chemically and metabolically stable dipeptide having potent sodium channel blocking activity

专利摘要:
CHEMICALLY AND METABOLICALLY STABLE DIPEPTIDE WITH POWERFUL SODIUM CHANNELING ACTIVITY. The invention relates to a very stable, selective and kidney-safe sodium channel blocker represented by the formula: (I) The invention also includes a variety of compositions, combinations and treatment methods using this sodium channel blocker inventive.
公开号:BR112013032771B1
申请号:R112013032771-5
申请日:2012-06-27
公开日:2021-01-12
发明作者:Michael Ross Johnson
申请人:Parion Sciences, Inc.；
IPC主号:

专利说明:

[0001] The present application claims priority for the application US Series No. 61 / 501,524, filed on June 27, 2011, the full text of which is hereby incorporated by reference. Field of the Invention
[0002] The present invention relates to the 3,5-diamino-6-chloro-N- (N- (4- (4 - ((S) -3- (dimethylamino) -4 - ((S ) -1- (dimethylamino) -6-guanidino-1-oxo-hexan-2-ylamino) -4-oxobutyl) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide (I). The present invention also includes a variety of treatment methods using this inventive sodium channel blocker. The present invention also relates to new compounds for the treatment of dry eye, particularly including 3,5-diamino-6-chloro-N- (N- (4- (4 - ((S) -3- (dimethylamino) - 4 - ((S) -1 - (dimethylamino) -6-guanidino-1-oxo-hexan-2-ylamino) -4-oxobutyl) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide (I) and their forms of pharmaceutically acceptable salt, useful as sodium channel blockers, compositions containing them, therapeutic methods and uses for them and processes for preparing them. Description of the Prior Art
[0003] The mucous surfaces at the interface between the environment and the body have developed innumerable “innate defenses”, that is, protection mechanisms. A primary form of such innate defense is to clean these surfaces with a liquid. Typically, the amount of the liquid layer on a mucous surface reflects the balance between epithelial fluid secretion, often reflecting the secretion of anions (C1- and / or HCO3-) coupled with water (and a cationic counterion), and the epithelial fluid absorption, often reflecting Na + absorption, coupled with water and a counter-anion (C1- and / or HCO3-). Many diseases of the mucous surfaces are caused by too little protective fluid on those mucous surfaces created by an imbalance between secretion (too little) and absorption (relatively too much). The defective salt transport processes that characterize these mucous dysfunctions reside in the epithelial layer of the mucosal surface.
[0004] One approach to replacing the protective liquid layer on mucous surfaces is to "rebalance" the system by blocking the Na + channel and liquid absorption. The epithelial protein that mediates the limiting step of the Na + and liquid absorption rate is the epithelial Na + channel (ENaC). ENaC is located on the apical surface of the epithelium, that is, the mucosal surface-environment interface. Therefore, to inhibit ENaC-mediated Na + and liquid absorption, an ENaC blocker of the amiloride class (which blocks from the extracellular domain of ENaC) must be distributed to the mucosal surface and, more importantly, be maintained in this site, to have therapeutic utility. The present invention describes diseases characterized by very little fluid on mucous surfaces and "topical" sodium channel blockers designed to exhibit the increased potency, reduced mucosal absorption, and the slow dissociation ("shutdown" or detachment) of the ENaC required for therapy of these diseases.
[0005] Chronic obstructive pulmonary diseases are characterized by dehydration of airway surfaces and the retention of mucus secretions in the lungs. Examples of such diseases include cystic fibrosis, chronic bronchitis, and primary or secondary ciliary dyskinesia. Such diseases affect approximately 15 million patients in the United States, they are the sixth leading cause of death. Other diseases of the airways or lungs characterized by the accumulation of retained mucus secretions include sinusitis (an inflammation of the paranasal sinuses associated with upper respiratory infection) and pneumonia.
[0006] Chronic bronchitis (CB), including the most common lethal genetic form of chronic bronchitis, cystic fibrosis (CF), are diseases that reflect the body's failure to clear mucus normally from the lungs, which essentially produces a chronic airway infection. In the normal lung, the primary defense against chronic infection of the intrapulmonary pathways (chronic bronchitis) is mediated by the continuous clearance of mucus from the surfaces of the bronchial airways. This health function effectively removes potentially harmful toxins and pathogens from the lung. Recent data indicate that the initial problem, that is, the “basic defect”, both in CB and CF, is the failure to clear mucus from the surfaces of the airways. The failure to clear mucus reflects an imbalance between the amount of fluid and mucin on the surfaces of the airways. This "liquid on the surfaces of the airways" (ASL) is mainly composed of salt and water in proportions similar to that of plasma (ie, isotonic). Mucin macromolecules organize themselves into a well-defined "mucus layer" that normally traps inhaled bacteria and is transported out of the lung via the actions of the cilia that touch a low-viscosity aqueous solution called "periciliary fluid" (PCL) . In the disease state, there is an imbalance in the amounts of mucus like ASL on the surfaces of the airways. This results in a relative reduction in ASL that leads to mucus concentration, a reduction in the lubricating activity of PCL, and a failure to clear mucus via ciliary activity to the mouth. The reduction in mechanical mucus clearance from the lung leads to chronic bacterial colonization of mucus adhering to airway surfaces. It is the chronic retention of bacteria, the lack of local antimicrobial substances to eliminate bacteria trapped in the mucus on a chronic basis, and the consequent chronic inflammatory responses of the body to this type of superficial infection that lead to CB and CF syndromes.
[0007] The current population affected in the United States is 12,000,000 patients with the acquired form (mainly from exposure to cigarette smoke) of chronic bronchitis and approximately 30,000 patients with the genetic form of cystic fibrosis. Approximately equal numbers of both populations are present in Europe. In Asia, there are few cases of CF but the incidence of CB is high and, as in the rest of the world, it is growing.
[0008] There is currently a great unmet medical need for products that specifically treat CB and CF at the level of the basic defect that causes these diseases. Current therapies for chronic bronchitis and cystic fibrosis focus on treating symptoms and / or the late effects of these diseases. Therefore, for chronic bronchitis, β-agonists, inhaled steroids, anticholinergic agents, and oral theo-phyllins and phosphodiesterase inhibitors are all in the development phase. However, none of these drugs effectively addresses the fundamental problem of failure to clear mucus from the lung. Similarly, in cystic fibrosis, the same spectrum of pharmacological agents is used. These strategies were complemented by more recent strategies designed to purify the CF lung of DNA ("Pulmozyme"; Genentech) that was deposited in the lung by neutrophils who tried unsuccessfully to eliminate the bacteria that grew in adherent mucus masses and through the use of inhaled antibiotics. ("TOBI") designed to increase the lung's own extermination mechanisms to get rid of mucus plaques adhering with bacteria. A general principle of the body is that if the initial lesion is not treated, in this case mucus retention / obstruction, bacterial infections become chronic and increasingly refractory to antimicrobial therapy. Therefore, an important unmet therapeutic need for pulmonary diseases CB and CF is an effective means of rehydrating airway mucus (ie, restoring / expanding the volume of ASL) and promoting its clearance, with bacteria, from the lung.
[0009] R.C. Boucher, in US 6,264,975, describes the use of sodium channel blockers to itpo pyrazinoylguanidine to hydrate mucous surfaces. These compounds, exemplified by the well-known diuretics amiloride, benzamyl, and phenamyl, are effective. However, these compounds suffer from the significant disadvantage that they are (1) relatively powerless, which is important because the mass of drug that can be inhaled by the lung is limited; (2) rapidly absorbed, which limits the drug's half-life on the mucosal surface; and (3) are freely dissociable from ENaC. The sum of these disadvantages incorporated in these well-known diuretics produces compounds with insufficient potency and / or effective half-life on mucous surfaces to have therapeutic benefit to hydrate mucous surfaces.
[0010] R. C. Boucher, in US Patent No. 6,926,911, suggests the use of relatively impotent sodium channel blockers such as amylo-ride, with osmolytes for the treatment of airway diseases. This combination does not offer any practical advantage over any of the treatments alone and is not clinically useful, see Donaldson et al., N Eng J Med., 2006; 353: 241-250. Amiloride has been found to block airway permeability to water and nullify the potential benefit of concomitant use of hypertonic saline and amiloride.
[0011] US Patent No. 5,817,028 to Anderson describes a method for causing airway narrowing (to assess susceptibility to asthma) and / or inducing sputum in individuals via inhalation of mannitol. It has been suggested that the same technique can be used to induce sputum and promote mucociliary clearance. Suggested substances include sodium chloride, potassium chloride, mannitol and dextrose.
[0012] Clearly, what is needed are drugs that are more effective in restoring mucus clearance from the lungs of patients with CB / CF. The value of these new therapies will be reflected in improvements in the quality and life span of populations with CF and with CB.
[0013] Other mucous surfaces both in and on the body exhibit subtle differences in the normal physiology of protective surface fluids on their surfaces but the pathophysiology of the disease reflects a common theme, that is, very little protective surface fluid. For example, in xerostomia (dry mouth) the oral cavity is depleted of fluid due to a failure of the sublingual and submandibular parotid glands to secrete fluid despite the continuous fluid absorption mediated by Na + transport (ENaC) from the oral cavity.
[0014] Similarly, dry keratoconjunctivitis (dry eye) is caused by the failure of the lacrimal glands to secrete liquid on the face of continuous Na + -dependent liquid absorption on conjunctival surfaces. Dry keratoconjunctivitis (KCS) or chronic dry eye disease (DED) is one of the most frequently diagnosed ophthalmic diseases, resulting in painful irritation, inflammation of the eye surface, and impaired vision. KCS / DED results from inadequate watery tear fluid in the eyes. The dry eye is one of the most frequently diagnosed ophthalmic diseases affecting more than 5 million people in the United States alone. The dry eye is a multifactorial disease, which results from a common etiology of insufficient tear film causing damage to the ocular surface and symptoms of ocular discomfort. The few therapies currently available, which include immunosuppressive agents and over-the-counter tear replacement, are not effective enough for many users or provide only temporary relief from the symptoms of dry eye. Therefore, the development of new agents to treat dry eye would bring tremendous benefit to the therapeutic environment. The volume of tear film on the ocular surface represents a balance between the production of tear fluid versus the loss of fluid via drainage, evaporation, or epithelial absorption. Similar to other epithelial tissues, the conjunctiva and corneal epithelium are able to regulate the hydration status of the mucosal surface by transporting active salt and water. The epithelial sodium channel (ENaC) is an essential regulator of sodium (and water) absorption in tissue numbers, including the eye. It is believed that inhibition of ENaC in the eye preserves tear secretions and maintains hydration of the ocular surface.
[0015] In rhinosinusitis, there is an imbalance, as in CB, between mucin secretion and relative depletion of ASL. Finally, in the gastrointestinal tract, failure to secrete C1- (and liquid) in the proximal small intestine, combined with increased absorption of Na + (and liquid) in the terminal ileum leads to distal intestinal obstruction syndrome (DIOS). In older patients, excessive Na + absorption (and volume) in the descending colon produces constipation and diverticulitis.
[0016] Published literature includes numerous patent applications and patents granted to Parion Sciences Inc., aimed at pyrazi-nilguanidine analogs as sodium channel blockers. Examples of such publications include PCT Publications No. WO2003 / 070182, WO2003 / 070184, WO2004 / 073629, WO2005 / 025496, WO2005 / 016879, WO2005 / 018644, WO2006 / 022935, WO2006 / 023573, WO2006 / 023617, WO2007 / 01817 , WO2007 / 146869, WO2008 / 031028, WO2008 / 031048, and in US Patents Nos 6858614, 6858615, 6903105, 7064129, 7186833, 7189719, 7192958, 7192959, 7192960, 7241766, 7247636, 7247637, 7317013, 7332496, 7368447, 7368450, 7368451, 7375102, 7388013, 7399766, 7410968, 7807834, 7842697, and 7868010.
[0017] There remains a need for new sodium channel blocking compounds with improved potency and efficacy in mucous tissues. There is also a need for new sodium channel blocking compounds that provide a therapeutic effect, but minimize or eliminate the appearance or progression of hyperkalaemia in the receptors. Summary of the Invention
[0018] It is an object of the present invention to offer a compound that is stable in liquid formulations suitable for topical administration.
[0019] It is an object of the present invention to offer a compound that is more potent in vivo and / or absorbed less rapidly from mucous surfaces, and / or that is reversible compared to known compounds such as amiloride, benzamyl and phenamyl.
[0020] It is another object of the present invention to provide compounds that are (1) absorbed less rapidly from mucous surfaces, especially from airway surfaces, compared to known compounds and; (2) when absorbed from mucous surfaces after administration to mucous surfaces, are largely not absorbed by the kidneys, thus avoiding renal renal effects such as hypercholemia.
[0021] It is another object of the present invention to provide treatment methods that take advantage of the pharmacological properties of the compounds described above.
[0022] In particular, it is an object of the present invention to offer treatment methods that depend on the rehydration of mucous surfaces.
[0023] In particular, it is an object of the present invention to offer methods of treating dry eye and related ophthalmic diseases.
[0024] The objectives of the present invention can be achieved with a pyrazinoylguanidine represented by the compound of formula (I):
[0025] The present invention also offers pharmaceutical compositions that comprise the compound described in this report. The present invention also offers a method for promoting hydration of mucous surfaces, comprising: administering an effective amount of compound I described in this report to an individual's mucosal surface. The present invention also offers a method for restoring mucosal defense, comprising: topically administer an effective amount of compound I described in this report to a mucosal surface of an individual in need of it. The present invention also offers a method for blocking ENaC, comprising: contact sodium channels with an effective amount of compound I described in this report. The present invention also offers a method for promoting clearance of mucus on mucous surfaces, comprising: administering an effective amount of compound I described in this report to an individual's mucosal surface. The present invention also offers a method for the treatment of chronic bronchitis, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating cystic fibrosis, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating rhinosinusitis, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating nasal dehydration, comprising: administering an effective amount of compound I described in this report to the nasal passages of an individual in need of it. In a specific modality, the nasal dehydration caused by the administration of dry oxygen to the individual. The present invention also offers a method for treating sinusitis, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating pneumonia, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating ventilator-induced pneumonia, comprising: administer an effective amount of compound I described in this report to an individual through a ventilator. The present invention also offers a method for treating asthma, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating primary ciliary dyskinesia, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating otitis media, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for inducing sputum for diagnostic purposes, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating chronic obstructive pulmonary disease, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating emphysema, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating dry eye, comprising: administering an effective amount of compound I described in this report to the eye of the individual in need. The present invention also offers a method for promoting hydration of the eye, comprising: administering an effective amount of compound I described in this report to the subject's eye. The present invention also offers a method for promoting hydration of the cornea, comprising: administering an effective amount of compound I described in this report to the subject's eye. The present invention also offers a method for treating Sjogren's disease, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating vaginal dryness, comprising: administering an effective amount of compound I described in this report to the vaginal tract of an individual in need of it. The present invention also offers a method for treating dry skin, comprising: administering an effective amount of compound I described in this report to the skin of an individual in need of it. The present invention also offers a method for treating dry mouth (xerostomia), comprising: administering an effective amount of compound I described in this report to the individual's mouth in need. The present invention also offers a method for treating distal intestinal obstruction syndrome, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating esophagitis, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating bronchiectasis, comprising: administering an effective amount of compound I described in this report to an individual in need of it. The present invention also offers a method for treating constipation, comprising: administering an effective amount of compound I described in this report to an individual in need of it. In one embodiment of this method, the compound is administered either orally or via a suppository or enema. The present invention also offers a method for treating chronic diverticulitis comprising: administering an effective amount of compound I described in this report to an individual in need of it.
[0026] It is another aspect of the present invention to offer treatments using the sodium channel blocker when administered with an osmotic enhancer. Therefore, such a sodium channel blocker of formula I when used in conjunction with osmolytes will provide a prolonged pharmacodynamic half-life on mucous surfaces compared to any of the compounds used alone.
[0027] It is another object of the present invention to offer treatments using together the sodium channel blocker of formula (I) and osmolytes which are absorbed less quickly from mucous surfaces, especially from airway surfaces.
[0028] It is another object of the invention to provide compositions that contain a sodium channel blocker of formula (I) and osmolytes.
[0029] The objectives of the invention can be achieved with a method of treating an improved disease by increased mucociliary clearance and mucosal hydration comprising administering an effective amount of the compound of formula (I) already defined in this report and an osmolyte to an individual in need of clearance mucociliary and mucosal hydration increased.
[0030] The objectives of the invention can also be achieved with a method of sputum induction for diagnostic purposes, comprising administering an effective amount of the compound of formula (I) already defined in this report and an osmolyte to an individual in need thereof.
[0031] The objectives of the invention can also be achieved with a method of treating anthrax, comprising administering an effective amount of the compound of formula (I) already defined in this report and an osmolyte to an individual in need thereof.
[0032] The objectives of the invention can also be achieved with a method of prophylactic, prophylactic after exposure, preventive or therapeutic treatment against diseases or conditions caused by pathogens, particularly pathogens that can be used in bioterrorism, comprising administering an effective amount of the compound of formula (I ) to an individual in need of it.
[0033] The objectives of the invention can also be achieved with a composition, comprising a compound of formula (I) already defined in this report and an osmotically active compound. Description of the Figures
[0034] Figure 1: Dose response curve for ENaC inhibition with P1046 (I) and amiloride. The potency of the P-1046 compared to that of the amylo-ride was generated in studies in the Ussing chamber using primary cultures of canine bronchial epithelial cells. P-1046 is 100 times more potent than amiloride as indicated by the shift to the left on the dose-response curve. Figure 2: The effects of P-1046 concentration (formula I, 15) on the production of tears in ExLac rats. The gross values of the production of PRT tears are shown in the left panel and those corrected for the baseline are shown in the right panel. At all concentrations, P-1046 increases tear production in ExLac rats 15 minutes to or slightly below the tear production values observed for normal rats. The effects are still observed 2 hours after application, except for the group that received a dose of 0.1 mM. n = 4 for all groups. Figure 3: The clearance of P-1046 (formula I, 15) from the ocular surface. The concentration of P-1046 was determined using the procedures described above by determining the mass of P-1046 extracted from each filament and the concentration determined based on the volume of fluid twisted over the filament. For all concentrations tested, P-1046 exhibits an apparent biphasic clearance, whereby most of the drug is cleared from the ocular surface within 30 minutes after application, followed by a slow clearance phase 30 to 120 minutes after application. application. Note that the concentration of P-1046 during the slow clearance phase for concentration = 1 mM is well above the IC50 for P-1046 (5.3 nM), thus providing a long-lasting increase in tear volume. Figure 4: Figure 4. The effects of 10 mM P-1046 (formula I, 15) on the production of tears in ExLac rats. The gross values of the production of PRT tears are shown in the left panel and those corrected for the baseline are shown in the right panel. A single 10 mM dose of P-1046 produces an increased tear volume compared to vehicle controls. Although the crude data on tear production is not significant beyond 60 minutes (reflecting the higher baseline values for control animals compared to animals treated with P1046), the corrected data for the baseline are statistically significant at all points in the time after application (p <0.03) n = 3 for both groups. Figure 5: HPLC analysis of P-1046 samples (formula I, 15) for solubility / stability on day 1 and 10. No degradation of P-1046 was observed after 10 days at 50 ° C in pH citrate buffer 4.2 with 2.8% NaCl. P-1046 was completely soluble in this buffer at the highest tested concentration of 8.8 mg / mL. Figure 6: Summary of ocular tolerability of P-1046. (A) Cumulative Draize scores for each dose group and each application interval (data are the sum of all Draize scores for the right eye). The two regimens of application for P-1046 show only slight increases in Draize scores that are not greater than those observed for animals treated with vehicle. For comparison, a compound identified as an irritant in the same study is shown in yellow. (B) The average blink rate for each dose group at each application interval. The two application regimes for P-1046 show only slight increases in some points, similar to animals treated with vehicles. These data suggest that P-1046 at 10 and 30 mM does not cause burning when instilled. Figure 7: Plasma levels of P-1046 after application to the eye. (A) Plasma levels of individual animals during and after 8 doses of 10 mM P-1046 (50 µl / dose). (B) Plasma levels of individual animals during and after 4 doses of 30 mM P-1046 (50 µl / dose). Detailed Description of the Invention
[0035] The present invention is based on the discovery that the compound of formula (I) is more potent and / or absorbed less quickly from mucous surfaces, especially from airway surfaces, compared to known sodium channel blockers such as amino acids. lord, benzamil, and fenamil. Therefore, the compound of formula (I) has a longer half-life on mucous surfaces when compared to these compounds.
[0036] The present invention is also based on the discovery that certain compounds covered by formula (I) are (1) absorbed less quickly from mucous surfaces, especially from ocular surfaces, when compared to known compounds and (2) when absorbed from mucous surfaces after administration to mucous surfaces, are excreted mainly by non-renal routes to minimize the chances of hyperkalaemia.
[0037] The present invention is also based on the discovery that certain compounds covered by formula (I) provide methods of treatment that take advantage of the pharmacological properties of the compounds described above.
[0038] In particular, the present invention is also based on the discovery that certain compounds covered by formula (I) rehydrate mucous surfaces.
[0039] In particular, the present invention is also based on the discovery that certain compounds covered by formula (I) are useful in the treatment of dry eye and related ophthalmic diseases.
[0040] Compound I described in this report can be prepared and used as the free base. Alternatively, the compound can be prepared and used as a pharmaceutically acceptable salt. Pharmaceutically acceptable salts are salts that preserve or improve the desired biological activity of the parent compound and do not impart undesired toxicological effects. Examples of such salts are (a) acid addition salts formed with inorganic acids, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, among others; (b) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic acid, 2-hydroxy-3-naphttoate, pamoate, acid salicylic, stearic acid, phthalic acid, mandelic acid, lactic acid, among others; and (c) salts formed from elemental anions, for example, chlorine, bromine, and iodine.
[0041] It should be noted that all enantiomers, diastereomers and racemic mixtures, tautomers, polymorphs, pseudopolymorphs and pharmaceutically acceptable salts of the compound within the scope of formula (I) are covered by the present invention. All mixtures of such enantiomers and diastereomers are within the scope of the present invention.
[0042] A compound of formula I and its pharmaceutically acceptable salts can exist as different polymorphs or pseudopolymorphs. As used in this report, crystalline polymorphism means the ability of a crystalline compound to exist in different crystal structures. Crystalline polymorphism can result from differences in crystal arrangement ("crystal packing") (or polymorphism in "packing polymorphism") or differences in arrangement between different confomers of the same molecule (conformational polymorphism). As used in this report, crystalline pseudopolymorphism means the ability of a hydrate or solvate of a compound to exist in different crystal structures. The pseudopolymorphs of the present invention may exist due to differences in the arrangement of the crystal (arrangement pseudopolymorphism) or due to differences in the arrangement between different comonomers of the same molecule ( conformational pseudopolymorphism.) The present invention comprises all polymorphs and pseudopo-limorphs of the compounds of formula I and their pharmaceutically acceptable salts.
[0043] A compound of formula I and its pharmaceutically acceptable salts can also exist as an amorphous solid. As used in this report, an amorphous solid is a solid in which there is no long-range order of the positions of the atoms in the solid. This definition also applies when the size of the crystal is two nanometers or less. Additives, including solvents, can be used to create the amorphous forms of the present invention. The present invention comprises all amorphous forms of the compounds of formula I-III and their pharmaceutically acceptable salts.
[0044] The compound of formula I can exist in different tauomeric forms. The person skilled in the art will recognize that guanidines can exist in tautomeric forms. By way of example and not by way of limitation, compounds of formula I can exist in various tautomeric forms as shown below:
[0045] All possible tautomeric forms of guanidines and acyl guanidines of all modalities of formula I are within the scope of the present invention.
[0046] "Enantiomers" refers to two stereoisomers of a compound that are specular images not superimposable of each other.
[0047] The stereochemical definitions and conventions used in this report generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. & Wi-len, S., Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., New York. Many organic compounds exist in optically active forms, that is, they have the ability to rotate the plane of plane-polarized light. When describing an optically active compound, the prefixes D and L or R and S are used to indicate the configuration of the molecule around its chiral centers. The prefixes del, D and L, or (+) and (-) are used to denote the rotation signal of the plane-polarized light by the compound, with S, (-), or 1 indicating that the compound is levogiratory whereas a compound with the prefix R, (+), or d is dextrogiratory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of each other. A specific stereoisomer can also be mentioned as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enanthiomers is called a racemic or racemate mixture, which can occur where there is no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.
[0048] A simple stereoisomer, for example, an enantiomer, substantially free of its stereoisomers can be obtained by resolving the racemic mixture using a method such as formation of diastereomers using optically active resolving agents ("Stereochemistry of Carbon Compounds," ( 1962) by EL Eliel, McGraw Hill; Lochmuller, CH, (1975) J. Chromatogr., 113: (3) 283-302). Racemic mixtures of chiral compounds of the invention can be separated and isolated by any suitable method, including: (2) formation of ionic diastereomeric salts with chiral compounds and separation by fractional crystallization or other methods, (2) formation of diastereomeric compounds with derivative reagents chiral, separation of diastereomers, and conversion to pure stereoisomers, and (3) separation of substantially pure or directly enriched stereoisomers under chiral conditions.
[0049] The term "diastereomer" refers to a stereoisomer with two or more centers of chirality and whose molecules are not mirror images of each other. Diastereomers have different physical properties, for example, melting points, boiling points, spectral properties, and reactivities. Mixtures of diastereomers can be separated by high-resolution analytical procedures such as electrophoresis and chromatography.
[0050] Without wishing to stick to any particular theory, we believe that the compound of formula (I) in vivo acts as a sodium channel blocker. By blocking the epithelial sodium channels present on the mucous surfaces, the compounds of formula (I) reduce the absorption of water by the mucous surfaces. This effect increases the volume of protective fluids on mucous surfaces, rebalances the system, and thus treats the disease.
[0051] The present invention also offers treatment methods that take advantage of the properties of the compounds described in this invention already discussed above. Therefore, individuals who can be treated by the methods of the present invention include, but are not limited to, patients with cystic fibrosis, primary ciliary dyskinesia, chronic bronchitis, bronchiectasis, chronic obstructive airway disease, artificially ventilated patients, patients with acute pneumonia, etc. The present invention can be used to obtain a sputum sample from a patient by administering the active compounds to at least one lung of the patient, and then inducing or collecting a sputum sample from that patient. Typically, the invention will be administered to respiratory mucosal surfaces via aerosol (liquid or dry powders) or washing.
[0052] Individuals who can be treated by the method of the present invention also include patients who receive supplemental oxygen via the nasal route (a regimen that tends to dry airway surfaces); patients affected by a disease or allergic response (for example, an allergic response to pollen, dust, animal hair or particles, insects or insect particles, etc.) that affects the surfaces of the nasal airways; patients affected by a bacterial infection, for example, staph infections such as Staphylococcus aureus infections, Hemophilus influenza infections, Streptococcus pneumoniae infections, Pseudomonas aeuriginosa infections, etc.) from nasal airway surfaces; patients affected by an inflammatory disease that affects the surfaces of the nasal airways; or patients affected by sinusitis (where the agent or active agents are administered to promote the drainage of secretions of congested mucus in the sinuses by administering an effective amount to promote the drainage of congested fluid in the sinuses), or in combination, rhinosinusitis. The invention can be administered to rhinosinusal surfaces by topical distribution, including aerosols and drops.
[0053] The present invention can be used to hydrate mucous surfaces other than the mucous surfaces of the airways. Such other mucous surfaces include gastrointestinal surfaces, oral surfaces, genitourethral surfaces, ocular surfaces or surfaces of the eye, inner ear and middle ear. For example, the active compounds of the present invention can be administered by any suitable means, including the local / topical, oral, or rectal route, in an effective amount.
[0054] The present invention relates mainly to the treatment of human subjects, but it can also be used to treat other mammals, such as dogs and cats, for veterinary purposes.
[0055] As discussed above, the compounds used to prepare the compositions of the present invention can be in the form of a pharmaceutically acceptable free base. Since the free base of the compound is generally less soluble than salt in aqueous solutions, compositions of the free base are employed to provide a more systematic release of the active agent into the lungs. An active agent present in the lungs in a particulate form that has not been dissolved in solution is not available to induce a physiological response, but serves as a deposit of bioavailable drug that gradually dissolves to form a solution.
[0056] Another aspect of the present invention relates to a pharmaceutical composition, comprising a compound of formula (I) in a pharmaceutically acceptable carrier (for example, an aqueous carrier solution). In general, the compound of formula (I) is included in the composition in an amount effective to inhibit the reabsorption of water by mucous surfaces.
[0057] Without wishing to stick to any particular theory, we believe that the sodium channel blockers of the present invention block the epithelial sodium channels present on mucous surfaces, the sodium channel blockers described in this invention reduce the absorption of salt and water by mucous surfaces . This effect increases the volume of protective fluids on mucous surfaces, rebalances the system, and therefore treats the disease. This effect is enhanced when used in combination with osmolytes.
[0058] The compounds of formula (I) can also be used in conjunction with osmolytes thereby decreasing the dose of the compound necessary to hydrate mucous surfaces. This important property means that the compound will have a lesser tendency to cause unwanted side effects by blocking the sodium channels located in non-target locations on the recipient's body, for example, in the kidneys when used in combination with an osmolyte.
[0059] Active osmolytes of the present invention are molecules or compounds that are osmotically active (i.e., they are "osmolytes"). The "osmotically active" compounds of the present invention are membrane-impermeable (i.e., essentially non-absorbable) on the epithelial surface of the airways or lungs. The terms "airway surface" and "pulmonary surface", as used in this report, include surfaces of the pulmonary airways such as the bronchi and bronchioles, alveolar surfaces, and nasal and sinus surfaces. The active compounds of the present invention can be ionic osmolytes (i.e., salts), or they can be non-ionic osmolytes (i.e., sugars, sugar alcohols, and organic osmolytes). We specifically intend that both racemic forms of the active compounds that are racemic in nature are included in the group of active compounds that are useful in the present invention. It should be noted that all racemates, enantiomers, diastereomers, tautomers, polymorphs and pseudopolymorphs and racemic mixtures of osmotically active compounds are covered by the present invention.
[0060] Active osmolytes useful in the present invention which are ionic osmolytes include any salt of a pharmaceutically acceptable anion and a pharmaceutically acceptable cation. Preferably, either the anion or cation (or both) are non-absorbable (that is, osmotically active and not subject to rapid active transport) in relation to the airway surfaces to which they are administered. Such compounds include, but are not limited to, anions and cations that are contained in salts marketed and approved by the FDA, see, for example, Remington: The Science and Practice of Pharmacy, Vol. II, p. 1457 (19th Ed. 1995), hereby incorporated by reference, and can be used in any combination including its conventional combinations.
[0061] Pharmaceutically acceptable osmotically active anions that can be used to carry out the present invention include, but are not limited to, acetate, benzenesulfonate, benzoate, bicarbonate, bitartarate, bromide, calcium edetate, cansilate (camphor sulfonate), carbonate, chloride, citrate, dihydrochloride, edetate , edisylate (1,2-ethanedisulfonate), stolate (lauryl sulfate), esylate (1,2-ethanedisulfonate), fumarate, gluceptate, gluconate, glutamate, glycolylsanylate (p-glycolamidophenylsonate), hexylresorcinate, dihydramine (N, N'-N'-N' (desidroabietil) ethylenediamine), hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, lactate, lactobionate, malate, maleate, mandelate, mesylate, methylbromide, methylnitrate, methylsulfate, mucate, NAPSI-broad, nitrate, nitrite, pamoate (embonate), pantothenate , phosphate or diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, sulfate, tannate, tartrate, theoclate (8-chloroteophyllinate), triethyliodide, bicarbonate, etc. Particularly preferred anions include chloride, sulfate, nitrate, gluconate, iodide, bicarbonate, bromide and phosphate.
[0062] Pharmaceutically acceptable cations that can be used to carry out the present invention include, but are not limited to, organic cations such as benzathine (N, N'-dibenzylethylenediamine), chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methyl D-glucamine), procaine, D-lysine, L-lysine, D-arginine, L-arginine, triethylammonium, N-methyl D-glycerol, among others. Particularly preferred organic cations are 3-carbon, 4-carbon, 5-carbon and 6-carbon organic cations. Metal cations useful in the practice of the present invention include, but are not limited to, aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, iron, ammonium, among others. Particularly preferred cations include sodium, potassium, choline, lithium, meglumine, D-lysine, ammonium, magnesium, and calcium.
[0063] Specific examples of osmotically active salts that can be used with the sodium channel blockers described in this report to carry out the present invention include, but are not limited to, sodium chloride, potassium chloride, choline chloride, choline iodide, lithium chloride , meglumine chloride, L-lysine chloride, D-lysine chloride, ammonium chloride, potassium sulfate, potassium nitrate, potassium gluconate, potassium iodide, ferric chloride, ferrous chloride, potassium bromide, etc. Whether a simple salt or a combination of different osmotically active salts can be used to carry out the present invention. Combinations of different salts are preferred. When different salts are used, one of the anion and the cation can be the same between the different salts.
[0064] Osmotically active compounds of the present invention also include non-ionic osmolytes such as sugars, sugar alcohols, and organic osmolytes. Sugars and sugar alcohols useful in the practice of the present invention include, but are not limited to, 3-carbon sugars (e.g., glycerol, dihydroxyacetone); 4-carbon sugars (for example, the D and L forms of erythrose, threose, and erythrulose); 5-carbon sugars (for example, the D and L forms of ribose, arabinose, xylose, lixose, psychosis, fructose, sorbose, and tagatose); and 6-carbon sugars (for example, the D and L forms of altose, alose, glucose, mannose, gulose, idose, galactose, and talose, and the D and L forms of allo-heptulose, allo-hepulose, gluco-heptulose , mano-heptulose, gulo-heptulose, acid-heptulose, galacto-heptulose, thallo-heptulose). Additional sugars useful in the practice of the present invention include raffinose, raffinose series oligosaccharides, and stachyose. The D and L forms of the reduced form of each sugar / sugar alcohol useful in the present invention are also active compounds within the scope of the invention. For example, glucose, when reduced, becomes sorbitol; within the scope of the present invention, sorbitol and other reduced forms of sugar / sugar alcohols (e.g., mannitol, dulcitol, arabitol) are therefore active compounds of the present invention.
[0065] The osmotically active compounds of the present invention further include the family of nonionic osmolytes called "organic osmolytes". The term "organic osmolytes" is generally used to indicate molecules used to control intracellular osmolality in the kidneys. See, for example, J. S. Handler et al., Comp. Biochem. Physiol, 117, 301306 (1997); M. Burg, Am. J. Physiol. 268, F983-F996 (1995), both of which are incorporated herein by reference. Although the inventor does not wish to adhere to any particular theory of the invention, it appears that these organic osmolytes are useful for controlling extracellular volume on airway / pulmonary surfaces. Organic osmolytes useful as active compounds in the present invention include, but are not limited to, three important classes of compounds: polyols (polyhydric alcohols), methylamines, and amino acids. Polyol-like organic osmolytes found useful in the practice of this invention include, but are not limited to, inositol, myo-inositol, and sorbitol. Methylamine-type organic osmolytes useful in the practice of this invention include, but are not limited to, choline, betaine, carnitine (L-, D- and DL forms), phosphoryl-choline, smooth-phosphorylcholine, glycerophosphorylcholine, creatine, and creatine phosphate. The amino acid-type organic osmolytes of the invention include, but are not limited to, the D- and L- forms of glycine, alanine, glutamine, glutamate, aspartate, proline and taurine. Additional osmolytes useful in the practice of the invention include thiulose and sarcosine. Organic mammalian osmolytes are preferred, with human organic osmolytes being more preferred. However, certain organic osmolytes are of bacterial, yeast, and marine origin, and these compounds are also active compounds useful in the scope of the present invention.
[0066] In certain circumstances an osmolyte precursor can be administered to the individual; therefore, these compounds are useful in the practice of the invention. The term "osmolyte precursor" as used in this report refers to a compound that is converted to an osmolyte by a metabolic step, whether catabolic or anabolic. The osmolyte precursors of this invention include, but are not limited to, glucose, glucose polymers, glycerol, choline, phosphatidylcholine, smooth phosphatidylcholine and inorganic phosphates, which are precursors of polyols and methylamines. Osmolyte precursors of the amino acid type within the scope of this invention include proteins, peptides, and polyamino acids, which are hydrolyzed to give osmolytic amino acids, and metabolic precursors that can be converted to osmolytic amino acids by a metabolic step such as transamination. For example, a precursor to the amino acid glutamine is poly-L-glutamine, and a glutamate precursor is poly-L-glutamic acid.
[0067] Also included within the scope of this invention are chemically modified osmolytes or osmolyte precursors. Such chemical modifications involve attaching an additional chemical group to the osmolyte (or precursor) that alters or intensifies the effect of the osmolyte or osmolyte precursor (for example, inhibits the degradation of the osmolyte molecule). Such chemical modifications have already been used with drugs or pro-drugs and are known in the literature. (See, for example, US Patent Nos. 4,479,932 and 4,540,564; Shek, E. et al., J. Med. Chem. 19: 113-117 (1976); Bodor, N. et al. , J. Pharm. Sci. 67: 1045-1050 (1978); Bodor, N. et al., J. Med. Chem. 26: 313-318 (1983); Bodor, N. et al., J. Pharm Sci. 75: 29-35 (1986), all of which are incorporated by reference.
[0068] In general, the osmotically active compounds of the present invention (both ionic and non-ionic) that do not promote, or actually deteriorate or retard, bacterial growth are preferred.
[0069] The compounds of formula (I) described in this invention and the osmotically active compounds disclosed in this report can be administered in any order and / or concomitantly to mucous surfaces such as the eye, nose, and airway surfaces including nasal passages, an individual's breasts and lungs by any suitable means known in the literature, such as nasal drops, mists, aerosols, continuous nighttime nasal cannulation etc. In one embodiment of the invention, the compounds of formula (I) and the osmotically active compounds of the present invention are administered concomitantly by transbronchoscopic washing. In a preferred embodiment of the invention, the compounds of formula (I) and the osmotically active compounds of the present invention are deposited on the surfaces of the pulmonary airways by administration by inhalation of respirable particles in a respirable aerosol comprised of the compounds of formula (I) and osmotically active compounds, where the compounds of formula (I) can precede or follow the independent distribution of an osmotically active compound within a sufficiently short time for their effects to be additive. Breathable particles can be liquid or solid. Numerous inhalers for delivering aerosol particles to an individual's lungs are known. In another preferred embodiment of the invention, compounds of formula (I) and osmotically active compounds can be given concomitantly as already defined in this report.
[0070] The compounds of formula (I) and the osmotically active compounds of the present invention are administered sequentially (in q order) or concomitantly to the individual in need thereof. As used in this report, the term "concurrently" means sufficiently close in time to produce a combined effect (that is, concurrently it may be simultaneously, or it may be two or more events occurring within a short period of time before or after each other ). Concomitantly, it also slows down the distribution of the compounds of formula (I) and osmolytes as a mixture or solution of the two components as well as when distributed from two different nebulizers. An example of this could be the distribution of compound 1 in a nebulizer and hypertonic saline solution in a second nebulizer connected by a T-shaped piece. When administered with other active agents, the active compounds of the present invention can function as a vehicle or vehicle for the other active agent, or they can simply be administered concurrently with the other active agent. The active compound of the present invention can be used as a dry or liquid vehicle to deliver other active ingredients to airway surfaces. Such other active agents can be administered for the treatment of the disease or disorder to which they are intended, in conventional manner and at conventional dosages, in combination with the active compounds of the present invention, which can be considered to function as a vehicle or vehicle for the other active agent. Such another active ingredient can be employed, particularly where hydration of airway surfaces (i.e., the activity of the osmotically active compounds of the present invention) facilitates the activity of the other active ingredient (for example, facilitating or improving the absorption of the active ingredient. , contributing to the mechanism of action of the other active principle, or by any other mechanisms). In a preferred embodiment of the invention, when the active compound of the present invention is administered concomitantly with another active agent, the active compound of the present invention has an additive effect in relation to the other active agent, that is, the desired effect of the other active agent is improved by concomitant administration of the active compounds of the present invention.
[0071] The compounds of formula (I) of the present invention are also useful for the treatment of airborne infections. Examples of airborne infections include, for example, RSV. The compounds of formula (I) of the present invention are also useful for the treatment of an anthrax infection. The present invention relates to the use of the compounds of formula (I) of the present invention for the prophylactic, prophylactic after exposure, preventive or therapeutic treatment against diseases or conditions caused by pathogens. In a preferred embodiment, the present invention relates to the use of the compounds of formula (I) for the prophylactic, prophylactic after exposure, preventive or therapeutic treatment against diseases or conditions caused by pathogens that can be used in bioterrorism.
[0072] In recent years, several research programs and measures of biodefense have been implemented to address concerns about the use of biological agents in acts of terrorism. These measures are intended to address concerns related to bioterrorism or the use of microorganisms or biological toxins to kill people, spread fear, and disrupt society. For example, the National Institute of Allergy and Infectious Diseases ("National Institute of Allergic and Infectious Diseases") (NIAID) has developed a Strategic Plan for Biodefense Research that outlines plans to address research needs in the broad field of bioterrorism and disease According to the plan, the deliberate exposure of the civilian population of the United States to Bacillus anthracis spores revealed a failure in the nation's overall preparedness against bioterrorism, and the report details that these attacks revealed a need still not satisfied with tests to quickly diagnose, vaccines and immunotherapies to prevent, and drugs and biologicals to cure diseases caused by bioterrorism agents.
[0073] Much of the focus of the various research efforts has turned to the study of the biology of pathogens identified as potentially dangerous as agents of bioterrorism, the study of the host's response against such agents, the development of vaccines against infectious diseases, the evaluation of therapeutics currently available and under investigation against such agents, and the development of diagnostics to identify signs and symptoms of threatening agents. Such efforts are commendable, but given the large number of pathogens that have been identified as potentially available for bioterrorism, these efforts have yet to provide satisfactory responses to possible bioterrorism threats. In addition, many of the pathogens identified as potentially dangerous as agents of bioterrorism do not offer adequate economic incentives for the development of therapeutic or preventive measures by the industry. In addition, even though preventive measures such as vaccines were available for each pathogen that can be used in bioterrorism, the cost of administering all of these vaccines to the general population is prohibitive.
[0074] Until convenient and effective treatments against any threat of bioterrorism are available, there is a great need for preventive, prophylactic or therapeutic treatments that can prevent or reduce the risk of infection by pathogens.
[0075] The present invention offers such methods of prophylactic treatment. In one aspect, the invention offers a method of prophylactic treatment comprising administering a prophylactically effective amount of the compounds of formula (I) to an individual in need of prophylactic treatment against an infection by one or more airborne pathogens. A particular example of an airborne pathogen is anthrax.
[0076] In another aspect, the invention offers a method of prophylactic treatment to reduce the risk of infection by an airborne pathogen that can cause disease in a human, said method comprising administering an effective amount of the compounds of formula (I) to the lungs of the human being who may be at risk of infection by the airborne pathogen but who is asymptomatic for the disease, where the effective amount of a sodium channel blocker and an osmolyte is sufficient to reduce the risk of infection in the being human. A particular example of an airborne pathogen is anthrax.
[0077] In another aspect, the invention offers a method of prophylactic treatment after exposure or therapeutic treatment for the treatment of an infection by an airborne pathogen comprising administering an effective amount of the compounds of formula (I) to the lungs of an individual with need for such treatment against an infection by an airborne pathogen. Pathogens that can be protected by the prophylactic treatment methods after exposure, rescue treatment and therapeutic treatment of the invention include all pathogens that can enter the body through the mouth, nose or nasal airways, thus proceeding to the lungs. Typically, pathogens are airborne pathogens, whether naturally occurring or by aerosolization. Pathogens can be naturally occurring or can be intentionally introduced into the environment after aerosolization or another method of introducing pathogens into the environment. Many pathogens that are not naturally transmitted by air have already been or can be aerosolized for use in bioterrorism. Pathogens for which the treatment of the invention may be useful include, but are not limited to, category A, B and C priority pathogens established by NIAID. These categories generally correspond to the lists compiled by the Centers for Disease Control and Prevention (CDC). As established by the CDC, category A agents are those that can be easily disseminated or transmitted from person to person, causing high mortality, with the potential to have a major impact on public health. Category B agents are next in terms of priority and include those that are moderately easy to spread and cause moderate morbidity and low mortality. Category C consists of emerging pathogens that can be built for mass dissemination in the future because of their bioavailability, are easy production and dissemination with the potential for high morbidity and mortality. Particular examples of these pathogens are anthrax and plague. Additional pathogens to protect or reduce the risk of infection include influenza viruses, rhinovirus, ade-novirus and respiratory syncytial viruses, among others. Another pathogen against which it is possible to protect is the coronavirus which is believed to be the cause of severe acute respiratory distress syndrome (SARS).
[0078] The compounds of the present invention can also be used in conjunction with a P2Y2 receptor agonist or a pharmaceutically acceptable salt thereof (also sometimes called the "active agent" in this report). The composition may further comprise a P2Y2 receptor agonist or a pharmaceutically acceptable salt thereof (also sometimes called an "active agent" in this report). The P2Y2 receptor agonist is typically included in an amount effective to stimulate chloride and water secretion from airway surfaces, particularly from nasal airway surfaces. Suitable P2Y2 receptor agonists are described in columns 9-10 of US Patents 6,264,975, US 5,656,256, and US 5,292,498, all of which are incorporated herein by reference.
[0079] Bronchodilators can also be used in combination with the compounds of the present invention. Such bronchodilators include, but are not limited to, β-adrenergic agonists including, but not limited to, epinephrine, isoproterenol, phenoterol, albutereol, terbutaline, pyrbuterol, bitolterol, metaproterenol, iosetarin, salmeterol xinafoate, as well as, but without anticholinergic agents including, but without, anticholinergic agents ipra-tropium bromide, as well as compounds such as theophylline and aminophylline. These compounds can be administered according to known techniques, either before or concurrently with the active compounds described in this invention.
[0080] Another aspect of the present invention relates to a pharmaceutical composition, comprising an active compound already described above in a pharmaceutically acceptable carrier (for example, an aqueous carrier solution). In general, the active compound is included in the composition in an amount effective to treat mucous surfaces, such as inhibiting water reabsorption by mucous surfaces, including airway and other surfaces.
[0081] The active compounds disclosed in this invention can be administered to mucosal surfaces by any suitable means, including topical, oral, rectal, vaginal, ocular and dermal, etc. For example, for the treatment of constipation, the active compounds can be administered orally or rectally to the gastrointestinal mucosal surface. The active compound can be combined with a pharmaceutically acceptable carrier in any suitable form, such as sterile or diluted physiological saline or topical solution, such as drops, tablets or the like for oral administration, as a suppository for rectal or genitourethral administration, etc. Ex-cipients can be included in the formulation to improve the solubility of the active compounds, as desired.
[0082] The active compounds disclosed in this invention can be administered to a patient's airway surfaces by any suitable means, including a spray, mist, or drops of the active compounds in a pharmaceutically acceptable carrier such as physiological or diluted saline solutions or distilled water. For example, the active compounds can be prepared as formulations and administered in the manner described in US Patent No. 5,789,391 issued to Jacobus, the description of which is incorporated herein in its entirety by reference.
[0083] Solid or liquid particulate active agents prepared for the practice of the present invention may, as noted above, include particles of respirable or non-respirable size, i.e., for respirable particles, particles of a sufficiently small size pass through the mouth and larynx by inhalation. and penetrate the bronchi and alveoli of the lungs, and for non-breathable particles, sufficiently large particles are retained in the passages of the nasal airways instead of passing through the larynx and penetrating the bronchi and alveoli of the lungs. In general, particles ranging from about 1 to 5 microns in size (more particularly, less than about 4.7 microns in size) are breathable. Particles of non-breathable size are larger than about 5 microns in size, up to the size of visible drops. Therefore, for nasal administration, a particle size in the range of 10-500 pm can be used to ensure its retention in the nasal cavity.
[0084] In the production of a formulation according to the invention, active agents or the physiologically acceptable free salts or bases thereof are typically mixed, inter alia, with an acceptable carrier. Naturally, the vehicle must be compatible with all other ingredients in the formulation and must not be harmful to the patient. The carrier must be solid or liquid, or both, and is preferably formulated with the compound as a unit dose formulation, for example, a capsule, which can contain 0.5% to 99% by weight of the active compound. One or more active compounds can be incorporated into the formulations of the invention, which formulations can be prepared by any of the well-known pharmacy techniques which essentially consists of mixing the components.
[0085] Compositions containing dry respirable or non-respirable particles of micronized active agent can be prepared by grinding the dry active agent with a pestle, then passing the micronized composition through a 400 mesh sieve to break or separate large agglomerates.
[0086] The particulate active agent composition may optionally contain a dispersant that serves to facilitate the formulation of an aerosol. A suitable dispersant is lactose, which can be mixed with the active agent in any desired proportion (for example, a 1 to 1 weight ratio).
[0087] The active compounds disclosed in this invention can be administered to airway surfaces including an individual's nasal passages, sinuses and lungs by a suitable means known in the literature, such as nasal drops, mists, etc. In an embodiment of the invention, the active compounds of the present invention are administered by transbroncoscopic lavage. In a preferred embodiment of the invention, the active compounds of the present invention are deposited on the surfaces of the lung airways by administering an aerosol suspension of respirable particles comprised of the active compound, which the individual inhales. Breathable particles can be liquid or solid. Numerous inhalers for delivering aerosol particles to an individual's lungs are known.
[0088] Inhalers such as those developed by Inhale Therapeutic Systems, Palo Alto, California, USA, may be employed, including, but not limited to, those disclosed in US Patent Nos. 5,740,794, 5,654,007, 5,458,135, 5,775,320 , and 5,785,049, all of which are incorporated by reference. The Applicant specifically intends that the descriptions of all patent references cited in this report are incorporated herein in their entirety for reference. Inhalers such as those developed by Dura Pharmaceuticals, Inc., San Diego, California, USA, may also be used, including, but not limited to, those disclosed in US Patent Nos. 5,622,166, 5,577,497, 5,645,051, and 5,492,112, all of which are incorporated by reference. In addition, inhalers such as those developed by Aradigm Corp., Hayward, California, USA, may be employed, including, but not limited to, those disclosed in US Patent Nos. 5,826,570, 5,813,397, 5,819,726, and 5,655 .516, all of which are incorporated by reference. Such devices are particularly suitable as dry particle inhalers.
[0089] Aerosols of liquid particles comprising the active compound can be produced by any means, such as by a pressure-driven aerosol nebulizer (L C Star) or an ultrasonic nebulizer (Pari eFlow). See, for example, US Patent No. 4,501,729, which is incorporated herein by reference. Nebulizers are commercially available devices that transform solutions or suspensions of the active ingredient into a therapeutic aerosol mist by accelerating compressed gas, typically air or oxygen, through a narrow venturi orifice or by ultrasonic agitation. Formulations suitable for use in nebulizers consist of the active ingredient in a liquid vehicle, the active ingredient comprising up to 40% w / w of the formulation, but preferably less than 20% w / w. The vehicle is typically water (and more preferably sterile water without pyrogen) or a dilute aqueous alcoholic solution. Perfluorocarbon-type vehicles can also be used. Optional additives include preservatives if the formulation is not sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents and surfactant.
[0090] Aerosols of solid particles comprising the active compound can also be produced with any solid particulate drug aerosol generator. Aerosol generators for administering solid particulate medications to an individual produce particles that are breathable, as explained above, and generate an aerosol volume containing a predetermined regulated dose of medication at a rate suitable for administration to man. An illustrative type of solid particulate aerosol generator is an insufflator. Formulations suitable for administration by insufflation include finely comminuted powders that can be delivered by means of an insufflator or taken to the nasal cavity by means of a sniff. In the insufflator, the powder (for example, a regulated dose of the same effective to perform the treatments described in this report) is contained in capsules or cartridges, typically made of gelatin or plastic, which are either perforated or opened in situ and the powder is distributed by air entrained through the device by inhalation or by a manually operated pump. The powder used in the insufflator consists only of the active ingredient or powder mixture 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. Regulated dose inhalers are pressurized aerosol dispensers, typically containing a suspension formulation or active ingredient solution in a liquefied propellant. During use, these devices discharge the formulation through a valve adapted to deliver a regulated volume, typically 10 to 150 µl, to produce a spray of fine particles containing the active ingredient. Suitable propellants include certain compounds of the chlorofluorocarbon type, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane and mixtures thereof. The formulation may additionally contain one or more cosolvents, for example, ethanol, surfactants, such as oleic acid or sorbitan trioleate, antioxidants and suitable flavoring agents.
[0091] The aerosol, whether formed from solid or liquid particles, can be produced by the aerosol generator at a rate of about 10 to 150 liters per minute, more preferably from 30 to 150 liters per minute, and even a partner molecule about 60 liters per minute. Aerosols containing larger amounts of medication can be administered faster.
[0092] The dosage of the active compounds disclosed in this report will vary depending on the condition being treated and the condition of the individual, but generally ranges from about 0.01, 0.03, 0.05, 0.1 to 1, 5, 10 or 20 mg of the pharmaceutical agent, deposited on the airway surfaces. The daily dose can be divided into one or more unit dose administrations. The goal is to achieve a concentration of pharmaceutical agents on the surfaces of the pulmonary airways between 10-9 - 104 M.
[0093] In another embodiment, they are administered by administering an aerosol suspension of respirable or non-respirable particles (preferably non-respirable particles) comprised of the active compound, which is inhaled by the individual through the nose. Breathable or non-breathable particles can be liquid or solid. The amount of active agent included can be an amount sufficient to achieve dissolved concentrations of active agent on the subject's airway surfaces from about 10-9, 10-8, or 10-7 to about 10-3, 10-2 , 10-1 moles / liter, and more preferably from about 10-9 to about 10-4 moles / liter.
[0094] The dosage of active compound will vary depending on the condition being treated and the condition of the individual, but it can usually be an amount sufficient to achieve dissolved concentrations of active agent on the surfaces of the individual's nasal airways of about 10-9, 10-8 , 10-7 to about 10-3, 10-2, or 10-1 moles / liter, and more preferably from about 10-7 to about 10-4 moles / liter. Depending on the solubility of the particular active compound formulation administered, the daily dose can be divided into one or more unit dose administrations. The daily dose by weight can vary from about 0.01, 0.03, 0.1, 0.5 or 1.0 to 10 or 20 milligrams of active agent particles for a human subject, depending on age and condition of the individual. A currently preferred unit dose is about 0.5 milligrams of active agent in a regimen of 2-10 administrations per day. The dosage can be presented as a pre-packaged unit by any suitable means (for example, encapsulated in a gelatin capsule).
[0095] In one embodiment of the invention, the particulate active agent composition may contain both a free base of the active agent and a pharmaceutically acceptable salt to provide both immediate and systematic release of the active agent for dissolution in mucus secretions from the nose. Such a composition serves to provide immediate relief to the patient, and systematic relief over time. Systematic relief, by decreasing the number of necessary daily administrations, is expected to increase patient collaboration during treatments with the active agent.
[0096] Pharmaceutical formulations suitable for administration to the airways include formulations of solutions, emulsions, suspensions and extracts. See, in general, J. Nairn, Solutions, Emulsions, Suspensions and Extracts, in Remington: The Science and Practice of Pharmacy, chap. 86 (19th ed. 1995), hereby incorporated by reference. Pharmaceutical formulations suitable for nasal administration can be prepared in the manner described in US Patent Nos. 4,389,393 issued to Schor, 5,707,644 issued to Illum, 4,294,829 granted to Suzuki, and 4,835,142 to Suzuki, the descriptions of which are here incorporated in their entirety as a reference.
[0097] Mists or aerosols of liquid particles comprising the active compound can be produced by any suitable means, such as by simple nasal spray with the active agent in a pharmaceutically acceptable aqueous vehicle, such as a sterile saline solution or sterile water. Administration can be done with a pressure-activated aerosol nebulizer or with an ultrasonic nebulizer. See, for example, US Patent Nos. 4,501,729 and 5,656,256, both of which are incorporated herein by reference. Formulations suitable for use in a nasal drops or spray bottle or 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. Typically the vehicle is water (and more preferably sterile water without pyrogen) or a dilute aqueous alcoholic solution, preferably made in 0.12% to 0.8% sodium chloride solution. Optional additives include preservatives if the formulation is not sterile, for example, methyl hydroxybenzoate, antioxidants, flavoring agents, volatile oils, buffering agents, osmotically active agents (for example, mannitol, xylitol, erythritol) and surfactants.
[0098] Compositions containing dry breathable or non-respirable particles of micronized active agent can be prepared by grinding the dry active agent in a pestle, then passing the micronized composition through a 400 mesh sieve to break or separate large pellets.
[0099] The particulate composition may optionally contain a dispersant which serves to facilitate the formulation of an aerosol. A suitable dispersant is lactose, which can be mixed with the active agent in any desired proportion (for example, a 1 to 1 weight ratio).
[0100] The compound of formula I can be synthesized according to procedures known in the literature. A representative synthesis procedure is shown in the diagram below:
[0101] These procedures are described, for example, in E.J. Cragoe, "The Synthesis of Amiloride and Its Analogs" (Chapter 3) in Amilori-de and Its Analogs, pp. 25-36, hereby incorporated by reference. Other methods for preparing the compounds are described, for example, in US patent 3,313,813, incorporated herein by reference. See in particular methods A, B, C, and D described in US patent 3,313,813. Added methods for preparing the intermediates used in the preparation of the compounds of the present invention are disclosed in US patents 7,064,129, US 6,858,615, US 6,903,105, WO 2004/073629, WO 2007/146869, and WO 2007/018640, all they are expressly incorporated by reference.
[0102] Several assays can be used to characterize the compounds of the present invention. Representative essays are discussed below. In vitro measurement of sodium channel blocking activity and its reversibility
[0103] An assay used to evaluate the mechanism of action and / or the potency of the compounds of the present invention involves the determination of luminal drug inhibition of epithelial sodium currents in the airways measured in short-circuit current (ISc) using epithelial monolayers of airways mounted on Ussing chambers. Cells obtained from the newly excised airways of man, dog, sheep or rodent are seeded in 0.4 micron porous Snapwell ™ Inserts (CoStar), grown under air-liquid interface (ALI) conditions in hormonally defined media, and analyzed for sodium transport activity (ISC) while bathed in Krebs Bicarbonate Ringer (KBR) in Using chambers. All test drug additions to the luminal bath with half-log dose addition protocols (from 1 x 10-11 M to 3 x 10-5 M), and the cumulative change in ISC (inhibition) were recorded. All drugs are prepared in dimethyl sulfoxide as stock solutions at a concentration of 1 x 10-2 M and stored at -20 ° C. Eight preparations are typically tested in parallel; two test preparations incorporate amiloride and / or benzamil as positive controls. After the maximum concentration (5 x 10-5 M) is administered, the luminal bath is exchanged three times with the KBR solution without drug, and the resulting ISC measured after each wash for approximately 5 minutes. Reversibility is defined as the percentage of return to baseline for the sodium stream after the third wash. All voltage clamps are collected via a computer interface and analyzed offline.
[0104] Dose-effect relationships for all compounds are considered and analyzed using the Prism 3.0 program. IC 50 values, maximum effective concentrations, and reversibility are calculated and compared with amiloride and benzamil as positive controls. The potency of the sodium channel blocking activity of representative compounds in cells newly excised from canine airways is shown in Table 2.
[0105] Bronchial cells (dog, man, sheep or rodent cells) are seeded at a density of 0.25 x 106 / cm2 over a collagen-coated Transwell-Col membrane with a growth area of 1.13 cm2 grown at an interface air-liquid in a hormonally defined medium that promotes a polarized epithelium. From 12 to 120 days after the development of an air-liquid interface (ALI) it is expected that the cultures are> 90% ciliated, and that mucins will accumulate on the cells. To guarantee the integrity of the primary airway epithelial cell preparations, the transepithelial resistance (Rt) and the differences in transepithelial potential (PD), which are indicators of the integrity of the polarized nature of the culture, are measured. Human cell systems are preferred for studies of apical surface absorption rates. The disappearance test is carried out under conditions that mimic "thin" films in vivo (~ 25 μl) and is initiated by the addition of experimental sodium channel blockers or positive controls (amiloride, benzamil, phenamil) to the apical surface at initial concentration of 10 μΜ. A series of samples (5 μl volume per sample) is collected at various points in time, including 0, 5, 20, 40, 90 and 240 minutes. Concentrations are determined by measuring the intrinsic fluorescence of each sodium channel blocker using a Fluorocount microplate fluorometer or HPLC. Quantitative analysis employs a standard curve generated from traditional reference materials of authentic concentration and known purity. The disappearance rate data analysis is performed using nonlinear regression, single-phase exponential decay (Prism V 3.0). 2. Confocal microscopy assay of amiloride congener absorption
[0106] Virtually all molecules similar to amiloride fluores-100 in the ultraviolet range. This property of these molecules can be used to directly measure cell refresh using x-z confocal microscopy. Equimolar concentrations of experimental compounds and positive controls including amiloride and compounds that demonstrate rapid absorption in the cell compartment (benzamyl and phenamyl) are placed on the apical surface of airway cultures on the platinum of the confocal microscope. Consecutive x-z images are obtained over time and the magnitude of fluorescence accumulating in the cell compartment is quantified and plotted as a change in fluorescence versus time. 3. In vitro tests of compound metabolism
[0107] Airway epithelial cells are able to metabolize drugs during the transepithelial absorption process. In addition, although less likely, it is possible that drugs can be metabolized on the epithelial surfaces of the airways by specific ectoenzyme activities. Perhaps more likely as an ectosurface event, the compounds can be metabolized by the infected secretions that occupy the lumens of the airways of patients with lung disease, for example, cystic fibrosis. For this reason, a series of tests is carried out to characterize the metabolism of the compound that results from the interaction of the test compounds with the human airway epithelium and / or human airway epithelial luminal products.
[0108] In the first series of assays, the interaction of the test compounds in KBR as an "ASL" stimulant is applied to the apical surface of human airway epithelial cells growing in the T-Col insert system. For most compounds, metabolism (production of new species) is tested using high performance liquid chromatography (HPLC) to resolve chemical species and the endogenous fluorescence properties of these compounds to estimate the relative amounts of the test compound and new metabolites. For a typical assay, a test solution (25 μL of KBR, containing 10 μM of test compound) is placed on the epithelial luminal surface. Sequential samples of 5 to 10 μl are obtained from the luminal compartments and for HPLC analysis of (1) the mass of test compound permeating the luminal bath into the serosal and (2) the potential formation of metabolites from the parent compound. In cases where the fluorescence properties of the test molecule are not suitable for such characterizations, radiolabeled compounds are used for these assays. From the HPLC data, the rate of disappearance and / or formation of new metabolite compounds on the luminal surface and the appearance of the test compound and / or a new metabolic in the basolateral solution is quantified. The data regarding the chromatographic mobility of new potential metabolites with reference to the parent compound are also quantified.
[0109] To analyze the potential metabolism of test compounds by CF sputum, a "representative" mixture of the expected sputum of CF obtained from 10 CF patients (under IRB approval) was collected. The sputum was solubilized in a 1: 5 mixture of KBR solution with vigorous stirring, after which the mixture was divided into a "clean" sputum aliquot and an aliquot was subjected to ultracentrifugation to obtain a "supernatant" aliquot ( clean = cell; supernatant = liquid phase). Typical studies of compound sputum metabolism by CF involve adding known masses of the test compound to "clean" CF sputum and aliquots of CF sputum "supernatant" incubated at 37 ° C, followed by sequential sampling of aliquots of each type of sputum to characterize the compound's stability / metabolism by HPLC analysis, as described above. As mentioned above, the analysis of the disappearance of the compound, the rates of formation of new metabolites, and the HPLC modalities of the new metabolites are then carried out. 4. Pharmacological effects and mechanism of action of the drug in animals
[0110] The effect of compounds to improve mucociliary clearance (MCC) can be measured using an in vivo model described by Sabater et al., Journal of Applied Physiology, 1999, pp. 2191-2196, hereby incorporated by reference. Methods
[0111] Animal preparation: Adult sheep (with weight ranging from 25 to 35 kg) were kept in an upright position in a special frame harness adapted for a modified shopping cart. The animals = heads were immobilized and local anesthesia of the nasal passage was induced with 2% lidocaine. The animals were then intubated through the nose with a 7.5 mm internal diameter endotracheal tube (ETT). The ETT wrist was placed just below the vocal cords and its position was checked with a flexible bronchoscope. After intubation, the animals were allowed to balance for approximately 20 minutes before beginning mucociliary clearance measurements. Radio-aerosol administration: Aerosols of human serum 99mTc-albumin (3.1 mg / ml; containing approximately 20 mCi) were generated using a Raindrop nebulizer that produces drops with an average aerodynamic diameter of 3.6 μm. The nebulizer was connected to a dosimetry system consisting of a solenoid valve and a source of compressed air (1,378 bar [20 psi]). The nebulizer outlet was directed to a plastic T connector; one end of which was connected to the endotracheal tube, and the other was connected to a plunger respirator. The system was activated for one second at the beginning of the respirator's inspiratory cycle. The respirator was adjusted to a tidal volume of 500 mL, an inspiratory to expiratory ratio of 1: 1, and a rate of 20 breaths per minute to maximize deposition in the central airway. The sheep inhaled the radiolabelled aerosol for 5 minutes. A gamma camera was used to measure the clearance of 99mTc-human serum albumin from the airways. The camera was positioned above the animal's back with the sheep in the natural vertical position supported on a cart so that the image field was perpendicular to the animal's spinal cord. External radiolabeled markers were placed on the sheep to ensure proper alignment of the gamma camera. All images were stored on a computer integrated with the gamma camera. A region of interest was drawn on the image corresponding to the sheep's right lung and the counts were recorded. The counts were corrected for decay and expressed as a percentage of radioactivity present in the baseline initial image. The left lung was excluded from the analysis because its contours were superimposed on the stomach and the counts can be swallowed and penetrate the stomach like radiolabelled mucus. Treatment Protocol (Evaluation of activity at t zero): A basal deposition image was obtained immediately after the administration of radioaerosol. At time zero, after baseline image acquisition, vehicle control (distilled water), positive control (amiloride), or experimental compounds were aerosolized from a 4 ml volume using a Pari LC JetPlus nebulizer for breathing animals freely. The nebulizer was activated by compressed air with a flow of 8 liters per minute. The solution distribution time was 10 to 12 minutes. The animals were extubated immediately after the distribution of the total dose to prevent false increases in the counts caused by the aspiration of the excess tracer from the ETT. Consecutive lung images were obtained at 15-minute intervals during the first 2 hours after application and hourly for the next 6 hours after application for a total observation period of 8 hours. An elimination period of at least 7 days separated the medication sessions with different experimental agents.
[0112] Treatment Protocol (Evaluation of activity in t 4 hours): The following variation of the treatment protocol was used to assess the durability of the response following a single exposure to control with vehicle (distilled water), positive control compounds (amiloride or ben -zamil), or investigative agents. At time zero, vehicle control (distilled water), positive control (amiloride), or investigational compounds were aerosolized from a volume of 4 ml using a Pari LC JetPlus nebulizer for animals breathing freely. The nebulizer was activated by compressed air with a flow of 8 liters per minute. The solution distribution time was 10 to 12 minutes. The animals were kept in an upright position in a special harness for 4 hours. At the end of the 4-hour period, the animals received a single dose of 99mTc-aerosolized human serum albumin (3.1 mg / ml; containing approximately 20 mCi) from a Raindrop nebulizer. The animals were extubated immediately after the distribution of the total dose of the radiotracerator. A basal deposition image was obtained immediately after the administration of radioaerosol. Consecutive lung images were obtained at 15-minute intervals during the first 2 hours after radiotracer administration (representing hours 4 to 6 after drug administration) and hourly for the next 2 hours after application for a period of total observation of 4 hours. An elimination period of at least 7 days separated the medication sessions with different experimental agents.
[0113] Statistics: The data were analyzed using SYSTAT for Windows, version 5. The data were analyzed using a repeated bilateral ANOVA (to assess the overall effects), followed by a paired t test to identify differences between specific pairs. Significance was recognized when P was less than or equal to 0.05. The slope values (calculated from data collected during the initial 45 minutes after application in the evaluation at t zero) for the mean MCC curves were calculated using least squares linear regression to assess differences in the initial rates during the clearance phase fast. EXAMPLES
[0114] Having described this invention in general, it is possible to understand it better with reference to certain specific examples which are offered by way of illustration only and are not intended to limit it in any way, unless otherwise specified. Preparation of sodium channel blockers
[0115] Materials and methods. All reagents and solvents were purchased from Aldrich Chemical Corp. and used without further purification. The proton and carbon NMR spectra were obtained on a Bruker AC 300 spectrometer at 300 MHz and 75 MHz, respectively. The proton spectra were referred to as tetramethylsilane as an internal standard and the carbon spectra were referenced to CDCl3, CD3OD, acetone-d6 or DMSO-d6 (purchased from Aldrich or Cambridge Isotope Laboratories, unless otherwise specified). The melting points were obtained in a Mel-Temp II device and were not corrected. The ESI mass spectra were obtained on a Shimadzu LCMS-2010 EV mass spectrometer. HPLC analyzes were obtained using a Waters XTerra RP C18 analytical column detected at 220 nm (unless otherwise specified) on a Shimadzu Prominence HPLC system. With a flow rate of 1.0 mL per minute, the following time program was used:
[0116] The following definitions of abbreviations apply unless otherwise noted.
[0117] Acetyl chloride (27.1 ml, 380.1 mmol) was added dropwise to MeOH (225 ml) at room temperature, and compound 1 (15.0 g, 54.3 mmol) was added. The resulting solution was refluxed overnight and concentrated, and then crystallized from hexanes (700 mL) to give the desired compound 2 (14.0 g, 89%) as an off-white solid: 1H NMR (300 MHz, CD3OD ) δ 7.07 (d, J = 8.7 Hz, 2H), 6.71 (d, J = 8.7 Hz, 2H), 4.02 (t, J = 6.6 Hz, 1H), 3.68 (s, 3H), 2.75-2.57 (m, 2H), 2.22-2.05 (m, 2H). Preparation of compound 3;
[0118] To a solution of compound 2 (14.0 g, 48.2 mmol) in Me-OH / H2O (140 mL / 70 mL) were added NaHCO3 (28.5 g, 337.4 mmol) and Boc2O (11.5 g, 53.0 mmol) at 0 ° C. The resulting mixture was allowed to warm to room temperature and stirred overnight. The reaction mixture was distributed between EtOAc (300 ml) and water (300 ml). The aqueous layer was separated and extracted with EtOAc (2 × 300 ml). The combined organic extracts were washed with brine, dried over Na2SO4 and concentrated to give compound 3 (13.5 g, 90%) as an off-white solid: H NMR (300 MHz, CD3OD) δ 6.98 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 8.4 Hz, 2H), 4.03 (t, J = 4.5 Hz, 1H), 3.68 (s, 3H), 2 , 65-2.40 (m, 2H), 1.92-1.82 (m, 1H), 1.44 (s, 9H). Preparation of compound 4;
[0119] To a solution of compound 3 (13.5 g, 43.6 mmol) in pyridine (150 mL) was added triflate (7.3 mL, 43.6 mmol) at 0 ° C, and the reaction mixture was stirred. at 0 ° C for 3 hours. After concentrating, the reaction mixture was distributed between EtOAc (300 ml) and water (300 ml). The aqueous layer was separated and extracted with EtOAc (2 × 300 ml). The combined organic extracts were washed with brine, dried over Na2SO4 and concentrated. The residue was purified by chromatography (silica gel, 95: 5 CHCl3 / MeOH) to give the desired compound 4 (16.0 g, 83%) as a brownish solid: 1H NMR (300 MHz, CDCl3) δ 7.26 -7.23 (m, 2H), 7.20-7.17 (m, 2H), 5.08 (br s, 1H), 4.36 (br s, 1H), 3.71 (s, 3H ), 2.73-2.67 (m, 2H), 2,212.13 (m, 1H), 1.99-1.87 (m, 1H), 1.45 (s, 9H). Preparation of compound 6;
[0120] To a solution of compound 4 (16.0 g, 36.2 mmol) in anhydrous CH3CN (160 mL) was added TEA (20.9 mL, 144.8 mmol), 10% (t-Bu) 3P in hexanes (1.4 g, 7.2 mmol), benzyl but-3-ynylcarbamate (5.8.8 g, 43.4 mmol) and CuI (340 mg, 1.8 mmol) at room temperature. The resulting mixture was degassed with argon for 3 minutes and Pd (PPh3) 4 (4.1 g, 3.6 mmol) was added quickly in one go. After degassing with argon for 5 minutes, the resulting mixture was heated to 60 ° C for 12 hours. The reaction mixture was concentrated in vacuo and the residue was purified by column (silica gel, 40:60 hexanes / EA) to give compound 6 (16.0 g, 70%) as a brown oil: 1H NMR (300 MHz , CDCl3) δ 7.36-7.28 (m, 8H), 7.07 (d, J = 8.4 Hz, 2H), 5.12 (br s, 4 H), 4.33 (br s , 1H), 3.71 (s, 3H), 3.46-3.39 (m, 2H), 2.68-2.59 (m, 4H), 2.14-2.20 (m, 1H ), 1.96-1.84 (m, 1H), 1.44 (s, 9H). Preparation of compound 7;
[0121] Compound 6 (8.0 g, 16.1 mmol) was dissolved in 4 N HCl in dioxane (60 ml) at room temperature and the resulting solution was stirred for 1 hour. After concentrating, the residue was crystallized from MTBE to hydrochloric acid salt 7 (6.75 g, 90%) as a white solid: 1H NMR (300 MHz, CD3OD) δ 7.32-7.28 ( m, 7H), 7.18 (d, J = 8.1 Hz, 2H), 5.08 (s, 2H), 4.04 (t, J = 6.3 Hz, 1H), 3.82 ( s, 3H), 3.31 - 3.29 (m, 2H), 2,822.67 (m, 2H), 2.58 (t, J = 6.9 Hz, 2H), 2.26-2.08 (m, 2H). Preparation of compound 8;
[0122] To a solution of compound 7 (6.75 g, 15.6 mmol) in Me-OH / AcOH (70 mL / 15 mL) was added 37% CH2O in H2O (25.2 mL, 312 mmol) and cyanoborohydride sodium (7.84 g, 124.8 mmol). The reaction mixture was stirred at room temperature overnight. After concentrating, the residue was purified by column chromatography (silica gel, 10: 1 CH2Cl2 / MeOH, 10: 1: 0.1 CHCl3 / MeOH / NH4OH) to give the desired compound 8 (3.8 g, 63% ) as an off-white solid: 1H NMR (300 MHz, CDCl3) δ 7.36-7.26 (m, 7H), 7.11 (d, J = 8.1 Hz, 2H), 5.11 (s, 2H), 3.69 (s, 3H), 3.45-3.39 (m, 2H), 3.10 (t, J = 7.5 Hz, 1H), 2.65-2.60 (m , 4H), 2.32 (s, 6H), 2.01-1.88 (m, 2H). Preparation of compound 9;
[0123] To a solution of methyl ester 8 (3.80 g, 8.9 mmol) in THF / MeOH / H2O (30 mL / 30 mL / 10 mL) was added NaOH (3.50 g, 89.0 mmol). The reaction mixture was stirred at room temperature overnight. After concentrating, the residue was dissolved in H2O (50 ml) and 1N aqueous HCl was added to adjust the pH to 5. The resulting precipitate was removed by filtration and dried to give the desired compound 9 (3.25 g , 90%) as an off-white solid: 1H NMR (300 MHz, CD3OD) δ 7.32-7.28 (m, 7H), 7.17 (d, J = 8.4 Hz, 2H), 5.08 (s, 2H), 3.46-3.42 (m, 1H), 3.45-3.39 (m, 2H), 2.83-2.70 (m, 8H), 2.58 (t , J = 6.9 Hz, 2H), 2,152.05 (m, 2H). Preparation of compound 11;
[0124] To a solution of compound 9 (1.80 g, 4.4 mmol) in anhydrous DMF (20 mL) were added HATU (3.3 g, 8.8 mmol) and DIPEA (4.0 mL, 22.0 mmol ). The resulting solution was added at room temperature for 0.5 hour and amine 10 (2.1 g, 5.2 mmol) was added. The reaction mixture was stirred at room temperature overnight and distributed between EtOAc (200 ml) and water (200 ml). The aqueous layer was separated and extracted with EtOAc (2 × 200 ml). The combined organic extracts were washed with brine, dried over Na2SO4 and concentrated. The residue was purified by column chromatography (silica gel, 95: 5 CHCl3 / MeOH) to give the desired compound 11 (1.70 g, 49%) as a white solid: 1H NMR (300 MHz, CDCl3) δ 7 , 36-7.28 (m, 7H), 7.10 (d, J = 8.1 Hz, 2H), 5.10 (s, 3H), 4.97-4.90 (m, 1H), 3.86 (t, J = 6.6 Hz, 2H), 3.45-3.35 (m, 2H), 3.12 (s, 3H), 3.09-3.00 (m, 2H) , 2.96 (s, 3H), 2.91-2.71 (m, 2H), 2.62 (t, J = 6.3 Hz, 2H), 2.30 (s, 6H), 1, 97-1.31 (m, 26H). Preparation of compound 12;
[0125] A suspension of compound 11 (1.7 g, 2.1 mmol) and 10% Pd / C (800 mg) in MeOH / AcOH (25 mL / 1.0 mL) was subjected to hydrogenation conditions (1 atm ) for one night at room temperature. The reaction mixture was filtered through celite and washed with MeOH. The filtrate was concentrated in vacuo and crystallized from MTBE to give acetic salt 12 (1.45g, 90%) as a colorless oil: 1H NMR (400 MHz, CD3OD) δ 7.12 (s, 4H), 4 , 97-4.90 (m, 2H), 3.90-3.85 (m, 2H), 3.29 (s, 3H), 2.95 (s, 3H), 2.91 (t, J = 7.6 Hz, 2H), 2.63 (t, J = 6.8 Hz, 2H), 2.57-2.50 (m, 8H), 1.95 (s, 6H), 2.02 -1.96 (m, 2H), 1.80-1.62 (m, 10H), 1.50 (s, 9H), 1.45 (s, 9H). Preparation of compound 14;
[0126] To a solution of acetic acid salt 12 (1.45 g, 2.1 mmol) and methyl 3,5-diamino-6-chloropyrazine-2-carbonylcarbamimidothioate hydrochloric acid salt (13, 1.28 g, 3, 3 mmol) in EtOH (20 mL) DIPEA (2.17 g, 16.8 mmol) was added at room temperature. The reaction mixture was heated to 70 ° C for 2 hours, and then cooled to room temperature and concentrated in vacuo. The residue was purified by column chromatography (silica gel, 9: 1 CH2Cl2 / MeOH, 80: 18: 2 CH-Cl3 / MeOH / NH4OH) to give the desired compound 14 (1.15 g, 68%) as a yellow solid: 1H NMR (400 MHz, CD3OD) δ 7.09 (s, 4H), 4.87-4.82 (m, 2H), 3.86 (t, J = 7.2 Hz, 2H) , 3.24-3.18 (m, 3H), 3.22 (s, 3H), 2.96-2.91 (m, 4H), 2.64-2.47 (m, 4H), 2 , 30 (t, 6H), 1.99-1.72 (m, 10 H), 1.55-1.32 (m, 20 H). Preparation of compound 15 [Formula I] - 3,5-diamino-6-chloro-N- (N- (4- (4 - ((S) -3- (dimethylamino) -4 - ((S) -1- (dimethylamino) -6-guanidino-1-oxo-hexan-2-ylamino) -4-oxobutyl) phenyl) butyl) carbamimidoyl) pyrazine-2-carboxamide;
[0127] To a solution of compound 14 (670 mg, 0.7 mmol) in CH2Cl2 (20 mL) was added TFA (5.0 mL) and the resulting solution was stirred at room temperature for 2 hours. The reaction mixture was concentrated in vacuo and transformed into an azeotrope with 1N aqueous HCl 3 times to give the hydrochloric acid salt of 15 (formula I, 600 mg, 99%) as a yellow hygroscopic solid: 1H NMR (400 MHz, D2O) δ 7.16-7.15 (m, 4H), 3,843.81 (m, 1H), 3.26 (br s, 2H), 3.13-3.11 (m, 5H), 2, 91-2.86 (m, 9H), 2.61-2.53 (m, 4H), 2.25-2.11 (m, 2H), 1.74-1.23 (m, 10 H) .
[0128] • Potência (IC50) em células epiteliais brônquicas caninas: 5,3 ± 3,3 nM (n=5) (Figura 1) • Reversão do efeito máxima em células epiteliais brônquicas caninas: 6,8 ± 5,1% (n=3) do efeito foram perdidos com 3 lavagens • Taxa de absorção através células epiteliais brônquicas humanas: 0,12 ± 0,05 nM/cm /min (n=3) • Metabolismo por células epiteliais brônquicas humanas: Nenhum metabólito detectado apical ou basolateral • Estabilidade no plasma humano: Nenhum metabolismo detectado depois de 4 horas • Durabilidade da retenção de líquido na superfície das vias aéreas por células epiteliais brônquicas caninas: 94 ± 5% (n=3) de fluido retido 8 horas depois da distribuição da fármaco; 56 ± 6% (n=2) de fluido retido 24 horas depois da distribuição da fármaco • Metabolismo em hepatócito de rato: Sem evidência de metabolismo significativo em toda a incubação de 24 horas • Ligação a proteínas plasmáticas humanas: 80 ± 5% (n=6) ligados a proteínas plasmáticas Summary of in vitro data for compound 15: • Potency (IC50) in canine bronchial epithelial cells: 5.3 ± 3.3 nM (n = 5) (Figure 1) • Reversal of the maximum effect in canine bronchial epithelial cells: 6.8 ± 5.1% (n = 3) of the effect was lost with 3 washes • Absorption rate through human bronchial epithelial cells: 0.12 ± 0.05 nM / cm / min (n = 3) • Metabolism by human bronchial epithelial cells: No apical or basolateral metabolites detected • Stability in human plasma: No metabolism detected after 4 hours • Durability of fluid retention on the surface of the airways by canine bronchial epithelial cells: 94 ± 5% (n = 3) of fluid retained 8 hours after drug delivery; 56 ± 6% (n = 2) of fluid retained 24 hours after drug delivery • Metabolism in rat hepatocyte: No evidence of significant metabolism throughout the 24-hour incubation • Binding to human plasma proteins: 80 ± 5% (n = 6) bound to plasma proteins
[0129] A dose-response curve of ENaC inhibition with P-1046 (I) and amiloride is shown in Figure 1.
[0130] Eye data and methods for compound 15 (formula I, also known as I, also known as P-1046, also known as 1046): The effects of P-1046 concentration (formula I, 15) on tear production in ExLac rats are shown in Figure 2. The clearance of P-1046 (formula I, 15) from the ocular surface is shown in Figure 3. The effects of 10 mM P-1046 (formula I, 15) on tear production in ExLac rats are shown in Figure 4.
[0131] Procedures for measuring phenol red filament (PRT) from tear production: Animal species / strain: Rats, Sprague Dawley (SD); lacrimal gland surgically removed (ExLac).
[0132] Number / sex of animal: 3-4 females / group Formulation of the test article: All stock solutions for each application arm were prepared at most 48 hours before the start of the study. The concentration of ENaC blockers in all test article solutions was confirmed by spectrophotometry.
[0133] Administration of the test article: Both ipsilateral and contralateral eyes were medicated with 5 l of the test article solution.
[0134] Phenol red filament test (PRT): Tear production was measured using a ZoneQuick cotton filament impregnated with phenol red dye. The folded end of the filament was kept in the lateral-ventral conjunctival sac bottom for 10 seconds. The length of the tear streak in the filament was determined by measuring the length of the filament that changes color from yellow to red. A stereomicroscope was used to aid an accurate measurement (recorded in millimeters) of the wick / color change. Procedure for P-1046 (compound 15 formula I, also known as I, also known as P-1046, also known as 1046): Extraction of PRT filaments:
[0135] 1. 200 µL de ACN a 70% (70% de ACN, 30% de H2O) foram introduzidos em tubos de ensaio que já contêm filamentos. 2. Os tubos da etapa 1 foram turbilhonados por 30 segundos e sonicados por 1 minuto para submergir completamente os filamentos. 3. As soluções de amostra foram incubadas por 4 horas à temperatura ambiente. 4. As amostras foram turbilhonadas mais uma vez por 30 segundos. 5. 75 µL de cada solução de amostra (da etapa 4) foram transferidos para uma placa UPLC de 96 poços com mais 75 µL de uma fase móvel A (5mM de formiato de amônio, 0,1% de ácido fórmico em H2O) acrescentados a cada poço com amostra. 6. Padrões analíticos (10 mM, 1mM, 100 M, 10 M, 1 M, 100 nM, Buffer) foram preparados de maneira idêntica às soluções de filamento. 7. As concentrações de fármaco de todas as amostras foram analisadas por UPLC. All PRT filaments were collected in eppen-dorf tubes and stored at -20 ° C until the moment of analysis of the drug concentration. P-1046 was extracted from the filaments and analyzed as follows: 1. 200 µL of 70% ACN (70% ACN, 30% H2O) were introduced into test tubes that already contain filaments. 2. The tubes in step 1 were swirled for 30 seconds and sonicated for 1 minute to completely submerge the filaments. 3. The sample solutions were incubated for 4 hours at room temperature. 4. The samples were swirled again for 30 seconds. 5. 75 µL of each sample solution (from step 4) was transferred to a 96-well UPLC plate with an additional 75 µL of a mobile phase A (5mM ammonium formate, 0.1% formic acid in H2O) added each well with sample. 6. Analytical standards (10 mM, 1 mM, 100 M, 10 M, 1 M, 100 nM, Buffer) were prepared in the same way as filament solutions. 7. The drug concentrations of all samples were analyzed by UPLC.
[0136] • Retirar uma porção da solução e diluir a mesma em uma proporção 1:10. Colocar esta solução a 50°C para o estudo de estabilidade acelerada por 10 dias. • Obter dados de estabilidade por análise por HPLC. 5. Preparar um tampão com 2,8% de NaCl e 25 mM de citrato, pH da solução 4,2. A osmolaridade da solução é aproximadamente 940 mOsM.6. Acrescentar uma quantidade apropriada da solução tampão a um composto de teste até atingir uma concentração aproximada de 10 mg/mL.7. Turbilhonar a solução por 15 segundos, sonicar por 30 segundos, turbilhonar novamente por 60 segundos e observar visualmente a solução.8. Calcular a concentração final da solução por meio de um es-pectrofotômetro. • Retirar uma porção da solução e diluir a mesma em uma proporção 1:10. Colocar esta solução a 50°C para o estudo de estabilidade acelerada por 10 dias. • Obter dados de estabilidade por análise por HPLC. Procedure for assessing solubility and stability: 1. Prepare a buffer with 2.8% NaCl and 25 mM citrate, pH of the solution 4.2. The osmolality of the solution is approximately 940 mOsM. 2. Add an appropriate amount of the buffer solution to a test compound until it reaches an approximate concentration of 10 mg / mL. 3. Swirl the solution for 15 seconds, sonicate for 30 seconds, swirl again for 60 seconds and visually observe the solution. 4. Calculate the final concentration of the solution using a spectrophotometer. • Remove a portion of the solution and dilute it 1:10. Place this solution at 50 ° C for the accelerated stability study for 10 days. • Obtain stability data by HPLC analysis. 5. Prepare a buffer with 2.8% NaCl and 25 mM citrate, pH of the solution 4.2. The osmolarity of the solution is approximately 940 mOsM. 6. Add an appropriate amount of the buffer solution to a test compound until it reaches an approximate concentration of 10 mg / mL. 7. Swirl the solution for 15 seconds, sonicate for 30 seconds, swirl again for 60 seconds and visually observe the solution. 8. Calculate the final concentration of the solution using a spectrophotometer. • Remove a portion of the solution and dilute it 1:10. Place this solution at 50 ° C for the accelerated stability study for 10 days. • Obtain stability data by HPLC analysis.
[0137] Figure 5 shows the HPLC analysis of the samples for the solubility / stability of P-1046 (formula I, 15) on day 1 and day 10. Summary of safety and tolerability for compound 15 (formula I, also known as I, also known as P-1046, A study of acute non-LPG eye toxicity in New Zealand white rabbits with P-1046 also known as 1046): • OBJECTIVE:
[0138] The purpose of this study was to assess eye tolerability and systemic exposure of P-1046, an epithelial sodium channel inhibitor (E-NaC), when administered as a topical instillation to New Zealand white rabbits. • METHODS:
[0139] The test article (P-1046) was presented with a light yellow white powder. The test articles were then prepared as dosage solutions for topical application to the eye. Twenty-eight male New Zealand white rabbits experimentally naive, approximately 4 months old at the start of the study and weighing 2.6-3.1 kilos at randomization were distributed into treatment groups as shown in the table below:
[0140] The animals were medicated with 50 µl of the test compound or control vehicle in the globe of the right eye and 50 µl of saline in the globe of the left eye eight times (approximately one hour between each dose) in groups 1 and 6 and four times (approximately twice between each dose) in group 7. Mortality and clinical observations were recorded daily. Body weights were recorded when ran-domination / selection and before sacrifice on day 2. To assess the blinking index, eye blinks in the right eye were only counted for 3 minutes before treatment started and after all treatments. doses. Cases of nerve contraction and kicking in the eye during the 3-minute blink observation period were recorded as clinical observations. Food consumption was recorded daily. The eyes were classified according to Draize before treatment, after each dose and on day 2 approximately 24 hours after application. Blood for the assessment of toxicokinetics was collected from the animals in groups 2-7 before the first dose, 30 minutes after the intermediate dose (4th dose in groups 3, 5, and 6; 2nd dose in groups 2, 4, and 7) and 30 , 60, 120 and 240 minutes after the final dose on day 1. Blood samples for evaluation of serum chemical parameters were collected before the start of treatment and 120 and 240 minutes after the final dose. Urine samples were collected over a period of 4 hours after the final dose. All animals were sacrificed after the end of the study procedures on day 2. • RESULTS AND CONCLUSIONS:
[0141] The animals under study appeared to be in good health, as assessed by clinical observations, body weights and food consumption.
[0142] Eye irritation was assessed by measuring the blinking index, Draize evaluation of the eyes and clinical observations. The irritation associated with each treatment is discussed below. Vehicle
[0143] The vehicle was well tolerated with only minimal transient eye irritation following application. The animals in the vehicle control group had conjunctival redness in both eyes (Draize score max = 1) and isolated incidences of nerve contraction and kicking in the eyes. The incidence of conjunctival redness was slightly higher in vehicle treated eyes (right eye) compared to saline treated eyes (left eye), which suggests that the observed irritation may have resulted from both the effects of the vehicle and the application procedure (Figure 6). All eyes looked normal on day 2. 10 and 30mM P-1046
[0144] P-1046 formulations were well tolerated. After application, conjunctival redness was observed, but the severity and incidence of this signal were comparable to that of vehicle controls. All eyes in the 10 mM group looked normal on day 2.
[0145] A summary of ocular tolerability to P-1046 is shown in Figure 6.
[0146] Figure 7 shows the plasma levels of P-1046 after application to the eye.

权利要求:
Claims (9)
[0001]
Compound, characterized by the fact that it is represented by formula (I):
[0002]
Compound according to claim 1, characterized in that it is an acid addition salt of an inorganic acid or an organic acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid , p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, malonic acid, sulfosalicylic acid, glycolic acid, 2-hydroxy-3-naphthoate, pamoate, salicylic acid, stearic acid, phthalic acid, mandelic acid, and lactic acid.
[0003]
Pharmaceutical composition, characterized in that it comprises the compound as defined in claim 1, and a pharmaceutically acceptable carrier.
[0004]
Composition, characterized by the fact that it comprises: the compound as defined in claim 1; and a P2Y2 receptor agonist.
[0005]
Composition, characterized by the fact that it comprises: the compound as defined in claim 1; and a bronchodilator.
[0006]
Process for the preparation of hydrochloric acid addition salt of a compound represented by formula (I)
[0007]
Process according to claim 6, characterized in that the treatment is carried out in CH2Cl2.
[0008]
Process according to claim 6, characterized in that the aqueous HCl is 1N aqueous HCl.
[0009]
Process according to claim 6, characterized in that it further comprises, subsequent to the treatment and before azeotroping, concentrating the resulting reaction mixture under vacuum.

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法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according art. 34 industrial property law|

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2018-10-16| B07D| Technical examination (opinion) related to article 229 of industrial property law|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2019-11-19| B07E| Notice of approval relating to section 229 industrial property law|

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2020-02-27| B06U| Preliminary requirement: requests with searches performed by other patent offices: suspension of the patent application procedure|

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2020-06-30| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application according art. 36 industrial patent law|

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2020-11-03| B09A| Decision: intention to grant|

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2021-01-12| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 27/06/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161501524P| true| 2011-06-27|2011-06-27|

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US61/501,524|2011-06-27|

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PCT/US2012/044372|WO2013003444A1|2011-06-27|2012-06-27|A chemically and metabolically stable dipeptide possessing potent sodium channel blocker activity|

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