MXPA00011495A - Therapeutic dinucleotide and derivatives - Google Patents

Abstract

The present invention relates to P1-(cytidine 5'-)-P4-(uridine 5'-)tetraphosphates and its salts, esters and amides, and formulations thereof which are highly stable and selective agonists of the P2Y2 and/or P2Y4 purinergic receptor. The compounds of the invention are useful in the treatment of chronic obstructive pulmonary diseases such as chronic bronchitis, primary ciliary dyskinesia, cystic fibrosis, as well as prevention of pneumonia due to immobility, and the induction of sputum and its expectoration. Furthermore, because of their general ability to clear retained mucus secretions and stimulate ciliary beat frequency, the compounds of the present invention are also useful in the treatment of sinusitis and otitis media.

Description

THERAPEUTIC DINUCLEOTIDE AND DERIVATIVES INTRODUCTION Technical Field This invention relates to a method for improving the elimination of secretions, by increasing the hydration of retained mucous secretions, stimulating the production of mucins, and increasing the frequency of ciliary rhythm when administering 10 P1- (cytidine 5'-) -P4- (uridine 5 '-) - tetraphosphate (CP4U) or the pharmaceutically acceptable esters, amides or salts thereof.

Background of the Invention Chronic Obstructive Pulmonary Disease (COPD) affects 1.5 million patients in the U.S. and it is the sixth leading cause of death. It is characterized by the retention of mucous secretions in the lungs, which results in progressive lung dysfunction over time. Several patients diagnosed with COPD have a disorder called chronic bronchitis (CB), and 600,000 patients are hospitalized every year due to an acute exacerbation of CB. Cystic fibrosis (CF) and primary ciliary dyskinesia (PCD) are other examples of lung disorders, which assume a clinical profile similar to COPD. Ciliary dyskinesia, either primary or secondary, results in retained secretions that can only be eliminated by coughing. The majority of patients "Jto * afatoAto- •" * - * * • "- * - - - • - - - with COPD use the cough to help eliminate secretions retained due to impaired mucociliary clearance Another disease state characterized by the accumulation of Mucous secretions retained is sinusitis.Sinusitis is an inflammation of the paranasal sinuses typically associated with an upper respiratory infection.It can occur either as a chronic or acute condition.It is the most common complaint of health care in this country, affecting an estimated 31 million people (A. Moss and V. Parsons, National Center for Health Statistics, 1986: 66-7, DHHS Publication No. (PHS) 86-1 588 (1985)) Otitis Media (OM) is a bacterial or viral infection of the middle ear, which primarily affects children under the age of 3. It is usually precipitated by an upper respiratory infection, which spreads to the middle ear through the nasopharynx and eustachian tube. -50 million official visits are made every year for the diagnosis and treatment of OM. At age three, about 75% of children will have had at least one episode of acute OM (J. Klein, Clin. Infect. Dis. 19,823-33 (1994)). Following proper treatment with antibiotics, fluid accumulated in the middle ear remains, causing hearing impairment and delays in cognitive development and potential language. The improved ability to eliminate secretions in the middle ear would reduce or eliminate significant otitis media sequelae. An additional disorder characterized by secretions retained is pneumonia. Patients who are immobilized for a variety of reasons are at high risk of developing pneumonia. Despite the extra vigilance and numerous interventions, pneumonia develops in more than 400,000 patients per year, with 5 mortality and significant morbidity. Patients requiring intubation and mechanical ventilation are at additional risk of ventilator-associated pneumonia (VAP) due to immobility and reduction in mucociliary clearance. The mortality rate for VAP can exceed 50% in more than 100,000 who develop VAP every year. There are also situations where it is therapeutically desirable to increase the drainage of the lacrimal system. When the tear drainage system is not functioning properly, the result can be excessive tearing (epiphora), discharge mucopurulent, and recurrent dacryocystitis. Current treatments for obstruction of the nasolacrimal duct are mainly aggressive surgical procedures, and researchers have tried to discover non-aggressive pharmaceutical treatments. Tear secretion can be stimulated from tissues lacrimal accessories through the mechanisms mediated by the purinergic receptor P2Y2 and / or P2Y similar to those that hydrate the airway epithelium. Dry eye disease is the general term for indications produced by abnormalities of precorneal tear fabric characterized by a reduction in the tear production or an increase in the evaporation of the fabric of < ^ *? JÍ * \ +. This is a tear, along with the ocular surface disease that results.

Currently, the pharmaceutical treatment of dry eye disease is mainly limited to the administration of artificial tears (saline) to temporarily rehydrate the eyes. However, the relief is short and frequent dose is necessary. Normally, mucous secretions are removed through the mucociliary clearance system (MCC). The MCC depends on the integrated action of three components: 1) mucous secretion by cup cells and submucosal glands; 2) the movement of the cilia in epithelial cells, which propel the mucus through the luminal surface; and 3) the transport of ion to and from the luminal epithelial cells that concomitantly controls the flow of water in the mucus. It is now known that nucleoside phosphates such as uridine 5'-triphosphate (UTP) modulate all the components of the MCC system. First, UTP has been shown to increase both the rate and total amount of mucin secretion by in vitro cup cells (M. Lethem et al., Amm J. Respir, Cell Mol. Biol. 9, 315-22. (1993)). Second, it has been shown that UTP increases the rate of cilia rhythm in airway epithelial cells in vitro (D. Drutz, er al., Drug Dev. Res. 37 (3), 185 (1996)). And third, it has been shown that UTP increases the secretion of Cl \ and therefore, the secretion of water from airway epithelial cells in vitro (S. Mason, et al., Br. J. Pharmacol., 103, 1649 -56 (1991)). In addition, it has been thought that ^^ MfeaÉÉíkáÜli-i release of the surfactant of Type II alveolar cells in response to UTP (Gobran, Am. J. Physiol. 267, L625-L633 (1 994)) contributes to the optimal functioning of the lungs and can help in maximizing MCC. It has been shown that UTP increases intracellular Ca ++ due to the stimulation of phospholipase C by the P2Y2 receptor (H. Brown, et al., Mol.Pharmacol. 40, 648-55 (1991)). The modulation of UTP's of all components of the mucociliary scaler system results in the improvement in mucociliary clearance of the lung in normal volunteers without any significant side effect (K. Olivier, et al., Am. J. Respir. Crit. Care Med. 154, 217-23 (1996)). In addition, UTP significantly improves the elimination of cough (elimination of secretions retained when coughing) in patients with PCD (P. Noone, et al., Am. J. Respir. Crit. Care Med 1 53, A530 (1996)). The dinucleotide, P \ P4-di (uridine-5 '-) tetraphosphate, has also been shown to increase sputum production in normal healthy volunteers, indicating an improvement of MCC. Due to the demonstrated ability of UTP's to increase the elimination of retained mucous secretions, applicants were motivated to investigate other nucleoside phosphates in order to maintain or improve therapeutic efficacy while increasing stability. During the course of this investigation, it was found that CP U different from the other dinucleoside tetraphosphate containing C2P cytidine possesses surprising potency at the P2Y2 and P2Y4 receptors. Additionally, it was observed that CP U has considerable and unexpectedly improved resistance towards degradation to . . .. »^ > . - .-. ..,. . ^ -. . . -, +. ~. ..... »... .-. ^ -,. .. ... . .-,. -. ......... biological, as evidenced by its stability in biological preparations. The present invention is based on the potency and increased duration of action of CP U in respiratory therapies due to its increased biological stability.

SUMMARY OF THE INVENTION A method is described for improving the elimination of secretion by hydrating the mucosa and increasing the frequency of ciliary rhythm in a subject in need of such treatment. The method comprises administering to the patient a compound of Formula I: Formula I 0 amides, esters or pharmaceutically acceptable salts thereof. The new amides and esters of the compound of the Formula 1 are also described. The compounds of Formula I are selective and highly stable agonists of the purinergic receptor P2Y2 and / or P2Y4; thus, they are useful in the treatment of chronic obstructive pulmonary diseases such as chronic bronchitis, PCD, and cystic fibrosis; They are also useful in the treatment of immobilized patients, who are at risk of developing pneumonia.

In addition, because of their general ability to clear retained mucous secretions and stimulate ciliary rhythm frequency, the compounds of the present invention are useful in the treatment of sinusitis and otitis media. The compounds of Formula I are also useful for facilitating the sputum of a sputum specimen for diagnostic purposes in patients at risk of lung cancer, pneumonia, and other infectious diseases. Additionally, it is postulated that the compounds of Formula I could be an adjunct in the treatment of asthma. They could also improve the performance of athletes by increasing the elimination of mucous secretions from the lungs. The compounds of Formula I may also be useful for wound healing.

DETAILED DESCRIPTION OF THE INVENTION The invention provides a method for eliminating retained mucosal secretion and improving ciliary rhythm frequency in a subject in need of such treatment. The method comprises administering to the patient a compound of Formula I. Formula I or amides, esters or pharmaceutically acceptable salts thereof.

The compounds of Formula I, including their salts, esters and amides, will be referred to hereafter as "the compounds of the present invention". The compounds of the present invention comprise pharmaceutically acceptable salts, such as, but not limited to, an alkali metal salt such as sodium or potassium; an alkaline earth metal salt such as magnesium or calcium; manganese; or a tetraalkyl ammonium or ammonium salt, ie, NX4 + (wherein X is C1-4 alkyl). Pharmaceutically acceptable salts are salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects. The salts of the present invention comprise mono-, di-, tri- and tetra- cations, which may contain a single cation or mixed cations. The present invention also provides prodrugs new acylated (e.g., esters and amides) of the compound described herein. Preferred esters of the invention include carboxylic acid esters, in which the carbonyl-free moiety of the ester grouping is selected from straight or branched chain alkyl such as, n-propyl, t-butyl; n-butyl, Alkoxyalkyl (for example methoxymethyl), aralkyl (for example, benzyl), aryloxyalkyl (for example, phenoxymethyl), and aryl (for example, phenyl); sulfonate esters such as alkyl- or aralkylsulfonyl (for example, methanesulfonyl); amino acid esters (for example, L-valyl or l-isoleucyl); acid esters dicarboxylic (for example, hemisuccinate). Phosphate esters ^ s ^^ wgtitejg can also be etherified by, for example, a C1-20 alcohol or by a glycerol of 2,3-di (C6-2) acyl. Any alkyl moiety present in such esters contains from 1 to 18 carbon atoms, particularly from 1 to 4 carbon atoms. Alkyl groups containing 3-1 8 carbon atoms may be saturated or unsaturated. Any aryl moiety present in such esters advantageously comprises an optionally substituted phenyl group, for example, by halogen, C 1-4 alkyl, C 1-4 alkoxy, nitro, or hydroxyl. The above-mentioned pharmaceutically acceptable amides of the invention include those derivatives wherein the amino group of cytosine is in the form of an amide, for example, NHCOR wherein R is C 1-6 alkyl or aryl (e.g., phenyl optionally substituted by halogen, C? -4 alkyl, C 1 alkoxy, nitro or hydroxyl). The compounds of the present invention are highly selective agonists of the purinergic receptor P2Y2 and P2Y4; thus, they are useful in the treatment of mammals including humans suffering from chronic obstructive pulmonary diseases such as chronic bronchitis, acute bronchitis, acute exacerbations of chronic bronchitis, PCD, cystic fibrosis, as well as the prevention of pneumonia due to immobility. In cases where the cilia are impaired or absent, the compounds of the present invention improve cough elimination. In addition, because of their general ability to eliminate retained mucous secretions and stimulate ciliary rhythm frequency, the compounds of the present invention are useful in the treatment of chronic and acute sinusitis and otitis media in mammals, including humans. By improving secretion elimination, the compounds are useful as protection before or after exposure to inhaled biological conflict agents. They can also be useful to improve lung representation by removing secretions from the lungs before obtaining the image. Due to their surprisingly increased biological stability, the compounds of the present invention provide increased duration of action for respiratory therapies. This improvement in stability offers advantages in the treatment of both chronic and acute respiratory disorders. The esters and amides of the compound of Formula I are useful for treating ophthalmic disorders such as dry eye and retinal detachment. They are also useful to increase drainage of the lacrimal system. Although the compounds of the present invention are primarily concerned with the treatment of human subjects, they can also be used for the treatment of other mammalian subjects such as dogs, cats and horses for veterinary purposes. The compounds of the present invention can be administered orally, topically, parenterally, by inhalation or spray, intra-operatively, rectally, or vaginally in single dose formulations containing conventional, non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term "topically" as used herein includes patches, gels, creams, ointments, liquid irrigation, or drops for the ears, eyes or nose. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular injections, intrasternal injection or infusion techniques. In another aspect of the invention, a pharmaceutical formulation comprising a compound of the present invention and a pharmaceutically acceptable carrier is provided. One or more compounds of the invention may be presented in association with one or more pharmaceutically acceptable non-toxic diluents or adjuvants or carriers and, if desired, other active ingredients. Such a carrier could be sugars, where the compounds can be intimately incorporated into the matrix through glazing or simply mixed with the carrier (eg, lactose, sucrose, trehalose, mannitol) or other excipients acceptable for respiratory or pulmonary delivery. The compounds of the present invention can be administered separately or together with mucolytics such as DNAse, or acetylcysteine, or with radiolabelled substances. The pharmaceutical compositions containing a compound of the present invention can be in a form suitable for oral use, for example, as tablets, lozenges, oily or aqueous suspensions, dispersible granules or powders, emulsion, hard or soft capsules, or syrups or elixirs. Compositions proposed for oral use can be prepared according to any method known for the subject for the manufacture of pharmaceutical compositions and such compositions can contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preservatives for the purpose of to provide pharmaceutically elegant and palatable preparations. The tablets contain the active ingredient in admixture with pharmaceutically acceptable non-toxic excipients, which are suitable for the manufacture of tablets. These excipients may be, for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate.; granulating agents and disintegrating agents, for example, corn starch, or alginic acid; binders, for example, starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may not be coated or may be coated by known techniques to delay disintegration or absorption in the gastrointestinal tract and thus provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed. Formulations for oral use can be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mix with water or a medium of oil, for example, peanut oil, liquid paraffin or olive oil. The aqueous formulations contain the active materials in admixture with excipients suitable for manufacture. Such excipients are dispersing agents, for example: sodium carboxymethylcellulose, methylcellulose and sodium alginate. The dispersing or wetting agents can be a naturally occurring phosphatide or condensation products of an oxide of allyl with fatty acids, or condensation products of ethylene oxide with long-chain aliphatic alcohols, or condensation products of ethylene oxide with partial fatty acid esters and hexitol, or condensation products of ethylene oxide with divided partial esters of fatty acids and hexitol anhydrides.

Those skilled in the art will recognize the various specific excipients and wetting agents comprised by the general description above. The aqueous formulations may also contain one or more preservatives, for example, ethyl, or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more agents flavors, and one or more sweetening agents, such as sucrose or saccharin. Dispersible granules and powders suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with an agent dispersant or humectant, dispersing agent and one or more A ^ ásfaa * ^ n ^ á ^ j & s'ife ^ tfe ^?. ^ Condoms. Suitable dispersants or humectants and dispersing agents are exemplified by those already mentioned above. Additional excipients, for example, sweeteners, flavors, and coloring agents, may also be present. The compounds of the present invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle. Advantageously, adjuvants such as local anesthetics, condoms and tampons can dissolve in the vehicle. The sterile injectable preparation can be a sterile injectable solution or suspension in a non-toxic parentally acceptable solvent or diluent. Among the vehicles and acceptable solvents that can be used are: sterile water, saline, or Ringer's solution. The compounds of the present invention can also be administered in the form of suppositories for administration by the ear, rectal or vaginal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient, which is solid at ordinary temperatures but liquid at body temperature and therefore melt in the body to release the drug. Such materials are cocoa butter and polyethylene glycols. The solutions of compounds of the present invention can be administered by intra-operative installation, using such methods such as irrigation, washing, topical application, injection or other method known to those skilled in the art. For the treatment of respiratory disorders, concentration levels of from about 10"7 M to about 10" 1 M, preferably 1 0"5 to 10 ~ 3 m, or doses of about 1 -400 mg can be used. Ophthalmic concentrations of 0.1 to 10.0% may be used The amount of active ingredient that can be combined with the carrier materials to produce a single dose form will vary depending on the host treated and the particular mode of administration. the specific dose level for any particular patient will depend on a variety of factors including the activity of the specific compound, age, body weight, general health, sex, diet, time of administration, route of administration, and rate of absorption, distribution, metabolism and excretion, drugs used in combination, and the type and severity of the recovery therapy of the particular disease The compounds comprised by the present invention can be prepared by condensation of uridine mono-, di-, or triphosphate, activated with a condensing agent such as, but not limited to, carbonyldiimidazole or dicyclohexylcarbodiimide, with mono-, di-, or triphosphate. of cytidine to form the desired dinucleotide tetraphosphate (Pi-iditidin 5 '-) P4- (uridine 5' -) tetraphosphate); or by condensation of a mono-, di-, or cytidine triphosphate similarly activated with mono-, di-, or uridine triphosphate. The - ** »analogous procedures produce N4-amido derivatives of (Pi- (cytidine 5 '-) P4- (uridine 5' -) tetraphosphate The nucleoside phosphates used as starting materials may be commercially available (Sigma) or made the corresponding nucleosides by methods well known to those skilled in the art Similarly, where nucleosides are not commercially available, they can be made by modification of other readily available nucleosides, or by synthesis of carbohydrate and heterocyclic precursors by methods by methods well known to those skilled in the art, those skilled in the art will recognize the additional synthetic methodologies, which may be employed to prepare pharmaceutically acceptable salts and acylated prodrugs of the compound of Formula I. In this manner, the salts can be prepared using resins of exchange of cation, esters and amides, for example, they can be made by reaction of the desired amino or hydroxy compound with the appropriate acid, activated with carbonyldiimidazole, dicyclohexylcarbodiimide or other suitable condensing agent, or with an acid anhydride or acid chloride with or without a basic catalyst such as a tertiary amine, salt of quaternary ammonium or other inorganic base. Those having experience in the art will recognize that the starting materials can be varied and additional steps used to produce the compounds comprised by the present invention, as demonstrated by the following examples.

In some cases the protection of certain reactive functionalities may be necessary to achieve some of the above transformations. In general, the need for such a protection group will be apparent to those skilled in the art of organic synthesis as well as the conditions necessary to incorporate and remove such groups. The invention is further illustrated by the following examples, which are not to be construed as limiting the invention in scope or spirit to the specific procedures described therein. EXAMPLE 1 Preparation of tetramonium salt, P1- (Citidine 5 '-) - P4- (uridine 5'-) tetraphosphate The tributylamine salt of uridine 5'-monosphate, was prepared by dissolving uridine 5'-monosphate, acid free (Sigma) (3.0g) with tributylamine (2.0 mL) in DMF to make a solution of 0.34 M.

An anhydrous DMF solution of tributylamine salt of uridine 5'-monosphate (5.6 ml 1.89 mmol, 0.34 M) was added to a 10 ml round base flask under N2 and carbonyldiimidazole (459 mg, 2.83 mmol) was added. ) and the solution was stirred at 25 ° C for 30 minutes. A tributylamine salt DMF solution of cytidine 5'-triphosphate, prepared by treating the trisodium salt with resin Dowex 50H4 followed by tributylamine in DMF was added and the reaction mixture was stirred at 65 ° C for 3 hours. The solution was evaporated in vacuo and purified twice by column chromatography (DEAE Sephadex: gradient of H20? 0.3M NH4HCO3). The pure fractions were concentrated in vacuo at 35 ° C, and H20 was added and re-evaporated ten times to obtain a white solid (203 mg): 1 H NMR (D 2 O, TMS) d 4.1 (m, br, 6H), 4.2 (m, 4H), 5.8 (m, 3H), 5.95 (m, 1 H), 5 7.7 (m, 2H): 31 P NMR (D2O, H3PO4 std) d -22.4 (m, 2P), - 10.6 (m, 2P); exact mass for C18H26N5? 22P4 (M-H) = 788.0020, found 787.9985. Example 2 Preparation of tetramonium salt, P1- (Cytidine 5 '-) - P4- (uridine 5'-10) tetraphosphate A solution of trisodium salt (5.86 g, 0 01 mol) of uridine 5'-triphosphate (UTP) ) in water (5mL) was placed in a column of strong cation exchange resin of BioRad AG-MP 50 in its pyridinium form (50 mL volume by mass) and eluted with water distilled (approximately 300 mL) in a flask containing tributylamine (5.55 g 0.03 mol). The suspension was stirred with ethanol (50 mL) and the mixture remained beside the refrigerator overnight. The solution was filtered to remove a little oily residue, evaporated to dryness under reduced pressure, and the residue was dried by 1 hour at 0.08 mm Hg at room temperature. The residue was dried by evaporation with 2 x 20 mL of anhydrous dimethylformamide (DMF) at 0.1 mm Hg. The anhydrous tributylamine salt was made up to 100 mL with anhydrous acetone to produce a concentrated solution (0.1 M in UTP). Dicyclohexylcarbodiimide (DCC) (Baker, 1.0 g, 5.0 mmol) was added to an aliquot of the previous UTP solution (10 mL, 1 mmol) ^ g * gjte * g ** j * and the solution was stirred at room temperature for 30 minutes. The deposited dicyclohexylurea was removed by filtration, the reaction mixture was extracted with hexane (100 mL), and the residue was dissolved in dry deuterated dimethylsulfoxide (DMSO-d6, 3.0 mL). This solution of 5'-cyclic uridine metaphosphate (UcTP) was added to the tributylamine salt of 5'-cytidine monophosphate (CMP), prepared by the addition of tribulamine (0.714 mL, 3 mmol) to CMP-free acid ( Sigma, 0.65 g, 2 mmol), and the suspension was stirred at 50 ° C for 24 hours. The reaction mixture was evaporated under high vacuum overnight, the residue was dissolved in water (5 mL) and separated by semipreparative ion exchange chromatography (Hamilton PRP X-100 column, eluting with 1.0 M sodium bicarbonate). Socratic ammonium, 8 mL / min 30 min, multiple injections of 500 μL). The dinucleotide tetraphosphate eluted between 31 and 37 min: fractions containing the product were repeatedly evaporated with water and the lyophilized residue to yield the main compound (59 mg) as a white solid. 1 H NMR D 2 O, d ppm tetramethylsilane: 4.10-4.13 (m, 6H) 4.17-4.26 (m, 4H); 5.81 5 (d, J = 7.8 Hz, 1 H); 5.82 (d, J = 5.0 Hz, 2H); 6,082 (d, J = 7.7 Hz, 1 H); 7.80 (d, J = 7.8 Hz, 1 H); 7.91 (d, J = 8.2 Hz, 1 H). 31 P NMR (D 2 O d of H 3 PO 4): -22.45 (m, 2P); -10.80 (m, 2P). Example 3 Pharmacological Activity As Measured by Inositol Phosphate Analysis The pharmaceutical utility of the compounds of this invention is indicated by the analysis of inositol phosphate for P2Y2 receptor activity and another P2Y. This widely used analysis, as described in E. Lazarowski, et al., Brit. J. Pharm. 1 16, 1619-27 (1995), depends on the measurement of inositol phosphate formation as a measurement of activity of the compounds that activate receptors bound through G-proteins to phospholipase C. The compounds of Formula I are tested for their ability to produce P2YL receptor activity P2Y2, P2Y and P2Y6 using the inositol phosphate assay as described by E. Lazarowski, et al., Brit. J. Pharm. 16, 1619-27 (1995). The results for CP U and all the cytidine containing dinucleotide, C2P4 are summarized in Table I below. Table I: Activity Summary * IA = Inactive (ie, without antagonist activity at 30 μM) In this analysis, CP4U showed surprising activity at the P2Y2 and P2Y4 receptors, compared to the whole cytidine dinucleotide analogue C2P4 Example 4 Induction of chloride secretion in vivo facilitates hydration of mucous airway secretions thickened in diseases, where patients will benefit from mobilization and elimination of such secretions. The activation of an apical chloride channel without CFTR induces the spillage of chloride ions and water that helps rehydrate the lung secretions (Boucher, U.S. Patent 5,292,498 and Boucher, et al, U.S. Patent 5,635, 160 and references in the I presented). Chloride Secretion in Human Nasal Respiratory Tract Cells Airway epithelial cells were segregated and isolated from freshly excised human nasal surgical specimens (Yankaskas, et al., Am. Rev. Respir. Dis. 132, 1281-1287 (1985) ). The confluent monolayers were grown on permeable collagen matrix supports in a medium supplemented with hormone F-12 (Wu, et al., Am. Rev. Respir Dis. 1 32, 3 1 1 -320 (1985)). The cells were incubated at 37 ° C and grew to confluence. The development of transepithelial resistance was monitored to determine the formation of tight junctions between the cells. After the formation of tight joints was confirmed, the matrix supports containing the crops were mounted in modified use chambers. The cultured human respiratory tract epithelium was mounted in use chambers with a submucosal Ringer bath of Krebs bicarbonate [KBR (in mM) 140 Na +, 120 CI "5.2 K +, 25 HCO3", 2.4 HPO -4, 0.4 HPO4- , 1 .1 Ca2 +, 1.2 Mg2 +, and 5.2 glucose]. The luminal surface is washed by KBr or by a high K +, CI "low (HKLC) Ringers [(in mM)] 40 Na +, 100 K", 4.5 Cl \ 120 gluconate, 25 HCO3", l £ < * ij ^ 2.4 HPO2J 0.4 HPO4 \ 1 .1 Ca2 +, 1.2 Mg2 +, and 5.2 glucose]. Polycarbonate utilization chambers were milled to fit the plastic suction cups that support the permeable collagen matrix, in which the cells grew. The bioelectric properties including Isc, transepithelial potential difference and resistance were monitored. The short circuit current (Isc) was measured with a digital voltmeter (UNC Electronics, Chapel Hill, NC) and marked on a band diagram recorder. The open circuit potential was recorded periodically, and the conductance was monitored in the voltage field mode by the current displacement in response to a voltage pulse of 10-mV. A stable baseline of / Sc was recorded and amiloride (100 μM) was added to the solution by bathing the apical surface to block the absorption of sodium. The residual lSc measured under these conditions was a good approximation of chloride secretion (Boucher, et al., J. Clin Invest. 78, 1245-1252 (1986); Willumsen, et al., Am. J. Physiol. 256, C226-233, C1 033-C1044, C1045-C1053 (1989)). After recording a stable baseline, a solution of the test compound was added to the chamber by bathing the apical surface of the apical culture. The change in lSc was recorded. The concentration response curves were obtained by the cumulative addition of high concentrations of the test compound in stages of logarithm 0.5. Figure I of Example 4 shows the change in chloride ion diffusion potentials (? SSc, change in short circuit potential) in human nasal epithelial cells when U2P4 or CP4U was added to the cell surface medium. The results show that CP4U is approximately ten times more potent than all the dinucleotide containing uridine, U2P in the chloride secretion analysis. FIGURE I [NUCLEOTIDO] EXAMPLE 5 Metabolic Stability as Measured by Incubation in Human Respiratory Tract Cells The compound of Formula I (CP U) was tested for its ability to resist metabolism by the enzymes present in the surface of bronchial epithelial cells (airway) - ^ the »- ^. cultured human and the results were compared to the dinucleotide U2P4 (P1, P4-Di (uridine 5 '-) tetraphosphate) and the nucleoside triphosphate, UTP (5'-uridine triphosphate). The epithelial cells grew as monolayers in an air-liquid interface in ALI medium. The analyzes were conducted in completely differentiated cells in the KBR-Ringer solution (pH 7.2) at 37 ° C with 0.1 mM nucleotide substrate. The aliquots of the airway surface fluid were removed at 5, 10, 15, 20, 40 and 60 minutes. The metabolism was stopped by cooking and filtering the aliquots, and the samples were analyzed by high performance liquid chromatography using the following conditions: column C-18 with a 55 min gradient of 1-0-100 mM KH2PO4 and constant 8 mM tetrabutyl ammonium hydrogen sulfate and 10% methanol). The results are presented in Figure II. The compound of Formula I, CP4U, showed unexpected stability in comparison to the other nucleotides, UTP and U2P. The CP4U was not metabolized for a period of 60 minutes, while the UTP and U2P4 were degraded by 1 00 and 40%, respectively.

FIGURE II Comparative rates of hydrolysis of CP4U, U2P4 and UTP by human bronchial epithelial cells -10 10 20 20 40 50 60 70 Time (inin) The compound that is the subject of the invention was tested in an animal model (L. Allegra, ef. Al., J. Applied Physiol., 55 (3) 726- 730 (1983), JR Sabater, ef. Al., Am. Respir. Crit. Care Med., 154, 341-345 (1996)). The nebulized compound stimulated the tracheal mucosal velocity (TMV) when compared to the vehicle control. TMV is a measure of mucociliary clearance in a single long airway. The invention, and the way and the process to elaborate and use it, now they are described in such exact, concise, clear and comp terms to allow any expert in the material, to which he belongs, to elaborate and use it. It is to be understood that the foregoing describes the preferred embodiments of the present invention and that the modifications may be made herein without departing from the spirit or scope of the present invention as set forth in the claims. To particularly point out and distinctly claim the subject subject observed as an invention, the following claims conclude this specification.

Claims (10)

1. CLAIMS 1. A method for improving secretion elimination and improving ciliary rhythm frequency in a mammal in need of such treatment, by administering to said mammal a compound of Formula I: Formula I or amides, esters and pharmaceutically acceptable salts thereof.
2. 2. A method for treating chronic obstructive pulmonary diseases in a mammal, by administering an effective amount of a compound of Formula I as described in claim 1 to improve removal and elimination of secretion.
3. 3. A method for treating chronic and acute sinusitis in a mammal, by administering an effective secretion elimination amount of a compound of Formula I as described in claim 1.
4. 4. A method for facilitating the sputum of a sputum specimen. deep in a mammal, by administering an amount of a compound of Formula I as described in claim 1, effective to facilitate the expectoration of a sample. i & *, *. . a fc. * "- & >
5. 5. A method for improving cough elimination by administering an effective amount of a compound of Formula I as described in claim 1.
6. 6. A method for improving lung representation by administering an effective amount of a compound of the Formula I as described in claim 1, effective to eliminate the secretions of the lungs before obtaining the image.
7. 7. A method for preventing pneumonia by administering an amount of a compound of Formula I effective to improve secretion elimination.
8. 8. A method for treating otitis media, by administering an effective amount of a compound of Formula I as described in claim 1.
9. 9. A method for improving the elimination of biologically disturbed respiratory agents, by administering an effective amount of a compound of Formula I as described in claim 1.
10. 10. A method for treating dry eye disease by administering an effective amount of an ester or amide of Formula I. 1 1. A method for treating retinal detachment, by administering an effective amount of an ester or amide of Formula I. 12. A pharmaceutical composition comprising a compound of Formula I, as described in claim 1 or a salt, ester. or pharmaceutically acceptable amide thereof together with a pharmaceutically acceptable carrier thereof. 13. An amide of the compound of Formula I according to claim 1 selected from the group consisting of derivatives characterized in that the amino group of cytosine is NHCOR wherein R is C? -6 alkyl, phenyl or phenyl substituted by halogen, C1-alkyl , C1- alkoxy, nitro or hydroxyl. 14. An ester of the compound of formula I according to claim 1 selected from the group consisting of n-propyl, t-butyl, methoxymethyl, benzyl, phenoxymethyl, phenyl, methanesulfonyl, L-valyl, L-isoleucyl or hemisuccinate. 15. Salt of tetramonium of P1- (Citidine-5 '-) P4- (uridine 5' -) tetraphosphate.