tetramethoxyhexyl) ((2S, 3R, 4R, 5R) 2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide, which falls within the scope of the general disclosure according to WO 2008/031028.

<img file="PL2723722T3_D0028.tif" />

Comparative Example 4

<img file="PL2723722T3_D0029.tif" />

(S) -3,5-diamino-6-chloro-N- (N- (4- (4- (2,3-diamino-3-oxopropoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide [0225] Compound according to Comparative Example 4 is shown on page 15 of US 2005/0080093 and as compound 2 on page 90 of WO 2008/031048 and as compound 2 on pages 42-43 of WO

2008/031028. To obtain useful activity in the treatment of cystic fibrosis and

COPD, the compound must have properties that lead to an increase in mucociliary clearance (MCC) at doses that do not increase plasma potassium, which ultimately results in hyperkalaemia, a serious and hazardous condition as a result of multiple dosing. Therefore this should not be allowed for this class of compounds that are known to increase plasma potassium if they are significantly excreted by the kidneys. To assess this potential, it is preferred that MCC activity occurs in vivo and does not increase the potassium plasma concentration at a useful dose. One model for assessing this issue is the sheep MCC model described below. Table 7 below shows that the value of ED₅₀ (AUC = 47%) for comparative example 1 in the sheep MCC model is approximately 3000 μM.

Table 7. Change in effect on MCC relative to substrate after 8 hours. in sheep based on 3 different indicators

<td>Table 1</td><td>slope (88.5 h)</td><td>AUC (% Cl * h)</td><td>clearance maximum (%)</td><td>Approximate ED value₅₀</td>

<td>300 gM (Ia)</td><td> 10,2 (100%)</td><td> 6,2 (100%)</td><td> 12,8 (100%)</td><td></td>

<td>30 gM (Ia)</td><td> 6,6 (65%)</td><td> 3,3 (53%)</td><td> 7,0 (55%)</td><td>2,4nmol / kg</td>

<td>3000 gM (example</td><td> 6,1 (60%)</td><td> 2,9 (47%)</td><td> 4 (31%)</td><td>240 nmol / kg</td>

<td>comparative 1)</td><td></td><td></td><td></td><td></td>

<td>Action</td><td></td><td></td><td></td><td></td>

<td>maximum</td><td> 10,2 (100%)</td><td> 6,2 (100%)</td><td> 12,8 (100%)</td><td></td>

[0226] From table 7 and figure 7 it appears that the ED value₅₀ comparative example 1 in the sheep MCC model is approximately 240 nmol / kg (3 mM) based on three different indicators (slope, AUC and maximum clearance). At this dose, which would be the clinically active dose, the compound of Comparative Example 1 causes an increase in plasma potassium, which will lead to hyperkalaemia with repeated dosing (Figure 8). This means that the compound of Comparative Example 1 is unacceptable for human use, while compound (Ia) provides a safe and effective MCC with a beneficial effect on a risk factor exceeding 1000 in this model.

[0227] To reduce the potential effect of this molecule on the kidneys, more lipophilic compounds have been studied. Comparative Example 2 in which two groups

100 hydrophilic in the compound of Comparative Example 1 replaced by two equal length lipophilic chains, gives compound of Comparative Example 2 whose potency is one order of magnitude less than Comparative Example 1 in vitro (Table 8), which means that it is unsuitable until a constant MCC value is obtained in vivo. Comparative Example 3, in which all oxygen atoms in connection with Comparative Example 1 were maintained and 5 methyl groups added on 5 additional hydroxyl groups, was characterized by a similar decrease in in vitro activity. It turned out, therefore, that under this structure it was not possible to obtain an active and safe molecule for the kidneys. Thus, it was surprisingly found that compound (Ia) retains in vitro activity equal to Comparative Example 1. Even more surprising and unexpected was that compound (Ia) had more than 100 times greater potency than Comparative Example 1 and did not cause an increase in plasma potassium at MCC-effective doses.

Table 8. Measurement of sodium channel blocking activity in vitro

<td>Relationship</td><td>IC50 (nM)</td>

<td>ia</td><td> 13,2</td>

<td>Comparative Example 1</td><td> 11,8</td>

<td>Comparative Example 2</td><td> 124,5</td>

<td>Comparative Example 3</td><td> 144,1</td>

<td>Comparative Example 4</td><td> 6,6</td>

[0228] Another compound that was extensively studied was the compound of Comparative Example 4, (S) -3,5-diamino-6-chloro-N- (N- (4- (4- (2,3-diamino3- oxopropoxy) phenyl) butyl) karbamimidoilo) pyrazine-2-carboxamide.

[0229] The disappearance of compound (Ia) at the apical surface and metabolism in the airway epithelium was evaluated in HBE and compared with Comparative Example 4 (Table 9). In these experiments, 25 g 25 μΜ of ENaC blocker solution was introduced on the top surface of HBE cells grown on the surface of air and liquid contact, and the concentration of the drug in the peak and basolateral range was measured within 2 h by

UPLC. After incubation of compound (Ia) on the apical surface (37 ° C) for 2 h, no metabolites on the apical or basolateral side were found,

101 and the basolateral side did not detect compound (Ia). In contrast, most of the compound of Comparative Example 4 was eliminated from the apical side and 83% was metabolized to the less active carboxylic acid, i.e. (S) -2-amino-3- (4- (4- (3- (3) , 5-diamino-65-chloropyrazine-2-carbonyl) guanidino) butyl) phenoxy) propane, whose structure is given below.

<img file="PL2723722T3_D0030.tif" />

Table 9. Peak atrophy and metabolism of compound (Ia) in HBE

<td>Relationship</td><td>% initial peak drug weight (parent compound and metabolite, 2 h)</td><td>% mass on the peak side as metabolites (2 h)</td><td>% of initial peak mass located on the basolateral side (2 h)</td><td>% basolateral mass as metabolites (2 h)</td>

<td>Relationship (Ia)</td><td> 80,7* ± 6,2%</td><td>lack</td><td>lack</td><td>lack</td>

<td>Comparative Example 4</td><td> 41,6 ± 7,6%</td><td> 83,0 ± 3,5%</td><td> 8,3 ± 0,2</td><td> 94,7 ± 1,0%</td>

<td colspan="5">The values ​​are mean ± SD (n). * indicates a significant difference (p &lt;0.05) relative to Comparative Example 4.</td>

[0230] Compound (Ia) has 10,000-fold greater potency against MCC in sheep than Comparative Example 4, and does not increase plasma K concentration, while the compound of Comparative Example 4 increases K concentration with an approximate ED50 of 3 mM (figures 9 and 10). This confirms once again the unique and unexpected potency and safety, i.e. the benefits associated with the compound Ia.

Table 10. MCC values ​​in sheep 4 h after dosing with vehicle, compound according to Comparative Example 4 or compound (Ia)

<td>Dose</td><td>Initial slope (4.0-4.5 h)</td><td>AUC (% Cl xh)</td><td>Maximum clearance</td>

<td>Comparative Example 4 (112 μδ / kg; 4 ml)</td><td> 32,2 ± 7,3* (6)</td><td> 14,1 ± 2,2* (6)</td><td> 22,9 ± 2,1* (6)</td>

<td>Comparative example 4 (11.2 μδ / kg; 4 ml)</td><td> 14,5 ± 1,3 (3)</td><td> 6,9 ± 1,0 (3)</td><td> 14,6 ± 0,9 (3)</td>

<td>Compound (Ia) (0.016 μδ / kg; 4 ml)</td><td> 33,3 ± 4,4* (4)</td><td> 14,4 ± 1,3*(4)</td><td> 25,5 ± 1,3* (4)</td>

<td>Substrate, H₂O (4 ml)</td><td> 17,2 ± 6,8 (8)</td><td> 7,3 ± 1,5 (8)</td><td> 12,2 ± 2,9 (8)</td>

102 [0231] It has now been shown that increased safety of compound (Ia) against the kidneys can be explained by a significant decrease in renal drug clearance. If it is possible to exclude this compound from the sodium channels in the kidneys, hyperkalaemia should be significantly reduced. After intravenous administration, 43% of the compound of Comparative Example 1 was recovered in the urine, while only 5% of the compound I was recovered in the urine. Even more radical is the surprising reduction of drug recovery in urine after aerosol administration directly into the lung. When the compound of Comparative Example 4 was administered to the sheep in the form of an aerosol for inhalation, 7% of the dose was recovered in urine, while only 0.07% of the dose of compound (Ia) aerosolized was recovered in urine. A reduced urinary clearance of the compound (10 to 100 fold) in combination with a significant reduction in the necessary dose, as described above, leads to an unexpected 100,000- to 1,000,000-fold difference in risk-benefit ratio.

Table 11. Urine excretion of compound (Ia) and compound according to Comparative Example 4.

<td>Mark</td><td>Comparative Example 4</td><td>Relationship (Ia)</td>

<td>Log D</td><td> 0,64</td><td> 0,2</td>

<td>IC50</td><td>6.6 ± 3.7 nM</td><td>13 ± 8 nM</td>

<td>Metabolism in human plasma</td><td>t1 / 2 = 37 min</td><td>Lack</td>

<td>Protein binding in human plasma</td><td> 76 ± 2%</td><td> 97 ± 2%</td>

<td>Urinary excretion of a given dose (sheep)</td><td> 7%</td><td> 0,07%</td>

[0232] Figure 9 is a graph of the percentage of mucus clearance versus time for compound (Ia), 3,5-diamino-6-chloro-Y- (Y- (4- (4- (220 (hexyl (( 25.3A, 4A, 5A) -2,3,4,5,6-pentahydroxyhexyl) amino) ethoxy) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide with hydrochloric acid, and Comparative Example 4 as described in the model MCC above. A similar percentage clearance in mucus was obtained for compound (Ia) at a dose of 7,000-fold lower than for comparative example 4.

Compound (Ia) provided maximum effect in a clinically relevant dose range.

103 [0233] Figure 10 shows a significant increase in plasma potassium level for an effective dose determined in sheep plasma treated with the compound of Comparative Example 4 in the MCC study (above) as a function of time. There was no effect on plasma potassium at any of the doses tested in sheep treated with compound (Ia).