US005656256A United States Patent 19 11 Patent Number: 5,656,256 Boucher et al. 45 Date of Patent: Aug. 12, 1997

54 METHODS OF TREATING LUNG DISEASE M.R. Knowles, et al; Activation by Extracellular Nucle BY AN AEROSOL CONTANING BENZAML otides of Chloride Secretion in the Airway Epithelia of ORPHENAML Patients with , NEngll Med325, pp 533-538 75) Inventors: Richard C. Boucher; Monroe Jackson (1991). Stutts, both of Chapel Hill, N.C. S.J. Mason et al; Regulation of transepithelial ion transport 73) Assignee: The University of North Carolina at and intracellular calcium by extracellular APT in human Chapel Hill, Chapel Hill, N.C. normal and cystic fibrosis airway epithelium, Br. J. Phar macol. 103, pp 1649-1656 (1991). 21 Appl. No.: 355,650 N.J. Willumsen and R.C. Boucher; Sodium transport and 22 Filed: Dec. 14, 1994 intracellular sodium activity in cultured human nasal epi (51 int. Cl. ...m. A61K9/12 thelium, Am. J. Physiol. (Cell Physiol 25) 256 52 U.S. Cl...... 424/45; 424/46; 514/851 C1033-C1053 (1989). 58) Field of Search ...... 424/45, 46; 514/851 56 References Cited Primary Examiner-Raj Bawa U.S. PATENT DOCUMENTS Attorney, Agent, or Firm-Bell, Seltzer, Park & Gibson, PA. 3,313,813 4/1967 Cragoe, Jr. et al...... 424/45 4,50,729 2/1985 Boucher et al...... 424/45 57 ABSTRACT 5,292,498 3/1994 Boucher, Jr...... 424/45 5,304,125 4/1994 Leith ...... 424/45 5,512,269 4/1996 Molina et al...... 424/45 Method of hydrating lung mucous secretions in the lungs of a subject are disclosed. The methods involve administering FOREIGN PATENT DOCUMENTS benzamil or phenamil to the lungs of the subject in an 0.451 130 10/1991 European Pat. Off.. amount effective to hydrate lung mucous secretions. The OTHER PUBLICATIONS administering step is preferably carried out by inhalation Dudeja, P.K. et al. (1994). Gastroenterology 106/1: administration. The method is useful in the treatment of 125-133. diseases such as cystic fibrosis and chronic bronchitis. Matalon, S. et al. (1993). Am. J. Phys. Lung Cell. Mol. Physiol. 264/4: 8-4. 32 Claims, 2 Drawing Sheets

RELATIVE POTENCES OF

AMLORIDE ANAOGS IN NORMAL HNE OO --AML A cut "A" - - O --PHEN 8. 1 -

. . . . A. . . . BENZ Af - i --V--EPA f I f l t ! t Viii.5LESS THAN O% I BY EPA i

LOG INHIBITOR HUAMSAR.ORG PLOT 2 U.S. Patent Aug. 12, 1997 Sheet 1 of 2 5,656.256

RELATIVE POTENCIES OF AMLORDE ANALOGS N NORMAL HNE OO --AMIL ...tv6"A"- -- O - - PHEN 8f 1. 1.t 8O A. . . . BENZ Af - l --V-- EPA f ip

LOG INHIBITOR HUAMSAR.ORG PLOT 2 FIG. I. U.S. Patent Aug. 12, 1997 Sheet 2 of 2 5,656,256

PERSISTANCE OF SODIUM ABSORPTION BLOCKADE AFTER WASHOUT ANGRIDE, BENZAML AND

5,656.256 1. 2 METHODS OF TREATING LUNG DSEASE cally acceptable carrier, (i) benzamil in an amount effective BYANAEROSOL CONTAINING BENZAML to inhibit the reabsorption of water from lung mucous ORPHENAML secretions; and (ii) UTP or an analog thereof in an amount effective to hydrate lung mucous secretions. These inventions were made with Government support 5 A fifth aspect of the present invention is a method of under grant number 22924 from the National Institutes of hydrating mucous secretions in the lungs of a subjectin need Health (NIH) Heart and Lung Institute. The Government has of such treatment. The method comprises administering certain rights to these inventions. phenamil to the lungs of the subject in an amount effective to hydrate lung mucous secretions. FIELD OF THE INVENTION 10 A sixth aspect of the present invention is a method of These inventions relate to a method of hydrating lung treating cystic fibrosis in a human subject in need of such mucous Secretions by administering benzamil to the lungs of treatment, comprising administering by inhalation an aero a subject, and a method of hydrating lung mucous secretions sol suspension of respirable particles to the respiratory by administering phenamil to the lungs of a subject. system of the subject, the particles comprised of phenamil, 15 the phenamil administered in an amount effective to hydrate BACKGROUND OF THE INVENTION retained lung mucous secretions in the lungs of the subject, In cystic fibrosis several functions of airway epithelia are whereby the retained mucous secretions are more easily abnormal, and deficiencies in both CL transport and Na transported from the lungs via mucociliary action. absorption are well documented. See, e.g. Knowles et al., 20 A seventh aspect of the present invention is the use of Science 221, 1067 (1983); Knowles et al., J. Clin. Invest. 71, phenamil for the manufacture of a medicament for carrying 1410 (1983). Regulation of ion transport might have poten out a therapeutic method of treatment as given above. tial therapeutic benefit in lung diseases characterized by An eighth aspect of the present invention is a pharma abnormalities in epithelial ion transport, e.g., cystic fibrosis. ceutical composition, comprising, together in a pharmaceu One therapeutic goal in cystic fibrosis and other pulmo 25 tically acceptable carrier, (i) phenamilin an amount effective nary diseases in which the water content of the mucous is to inhibit the reabsorption of water from lung mucous altered is to hydrate the lung mucous secretions, so that the secretions; and (ii) UTP or an analog thereof in an amount secretions may be thereafter more easily removed from the effective to hydrate lung mucous secretions. lungs by mucociliary action or simple coughing. The use of BRIEF DESCRIPTION OF THE DRAWTNGS aerosolized to hydrate mucous secretions is 30 described in U.S. Pat. No. 4,501.729. Amiloride appears to FIG. 1 shows the log concentration-effect curves block Nareabsorption by airway epithelial cells, and there (percentage change in from basal levels) of amiloride, fore inhibits water absorption from the mucous. While an benzamil, phenamil and 5-(N.N.-hexamethylene)amiloride important breakthrough in providing treatments for cystic (or EIPA) applied to the apical surface of human nasal fibrosis, a potential problem with amiloride treatments is the 35 epithelium. relatively short duration of action of amiloride. FIG. 2 shows a comparison of the persistence of the Na A different therapeutic approach for hydrating lung absorption blockade after the washout of amiloride, ben mucous secretions is exemplified by techniques that involve zamil and phenamil. The time needed for the washout of the administration of ATP or UTP, which appear to stimulate phenamil and benzamil as compared to the time needed to chloride secretion from respiratory epithelial cells. See, e.g., 40 wash out amidoride is illustrated. U.S. Pat. No. 5.292.498 to Boucher. DETALED DESCRIPTION OF THE In view of the large numbers of people afflicted with INVENTION cystic fibrosis, there is an ongoing need for new methods for providing methods of hydrating lung mucous secretions and The method of the present invention may be used to 45 remove mucous secretions retained in the lungs of a subject thereby facilitating lung mucous clearance. for any reason, including (but not limited to) retention of SUMMARY OF THE INVENTION secretions arising from airway diseases such as cystic fibrosis, chronic bronchitis, asthma, and bronchiectasis. Two A first aspect of the present invention is a method of compounds, phenamil and benzamil, were identified as hydrating mucous secretions in the lungs of a subjectin need particularly potent blockers of airway epithelial Na' of such treatment. The method comprises administering 50 channels, having K.'s of <107M in human airway epithelial benzamil to the lungs of the Subject in an amount effective preparations. The novel features of benzamil and phenami to hydrate lung mucous secretions. as compared to amiloride are that these compounds are A second aspect of the present invention is a method of 1-1.5 log-concentration units more potent than amiloride. treating cystic fibrosis in a human subject in need of such 55 Additionally, they appear to bind more avidly to the Na treatment, comprising administering by inhalation an aero channel and thus have longer durations of action during sol suspension of respirable particles to the respiratory intermittent dose regimens. system of the subject, the particles comprised of benzamil, The method of the present invention can be used to the benzamil administered in an amount effective to hydrate facilitate (i.e., enhance, speed, assist) the clearance of retained lung mucous secretions in the lungs of the subject, mucous secretions from the lungs of a subject in need of whereby the retained mucous secretions are more easily such treatment for any reason, including (but not limited to) transported from the lungs via mucociliary action. retained secretions arising from airway diseases such as A third aspect of the present invention is the use of cystic fibrosis, chronic bronchitis, asthma, bronchiectasis, benzamil for the manufacture of a medicament for carrying post-operative atelectasis (plugging of airways with retained out a therapeutic method of treatment as given above. 65 secretions after surgery), and Kartagener's syndrome. A fourth aspect of the present invention is a pharmaceu The present invention is concerned primarily with the tical composition, comprising, together in a pharmaceuti treatment of human subjects, but may also be employed for 5,656.256 3 4 the treatment of other mammalian subjects, such as dogs and less than about 4.7 microns in size) are respirable. Particles cats, for veterinary purposes. of non-respirable size which are included in the aerosol tend Benzamil (also known as 3,5-diamino-6-chloro-N- to be deposited in the throat and swallowed, and the quantity (benzylaminoaminomethylene)pyrazinecarboxamide) and of non-respirable particles in the aerosol is preferably mini 5 mized. For nasal administration, a particle size in the range phenamil (also known as 3.5-diamino-6-chloro-N- of 10-500 m is preferred to ensure retention in the nasal (phenylaminoaminomethylene)pyrazinecarboxamide) are cavity. known compounds and are disclosed in U.S. Pat. No. 3.313,813 to E. Cragoe (applicant specifically intends that The dosage of active compound will vary depending on the disclosure of this and all other patents cited herein be the condition being treated and the state of the subject, but 10 generally may be an amount sufficient to achieve dissolved incorporated herein by reference). concentrations of active compound on the airway surfaces of The terms “benzamil” and “phenami” as used herein, the subject of from about 107 to about 10 Moles/liter, and include the pharmaceutically acceptable salts thereof, such more preferably from about 10 to about 3x10 Moles/ as (but not limited to) benzamil hydrochloride or phenamil liter. Depending upon the solubility of the particular formu hydrochloride. Pharmaceutically acceptable salts are salts 15 lation of active compound administered, the daily dose may that retain the desired biological activity of the parent be divided among one or several unit dose administrations. compound and do not impart undesired toxicological effects. The daily dose by weight may range from about 1 to 20 Examples of such salts are (a) acid addition salts formed milligrams of respirable benzamil or phenamil particles for with inorganic acids, for example hydrochloric acid, hydro a human subject, depending upon the age and condition of bromic acid, sulfuric acid, phosphoric acid, nitric acid and the subject. A currently preferred unit dose is about 2 the like; and salts formed with organic acids such as, for 20 milligrams of respirable benzamil or phenamil particles example, acetic acid, oxalic acid, tartaric acid, succinic acid, given at a regimen of four administrations per day. The maleic acid, fumaric acid, gluconic acid, citric acid, malic dosage may be provided as a prepackaged unit by any acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, Suitable means (e.g., encapsulating in a gelatin capsule). alginic acid, polyglutamic acid, naphthalenesulfonic acid, 25 In the manufacture of a formulation according to the methanesulfonic acid, p-toluenesulfonic acid, naphthalene invention, active agents or the physiologically acceptable disulfonic acid, polygalacturonic acid, and the like; and (b) salts or free bases thereof are typically admixed with, inter salts formed from elemental anions such as chlorine, alia, an acceptable carrier. The carrier must, of course, be bromine, and iodine. acceptable in the sense of being compatible with any other Benzamil or phenamil used to prepare compositions for 30 ingredients in the formulation and must not be deleterious to the present invention may alternatively be in the form of a the patient. The carrier may be a solid or a liquid, or both, pharmaceutically acceptable free base of benzamil or phe and is preferably formulated with the compound as a unit namil. Because the free base of the compound is less soluble dose formulation, for example, a capsule, which may con than the salt, free base compositions are employed to pro tain from 0.5% to 99% by weight of the active compound. vide more sustained release of benzamil or phenami to the 35 One or more active compounds may be incorporated in the lungs. Benzamil or phenamil present in the lungs in particu formulations of the invention, which formulations may be late form which has not gone into solution is not available prepared by any of the well-known techniques of pharmacy to induce a physiological response, but serves as a depot of consisting essentially of admixing the components. bioavailable drug which gradually goes into solution. Aerosols of liquid particles comprising the active com The active compounds disclosed herein may be adminis 40 pound may be produced by any suitable means, such as with tered to the lungs of a patient by any suitable means, but are a pressure-driven aerosol nebulizer or an ultrasonic nebu preferably administered by administering an aerosol suspen lizer. See, e.g., U.S. Pat. No. 4.501,729. Nebulizers are sion of respirable particles comprised of the active commercially available devices which transform solutions compound, which the subject inhales. The respirable par or suspensions of the active ingredient into a therapeutic ticles may be liquid or solid. The quantity of benzamil or 45 aerosol mist either by means of acceleration of compressed phenamil included may be an amount sufficient to achieve gas, typically air or oxygen, through a narrow venturi orifice dissolved concentrations of benzamil or phenamil on the or by means of ultrasonic agitation. Suitable formulations airway surfaces of the subject of from about 107 to about for use in nebulizers consist of the active ingredient in a 10 Moles/liter, and more preferably from about 10 to liquid carrier, the active ingredient comprising up to 40% about 10 Moles/liter. 50 w/w of the formulation, but preferably less than 20% w/w. In one embodiment of the invention, the particulate ben The carrier is typically water (and most preferably sterile, Zamil or phenamil composition may contain both a free base pyrogen-free water) or a dilute aqueous alcoholic solution, of phenamil or benzamil and a pharmaceutically acceptable preferably made isotonic with body fluids by the addition of, salt such as benzamil hydrochloride or phenamil hydrochlo for example, sodium chloride. Optional additives include ride to provide both early release of and sustained release of 55 preservatives if the formulation is not made sterile, for benzamil or phenamil for dissolution into the mucous secre example, methyl hydroxybenzoate, antioxidants, flavoring tions of the lungs. Such a composition serves to provide both agents, volatile oils, buffering agents and surfactants. early relief to the patient, and sustained relief over time. Aerosols of solid particles comprising the active com Sustained relief, by decreasing the number of daily admin pound may likewise be produced with any solid particulate istrations required, is expected to increase patient compli medicament aerosol generator. Aerosol generators for ance with a course of benzamil or phenamil treatments. administering solid particulate medicaments to a subject Solid or liquid particulate benzamil or phenamil prepared produce particles which are respirable, as explained above, for practicing the present invention should include particles and generate a volume of aerosol containing a predeter of respirable size: that is, particles of a size sufficiently small mined metered dose of a medicament at a rate suitable for to pass through the mouth and larynx upon inhalation and 65 human administration. One illustrative type of solid particu into the bronchi and alveoli of the lungs. In general, particles late aerosol generator is an insufflator. Suitable formulations ranging from about 1 to 5 microns in size (more particularly, for administration by insufflation include finely comminuted 5,656.256 5 powders which may be delivered by means of an insufflator or taken into the nasal cavity in the manner of a Snuff. In the insufflator, the powder (e.g., a metered dose thereof effective to carry out the treatments described herein) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either pierced or opened in situ and the powder delivered by air drawn through the device upon inhalation or by means of a manually-operated pump. The powder employed in the insufflator consists either solely of the O active ingredient or of a powder blend comprising the active ingredient, a suitable powder diluent, such as lactose, and an optional surfactant. The active ingredient typically com wherein: prises from 0.1 to 100 w/w of the formulation. A second type X, X, and X are eachindependently either O(i.e., OH) of illustrative aerosol generator comprises a metered dose 15 or S (i.e., SH). Preferably X and X are O. inhaler. Metered dose inhalers are pressurized aerosol R is O., imido, methylene, dihalomethylene (e.g., dispensers, typically containing a suspension or solution dichloromethylene, difluoromethylene). Preferably, R is formulation of the active ingredient in a liquified propellant. OXygen. R is H or Br. Preferably, R is H. During use these devices discharge the formulation through A particularly preferred compound of Formula () above a valve adapted to deliver ametered volume, typically from is the UTP analog uridine 5'-O-(3-thiotriphosphate) (or 10 to 150 pil to produce a fine particle spray containing the “UTPS"). active ingredient. Suitable propellants include certain chlo The present invention is explained in greater detail in the rofluorocarbon compounds, for example, Examples which follow. These examples are intended as dichlorodifluoromethane, trichlorofluoromethane, dichlo 25 illustrative of the invention, and are not to be taken as rotetrafluoroethane and mixtures thereof. The formulation limiting thereof. Amiloride was obtained from Sigma may additionally contain one or more co-solvents, for Chemicals (St. Louis, Mo.); benzamil and phenamil were a example, , surfactants, such as oleic acid or sorbitan gift from Dr. Thomas Kleyman of the University of Penn trioleate, antioxidants and suitable flavoring agents. sylvania. The composition of Krebs bicarbonate Ringer's 30 solution (KBR) was 140 milliMolar (mM) Na, 120 mM C1, 52 mMK, 25 mMHCO, 2.4 mM HPO, 0.4 mM Compositions containing respirable dry particles of HPO, 1.1 mM Ca", and 5 mM glucose. micronized benzamil or phenamil may be prepared by grinding the dry phenamil or benzamil with a mortar and EXAMPLE 1. 35 pestle, and then passing the micronized composition through Human Nasal Epithelium (HNE) Cultures a 400 mesh screen to breakup or separate out large agglom Nasal specimens are obtained from human subjects and erates. are typically inferior turbinates removed for sleep apnea syndromes or plastic reconstruction. The cell culture proce The aerosol, whether formed from solid or liquid dures used in this example are performed as described in particles, may be produced by the aerosol generator at a rate Willumsen, N.J., et al., Am. J. Physiol. 256:C1033-C1044 of from about 10 to 150 liters per minute, more preferably (Cell. Physiol. 25) (1989) and Yankaskas, J. R. et al., Am. from about 30 to 150 liters perminute, and most preferably Rev. Respir: Dis. 132:1281-1287 (1985). Cells from freshly about 60 liters per minute. Aerosols containing greater excised specimens are protease isolated with protease XIV amounts of medicament may be administered more rapidly. 45 (Sigma, St, Louis, Mo.), concentrated, and plated on col lagen membranes in the bottom of plastic-tissue culture cups. The cells are fed for 5 days with serum-free F-12 The particulate benzami or phenamil composition may medium containing the following additives (F-12/7X): optionally contain a dispersant which serves to facilitate the insulin, epidermal growth factor, cholera toxin, transferrin, formation of an aerosol. A suitable dispersant is lactose, 50 hydrocortisone, triiodothyronine, and endothelial cell which may be blended with the benzamil orphenamilin any growth substance. Subsequently, they are fed with F-12/7X suitable ratio (e.g., a 1 to 1 ratio by weight). media supplemented (1:1) with 3T3 fibroblast-conditioned media containing 1% fetal bovine serum. After the fifth day If desired, the benzamil or phenamil may be concurrently in culture, the transepithelial potential difference (PD) administered with UTP or an analog thereof (including the 55 developed by the culture is measured daily to detect the pharmaceutically acceptable salts thereof) in an amount occurrence of confluency. Cell preparations are routinely effective to stimulate chloride secretion from respiratory studied within 1 day of the development of the maximal PD. epithelial cells (and thereby further hydrate the lung mucous EXAMPLE 2 secretions), and formulations containing benzanil or phe namil may also contain UTP or an analog thereof in an Electrophysiological Measurements amount effective to stimulate chloride secretion from respi The transepithelial electrophysiological techniques used ratory epithelial cells. UTP and analogs thereof that may be in this example have been described in Willumsen, N.J., et used to carry out this technique are disclosed in U.S. Pat. No. al., Am. J. Physiol. (Cell Physiol. 25). 256:C1033-C1053 5,292.498 to Boucher. In general, such compounds are of the 65 (1989). structure of Formula (I) below, or a pharmaceutically accept The tissue preparation described in Example 1 is mounted able salt (as given above) thereof: in a modified (superfusion, not recirculating), miniature 5,656.256 7 8 Ussing chamber interfaced to a voltage clamp that measures compartment, and (2) binding of drug to the target site transepithelial PD and the PD response to constant current within the airway epithelium. This Example illustrates the (I) pulses. The chamber contains 1 ml of bathing solution for contribution of binding of drug to the target site (2) to the each (apical; basolateral) surface of the preparation. The duration of drug action. solution used as the vehicle for drug delivery is a Krebs bicarbonate Ringer solution (KBR) which approximates the A protocol was designed to measure the duration of drug ionic composition of plasma. This solution is warmed (37) action after removal of drug from the airway surface liquid and gassed (95% oxygen, 5% CO2) to maintain pH 7.4. The compartment. For this protocol, human airway epithelial cultured cells are superfused on both surfaces with KBR. Drugs are delivered by adding drug selectively to the apical 10 preparations as described in Example 1 are mounted in or basolateral perfusate and monitoring the preparation for 5 modified Ussing chambers and interfaced to voltage clamps minutes with a drug expected to affect transepithelial sodium as described above in Example 2. Basal measurements of I transport (here, amiloride, benzamil, phenamil or 5-(N.N.- in KBR are made and the steady state response to a 5 minute hexamethylene)amiloride (EIPA)). administration of a single maximal effective concentration The measurement of sodium transport rates is performed 15 (10M) of drug delivered to the luminal surface is mea by recording the spontaneous transepithelial PD (V) and sured. Following this step, administration of the drug is responses of the PD to constant current pulses. From the stopped, the lumen is perfused with standard KBR solutions, relationship between V, and induced V, deflections, the and the time required for I to return to baseline or basal transepithelial resistance (R) is calculated. The short-circuit levels is measured. The percent washout time for each drug current (I), or measure of sodium transport rate, is deter is calculated as: mined as I=V/R. Measurements of transepithelial unidi rectional isotopic Na+ fluxes, in cultures matched on the Tbx - Tax basis of V, and R (<25% difference), mounted in Ussing - ThaRe- - Toda X 00 chambers, bathed by KBR, gassed with a 95% O-5% CO gas mixture, and warmed to 37° C., confirmed that I is a 25 measure of Na' transport. where T-time to return to basal I after cessation of drug Each cultured human airway epithelial preparation is administration; T-time at which administration of drug exposed to different concentrations (10M-10M) of a ceases; X=test drug (benzamil or phenamil); and Sodium-channel blocking drug on either the basolateral or A=amiloride. apical surface for the dose response studies. To construct 30 concentration-effect relationships of the response to the FIG. 2 illustrates a comparison of the persistence of the drugs, it was assumed that the same maximum response to Na" absorption blockade after the washout of amiloride, a drug could be induced from each tissue culture preparation benzamil and phenamil. The time needed for the washout of from the same individual. phenamil and benzamil as compared to the time needed to 35 wash out amiloride is shown. Benzamil and phenamil have Comparative Example A a significantly longer duration of activity than amiloride, Effects of Benzamil and Phenamil on Sodium Absorption as with benzanil and phenamil remaining effective in sodium Compared with Amiloride absorbing channels almost twice as long as amiloride. FIG. 1 shows the log concentration-effect curves 40 (percentage of maximum inhibition of sodium absorption as The foregoing Examples are illustrative of the present a function of the log of drug concentration) of amiloride, invention, and are not to be construed as limiting thereof. benzamil, phenamil and 5-(N.N.-hexamethylene)amiloride The invention is defined by the following claims, with (or EIPA) applied to the apical surface of human nasal equivalents of the claims to be included therein. epithelium. Data points represented by inverted triangles 45 That which is claimed is: indicate the effect of EPA on sodium absorption; upright triangles indicate benzamil; circles represent phenamii and 1. A method of hydrating mucous secretions in the lungs squares represent amiloride. of a subject in need of such treatment, comprising admin These results illustrate the comparative effects of istering benzamil to the lungs of the subject in an amount amiloride, benzamil and phenamil on the steady-state inhi 50 effective to hydrate lung mucous secretions, wherein said bition of Na" transport rates by human nasal epithelia. benzamil comprises respirable particles having a particle Sodium uptake is inhibited less than 10% by EIPA; that is, size within the range of about 1 to 5 microns. EIPA has very little effect on sodium transport rate. 2. A method according to claim 1, wherein said benzamil Amiloride appears to be a potent blocker of apical Na" is administered by delivering an aerosol suspension of channels in Na'-absorbing epithelia, but is significantly less 55 respirable particles comprised of benzami to the lungs of potent than benzamil or phenamil, which achieve the same said subject. level of complete blocking at approximately 3. A method according to claim 2, wherein said particles one log concentration less. are selected from the group consisting of solid particles and EXAMPLE 3 liquid particles. 60 4. A method according to claim 1, wherein said benzamil Comparative Example B is administered in an amount sufficient to achieve concen Persistence of Efficacy of Benzamil, Phenamil and trations of benzamil on the airway surfaces of said subject of Amiloride from about 107 to about 10 Moles/liter. In vivo, drug is delivered to the lungs as a single bolus. 65 5. A method according to claim 1, further comprising Hence, the duration of drug action in vivo will reflect (1) the concurrently administering to said subject a compound of retention of the drug in the airway surface liquid Formula (I), or pharmaceutically acceptable salt thereof: 5,656.256 10 a compound of Formula (I), or pharmaceutically accept able salt thereof:

O 5

O wherein: X, X and X are each independently selected from the group consisting of OH and SH; 15 R is selected from the group consisting of O, imido, wherein: methylene, and dihalomethylene; and X, X, and X are each independently selected from the R is selected from the group consisting of H and Br; group consisting of OH and SH; in an amount effective to stimulate chloride secretion into said mucous from respiratory epithelial cells. 20 R is selected from the group consisting of O, imido, 6. A method of treating cystic fibrosis in a human subject methylene, and dihalomethylene; and in need of such treatment, comprising administering by R is selected from the group consisting of H and Br; inhalation an aerosol suspension of respirable particles hav in an amount effective to hydrate lung mucous secretions. ing a particle size within the range of about 1 to 5 microns to the respiratory system of said subject, said particles 25 11. A pharmaceutical composition according to claim 10, comprised of benzamil, said benzamil administered in an wherein said carrier is selected from the group consisting of amount effective to hydrate retained lung mucous secretions solid carriers and liquid carriers. in the lungs of said subject, whereby the retained mucous 12. A pharmaceutical composition according to claim 10, secretions are more easily transported from the lungs via wherein said compound of Formula (I) is selected from the mucociliary action. 30 7. A method according to claim. 6, wherein said particles group consisting of uridine 5'-triphosphate, uridine 5'-O-(3- are selected from the group consisting of solid particles and thiotriphosphate), and the pharmaceutically acceptable salts liquid particles. thereof. 8. A method according to claim 6, wherein said benzamil 13. A pharmaceutical composition useful for hydrating is administered in an amount sufficient to achieve concen trations of benzami on the airway surfaces of said subject of 35 mucous secretion in the lungs of a subject in need of such from about 107 to about 10 Moles/liter. treatment, said composition comprising aerosolizable and 9. A method according to claim 6, further comprising respirable solid particles, said solid particles comprising concurrently administering to said subject a compound of benzamil having a particle size within the range of from Formula (I), or pharmaceutically acceptable salt thereof: about 1 to 5 microns. 40 14. A pharmaceutical composition according to claim 13, O said solid particles further comprising a pharmaceutically RN acceptable carrier. 15. A composition according to claim 13, wherein said O O O 4. 45 O N composition further comprises a propellant. HO-P-R-P-O-P-O-CH O 16. Amethod of hydrating mucous secretions in the lungs X X X3 H of a subject in need of such treatment, comprising admin H istering phenamil to the lungs of the subject in an amount y B O H effective to hydrate lung mucous secretions, wherein said OH, OH 50 phenamii comprises respirable particles having a particle size within the range of about 1 to 5 microns. wherein: 17. A method according to claim 16, wherein said phe X, X and X are each independently selected from the namilis administered by delivering an aerosol suspension of group consisting of OH and SH; 55 respirable particles comprised of phenamil to the lungs of R is selected from the group consisting of O, imido, said subject. methylene, and dihalomethylene; and 18. A method according to claim 17, wherein said par R is selected from the group consisting of H and Br; ticles are selected from the group consisting of solid par in an amount effective to stimulate chloride secretion into ticles and liquid particles. said mucous from respiratory epithelial cells. 10. A pharmaceutical composition, comprising, together 19. A method according to claim 16, wherein said phe in a pharmaceutically acceptable carrier: nami is administered in an amount sufficient to achieve benzamil in an amount effective to inhibit the reabsorp concentrations of phenamil on the airway surfaces of said tion of water from lung mucous secretions, wherein subject of from about 107 to about 10 Moles/liter. said benzamil comprises respirable particles having a 65 20. A method according to claim 16, further comprising particle size within the range of about 1 to 5 microns; concurrently administering to said subject a compound of and Formula (I), or pharmaceutically acceptable salt thereof: 5,656.256 11 12 a compound of Formula (), or pharmaceutically accept O able salt thereof:

RN O O O O 4. O N R2N HO-P-R-P-O-P-O-CH2 O O O O X X X H I es N H HO-P-R-P-O-P-O-CH, O y 8 H X X X3 H OH, OH H y B C. H wherein: OH, OH X, X, and X are each independently selected from the group consisting of OH and SH; 15 wherein: R is selected from the group consisting of O, imido, X, X2, and X are each independently selected from the methylene, and dihalomethylene; and group consisting of OH and SH; R is selected from the group consisting of H and Br; R is selected from the group consisting of O, imido, in an amount effective to stimulate chloride secretion into methylene, and dihalomethylene; and said mucous from respiratory epithelial cells. 20 R is selected from the group consisting of H and Br; in 21. A method of treating cystic fibrosis in a human subject an amount effective to hydrate lung mucous secretions. in need of such treatment, comprising administering by 26. A pharmaceutical composition according to claim 25, inhalation an aerosol suspension of respirable particles hav ing a particle size within the range of about 1 to 5 microns wherein said carrier is selected from the group consisting of to the respiratory system of said subject, said particles 25 solid carriers and liquid carriers. comprised of phenamil, said phenamil administered in an 27. A pharmaceutical composition according to claim 25, amount effective to hydrate retained lung mucous secretions wherein said compound of Formula (I) is selected from the in the lungs of said subject, whereby the retained mucous group consisting of uridine 5'-triphosphate, uridine 5'-O-(3- secretions are more easily transported from the lungs via thiotriphosphate), and the pharmaceutically acceptable salts thereof. mucociliary action. 30 22. A method according to claim 21, wherein said par 28. A pharmaceutical composition useful for hydrating ticles are selected from the group consisting of solid par mucous secretion in the lungs of a subject in need of such ticles and liquid particles. treatment, said composition comprising aerosolizable and 23. A method according to claim 21, wherein said phe respirable solid particles, said solid particles comprising namil is administered in an amount sufficient to achieve phenamil having a particle size within the range of from concentrations of phenamil on the airway Surfaces of said 35 about 1 to 5 microns. subject of from about 107 to about 10 Moles/liter. 29. A pharmaceutical composition according to claim 28, 24. A method according to claim 21, further comprising said solid particles further comprising a pharmaceutically concurrently administering to said subject a compound of acceptable carrier. Formula (I), or pharmaceutically acceptable salt thereof: 30. A composition according to claim 28, wherein said 40 composition further comprises a propellant. O 31. A method of hydrating mucous secretions in the lungs of a subject in need of such treatment, comprising admin istering benzamil to the lungs of the subject in an amount effective to hydrate lung mucous secretions; 45 wherein said benzamil is delivered by administering an aerosol of respirable particles containing said benzamil to the lungs of said subject; wherein said aerosol is comprised of particles having a 50 particle size within the range of from about 1 to 5 microns; wherein said benzamil is administered in a dose range of wherein: about 1 to 20 mg; X, X and X are each independently selected from the and wherein Said benzanil is administered in an amount group consisting of OH and SH; 55 Sufficient to achieve concentrations of benzamil on the R is selected from the group consisting of O, imido, airway surfaces of said subject of from about 107 methylene, and dihalomethylene; and moles/liter to about 10 moles/liter. R is selected from the group consisting of H and Br; 32. A method of hydrating mucous secretions in the lungs in an amount effective to stimulate chloride secretion into of a Subject in need of Such treatment, comprising admin said mucous from respiratory epithelial cells. istering phenamil to the lungs of the Subject in an amount 25. A pharmaceutical composition, comprising, together effective to hydrate lung mucous secretions; in a pharmaceutically acceptable carrier: wherein said phenami is delivered by administering an phenamil in an amount effective to inhibit the reabsorp aerosol of respirable particles containing said phenamil tion of water from lung mucous secretions, wherein to the lungs of said subject; said phenamil comprises respirable particles having a 65 wherein said aerosol is comprised of particles having a particle size within the range of about 1 to 5 microns; particle size within the range of from about 1 to 5 and microns; 5,656.256 13 14 wherein said phenamil is administered in a dose range of airway surfaces of said subject of from about 107 about 1 to 20 mg; moles/liter to about 10 moles/liter. and wherein said phenamil is administered in an amount sufficient to achieve concentrations of phenamil on the : e :