| PATENT ASSIGNEE'S COUNTRY | USA |
| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | 07.11.2000 |
| PATENT TITLE |
Uridine 5'-diphosphate and analogs useful for treating lung diseases |
| PATENT ABSTRACT | Compounds of Formula I: ##STR1## wherein: X.sub.1, and X.sub.2 are each independently either O.sup.- or S.sup.- ; X.sub.3 and X.sub.4 are each independently either --H or --OH, with the proviso that X.sub.3 and X.sub.4 are not simultaneously --H; R.sub.1 is selected from the group consisting of O, imido, methylene and dihalomethylene; R.sub.2 is selected from the group consisting of H, halo, alkyl, substituted alkyl, alkoxyl, nitro and azido; R.sub.3 is selected from the group consisting of H, alkyl, acyl, aryl, and arylalkyl; and R.sub.4 is selected from the group consisting of --OR', --SR', --NR', and --NR'R", wherein R' and R" are independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, alkoxyl, and aryloxyl, with the proviso that R' is absent when R.sub.4 is double bonded from an oxygen or sulfur atom to the carbon at the 4-position of the pyrimidine ring, are used in methods of hydrating lung mucus secretions and treating lung disorders such as cystic fibrosis, ventilator-associated pneumonia, chronic bronchitis, chronic obstructive pulmonary disorder and primary ciliary dyskinesia. Pharmaceutical compositions containing compounds of Formula I, and novel compounds of Formula I are also described |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | August 28, 1998 |
| PATENT REFERENCES CITED |
Eric A. Barnard; Hexokinases from Yeast, Methods in Enzymology 42:6-20 (1975). Brown et al.; Evidence that UTP and ATP Regulate Phospholipase C through a Common Extracellular 5'-Nucelotide Receptor in Human Airway Epithelial Cells, Molecular Pharmacology 40:648-655. Chang et al.; Molecular Cloning and Functional Analysis of a Novel P.sub.2 Nuecloetide Receptor, The Journal of Biological Chemistry, 270, No. 44:26152-26158. S.H. Donaldson et al; In Vivo Regulation of ATP Levels in Human Nasal Epithelia, Pediatric Pulmonology Supp.; 13, 290 (Abstract No. 286) (1996). Erb et al; Site-directed Mutagenesis of P.sub.2u Purinoceptors, The Journal of Biological Chemistry 270:4185-4188 (1995). Goody et al; Thiophasphate Analogs of Nucleoside Di- and Triphosphates, Journal of the American Chemical Society 93:6252-6258 (1971). Knowles et al; Activation by Extracellular Nucleotides of Chloride Secretion in the Airway Epithelia of Patients with Cystic Fibrosis, New England Journal of Medicine, 325:533-538 (Aug. 22, 1991). Knowles et al; Extracellular ATP and UTP Induce Chloride Secretion in Nasal Epithelia of Cystic Fibrosis Patients and Normal Subject in vivo, Chest 101:60S-63S (Mar. 1992 Supplement). Knowles et al; Abnormal Ion Permeation Through Cystic Fibrosis Respiratory Epithelium Science 221:1067-1069 (1983). Knowles et al; Relative Ion Permeability of Normal and Cystic Fibrosis Nasal Epithelium, J. Clinical Invest. 71:1410-1417 (May 1983). Larsen et al; Cation Transport by Sweat Ducts in Primary Culture, Ionic Mechanism of Cholinergically Evoked Current Oscillations Journal of Physiology 424:109-131 (1990). Lazarowski et al; Identification of a Uridine Nucleotide-selective G-protein-linked Receptor that Activates Phospholipase C, The Journal of Biological Chemistry 269(11830-11836) (1994). Lazarowski et al; Pharmacological selectivity of the cloned human P.sub.2u -puriniceptor potent activation by diadenosine tetraphosphate Br. J. Pharmacol. 116:1619-1627 (1995). Lazarowski et al; Enzymatic synthesis of UTP.sub..gamma. S, a potent hydrolysis resistant agonist of P.sub.2u- purinoceptors, British Journal of Pharmacology 117:203-209 (1996). Lazarowski et al; Mucosal-Restricted P.sub.2u -Receptor Independent Action of UDP on Human Nasal Epithelial Cells; Pediatric Pulmonology Supp 13, 290(Abstract No. 292)(1996). Nakahata et al.; Regulation of inositol trisphosphate accumulation by muscarinic cholinergic and H.sub.1 -histamine receptors on human astrocytoma cells, Biochem J. 241:337-344 (1987). Mason et al; Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium Br. J. Pharmacol. 103:1649-1656 (1991). Nicholas et al; Uridine Nucleotide Selectivity of Three Phospholipase C-Activating P.sub.2 Receptors: Identification of a UDP-Selective, a UTP-Selective, and an ATP- and UTP-Specific Receptor Molecular Pharmacology, 50:224-229 (1996). Olivier et al.; Acute Safety and Effects on Mucociliary Clearance of Aerosolized Uridine 5'-Triphosphate .+-.Amiloride in Normal Human Adults Am j Respir Crit Care Med 154:217-223 (1996). Paradiso et al; Membrane-restricted regulation of Ca.sup.2+ release and influx in polarized epithelia Nature 377:643-646 (Oct. 19, 1995). Parr et al; Cloning and expression of a human P.sub.2u nucleotide receptor, a target for cystic fibrosis pharmacotherapy Proc. Natl. Acad. Sci. USA 91:3275-3279 (Apr. 1994). Stutts et al; Multiple modes of regulation of airway epithelial chloride secretion by extracellular ATP American Physiological Society 267:C1442-C1451(1994). Wu et al; Growth and Differentiation of Human Nasal Epithelial Cells in Culture, Am Rev Respir Dis 132:311-320 (1985). |
| PATENT GOVERNMENT INTERESTS |
GOVERNMENT SUPPORT This invention was made with United States government support under grant number # 2PO1 HL32322-11A1 from the National Institutes of Health. The United States government has certain rights in this invention. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
That which is claimed: 1. A pharmaceutical composition, comprising, together in a pharmaceutically acceptable carrier, a compound of Formula I: ##STR4## wherein: X.sub.1, and X.sub.2 are each independently either O.sup.- or S.sup.- ; X.sub.3 and X.sub.4 are each independently either --H or --OH, with the proviso that X.sub.3 and X.sub.4 are not simultaneously --H; R.sub.1 is selected from the group consisting of O, imido, methylene and dihalomethylene; R.sub.2 is selected from the group consisting of H, halo, alkyl, substituted alkyl, nitro and azido; R.sub.3 is selected from the group consisting of H, alkyl, acyl, aryl, and arylalkyl; and R.sub.4 is selected from the group consisting of --OR', --SR', --NR', and --NR'R", wherein R' and R" are independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, alkoxyl, and aryloxyl, and with the proviso that R' is absent when R.sub.4 is double bonded from an oxygen or sulfur atom to the carbon at the 4-position of the pyrimidine ring; or a pharmaceutically acceptable salt thereof, in an amount effective to hydrate lung mucus secretions, with the proviso that said compound of Formula I is not uridine 5'-diphosphate. 2. A pharmaceutical composition according to claim 1 wherein said carrier is selected from the group consisting of solid carriers and liquid carriers. 3. A pharmaceutical composition according to claim 1 wherein said compound of Formula (I) is selected from the group consisting of; uridine 5'-O-(2-thiodiphosphate); 2-deoxyuridine 5'-diphosphate; and 4-mercaptouridine 5'-diphosphate, and the pharmaceutically acceptable salts thereof. 4. A pharmaceutical composition according to claim 1 wherein said compound of Formula (I) is selected from the group consisting of 3'-deoxyuridine-5'-diphosphate; 5-(1-phenylethynyl)-uridine 5'-diphosphate; 5-methyluridine 5'-diphosphate; 4-hexylthiouridine 5'-diphosphate; 4-mercaptouridine 5'-diphosphate; 4-methoxyuridine 5'-diphosphate; 4-hexyloxyuridine 5'-diphosphate; N,N-dimethylcytidine 5'-diphosphate; N-hexylcytidine 5'-diphosphate; and the pharmaceutically acceptable salts thereof. 5. A pharmaceutical composition according to claim 1, wherein said composition further comprises a propellant. 6. A compound according to Formula I: ##STR5## wherein: X.sub.1, and X.sub.2 are each independently either O.sup.- or S.sup.- ; X.sub.3 and X.sub.4 are each independently either --H or --OH, with the proviso that X.sub.3 and X.sub.4 are not simultaneously --H; R.sub.1 is selected from the group consisting of O, imido, methylene and dihalomethylene; R.sub.2 is selected from the group consisting of H, halo, alkyl, substituted alkyl, alkoxyl, nitro and azido; R.sub.3 is selected from the group consisting of H, alkyl, acyl, aryl, and arylalkyl; and R.sub.4 is selected from the group consisting of --OR', --SR', --NR', and --NR'R", wherein R' and R" are independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, alkoxyl, and aryloxyl, and with the proviso that R' is absent when R.sub.4 is double bonded from an oxygen or sulfur atom to the carbon at the 4-position of the pyrimidine ring; and; with the proviso that said compound of Formula I is not a compound selected from the group consisting of uridine 5'-diphosphate, 2-deoxyuridine 5'-diphosphate, uridine 5'-O-(2-thiodiphosphate), and 4-mercaptouridine 5'-diphosphate. 7. A compound according to claim 6, wherein said compound of Formula I is 3'-deoxyuridine-5'-diphosphate. 8. A compound according to claim 6, wherein said compound of Formula I is 5-(1-phenylethynyl)-uridine 5'-diphosphate. 9. A compound according to claim 6, wherein said compound of Formula I is 5-methyluridine 5'-diphosphate. 10. A compound according to claim 6, wherein said compound of Formula I is 4-hexylthiouridine 5'-diphosphate. 11. A compound according to claim 6, wherein said compound of Formula I is 4-mercaptouridine 5'-diphosphate. 12. A compound according to claim 6, wherein said compound of Formula I is 4-methoxyuridine 5'-diphosphate. 13. A compound according to claim 6, wherein said compound of Formula I is 4-hexyloxyuridine 5'-diphosphate. 14. A compound according to claim 6, wherein said compound of Formula I is N,N-dimethylcytidine 5'-diphosphate. 15. A compound according to claim 6, wherein said compound of Formula I is N-hexylcytidine 5'-diphosphate. 16. A compound according to claim 6, wherein said compound of Formula I is N-cyclopentylcytidine 5'-diphosphate |
| PATENT DESCRIPTION |
FIELD OF THE INVENTION This invention relates to methods of treating lung disease, and novel compounds and pharmaceutical compositions useful therefor. BACKGROUND OF THE INVENTION One therapeutic goal in cystic fibrosis and other pulmonary diseases in which the water content of lung mucus is altered is to hydrate the lung mucus secretions, so that the secretions may thereafter be more easily removed from the lungs by mucociliary action or simple coughing. For example, the use of aerosolized amiloride to hydrate mucus secretions is described in U.S. Pat. No. 4,501,729 to Boucher et al. Amiloride appears to block Na.sup.+ reabsorption by airway epithelial cells, and therefore inhibits water absorption from the mucus. While an important breakthrough in providing treatments for cystic fibrosis, a potential problem with amiloride as a therapeutic is its relatively short duration of action. In certain lung diseases (e.g., cystic fibrosis), several functions of airway epithelia are abnormal, and deficiencies in both Cl.sup.- transport and Na.sup.+ absorption are well documented. See, e.g. Knowles et al., Science 221, 1067 (1983); Knowles et al., J. Clin. Invest. 71, 1410 (1983). Regulation of ion transport is thus thought to have potential therapeutic benefit in lung diseases characterized by abnormalities in epithelial ion transport. Confirmation of the presence of P2Y.sub.2 (P.sub.2U -purinergic) receptors on the apical surface of human airway epithelial cells raised the possibility that aerosolized nucleotides might be used therapeutically to induce Cl.sup.- secretion in individuals with cystic fibrosis or other airway diseases. Accordingly, a different therapeutic approach for hydrating lung mucus secretions is exemplified by techniques that involve the administration of ATP or UTP, which appear to stimulate chloride secretion from respiratory epithelial cells. See, e.g., U.S. Pat. No. 5,292,498 to Boucher. Existence of a G-protein-coupled receptor that selectively recognizes uridine 5'-diphosphate (UDP) was originally established in studies of a receptor natively expressed by C6-2B rat glioma cells. E. R. Lazarowski et al. J. Biol. Chem. 269, 11830-11836 (1994). The P2Y.sub.6 receptor was recently cloned by K. Chang et al., J. Biol. Chem. 270, 26152-26158 (1995). This receptor was subsequently shown to be selectively activated by UDP, and to be the UDP receptor natively expressed in C6-2B cells. R. A. Nicholas et al., Mol. Pharmacol. 50, 224-229 (1996). The failure to identify this receptor in previous studies of mammalian tissues likely has been a consequence of the lack of availability of potent selective agonists for uridine nucleotide receptors, and the low chemical and metabolic stability of the available nucleotides. It was originally reported that UDP stimulated inositol phosphate accumulation in human airway epithelial cells by low potency activation of the P2Y.sub.2 receptor. E. R. Lazarowski et al. Br. J. Pharmacol. 116, 1619-1627 (1995); H. A. Brown et al., Mol. Pharmacol. 40, 648-655 (1991). However, it has been recently demonstrated that UDP is in fact not an agonist at the P2Y.sub.2 receptor (Nicholas et al., supra) and that the previously observed effect of UDP at P2Y.sub.2 receptors can be explained by the presence of small amounts of contaminating UTP in UDP solutions and/or by conversion of UDP to UTP by cell surface nucleoside diphosphokinase. Despite the evidence related to the P2Y.sub.6 receptor and its relationship to UDP, it has heretofore not been recognized that this relationship may be useful in the treatment of airway disease. SUMMARY OF THE INVENTION The present inventors have discovered that the P2Y.sub.6 receptor, which selectively recognizes UDP as a potent agonist, also exists in airway tissue. The association of the P2Y.sub.6 receptor with increases in Cl.sup.- secretion indicates that UDP and other receptor-selective drugs that derive from this molecule are of therapeutic value in the treatment of a variety of airway diseases. Accordingly, a first aspect of the present invention relates to a method of hydrating mucus secretions in the lungs of a subject in need of such treatment. The method comprises administering to the lungs of the subject a compound of Formula I below, or a pharmaceutically acceptable salt thereof (hereinafter referred to as the "active compound"), in an amount effective to hydrate lung mucus secretions: ##STR2## wherein: X.sub.1, and X.sub.2 are each independently either O.sup.- or S.sup.- ; X.sub.3 and X.sub.4 are each independently either --H or --OH, with the proviso that X.sub.3 and X.sub.4 are not simultaneously --H; R.sub.1 is selected from the group consisting of O, imido, methylene, and dihalomethylene (e.g., dichloromethylene, difluoromethylene); R.sub.2 is selected from the group consisting of H, halo, alkyl, substituted alkyl, alkoxyl, nitro and azido; R.sub.3 is selected from the group consisting of H, alkyl, acyl (including arylacyl), and arylalkyl; and R.sub.4 is selected from the group consisting of --OR', --SR', NR', and NR'R", wherein R' and R" are independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, alkoxyl, and aryloxyl, and with the proviso that R' is absent when R.sub.4 is double bonded from an oxygen or sulfur atom to the carbon at the 4-position of the pyrimidine ring. The method of the present invention may further comprise the step of concurrently administering amiloride, benzamil, or phenamil to the subject in an amount effective to inhibit the reabsorption of water from lung mucus secretions. The method of the present invention is useful in treating several disorders of the lung, including but not limited to, cystic fibrosis, chronic bronchitis, chronic obstructive pulmonary disorder (COPD), primary ciliary dyskinesia, and ventilator-associated pneumonia (VAP). A second aspect of the present invention is a pharmaceutical composition containing the active compounds disclosed herein, in an amount effective to hydrate lung mucus secretions, in a pharmaceutically acceptable carrier. Novel compounds useful in the treatment of lung disorders are a third aspect of the present invention. These compounds have the structure of Formula I as set forth above, with the proviso that such novel compounds do not include the known compounds uridine 5'-diphosphate (or UDP), 2-deoxyuridine 5'-diphosphate (or dUDP), uridine 5'-O-(2-thiodiphosphate) (or UDP-.beta.-S), and 4-mercaptouridine 5'-diphosphate (or 4-mercaptoUDP). Novel compounds of the present invention include, but are not limited to, 3'-deoxyuridine 5'-diphosphate; 5-bromouridine 5'-diphosphate; 5-(1-phenylethynyl)-uridine 5'-diphosphate; 5-methyluridine 5'-diphosphate; 4-hexylthiouridine 5'-diphosphate; 4-methoxyuridine 5'-diphosphate; 4-(N-morpholino)uridine 5'-diphosphate; 4-hexyloxyuridine 5'-diphosphate; N,N-dimethylcytidine 5'-diphosphate; N-hexylcytidine 5'-diphosphate; and N-cyclopentylcytidine 5'-diphosphate. A fourth aspect of the present invention is the use of the active compounds described herein for the manufacture of a medicament for the therapeutic hydration of mucus secretions in the lungs of a subject in need of such treatment. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing the time course for the conversion of [.sup.3 H]UTP to [.sup.3 H]UDP in the presence of 10 units/mL hexokinase (HK). The data is expressed as the percentage of [.sup.3 H]uridine triphosphate converted to [.sup.3 H]uridine diphosphate with open circles (.largecircle.) indicating the relative amount of [.sup.3 H]uridine diphosphate, while filled-in circles (.circle-solid.) indicate the related amount of [.sup.3 H]uridine triphosphate. The data indicate the mean value of one experiment representative of two independent experiments performed with duplicate samples that differed by less than 20%. FIG. 2. illustrates, by means of three separate HPLC traces, the metabolism of [.sup.3 H]UTP and [.sup.3 H]UDP by human nasal epithelial cells. Each trace represents the results of experiments in which confluent, polarized human airway epithelial cells were incubated for 20 minutes at 37.degree. C. in the presence of 1 .mu.M (0.2 .mu.Ci) of [.sup.3 H]UTP (FIG. 2A), [.sup.3 H]UDP (FIG. 2B), and [.sup.3 H]UDP combined with 100 .mu.M ATP (FIG. 2C). The traces shown in FIGS. 2A, 2B and 2C are representative of at least three independent experiments performed with duplicates. In each trace, the X-axis of the trace indicates elution time in minutes, while the Y-axis indicates the concentration of [.sup.3 H] radioactivity in units of cpm.times.10.sup.-3. FIG. 3 is a schematic representation of the effects of UTP and UDP on [.sup.3 H]inositol phosphate formation in relation to either the mucosal or serosal cell surface. FIG. 3 also illustrates the effects if UTP and UDP on intracellular calcium mobilization in polarized human nasal epithelial cells. Confluent cells were loaded with [.sup.3 H]myo-inositol and preincubated with LiCl (FIG. 3A), or with Fura-2 (FIG. 3B), as described below in Examples 2 and 3. The cells were challenged with 100 .mu.M of either UTP (left-hand pair of data bars) or UDP (right-hand set of data bars), added to either the serosal (open bars) or the mucosal (filled-in bars) medium. The data in FIG. 3A are shown as the concentration of [.sup.3 H]inositol phosphate in units of cpm.times.10.sup.-3 and represent the medium (.+-.S.E.M.) from three experiments performed with triplicates. The data in FIG. 3B are shown as .DELTA.Ca.sup.2+.sub.i in units of .mu.M, and represent the medium (.+-.S.E.M.) from fourteen individual experiments. FIG. 4 consists of representative tracings of UDP- and UTP-promoted changes in intracellular Ca.sup.2+ concentration and Cl.sup.- diffusion potentials in human nasal epithelial cells. The upper tracings show changes in intracellular Ca.sup.2+ concentration on the serosal (left-hand tracing) and mucosal (right-hand tracing) after the addition of UDP and UTP to the cell surface medium, as indicated. The lower tracings illustrate changes of transepithelial potential difference (.DELTA.TEP) on the serosal (left-hand tracing) and mucosal (right-hand tracing) after the addition of UDP and UTP to the cell surface medium, as indicated. The data are representative of at least eight independent experiments. FIG. 5 is a graph illustrating the concentration-response relationship for mucosal UDP- and UTP-stimulated [.sup.3 H]inositol phosphate formation in human nasal epithelial cells. The log-concentration of either UTP (filled-in circles, .circle-solid.) or UDP (filled-in squares, .box-solid.) is indicated on the x-axis of the graph. The concentration of [.sup.3 H]inositol phosphates in units of cpm.times.10.sup.-3 M is indicated on the y-axis if the graph. The data represent the mean value (.+-.S.E.M.) from three independent experiments performed with triplicate samples. FIG. 6 is a graph illustrating the effects on mucociliary clearance in sheep after the administration of either a saline control (circles, .circle-solid.) or UDP (squares, .box-solid.). Time in minutes after administration of the compound is indicated on the x-axis of the graph, while percentage retention of mucus is indicated on the y-axis. FIG. 7 is a graph illustrating the effects on tracheal mucus velocity (TMV) in sheep after the administration of either a saline control (open diamond .diamond.) or UDP (closed circle, .circle-solid.). Time in minutes after administration of the compound is indicated on the x-axis of the graph, while TMV measured as a percentage of baseline is indicated on the y-axis. DETAILED DESCRIPTION OF THE INVENTION The methods and pharmaceutical formulations of the present invention can be used to facilitate (i.e., enhance, speed, assist) the clearance of mucus secretions from the lungs of a subject in need of such treatment for any reason, including (but not limited to) retained secretions arising from airway diseases such as cystic fibrosis, chronic bronchitis, chronic obstructive pulmonary disorder (COPD), ventilator-associated pneumonia (VAP), primary ciliary dyskinesia, asthma, bronchiectasis, post-operative atelectasis (plugging of airways with retained secretions after surgery), and Kartagener's syndrome. The present invention is concerned primarily with the treatment of human subjects, but may also be employed for the treatment of other mammalian subjects, such as dogs and cats, for veterinary purposes. The methods of the present invention include the administration of compounds of Formula I, while pharmaceutical compositions of the present invention comprise compounds of Formula I. As used herein, a compound of Formula I is as follows: ##STR3## wherein: X.sub.1, and X.sub.2 are each independently either O.sup.- or S.sup.- ; X.sub.3 and X.sup.4 are each independently either --H or --OH, with the proviso that X.sub.3 and X.sup.4 are not simultaneously --H; R.sub.1 is selected from the group consisting of O, imido, methylene, and dihalomethylene (e.g., dichloromethylene, difluoromethylene); R.sub.2 is selected from the group consisting of H, halo, alkyl, substituted alkyl, alkoxyl, nitro and azido; R.sub.3 is selected from the group consisting of H, alkyl, acyl (including arylacyl), and arylalkyl; and R.sub.4 is selected from the group consisting of --OR', --SR', NR', and NR'R", wherein R' and R" are independently selected from the group consisting of H, alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, alkoxyl, and aryloxyl, and with the proviso that R' is absent when R.sub.4 is double bonded from an oxygen or sulfur atom to the carbon at the 4-position of the pyrimidine ring. As used herein the term "alkyl" refers to C.sub.1-10 inclusive, linear, branched, or cyclic, saturated or unsaturated (i.e., alkenyl and alkynyl) hydrocarbon chains, including for example, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, octyl, ethenyl, propenyl, butenyl, pentenyl, hexenyl, octenyl, butadienyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, and allenyl groups. As used herein, the term "acyl" refers to an organic acid group wherein the --OH of the carboxyl group has been replaced with another substituent (i.e., as represented by RCO--, wherein R is an alkyl or an aryl group). As such, the term "acyl" specifically includes arylacyl groups. Specific examples of acyl groups include acetyl and benzoyl. As used herein, the term "aryl" refers to 5 and 6-membered hydrocarbon and heterocyclic aromatic rings. Specific examples of aryl groups include but are not limited to cyclopentadienyl, phenyl, furan, thiophene, pyrrole, pyran, pyridine, imidazole, isothiazole, isoxazole, pyrazole, pyrazine, pyrimidine, and the like. The term "alkoxyl" as used herein refers to C.sub.1-10 inclusive, linear, branched, or cyclic, saturated or unsaturated oxo-hydrocarbon chains, including for example methoxy, ethoxy, propoxy, isopropoxy, butoxy, t-butoxy, and pentoxy. The term "aryloxyl" as used herein refers to phenyloxyl or hexyloxyl, and alkyl, halo, or alkoxyl substituted phenyloxyl or hexyloxyl. As used herein, the terms "substituted alkyl" and "substituted aryl" include alkyl and aryl groups, as defined herein, in which one or more atoms or functional groups of the aryl or alkyl group are replaced with another atom or functional group, including for example, halogen, aryl, alkyl, alkoxy, hydroxy, nitro, amino, alkylamino, dialkylamino, sulfate, and mercapto. The terms "halo," "halide," or "halogen" as used herein refer to fluoro, chloro, bromo, and iodo groups. Compounds illustrative of the compounds of Formula (I) above include: uridine 5'-diphosphate (also referred to herein as UDP); uridine 5'-O-(2-thiodiphosphate) (also referred to herein as UDP-.beta.-S); 2-deoxyuridine 5'-diphosphate (also referred to herein as dUDP); 3'-deoxyuridine 5'-diphosphate (also referred to herein as 3'-deoxyUDP); 5-bromouridine 5'-diphosphate (also referred to herein as 5-BrUDP); 5-(1-phenylethynyl)-uridine 5'-diphosphate (also referred to herein as 5-(1-phenylethynyl)UDP); 5-methyluridine 5'-diphosphate (also referred to herein as 5-methylUDP); 4-hexylthiouridine 5'-diphosphate (also referred to herein as 4-hexylthioUDP); 4-mercaptouridine 5'-diphosphate (also referred to herein as 4-mercaptoUDP); 4-methoxyuridine 5'-diphosphate (also referred to herein as 4-methoxyUDP); 4-(N-morpholino)uridine 5'-diphosphate (also referred to herein as 4-(N-morpholino)UDP; 4-hexyloxyuridine 5'-diphosphate (also referred to herein as 4-hexyloxyUDP); N,N-dimethylcytidine 5'-diphosphate (also referred to herein as N,N-dimethylCDP); N-hexylcytidine 5'-diphosphate (also referred to herein as N-hexylCDP); and N-cyclopentylcytidine 5'-diphosphate (also referred to herein as N-cyclopentylCDP). Certain compounds of Formula I (e.g., UDP, dUDP, UDP-.beta.-S, and 4-mercaptoUDP) are known and may be made in accordance with known procedures or variations thereof, which will be apparent to those skilled in the art. For example, the identification and preparation of certain thiophosphate analogues of nucleoside diphosphates (such as UTP-.beta.-S) are set forth in U.S. Pat. No. 3,846,402 to Eckstein et al., and in R. S. Goody and F. Eckstein, J. Am. Chem. Soc. 93, 6252-6257 (1971). Alternatively, UDP, dUDP, and other analogs thereof are also commercially available from vendors such as Sigma (St. Louis, Mo.) and Pharmacia (Uppsala, Sweden). Other compounds of Formula I useful in the present invention (e.g., 5-(1-phenylethynyl)UDP; 3'-deoxyUDP, 5-methylUDP; 4-hexylthioUDP; 4-methoxyUDP; 4-hexyloxyUDP; 4-(N-morpholino)UDP; N,N-dimethylCDP; N-hexylCDP; and N-cyclopentylCDP) are novel compounds disclosed for the first time herein, and as such are claimed accordingly in the appended claims. For the sake of simplicity, Formula I herein illustrates uridine diphosphate active compounds in the naturally occurring D configuration, but the present invention also encompasses compounds in the L configuration, and mixtures of compounds in the D and L configurations, unless otherwise specified. The naturally occurring D configuration is preferred. The active compounds of Formula (I) may be administered by themselves or in the form of their pharmaceutically acceptable salts, e.g., an alkali metal salt such as sodium or potassium, an alkaline earth metal salt, or an ammonium and tetraalkyl ammonium salt, NX.sup.4 + (wherein X is a Cl.sub.4 alkyl group). Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. Active compounds of the present invention may optionally be administered in conjunction with other compounds useful in the hydration of lung mucus secretions or useful in the facilitation of the removal of lung mucus secretions. Such compounds (herein referred to as "supplemental compounds") include, but are not limited to, benzamil, phenamil, and amiloride. Amiloride (also known as 3,5,-diamino-6-chloro-N-(diaminomethylene)pyrazinecarboxamide), benzamil (also known as 3,5-diamino-6-chloro-N-(benzylaminoaminomethylene)pyrazinecarboxamide) and phenamil (also known as 3,5-diamino-6-chloro-N-(phenylaminoaminomethylene)pyrazinecarboxamide) are known compounds and are disclosed in U.S. Pat. No. 3,313,813 to E. Cragoe. The terms "amiloride," "benzamil," and "phenamil," as used herein include the pharmaceutically acceptable salts thereof, such as (but not limited to) amiloride hydrochloride, benzamil hydrochloride or phenamil hydrochloride. Amiloride, benzamil or phenamil used to prepare compositions for the present invention may alternatively be in the form of a pharmaceutically acceptable free base of amiloride, benzamil or phenamil. In one embodiment of the invention, the supplemental compound is concurrently administered with the active compound or compounds of the present invention. As used herein, the word "concurrently" means sufficiently close in time to produce a combined (e.g., additive or synergistic) effect. In other words, concurrently may be defined as simultaneously, or it may be defined as two or more events occurring within a short time period before or after each other. The active and supplemental compounds described herein may be administered to the lungs of a patient by any suitable means, but are preferably administered by administering an aerosol suspension of respirable particles comprised of the active compound, which the subject inhales. The active compound can be aerosolized in a variety of forms, such as, but not limited to, dry powder inhalants, metered dose inhalants, or liquid/liquid suspensions. The respirable particles may be liquid or solid. The quantity of active compound included may be an amount sufficient to achieve dissolved concentrations of active compound on the airway surfaces of the subject of from about 10.sup.-9 to about 10.sup.-1 Moles/liter, and more preferably from about 10.sup.-6 to about 10.sup.-4 Moles/liter. The particulate pharmaceutical composition may optionally be combined with a carrier to aid in dispersion or transport. A suitable carrier such as a sugar (i.e., lactose, sucrose, trehalose, mannitol) may be blended with the active compound or compounds in any suitable ratio (e.g., a 1 to 1 ratio by weight). Solid or liquid particulate forms of the active compound prepared for practicing the present invention should include particles of respirable size: that is, particles of a size sufficiently small to pass through the mouth and larynx upon inhalation and into the bronchi and alveoli of the lungs. In general, particles ranging from about 1 to 10 microns in size are within the respirable range. Particles of non-respirable size which are included in the aerosol tend to be deposited in the throat and swallowed, and the quantity of non-respirable particles in the aerosol is preferably minimized. The dosage of active compound will vary depending on the condition being treated and the state of the subject, but generally may be an amount sufficient to achieve dissolved concentrations of active compound on the airway surfaces of the subject of from about 10.sup.-9 to about 10.sup.-1 Moles/liter, and more preferably from about 10.sup.-6 to about 10.sup.-4 Moles/liter. Depending upon the solubility of the particular formulation of active compound administered, the daily dose may be divided among one or several unit dose administrations. The daily dose by weight will depend upon the age and condition of the subject. Such a daily dose may be as low as 1 mg per day, under certain circumstances may be as low as 0.5 mg per day, and may even be as low as 0.1 mg/day. The daily dose of the active compounds may also be as high as 200 mg/day, under certain conditions may be as high as 500 mg/day, and may even be as high as 1000 mg/day. The doses of the active compounds may be provided as one or several prepackaged units. In the manufacture of a formulation according to the invention, active compounds of the present invention or the pharmaceutically acceptable salts or free bases thereof are typically admixed with, inter alia, an acceptable carrier. The carrier must, of course, be acceptable in the sense of being compatible with any other ingredients in the formulation and must not be deleterious to the patient. The carrier may be a solid or a liquid, or both, and is preferably formulated with the compound as a unit-dose formulation which may contain from 0.5% to 99% by weight of the active compound. One or more active compounds may be incorporated in the formulations of the invention, which formulations may be prepared by any of the well-known techniques of pharmacy consisting essentially of admixing the components. Aerosols of liquid particles comprising the active compound may be produced by any suitable means, such as with a pressure-driven aerosol nebulizer or an ultrasonic nebulizer. See, e.g., U.S. Pat. No. 4,501,729. Nebulizers are commercially available devices which transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist either by means of acceleration of compressed gas, typically air or oxygen, through a narrow venturi orifice or by means of ultrasonic agitation. Suitable formulations for use in nebulizers consist of the active ingredient in a liquid carrier, the active ingredient comprising up to 40% w/w of the formulation, but preferably less than 20% w/w. The carrier is typically water (and most preferably sterile, pyrogen-free water) or a dilute aqueous alcoholic solution, preferably made isotonic but may be hypertonic with body fluids by the addition of, for example, sodium chloride. Optional additives include preservatives if the formulation is not made sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents and surfactants. Aerosols of solid particles comprising the active compound may likewise be produced with any solid particulate medicament aerosol generator. Aerosol generators for administering solid particulate medicaments to a subject produce particles which are respirable, as explained above, and generate a volume of aerosol containing a predetermined metered dose of a medicament at a rate suitable for human administration. One illustrative type of solid particulate aerosol generator is an insufflator. Suitable formulations for administration by insufflation include finely comminuted 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 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 comprises from 0.1 to 100 w/w of the formulation. A second type of illustrative aerosol generator comprises a metered dose inhaler. Metered dose inhalers are pressurized aerosol dispensers, typically containing a suspension or solution formulation of the active ingredient in a liquified propellant. During use these devices discharge the formulation through a valve adapted to deliver a metered volume, typically from 10 to 200 .mu.l, to produce a fine particle spray containing the active ingredient. Suitable propellants include certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation may additionally contain one or more co-solvents, for example, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents. Any propellant may be used in carrying out the present invention, including both chlorofluorocarbon-containing propellants and non-chlorofluorocarbon-containing propellants. Thus, fluorocarbon aerosol propellants that may be employed in carrying out the present invention including fluorocarbon propellants in which all hydrogens are replaced with fluorine, chlorofluorocarbon propellants in which all hydrogens are replaced with chlorine and at least one fluorine, hydrogen-containing fluorocarbon propellants, and hydrogen-containing chlorofluorocarbon propellants. Examples of such propellants include, but are not limited to: CF.sub.3 --CHF--CF.sub.2 H; CF.sub.3 --CH.sub.2 --CF.sub.2 H; CF.sub.3 --CHF--CF.sub.3 ; CF.sub.3 --CH.sub.2 --CF.sub.3 ; CF.sub.3 --CHCl--CF.sub.2 Cl; CF.sub.3 --CHCl--CF.sub.3 ; cy-C(CF.sub.2).sub.3 --CHCl; CF.sub.3 --CHCl--CH.sub.2 Cl; CF.sub.3 --CHF--CF.sub.2 Cl; CF.sub.3 --CHCl--CFHCl; CF.sub.3 --CFCl--CFHCl; CF.sub.3 --CF.sub.2 --CF.sub.2 H; CF.sub.3 --CF.sub.2 --CH.sub.3 ; CF.sub.2 H--CF.sub.2 --CFH.sub.2 ; CF.sub.3 --CF.sub.2 --CFH.sub.2 ; CF.sub.3 --CF.sub.2 --CH.sub.2 Cl; CF.sub.2 H--CF.sub.2 --CH.sub.3 ; CF.sub.2 H--CF.sub.2 --CH.sub.2 Cl; CF.sub.3 --CF.sub.2 --CF.sub.2 --CH.sub.3 ; CF.sub.3 --CF.sub.2 --CF.sub.2 H; CF.sub.3 --CHF--CHF--CF.sub.3 ; CF.sub.3 --O--CF.sub.3 ; CF.sub.3 --O--CF.sub.2 H; CF.sub.2 H--H--O--CF.sub.2 H; CF.sub.2 H--O--CFH.sub.2 ; CF.sub.3 --O--CH.sub.3 ; CF.sub.3 --O--CF.sub.2 --CF.sub.2 H; CF.sub.3 --O--CF.sub.2 --O--CF.sub.3 ; cy--CF.sub.2 --CF.sub.2 --O--CF.sub.2 --; cy--CHF--CF.sub.2 --O--CF.sub.2 --; cy--CH.sub.2 --CF.sub.2 --O--CF.sub.2 --; cy--CF.sub.2 --O--CF.sub.2 --; CF.sub.3 --O--CF.sub.2 --Br; CF.sub.3 --O--CF.sub.2 --Br; and mixtures thereof, where "cy" denotes a cyclic compound in which the end terminal covalent bonds of the structures shown are the same so that the end terminal groups are covalently bonded together. Particularly preferred are hydrofluoroalkanes such as 1,1,1,2-tetrafluoroethane (propellant 134a) and heptafluoropropane (propellant 227). A stabilizer such as a fluoropolymer may optionally be included in formulations of fluorocarbon propellants, such as described in U.S. Pat. No. 5,376,359 to Johnson. Compositions containing respirable dry particles of micronized active compound of the present invention may be prepared by grinding the dry active compound with, e.g., a mortar and pestle or other appropriate grinding device, and then passing the micronized composition through a 400 mesh screen to break up or separate out large agglomerates. The aerosol, whether formed from solid or liquid particles, may be produced by the aerosol generator at a rate of from about 10 to 150 liters per minute. Aerosols containing greater amounts of medicament may be administered more rapidly. Typically, each aerosol may be delivered to the patient for a period from about 30 seconds to about 20 minutes, with a delivery period of about five to ten minutes being preferred. The particulate composition comprising the active compound may optionally contain a carrier which serves to facilitate the formation of an aerosol. A suitable carrier is lactose, which may be blended with the active compound in any suitable ratio. Again, other therapeutic compounds such as amiloride, benzamil or phenamil may also be included. If desired, the active compounds of the present invention may be concurrently administered with uridine 5'-triphosphate (UTP) or an analog thereof (including the pharmaceutically acceptable salts thereof), in an amount effective to stimulate chloride secretion from respiratory epithelial cells (and thereby further hydrate the lung mucus secretions). Formulations containing amiloride, benzamil or phenamil may also contain UTP or an analog thereof in an amount effective to stimulate chloride secretion from respiratory epithelial cells. UTP and analogs thereof that may be used to carry out this technique are disclosed in U.S. Pat. No. 5,292,498 to Boucher. The present invention is explained in greater detail in the Examples which follow. These examples are intended as illustrative of the invention, and are not to be taken as limiting thereof. In the following Examples, UTP and ATP were obtained from Pharmacia (Uppsala, Sweden), and UDP and hexokinase were from Boehringer Mannheim (Indianapolis, Ind.). [.sup.3 H]myo-inositol (20 Ci/mmol) was from ARC (St. Louis, Mo.) and [.sup.3 H]UTP and [.sup.3 H]ATP (97% and 99% pure, respectively) (17-20 Ci/mmol) were from Amersham (Arlington Heights, Ill.). The miniature perfusion chamber described herein was graciously provided by the staff of E. Larsen's laboratory (Zoophysiological Laboratory A, August Krogh Institute, University of Copenhagen, Denmark). Abbreviations used in the following Examples are as follows: .degree.C. means degrees in Centigrade; h means hours; min means minutes; sec means seconds, nm means nanometers; g means grams, ng means nanograms, mg means milligrams, L means liter, mL means milliliter, mmole means millimoles, .mu.mole means micromoles, Ci means curies, .mu.Ci means microcuries; and DMEM means Dulbecco's Modified Eagle's Medium. |
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