Treatments for pulmonary fibrosis

Abstract

The present invention provides a method for treating pulmonary fibrosis using an arylpiperazine derivative. The method comprises the step of administering an effective amount of a compound of formula I to a pulmonary fibrosis patient in need thereof, wherein the compound of formula I is an arylpiperazine derivative.

Description

Technical Field

[0001] The present invention relates to methods of treating pulmonary fibrosis using arylpiperazine derivatives.

[0002] background

[0003] Pulmonary fibrosis (PF) is a progressive respiratory disorder characterized by scarring and thickening of the lung lining, resulting in an irreversible loss of the ability to transport and exchange oxygen. As lung tissue becomes scarred, it becomes stiffer, making it harder for the lungs to inflate and deflate. When this happens, less oxygen is transferred to the bloodstream, making breathing more difficult. As PF worsens, individuals become progressively weaker and short of breath, and this damage can eventually lead to death. If the cause of PF cannot be clearly identified, the condition is called idiopathic pulmonary fibrosis (IPF).

[0004] IPF is a chronic disease that destroys the interstitial spaces within the lungs and is the most common form of diffuse parenchymal lung disease. It is increasingly understood that IPF is the result of an irreversible cascade of fibrosis and abnormal wound healing. The course of pulmonary fibrosis and the severity of symptoms can vary significantly between individuals. The main signs and symptoms of PF include shortness of breath (dyspnea), dry cough, fatigue, unexplained weight loss, muscle and joint pain, and widening and rounding of the tips of the fingers or toes (clubbing). Co-morbidities are common in patients with PF. The most common co-morbidities are emphysema (often clinically referred to as CPFE: combined pulmonary fibrosis and emphysema), pulmonary hypertension (PH), venous thromboembolism, lung cancer, gastroesophageal reflux disease (GERD), cardiovascular disease, diabetes, and neuropsychiatric symptoms such as psychosis, depression, anxiety, and cognitive deficits.

[0005] The exact prevalence of IPF worldwide is unknown, but the American Lung Association estimates that IPF affects approximately 140,000 Americans each year and kills approximately 40,000 people each year. IPF usually develops in people aged ≥50 years (range 40-70 years), affecting more men than women. The median survival after initial diagnosis is 2 to 3 years. Approximately two-thirds of people with IPF die within 5 years, and the risk of death increases with age.

[0006] Although many advances have been made in recent years, especially those related to the molecular genetics and cell biology of pulmonary fibrosis (PF), the pathogenesis of PF is still not fully understood. Injury and destruction of epithelial or endothelial cells during normal wound healing leads to the development of pulmonary fibrosis (Wynn 2011). The morphological hallmarks of pulmonary fibrosis are alveolar fibrosis, vascular fibrosis, blood clotting and coagulation, lung inflammation, and respiratory resistance (Wynn 2011). Serotonin (5-HT) and key serotonin receptors have been reported to play a key role in the pathobiology of PF. Elevated serotonin levels in epithelial and endothelial cells of patients with PF as well as serotonin 5-HT have been reported. 2A , 5-HT 2B and 5-HT7 receptor expression. 2A Receptor regulation can regulate blood coagulation and clotting, proliferation and vasodilation. 5-HT 2B Receptor regulation plays a central role in the regulation of fibrosis and proliferation, and the 5-HT7 receptor has been reported to regulate inflammatory cytokines and chemokines (Lofdhal 2016, Mann 2013, and Dees 2011).

[0007] Despite an increasing understanding of the pathobiology of pulmonary fibrosis (PF), the prognosis for patients remains poor. PF is irreversible, and no current therapy can stop or significantly slow disease progression. Typically, treatment strategies for PF are aimed at improving quality of life (i.e., relieving disease signs / symptoms) or attempting to limit further inflammation and scarring. Anti-inflammatory drugs, including corticosteroids and cytotoxic agents, have also been used, although there is no evidence of a benefit on long-term survival. Pirfenidone and nintedanib are two FDA-approved drugs for the management of IPF. Both pirfenidone and nintedanib have been reported to reduce fibrotic tissue in the lungs of patients with PF to some extent, but the treatments are far from optimal. There is an urgent need for more effective and tolerable next-generation therapies or treatments that can significantly delay the progression of pulmonary fibrosis, even if they cannot provide a cure and improve the patient's overall quality of life (QOL).

[0008] BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Depicted is the schedule for various treatments of animals during a bleomycin-induced IPF study.

[0010] Figure 2Survival curves of animals from days 1-10 (A) and from days 11-21 (B) and body weight (C) are shown for sham, BLM-induced and treated groups. BLM: bleomycin; sham: animals treated with vehicle and not induced. *P<0.05 BLM+Veh; compared to sham. **P<0.05; compared to BLM+Veh.

[0011] Figure 3 Hemodynamic and cardiac parameters (AC) and systemic arterial pressure (DF) measured on day 21 are shown. BLM: Bleomycin; Sham: Animals treated with vehicle and not induced. *P<0.05 BLM+Veh; compared to Sham. #P<0.05; compared to BLM+Veh. ##P<0.05; compared to BLM+Veh.

[0012] Figure 4 Respiratory resistance (A) and lung hydroxyproline (B) measured on day 21 are shown. BLM: bleomycin; sham: animals treated with vehicle and not induced. *P<0.05, BLM+Veh; compared to sham. ***P<0.001; compared to sham. #P<0.05; compared to BLM+Veh. ##P<0.01; compared to BLM+Veh.

[0013] Figure 5 Parameters reflecting lung edema on day 21 are shown, including lung weight (A), BALF cell count (B), and BALF total protein (C). BALF: bronchoalveolar lavage; BLM: bleomycin; sham: animals treated with vehicle and not induced. **P<0.01, BLM+Veh; compared to sham. ***P<0.001; compared to sham. #P<0.05; compared to BLM+Veh.

[0014] Figure 6 Morphological changes revealed by H&E staining and Ashcroft score (A, C) are shown. Masson trichrome staining (B, D) induced by BLM in rats at day 21 reflects the deposition of collagen. BLM: Bleomycin; Sham: Animals with vehicle and not induced. BLM+RP5063 (all animals). ***P<0.001; compared to Sham. ###P<0.001; compared to BLM+Veh.

[0015] Figure 7The effects of BLM induction on blood oxygen saturation (A) and blood lactate levels (B) measured on day 21 are shown. BLM: Bleomycin; Sham: Animals treated with vehicle and not induced. **P<0.01; compared to Sham. #P<0.05; compared to BLM+Veh. ##P<0.01; compared to BLM+Veh.

[0016] Figure 8 BALF cytokine levels at day 21 are shown: MIP1 (A); MCP1 (B); IL6 (C); IP10 (D); and RANTES (E). BLM: Bleomycin; Sham: Animals treated with vehicle and not induced. *P<0.05; compared to Sham. ***P<0.001; compared to Sham. #P<0.05; compared to BLM+Veh. ##P<0.01; compared to BLM+Veh. ###P<0.001; compared to BLM+Veh. DETAILED DESCRIPTION OF THE INVENTION

[0018] definition

[0019] "Alkyl" or "hydrocarbon" refers to a saturated, branched or straight chain or cyclic monovalent hydrocarbon radical derived by removing a hydrogen atom from a single carbon atom of a parent alkane. Typical hydrocarbon radicals include, but are not limited to, methyl; ethyl; propyl, such as prop-1-yl, prop-2-yl, cycloprop-1-yl; butyl, such as but-1-yl, but-2-yl, 2-methyl-prop-1-yl, 2-methyl-prop-2-yl, cyclobut-1-yl, etc. Preferably, the hydrocarbon radical contains 1-20 carbon atoms, more preferably, 1-10, or 1-6, or 1-4 carbon atoms.

[0020] "Alkenyl" refers to an unsaturated branched, straight chain or cyclic alkyl radical having at least one carbon-carbon double bond derived by removing a hydrogen atom from a single carbon atom of a parent alkene. The radical may be in a cis or trans configuration relative to the double bond. Typical alkenyl groups include, but are not limited to, vinyl; propenyl, such as prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl, cycloprop-1-en-1-yl, cycloprop-2-en-1-yl; butenyl, such as but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, but-1,3-dien-1-yl, but-1,3-dien-2-yl, cyclobut-1-en-1-yl, cyclobut-1-en-3-yl, cyclobut-1,3-dien-1-yl, etc.; etc.

[0021] "Alkynyl" refers to an unsaturated branched, straight chain or cyclic alkyl radical having at least one carbon-carbon triple bond derived by removing a hydrogen atom from a single carbon atom of a parent alkyne. Typical alkynyl groups include, but are not limited to, ethynyl; propynyl, such as prop-1-yn-1-yl, prop-2-yn-1-yl, etc.; butynyl, such as but-1-yn-1-yl, but-1-yn-3-yl, but-3-yn-1-yl, etc.; and the like.

[0022] "Acyl" refers to the group -C(O)R, wherein R is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arene, heteroalkyl, heteroaryl, heteroarene, as defined herein, optionally substituted with one or more substituents as defined herein. Representative examples include, but are not limited to, formyl, acetyl, cyclohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.

[0023] "Acylamino" means an "amide" as defined herein.

[0024] "Alkylamino" refers to a group -NHR, wherein R is an alkyl group as defined herein, or a cycloalkyl group, optionally substituted with one or more substituents as defined herein. Representative examples include, but are not limited to, methylamino, ethylamino, 1-methylethylamino, cyclohexylamino, and the like.

[0025] "Alkyloxy" refers to a group -OR, wherein R is an alkyl group as defined herein, or a cycloalkyl group, optionally substituted with one or more substituents as defined herein. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclohexyloxy, and the like.

[0026] "Alkyloxycarbonyl" refers to the group -C(O)-alkoxy where alkoxy is as defined herein.

[0027] "Alkylsulfonyl" refers to the group -S(O)2R, wherein R is an alkyl group as defined herein, or a cycloalkyl group, optionally substituted with one or more substituents as defined herein. Representative examples include, but are not limited to, methylsulfonyl, ethylsulfonyl, propylsulfonyl, butylsulfonyl, and the like.

[0028] "Alkylsulfinyl" refers to the group -S(O)R, wherein R is an alkyl or cycloalkyl group as defined herein, optionally substituted with one or more substituents as defined herein. Representative examples include, but are not limited to, methylsulfinyl, ethylsulfinyl, propylsulfinyl, butylsulfinyl, and the like.

[0029] "Hydrocarbylthio" refers to a group -SR, wherein R is a hydrocarbon or cycloalkyl group as defined herein, optionally substituted with one or more substituents as defined herein. Representative examples include, but are not limited to, methylthio, ethylthio, propylthio, butylthio, and the like.

[0030] "Amide" or "acylamino" refers to the group -NR'C(O)R", wherein R' and R" are each independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, aromatic hydrocarbon, heteroalkyl, heteroaryl, heteroaromatic hydrocarbon, as defined herein, optionally substituted with one or more substituents as defined herein. Representative examples include, but are not limited to, formylamino, acetylamino, cyclohexylcarbonylamino, cyclohexylmethylcarbonyl-amino, benzoylamino, benzylcarbonylamino, and the like.

[0031] "Amino" refers to the group -NH2.

[0032] "Aryl" refers to a monovalent aromatic hydrocarbon radical derived by removing one hydrogen atom from a single carbon atom of a parent aromatic ring system. Typical aryl groups include, but are not limited to, radicals derived from aceanthrylene, acenaphthylene, acephenanthrylene, anthracene, azulene, benzene, chrysene, coronene, fluoranthene, fluorine, hexacene, hexaphene, hexalene, as-indacene, s-indacene, indane, indene, naphthalene, octacene, octaphene , octalene, ovalene, pentane-2,4-diene, pentacene, pentalene, pentaphene, perylene, phenalene, phenanthrenes, picene, pleidene, pyrene, pyranthrene, rubicene, triphenylene, trinaphthalene, etc. Preferably, the aryl group contains 6 to 20 carbon atoms, more preferably 6 to 12 carbon atoms.

[0033] "Aromatic hydrocarbon group" refers to an acyclic hydrocarbon group in which a hydrogen atom (usually a terminal or sp 3 One of the carbon atoms) is substituted by an aryl group. Typical aromatic hydrocarbon groups include, but are not limited to, benzyl, 2-phenylethyl-1-yl, naphthylmethyl, 2-naphthylethyl-1-yl, naphthylbenzyl, 2-naphthylphenylethyl-1-yl, etc. Preferably, the aromatic hydrocarbon group is (C6-C 30 ) aromatic hydrocarbon group, for example, the hydrocarbon group portion of the aromatic hydrocarbon group is (C1-C10 ) and the aryl moiety is (C6-C 20 ), more preferably, the aromatic hydrocarbon group is (C6-C 20 ) aromatic hydrocarbon group, for example, the hydrocarbon portion of the aromatic hydrocarbon group is (C1-C8) and the aryl portion is (C6-C 12 ).

[0034] "Aryloxy" refers to the group -O-aryl wherein aryl is as defined herein optionally substituted by one or more substituents as defined herein.

[0035] "Aryloxycarbonyl" refers to the group -C(O)-O-aryl wherein aryl is as defined herein optionally substituted by one or more substituents as defined herein.

[0036] "Carbamoyl" refers to the group -C(O)NRR wherein each R group is independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined herein, optionally substituted with one or more substituents as defined herein.

[0037] "Carbamate" refers to the group -NR'C(O)OR", wherein R' and R" are each independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined herein, optionally substituted with one or more substituents as defined herein. Representative examples include, but are not limited to, methylcarbamate (-NHC(O)OCH3), ethylcarbamate (-NHC(O)OCH2CH3), benzylcarbamate (-NHC(O)OCH2C6H5), and the like.

[0038] "Carbonate" refers to the group -OC(O)OR, where R is alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, as defined herein, optionally substituted with one or more substituents as defined herein. Representative examples include, but are not limited to, methyl carbonate (-C(O)OCH3), cyclohexyl carbonate (-C(O)OC6H 11 ), phenyl carbonate (-C(O)OC6H5), benzyl carbonate (-C(O)OCH2C6H5), etc.

[0039] "Carboxy" refers to the group -C(O)OH.

[0040] "Cyano" refers to the radical -CN.

[0041] "Cycloalkyl" refers to a substituted or unsubstituted cyclic hydrocarbon group. Typical cycloalkyl groups include, but are not limited to, groups derived from cyclopropane, cyclobutane, cyclopentane, cyclohexane, etc. In a preferred embodiment, the cycloalkyl group is (C3-C 10)cycloalkyl, more preferably (C3-C7)cycloalkyl.

[0042] "Cycloheteroalkyl" refers to a saturated or unsaturated cyclic hydrocarbon group in which one or more carbon atoms (and any associated hydrogen atoms) are independently replaced by the same or different heteroatoms. Typical heteroatoms replacing carbon atoms include, but are not limited to, N, P, O, S, Si, and the like. Where a particular level of saturation is to be achieved, the term "cyclohetero" or "cycloheteroalkenyl" is used. Typical cycloheteroalkyl groups include, but are not limited to, groups derived from epoxides, imidazolidines, morpholines, piperazines, piperidines, pyrazolidines, pyrrolidines, quinuclidines, and the like.

[0043] "Halogen" refers to fluorine, chlorine, bromine, or iodine.

[0044] "Heteroaryl" refers to a monovalent heteroaromatic group derived by the removal of one hydrogen atom from a single atom of a parent heteroaromatic ring system. Typical heteroaryl groups include, but are not limited to, groups derived from acridine, indole, carbazole, carboline, chromane, chromene, benzodihydronaphthalene, furan, imidazole, indazole, indole, indoline, indolizine, isobenzofuran, isochromene, isoindole, isoindoline, isoquinoline, isothiazole, isoxazole, naphthyridine, oxadiazole, oxazole, piperidine, phenanthridine, phenanthroline, phenazine, phthalazine, piperidine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, tetrazole, thiadiazole, thiazole, thiophene, triazole, xanthene, and the like. Preferably, the heteroaryl group is a 5-20 membered heteroaryl group, wherein a 5-10 membered heteroaryl group is particularly preferred. Preferred heteroaryl groups are groups derived from thiophene, pyrrole, benzothiophene, benzofuran, indole, pyridine, quinoline, imidazole, oxazole and pyrazine.

[0045] "Heteroaryloxycarbonyl" refers to the group -C(O)-OR wherein R is heteroaryl as defined optionally substituted with one or more substituents as defined herein.

[0046] "Heteroarylalkyl" refers to an acyclic hydrocarbon radical in which one of the hydrogen atoms (usually terminal or sp 3 Preferably, the heteroarylalkyl group is a heteroarylalkyl group of 6-30 carbon atoms, for example, the alkyl portion of the heteroarylalkyl group is 1-10 members and the heteroaryl portion is a 5-20 member heteroaryl, more preferably a 6-20 member heteroarylalkyl group, for example, the alkyl portion of the heteroarylalkyl group is 1-8 members and the heteroaryl portion is a 5-12 member heteroaryl.

[0047] "Hydroxy" refers to the radical -OH.

[0048] "Oxo" refers to a divalent radical =0.

[0049] "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0050] "Pharmaceutically acceptable salts" refer to salts of the compounds of the present invention that are pharmaceutically acceptable and possess the desired pharmacological activity of the parent compound. Such salts include: (1) acid addition salts, formed with inorganic acids (such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc.); or with organic acids (such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid) , camphorsulfonic acid, 4-methylbicyclo[2,2,2]-oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid, laurel sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, etc.); or (2) salts formed when the acidic protons present in the parent compound are replaced by metal ions (such as alkali metal ions, alkaline earth metal ions, or aluminum ions); or coordination complexes with organic bases (such as ethanolamine, diethanolamine, triethanolamine, N-methylglucamine, etc.).

[0051] "Pharmaceutically acceptable carrier" refers to a diluent, adjuvant, excipient or vehicle with which a compound of the present invention is administered.

[0052] "Phosphate" refers to the group -OP(O)(OR')(OR"), wherein R' and R" are each independently hydrogen, hydrocarbyl, cyclohydrocarbyl, cycloheterohydrocarbyl, aryl, arene, heterohydrocarbyl, heteroaryl, heteroarene, as defined herein, optionally substituted with one or more substituents as defined herein.

[0053] "Phosphate" refers to the group -P(O)(OR')(OR"), wherein R' and R" are each independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, aromatic hydrocarbon, heteroalkyl, heteroaryl, heteroaromatic hydrocarbon, as defined herein, optionally substituted with one or more substituents as defined herein.

[0054] "Prevent" or "prevention" refers to reducing the risk of developing a disease or disorder (i.e., causing at least one clinical symptom of the disease not to develop in a patient who may be exposed or susceptible to the disease but does not yet experience or display symptoms of the disease).

[0055] "Racemate" refers to an equimolar mixture of enantiomers of a chiral molecule.

[0056] "Substituted" refers to a group in which one or more hydrogen atoms are independently replaced by the same or different substituents. Typical substituents include, but are not limited to, -X, -R 54 , -O - , = O, -OR 54 , -SR 54 ,-S,=S,-NR 54 R 55 , =NR 54 , -CX3, -CF3, -CN, -OCN, -SCN, -NO, -NO2, =N2, -N3, -S(O)2O - , -S(O)2OH, -S(O)2OR 54 , -OS(O)2O 31 , -OS(O)2R 54 ,-P(O)(O-)2,-P(O)(OR 14 )(O 31 ), -OP(O)(OR 54 )(OR 55 ), -C(O)R 54 , -C(S)R 54 , -C(O)OR 54 , -C(O)NR 54 R 55 , -C(O)O - , -C(S)OR 54 , -NR 56 C(O)NR 54 R 55 , -NR 56 C(S)NR 54 R 55 , -NR 57 C(NR 56 )NR 54 R 55 , and -C(NR 56 )NR 54 R 55 , wherein each X is independently halogen; R 54 , R 55 , R 56 and R 57 are each independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aromatic alkyl, substituted aromatic alkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroaromatic alkyl, substituted heteroalkyl, -NR 58 R59 , -C(O)R 58 or -S(O)2R 58 or optionally R 58 and R 59 Together with the atoms to which they are attached, they form a cycloheteroalkyl or substituted cycloheteroalkyl ring; and R 58 and R 59 and R is independently hydrogen, hydrocarbyl, substituted hydrocarbyl, aryl, substituted aryl, arene, substituted arene, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroalkyl, substituted heteroalkyl, heteroaryl, substituted heteroaryl, heteroaralkyl, substituted heteroaralkyl.

[0057] "Sulfate" refers to the group -OS(O)(O)OR where R is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arene, heteroalkyl, heteroaryl, heteroaralkyl, as defined herein, optionally substituted with one or more substituents as defined herein.

[0058] "Sulfonamide" refers to the group -S(O)(O)NR'R", wherein R' and R" are independently hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arene, heteroalkyl, heteroaryl, heteroaralkyl, as defined herein, optionally substituted with one or more substituents as defined herein, or optionally R' and R" together with the atoms to which they are both attached form a cycloheteroalkyl or substituted cycloheteroalkyl ring. Representative examples include, but are not limited to, azetidinyl, pyrrolidinyl, piperidinyl, morpholinyl, 4-(NR"')-piperazinyl or imidazolyl, wherein said groups are optionally substituted with one or more substituents as defined herein. R"' is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arene, heteroalkyl, heteroaryl, heteroaralkyl, as defined herein, optionally substituted with one or more substituents as defined herein.

[0059] "Sulfonate" refers to the group -S(O)(O)OR where R is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arene, heteroalkyl, heteroaryl, heteroarene, as defined herein, optionally substituted with one or more substituents as defined herein.

[0060] "Thio" refers to the group -SH.

[0061] "Thioether" refers to a group -SR where R is alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl as defined herein, optionally substituted with one or more substituents as defined herein.

[0062] In one embodiment, "treating" or "treating" any disease or disorder refers to ameliorating the disease or disorder (i.e., arresting or alleviating the development of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to improving at least one physical parameter that may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to inhibiting a disease or disorder physically (e.g., stabilization of discernible symptoms), physiologically (e.g., stabilization of a physical parameter), or both.

[0063] "Therapeutically effective amount" refers to the amount of a compound that, when administered to a patient for treating a disease, is sufficient to affect the treatment of that disease. The "therapeutically effective amount" will vary depending on the compound, the disease, and the severity and age, weight, etc. of the patient to be treated, and can be determined by one skilled in the art without undue experimentation.

[0064] The present invention relates to a method for treating pulmonary fibrosis.

[0065] Compounds useful in the present invention

[0066] The compounds of formula (I) can be used in the present invention:

[0067]

[0068] in:

[0069] A is -O-(CH2) n -,-(CH2) n -,-S-(CH2) n -,-S(O)(O)-(CH2) n -, -NH-(CH2) n -,-CH2-O-(CH2) n -,-(CH2) n -O-CH2-CH2-, -CH2-S-(CH2) n -,-(CH2) n -S-CH2-CH2-, -CH2-S(O)(O)-(CH2) n -,-(CH2) n -S(O)(O)-CH2-CH2-, -OC(O)-(CH2) n -,-SC(O)-(CH2) n -,-NH-C(O)-(CH2) n -,-CH2-C(O)-O-(CH2) n -,-CH2-C(O)-NH-(CH2) n -,-CH2-C(O)-S-(CH2) n-,-(CH2) n -C(O)-O-CH2-CH2-, -(CH2) n -C(O)-NH-CH2-CH2-, -(CH2) n -C(O)-S-CH2-CH2-, -CH2-OC(O)-(CH2) n -,-CH2-NH-C(O)-(CH2) n -,-CH2-SC(O)-(CH2) n -,-(CH2) n -OC(O)-CH2-CH2-, (CH2) n -NH-C(O)-CH2-CH2-, or (CH2) n -SC(O)-CH2-CH2-, wherein n is an integer from 1 to 7, preferably n is from 2 to 5, for example n is 4;

[0070] B is O, S, S(O)(O), or NR 5 ;and

[0071] R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 and are independently hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, aromatic alkyl, substituted aromatic alkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl, substituted cycloheteroalkyl, heteroaryl, substituted heteroaryl, heteroaromatic alkyl, substituted heteroaromatic alkyl, acylalkyloxycarbonyl, acyloxyalkyloxycarbonyl, acylalkyloxycarbonylamino, acyloxyalkyloxycarbonylamino, alkyloxy, alkyloxycarbonyl, alkyloxycarbonylalkyloxy, alkyloxycarbonylalkyl amino, alkylsulfinyl, alkylsulfonyl, alkylthio, amino, alkylamino, arylalkylamino, dialkylamino, aryloxy, aryloxycarbonylalkyloxy, aryloxycarbonylalkylamino, aryloxycarbonyl, aryloxycarbonylalkyloxy, aryloxycarbonylalkylamino, carboxyl, carbamoyl, carbamate, carbonate, cyano, halogen, heteroaryloxycarbonyl, hydroxy, phosphate, phosphonate, sulfate, sulfonate, or sulfonamide, wherein R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 and R 8 and A may be optionally substituted with isotopes, including but not limited to 2 H (deuterium),3 H (tritium), 13 C, 36 Cl, 18 F, 15 N, 17 Oh, 18 Oh, 31 P, 32 P, and 35 S; preferred 2 H (deuterium);

[0072] or a pharmaceutically acceptable salt, racemate or diastereomeric mixture thereof.

[0073] In one aspect of the invention, A is -O-(CH2) n -.

[0074] In one aspect of the invention, A is -(CH2) n -.

[0075] In another aspect of the invention, A is -S-(CH2) n -,-CH2-O-(CH2) n -,-(CH2) n -O-CH2-CH2-, -CH2-S-(CH2) n -, or -(CH2) n -S-CH2-CH2-; preferably A is -O-(CH2) n -.

[0076] In another aspect of the invention, A is -NH-C(O)-(CH2) n -,-CH2-NH-C(O)-(CH2) n -,-CH2-C(O)-NH-(CH2) n -or-(CH2) n -C(O)-NH-CH2-CH2-.

[0077] In another aspect of the invention, B is O.

[0078] In another aspect of the present invention, R 3 , R 4 , R 6 , R 6 , and R 8 It's H.

[0079] In a preferred embodiment, A is -O-(CH2) n - or -NH-C(O)-(CH2) n -, n=2-5.

[0080] In a preferred embodiment, B is O.

[0081] In a preferred embodiment, R 3 , R 4 , R 6 , R 6 , and R 8 It's H.

[0082] In a preferred embodiment, R 1 and R 2 Each is independently H, halogen (eg, chlorine), halogenated hydrocarbon group, or hydrocarbon group (eg, methoxy or ethoxy); preferably halogen or hydrocarbon group.

[0083] Preferred compounds of formula I include, for example, the following compounds AD and their hydrochlorides.

[0084]

[0085] 6-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2H-benzo[b][1,4]oxazin-3(4H)-one (brilaroxazine, or compound A);

[0086]

[0087] 6-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-2H-benzo[b][1,4]oxazin-3(-4H)-one (Compound B);

[0088]

[0089] Compounds useful in the present invention further relate to enantiomerically separated compounds of formula I. The separated enantiomeric forms of the compounds of formula I are substantially free of each other (i.e., enantiomeric excess). In other words, the "R" form of the compound is substantially free of the "S" form of the compound, and therefore, the "R" form is in enantiomeric excess. In contrast, the "S" form of the compound is substantially free of the "R" form of the compound, and therefore, the "S" form is in enantiomeric excess. In one embodiment of the present invention, the separated enantiomeric compound is in at least about 80% enantiomeric excess. Thus, for example, the compound is in at least about 90% enantiomeric excess, preferably at least about 95% enantiomeric excess, more preferably at least about 97% enantiomeric excess, or further preferably at least 99% or greater than 99% enantiomeric excess.

[0090] Compounds of Formula I can be synthesized according to US Pat. No. 8,188,076, which is incorporated herein in its entirety.

[0091] Treatments for pulmonary fibrosis

[0092] The present invention relates to a method for treating pulmonary fibrosis (PF) and idiopathic pulmonary fibrosis (IPF). If the cause of PF cannot be clearly identified, this condition is referred to as IPF. Although the causes of PF and IPF may be different, the signs and symptoms of PF and IPF are the same, and the present invention can effectively treat PF and IPF, regardless of the cause. The method includes the step of administering an effective amount of a compound of formula I to a patient suffering from pulmonary fibrosis. The compound of formula I can reduce fibrosis in the patient's pulmonary artery (the blood vessel leading from the heart to the lungs) or alveoli and treat pulmonary fibrosis. The treatment can also reduce disease complications, such as lung inflammation, shortness of breath, pain crisis (crisis), pneumonia, and increase survival.

[0093] In one embodiment, the method treats pulmonary fibrosis in a subject having chronic obstructive pulmonary disease (COPD), pulmonary arterial hypertension (PAH), sickle cell disease (SCD), scleroderma, or lung cancer.

[0094] In one embodiment, the method treats comorbid psychiatric disorders, such as psychosis, depression, and mood symptoms, in a patient with pulmonary fibrosis. In another embodiment, the method treats anxiety in a patient with pulmonary fibrosis.

[0095] When used to treat pulmonary fibrosis, one or more compounds of Formula I may be administered to a patient alone or in combination with other agents. The patient may be an animal, preferably a mammal, more preferably a human.

[0096] The compound of formula I is preferably administered orally. The compound of formula I may also be administered by any other convenient route, for example, by infusion or bolus injection, by absorption of epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.). It may be systemic or topical. A variety of delivery systems (e.g., encapsulated in liposomes, microparticles, microcapsules, capsules, etc.) known to be useful for administering the compounds and / or compositions of the present invention are known. Methods of administration include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, oral, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by inhalation, or locally, particularly to ears, noses, eyes, or skin. Inhalation or transdermal administration may be preferred for young children.

[0097] The compounds of formula I may be delivered by a sustained release system, preferably an oral sustained release system. In one embodiment, a pump may be used (see, Langer, supra; Seffon, 1987, CRC Crit. Ref Biomed. Eng. 14:201; Saudek et al., 1989, N. Engl. J. Med. 321:574).

[0098] In one embodiment, polymeric materials can be used (see "Medical Applications of Controlled Release", Langer and Wise (Eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In a preferred embodiment, polymeric materials are used for oral sustained release delivery. Preferred polymers include sodium carboxymethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose and hydroxyethyl cellulose (most preferably hydroxypropyl methylcellulose). Other preferred cellulose ethers have been described in the art (Bamba et al., Int. J. Pharm., 1979, 2, 307).

[0099] In one embodiment, enteric coating formulations can be used for oral sustained release administration. Preferred coating materials include polymers with pH-dependent solubility (i.e., pH-controlled release), polymers with slow or pH-dependent swelling, dissolution or erosion rates (i.e., time-controlled release), polymers that are degraded by enzymes (i.e., enzyme-controlled release), and polymers that form strong layers that are destroyed by increased pressure (i.e., pressure-controlled release).

[0100] In another embodiment, an osmotic delivery system is used for oral sustained release administration (Verma et al., Drug Dev. Ind. Pharm., 2000, 26: 695-708). In a preferred embodiment, Osmotic delivery systems are used in oral sustained-release delivery devices (see, eg, Theeuwes et al., US Pat. No. 3,845,770; and Theeuwes et al., US Pat. No. 3,916,899).

[0101] In yet another embodiment, a controlled release system can be placed near the target of the compounds and / or compositions of the invention, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, "Medical Applications of Controlled Release," supra, vol. 2, pp. 115-138 (1984)). Other controlled release systems discussed in Langer, 1990, Science 249: 1527-1533 can also be used.

[0102] Compounds of formula I may be cleaved chemically and / or enzymatically. One or more enzymes present in the stomach, intestinal lumen, intestinal tissue, blood, liver, brain or any other suitable tissue of a mammal may enzymatically cleave the compounds and / or compositions of the present invention.

[0103] Pharmaceutical preparations of the present invention

[0104] The present invention relates to a pharmaceutical preparation for the treatment of pulmonary fibrosis. The pharmaceutical preparation comprises a therapeutically effective amount of one or more compounds of formula I (preferably in purified form), and an appropriate amount of a pharmaceutically acceptable carrier. When applied to a patient, the pharmaceutical preparation is preferably sterile. When the compound of the present invention is administered intravenously, water is a preferred carrier. Saline solutions and aqueous glucose solutions and glycerol solutions can also be used as liquid carriers, particularly for injection solutions. Suitable pharmaceutical carriers also include excipients, such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glyceryl monostearate, talc, sodium chloride, skimmed milk powder, glycerol, propylene, ethylene glycol, water, ethanol, etc. This reagent or pH buffer. In addition, adjuvants, stabilizers, thickeners, lubricants and colorants can be used.

[0105] Pharmaceutical compositions comprising the compounds of the invention can be prepared by conventional mixing, dissolving, granulating, diafiltration, and emulsification, encapsulation, embedding or lyophilization processes. Pharmaceutical compositions can be prepared in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that help process the compounds of the invention into pharmaceutically acceptable preparations. Suitable formulations depend on the selected route of administration.

[0106] The composition of the present invention can take the form of solution, suspension, emulsion, tablet, pill, granule, and capsule, containing liquid, powder, sustained-release preparation, suppository, emulsion, aerosol, spray, suspension, or any other form suitable for use. In one embodiment, the pharmaceutically acceptable carrier is a capsule (see, for example, Grosswald et al., U.S. Patent No. 5,698,155). Other examples of suitable pharmaceutical carriers have been described in the art (see Remington Pharmaceutical Sciences, Fairfield School of Pharmacy and Science, 17th edition, 1985). The preferred composition of the present invention is prepared for oral delivery, particularly for oral sustained-release administration.

[0107] The form of the composition for oral delivery can be, for example, in the form of tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs. The composition for oral administration may include one or more optional agents, such as sweeteners, such as fructose, aspartame or saccharin; flavoring agents, such as peppermint, wintergreen oil, or cherry coloring agents and preservatives, to provide pharmaceutically palatable preparations. In addition, in the case of tablet or pill form, the composition can be coated to delay disintegration and absorption in the gastrointestinal tract, thereby providing a sustained effect over an extended period of time. Selective permeable membranes surrounding osmotic active driving compounds are also suitable for oral administration compounds of the present invention. In these latter platforms, the liquid from the surrounding capsule environment is absorbed by the driving compound, which swells to displace the agent or agent composition through the hole. In contrast to the sharp curve of the immediate release preparation, these delivery platforms can provide a substantially zero-order delivery curve. Delay materials, such as glyceryl monostearate or glyceryl stearate can also be used. Oral compositions can include standard carriers such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Such carriers are preferably of pharmaceutical grade.

[0108] For oral liquid preparations, such as suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, saline, alkylene glycols (such as propylene glycol), polyalkylene glycols (such as polyethylene glycol) oils, alcohols, slightly acidic buffers of pH 4 to pH 6 (e.g., acetate, citrate, ascorbate, concentration of about mM to about 50 mM), etc. In addition, flavoring agents, preservatives, colorants, bile salts, acylcarnitines, etc. may be added.

[0109] Compositions administered by other routes may also be considered. For buccal administration, the composition may be in the form of tablets, lozenges, etc., prepared in a conventional manner. Liquid drug formulations suitable for sprayers and liquid spray devices and EHD aerosol devices typically include a compound of the present invention and a pharmaceutically acceptable carrier. Preferably, the pharmaceutically acceptable carrier is a liquid, such as an alcohol, water, polyethylene glycol, or a perfluorocarbon. Optionally, another material may be added to change the aerosol properties of a solution or suspension of the compound of the present invention. Preferably, the material is a liquid, such as an alcohol, ethylene glycol, polyethylene glycol, or a fatty acid. Other methods for preparing liquid drug solutions or suspensions suitable for aerosol devices are known to those skilled in the art (see, for example, Biesalski, U.S. Pat. No. 5,112,598; Biesalski, U.S. Pat. No. 5,556,611). The compounds of the present invention may also be formulated into rectal or vaginal compositions, such as suppositories or retention enemas, for example containing conventional suppository bases, such as cocoa, butter, or other glycerides. In addition to the preparations described above, the compounds of this invention can also be formulated into depot preparations. Such depot preparations can be used by implantation (e.g., subcutaneous or intramuscular) or intramuscular injection. Therefore, for example, the compounds of this invention can be prepared together with suitable polymerization or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as a sparingly soluble derivative, for example, as a sparingly soluble salt.

[0110] Therapeutic dose

[0111] The amount of the compound of formula I administered depends, among other factors, on the subject being treated, the subject's weight, the severity of the disease, the mode of administration, and the judgment of the prescribing physician. For example, the dosage can be delivered in a pharmaceutical composition by single administration, multiple administration, or controlled release. In one embodiment, the compounds of this invention are delivered by oral sustained-release administration. In one embodiment, the compounds of this invention are administered twice a day, preferably once a day. The dosage can be repeated intermittently, can be provided alone or in combination with other drugs, and can be continued to the time required for the effective treatment of the disease state or disorder.

[0112] The compound of formula I may be administered in the range of 0.1 mg to 500 mg per day, preferably 1 mg to 100 mg, for example 5 mg, 10 mg, 15 mg, 20 mg, 25 mg, 35 mg or 50 mg per day, and preferably 10 mg per day.

[0113] Combination therapy

[0114] In certain embodiments of the invention, the compounds of the invention can be used in combination therapy with at least one other therapeutic agent. The compound of formula I and the therapeutic agent can act additively or synergistically. In one embodiment, the compound of formula I is administered simultaneously with another therapeutic agent, which can be part of the same composition of the compound of formula I. In another embodiment, a composition comprising a compound of the invention is administered before or after administration of another therapeutic agent.

[0115] The present invention is effective for treating pulmonary fibrosis. The compound of formula I has an effect on serotonin 5-HT 2A Receptor (Compound A, Ki = 2.5 nM, see Example 1), 5-HT 2B The compound of formula I has strong binding affinity to dopamine (D2) and serotonin (5-HT7). 1A ) exhibits partial agonist activity on the key subtype of serotonin and exhibits antagonist activity on the serotonin 5-HT6 receptor. In addition, in the bleomycin-induced pulmonary fibrosis rat model, the compound of formula I (Compound A) shows efficacy in treating pulmonary fibrosis (Example 2).

[0116] The invention is illustrated by the following examples. Example

[0117] Example 1. In vitro pharmacology results

[0118] Two arylpiperazine derivatives of formula (I), compound A and compound B, were tested in in vitro pharmacological experiments to evaluate their effects on dopamine, D 2S , serotonin, 5-HT 1A , 5-HT 2A , 5-HT 2B , 5-HT6 and 5-HT7 receptor activity. This article describes the in vitro experimental protocols and references.

[0119] Dopamine, D 2S Radioligand binding assay

[0120] Materials and methods:

[0121] Receptor source: human recombinant D 2S Mammalian cells expressing

[0122] Radioligand: [ 3 H] spiroperidol (20-60 Ci / mmol) or [3H]-7-hydroxy DPAT, 1.0 nM

[0123] Control compound: Haloperidol or Chlorpromazine

[0124] Incubation conditions: 60 minutes at 25°C in 50 mM TRIS-HCl (pH 7.4) containing 120 mM NaCl, 5 mM KCl, 5 mM MgCl2, 1 mM EDTA. The reaction was terminated by rapid vacuum filtration onto a glass fiber filter. The radioactivity captured on the filter was determined and compared to the control value to determine any interaction between the test compound and the cloned dopamine-D2 short binding site (Literature references: Jarvis, KR et al. Journal of Receptor Research 1993, 13 (1-4), 573-590; Gundlach, AL et al., Life Sciences 1984, 35, 1981-1988.)

[0125] Serotonin, 5HT 1A Radioligand binding assay

[0126] Materials and methods:

[0127] Receptor source: human recombinant 5-HT 1A Mammalian cells expressing

[0128] Radioligand: [ 3 H]-8-OH-DPAT (221Ci / mmol)

[0129] Reference compound: 8-OH-DPAT

[0130] Incubation conditions: 1 hour at room temperature in 50 mM TRIS-HCl (Ph7.4) containing 10 mM MgSO4, 0.5 mM EDTA and 0.1% ascorbic acid. The reaction was terminated by rapid vacuum filtration onto a glass fiber filter. The radioactivity captured on the filter was determined and compared to the control value to determine the interaction between the test compound and the cloned serotonin-5HT 1A Any interaction between binding sites (references: Hoyer, D. et al. Eur. Journal Pharmacol. 1985, 118, 13-23; Schoeffter, P. and Hoyer, D. Naunyn-Schmiedeberg's Arch. Pharmac. 1989, 340, 135-138).

[0131] Serotonin, 5HT 2A Radioligand binding assay

[0132] Materials and methods:

[0133] Receptor source: human cerebral cortex or human recombinant 5-HT 2AMammalian cells expressing

[0134] Radioligand: [ 3 H]-Ketanserin (60-90Ci / mmol)

[0135] Control compound: Ketanserin

[0136] Incubation conditions: 90 minutes at room temperature in 50 mM TRIS-HCl (pH 7.6). The reaction was terminated by rapid vacuum filtration onto glass fiber filters. The radioactivity captured on the filter was determined and compared to the control value to determine the interaction of the test compound with serotonin-5HT. 2A Any interaction between binding sites (References: Leysen, JE et al. Mol. Pharmacol. 1982, 21, 301-314; Martin, GR and Humphrey, PP A Neuropharmacol. 1994, 33 (3 / 4), 261-273.).

[0137] Serotonin, 5HT 2a Radioligand binding assay

[0138] Materials and methods:

[0139] Receptor source: human recombinant 5-HT 2B CHO-K1 cells expressing

[0140] Radioligand: 1.20 nM [3H] lysergic acid diethylamide (LSD)

[0141] Control compound: Ketanserin

[0142] Incubation conditions: 90 minutes at room temperature in 50 mM TRIS-HCl (pH 7.6). The reaction was terminated by rapid vacuum filtration onto glass fiber filters. The radioactivity captured on the filter was determined and compared to the control value to determine the interaction of the test compound with serotonin-5HT. 2B Any interactions between binding sites.

[0143] Serotonin, 5HT6 radioligand binding assay

[0144] Materials and methods:

[0145] Receptor source: mammalian cells expressing human recombinant 5-HT6

[0146] Radioligand: [125I]SB258585, 15 nM or [ 3 H]LSD, 2 nM

[0147] Control compound: Methiothepin or serotonin

[0148] Incubation conditions: 1 hour at room temperature in 50 mM TRIS-HCl (pH 7.4) containing 10 mM MgSO4, 0.5 mM EDTA and 0.1% ascorbic acid. The reaction is terminated by rapid vacuum filtration onto a glass fiber filter. The radioactivity captured on the filter is determined and compared with the control value to determine any interaction between the test compound and the cloned serotonin-5HT6 binding site (reference: Gonzalo, R., et al., Br. J. Pharmacol., 2006 (148), 1133-1143).

[0149] Serotonin, 5HT7 radioligand binding assay

[0150] Materials and methods:

[0151] Receptor source: CHO cells expressing human recombinant 5-HT7

[0152] Radioligand: [3H]Lysergic acid diethylamide (LSD), 4 nM

[0153] Control compound: serotonin

[0154] Incubation conditions: 90 minutes at room temperature in 50 mM TRIS-HCl (pH 7.6). The reaction is terminated by rapid vacuum filtration onto a glass fiber filter. The radioactivity captured on the filter is determined and compared to the control value to determine any interaction between the test compound and the serotonin-5HT7 binding site.

[0155] Radioligand binding experiments were performed at 6 to 10 different concentrations, testing concentrations were 0.1 nM, 0.3 nM, 1 nM, 10 nm, 30 nM, 100 nM, 300 nM and 1000 nM.

[0156] In the following table the in vitro pharmacological activities of selected compounds A and B using radioligand binding assays are reported. Compound A (brilaroxazine) = 6-(4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butoxy)-2H-benzo[b][1,4]oxazin-3(4H)-one hydrochloride. Compound B = 6-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butoxy)-2H-benzo[b][1,4]oxazin-3(-4H)-one hydrochloride.

[0157]

[0158] Example 2. Evaluation of the effect of compound A (RP5063) in a rat model of bleomycin (BLM)-induced pulmonary fibrosis

[0159] Materials and methods

[0160] Study Animals: This study involved 34 male Sprague Dawley rats (weight: 275-300 g; age: 8 weeks; Charles River Laboratories, Quebec, Canada) randomly divided equally into four groups according to their weight. Study Design: The aim of this parallel design study was to evaluate the effects of Compound A (RP5063) on functional, histological, and pathophysiological parameters in the BLM-induced model, starting on days 1 and 10 after BLM induction.

[0161] Study Methods: On day 0, animals in Group 1 (sham treatment; N=5) received a single intratracheal administration of vehicle, 0.250 mL of vehicle 0.9% saline solution ( Figure 1 ). Animals in Groups 2 to 4 (n=9, 10, 10, respectively) received a single intratracheal instillation of 0.250 mL (5 U / kg-3.33 mg / kg) bleomycin sulfate (Cayman Chemicals, Ann Arbor, Michigan). Vehicle was administered to Groups 1, 2, and 4 from Day 1 to Day 10. Compound A (RP5063) was administered to Group 3 at 15 mg / kg twice a day (bid) per gavage from Day 1 to Day 20 as treatment (Group 3, RPT). Vehicle administration continued to Group 1 (sham) and Group 2 (BLM) until Day 20. Group 4 received Compound A (RP5063) at 15 mg / kg twice a day (bid) per gavage from Day 10 to Day 20 as intervention treatment (Group 4, RPI). During the treatment period, rats were provided with food and water ad libitum. Animal behavior, general health, and survival were monitored daily. Body weight and food intake were also measured.

[0162] The solutions used were prepared by dissolving 750 mg of Compound A (RP5063) in 500 mL of sterile 5% glucose solution to obtain a 1.5 mg / mL solution. For bleomycin, 50 mg of the drug was weighed and dissolved in 12.5 mL of sterile 0.9% saline solution to obtain a 4 mg / mL solution. The vehicle was produced by dissolving 50 g of glucose in 1 L of water to produce a 5% solution.

[0163] On day 21, the animals were anesthetized and implanted with the device. Hemodynamic parameters (systemic arterial blood pressure, heart rate, and blood oxygen saturation) were recorded continuously for at least 5 minutes. At the end of the recording, blood samples were collected. After the animals were bled, the pulmonary circulation was flushed with 0.9% NaCl, and all tissues (lungs, trachea, and heart) were collected from the chest cavity for further analysis.

[0164] Parameters measured on the day of surgery: Cardiac activity was recorded continuously during surgery. Cardiac activity was monitored using three electrocardiogram (ECG) contact electrodes, placed in a lead-I / II configuration and connected to an IsoDam8 differential amplifier. Heart rate (HR) was recorded using a repeating system: from the ECG recordings (RR intervals) and using an N-595 pulse oximeter attached to the left forepaw of the animal. Heart rate values ​​from the pulse oximeter were measured in pulses / minute (bpm) using cursor readouts in Clampfit 10.2.0.14CA. Blood oxygen saturation (SpO2) was measured using the pulse oximeter signal attached to the left forepaw of the animal.

[0165] Systemic arterial blood pressure (SAP) was continuously monitored using an intra-arterial fluid-filled catheter connected to a pressure transducer, with diastolic and systolic values ​​measured in mmHg. Mean SAP (mSAP) and pulse pressure (PP) were calculated using the following formulas: 1-mSAP = systemic diastolic pressure + ([systemic systolic pressure - systemic diastolic pressure] / 3); and 2-PP = systemic systolic pressure - systemic diastolic pressure. Pulse pressure was calculated as the difference between the systolic and diastolic pressure readings.

[0166] After harvesting the trachea, it was connected to the cannula of the perfusion system. Clamp the left lung, and 5mL of cold PBS solution was injected into the trachea to obtain right lobe fluid (BALF) for bronchoalveolar lavage, and collected for further analysis (total cell count and cytokine measurement). After collecting the BALF sample, it was centrifuged at 1200rpm for 10 minutes at 4°C. The supernatant was frozen at -80°C until cytokine analysis was performed. The cells were then resuspended in PBS and counted with a hemocytometer.

[0167] Organ weights were expressed as relative percentages and calculated as follows: relative organ weight = (organ weight x 100) / body weight.

[0168] Histological preparation and classification: For each rat, the left lung lobe was harvested and perfused and fixed with 10% neutral buffered formalin. Transverse sections of the left middle lobe were cut, placed in 10% NBF and transferred to the Institute of Immunology and Cancer Research (Montreal, Quebec, Canada). The tissue was embedded, sliced ​​(5-μm thick), fixed, and stained routinely. Staining included hematoxylin and eosin (H&E) and Ashcroft scoring, and finally Masson trichrome staining for fibrosis quantification. Slides containing fixed and stained tissues were observed at 20x magnification (Eclipse T100 microscope, Nikon). At least five non-overlapping fields / lungs were selected for microscopic imaging (Nikon DS-Fil digital camera equipped with Nikon NIS Elements 4.30, Nikon).

[0169] H&E slides were used to assess the alveolar septa and lung architecture. Tissues were scored using a modified Ashcroft scale based on the average of five non-overlapping fields selected previously. Masson trichrome-stained slides were also viewed using a scanner to determine the percentage of fibrotic tissue on the sections.

[0170] Hydroxyproline determination: Hydroxyproline (a biomarker of PF) content was determined using a colorimetric assay kit (CellBiolabs Inc, CA, USA). Part of the right lobe was removed and homogenized in 0.1 mL of water. The supernatant was hydrolyzed in 0.1 mL of 10 N HCl at 120 °C for 6 hours. After adding 5 mg of activated carbon, the sample was centrifuged at 10,000 rpm for 5 minutes, and the supernatant was transferred to a new test tube and processed according to the experimental instructions. The absorbance was measured at 540 nm, and the amount of hydroxyproline was determined and calibrated for protein content.

[0171] Cytokine quantification: On day 21, BALF samples were collected immediately after bleeding of the animals. For all groups, half of each sample was saved for analysis of the following cytokines: (1) macrophage inflammatory protein 1 (MIP1); (2) monocyte chemoattractant protein 1 (MCP1); (3) interleukin (IL)-6; (4) interferon gamma-induced protein 10 (IP10); and (5) RANTES. Cytokine analysis was performed using the Luminex assay (Eve Technologies, Calgary, Alberta).

[0172] Analysis and Statistics: The primary outcomes involved survival and body weight. Other parameters of interest included cardiopulmonary and pressure parameters at the time of surgery, tissue weights, histological samples, bronchoalveolar lung lavage fluid cell counts, hydroxyproline levels, and cytokines. Results are expressed as mean ± SEM. Normally distributed data were compared using ANOVA followed by Fisher post hoc tests to assess differences between BLM groups using Graph Pad Prism software, version 7.0 for Mac (San Diego, CA, USA). Comparisons included sham versus BLM and treatment versus BLM. No treatment differences were compared. Differences were considered statistically significant when P values ​​were less than 0.05.

[0173] result

[0174] Animal survival and body weight: The survival rate of the 19 animals in Groups 2 and 4 was 82% on day 10 during this period ((P < .05, sham treatment). In contrast, the survival rate of the uninduced and interventional therapy (RPI) animals (Groups 1 and 3, respectively) was 100% (P < .05, BLM) ( Figure 2 A). On day 21, the survival rate of BLM (Group 2) decreased to 62% (P < .05, sham), while the survival rates of RPT and RPI were 90% and 89.5%, respectively (P < .05, BLM) ( Figure 2 B), persistence of animals in the sham-treated group was 100%.

[0175] The corresponding value to the survival rate is the body weight ( Figure 2 C). Three weeks after BLM induction, the body weight of the sham-treated group increased by about 50%; while that of the BLM-treated rats was significantly lower (P < 0.05, sham). RPT significantly attenuated BLM-induced body weight loss at day 21 compared with the BLM-treated rats (P < 0.01, BLM). RPT administration starting from day 10 after BLM induction slightly increased body weight compared with BLM-treated rats at day 21.

[0176] Hemodynamic and Cardiac Effects: Hemodynamic parameters were recorded on day 21 ( Figure 3 Animals in the BLM experienced an arterial pulse pressure ( Figure 3 A) and cardiac output ( Figure 2B) was significantly affected (P < 0.05, sham treatment). Ventricular dysfunction (lack of left ventricular preload) and hypovolemia may be responsible for these observations and pulse pressure. Animals in RPT experienced improved arterial pulse pressure (P < 0.05, BLM) and were at a similar level to that experienced by the sham group. In addition, these animals had restored cardiac output compared to BLM (P < 0.01). RPI slightly restored arterial pulse pressure and cardiac output. Heart rate was relatively similar in all groups ( Figure 3 C).

[0177] About systemic arterial pressure ( Figure 3 DF), although no differences between the groups were observed, animals in the BLM group showed a non-significant decrease in systemic arterial pressure; this effect may be the result of reduced cardiac output.

[0178] Parameters reflecting lung fibrosis and function: Pulmonary fibrosis is known to cause respiratory resistance; resistance to airflow through the airways during inhalation and exhalation. This resistance reduces gas exchange (O2-CO2) in the alveoli and leads to a shortened lifespan. BLM animals showed a significant increase in respiratory resistance (P<0.001, sham treatment) ( Figure 4 A). Animals in the RPT group showed a significant decrease in this parameter (P < 0.05, BLM), while animals in the RPI group showed an improvement (P = 0.10, BLM). Hydroxyproline content in the lungs was also measured to assess the presence of pulmonary fibrosis ( Figure 4 B) and mirror the changes observed in respiratory resistance. Hydroxyproline concentrations were twofold higher in BLM animals (P < 0.05, sham). Hydroxyproline concentrations were significantly reduced in the RPT group (P < 0.05, BLM) and the RPI group (P < 0.01, BLM).

[0179] Animals in the BLM group had significantly (P < 0.01, sham) higher lung weights, reflecting the lung changes caused by BLM, indicating the presence of edema ( Figure 5 A). Lung weights were significantly reduced in RPT animals (P < 0.05, BLM). Lung weights were also reduced in the RPI group.

[0180] Total cell counts were obtained from BALF of the right lobe of the lung (inflammatory, Figure 5 B) and total protein content (edema, Figure 5 C) to enhance lung weight measurement and reflect the presence of pulmonary edema. BLM animals showed a significant increase in both cell counts and protein levels (P < 0.01, sham). RPT animals showed a reversal in both cell counts and protein levels (P < 0.05 for both, BLM). RPI animals showed a significant decrease in total cell counts (P < 0.05, BLM) and showed numerical improvement in the decrease in BALF protein concentration.

[0181] Staining of lung tissues provided additional evidence that BLM-induced lung fibrosis developed and was attenuated with RP5063 treatment. As reflected by H&E staining ( Figure 6 ), BLM group lung tissue samples ( Figure 6 Ashcroft scores in A, C) were significant (P < 0.001, sham treatment). Samples obtained from RPT animals showed a significant reduction in these fibrotic changes in the lung parenchyma during treatment ( Figure 6 C), (P < 0.001, BLM). Pulmonary fibrosis is characterized by excessive collagen distribution in the lung, as reflected by the percentage of collagen area measured by Masson's trichrome staining. On day 21, stained samples from BLM animals showed a significant increase in the percentage of collagen distribution in the lung (P < 0.001, sham treatment) ( Figure 6 B, D). Therapeutic intervention with RP5063 (RPT) significantly reduced these changes (P<0.001, BLM). These results correlate well with the hydroxyproline concentration quantification presented previously.

[0182] On day 21, cardiopulmonary capacity, blood oxygen saturation ( Figure 7 A) and blood lactate levels ( Figure 7 B). BLM animals showed a significant decrease in saturation and a significant increase in blood lactate levels (P < 0.01 for both, sham treatment). RPT group animals showed normalization of blood oxygen levels (P < 0.05, BLM). RPI animals had improved saturation levels and were numerically superior to the BLM group. Although treatment caused a decrease in blood lactate, a biomarker of PF, this was significant for both RPT and RPI (P < 0.01, P < 0.05, respectively, vs. BLM).

[0183] BLM-induced inflammatory and fibrotic cytokines: To evaluate the effects of treatment on BLM-induced inflammatory and fibrotic cytokines, BALF was analyzed starting at day 21 to quantify the levels of MIP1, MCP1, IL-6, IP10, and RANTES ( Figure 8 AE). Animals in the BLM group showed significant increases in all five cytokine levels (P < 0.05 for MIP1 and MCP1; P < 0.01 for IP10 and RANTES, sham treatment). Although RPT slightly reduced the production of the cytokine MIP-1, no treatment statistically reversed the production of MIP-1 ( Figure 8 A). The group of animals treated with RP5063 showed a decrease in MCP-1 concentration ( Figure 8B), showing a significant decrease in the RPI group (P < 0.05, BLM). Animals in both treatment groups had numerically reduced IL6 levels (RPT, RPI). Figure 8 C). Animals in the RPT and RPI groups showed a significant decrease in IP10 ( Figure 8 Finally, the levels of RANTES cytokines were significantly decreased in both RP5063-treated animals ( Figure 8 E) (P<0.01, BLM).

[0184] in conclusion

[0185] RP5063 (Compound A, brilaroxazine) was able to significantly impact all important biomarkers and practical endpoints described in this study, according to de facto standards for demonstrating therapeutic efficacy in IPF. RP5063 treatment reduced BLM-induced lung fibrosis, inflammation, and ECM deposition (collagen), and improved cardiac and lung function in rodents. RP5063 reduced IPF progression both as a preventive treatment (RPT) starting on day 1 and as an intervention treatment (RPI) starting on day 10 after BLM induction. Positive effects on body weight, survival, pulmonary edema, profibrotic cytokine production, hydroxyproline content, and respiratory resistance, and cardiopulmonary capacity provide supportive evidence that RP5063 affects functional and pathological effects associated with IPF. The overall effects in this translational animal model suggest that RP5063 is an effective therapy for the treatment of IPF and PF.

[0186] It will be appreciated that the foregoing describes preferred embodiments of the invention and that modifications may be made without departing from the scope of the invention as set forth in the claims.

Claims

1. Use of compound A or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating pulmonary fibrosis in a subject, Compound A.

2. The use according to claim 1, wherein The compound A is in the form of hydrochloride.

3. The use according to claim 1, wherein The compound A is administered in the form of a pharmaceutical composition, which contains a pharmaceutically acceptable carrier or excipient.

4. The use according to claim 1, wherein The compounds are administered orally. The use according to claim 1 , wherein the pulmonary fibrosis is idiopathic pulmonary fibrosis.

6. The use according to claim 1, wherein the pulmonary fibrosis is pulmonary fibrosis in a subject suffering from chronic obstructive pulmonary disease.

7. The use according to claim 1, wherein the pulmonary fibrosis is pulmonary fibrosis in a subject suffering from sickle cell disease.

8. The use of claim 1, wherein the pulmonary fibrosis is pulmonary fibrosis in a subject suffering from scleroderma.

9. The use of claim 1, wherein the pulmonary fibrosis is pulmonary fibrosis in a subject suffering from lung cancer.

Images