EA044096B1 - USES OF LYSILOXIDASE-LIKE PROTEIN INHIBITOR 2 - Google Patents

Description

Field of technology to which the present invention relates

Disclosed herein are methods of using a lysyl oxidase-like protein 2 (LOXL2) inhibitor in the treatment or prevention of conditions, diseases, or disorders associated with LOXL2 activity.

BACKGROUND OF THE INVENTION

Lysyl oxidase-like protein 2 (LOXL2) is an amine oxidase enzyme that catalyzes the cross-linking of extracellular matrix proteins. LOXL2 is also involved in intracellular processes such as mediating epithelial-mesenchymal transition of cells. LOXL2 signaling has been implicated in, for example, fibrotic diseases and cancer. There is an unmet medical need for therapeutic agents that can provide benefit to patients with fibrotic diseases and cancer.

Brief Disclosure of the Present Invention

Disclosed herein is the use of a small molecule inhibitor of LOXL2 in the treatment of a disease or condition in a mammal that will benefit from inhibition or reduction of LOXL2 activity, wherein the small molecule inhibitor of LOXL2 is more selective for inhibiting or binding to LOXL2 than for LOX. In accordance with some embodiments, the small molecule inhibitor of LOXL2 is at least 10 times more selective for inhibiting or binding to LOXL2 than for LOX. In accordance with some embodiments, the small molecule LOXL2 inhibitor is at least 10-fold, at least 20-fold, at least 30-fold, at least 40-fold, at least 50-fold, at least 60-fold, at least 70-fold, at least 80-fold, at least 90-fold, or greater than 100-fold more selective for inhibiting or binding to LOXL2 than LOX. In accordance with some embodiments, the small molecule inhibitor of LOXL2 is at least 100 times more selective for inhibiting or binding to LOXL2 than for LOX. In accordance with some embodiments, the small molecule LOXL2 inhibitor is at least 100-fold, at least 120-fold, at least 140-fold, at least 160-fold, at least 180-fold, at least 200-fold, at least 250-fold, at least 300-fold, at least 350-fold, or at least 400-fold more selective for LOXL2 than for LOX. In accordance with some embodiments, the small molecule inhibitor of LOXL2 is at least 400 times more selective for LOXL2 than for LOX.

In one aspect, the disease or condition is a lung disease, liver disease, kidney disease, cardiac fibrosis, ocular fibrosis, ear fibrosis, myelofibrosis, scleroderma, cancer, an autoimmune disease or condition, an inflammatory disease or condition, or a combination thereof.

In one aspect, the small molecule LOXL2 inhibitor is trans-(3-((4(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, the small molecule LOXL2 inhibitor is (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3fluoro-4 -hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof, (S,S)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl) (3-fluoro-4-hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof or a mixture thereof. In some embodiments, the small molecule LOXL2 inhibitor is (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro -4hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, (R,R)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1 -yl)methanone practically does not contain (S,S)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4hydroxypyrrolidin-1- yl)methanone or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, a pharmaceutically acceptable salt of (R,R)-trans-(3-((4(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4- hydroxypyrrolidin-1-yl)methanone is obtained from (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4hydroxypyrrolidin- 1-yl)methanone and an acid selected from the group consisting of hydrogen chloride

- 1 044096 nic acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, metaphosphoric acid, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L), benzenesulfonic acid, benzoic acid, camphoric acid (+), camphor10-sulfonic acid (+), capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid , glucoheptanoic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, hydroxysuccinic acid (-L), malonic acid, mandelic acid (DL), methanesulfonic acid, monomethyl fumarate, naphthalene-1,5-disulfonic acid, naphthalene-2sulfonic acid, nicotinic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid acid, phosphoric acid, propionic acid, pyroglutamic acid (L), salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid (+L), thiocyanic acid, toluenesulfonic acid (p) and undecenoic acid. According to some embodiments, (R,R)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1 -yl)methanone is used in the form of mesylate salt, hydrochloride salt, sulfate salt, maleate salt, phosphate salt, L tartrate salt, fumarate salt, succinate salt, citrate salt or acetate salt. According to some embodiments, (R,R)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1 -yl)methanone is used in the form of mesylate salt.

In some embodiments, the small molecule LOXL2 inhibitor is (S,S)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro4 -hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, (S,S)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1 -yl)methanone practically does not contain (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4hydroxypyrrolidin-1- yl)methanone or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, a pharmaceutically acceptable salt of (S,S)-trans-(3-((4(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4- hydroxypyrrolidin-1-yl)methanone is obtained from (S,S)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4hydroxypyrrolidin- 1-yl)methanone and an acid selected from the group consisting of hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, metaphosphoric acid, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid , 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L), benzenesulfonic acid, benzoic acid, camphoric acid (+), camphor10-sulfonic acid (+ ), capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptanoic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, hydroxysuccinic acid (- L), malonic acid, mandelic acid (DL), methanesulfonic acid, monomethyl fumarate, naphthalene-1,5-disulfonic acid, naphthalene-2sulfonic acid, nicotinic acid , oleic acid, oxalic acid, palmitic acid, pamic acid, phosphoric acid, propionic acid, pyroglutamic acid (L), salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid (+ L), thiocyanic acid, toluenesulfonic acid (p) and undecenoic acid. According to some embodiments, (S,S)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1 -yl)methanone is used in the form of mesylate salt, hydrochloride salt, sulfate salt, maleate salt, phosphate salt, L tartrate salt, fumarate salt, succinate salt, citrate salt or acetate salt. According to some embodiments, (S,S)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1 -yl)methanone is used in the form of mesylate salt.

Disclosed herein is the compound (R,R)-trans-(3-((4-(aminomethyl)-6

- 2 044096 (trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof, (S,S)-trans-(3-( (4-(aminomethyl)-6-(trifluoromethyl)pyridin-2yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof, or a mixture thereof. According to some embodiments, (R,R)-trans-(3-((4(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1 -yl)methanone or a pharmaceutically acceptable salt or solvate thereof, (S,S)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro- 4-hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof or a mixture thereof is used in the treatment or prevention of diseases or conditions that are associated with LOXL2 activity. In some embodiments, (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidine-1- yl)methanone or a pharmaceutically acceptable salt or solvate thereof, (S,S)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro- 4hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof, or a mixture thereof, is used in the treatment or prevention of diseases or conditions that are described herein. In some embodiments, the hydrochloride salt of (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4hydroxypyrrolidine) is used -1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof, (S,S)trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3- fluoro-4-hydroxypyrrolidin1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof or a mixture thereof. In some embodiments, the mesylate salt of (R,R)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4- hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof, (S,S)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2yl)oxy)phenyl)(3- fluoro-4-hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof or a mixture thereof.

Disclosed herein is the compound (R,R)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl )methanone (compound I) or a pharmaceutically acceptable salt or solvate thereof. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in the treatment or prevention of diseases or conditions that are associated with LOXL2 activity. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in the treatment or prevention of diseases or conditions that are described herein. In accordance with some embodiments, the hydrochloride salt of Compound I (ie, Compound 1) is used. In accordance with some embodiments, the mesylate salt of Compound I (ie, Compound 2) is used.

In one aspect, described herein is a method of treating or preventing the development of a lung disease in a mammal, comprising administering to the mammal a compound (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridine-2- yl)oxy)phenyl)(3-fluoro-4hydroxypyrrolidin-1-yl)methanone (compound I) or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, the compound (R,R)-trans-(3-((4(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin- 1-yl)methanone practically does not contain (S,S)-trans-(3 -((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3 fluoro-4-hydroxypyrrolidin- 1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, the compound (R,R)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin- 1-yl)methanone is administered to the mammal in the form of the mesylate salt (compound 2) or its solvate.

In accordance with some embodiments, the lung disease is pulmonary fibrosis. In accordance with some embodiments, the lung disease is interstitial lung disease (ILD). In some embodiments, the lung disease is idiopathic interstitial pneumonia, interstitial lung disease associated with diffuse connective tissue disease (CTDILD), sarcoidosis, hypersensitivity pneumonitis, iatrogenic pneumonitis/fibrosis (drug-induced ILD, radiation injury), eosinophilic ILD (eg , eosinophilic pneumonia), occupational lung disease, hereditary pulmonary fibrosis, Hermansky-Pudlak syndrome) or pulmonary Langerhans cell histiocytosis. In some embodiments, the lung disease is idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP), respiratory bronchiolitis associated with interstitial lung disease (RBILD), desquamative interstitial pneumonia (DIP) , acute interstitial pneumonia (AIP) or lymphoid interstitial pneumonia (LIP). In accordance with some embodiments, the lung disease is idiopathic pulmonary fibrosis (IPF). In accordance with some embodiments, the compound reduces serum levels of LOXL2 (sLOXL2) in a mammal. In accordance with some embodiments, the connection slows the decline in function

- 3 044096 lung, reduces the frequency of exacerbations of lung disease, improves survival of a mammal with lung disease, or a combination thereof.

In accordance with some embodiments of treating or preventing the development of a lung disease, the method further comprises administering at least one additional therapeutic agent to the mammal. In some embodiments, the at least one additional therapeutic agent is a pneumonia vaccination, a cough suppressant drug, a corticosteroid, an immunosuppressive agent, N-acetylcysteine (NAC), pirfenidone, nintedanib, or combinations thereof. In accordance with some embodiments, the mammal is a human. In accordance with some embodiments, the person is an adult.

In accordance with some embodiments, the lung disease is pulmonary alveolar proteinosis (PAP). In accordance with some embodiments of treating or preventing the development of a lung disease (eg, PAP), the method further comprises whole lung lavage, administration of at least one additional therapeutic agent, or a combination thereof. In accordance with some embodiments, the at least one additional therapeutic agent is a corticosteroid, a mucolytic agent, or a proteinase inhibitor.

In accordance with some embodiments of treating or preventing the development of a lung disease, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered systemically to a mammal. In some embodiments of treating or preventing the development of a lung disease, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered to a mammal orally, by injection, or intravenously. In some embodiments of treating or preventing the development of a lung disease, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered to a mammal in the form of an oral solution, oral suspension, powder, pill, tablet, or capsule. In some embodiments, for treating or preventing the development of a lung disease, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered directly to the lungs of a mammal.

In some embodiments of treating or preventing the development of a lung disease, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered directly into the lungs of a mammal using a nebulizer, metered dose inhaler, or dry powder inhaler.

In one aspect, described herein is a method of treating or preventing the development of liver disease in a mammal, comprising administering to the mammal a compound (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridine-2- yl)oxy)phenyl)(3-fluoro-4hydroxypyrrolidin-1-yl)methanone (compound I) or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, the compound (R,R)-trans-(3-((4(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin- 1-yl)methanone practically does not contain (S,S)-trans-(3 -((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3 fluoro-4-hydroxypyrrolidin- 1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, the compound (R,R)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin- 1-yl)methanone is administered to the mammal in the form of the mesylate salt (compound 2) or its solvate.

In accordance with some embodiments, the liver disease is a fibrotic liver disease. In some embodiments, the liver disease is a fibrotic liver disease caused by infection with hepatitis C virus (HCV), non-alcoholic steatohepatitis (NASH), primary sclerosing cholangitis (PSC), cirrhosis, liver fibrosis, trypsin inhibitor alpha 1 deficiency disease, hereditary hemochromatosis, Wilson's disease, steatohepatitis and cirrhosis, which are associated with infection with hepatitis B virus (HBV) and HIV, and associated conditions such as chronic viral hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC) or biliary cirrhosis. In some embodiments, the liver disease is a fibrotic liver disease caused by infection with hepatitis C virus, nonalcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), Wilson's disease, and biliary primary cirrhosis or sclerosing cholangitis. In accordance with some embodiments, the liver disease is a human fibrotic liver disease caused by non-alcoholic fatty liver disease (NAFLD). According to some embodiments, the liver disease is a fibrotic human liver disease caused by a viral disease or liver condition. In some embodiments, the liver disease is liver fibrosis and the mammal is a human diagnosed with NASH. In some embodiments, the liver disease is liver fibrosis and the mammal is a human diagnosed with primary sclerosing cholangitis (PSC). In accordance with some embodiments, the liver disease is cirrhosis due to NASH.

In accordance with some embodiments of treating or preventing the development of liver disease, the method further comprises administering at least one additional therapeutic agent to the mammal. In accordance with some embodiments, the at least one additional therapeutic agent is selected from the group consisting of PPAR agonists, incretin, Glut2-I, FXR agonists, antioxidants, GLP-1 modulators, SGLT-2 inhibitors, bile acids, caspase protease inhibitors , synthetic fatty acid/bile acid conjugates, dual CCR2/CC5 antagonists, immunomodulators, sirtuin stimulators, fatty acid inhibitor, DGAT1 inhibitors, CD3 antigens, PDE-4 modulators, AMPK stimulators, ROCK2 inhibitors, ASBT inhibitors, ASK1 inhibitors, TLR-antagonists 4, THR-beta antagonists, cathepsin B inhibitors, galectin-3 modulators and combinations thereof.

In accordance with some embodiments of treating or preventing the development of liver disease, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered systemically to a mammal. In some embodiments, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered to a mammal orally, by injection, or intravenously. In some embodiments, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered to a mammal in the form of an oral solution, oral suspension, powder, pill, tablet, or capsule.

In one aspect, described herein is a method of treating or preventing the development of renal fibrosis, cardiac fibrosis, ocular fibrosis, ear fibrosis, myelofibrosis or scleroderma in a mammal, comprising administering to the mammal a compound (R,R)-trans-(3-((4( aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone (compound I) or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, the compound (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidine-1 -yl)methanone practically does not contain (S,S)-trans-(3-((4(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1 -yl)methanone or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, the compound (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3fluoro-4-hydroxypyrrolidine-1 -yl)methanone is administered to the mammal in the form of the mesylate salt (compound 2) or its solvate.

In accordance with some embodiments, the myelofibrosis is primary myelofibrosis or secondary myelofibrosis. In some embodiments, the myelofibrosis is a primary, post-polycythemia vera or post-essential thrombocythemia myelofibrosis. In accordance with some embodiments of treating or preventing the development of myelofibrosis, the method further comprises administering at least one additional therapeutic agent to the mammal. In accordance with some embodiments, the at least one additional therapeutic agent is ruxolitinib.

In accordance with some embodiments, scleroderma is limited systemic sclerosis or diffuse systemic sclerosis.

According to some embodiments, ocular fibrosis includes fibrosis of the vitreous, iris, ciliary body, lens, choroid, retinal pigment epithelium, cornea, retina, or combinations thereof.

According to some embodiments, ocular fibrosis is a consequence of eye surgery.

In some embodiments, the mammal is diagnosed with glaucoma, age-related macular degeneration (AMD), choroidal neovascularization (CNV), corneal degeneration, dry eye syndrome, keratitis, corneal ulcers, retrolethal fibroplasia (ROP), pterygium, cataracts, diabetic retinopathy with retinal edema and neovascularization, proliferative vitreoretinopathy (PVR), retinal detachment, macular edema.

In accordance with some embodiments of treating or preventing the development of a fibrotic disease, the method comprises administering at least one additional therapeutic agent to a mammal.

In accordance with some embodiments, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered systemically to a mammal. In accordance with some embodiments, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered to a mammal orally, by injection, or intravenously.

In one aspect, described herein is a method of treating or preventing the development of cancer in a mammal, comprising administering to the mammal a compound (R,R)-trans(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl) oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-5 044096 yl)methanone (compound I) or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, the compound (R,R)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin- 1-yl)methanone practically does not contain (S,S)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4hydroxypyrrolidin-1 -yl)methanone or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, the compound (R,R)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin- 1-yl)methanone is administered to the mammal in the form of the mesylate salt (compound 2) or its solvate.

In accordance with some embodiments, the cancer is breast cancer, colorectal cancer, stomach cancer, head and neck cancer, lung cancer, melanoma, or combinations thereof. In some embodiments, the cancer is colorectal cancer, esophageal tumors, oral squamous cell carcinomas, laryngeal squamous cell carcinomas, and head and neck squamous cell carcinomas.

In accordance with some embodiments of treating or preventing the development of cancer, the method further comprises administering at least one additional therapeutic agent to the mammal. In accordance with some embodiments, the at least one additional therapeutic agent is an anticancer agent.

In accordance with some embodiments, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered systemically to a mammal. In accordance with some embodiments, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered to a mammal orally, by injection, or intravenously.

In one aspect, described herein is a method of treating or preventing the development of an autoimmune disease or condition or an inflammatory disease or condition in a mammal, comprising administering to the mammal a compound (R,R)-trans-(3-((4(aminomethyl)-6-( trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone (compound I) or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, the compound (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidine-1 -yl)methanone practically does not contain (S,S)-trans-(3-((4(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1 -yl)methanone or a pharmaceutically acceptable salt or solvate thereof. According to some embodiments, the compound (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3fluoro-4-hydroxypyrrolidine-1 -yl)methanone is administered to the mammal in the form of the mesylate salt (compound 2) or its solvate.

In some embodiments, the autoimmune disease or condition is rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, osteoarthritis, Still's disease, lupus, diabetes mellitus, myasthenia gravis, Hashimoto's thyroiditis, Ord's thyroiditis, Graves' disease, Sjögren's syndrome, multiple sclerosis , Guillain-Barre syndrome, acute disseminated encephalomyelitis, Addison's disease, opso-myoclonal syndrome, ankylosing spondylitis, antiphospholipid antibody syndrome, aplastic anemia, autoimmune hepatitis, celiac enteropathy, Goodpasture's syndrome, idiopathic thrombocytopenic purpura, optic neuritis, scleroderma, biliary primary qi rroz liver, Reiter's syndrome, Takayasu's syndrome, temporal arteritis, autoimmune hemolytic anemia due to warm antibodies, Wegener's granulomatosis, psoriasis, alopecia universalis, Behçet's disease, chronic fatigue, autonomic dystonia, endometriosis, interstitial cystitis, neuromyotonia, scleroderma or vulvodynia. In some embodiments, the autoimmune disease or condition is rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, osteoarthritis, or ankylosing spondylitis.

In some embodiments, the inflammatory disease or condition is asthma, inflammatory bowel disease, appendicitis, blepharitis, bronchiolitis, bronchitis, bursitis, cervicitis, cholangitis, cholecystitis, colitis, conjunctivitis, cystitis, dacryoadenitis, dermatitis, dermatomyositis, encephalitis, endocarditis , endometritis, enteritis, enterocolitis, epicondylitis, epididymitis, fasciitis, fibrositis, gastritis, gastrointestinal tract, hepatitis, hidradenitis suppurativa, laryngitis, mastitis, meningitis, osteomyelitis, myocarditis, myositis, nephritis, oophoritis, orchitis, osteitis, otitis media, pancreatitis, mumps, pericarditis, peritonitis, pharyngitis, pleurisy, phlebitis, pneumonitis, pneumonia, proctitis, prostatitis, pyeloniphritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis, tendinitis, tonsillitis, uveitis, vaginitis, vasculitis or vulvitis.

In accordance with some embodiments of treating or preventing the development of an autoimmune disease or condition or an inflammatory disease or condition, the method further comprises administering at least one additional therapeutic agent to the mammal. In accordance with some embodiments, the mammal

- 6 044096 is a person.

In accordance with some embodiments, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered systemically to a mammal. In some embodiments, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered to a mammal orally, by injection, or intravenously. In some embodiments, the compound, or a pharmaceutically acceptable salt or solvate thereof, is administered to a mammal in the form of an oral solution, oral suspension, powder, pill, tablet, or capsule.

In one aspect, described herein is a pharmaceutical composition comprising Compound I or a pharmaceutically acceptable salt or solvate thereof and at least one pharmaceutically acceptable excipient.

In accordance with some embodiments, the pharmaceutical composition is formulated for administration to a mammal by intravenous administration, subcutaneous administration, oral administration, inhalation, nasal administration, dermal administration, or ocular administration. In accordance with some embodiments, the pharmaceutical composition is formulated for administration to a mammal by intravenous administration, subcutaneous administration, or oral administration. In accordance with some embodiments, the pharmaceutical composition is formulated for oral administration to a mammal. In accordance with some embodiments, the pharmaceutical composition is in the form of a tablet, pill, capsule, liquid, suspension, gel, dispersion, solution, emulsion, ointment, or lotion. In accordance with some embodiments, the pharmaceutical composition is in the form of a tablet, pill, or capsule.

In one aspect, described herein is a method of treating a disease or condition in a mammal that will benefit from inhibiting or reducing the activity of lysyl oxidase-like protein 2 (LOXL2), comprising administering Compound I or a pharmaceutically acceptable salt or solvate thereof to a mammal in need thereof. . In accordance with some embodiments, the disease or condition is fibrosis or cancer. According to some embodiments, fibrosis includes pulmonary fibrosis, liver fibrosis, renal fibrosis, cardiac fibrosis, peritoneal fibrosis, ocular fibrosis, ear fibrosis, or cutaneous fibrosis. In accordance with some embodiments, the fibrosis is myelofibrosis.

In one aspect, described herein is a method of treating or preventing the development of any of the diseases or conditions described herein, comprising administering a therapeutically effective amount of Compound I, or a pharmaceutically acceptable salt or solvate thereof, to a mammal in need thereof.

In one aspect, described herein is a method of treating or preventing the development of fibrosis in a mammal comprising administering a therapeutically effective amount of Compound I or a pharmaceutically acceptable salt or solvate thereof to a mammal in need thereof. In other embodiments, fibrosis is treatable with a LOXL2 inhibitor. In accordance with some embodiments, the fibrosis is pulmonary fibrosis. In accordance with some embodiments, the method further comprises administering a second therapeutic agent to the mammal in addition to Compound I or a pharmaceutically acceptable salt or solvate thereof.

According to any of the above-described aspects, further embodiments are provided wherein an effective amount of Compound I or a pharmaceutically acceptable salt or solvate thereof: (a) is administered systemically to a mammal; and/or (b) administered orally to the mammal; and/or (c) administered intravenously to the mammal; and/or (d) administered by inhalation; and/or (f) administered by nasal administration; and/or (f) administered to a mammal by injection; and/or (g) administered topically to the mammal; and/or (h) administered by ocular administration; and/or (i) administered rectally to the mammal; and/or (j) administered non-systemically or locally to the mammal.

According to any of the above-described aspects, further embodiments are provided comprising single administrations of an effective amount of Compound I or a pharmaceutically acceptable salt or solvate thereof, including further embodiments wherein Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a mammal once daily; Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a mammal multiple times over the course of one day. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered according to a continuous dosing schedule. In accordance with some embodiments, the compound is administered according to a continuous daily dosing schedule.

According to any of the above-described aspects involving the treatment of a disease or condition, additional embodiments are provided that provide for the administration of at least one additional agent in addition to the administration of Compound I or a pharmaceutically acceptable salt or solvate thereof. According to various embodiments,

- 7 044096 each agent is administered in any order, including simultaneous administration.

In accordance with any of the embodiments disclosed herein, the mammal is a human.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a human. In accordance with some embodiments, Compound I, or a pharmaceutically acceptable salt or solvate thereof, is administered orally. In accordance with some embodiments, oral administration is via tablets containing Compound I or a pharmaceutically acceptable salt or solvate thereof.

Articles that include packaging material, a compound described herein or a pharmaceutically acceptable salt thereof in the packaging material, and a label stating that Compound I or a pharmaceutically acceptable salt or solvate thereof is used to inhibit the activity of LOXL2 or to treat, prevent or attenuate one or more symptoms of a disease or condition that will benefit from inhibiting or reducing LOXL2 activity.

Other objects, features and advantages of the compounds, methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments, are given by way of illustration only, since from the present detailed description, various changes and modifications will be apparent to one skilled in the art within the spirit and scope of the present inventions.

Brief description of drawings

In fig. Figure 1 shows Ashcroft scores derived from histopathological analysis of trichrome-stained lung sections reflecting pulmonary fibrosis in a prophylactic 14-day Rac-1 dose-response study in a mouse model of bleomycin-induced pulmonary fibrosis (*p<0.05; * *p<0.01; ***p<0.001). From these data it is clear that Rac-1 reduced fibrosis in a dose-dependent manner and that 30 mg/kg QD is the minimum dose to achieve maximum antifibrotic efficacy.

In fig. Figure 2 shows Ashcroft scores obtained from histopathological analyzes of trichrome-stained lung sections in a mouse model of bleomycin-induced pulmonary fibrosis. Compounds 1, Rac-1 and Ent-1 were administered at a dose of 60 mg/kg QD in both prophylactic (profile) and therapeutic (therapeutic) modes (*p <0.05, **p <0.01, ****p <0.0001).

In fig. 3 shows Ashcroft histopathological scores reflecting pulmonary fibrosis in a 28-day recovery study in which Compound 1 was administered at 60 mg/kg QD starting on day 14 post-bleomycin.

In fig. Figure 4 shows the histopathological Ashcroft score reflecting pulmonary fibrosis in a 14-day prophylactic study comparing 60 mg/kg QD, 60 mg/kg Q2D and 60 mg/kg Q3D of Rac-1 compound on mouse model of bleomycin-induced pulmonary fibrosis (**p <0.01; ****p <0.0001).

In fig. 5 shows the histopathological Ashcroft score reflecting pulmonary fibrosis in a 14-day prophylaxis study comparing 60 mg/kg QD compound 1 with 30 mg/kg rAB0023, an anti-LOXL2 antibody.

In fig. Figure 6a shows glomerulosclerosis (left) and interstitial fibrosis (right) scores reflecting renal fibrosis in a Col4A3-deficient mouse model of Alport syndrome and chronic kidney disease. Samples were collected at 7 weeks of age following oral administration of Compound 1 at a 30 mg/kg QD dose starting at 2 or 5 weeks of age (**p < 0.01).

In fig. 6b shows tumor volume in an orthotopic human breast cancer model with MDA-MB-435-GFP cells implanted into the mammary fat pads of nude mice. Tumor volumes were measured weekly during the 4-week study (***p < 0.001, ****p < 0.0001).

In fig. Figure 7 shows the proportion of collagen area in Mdr2KO mice as measured by the percentage of positive picrosirius red staining in the liver. Mice were orally treated with Compound 1 at 30 or 60 mg/kg QD starting at 6 weeks of age. Samples were collected at 12 weeks of age.

In fig. Figure 8 shows the collagen area fraction in a mouse model of thioacetimide (TAA)-induced liver fibrosis. Mice were orally treated with compound 1 at a dose of 30 mg/kg QD starting 3 or 6 weeks after the onset of TAA. Samples were collected at 12 weeks of age after initiation of TAA.

In fig. 9 shows plasma LOXL2 concentrations measured in healthy male and female subjects and scleroderma patients (n=10 each) measured using a biotin-labeled LOXL2 inhibitor and proprietary Erenna®-based assay (*p=0 .04 unpaired t-test in SSc (female) versus healthy controls (female)).

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Detailed Disclosure of the Present Invention

Lysyl oxidase-like protein 2 (LOXL2) is a member of the lysyl oxidases (LOX) family, which includes Cu ²⁺ -dependent and lysine tyrosylquinoline (LTQ)-dependent amine oxidases. The family includes five genes: lox (LOX), loxl1 (lysyl oxidase-like protein-1, LOXL1), loxl2 (LOXL2), loxl3 (lysyl oxidase-like protein-3, LOXL3) and loxl4 (lysyl oxidase-like protein-4, LOXL4) . The LOX family is known for its ability to catalyze the oxidative deamination of the amino group of lysines and hydroxylysines in collagen and elastin, promoting the cross-linking of these molecules. Cross-linking of collagen and elastin is essential for maintaining the tensile strength of the extracellular matrix.

The development of pathological stroma plays an important role in the disease. The pathological stroma consists of activated stromal cells, collagen matrix, growth factors and angiogenic structures. During pathological conditions such as fibrogenesis, fibroblasts are recruited and activated, resulting in a microenvironment that promotes increased synthesis and deposition of extracellular matrix proteins, leading to the development of fibrosis.

Disease-associated activation of fibroblasts in fibrotic diseases and cancer leads to extracellular matrix remodeling, which ultimately leads to excessive deposition of extracellular matrix proteins, including collagen I and III, increased cross-linking of newly deposited collagen, and increased tissue stiffness. In addition, activated fibroblasts express numerous proangiogenic, provasculogenic and proliferative growth factors and cytokines, such as transforming growth factor beta (TGF-β), connective tissue growth factor (CTGF), stromal cell-derived factor 1 (SDF-1) and endothelial growth factor blood vessels (VEGF), thereby playing an important role in paracrine signaling during disease progression. Impairing the development of this pathological stroma by inhibiting the activation and recruitment of fibroblasts and/or their signaling pathways represents a new therapeutic strategy for fibrotic disease.

Despite similar catalytic activity, each lysyl oxidase enzyme has been reported to have unique expression and functional activity. LOXL2 plays a central role in the development of pathological stroma in fibrotic diseases by activating and recruiting fibroblasts to the pathological site.

LOXL2 has been demonstrated to have intracellular functions in addition to its role in extracellular matrix remodeling. LOXL2 positively regulates the epithelial-mesenchymal transition (EMT) transducer Snail1, promoting the stability and functional activity of Snail1. LOXL2 makes a positive contribution to the activation of the focal adhesion kinase (FAK) signaling pathway and is involved in the organization of focal adhesion complexes. LOXL2 gene silencing results in reacquisition of polarity in epithelial cells and reduces the migratory and invasive ability of mammary cell lines. Modulation of cell adhesion and cell polarity has been reported to be mediated by intracellular LOXL2. LOXL2 transcriptionally represses E-cadherin as well as tight junction and cell polarity genes through Snail1-dependent and Snail1-independent mechanisms. More recently, LOXL2 has been described to associate with chromatin and has been reported to be involved in trimethyldeamination of histone H3, a function that is dependent on the catalytic domain of LOXL2.

In accordance with some embodiments, the methods disclosed herein are methods of inhibiting intracellular LOXL2. In accordance with some embodiments, the methods disclosed herein are methods of inhibiting extracellular (secreted) LOXL2. In accordance with some embodiments, the methods disclosed herein are methods of inhibiting extracellular and intracellular LOXL2.

Fibrosis.

LOXL2 is involved in fibrotic processes. Fibrous processes involve excessive deposition of extracellular matrix components such as collagen, which alters the physical, biochemical, and biomechanical properties of the matrix, leading to organ malfunction and organ failure. Tissue fibrosis is also associated with cancer progression through direct stimulation of cellular transformation and metastasis. Tumors are typically stiffer than normal tissue, and tumor stiffness influences tumor metastasis.

Excessive activity of the LOXL2 enzyme is associated with increased tumor stiffness. Elevated LOXL2 levels are also associated with fibrotic liver lesions in patients suffering from Wilson's disease, primary biliary cirrhosis and NASH. In addition, administration of the LOXL2-specific monoclonal antibody AB0023 was effective in reducing disease severity in a fibrosis model. AB0023 has been shown to inhibit the production of growth factors and cross-linked collagen matrix and TGF-beta signaling.

LOXL2 promotes the cross-linking of type I collagen and is the main regulator of fibrogenesis of various etiologies and in various organs. Circulating LOXL2 levels correlate with stage

- 9 044096 fibrosis. LOXL2 is a major target of the pathway in fibrotic disease. Mehal et al. Expressway to the core of fibrosis, Nat Med.2011. 17: 552-553.

In healthy adult tissues, there is only negligible expression of LOXL2, and under normal (eg, disease-free) conditions, the amount of circulating LOXL2 is low. In certain disease states, circulating levels of LOXL2 are elevated. For example, LOXL2 may be elevated in the serum of patients with pulmonary fibrosis and chronic liver disease, such as those with chronic hepatitis C, with higher levels in patients with more advanced fibrosis. Detection of circulating LOXL2 is useful in determining whether an individual has a disease that results in elevated levels of circulating LOXL2. These diseases include fibrosis and cancer.

Circulating LOXL2 levels were found to correlate with the stage of fibrosis. It has also been discovered that the level of circulating LOXL2 can serve as an indicator of whether an individual suffering from fibrosis is responsive to treatment for fibrosis, and provide other prognostic and predictive information regarding the disease, such as the likelihood of a particular expected result, outcome or event, such as disease outcome or susceptibility of the disease to treatment.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof for treating or preventing the development of fibrosis in a mammal.

Fibrosis as used herein refers to the accumulation of extracellular matrix components that occurs following injury, inflammation, tissue repair, immunological reactions, cellular hyperplasia and neoplasia.

In accordance with some embodiments, disclosed herein is a method of reducing fibrosis in tissue, comprising contacting a fibrotic cell or tissue with a compound disclosed herein in an amount sufficient to reduce or inhibit fibrosis. In accordance with some embodiments, fibrosis includes a fibrotic condition.

According to some embodiments, fibrosis includes pulmonary fibrosis, liver fibrosis, renal fibrosis, cardiac fibrosis, peritoneal fibrosis, ocular fibrosis, or cutaneous fibrosis. In accordance with some embodiments, the fibrosis is pulmonary fibrosis. In accordance with some embodiments, the fibrosis is liver fibrosis. In accordance with some embodiments, the fibrosis is renal fibrosis. In accordance with some embodiments, the fibrosis is cardiac fibrosis. In accordance with some embodiments, the fibrosis is peritoneal fibrosis. In accordance with some embodiments, the fibrosis is fibrosis of the eye. In accordance with some embodiments, the fibrosis is dermal fibrosis.

In accordance with some embodiments, reducing fibrosis or treating a fibrotic condition includes reducing or inhibiting one or more of the formation or deposition of extracellular matrix proteins; number of profibrotic cell types (eg number of fibroblasts or immunocytes); the content of cellular collagen or hydroxyproline in fibrous lesions; fibrogenic protein expression or activity; or reducing fibrosis associated with the inflammatory response.

In accordance with some embodiments, the fibrotic condition is a fibrotic condition of the lung.

In accordance with some embodiments, the fibrotic condition is a fibrotic condition of the liver.

In accordance with some embodiments, the fibrotic condition is a fibrotic condition of the heart.

In accordance with some embodiments, the fibrotic condition is a fibrotic condition of the kidney.

In accordance with some embodiments, the fibrotic condition is a fibrotic condition of the skin.

In accordance with some embodiments, the fibrotic condition is a fibrotic condition of the eye.

In accordance with some embodiments, the fibrotic condition is a fibrotic condition of the gastrointestinal tract.

In accordance with some embodiments, the fibrotic condition is a fibrotic condition of the bone marrow.

According to some embodiments, the fibrotic condition is a fibrotic condition of the ear.

In accordance with some embodiments, the fibrotic condition is idiopathic. In some embodiments, the fibrotic condition is associated with (e.g., developed from) a disease (e.g., an infectious disease, an inflammatory disease, an autoimmune disease, a malignant or cancerous disease, and/or a connective tissue disease). ; toxin; stroke (eg, environmental hazards (eg, asbestos, coal dust, polycyclic aromatic hydrocarbons), cigarette smoking, wound); medical treatment (such as surgery, chemotherapy or radiation) or a combination of these.

In accordance with some embodiments, disclosed herein is a method of treating or preventing the development of fibrosis in a mammal, comprising administering a LOXL2 inhibitor described herein or a pharmaceutically acceptable salt thereof to a mammal in need thereof.

In accordance with some embodiments, disclosed herein is a method of improving lung function in a mammal comprising administering a LOXL2 inhibitor described herein or a pharmaceutically acceptable salt thereof to a mammal in need thereof. In accordance with some embodiments, the mammal has been diagnosed with pulmonary fibrosis.

In accordance with some embodiments, disclosed herein is a method of treating idiopathic pulmonary fibrosis in a mammal, comprising administering a LOXL2 inhibitor described herein or a pharmaceutically acceptable salt thereof to a mammal in need thereof.

In accordance with some embodiments, disclosed herein is a method of controlling abnormal accumulation or activation of cells, fibronectin, collagen, or increased fibroblast tissue recruitment in a mammal, comprising administering a LOXL2 inhibitor described herein or a pharmaceutically acceptable salt thereof to the mammal in need thereof. In accordance with some embodiments, abnormal accumulation or activation of cells, fibronectin, collagen, or increased recruitment of fibroblasts to tissue results in fibrosis.

In accordance with some embodiments, disclosed herein is a method of treating or preventing the development of scleroderma in a mammal, comprising administering a LOXL2 inhibitor described herein or a pharmaceutically acceptable salt thereof to a mammal in need thereof.

In accordance with some embodiments, disclosed herein is a method of reducing unwanted or abnormal skin thickening in a mammal comprising administering to the mammal in need a LOXL2 inhibitor described herein or a pharmaceutically acceptable salt thereof. In accordance with some embodiments, the skin thickening is associated with scleroderma.

In accordance with some embodiments, described herein is a method of controlling abnormal accumulation or activation of cells, fibronectin, collagen, or increased fibroblast recruitment in tissues of a mammal, comprising administering to the mammal in need thereof a LOXL2 inhibitor described herein or a pharmaceutically acceptable salt thereof. In accordance with some embodiments, abnormal accumulation or activation of cells, fibronectin, collagen, or increased recruitment of fibroblasts in skin tissue results in fibrosis. In accordance with some embodiments, described herein is a method of reducing hydroxyproline in tissues of a mammal with fibrosis by administering to the mammal in need a LOXL2 inhibitor described herein or a pharmaceutically acceptable salt thereof.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in treating or preventing the development of fibrosis or a fibrotic disease or condition in a mammal. In some cases, the disease or condition is associated with desmoplasia. Fibrosis may involve an abnormal accumulation of fibrous tissue that may occur, for example, as part of the wound healing process in damaged tissue, which may be caused, for example, by physical trauma, inflammation, infection, exposure to toxins, and other causes. Examples of fibrosis include skin scarring, keloids, liver fibrosis, pulmonary fibrosis, renal fibrosis, glomerular sclerosis, tubulointerstitial fibrosis, and scleroderma.

In one aspect, provided herein are methods of using Compound I or a pharmaceutically acceptable salt or solvate thereof to prevent the development and/or treat a condition, disease or disorder associated with LOXL2 activity. In accordance with some embodiments, the methods disclosed herein comprise administering Compound I, or a pharmaceutically acceptable salt or solvate thereof, to a subject with a disease, condition or disorder described herein. In some embodiments, the methods disclosed herein comprise administering Compound I or a pharmaceutically acceptable salt or solvate thereof to a subject suspected of having or developing a disease, condition or disorder described herein.

In one aspect, provided herein are methods of treating or preventing the development of one or more signs, symptoms or complications in a subject caused by a disease, condition or disorder described herein, the methods comprising administering Compound I or a pharmaceutically acceptable salt or solvate thereof subject.

In one aspect, provided herein are methods of preventing the development of a condition, disease, or disorder associated with LOXL2 activity, the methods comprising administering Compound I or a pharmaceutically acceptable salt or solvate thereof in combination with another preventative therapy.

In one aspect, provided herein are methods of treating a condition, disease, or disorder associated with LOXL2 activity, the methods comprising administering Compound I or a pharmaceutically acceptable salt or solvate thereof in combination with another treatment agent.

In one aspect, provided herein are methods for mitigating, relieving and/or suppressing a sign, symptom or complication of a condition, disease or disorder associated with LOXL2 activity, the methods comprising administering Compound I or a pharmaceutically acceptable salt or solvate thereof.

In one aspect, provided herein are methods for mitigating, relieving and/or eliminating a sign, symptom or complication of a condition, disease or disorder associated with LOXL2 activity, the methods comprising administering Compound I or a pharmaceutically acceptable salt or solvate thereof in combination with one or several additional therapies.

In accordance with some embodiments, administration of Compound I or a pharmaceutically acceptable salt or solvate thereof, or administration of a pharmaceutical composition comprising Compound I or a pharmaceutically acceptable salt or solvate thereof described herein, involves administering Compound I or a pharmaceutically acceptable salt or solvate thereof therapeutically effective dose. In some embodiments, the therapeutically effective dose is from about 0.01 mg to 5000 mg. For example, a therapeutic dose is from about 1 mg to about 5000 mg, from about 50 mg to about 4000 mg, from about 50 mg to about 4000 mg, from about 150 mg to about 4000 mg, from about 250 mg to about 2000 mg, from about 50 mg to about 1000 mg or any integer between the above values. In accordance with some embodiments, the therapeutically effective dose is administered continuously. In some embodiments, the therapeutically effective dose is administered 4 times a day, 3 times a day, 2 times a day, once a day, 6 times a week, 5 times a week, 4 times a week, 3 times a week, twice a week, once a week or less. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered for a therapeutically effective time interval by any of the methods described herein. In some cases, a therapeutically effective time interval is the time required to reduce the intensity or eliminate one or more signs or symptoms of a disease, condition or disorder described herein. For example, a therapeutically effective time interval is from 1 day to 1 year. The above examples of therapeutic doses are not limiting. Additional treatment regimens are further described elsewhere in this document.

Pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a specific form of chronic, progressive, fibrosing interstitial pneumonia of unknown etiology that is limited to the lungs. More than one hundred different forms of interstitial lung disease (ILD) have been described. These diffuse infiltrative lung disorders are usually characterized by the presence of inflammation and altered interstitial lung tissue. Histopathological changes in the lungs of patients with ILD can range from granulomatous inflammation without parenchymal fibrosis in patients with sarcoidosis to extensive pulmonary fibrosis with structural deformation of the lung in patients with idiopathic pulmonary fibrosis (IPF). Some forms of ILD have been associated with certain genetic abnormalities (eg, Hermansky-Pudlak syndrome, hereditary pulmonary fibrosis), and a number of gene variants have been associated with an increased risk of developing ILD disorders such as IPF, sarcoidosis, or chronic berylliosis (CBD).

Interstitial lung disease may also complicate connective tissue diseases (CTD), and histopathological changes in the lungs may have features consistent with the pattern of usual interstitial pneumonia (UIP) or nonspecific interstitial pneumonia (NSIP) in CTD-associated ILD.

In some embodiments, interstitial lung disease includes, but is not limited to, idiopathic interstitial pneumonia, interstitial lung disease associated with diffuse connective tissue disease (CTD-ILD), sarcoidosis, hypersensitivity pneumonitis, iatrogenic pneumonitis/fibrosis (drug-induced ILD). , radiation injury), eosinophilic ILD (eg, eosinophilic pneumonia), occupational lung disease, hereditary pulmonary fibrosis, Hermansky-Pudlak syndrome), or pulmonary Langerhans cell histiocytosis. In some embodiments, idiopathic interstitial pneumonia includes, but is not limited to, idiopathic pulmonary fibrosis (IPF), nonspecific interstitial pneumonia (NSIP), cryptogenic organizing pneumonia (COP), respiratory bronchiolitis associated with interstitial lung disease (RBILD), desquamative interstitial pneumonia (DIP), acute interstitial pneumonia (AIP), lymphoid interstitial pneumonia (LIP).

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal lung disease involving damage to airway epithelial cells, fibroblast activation and proliferation, and excessive deposition of collagen and other extracellular matrix (ECM) components. These modifications in ECM composition and organization alter the biomechanical properties of the lung parenchyma and increase local stress, which is critical in the pathogenesis of IPF disease.

One important driver of matrix tension is lysyl oxidase-like protein 2 (LOXL2), an enzyme that catalyzes the covalent cross-linking of ECM molecules, including fibrillar collagens. LOXL2 protein expression is observed in fibroblastic lesions and collagenous areas of diseased lung tissue in IPF, with relatively little expression in healthy lung tissue (Barry-Hamilton et al. Nat Med 2010; 16:1009–1017). LOXL2 has also been localized to the perimeter of the active, diseased zone in liver fibrosis and is considered a major driver in fibrosis (Mehal et al., Nat Med 2011; 17:552-553). LOXL2 is associated with areas of active fibrogenesis in affected tissues.

IPF is a specific form of chronic, progressive, fibrosing interstitial pneumonia of unknown etiology, occurring predominantly in the elderly, limited to the lungs and associated with a histopathological and/or radiological pattern of UIP. This disease is characterized clinically by progressive deterioration of dyspnea and pulmonary function, and pathologically by the formation of scar tissue in the lungs in the absence of any known provocation. Patients with IPF symptoms typically range in age from 40 to 70 years, with a median age of onset of 66 years.

IPF is characterized by a decline in lung function over time. The most noticeable symptoms of IPF are exercise-induced shortness of breath and a chronic dry cough, which interfere with patients' daily activities. In addition to limiting pulmonary function defects, other common clinical signs of IPF include moist rales in the lower respiratory tract and hypoxemia-induced clubbing. Retrospective studies suggest that symptoms precede diagnosis of IPF by 6 months to 2 years. Symptoms appear slowly, but over months or years, symptoms worsen and lung function slowly declines, leading to hypoxia and eventually death from respiratory failure. There are 3 possible clinical courses for IPF: a) slow physiological deterioration with increasing severity of dyspnea, which is the most common; b) rapid deterioration and progression to death; or c) periods of relative stability interspersed with periods of acute deterioration in respiratory function, which sometimes occur during hospitalization for respiratory failure. The median survival time for IPF is estimated to be 2 to 5 years from diagnosis.

Although IPF is considered a disorder of unknown etiology by definition, a number of potential risk factors have been identified. Cigarette smoking is strongly associated with IPF. In addition, various other environmental and occupational exposures to metal dust, wood dust, agricultural work, hairdressing, stone cutting/polishing, animal husbandry, and plant/animal dust have been found to be associated with an increased risk of developing IPF.

Usual interstitial pneumonia has a histological/radiological pattern associated with IPF. The histologic appearance of UIP consists of normal lung interspersed with patches of dense fibrosis.

Hamman-Rich syndrome is also known as acute interstitial pneumonia or AIP and has a rapid clinical course (days to weeks), a high mortality rate, and a characteristic histopathological appearance on biopsy; the predominant histopathological feature is diffuse alveolar damage (DAD).

The median age of AIP in published cases is 50 years. The management of patients with AIP/DAD often involves immunosuppressive treatment, usually in the form of high-dose intravenous corticosteroids. In contrast, in IPF, immunosuppressive therapy is known to be ineffective as maintenance therapy.

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ILD can be diagnosed in patients under the age of 18 and is known as childhood interstitial lung disease (chILD). Immunocompetent children with ILD are usually treated with some type of immunosuppressive therapy, most often corticosteroids. Fibrosis is believed to be a consequence of constant inflammation. In general, chILD can be divided into 2 main categories: diseases that appear soon after birth, and diseases that develop after age 2 years. Diseases that appear after the age of 2 years are usually treated with anti-inflammatory therapy, in particular corticosteroids. In contrast, diseases younger than 2 years of age tend to be air space-filling diseases with associated interstitial fibrosis (ie, not similar to adult ILD) and tend to have a poor prognosis compared with older children with chILD. Patients with chILD <2 years of age include a subset of infants with mutations in the ATP-binding cassette transporter A3 (ABCA3) and surfactant protein C. Although many surfactant protein mutations are fatal from acute neonatal respiratory failure, some patients with fewer severe mutations develop chronic interstitial lung disease, which is not treatable with standard therapy.

In accordance with some embodiments, described herein is a method of treating pulmonary fibrosis in a mammal comprising administering a selective LOXL2 inhibitor to the mammal in need thereof. In accordance with some embodiments, the selective LOXL2 inhibitor is Compound I or a pharmaceutically acceptable salt or solvate thereof. In some embodiments, the selective LOXL2 inhibitor is a hydrochloride salt of Compound I. In some embodiments, the selective LOXL2 inhibitor is a mesylate salt of Compound I.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in the treatment or prevention of the development of a pulmonary fibrosis-related disease or condition. Lung fibrosis includes a large number of syndromes and diseases. Illustrative diseases include idiopathic pulmonary fibrosis (IPF), idiopathic interstitial pneumonia, and acute respiratory distress syndrome (ARDS). Pulmonary fibrosis also includes, but is not limited to, cryptogenic fibrosing alveolitis, chronic fibrosing interstitial pneumonia, interstitial lung disease (ILD), and diffuse parenchymal lung disease (DPLD).

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in the treatment or prevention of pulmonary fibrosis.

The pathogenesis of most pulmonary fibrosis, including the aforementioned diseases, is not well understood, but all are characterized by an influx of inflammatory cells and a subsequent increase in the synthesis and deposition of collagen-rich extracellular matrix.

IPF is characterized by inflammation and eventual fibrosis of lung tissue; although these two symptoms can also be dissociated. The etiology of IPF is unknown; it can occur either due to an autoimmune disorder or as a result of infection. Symptoms of IPF include dyspnea (ie, shortness of breath), which becomes a major symptom as the disease progresses, and a dry cough. Death may result from hypoxemia, right heart failure, heart attack, pulmonary embolism, stroke, or lung infection that may be caused by this disease.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in treating or preventing the development of a fibrotic lung condition. In some embodiments, the fibrotic lung condition is selected from one or more of pulmonary fibrosis, idiopathic pulmonary fibrosis (IPF), usual interstitial pneumonitis (UIP), interstitial lung disease, cryptogenic fibrosing alveolitis (CFA), bronchiolitis obliterans, or bronchiectasis. In some embodiments, pulmonary fibrosis develops due to a disease, a toxin, a stroke, medical treatment, or a combination thereof. In accordance with some embodiments, pulmonary fibrosis is associated with one or more of a disease process, such as asbestosis and silicosis; harmful production factor; environmental pollution; smoking cigarettes; connective tissue-related autoimmune disorders (eg, rheumatoid arthritis, scleroderma and systemic lupus erythematosus (SLE)); connective tissue disorder such as sarcoidosis; an infectious disease, for example an infection, in particular a chronic infection; medical treatment, including, without limitation, radiation therapy and drug therapy, such as chemotherapy (eg, treatment with bleomycin, methotrexate, amiodarone, busulfan and/or nitrofurantoin). In accordance with some embodiments, the fibrotic lung condition treated using the methods of the present invention is associated with (eg, developed in the background of) anticancer treatment, for example, treatment of cancer (eg, squamous cell carcinoma, testicular cancer, Hodgkin's disease with bleomycin ).

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In people with IPF, higher serum LOXL2 (sLOXL2) levels are associated with an increased risk of IPF disease progression (Chien et al. Eur Respir J 2014;43:1430-1438). In accordance with some embodiments, sLOXL2 levels predict the response of a patient with IPF to targeted therapy with a selective LOXL2 inhibitor (eg, Compound I or a pharmaceutically acceptable salt or solvate thereof). Patients with high baseline LOXL2 levels are at increased risk of poor IPF outcome. In some embodiments, Compound I, or a pharmaceutically acceptable salt or solvate thereof, is used to treat a fibrotic disease or condition in a human in which the level of sLOXL2 is at least 2-fold, at least 4-fold, at least 6-fold , at least 8-fold, at least 10-fold, at least 20-fold, at least 50-fold, or at least 100-fold higher than the sLOXL2 level of a person without a fibrotic disease or condition. In accordance with some embodiments, the fibrotic disease or condition is pulmonary fibrosis. In accordance with some embodiments, the fibrotic disease or condition is IPF.

In some embodiments, the severity of idiopathic pulmonary fibrosis is assessed by assessing symptoms, testing pulmonary function, exercise capacity, lung structure using CT scans, and using the St. George's Respiratory Questionnaire (SGRQ).

Pulmonary function tests (PFTs) are an important tool in assessing the severity of IPF. The easiest test to perform is spirometry, which involves maximal exhalation through a mouthpiece after maximal inhalation. The result is forced vital capacity (FVC). This is the amount of air exhaled after maximum inhalation.

Results are compared to normal rates, adjusted for age, sex, and race. The results are displayed as air volume as well as predicted percentage. A normal result is approximately 80% of predicted or higher. There is no single agreed upon threshold for staging IPF by FVC, but many clinicians use the following: mild IPF at approximately >75% of predicted FVC, moderate IPF at approximately 50-75% of predicted FVC, severe IPF at approximately 25-49% of predicted FVC and very severe IPF at approximately <25% of predicted FVC. More important than the specific FVC value is the change in FVC over time. A >5-10% decrease in FVC is associated with an increased risk of death.

In accordance with some embodiments, administration of Compound I or a pharmaceutically acceptable salt or solvate thereof to a human with pulmonary fibrosis increases the human's FVC. In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof increases the FVC of a person with pulmonary fibrosis by about 5%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30 %, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80 %, about 85%, about 90%, about 95%, about 100%, or more than 100%.

Diffusion capacity is another type of pulmonary function test. It is an indicator of how gas exchange occurs through the lungs. Results are presented as a percentage of predicted values. Lower values indicate more advanced stages of the disease. Values less than 40% are associated with worse survival. Decreased diffusion capacity is also associated with worse outcome. The diffusing capacity of the lungs for carbon monoxide (DLCO) determines how much oxygen passes from the alveoli of the lungs to the bloodstream.

In accordance with some embodiments, administration of Compound I or a pharmaceutically acceptable salt or solvate thereof to a human with pulmonary fibrosis increases DLCO. In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof increases the DLCO of a person with pulmonary fibrosis by about 5%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30 %, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80 %, about 85%, about 90%, about 95%, about 100%, or more than 100%.

The six-minute walk test measures exercise capacity (distance traveled, oxygen saturation during exercise, and heart rate and blood pressure).

High-resolution CT scans can assess the structural extent of fibrosis—how much fibrosis is present. More severe fibrosis on radiographs is associated with poorer outcomes. Increasing degree of fibrosis over time is also associated with poorer outcomes.

Other factors associated with a poorer prognosis include advanced age, gender, recording

- 15 044096 history of heavy smoking, underweight, development of pulmonary hypertension and exacerbation of the underlying disease. Patients with IPF are also at increased risk of developing lung cancer, which has a strong impact on prognosis.

The St George's Respiratory Questionnaire (SGRQ) provides a quotient designed to measure and quantify health status in patients with chronic airflow limitation. It has been shown to correlate well with established measures of symptom level, disease activity, and disability (Jones et al., The St. George's Respiratory Questionnaire. Resp Med 1991;85 (suppl B):2531; Jones et al., A self- complete measure of health status for chronic airflow limitation. Am Rev Respir Dis 1992; 145; 1321-1327; Barr et al., American translation, modification, and validation of the St. George's Respiratory Questionnaire. Clin Ther. 2000 Sep, 22( 9): 1121-45).

The SGRQ is self-administered. The first part of the SGRQ (Symptoms) assesses symptomatology, including frequency of cough, sputum production, wheezing, shortness of breath, and the duration and frequency of episodes of shortness of breath or wheezing. The assessment is repeated at 1, 3 or 12 month recall. The second part consists of two components: Activity and Impact. The Activities section focuses on activities that cause shortness of breath or are limited due to shortness of breath. The Impact section covers a range of factors, including impact on employment, health management, panic, disease-specific symptoms, medication requirements, side effects from prescribed therapies, health predictions, and disruption to daily life.

Scores range from 0 to 100, with higher scores indicating more limitations. Based on empirical data and patient interviews, a mean change score of 4 is associated with a marginally effective treatment, 8 with a moderately effective change, and 12 with a highly effective change (Jones PW., Eur Respir J 1994, 7:55-62 ; Jones PW. Eur Respir J. 2002 Mar, 19(3):398-404).

In accordance with some embodiments, administration of Compound I or a pharmaceutically acceptable salt or solvate thereof to a human with pulmonary fibrosis results in a decrease in the SGRQ score. In accordance with some embodiments, the SGRQ score is reduced by at least 1 unit, by at least 2 units, by at least 3 units, by at least 4 units, by at least 5 units, by at least 6 units, at least 7 units, at least 8 units, at least 9 units, at least 10 units, at least 11 units, at least 12 units or more than 12 units.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used to slow the decline of lung function in a person with pulmonary fibrosis. In accordance with some embodiments, Compound I, or a pharmaceutically acceptable salt or solvate thereof, is used to reduce the frequency of exacerbations of disease in a person with pulmonary fibrosis. In accordance with some embodiments, Compound I, or a pharmaceutically acceptable salt or solvate thereof, is used to prolong survival of a person with pulmonary fibrosis. In some embodiments, Compound I, or a pharmaceutically acceptable salt or solvate thereof, is used to slow the decline of lung function, reduce the incidence of disease exacerbations, and increase survival in a person with pulmonary fibrosis.

As the normal lung is replaced by scar tissue, the lungs' ability to exchange gas and deliver oxygen to the blood is impaired. If enough of the lungs are involved, this can cause low oxygen levels in the blood. This is called hypoxemia or hypoxia. Blood oxygen levels are measured in two ways.

Non-invasive oxygen measurements are made using a pulse oximeter. A pulse oximeter reads saturation, which measures the percentage of hemoglobin that carries oxygen. Normal values are between 96-100%.

A more accurate way to measure the amount of oxygen in the blood is using arterial blood gas. To do this, you need to insert a needle into the artery of the wrist and withdraw a few milliliters of blood. The oxygen pressure is then measured directly.

In some embodiments, oxygen is administered to a subject when saturation values are less than 88-89%, either at rest, during activity, or during sleep. Resting oxygen saturation values are generally higher than oxygen saturation during exercise. Oxygen saturation during sleep usually falls in between.

Oxygen is supplied from reservoirs or concentrators through a nasal cannula. Typical flow rates start at 2 liters per minute, but can be increased if necessary. Advanced delivery systems, such as the Oximizer pendant, can improve oxygen delivery to patients who require high flow rates.

In some embodiments, gastroesophageal reflux disease (GERD) plays a role in the progression of IPF. In accordance with some embodiments, an acid suppressing therapeutic agent is administered together with Compound I or a pharmaceutically acceptable salt or solvate thereof. Acid-suppressing therapeutic agents include, but are not limited to, H2 blockers (eg, cimetidine, famotidine, lafutidine, nizatidine, ranitidine, roxatidine, thiotidine) and proton pump inhibitors (eg, omeprazole).

In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in conjunction with vaccination against pneumonia. Suitable vaccines include, but are not limited to, polysaccharide vaccines and conjugate vaccines. The polysaccharide vaccine most commonly used today (PneumoVax) consists of purified polysaccharides from 23 serotypes (1, 2, 3, 4, 5, 6b, 7F, 8, 9N, 9V, 10A, 11A, 12F, 14, 15B, 17F , 18C, 19F, 19A, 20, 22F, 23F and 33F). Conjugate vaccines consist of capsular polysaccharides covalently linked to diphtheria toxoid CRM197. An example of a conjugate vaccine is Prevnar 13. PneumoVax is given as 2 doses separated by at least 5 years and separated from Prevnar by at least one year. Prevnar is given as a single dose.

In some embodiments, pulmonary rehabilitation is administered together with Compound I or a pharmaceutically acceptable salt or solvate thereof. Pulmonary rehabilitation is a structured exercise program that focuses on both aerobic and strength training.

In accordance with some embodiments, one or more cough suppressant drugs are administered together with Compound I or a pharmaceutically acceptable salt or solvate thereof. Cough can be one of the most bothersome symptoms of IPF. Cough medications include, but are not limited to, expectorants, antitussives or cough suppressants, antihistamines, decongestants, steroids, benzonates, thalidomide, cannabinoids, honey and sugar syrups.

Expectorants include, but are not limited to, acetylcysteine and guaifenesin.

Antitussives or cough suppressants include, but are not limited to, codeine, pholcodine, dextromethorphan, noscapine, and butamirate.

Antihistamines include, but are not limited to, mepyramine (pyrilamine), antazoline, diphenhydramine, paracarbinoxamine, doxylamine, clemastine, dimenhydrinate, pheniramine, chlorphenamine (chlorpheniramine), dexchlorpheniramine, brompheniramine, triprolidine, cetirizine, cyclizine, chlorcyclizine, hydroxyzine, meclizine, loratadine, deslorata ding, promethazine, alimemazine (trimeprazine), cyproheptadine, azatadine, ketotifen, arivastine, astemizole, cetirizine, mizolastine, terfenadine, azelastine, levocabastine, olopatadine, levocetirizine, fexofenadine.

Decongestants include, but are not limited to, ephedrine.

Steroids include, but are not limited to, betamethasone, prednisolone, aclomethasone, aldosterone, amcinonide, beclomethasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortisone, cortivasol, deflazacort, deoxycorticosterone, desonide, deoxymethasone, deoxycortone, xamethasone, diflorazon, diflucortolone , difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin, fluocortolone, fluorometholone, fluperolone, fluprednidene, fluticasone, formocortal, galcinonide, halomethasone, hydrocortisone/cortisol, hydro cortisone aceponate, hydrocortisone buteprate, hydrocortisone butyrate, loteprednol , medrisone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisone/prednisolone, rimexolone, thixocortol, triamcinolone and ulobetazole.

Cannabinoids include, but are not limited to, cannabis, marinol, and dronabinol.

In accordance with some embodiments, honey or sugar syrups soothe cough.

In accordance with yet another embodiment described herein, Compound I or a pharmaceutically acceptable salt or solvate thereof is co-administered with at least one agent used in the treatment of respiratory conditions. Drugs used in the treatment of respiratory conditions include, but are not limited to, bronchodilators (e.g., sympathomimetic agents and xanthine derivatives), leukotriene receptor antagonists, leukotriene inhibitors, leukotriene modulators, nasal decongestants, respiratory enzymes, pulmonary surfactants, antihistamines (e.g., mepyramine (pyrilamine) , antazoline, diphenhydramine, carbinoxamine, doxylamine, clemastine, dimenhydrinate, pheniramine, chlorphenamine (chlorpheniramine), dexchlorpheniramine, brompheniramine, triprolidine, cetirizine, cyclizine, chlorcyclizine, hydroxyzine, meclizine, loratadine, desloratidine, promethazine, alimemazine (trimeprazine), cyproheptadine, azatadine , ketotifen, arivastine, astemizole, cetirizine, mizolastine, terfenadine, azelastine, levocabastine, olopatadine, levocetirizine, fexofenadine), mucolytics, corticosteroids, anticholinergics, antitussives, analgesics, expectorants, albuterol, ephedrine, epinephrine, formoterol, metaproterene ol, terbutaline, budesonide, ciclesonide, dexamethasone, flunisolide, fluticasone propionate, triamcinolone acetonide, ipratropium bromide, pseudoephedrine, theophylline, montelukast, zafirlukast, ambrisentan, bosentan, enrasentan, sitaxentan, tezosentan, iloprost, treprostinil, pirfenidone, nintedanib, inhibitors 5-lipoxygenase-activating protein (FLAP) ), FLAP modulators and 5-LO inhibitors.

- 17 044096

In accordance with a specific embodiment described herein, Compound I or a pharmaceutically acceptable salt or solvate thereof is co-administered with at least one anti-inflammatory agent. In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is co-administered with at least one additional agent selected, but not limited to, epinephrine, isoproterenol, orciprenaline, bronchodilators, glucocorticoids, leukotriene modifiers, mast cell stabilizers, xanthines, anticholinergics agents, β-2-agonists, FLAP inhibitors, FLAP modulators or 5-LO inhibitors. β-2 agonists include, but are not limited to, short-acting β-2 agonists (e.g., salbutamol (albuterol), levalbuterol, terbutaline, pirbuterol, procaterol, metaproterenol, fenoterol, and bitolterol mesylate) and long-acting β-2 agonists (e.g., salmeterol, formoterol , bambuterol and clenbuterol). FLAP inhibitors and/or FLAP modulators include, but are not limited to, 3-[3-tert-butylsulfanyl-1-[4-(6-methoxy-pyridin-3-yl)-benzyl]-5(pyridin-2-ylmethoxy)-1 N-indol-2-yl]-2,2-dimethylpropionic acid, 3-[3-tert-butylsulfanyl-1 [4-(6-ethoxy-pyridin-3-yl)-benzyl]-5-(5 -methyl-pyridin-2-ylmethoxy)-III-indol-2-yl]-2,2-dimethylpropionic acid, MK-886, MK-0591, BAY-x1005, MN-001 and compounds that can be found in US 2007 /0225285, US 2007/0219206, US 2007/0173508, US 2007/0123522 and US 2007/0105866 (each of which is therefore incorporated herein by reference). Glucocorticoids include, but are not limited to, beclomethasone, budesonide, ciclesonide, fluticasone, and mometasone. Anticholinergic drugs include, but are not limited to, ipratropium and tiotropium. Mast cell stabilizers include, but are not limited to, chromoglycate and nedocromil. Xanthines include, but are not limited to, amminophylline, theobromine and theophylline. Leukotriene antagonists include, but are not limited to, montelukast, tomelukast, pranlukast, and zafirlukast. 5-LO inhibitors include, but are not limited to, zileuton, VIA-2291 (ABT761), AZ-4407 and ZD-2138 and compounds which can be found in US 2007/0149579, WO2007/016784.

In one aspect, Compound I or a pharmaceutically acceptable salt or solvate thereof is co-administered with one or more agents used to treat asthma, including, but not limited to, combination inhalation agents (fluticasone and salmeterol oral inhalation formulation (eg, Advair)); inhaled beta-2 agonists (albuterol inhalation; albuterol nebulizer solution; formoterol; isoproterenol oral inhalation; levalbuterol; metaproterenol inhalation; pirbuterol acetate oral inhalation; salmeterol inhalation aerosol; salmeterol inhalation powder; inhalation terbutaline); inhaled corticosteroids (beclomethasone oral inhalation; budesonide inhalation solution; budesonide inhalation; flunisolide oral inhalation; fluticasone inhalation aerosol; fluticasone oral inhalation powder; mometasone inhalation powder; triamcinolone oral inhalation); leukotriene modifiers (montelukast; zafirlukast; zileuton); mast cell stabilizers (inhalation drug cromolyn; oral inhalation drug nedocromil); monoclonal antibodies (omalizumab); oral beta-2 agonists (albuterol oral syrup; albuterol oral tablets; metaproterenol; terbutaline); bronchodilator (aminophylline; oxtriphylline; theophylline).

In one aspect, Compound I or a pharmaceutically acceptable salt or solvate thereof is co-administered with one or more allergy treatments, including, without limitation, antihistamine and decongestant combinations (cetirizine and pseudoephedrine; desloratadine and pseudoephedrine ER; fexofenadine and pseudoephedrine; loratadine and pseudoephedrine); antihistamines (azelastine nasal spray; brompheniramine; brompheniramine oral suspension; carbinoxamine; cetirizine; chlorpheniramine; clemastine; desloratadine;

dexchlorpheniramine ER; dexchlorpheniramine oral syrup; oral diphenhydramine; fexofadadine; loratadine; promethazine); decongestants (pseudoephedrine); leukotriene modifiers (montelukast; montelukast granules); nasal anticholinergics (ipratropium); nasal corticosteroids (beclomethasone nasal inhalation; budesonide nasal inhalation; flunisolide inhalation; fluticasone nasal inhalation; mometasone nasal spray; triamcinolone nasal inhalation; triamcinolone spray); nasal decongestants (phenylephrine); nasal mast cell stabilizers (cromolyn nasal spray).

In one aspect, Compound I or a pharmaceutically acceptable salt or solvate thereof is co-administered with one or more agents used to treat chronic obstructive pulmonary disease (COPD), including, but not limited to, anticholinergics (ipratropium bromide oral inhalation agent); combination inhalation drugs (albuterol and ipratropium (eg, Combivent, Duoneb); fluticasone and salmeterol oral inhalation drug (eg, Advair)); corticosteroids (dexamethasone tablets; fludrocortisone acetate; hydrocortisone tablets; methylprednisolone; liquid prednisolone; pen

- 18 044096 oral prednisone; oral triamcinolone); inhaled beta-2 agonists (albuterol inhalation; albuterol nebulizer solution; formoterol; isoproterenol oral inhalation; levalbuterol; metaproterenol inhalation; pirbuterol acetate oral inhalation; salmeterol inhalation aerosol; salmeterol inhalation powder; inhalation terbutaline drug); inhaled corticosteroids (beclomethasone oral inhalation; budesonide inhalation solution; budesonide inhalation; flunisolide oral inhalation; fluticasone inhalation aerosol; fluticasone oral inhalation powder; triamcinolone oral inhalation); mucolytics (guaifenesin); oral beta-2 agonists (albuterol oral syrup; albuterol oral tablets; metaproterenol; terbutaline); bronchodilator (aminophylline; oxtriphylline; theophylline).

In accordance with one embodiment, Compound I or a pharmaceutically acceptable salt or solvate thereof is co-administered with inhaled corticosteroids.

In accordance with some embodiments, an immunosuppressive agent is administered together with Compound I or a pharmaceutically acceptable salt or solvate thereof. Immunosuppressive agents include, but are not limited to, prednisone and azathioprine.

In some embodiments, low doses of prednisone are administered together with Compound I or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, N-acetylcysteine (NAC) is co-administered with Compound I or a pharmaceutically acceptable salt or solvate thereof.

In some embodiments, Compound I, or a pharmaceutically acceptable salt or solvate thereof, is used in combination with another therapeutic agent that is useful in the treatment of pulmonary fibrosis, such as IPF. In accordance with some embodiments, therapeutic agents that are useful in treating pulmonary fibrosis such as IPF include agents that slow the decline of lung function over time. Therapeutics that slow the decline of lung function over time include, but are not limited to, pirfenidone and nintedanib. Additional therapeutic agents are provided, such as imatinab and other tyrosine kinase inhibitors, PBI-4050, recombinant pentraxin-2/SAP (PRM151), aerosolized IFN-γ, inhibitors of CTGF activity (FG-3019), LPA receptor antagonists (BMS986020, SAR100842), autotaxin inhibitors (GLPG-1690, PAT-409), galectin-3 inhibitors (TD 139), tipelukast (MN-001), integrin antagonists (STX-100/BG00011, GSK3008348), PI3K inhibitors (GSK2126458), JNK inhibitors (JNK -90001), ROCK inhibitors (KD025), anti-IL-13 compounds (tralokinumab, lebrikizumab, QAX-576), CCL2 antagonists, CCL2ta (CNTO888), CCR2 antagonists (cenicriviroc), anti-CD20 compounds (rituximab), anticoagulants (dabigatran) , collagen V-based therapies (IW001) and ASK1 inhibitors (GS4997).

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in combination with pirfenidone. In some embodiments, pirfenidone is co-administered with Compound I or a pharmaceutically acceptable salt or solvate thereof up to a maximum daily dose of 2403 mg.

In accordance with some embodiments, Compound I, or a pharmaceutically acceptable salt or solvate thereof, is used in combination with nintedanib. In some embodiments, nintedanib is co-administered with Compound I or a pharmaceutically acceptable salt or solvate thereof, up to a maximum daily dose of 300 mg.

The individual compounds of such combinations are administered either sequentially or simultaneously in separate or combination pharmaceutical preparations. In accordance with one embodiment, the individual compounds will be administered simultaneously in a combination pharmaceutical preparation. Suitable dosages of known therapeutic agents will be apparent to those skilled in the art.

The combinations mentioned herein are conveniently provided for use in the form of pharmaceutical compositions together with pharmaceutically acceptable diluent(s) or carrier(s).

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to an adult with pulmonary fibrosis. In accordance with some embodiments, the pulmonary fibrosis is IPF. In some embodiments, the adult's age is >18 years, >20 years, >25 years, >30 years, >35 years, >40 years, >45 years, >50 years, >55 years, >60 years , >65 years, >70 years, >75 years, >80 years, or >85 years. In some embodiments, the adult is 30 to 85 years old, 35 to 85 years old, 40 to 85 years old, 45 to 85 years old, or 40 to 80 years old.

Idiopathic pulmonary fibrosis most often affects adult men rather than women. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to an adult male with pulmonary fibrosis.

In accordance with some embodiments, Compound I or pharmaceutically

- 19 044096 an acceptable salt or solvate is administered to a child with ILD. The child's age is <18 years. In chronic ILD, children aged 0 to 2 years have a different spectrum of conditions compared to children aged 2 to 18 years. Genetic and developmental disorders affecting the airways are more likely to occur in children aged 0 to 2 years. After 2 years of age, disease in children tends to somewhat resemble IPF in adults (ie, more fibrotic). There is also a subgroup of infants with recessive ABCA3 mutations, which cause childhood interstitial lung disease.

In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a child with ILD, wherein the child's age is <18 years, 0 to <2 years, or >2 to <18 years.

Pulmonary alveolar proteinosis (PAP).

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment or prevention of pulmonary alveolar proteinosis (PAP). Pulmonary alveolar proteinosis (PAP) is a lung disease in which abnormal accumulation of pulmonary surfactant phospholipids and protein components occurs in the alveoli adjacent to the gas exchange zone. PAP may occur in a primary form or secondary to malignancy (especially myeloid leukemia), pulmonary infection, or exposure to environmental dust or chemicals. Rare hereditary forms have also been identified, suggesting a genetic component in some cases.

In accordance with some embodiments, LOXL2 is expressed in PAP tissue, but not in normal lung tissue. In accordance with some embodiments, LOXL2 promotes the development of PAP.

There are two forms of RAP: (1) primary (idiopathic) and (2) secondary (due to pulmonary infections, hematologic malignancies, and inhalation of mineral dusts such as silica, titanium oxide, aluminum, and insecticides). The incidence of RAR is increased in patients with hematologic malignancies and AIDS, suggesting an association with immune dysfunction.

The alveoli in RAP are filled with proteinaceous material, which has been carefully analyzed and determined to be normal surfactant, consisting of lipids and surfactant-associated proteins A, B, C and D (SP-A, SP-C, SP-D). There is evidence of disruption of the homeostatic mechanism of either surfactant production or clearance by alveolar macrophages and ciliated epithelium. A clear relationship between PAP and impaired macrophage maturation has been demonstrated.

The incidence for men is 4 times higher than for women. At the onset of the disease, the patient is usually 20-50 years old.

Patients with RAD typically have a gradual onset of symptoms including, but not limited to, persistent dry cough (or poor sputum production), progressive shortness of breath, fatigue and malaise, weight loss, intermittent low-grade fever and/or night sweats, pleuritic chest pain, cyanosis and hemoptysis.

The etiology of RAR is unknown. Causes may include inhalation of silica dust (acute silicoproteinosis), exposure to insecticides, aluminum dust, titanium dioxide and other inorganic contaminants, hematologic malignancies, myeloid disorders, lysinuric protein intolerance, HIV infection (AIDS), a case report of leflunomide, and disease-modifying therapy rheumatoid arthritis. Various factors include hypersensitivity pneumonitis, lung cancer, non-small cell lung cancer, oat cell lung cancer (small cell), Pneumocystis carinii pneumonia, pulmonary edema, and cardiogenic sarcoidosis.

There is no specific therapy for RAP. The standard of care is serial lavage of the entire lung. Control of PAP depends on disease progression, co-infections, and the degree of physiological impairment. The standard of care for PAP is mechanical removal of lipoprotein material through whole-lung lavage, which is often repeated. Historically, patients have been treated with systemic steroids, mucolytics (aerosol), and proteinase (aerosol) without much success. In secondary RAD, appropriate treatment of the underlying cause is also necessary. Congenital PAP responds favorably to lung transplantation.

Lung transplantation is the preferred treatment in patients with congenital RAP and in adult patients with end-stage interstitial fibrosis. The main complications are lung infections from N. asteroides, Pneumocystis carinii and/or intracellular Mycobacterium avium. Pulmonary fibrosis can also complicate RAR.

Liver disease.

Lysyl oxidase-like protein 2 (LOXL2) is expressed in fibrotic human liver tissue, where it cross-links collagen and other matrix components, resulting in increased stiffness, fibroblast activation, and a dynamic process of matrix remodeling and fibrogenesis. LOXL2 is expressed in fibrous liver tissue resulting from human diseases of various etiologies, including hepatitis C, non-alcoholic steatohepatitis (NASH), alcoholic steatohepatitis (ASH), Wilson's disease and primary biliary cirrhosis, in addition to murine models of sclerosing cholangitis .

Chronic liver diseases affect liver tissue in various ways, such as fibrosis and steatosis.

Any prolonged attack on the liver will cause inflammation, which then leads to the formation of fibrous scar tissue in the liver, creating liver fibrosis. Thus, this fibrosis is a scarring process that will replace damaged liver cells. The extent of this fibrosis can vary and is described in several stages. A normal liver is at a stage between F0 and F1. Stage F2 denotes mild fibrosis, and F3 is severe fibrosis. Cirrhosis is defined at stage F4, when scar tissue is already present throughout the liver.

Fibrosis disorganizes the structure of the liver both anatomically and functionally. When fibrosis reaches the stage of cirrhosis, it is initially completely asymptomatic; This is the stage of compensated cirrhosis, i.e. without complications. Then cirrhosis of the liver decompensates, and liver complications appear. Liver complications include, but are not limited to, portal hypertension, which occurs secondary to liver fibrosis (which obstructs venous circulation and causes increased portal vein pressure), ascites (which is the formation of fluid in the abdominal cavity that can become infected), icterus (jaundice) , hepatic encephalopathy (which corresponds to neurological disorders resulting from the accumulation of toxins that are not broken down by the liver), primary liver cancer (which is a final complication and may also be called hepatocellular carcinoma).

The degree of fibrosis is an important prognostic parameter. The severity of fibrosis is one of the factors influencing diagnosis and treatment decisions, as well as a criterion for monitoring the progress of the disease and the effectiveness of therapy.

Hepatic steatosis is the accumulation of fat in the liver, becoming fatty liver. This corresponds to the accumulation of lipids (triglycerides) in liver cells (hepatocytes) and may complicate alcohol intoxication or metabolic disorders such as type 2 diabetes, obesity and dyslipemia. Such steatosis can be either isolated, making it pure steatosis, or associated with hepatitis, making it non-alcoholic steatohepatitis (NASH). Steatosis and NASH form non-alcoholic fatty liver disease (NAFLD). These are usually asymptomatic conditions, but are now becoming more common due to the increasing number of overweight patients.

In some cases, steatosis can develop into fibrosis, which can lead to cirrhosis.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in treating or preventing the development of a liver disease or condition. In some embodiments, such as a fibrotic disease or liver condition is liver (hepatic) fibrosis, for example, any liver fibrosis, regardless of the underlying liver disease. Liver fibrosis and fibrosis-related liver diseases include, but are not limited to, liver disease due to hepatitis C virus (HCV) infection, non-alcoholic steatohepatitis (NASH), primary sclerosing cholangitis (PSC), cirrhosis, liver fibrosis and portal hypertension, and may also include biliary primary Liver cirrhosis (LLC), autoimmune hepatitis, alcoholic cirrhosis, trypsin inhibitor alpha 1 deficiency disease, hereditary hemochromatosis, Wilson's disease, steatohepatitis and cirrhosis, which are associated with infection with hepatitis B virus (HBV) and HIV, and associated conditions such as such as chronic viral hepatitis, non-alcoholic fatty liver disease (NAFLD), alcoholic steatohepatitis (ASH), non-alcoholic steatohepatitis (NASH), primary biliary cirrhosis (PBC), biliary cirrhosis, primary sclerosing cholangitis and autoimmune hepatitis. In some embodiments, the disease or condition is nonalcoholic steatohepatitis (NASH), biliary primary cirrhosis (PBC), or primary sclerosing cholangitis (PSC). In accordance with some embodiments, the disease or condition is a viral disease or liver condition that is acute or chronic. Exemplary diseases and conditions include disease caused by hepatitis C virus (HCV), hepatitis B virus (HBV), with or without liver damage caused by HCV infection or HBV infection. Thus, the proposed methods are methods of antifibrotic therapy in patients with liver disease such as viral hepatitis. In some aspects, the liver disease is a compensated liver disease. On the other hand, it is a decompensated liver disease, such as liver disease associated with ascites, esophageal varices, encephalopathy and/or jaundice.

Liver (hepatic) fibrosis is implicated in the pathology of numerous liver diseases and may occur as part of the wound healing process of chronic liver injury, as a complication of hemochromatosis, Wilson's disease, alcoholism, schistosomiasis, viral hepatitis, bile duct obstruction.

- 21 044096 ducts, exposure to toxins and metabolic disorders. Liver fibrosis is characterized by the accumulation of extracellular matrix, which can be qualitatively distinguished from that in normal liver. If left uncontrolled, liver fibrosis progresses to cirrhosis (defined by the presence of encapsulated nodules), liver failure, and death. Chronic liver injury from sources including parasites and viral infection (eg, hepatitis B virus (HBV), HCV, human immunodeficiency virus (HIV), schistosomiasis) or prolonged stress from alcohol use typically results in liver remodeling, presumably encapsulation damaged area and protecting the remaining liver tissue from damage. (Li and Friedman, Gastroenterol. Hepatol. 14:618-633, 1999). Liver fibrosis results in changes in the extracellular matrix, including a 3- to 10-fold increase in total collagen content and replacement of low-density basement membrane with high-density matrix, which impairs the metabolic and synthetic function of hepatocytes, hepatic stellate cells, and endothelial cells. Activation of hepatic stellate cells (HSC) is a central event leading to liver fibrosis. HSC activation involves two stages: initiation (pre-inflammatory stage) and persistence, which also includes several changes: proliferation, chemotaxis, fibrogenesis, contractility, matrix degradation, loss of retinoids, WBC chemoattractants and cytokine release (Girogescu, M., Non-invasive Biochemical Markers of Liver Fibrosis, J. Gashointestin. Liver Dis., 15(2): 149-159 (2006)).

Gradual accumulation of collagen in the liver parenchyma is the last common pathway of chronic liver disease. This progressive accumulation of fibrosis can eventually lead to cirrhosis and end-stage liver disease. LOXL2 expression is increased in diseased liver tissue.

During liver injury, a process called activation occurs in which HSCs undergo transformation from retinoid-rich pericyte-like cells to myofibroblast-like cells. Highly activated HSCs are morphologically indistinguishable from myofibroblasts.

Activated HSCs express collagen I and other extracellular matrix genes and are quantitatively the major source of matrix that accumulates during fibrosis. During activation, HSCs enter the cell cycle, resulting in an overall increase in HSC numbers in addition to changes in HSC gene expression and consequent accumulation of hepatic matrix.

Recovery from liver fibrosis involves remodeling of excess liver matrix, resulting in restoration of near-normal liver structure. An element of this repair process is apoptosis of activated HSCs.

Therapeutic strategies for liver fibrosis include addressing the underlying cause (eg, toxin or infectious agent), suppressing inflammation (eg, corticosteroids, IL-1 receptor antagonists, or other agents), downregulating stellate cell activation with (eg, gamma -interferon or antioxidants), stimulation of matrix degradation or stimulation of stellate cell apoptosis. Treatments are needed that target the underlying biochemical process rather than simply suppressing inflammation. Embodiments of the presented methods solve this problem.

In accordance with some embodiments, a regimen for treating liver fibrosis with Compound I or a pharmaceutically acceptable salt or solvate thereof comprises eliminating the underlying cause of fibrosis (if known), suppressing inflammation, downregulating stellate cell activation, promoting matrix degradation, promoting stellate cell apoptosis, or their combinations.

There are a number of standardized scoring systems that quantify the extent and severity of liver fibrosis. These include METAVIR, Knodell, Scheuer, Ludwig and Ishak rating systems. Individuals with liver fibrosis include individuals with any degree or severity of liver fibrosis as measured by any of the METAVIR, Knodell, Scheuer, Ludwig, and Ishak scoring systems.

The METAVIR scoring system is based on the analysis of various features of liver biopsy, including fibrosis (portal fibrosis, centrilobular fibrosis and cirrhosis), necrosis (fine-focal or lobular necrosis, acidophilic retraction and ballooning degeneration), inflammation (portal intestinal inflammation, portal lymphoid aggregates and dissemination portal inflammation), bile duct changes and the Knodell index (assessing periportal necrosis, lobular necrosis, portal inflammation, fibrosis and overall disease activity). The definitions of each stage in the METAVIR system are as follows: 0 - no fibrosis; score: 1 - star-shaped expansion of the portal tract, but without the formation of bridges; score: 2 - expansion of the portal tract with the formation of rare bridges; score: 3 - numerous bridges without cirrhosis; and score: 4 - cirrhosis.

The Knodell scoring system, also called the Histologic Activity Index, classifies specimens based on scores into four categories of histologic features: I) periportal and/or bridging necrosis; II) intralobular degeneration and focal necrosis; III) portal inflammation; and IV) fibrosis. In the Knodell staging system, scores are as follows: 0, no fibrosis; 1 - mild fibrosis (fibrous portal expansion); 2 - moderate fibrosis; 3 - heavy

- 22 044096 fibrosis (bridging fibrosis); and a score of 4 is cirrhosis. The higher the score, the more severe the damage to liver tissue. In accordance with some embodiments, the assessment includes analysis of the total Knodell Necroinflammatory Index and/or its individual components, such as the Knodell Inflammation Score and/or the Knodell Necrosis Score.

In the Scheuer grading system, the grades are as follows: 0 - no fibrosis; 1 - enlarged fibrous portal tracts; 2 - periportal or porto-portal bridges, but intact structure; 3 - fibrosis with structural distortion, but without obvious cirrhosis; 4 - probable or definite cirrhosis.

In the Ishak scoring system, stage 0 - no fibrosis; stage 1 - fibrous expansion of some portal areas with or without short fibrous bridges; stage 2 - fibrous expansion of most portal areas with or without short fibrous bridges; stage 3 fibrous expansion of most portal areas with periodic formation of portoportal bridges (P-P); stage 4 - fibrous expansion of the portal zones with pronounced formation of bridges (P-P), as well as portal-central (P-C); stage 5 - pronounced formation of bridges (R-R and/or R-C) with random nodes (incomplete cirrhosis); stage 6 - cirrhosis, probable or definite.

In some aspects, liver disease or fibrosis is assessed by determining a Model for End-Stage Liver Disease (MELD) score. In some aspects, the methods predict or determine the probability that an individual has or at least has a particular MELD score.

In some aspects, the liver disease is a compensated or decompensated liver disease. For example, decompensated liver disease may be associated with ascites, esophageal varices, encephalopathy, and/or jaundice.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of liver fibrosis. In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of liver fibrosis in humans with NASH. In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of liver fibrosis in humans with PSC.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of NASH-associated liver fibrosis in humans. In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of advanced liver fibrosis, but not NASH-associated cirrhosis, in humans. In accordance with some embodiments, the treatment includes increasing event-free survival (EFS). EFS represents the time for progression to cirrhosis. In some embodiments, adults with liver fibrosis have chronic liver disease due to NASH and Ishak stage 3-4 fibrosis based on liver biopsy results. In some embodiments, adults with advanced liver fibrosis have chronic liver disease due to NASH and Ishak stage 3-4 fibrosis based on liver biopsy results.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in preventing the progression of liver fibrosis. In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in preventing the progression of liver fibrosis in subjects with primary sclerosing cholangitis (PSC). In accordance with some embodiments, the subjects are adult subjects (age >18) with chronic cholestatic liver disease.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of cirrhosis due to NASH.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of compensated cirrhosis due to NASH.

In some embodiments, treatment includes reversing cirrhosis due to NASH, reversing fibrosis, reducing hepatic venous pressure gradient (HVPG), and/or increasing event-free survival (EFS). EFS represents the time to first liver-related complication or death. Liver-related complications include any of the following: liver transplantation, qualification for liver transplantation (MELD > 15), complications indicating hepatic decompensation, esophageal variceal bleeding, ascites, hepatic encephalopathy, newly diagnosed varices in a subject without previous varices extensions.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of liver fibrosis in virus-infected humans. In some embodiments, the virus is a human immunodeficiency virus (HIV) or hepatitis C virus (HCV) or an HIV/HCV mixed infection.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of NASH.

In a model of liver fibrosis, treatment with Compound I or a pharmaceutically acceptable salt or solvate thereof significantly reduced fibrosis as assessed by the proportion of areas staining positive for Picrosirius red (collagen), while only a trend towards reduction was observed with rAB0023.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in combination with a second therapeutic agent for the treatment of liver disease in a mammal. In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in combination with a second therapeutic agent for the treatment of liver disease in a mammal, wherein the second therapeutic agent is selected from the group consisting of PPAR agonists, incretins, Glut2 -I, FXR agonists, antioxidants, GLP-1 modulators, SGLT-2 inhibitors, bile acids, caspase protease inhibitors, ACC inhibitors, synthetic fatty acid/bile acid conjugates, CCR2/CC5 dual antagonists, immunomodulators, sirtuin stimulators, fatty acid inhibitor , DGAT1 inhibitors, CD3 antigens, PDE-4 modulators, AMPK stimulators, ROCK2 inhibitors, ASBT inhibitors, ASK1 inhibitors, JNK inhibitors, TLR-4 antagonists, THR-beta antagonists, cathepsin B inhibitors, galectin-3 modulators, anti-miR compounds 21 and their combinations.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in combination with a second therapeutic agent for the treatment of liver disease in a mammal, wherein the second therapeutic agent is selected from PPAR agonists (e.g., saroglitazar, pioglitazone, GFT 505), incretins, Glut2-I, FXR agonists (e.g. fexaramine, PX 102, PX 104, obeticholic acid (OCA), chenodeoxycholic acid (CDCA), GW4064, WAY-362450 (FXR-450 or XL335)), antioxidants ( eg, cysteine depleting agents, vitamin E, RP103, mitoquinone), GLP-1 modulators (eg, liraglutide), SGLT-2 inhibitors (eg, renogliflozin etabonate), bile acids (eg, ursodeoxycholic acid), caspase protease inhibitors (eg, emricasan ethyl ester icosapent), acetyl-CoA carboxylase inhibitor, ACC inhibitors (e.g. NDI-010976), synthetic fatty acid/bile acid conjugates (e.g. ararrichol), dual CCR2/CC5 antagonists (e.g. cenicriviroc), immunomodulators ( e.g. IMM 124E), sirtuin stimulators (e.g. MB 12065), fatty acid inhibitors (e.g. oltipraz), DGAT1 inhibitors (e.g. pridigastat), CD3 antigens (e.g. TRX 318), PDE-4 modulators (e.g. roflurnilast) , AMPK activator (eg, MB 11055), ROCK2 inhibitors (eg, KD 025), ASBT inhibitors (eg, SFIP 626), ASK1 inhibitors (eg, GS-4997), TLR-4 antagonists (eg, JKB-121), THR-beta antagonists (eg, MGL-3195), cathepsin B inhibitors (eg, SHP 626, VBy-376), galectin-3 modulators (eg, GR MD 02, LGPC-1010), NC 101, DUR-928, DWP -10292, anti-miR-21 compounds (RG-012) and combinations thereof.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in combination with a second therapeutic agent for treating liver disease in a mammal, wherein the second therapeutic agent is a PPAR (α,γ) agonist or a PPAR agonist (α,δ).

Kidney fibrosis.

In accordance with some embodiments, the disease or condition is or is associated with renal fibrosis. Like liver fibrosis, kidney fibrosis can result from a variety of kidney diseases and injuries. Examples of such diseases and injuries include chronic kidney disease, metabolic syndrome, vesicoureteral reflux, renal tubulointerstitial fibrosis, IgA nephropathy, diabetes mellitus (including diabetic nephropathy), Alport syndrome, HIV-associated nephropathy, subsequent glomerular nephritis (GN), including without limitations glomerular nephritis and focal segmental glomerulosclerosis and membranous glomerulonephritis, mesangiocapillary GN and subsequent interstitial fibrosis and tubular atrophy (IFTA), including without limitation recovery from acute kidney injury (AKI), acute obstructive nephropathy and drug-induced fibrosis.

It is already generally accepted that the metabolic syndrome is a group of pathologies that includes signs of diabetes mellitus, such as insulin resistance, as well as central or visceral obesity and hypertension. In almost all cases, glucose dysregulation results in stimulation of cytokine release and increased extracellular matrix deposition. Additional factors contributing to the development of chronic kidney disease, diabetes mellitus, metabolic syndrome, and glomerular nephritis include hyperlipidemia, hypertension, hyperglycemia, and proteinuria, all of which lead to further kidney damage and further stimulation of extracellular matrix deposition. Thus, regardless of the underlying cause, kidney damage can lead to renal fibrosis and concomitant loss of kidney function. Schena, F. and Gesualdo, L., Pathogenic Mechanisms of Diabetic Nephropathy, J. Am. Soc. Nephrol., 16: S30-33 (2005); WhaleyConnell, A., and Sower, J. R., Chronic Kidney Disease and the Cardiometabolic Syndrome, J. Clin. Hypert., 8(8): 546-48 (2006)).

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in combination with a second therapeutic agent for the treatment of kidney disease in a mammal, wherein the second therapeutic agent is selected from antihypertensive agents, including, but not limited to, angiotensin receptor blockers ( ARB), angiotensin-converting enzyme (ACE) inhibitors and calcium channel blockers, sodium-glucose transporter-2/gliflozin (SGLT2) inhibitors (eg, empagliflozin or canaglifozin), diuretics (thiazide diuretic) or new in development diabetes therapy kidney disease, such as PKC inhibitors (eg, ruboxistaurine), endothelin receptor antagonists (atrasentan), allopurinols (xanthine oxidase), steroidal mineralcorticoid receptor antagonists (eg, finerenone), anti-AGE therapeutics (eg, PYR-311), Janus inhibitors kinases (eg, aricitinib), DPP-4 inhibitors (gliptins such as saxagliptin, vildagliptin, linagliptin or sitagliptin), GLP1 receptor antagonists (eg, liraglutide or dulaglutide), anti-inflammatory drugs such as pentoxifylline. In accordance with some embodiments, the mammal is on dialysis. In accordance with some embodiments, the mammal is not on dialysis.

In fig. Figure 6A shows that lysyl oxidase-like protein-2 contributes to the progression of renal Alport syndrome. In this model of renal fibrosis, treatment with compound 1 significantly reduced both tubulointerstitial fibrosis and glomerulosclerosis. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in the treatment of kidney disease. In accordance with some embodiments, the kidney disease is renal fibrosis. In accordance with some embodiments, the kidney disease is renal Alport syndrome. In accordance with some embodiments, the kidney disease is a chronic kidney disease.

Myelofibrosis.

In accordance with some embodiments, the disease or condition is or is associated with myelofibrosis. Pathogenic processes in primary myelofibrosis include primary megakaryocyte-weighted clonal myeloproliferation and a paraneoplastic stromal reaction, which includes bone marrow fibrosis, osteosclerosis, angiogenesis, and extramedullary hematopoiesis. The bone marrow response includes excess deposition of extracellular matrix proteins such as fibrillar collagen, hypocellularity, activation and recruitment of bone marrow fibroblasts, excess production of cytokines and growth factors, and other changes that result in decreased hematopoietic capacity. Secondary myelofibrosis may result from polycythemia vera or essential thrombocythemia.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of myelofibrosis.

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of primary post-polycythemia vera or post-essential thrombocythemia myelofibrosis. In some embodiments, treatment includes a reduction in bone marrow fibrosis, clinical improvement, partial remission, or complete remission.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used either alone or in combination with ruxolitinib.

Scleroderma.

Scleroderma or systemic sclerosis is a potentially fatal autoimmune disease of unknown etiology, characterized by progressive multiorgan fibrosis that is largely resistant to currently available pharmacological therapies. Systemic sclerosis is believed to be caused by tissue damage, in response to which impaired wound healing processes are believed to contribute to the development of fibrosis. Patients with scleroderma have elevated plasma LOXL2 concentrations compared to healthy subjects (Figure 9).

- 25 044096

In accordance with some embodiments, disclosed herein is the use of Compound I or a pharmaceutically acceptable salt or solvate thereof in the treatment of scleroderma. There are two main forms of scleroderma: limited systemic sclerosis (also known as focal scleroderma or cutaneous scleroderma) and diffuse systemic sclerosis. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used for the treatment of limited systemic sclerosis. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used for the treatment of diffuse systemic sclerosis.

In some cases, scleroderma occurs in families. Therefore, in some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered prophylactically to asymptomatic family members, wherein at least one family member has been diagnosed with scleroderma.

In accordance with some embodiments, scleroderma is a manifestation of another disease or condition. To such individuals who have been diagnosed with another disease or condition, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered prophylactically to prevent the occurrence of scleroderma.

As used herein, the term limited systemic scleroderma means a disorder characterized by thickening and hardening of the skin and subcutaneous tissues due to excessive collagen deposition. This is often accompanied by the following: calcification, Raynaud's phenomenon, esophageal dysfunction, sclerodactyly and telangiectasia. In addition, an individual suffering from limited systemic sclerosis may have pulmonary arterial hypertension.

As used herein, the term diffuse systemic scleroderma refers to a disorder of the skin and internal organs characterized by thickening and hardening of the skin and subcutaneous tissues due to excessive collagen deposition. In some cases, diffuse systemic scleroderma is accompanied by Raynaud's phenomenon and calcification.

In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in the treatment or prevention of the development in a mammal of any of the following: localized cutaneous scleroderma, localized focal scleroderma, lichen sclerosus and atrophic overlay, generalized focal scleroderma, Pasini and Pierini atrophoderma , pansclerotic focal scleroderma, deep focal scleroderma, linear scleroderma, systemic scleroderma, CREST syndrome, sclerodactyly, systemic sclerosis, progressive systemic sclerosis.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to treat or prevent the development of Dupuytren's disease. Dupuytren's disease is a condition in which the tissue under the skin of the palm thickens and contracts so that the tendons connected to the fingers cannot move freely. Dupuytren's disease results from abnormal fibrosis of the palmar fascia.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a mammal to treat or prevent the development of capsular contracture. Capsular contracture is an abnormal response of the immune system to foreign materials. When breast implants or any other foreign objects such as artificial joint replacements are placed into the body, the body forms a lining around it. Capsular contracture is characterized by the formation of capsules of collagen fibers around a foreign body. In some cases, capsular contracture occurs as a result of an abnormal immune response to breast implants and artificial joint replacements. In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered before, during, or concurrently with breast augmentation surgery. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered before, during, or concurrently with implantation of the artificial joint.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered for the treatment of radiation skin syndrome. As used herein, radiation skin syndrome refers to the pathophysiological reactions of the skin and skin appendages to significant levels of ionizing radiation. In some cases, an individual with radiation skin syndrome will have abnormal skin fibrosis. In accordance with some embodiments, a drug disclosed herein is used to treat radiation skin syndrome in which the individual in need has unwanted/abnormal skin fibrosis.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a mammal to treat scarring. As used herein, the term scarring refers to the formation of a scar. In one aspect, the scar is a hypertrophic scar, or a keloid scar, or a scar that is

- 26 044096 a consequence of acne. In some cases, the scar is an area of fibrous tissue that results from overproduction of collagen. In some cases, wound healing involves the migration of fibroblasts to the site of injury. In some cases, fibroblasts deposit collagen. In some cases, fibroblasts deposit excess collagen at the wound site, resulting in scar formation.

In accordance with some embodiments, the scarring occurs as a result of trauma (eg, surgery). In accordance with some embodiments, a drug disclosed herein is administered before, after, or concurrently with surgery.

In accordance with some embodiments, the scarring occurs as a result of a burn. In accordance with some embodiments, a drug disclosed herein is administered when treating an individual for a burn.

In some embodiments, a drug disclosed herein is administered before, after, or concurrently with a scar correction procedure.

Fibrosis of the eye.

In healthy eye tissues, lysyl oxidase activity is present in the vitreous, iris, ciliary body, lens, choroid, retinal pigment epithelium, and retina. Most blinding eye diseases are associated with disruption of the structure of ocular tissue caused by extravasation and fibrosis (Friedlander M. Fibrosis and diseases of the eye. J Clin Invest. 2007; 117:576586). Progressive fibrosis is not only associated with the pathogenesis of glaucoma, but can also be a consequence of surgical treatment to reduce glaucoma. Poor surgical outcome is indeed characterized by an excessive postoperative wound healing response followed by scarring.

Targeting LOXL2 via an inhibitory monoclonal antibody reduced pathological angiogenesis, inflammation, and fibrosis in a rabbit model of anti-glaucomatous surgery. (Van Bergen T, Marshall D, Van de Veire S, et al. The role of LOX and LOXL2 in scar formation after glaucoma surgery. Invest Ophthalmol Vis Sci. 2013;54:5788-5796). Additionally, targeting LOXL2 reduced angiogenesis and inflammation, in addition to fibrosis, in an animal model of CNV-related AMD (Van Bergen et al. The role of LOX and LOXL2 in the pathogenesis of an experimental model of choroidal neovascularization. Invest Ophthalmol Vis Sci. 2015; 56:5280-5289).

In accordance with some embodiments, LOXL2 contributes to the wound healing response in ocular tissue.

In some cases, corneal scarring is caused by damage to the cornea (abrasion, tear, burns, or disease). Superficial abrasions heal well and do not leave scars. Deeper abrasions and ulcerations/tears result in loss of corneal tissue, which is replaced by scar tissue. The proliferation of new blood vessels in the clear cornea promotes the healing process. Aberrant wound healing results in vision loss.

In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a mammal to reduce or inhibit fibroblast proliferation and/or increase fibroblast apoptosis associated with fibrotic disorders, inflammation and/or proliferative disorders of the eye. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof reduces, attenuates, or inhibits fibrosis and/or aberrant wound healing in ocular tissues.

In another aspect, Compound I or a pharmaceutically acceptable salt or solvate thereof is used to enhance corneal desensitization caused by corneal surgery such as laser in situ keratoplasty (LASIK) or cataract surgery, corneal desensitization caused by corneal degeneration, and associated This is a symptom of dry eye.

In yet another aspect, Compound I or a pharmaceutically acceptable salt or solvate thereof is used to reduce, attenuate, or inhibit aberrant wound healing and/or scarring of ocular tissue (eg, cornea or retina). In some cases, scarring is the result of a disease such as keratitis (such as inflammation caused by herpes simplex or syphilis). In some cases, corneal scarring is caused by surgical procedures including, for example, corneal graft, corneal graft, trabeculectomy and/or radiation eye surgery. In some cases, corneal damage results from laser in situ keratoplasty (LASIK). In some cases, corneal scarring results from corneal ulcers. In some cases, proliferative membranes of the posterior segment of the eye cause deposition of mutilating fibrous tissue and subsequent formation of scar tissue. In some cases, thinning and scarring of the retina occurs due to structural changes in the retina caused by chronic cystoid macular edema (chronic CME). In some cases, disorganized growth of retinal blood vessels in premature babies leads to scarring and/or detachment of the retina (retinopathy of prematurity (ROP)). In some cases, bleeding, extravasation, and scarring caused by abnormal blood vessel growth (choroidal neovascularization) due to wet age-related macular degeneration (wet AMD) leads to permanent photoreceptor damage and rapid vision loss if left untreated.

Examples of disorders associated with aberrant wound healing and/or scarring of ocular tissue that, in some embodiments, are treated with Compound I or a pharmaceutically acceptable salt or solvate thereof include episcleral fibrosis leading to the adverse outcome of blister formation (trabeculectomy) post-filtration surgery glaucoma, pterygia (including post-operative wound healing/scarring), cataracts (post-surgical scarring), corneal scarring, scarring associated with ocular cicatricial pemphigoid, glaucoma filtration surgery (trabeculectomy), fibrosis associated with keratoprosthetic procedure, wet age-related degeneration maculae with neovascularization and foveal thickening, diabetic retinopathy with retinal edema (and neovascularization), proliferative vitreoretinopathy (PVR), prevention and treatment of photocoagulation-associated macular thickening, retinopathy of prematurity (ROP), (primary) retinal detachment, chronic macular retinal edema , chronic cystoid macular edema, postoperative macular edema, macular edema associated with hereditary retinal disease.

Fibrosis of the ear.

In accordance with some embodiments, the disease or condition is or is associated with ear fibrosis. Like liver fibrosis, ear fibrosis can result from various ear diseases and injuries. Fibrosis can occur in both the middle ear and the inner ear. Inflammation in the middle ear can lead to fibrosis of the middle ear canal, and this is characterized by the formation of fibrous tissue in the bony external auditory canal (Ishii, Fluid and Fibrosis in the Human Middle Ear, Am. J. Otolaryngol, 1985: 6: 196-199). Fibrosis of the inner ear includes disorders in which there is dysfunction of the cochlear labyrinth strip as a result of thickening of the membrane. These diseases include Alport syndrome, lupus and diabetes. Type IV collagen disorder (which can be seen in patients with Alport syndrome) is associated with sensorineural hearing loss with structural changes in the connective tissue and micromechanics of the inner ear. Detailed assessments of basement membrane morphology were obtained in a mouse model of Alport syndrome, and a clear thickening of the basement membrane of the stria vascularis was observed (Cosgrove, Ultrastructural, physiological, and molecular defects in the inner ear of a gene-knockout mouse model for autosomal Alport syndrome. Hear Res. 1998;121:84-98).

Cancer.

LOXL2 has been shown to be involved in signaling associated with cancer cell growth, adhesion, motility, and invasion. Specifically, LOXL2 induces epithelial-mesenchymal transition (EMT) of cells, promoting tumor invasion. LOXL2 is also upregulated in hypoxic tumor environments, leading to enhanced tumor cell invasion. LOXL2 has also been shown to promote angiogenesis in a hypoxic tumor environment.

Increased LOXL2 expression is associated with poor prognosis in patients with colon, esophageal, oral squamous cell carcinomas, laryngeal squamous cell carcinomas, and head and neck squamous cell carcinomas. LOXL2 has been suggested to be involved in the development of forms of breast, colon, gastric, head and neck, lung and melanoma cancers.

In accordance with some embodiments, methods of treating cancer with a compound disclosed herein are disclosed herein.

As used herein, the term cancer refers to the abnormal growth of cells that have a tendency to proliferate uncontrollably and, in some cases, metastasize (spread). Types of cancer include, but are not limited to, solid tumors (such as tumors of the bladder, bowel, brain, breast, endometrium, heart, kidney, lung, liver, uterus, lymphatic tissue (lymphoma), ovary, pancreas, or other endocrine organ (thyroid) gland), prostate, skin (melanoma or basal cell carcinoma) or hematological tumors (such as leukemia and lymphoma) at any stage of the disease with or without metastases.

In accordance with some embodiments, the disease or condition is or is associated with cancer or a tumor. Thus, in accordance with some embodiments, the subject is a cancer patient. Such diseases and conditions and forms of cancer include carcinomas, sarcomas, benign tumors, primary tumors, metastatic tumors, solid tumors, non-solid tumors, blood tumors, leukemias and lymphomas, as well as primary and metastatic tumors.

Carcinomas include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell lung carcinoma, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, carcinoma oma greasy glands, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteo gene carcinoma, epithelial carcinoma and carcinoma of the nasopharynx, etc.

Sarcomas include, but are not limited to, fibrosarcoma, malignant myxoma, liposarcoma, chondrosarcoma, chordoma, osteosarcoma, osteosarcoma, angiosarcoma, endothelial sarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing sarcoma, leiomyosarcoma, rhabdomyosarcoma and other soft tissue sarcomas.

Solid tumors include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, and retinoblastoma.

Leukemias include, without limitation, a) chronic myeloproliferative syndromes (neoplastic disorders of multipotent hematopoietic stem cells); b) acute myelogenous leukemia (neoplastic transformation of a multipotential hematopoietic stem cell or hematopoietic cell with limited clonal potential); c) chronic lymphocytic leukemias (CLL; clonal proliferation of immunologically immature and functionally incompetent small lymphocytes), including B-cell CLL, T-cell CLL, prolymphocytic leukemia and hairy cell leukemia; and d) acute lymphoblastic leukemia (characterized by the accumulation of lymphoblasts). Lymphomas include, but are not limited to, B-cell lymphomas (eg, Burkitt's lymphoma), Hodgkin's lymphoma, and the like.

Benign tumors include, for example, hemangiomas, hepatocellular adenoma, cavernous hemangioma, focal nodular hyperplasia, acoustic neuromas, neurofibroma, bile duct adenoma, bile duct cystanoma, fibroma, lipomas, leiomyomas, mesotheliomas, teratomas, myxomas, nodular regeneration active hyperplasia, trachoma and pyogenic granulomas.

Primary and metastatic tumors include, for example, lung cancer (including but not limited to lung adenocarcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, non-small cell carcinoma, small cell carcinoma, mesothelioma), breast cancer (including but not limited to ductal carcinoma, lobular carcinoma, edema - infiltrative breast cancer, clear cell carcinoma, mucus-forming carcinoma), colorectal cancer (including without limitation colorectal cancer, rectal cancer), anal cancer, pancreatic cancer (including without limitation pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors), prostate cancer, ovarian carcinoma (including without limitation epithelial ovarian carcinoma or stromal tumor of the superficial epithelium, including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma, germinal cord stromal tumor), liver and bile duct cancer (including without limitation hepatocellular carcinoma, cholangiocarcinoma, hemangioma), esophageal carcinoma (including without limitation esophageal adenocarcinoma and squamous cell carcinoma), non-Hodgkin's lymphoma, bladder carcinoma, uterine carcinoma (including without limitation endometrial adenocarcinoma, uterine papillary serous carcinoma, uterine clear cell carcinoma, uterine sarcomas and leiomyosarcoma, mixed müllerian ovs tumors), glioma, glioblastoma, medulloblastoma and other brain tumors, forms of kidney cancer (including but not limited to renal cell carcinoma, clear cell carcinoma, Wilm's tumor), head and neck cancer (including but not limited to squamous cell carcinomas), stomach cancer (including but not limited to adenocarcinoma stomach, gastrointestinal stromal tumor), multiple myeloma, testicular cancer, germinoma, neuroendocrine tumor, cervical cancer, carcinoids of the gastrointestinal tract, breast and other organs, and signet ring cell carcinoma.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered for the treatment of skin cancer. Skin cancers include melanoma, squamous cell carcinoma and basal cell carcinoma.

In one aspect, Compound I or a pharmaceutically acceptable salt or solvate thereof reduces, attenuates, or inhibits cell proliferation and/or fibrosis associated with various forms of cancer.

In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered for the treatment of melanoma, cutaneous T-cell lymphoma, mycosis fungoides, Merkel cell carcinomas, head and neck carcinomas, solar keratoses, squamous cell or basal cell carcinomas at any stage diseases with or without metastases.

Rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder in which

- 29 044096 rum The body's immune system attacks joints and additional organs such as skin, eyes, lungs and blood vessels. RA is a chronic inflammatory disease characterized by synovial hyperplasia composed of infiltrated immunocytes and resident synovial fibroblasts (SF). Rheumatoid arthritis synovial fibroblasts (RASF) are found in RA-affected synovium, are key players in joint destruction, and are capable of migrating in vitro and in vivo. Various cytokines from infiltrated immunocytes induce SF proliferation and activation. Activated SF in turn creates a pathogenic stroma to maintain chronic inflammation, leading to cartilage and bone destruction.

RASF differ from healthy synovial fibroblasts in their morphology and aberrant gene expression. RASF are characterized by the expression of antiapoptotic molecules, proto-oncogenes, and the absence of expression of tumor suppressor genes. Due to its ability to produce proinflammatory cytokines and chemokines, RASF further attracts inflammatory cells of the immune system to the synovium. In addition, RASF produces enzymes such as matrix metalloproteinases (MMPs) that promote cartilage penetration and breakdown.

LOXL2 is expressed and secreted in RASF and is activated by TNF-α and IL-1e. Knockdown of LOXL2 and anti-LOXL2 antibodies attenuated collagen deposition in RASF. In addition, LOXL2 knockdown reduced RASF proliferation and invasion. For example, the LOXL inhibitor β-aminopropionitrile (BAPN) attenuates collagen-induced arthritis (CIA) in an in vivo mouse model of CIA (Tetsuya Saito, et al., Roles of collagen crosslinking enzyme, lysyl oxidase-like 2, in rheumatoid novial fibroblasts, Keystone Symposia on Molecular and Cellular Biology, February 7-11, 2016, abstract Q3 3014).

LOXL2 is involved in activated RASF phenotypes and represents a therapeutic target in RA. The aggressive, invasive phenotype of RASF appears in the early stages of RA as a consequence of sustained cell activation. Several key factors in the pathogenesis of RA, including proinflammatory cytokines, innate immunity, and matrix degradation products, critically enhance RASF activation.

In accordance with some embodiments, described herein is the use of a LOXL2 inhibitor (eg, Compound I or a pharmaceutically acceptable salt or solvate thereof) in the treatment of RA. In accordance with some embodiments, described herein is the use of a LOXL2 inhibitor (eg, Compound I or a pharmaceutically acceptable salt or solvate thereof) in the treatment of RA in combination with one or more additional agents used to treat RA.

Juvenile idiopathic arthritis.

Juvenile idiopathic arthritis (JIA), also known as juvenile rheumatoid arthritis (JRA), is the most common form of arthritis in children and adolescents. Juvenile in this context means beginning before the age of 16. In accordance with some embodiments, described herein is the use of a LOXL2 inhibitor (eg, Compound I or a pharmaceutically acceptable salt or solvate thereof) in the treatment of JIA. In accordance with some embodiments, described herein is the use of a LOXL2 inhibitor (eg, Compound I or a pharmaceutically acceptable salt or solvate thereof) in the treatment of JIA in combination with one or more additional agents used to treat JIA.

Osteoarthritis.

Osteoarthritis (OA) is a type of joint disease that results from the destruction of articular cartilage and underlying bone. LOXL2 has been shown to be highly expressed in the damaged area of OA cartilage (T. Sato et al., Arthritis & Rheumatism, Vol. 54, No. 3, pp 808-817 (2006)). In accordance with some embodiments, described herein is the use of a LOXL2 inhibitor (eg, Compound I or a pharmaceutically acceptable salt or solvate thereof) in the treatment of osteoarthritis. In accordance with some embodiments, described herein is the use of a LOXL2 inhibitor (eg, Compound I or a pharmaceutically acceptable salt or solvate thereof) in the treatment of osteoarthritis in combination with one or more additional agents used to treat osteoarthritis.

Psoriatic arthritis.

Psoriatic arthritis is a type of inflammatory arthritis that up to 30 percent of people with psoriasis will develop. Psoriatic arthritis is classified as a seronegative spondyloarthropathy and is therefore more common in patients with the HLA-B27 tissue type. In accordance with some embodiments, the use of a LOXL2 inhibitor in the treatment of psoriatic arthritis is described herein. In accordance with some embodiments, described herein is the use of a LOXL2 inhibitor (eg, Compound I or a pharmaceutically acceptable salt or solvate thereof) in the treatment of psoriatic arthritis in combination with one or more additional agents used to treat psoriatic arthritis.

- 30 044096

Ankylosing spondylitis.

Ankylosing spondylitis (also known as ankylosing spondylitis, ankylosing spondylitis, Marie Strumpel's disease or AS) is a chronic inflammatory disease of the axial skeleton. In accordance with some embodiments, the use of a LOXL2 inhibitor in the treatment of ankylosing spondylitis is described herein. In accordance with some embodiments, described herein is the use of a LOXL2 inhibitor (eg, Compound I or a pharmaceutically acceptable salt or solvate thereof) in the treatment of ankylosing spondylitis in combination with one or more additional agents used to treat ankylosing spondylitis.

(R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4 hydroxypyrrolidin-1-yl)methanone (compound I ).

Compound I, or (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl) methanone, or (3-((4-(aminomethyl)-6(trifluoromethyl)-pyridin-2-yl)oxy)phenyl)(3R,4R)-3-fluoro-4-hydroxypyrrolidin-1-yl)methanone, or any other similar name refers to a compound with the following structure:

According to some embodiments, Compound I is substantially free of the (S,S)-isomer (i.e., Compound I is substantially free of (S,S)-trans-(3-((4-(aminomethyl)-6( trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone, or (3-((4(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl) oxy)phenyl)(3S,4S)-3-fluoro-4-hydroxypyrrolidin-1yl)methanone, or any other similar name).

Substantially free of an enantiomer means that the specified enantiomer is absent or that it constitutes less than 5%, less than 4%, less than 3%, less than 2% or less than 1% of the specified enantiomer.

Compound Ent-I, or (S,S)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl )methanone, or (3-((4-(aminomethyl)-6(trifluoromethyl)-pyridin-2-yl)oxy)phenyl)(3S,4S)-3-fluoro-4-hydroxypyrrolidin-1-yl)methanone, or any other similar name refers to a compound with the following structure:

In some embodiments, instead of Compound I, a racemic mixture of trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4hydroxypyrrolidine-1- yl)methanone. Racemic compound I (compound Rac-I) is represented as follows:

Compound I is a potent suicide inhibitor of LOXL2. Compound I is a high affinity, selective, irreversible, small molecule inhibitor of LOXL2. In some embodiments, the aminomethylpyridine moiety of Compound I reacts with the active site of the enzyme to form a time-dependent, pseudo-irreversible inhibitory complex. The results of kinetic studies indicate that the two enantiomers of compound I (i.e. (R,R) and (S,S)) are very similar to each other and to the racemic compound I in pharmacological and pharmacokinetic profile. Compound I was more potent than the (S,S) isomer in in vitro assays. In some embodiments, Compound I was less than 2-fold more potent than the (S,S) isomer in in vitro assays.

In accordance with some embodiments, Compound I specifically inhibits

- 31 044096 and/or contacts LOXL2. In accordance with some embodiments, Compound I does not substantially inhibit and/or bind to any other lysyl oxidase. Other lysyl oxidases include LOX, LOXL1, LOXL3 and LOXL4. In accordance with some embodiments, Compound I is specific for LOXL2. In accordance with some embodiments, Compound I inhibits LOXL2 activity and substantially does not inhibit LOX activity. In accordance with some embodiments, Compound I inhibits the activity of LOXL2 and substantially does not inhibit the activity of another lysyl oxidase-like protein.

As used herein, the term selective LOXL2 inhibitor means a small molecule inhibitor of LOXL2 that substantially does not inhibit and/or bind to any other lysyl oxidase. Other lysyl oxidases include LOX, LOXL1, LOXL3 and LOXL4. In accordance with some embodiments, the selective LOXL2 inhibitor substantially does not inhibit and/or bind to LOX or LOXL3. In accordance with some embodiments, the selective LOXL2 inhibitor is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times times, at least 100 times, at least 120 times, at least 140 times, at least 160 times, at least 180 times, at least 200 times, at least 250 times, at least 300 times, at least 350 times, at least 400 times, at least 450 times, at least 500 times, at least 550 times, at least 600 times, according to at least 650-fold, at least 700-fold, at least 800-fold, at least 900-fold, or at least 1000-fold more selective for LOXL2 than for LOX. In accordance with some embodiments, the selective LOXL2 inhibitor is at least 400 times more selective for LOXL2 than for LOX. In accordance with some embodiments, the selective LOXL2 inhibitor is at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 20 times, at least 30 times, at least 40 times, at least 50 times, at least 60 times, at least 70 times, at least 80 times, at least 90 times times, at least 100 times, at least 120 times, at least 140 times, at least 160 times, at least 180 times, at least 200 times, at least 250 times, at least 300 times, at least 350 times, at least 400 times, at least 450 times, at least 500 times, at least 550 times, at least 600 times, according to at least 650-fold, at least 700-fold, at least 800-fold, at least 900-fold, or at least 1000-fold more selective for LOXL2 than for LOXL3. In accordance with some embodiments, the selective LOXL2 inhibitor is at least 5 times more selective for LOXL2 than for LOXL3.

In accordance with any of the embodiments disclosed herein (including methods, uses, preparations, combination therapy, etc.), Compound I or a pharmaceutically acceptable salt or solvate thereof is replaced by a) Compound I or a pharmaceutically acceptable salt or solvate thereof with lower chiral purity; b) (S,S)-trans-(3-((4-(aminomethyl)-6(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof of any optical purity or c) a racemic mixture of trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4- hydroxypyrrolidin1-yl)methanone or a pharmaceutically acceptable salt or solvate thereof.

The term pharmaceutically acceptable salt, as applied to Compound I, means a salt of Compound I that does not cause significant irritation to the mammal to which it is administered and substantially does not affect the biological activity and properties of the compound. Handbook of Pharmaceutical Salts: Properties, Selection and Use. International Union of Pure and Applied Chemistry, Wiley-VCH 2002. S.M. Berge, L.D. Bighley, D.C. Monkhouse, J. Pharm. Sci. 1977, 66, 1-19. P. H. Stahl and C. G. Wermuth, editors, Handbook of Pharmaceutical Salts: Properties, Selection and Use, Weinheim/Zurich:Wiley-VCH/VHCA, 2002. Pharmaceutical salts are generally more soluble and more readily soluble in gastric and intestinal juices than nonionic compounds and are therefore suitable in solid dosage forms. In addition, since their solubility is often pH dependent, selective dissolution in one part or another of the digestive tract is possible, and this possibility can be manipulated as an aspect of the sustained and delayed release properties. Also, since the salt-forming molecule can be in equilibrium with the neutral form, passage through biological membranes can be controlled.

It should be understood that reference to a pharmaceutically acceptable salt includes solvent-added forms (solvates). Solvates contain either stoichiometric or non-stoichiometric amounts of solvent and are formed by the formation or isolation of the product with pharmaceutically acceptable solvents such as water, ethanol, methyl tert-butyl ether, isopropanol, acetonitrile, heptane, and the like. In one aspect, the solvates are prepared using

- 32 044096 without limitation of solvent(s) of the 3rd class. Categories of solvents are defined, for example, in the International Conference on Harmonization of Technical Requirements for Registration of Medicinal Products for Human Use (ICH), Impurities: Guidelines for Residual Solvents, Q3C(R3), (November 2005). If the solvent is water, hydrates are obtained, or alcoholates are obtained if the solvent is alcohol. In accordance with one embodiment, solvates of Compound I or its pharmaceutically acceptable salts are conveniently prepared or formed during the preparation of Compound I or its pharmaceutically acceptable salts. In addition, compound I or its pharmaceutically acceptable salts exists in unsolvated form. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt thereof is hydrated.

A wide range of pharmaceutically acceptable salts are prepared from compound I, which includes:

salts obtained by treating compound I (i.e. in free base form) with an inorganic acid. Inorganic acids include, but are not limited to, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and metaphosphoric acid;

salts obtained by treating compound I (i.e. in free base form) with an organic acid. Organic acids include, but are not limited to, 1-hydroxy-2-naphthoic acid, 2,2-dichloroacetic acid, 2-hydroxyethanesulfonic acid, 2-oxoglutaric acid, 4-acetamidobenzoic acid, 4-aminosalicylic acid, acetic acid, adipic acid, ascorbic acid (L), aspartic acid (L), benzenesulfonic acid, benzoic acid, camphoric acid (+), camphor-10-sulfonic acid (+), capric acid (decanoic acid), caproic acid (hexanoic acid), caprylic acid (octanoic acid), carbonic acid acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfuric acid, ethane-1,2-disulfonic acid, ethanesulfonic acid, formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptanoic acid (D), gluconic acid (D), glucuronic acid (D), glutamic acid, glutaric acid, glycerophosphoric acid, glycolic acid, hippuric acid, isobutyric acid, lactic acid (DL), lactobionic acid, lauric acid, maleic acid, hydroxysuccinic acid (-L), malonic acid, mandelic acid acid (DL), methanesulfonic acid, monomethyl fumarate, naphthalene-1,5-disulfonic acid, naphthalene-2-sulfonic acid, nicotinic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, phosphoric acid, propionic acid, pyroglutamic acid ( - L), salicylic acid, sebacic acid, stearic acid, succinic acid, sulfuric acid, tartaric acid (+ L), thiocyanic acid, toluenesulfonic acid (p) and undecenoic acid.

In any of the embodiments disclosed herein (including methods, uses, formulations, combination therapies, etc.), a hydrochloride salt of Compound I or a solvate thereof is used. The hydrochloride salt of compound I (i.e. compound 1) has the following structure:

The (S,S)-e nanomer of compound 1 (compound Ent-1) has the following structure:

Racemic compound 1 (compound Rac-1) is represented as follows:

mixture. 1:1

In any of the embodiments disclosed herein (including methods, uses, formulations, combination therapy, etc.), a methane sulfonic acid salt of Compound I or a solvate thereof is used. Compound I methanesulfonate (compound 2), or the mesylate salt of compound I, or any other similar name, has the following structure:

The (S,S)-e nanomer of compound 2 (compound Ent-2) has the following structure:

Racemic compound 2 (compound Rac-2) is represented as follows:

In accordance with some embodiments, Compound I described herein is prepared as a chloride salt, sulfate salt, bromide salt, mesylate salt, maleate salt, citrate salt, or phosphate salt. In accordance with some embodiments, Compound I described herein is obtained as a hydrochloride salt. In accordance with some embodiments, Compound I described herein is obtained as a mesylate salt.

It should be understood that reference to a pharmaceutically acceptable salt includes solvent-added forms. In accordance with some embodiments, the solvates contain either stoichiometric or non-stoichiometric amounts of solvent and are formed by a crystallization process with pharmaceutically acceptable solvents such as water, ethanol, and the like. If the solvent is water, hydrates are obtained, or alcoholates are obtained if the solvent is alcohol. The solvates of the compounds described herein are conveniently prepared or formed by the processes described herein. In addition, the compounds presented herein optionally exist in unsolvated as well as solvated form.

In accordance with any of the embodiments disclosed herein (including methods, uses, preparations, combination therapy, etc.), amorphous compound I is used. In accordance with any of the embodiments disclosed herein (including methods, uses, drugs, combination therapy, etc.), a crystalline compound I is used. In accordance with any of the embodiments disclosed herein (including methods, uses, drugs, combination therapy, etc.), a partially crystalline compound I is used.

In some embodiments, in accordance with any of the embodiments disclosed herein (including methods, uses, formulations, combination therapies, etc.), Compound I or a pharmaceutically acceptable salt thereof is replaced by an active metabolite of Compound I or pharmaceutically acceptable salt.

In some embodiments, in accordance with any of the embodiments disclosed herein (including methods, uses, formulations, combination therapies, etc.), Compound I or a pharmaceutically acceptable salt thereof is replaced by a prodrug of Compound I or a pharmaceutically acceptable salt thereof. acceptable salt.

In some embodiments, in accordance with any of the embodiments disclosed herein (including methods, uses, formulations, combination therapies, etc.), Compound I or a pharmaceutically acceptable salt thereof is replaced by a deuterated analogue of Compound I or pharmaceutically acceptable salt.

In accordance with some embodiments, compound I has an isotopic tag in which one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number that is typically found in nature. Examples of isotopes that may be included in the present compounds include isotopes of hydrogen, carbon,

- 34 044096 nitrogen, oxygen, fluorine and chlorine, such as, for example, ² H, ³ H ^{, 13} C, ¹⁴ C, ¹⁵ N, 18 O, ¹⁷ 0, ³⁵ S, ¹⁸ F, ³⁶ Cl. In one aspect, the isotopically labeled compounds described herein, such as those incorporating radioactive isotopes such as ³ H and ¹⁴ C, are useful in drug and/or substrate tissue distribution assays. In one aspect, substitution with isotopes such as deuterium provides certain therapeutic benefits due to greater metabolic stability, such as, for example, increased in vivo half-life or reduced dosage requirements.

According to additional or other embodiments, Compound I is metabolized upon administration to a mammal to form a metabolite, which is then used to obtain the desired effect, including the desired therapeutic effect.

A metabolite of a compound disclosed herein is a derivative of such a compound that is formed during the metabolization of the compound. The term active metabolite refers to a biologically active derivative of a compound that is formed during the metabolization of the compound. As used herein, the term metabolized refers to the sum of the processes (including, without limitation, hydrolysis reactions and enzyme-catalyzed reactions) by which a particular substance is changed by the body. Thus, enzymes can produce specific structural changes in a compound. For example, cytochrome P450 catalyzes a series of oxidation and reduction reactions, while uridine diphosphate glucuronyl transferases catalyze the transfer of an activated glucuronic acid molecule to aromatic alcohols, aliphatic alcohols, carboxylic acids, amines, and free sulfhydryl groups. Metabolites of the compounds disclosed herein are optionally identified either by administering the compounds to a host and analyzing tissue samples taken from the host, or by incubating the compounds with liver cells in vitro and analyzing the resulting compounds.

Some terminology.

Unless otherwise specified, the following terms as used in this application have the definitions given below. The use of the term including, as well as other forms such as include, includes and included, is not limiting. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

As used herein, acceptable in relation to a drug, composition or ingredient means that there is no persistent harmful effect on the general health of the subject being treated.

As used herein, the term modulate means to interact with a target, either directly or indirectly, to change the activity of the target, including, for example, to enhance the activity of the target, to inhibit the activity of the target, to limit the activity of the target, or to expand the activity of the target.

As used herein, the term modulator refers to a molecule that either directly or indirectly interacts with a target. Interactions include, but are not limited to, agonist, partial agonist, inverse agonist, antagonist, disruptor, or combinations thereof. In accordance with some embodiments, the modulator is an antagonist. In accordance with some embodiments, the modulator is a destructor.

As used herein, the terms introduce, process of introduction, administration, etc. refer to methods that can be used to effect delivery of compounds or compositions to the desired site of biological action. These routes include, but are not limited to, oral routes, intraduodenal routes, parenteral injection (including intravenous, subcutaneous, intraperitoneal, intramuscular, intravascular or infusion), topical application and rectal administration. Those skilled in the art will be familiar with administration techniques that can be used with the compounds and methods described herein. In accordance with some embodiments, the compounds and compositions described herein are administered orally.

As used herein, the terms co-administration or the like. are intended to cover the administration of selected therapeutic agents to a single patient and are intended to include treatment regimens whereby the agents are administered by the same or a different route of administration or at the same or different times.

As used herein, the terms effective amount or therapeutically effective amount refer to a sufficient amount of an agent or compound administered that will reduce to some extent one or more symptoms of the disease or condition being treated. The result includes the reduction and/or mitigation of signs, symptoms or causes of disease or any other desired change in the biological system. For example, an effective amount for therapeutic applications is the amount of a composition containing a compound disclosed herein necessary to achieve clinically

- 35 044096 significant reduction in the intensity of symptoms of the disease. The appropriate effective amount in any particular case is not necessarily determined by techniques such as dose escalation studies.

As used herein, the terms enhance or intensify mean to increase or prolong either the strength or duration of the desired effect. Thus, with regard to enhancing the effect of therapeutic agents, the term enhancement refers to the ability to increase or prolong, either in strength or duration, the effect of other therapeutic agents on the system. As used herein, the term potentiator-effective amount refers to an amount sufficient to enhance the effect of another therapeutic agent in the desired system.

As used herein, the term pharmaceutical combination means a product that is obtained by mixing or combining more than one active ingredient and includes both fixed and non-fixed combinations of active ingredients. The term fixed combination means that the active ingredients, for example, both a compound described herein or a pharmaceutically acceptable salt thereof, and a co-administered agent are administered to a patient simultaneously in the form of a single unit or a single dose. The term "unfixed combination" means that the active ingredients, for example, a compound described herein or a pharmaceutically acceptable salt thereof, and a co-administered agent are administered to a patient in separate units simultaneously, in parallel or sequentially without any specific intervening time frame, and such administration provides effective levels two compounds in the patient's body. The latter also applies to mixed therapy, such as the administration of three or more active ingredients.

The terms set and product are used as synonyms.

The term subject or patient includes mammals. Examples of mammals include, but are not limited to, any member of the class Mammals: humans, non-human primates such as chimpanzees and other apes and apes; farm animals such as cattle, horses, sheep, goats, pigs; pets such as rabbits, dogs and cats; laboratory animals, including rodents such as rats, mice and guinea pigs, etc. In one aspect, the mammal is a human.

As used herein, the terms treat, treatment process, or treatment include alleviating, reducing, or ameliorating at least one symptom of a disease or condition, preventing the onset of additional symptoms, inhibiting a disease or condition, e.g., reversing the progression of a disease or condition, alleviating a disease or condition, inducing regression of a disease or condition, alleviation of a condition caused by a disease or condition, or cessation of symptoms of a disease or condition, either for prophylactic and/or therapeutic purposes.

Pharmaceutical compositions.

In accordance with some embodiments, the compounds described herein are included in pharmaceutical compositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into compositions that are used for pharmaceutical purposes. The required drug depends on the chosen route of administration. Brief descriptions of the pharmaceutical compositions described herein can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), which are incorporated herein by reference for each disclosure.

In accordance with some embodiments, the compounds described herein are administered either alone or in combination with pharmaceutically acceptable carriers, excipients, or diluents in a pharmaceutical composition. The compounds and compositions described herein can be administered by any method that allows delivery of the compounds to the site of action. Such routes include, but are not limited to, delivery via enteral routes (including oral, gastric or duodenal feeding tubes, rectal suppositories, and rectal enema), parenteral routes (injection or infusion, including intraarterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural and subcutaneous), inhalation, transdermal, transmucosal, sublingual, buccal and topical (including epithelial, skin, enema, eye drops, ear drops, intranasal, vaginal) administration, although the most appropriate route may depend, for example, on the condition and impairment of the patient taking the drug. By way of example only, the compounds described herein can be administered locally to the area in need of treatment, for example, by local infusion during surgery, topical application such as creams or ointments, injection, catheter or implant. Administration can also be done by direct injection into the affected tissue or organ.

In accordance with some embodiments, pharmaceutical compositions suitable for oral administration are presented in the form of individual units, such as capsules, sachets or tablets, each containing a predetermined amount of the active ingredient; in the form of powder or granules; in the form of a solution or suspension in an aqueous liquid or a non-aqueous liquid; or in the form of an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. In accordance with some embodiments, the active ingredient is presented in the form of a bolus, electuary, or paste.

Pharmaceutical compositions that can be administered orally include tablets, hard capsules made from gelatin, and soft capsules made from gelatin and a plasticizer such as glycerin or sorbitol. Tablets may be prepared by compression or molding, optionally with one or more additional ingredients. Compressed tablets can be prepared by compressing in a suitable device the active ingredient in bulk form such as powder or granules, optionally mixed with binders, inert diluents or lubricants, surfactants or dispersants. Molded tablets can be prepared by molding in a suitable device a mixture of a powdered compound moistened with an inert liquid diluent. In some embodiments, the tablets are coated or scored and formulated to provide slow or controlled release of the active ingredient. All drugs for oral administration must be in dosages suitable for such administration. Hard capsules may contain the active ingredients in admixture with excipients such as lactose, binders such as starches and/or lubricants such as talc or magnesium stearate, and optionally stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids such as fatty oils, liquid paraffin or liquid polyethylene glycols. In accordance with some embodiments, stabilizers are added. The dragee cores are provided with suitable shells. For this purpose, concentrated sugar solutions can be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol and/or titanium dioxide, lacquer solutions and suitable organic solvents or mixtures of solvents. Dyes or pigments may be added to tablets or dragee shells for identification or to characterize different dosage combinations of the active compound.

In accordance with some embodiments, the pharmaceutical compositions are formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion. Injectable preparations may be presented in single dosage form, such as ampoules, or in multi-dose containers with the addition of a preservative. The compositions may take forms such as suspensions, solutions or emulsions in oily or aqueous dosage form excipients, and may contain auxiliary agents such as suspending, stabilizing and/or dispersing agents. The compositions may be presented in single-dose or multi-dose containers, such as hermetically sealed ampoules and vials, and may be stored in powder form or in a freeze-dried (lyophilized) state, requiring only the addition of a sterile liquid carrier, e.g. , saline solution or sterile pyrogen-free water. Injectable solutions and suspensions for immediate administration can be prepared from sterile powders, granules and tablets of the type previously described.

Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous (oily) sterile injection solutions of the active compounds, which may contain antioxidants, buffers, bacteriostatic agents and solutes that render the preparation isotonic with the blood of the intended patient taking the drug; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickening agents. Suitable lipophilic solvents or excipients for dosage forms include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethylcellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds, allowing highly concentrated solutions to be obtained.

Pharmaceutical compositions can also be formulated as a depot preparation. Such long-acting drugs can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (eg, as an emulsion in a suitable oil) or ion exchange resins, or as sparingly soluble derivatives, e.g.

- 37 044096 salt.

For buccal or sublingual administration, the compositions can be formulated into tablets, lozenges, lozenges or gels formulated in a conventional manner. Such compositions may contain the active ingredient in a flavored base such as sucrose and gum acacia or tragacanth.

The pharmaceutical compositions can also be formulated into rectal compositions such as suppositories or retention enemas, for example, containing traditional suppository bases such as cocoa butter, polyethylene glycol or other glycerides.

Pharmaceutical compositions can be applied topically, i.e. through non-systemic use. This involves applying the compound of the present invention externally to the epidermis or oral cavity and instilling such compound into the ear, eye and nose so that the compound does not enter the bloodstream to a significant extent. In contrast, systemic administration refers to oral, intravenous, intraperitoneal and intramuscular administration.

Pharmaceutical compositions suitable for topical administration include liquid or semi-liquid preparations suitable for penetration through the skin into the site of inflammation, such as gels, liniments, lotions, creams, ointments or pastes, and drops suitable for administration to the eye, ear or nose. The active ingredient may constitute, in the case of topical application, from 0.001 to 10% by weight, for example from 1 to 2% by weight of the preparation.

Pharmaceutical compositions for administration by inhalation are conveniently delivered from an insufflator, pressurized spray packs, or other convenient aerosol spray delivery means. The pressurized packages may contain a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by incorporating a valve to deliver the metered quantity. Alternatively, for administration by inhalation or insufflation, the pharmaceutical preparations can be formulated as a dry powder composition, for example, a powder mixture of the compound and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form, for example, in capsules, cartridges, gelatin or blister packs, from which the powder can be administered using an inhaler or insufflator.

It should be understood that in addition to the ingredients, particularly those mentioned above, the compounds and compositions described herein may include other agents conventional in the art, taking into account the type of preparation in question, for example, agents that are suitable for oral administration may include flavoring agents .

Methods of dosing and treatment regimens.

In accordance with one embodiment, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in the preparation of medicaments for the treatment of diseases or conditions in a mammal that will benefit from inhibition or reduction of LOXL2 activity. Methods of treating any of the diseases or conditions described herein in a mammal in need of such treatment involve administering pharmaceutical compositions that include to said mammal Compound I or a pharmaceutically acceptable salt or solvate, active metabolite, prodrug thereof in therapeutically effective amounts.

In accordance with certain embodiments, compositions containing the compound(s) described herein are administered for the purpose of prophylactic and/or therapeutic treatment. For certain therapeutic purposes, the compositions are administered to a patient already suffering from a disease or condition in an amount sufficient to cure or at least partially prevent the development of at least one of the symptoms of the disease or condition. Amounts effective for such use depend on the severity and course of the disease or condition, prior therapy, the patient's medical condition, weight and response to medications, and the judgment of the attending physician. Therapeutically effective amounts are not necessarily determined by methods involving, without limitation, dose escalation and/or dose varying clinical studies.

For prophylactic purposes, compositions containing Compound I or a pharmaceutically acceptable salt or solvate thereof are administered to a patient susceptible to or otherwise at risk for developing a particular disease, disorder or condition. Such amount is defined as a prophylactically effective amount or a prophylactically effective dose. In such use, the exact amounts also depend on the patient's health, weight, and the like. When used in patients, the amounts effective for such use will depend on the severity and course of the disease, disorder or condition, prior therapy, the patient's medical condition and response to medications, and the judgment of the attending physician. In one aspect, the methods of prophylactic treatment comprise administering to a mammal that has previously experienced at least one symptom of the disease being treated and that is currently in remission, a pharmaceutical composition containing Compound I or a pharmaceutically acceptable salt or solvate thereof, to prevent recurrence of symptoms of a disease or condition.

In certain embodiments, in which the patient's condition does not improve, at the physician's discretion, administration of Compound I or a pharmaceutically acceptable salt or solvate thereof is administered on an ongoing basis, that is, over an extended period of time, including the entire lifespan of the patient, for the purpose of mitigating or otherwise controlling or limiting the symptoms of a disease or condition of a patient.

In accordance with certain embodiments, in which the patient's condition does improve, the dose of the administered drug is temporarily reduced or temporarily suspended for a certain period of time (ie, a drug holiday). According to specific embodiments, the duration of the drug holiday ranges from 2 days to 1 year, including, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days or more than 28 days. The dose reduction during drug holidays is, for example, 10-100%, including, for example, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 and 100%.

After the patient's condition improves, if necessary, a maintenance dose is administered. Then, in accordance with specific embodiments, the dose or frequency of administration, or both, is reduced, depending on the symptoms, to a level at which the improved status of the disease, disorder or condition is maintained. However, in certain embodiments, any recurrence of symptoms requires the patient to receive intermittent treatment on a long-term basis.

In one aspect, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered daily to individuals in need of therapy with Compound I or a pharmaceutically acceptable salt or solvate thereof. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered once daily. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered twice daily. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered three times daily. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered once every two days. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered twice a week.

Typically, dosages of Compound I or a pharmaceutically acceptable salt or solvate thereof used to treat the diseases or conditions described herein in humans will typically range from about 0.1 mg to about 10 mg/kg body weight per dose. In accordance with one embodiment, the required dose is conveniently presented as a single dose or divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example, as two, three, four or more sub-doses per day. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is conveniently presented in divided doses that are administered simultaneously (or over a short period of time) once daily. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is conveniently presented in divided doses that are administered in equal portions twice daily.

In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered orally to a human at a dose of about 0.1 mg to about 10 mg/kg body weight per dose. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a human subject according to a continuous daily dosing schedule.

The term continuous dosing schedule refers to the administration of a specific therapeutic agent at regular intervals. In accordance with some embodiments, a continuous dosing schedule refers to the administration of a particular therapeutic agent at regular intervals without any drug holiday for the particular therapeutic agent. In accordance with some other embodiments, a continuous dosing schedule refers to the administration of a particular therapeutic agent in cycles. In accordance with some other embodiments, a continuous dosing schedule refers to the administration of a particular therapeutic agent in cycles of drug administration followed by drug holidays (eg, a washout period or other such period of time when no drug is administered) for the particular therapeutic agent. For example, in accordance with some embodiments, the therapeutic agent is administered once a day, twice a day, three times a day, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, seven times a week, once every two days, once every three days, once every four days, daily for a week followed by a week without administration of a therapeutic agent, daily for two weeks followed by one or two weeks without administration of a therapeutic agent, daily for three weeks followed by one, two or three weeks without administration of a therapeutic agent, daily for four weeks followed by one, two, three or four weeks without administration of a therapeutic agent, weekly administration of a therapeutic agent with followed by a week without administration of a therapeutic agent, or two weeks of administration of a therapeutic agent followed by two weeks without administration of a therapeutic agent. In accordance with some embodiments, daily administration is performed at one time per day. In accordance with some embodiments, daily administration is carried out twice a day. In accordance with some embodiments, daily administration is carried out three times a day. In accordance with some embodiments, the daily administration is more than three times a day.

The term continuous daily dosing schedule refers to the daily administration of a specific therapeutic agent each day at approximately the same time. In accordance with some embodiments, daily administration is performed at one time per day. In accordance with some embodiments, daily administration is carried out twice a day. In accordance with some embodiments, daily administration is carried out three times a day. In accordance with some embodiments, the daily administration is more than three times per day.

In accordance with some embodiments, an amount of Compound I or a pharmaceutically acceptable salt or solvate thereof is administered once daily. In accordance with some other embodiments, an amount of Compound I or a pharmaceutically acceptable salt or solvate thereof is administered twice daily. In accordance with some other embodiments, an amount of Compound I or a pharmaceutically acceptable salt or solvate thereof is administered three times daily.

In certain embodiments, when a person does not experience improvement in the disease or condition, the daily dose of Compound I or a pharmaceutically acceptable salt or solvate thereof is increased. In some embodiments, the once-daily dosing schedule is changed to a twice-daily dosing schedule. In accordance with some embodiments, a three times daily dosing schedule is used to increase the administered amount of Compound I or a pharmaceutically acceptable salt or solvate thereof. In accordance with some embodiments, the frequency of administration by inhalation is increased to repeat high Cmax levels on a more regular basis. In accordance with some embodiments, the frequency of administration is increased to maintain or more regularly expose Compound I or a pharmaceutically acceptable salt or solvate thereof. In accordance with some embodiments, the frequency of administration is increased to repeat high Cmax levels on a more regular basis and maintain or more regular exposure to Compound I or a pharmaceutically acceptable salt or solvate thereof.

According to any of the above-described aspects, further embodiments are provided wherein an effective amount of Compound I or a pharmaceutically acceptable salt or solvate thereof: (a) is administered to a mammal systemically; and/or (b) administered orally to the mammal; and/or (c) administered intravenously to the mammal; and/or (d) administered to a mammal by injection; and/or (e) administered topically to the mammal; and/or (f) administered non-systemically or locally to a mammal.

According to any of the above-described aspects, further embodiments are provided comprising single administrations of an effective amount of Compound I or a pharmaceutically acceptable salt or solvate thereof, including further embodiments wherein (i) Compound I or a pharmaceutically acceptable salt or solvate thereof is administered once every day; or (ii) Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a mammal multiple times over the course of one day.

According to any of the above-described aspects, further embodiments are provided comprising multiple administrations of an effective amount of Compound I or a pharmaceutically acceptable salt or solvate thereof, including further embodiments wherein (i) Compound I or a pharmaceutically acceptable salt or solvate thereof is administered continuously or periodically: as a single dose; (ii) the time between multiple administrations is 6 hours; (iii) Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to the mammal once every 8 hours; (iv) Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to the mammal once every 12 hours; (v) Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to the mammal once every 24 hours. In accordance with other or alternative embodiments, the method involves a drug holiday, wherein the administration of Compound I or a pharmaceutically acceptable salt or solvate thereof is temporarily suspended or the administered dose of Compound I or a pharmaceutically acceptable salt or solvate thereof is temporarily reduced; at the end of the dosage, the dose of compound I or its pharmaceutically acceptable salt or solvate is resumed. In accordance with one embodiment, the duration of the drug holiday varies from 2 days to 1 year.

- 40 044096

Typically, doses used to treat an adult tend to range from 1 mg to 5000 mg per day. In some embodiments, dosages used to treat an adult are from about 1 mg to about 4000 mg per day, from about 150 mg to about 4000 mg per day, from about 50 mg to about 2000 mg per day, from about 100 mg to about 2000 mg per day, or about 150 mg to about 2000 mg per day. In some embodiments, 50, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050 are administered to an adult human. 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950 or 2000 mg of compound I. According to some option ami implementation, the required dose for convenience, it is presented as a single dose or divided doses administered simultaneously or at appropriate intervals, for example, as two, three, four or more sub-doses per day.

In accordance with some embodiments, the daily dose or amount of active substance in the dosage form is lower or higher than the ranges specified herein, taking into account a number of variables with respect to the individual treatment regimen. In accordance with various embodiments, daily and unit doses are varied depending on a number of variables, including, but not limited to, the disease or condition being treated, the route of administration, the requirements of the individual subject, the severity of the disease or condition being treated, the characteristics (eg, weight) of the individual, and the specific additional therapeutic agents administered (as appropriate) and the judgment of the practitioner.

The toxicity and therapeutic efficacy of such therapeutic regimens are determined using standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, determination of LD ₅₀ and ED50. The dose ratio between toxic and therapeutic effects is the therapeutic index and is expressed as the ratio between LD ₅₀ and ED ₅₀ . In accordance with certain embodiments, data obtained from cell culture assays and animal studies are used in formulating a therapeutically effective daily dosage range and/or a therapeutically effective unit dose amount for use in mammals, including humans. In accordance with some embodiments, the daily dose amount of Compound I or a pharmaceutically acceptable salt or solvate thereof lies within a circulating concentration range that includes the ED ₅₀ with minimal toxicity. In accordance with certain embodiments, the daily dosage range and/or unit dose size varies within such range depending on the dosage form used and the route of administration used.

In some embodiments, following administration of a therapeutically effective dose of Compound I or a pharmaceutically acceptable salt or solvate thereof to a subject, the No Observable Adverse Effect Level (NOAEL) is at least 1, 10, 20, 50, 100, 500, or 1000 milligrams of compound I or its pharmaceutically acceptable salt or solvate per kilogram of body weight (mg/kg). In some examples, the 7-day NOAEL for a rat administered Compound I or a pharmaceutically acceptable salt or solvate thereof is at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 mg/day. kg. In some examples, the 7-day NOAEL for a dog administered Compound I or a pharmaceutically acceptable salt or solvate thereof is at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500 mg/kg.

Methods of combined treatment.

In certain cases, it is advisable to administer Compound I or a pharmaceutically acceptable salt or solvate thereof in combination with one or more other therapeutic agents.

In one embodiment, the therapeutic efficacy of Compound I or a pharmaceutically acceptable salt or solvate thereof is increased by administration of an adjuvant (i.e., the adjuvant alone has minimal therapeutic effect, but when combined with another therapeutic agent, the overall therapeutic effect for the patient is increased) . Or, in accordance with some embodiments, the effect experienced by the patient is enhanced by administering Compound I or a pharmaceutically acceptable salt or solvate thereof with another agent (which also includes a therapeutic regimen) that also produces a therapeutic effect.

In accordance with one specific embodiment, Compound I or a pharmaceutically acceptable salt or solvate thereof is co-administered with a second therapeutic agent, wherein Compound I or a pharmaceutically acceptable salt or solvate thereof and the second therapeutic agent modulate various aspects of the disease, disorder or condition being treated, such that thus providing a greater overall effect than when administering either therapeutic agent alone.

In any case, regardless of the disease, disorder or condition being treated, the overall effect experienced by the patient is simply the addition of the effects of the two therapeutic agents, or the patient experiences a synergistic effect.

- 41 044096

In certain embodiments, if Compound I or a pharmaceutically acceptable salt or solvate thereof is administered in combination with one or more additional agents, such as an additional therapeutically effective drug, an adjuvant, or the like, in a pharmaceutical composition and/or treatment regimen Various therapeutically effective doses of Compound I or a pharmaceutically acceptable salt or solvate thereof will be used. Therapeutically effective doses of drugs and other agents for use in combination treatment regimens are not necessarily determined by means similar to those set forth above for the active substances themselves. In addition, the prevention/treatment methods described herein involve the use of intermittent dosing, i.e., administration of more frequent, lower doses to minimize toxic side effects. In accordance with some embodiments, the combination treatment regimen includes treatment regimens in which administration of Compound I or a pharmaceutically acceptable salt or solvate thereof is initiated before, during, or after treatment with a second agent described herein and is continued until some point during the course of treatment. treatment with a second agent or after cessation of treatment with a second agent. It also includes treatment in which Compound I or a pharmaceutically acceptable salt or solvate thereof and a second agent used in combination are administered simultaneously or at different times and/or at decreasing or increasing intervals during the treatment period. Combination treatment further includes intermittent treatment that is started and stopped at different points in time to facilitate clinical management of the patient.

It will be understood that the dosage regimen for treating, preventing the development or amelioration of the condition(s) for which relief is desired will be modified according to various factors (eg, disease, disorder or condition from which the subject is suffering, age, weight , gender, diet and health status of the subject). Thus, in some cases, the dosage regimen actually used varies and, in some embodiments, deviates from the dosage regimens that are set forth herein.

In the case of the combination therapies described herein, the dosages of the co-administered compounds will vary depending on the type of drug used together, the specific drug used, the disease or condition being treated, and so on. According to additional embodiments, when coadministered with one or more other therapeutic agents, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered either simultaneously with one or more other therapeutic agents or sequentially.

In combination therapies, multiple therapeutic agents (one of which is Compound I or a pharmaceutically acceptable salt or solvate thereof) are administered in any order or even simultaneously. If administration is simultaneous, multiple therapeutic agents are, for example, administered in a single, unitary form or in multiple forms (eg, as a single pill or as two separate pills).

Compound I or a pharmaceutically acceptable salt or solvate thereof, as well as combination therapies, are administered before, during or after the onset of a disease or condition, and the intervals between administration of a composition containing Compound I or a pharmaceutically acceptable salt or solvate thereof vary. Thus, in accordance with one embodiment, Compound I or a pharmaceutically acceptable salt or solvate thereof is used as a prophylactic agent and is continuously administered to subjects prone to developing conditions or diseases for the purpose of preventing the occurrence of the disease or condition. According to another embodiment, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a subject during or as soon as possible after the onset of symptoms. In certain embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered as soon as practicable upon discovery or suspicion of the disease or condition and for a period of time necessary to treat the disease. In accordance with some embodiments, the duration required for treatment varies, and the duration of treatment is adjusted according to the specific needs of each subject. For example, in certain embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof, or a preparation containing Compound I or a pharmaceutically acceptable salt or solvate thereof, is administered for at least 2 weeks, from about 1 month to about 5 years.

Illustrative means for use in combination therapy.

In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered in combination with a chemotherapeutic agent, a hormone blocking therapeutic agent, a radiation therapy agent, a monoclonal antibody, or a combination thereof.

In accordance with certain embodiments, at least one additional

- 42 044096 The new therapeutic agent is administered simultaneously with the compound or a pharmaceutically acceptable salt or solvate thereof. In accordance with certain embodiments, the at least one additional therapeutic agent is administered less frequently than Compound I or a pharmaceutically acceptable salt or solvate thereof. In accordance with certain embodiments, at least one additional therapeutic agent is administered more frequently than Compound I or a pharmaceutically acceptable salt or solvate thereof. In accordance with certain embodiments, at least one additional therapeutic agent is administered prior to administration of Compound I or a pharmaceutically acceptable salt or solvate thereof. In accordance with certain embodiments, the at least one additional therapeutic agent is administered after administration of Compound I or a pharmaceutically acceptable salt or solvate thereof.

Hormone blocking therapy uses medications that block the production of estrogen or block estrogen receptors. In accordance with some embodiments, the hormone blocking therapy involves the use of estrogen receptor modulators and/or aromatase inhibitors. Estrogen receptor modulators include triphenylethylene derivatives (eg, tamoxifen, toremifene, droloxifene, 3-hydroxytamoxifen, idoxifene, TAT-59 (a phosphorylated derivative of 4-hydroxytamoxifen) and GW5638 (a carboxylic acid derivative of tamoxifen)); nonsteroidal estrogen receptor modulators (eg, raloxifene, LY353381 (SERM3) and LY357489); steroidal estrogen receptor modulators (eg, ICI-182780). Aromatase inhibitors include steroidal aromatase inhibitors and non-steroidal aromatase inhibitors. Steroidal aromatase inhibitors include, but are not limited to, exemestane. Non-steroidal aromatase inhibitors include, but are not limited to, anastrozole and letrozole.

Chemotherapy involves the use of anti-cancer drugs.

In some embodiments, anticancer agents for use in combination with Compound I or a pharmaceutically acceptable salt or solvate thereof include one or more of the following: abiraterone, abarelix, abraxane, adriamycin, actinomycin, acivicin, aclarubicin, acodazole hydrochloride, acronine, adozelesin, aldesleukin, alemtuzumab, allopurinol, alitretinoin, altetamine, ametantrone acetate, aminoglutethimide, aminolevulinic acid, amifostina, amsacrine, anastrozole, anthramycin, aprepitant, arsenic trioxide, asparaginase, asperlin, azacitidine, azetepa, azotomycin, batimastat, bendamustine hydrochloride, benzodepa, bevacizumab, bexarotene, bicalutamide, bisantrene hydrochloride, bisnafide dimesylate, biselezin, bleomycin, bleomycin sulfate, bortezomib, brequinar sodium, bropyrimine, busulfan, cactinomycin, calusterone, caracemide, carbetimer, carboplatin, carmustine, carubicin hydrochloride, carzel ezin, capecitabine, cedefingol, cetuximab , chlorambucil, cirolemycin, cisplatin, cladribine, clofarabine, crisnathol mesylate, cyclophosphamide, cytarabine, dacarbazine, dasatinib, daunorubicin hydrochloride, dactinomycin, darbepoetin alfa, decitabine, degarelix, denileukin diftitox, dexormaplatin, dexrazox ana hydrochloride, desaguanine, desaguanine mesylate, diaziquon, docetaxel , doxorubicin, doxorubicin hydrochloride, droloxifene, droloxifene citrate, dromostanolone propionate, duazomycin, edatrexate, eflornithine hydrochloride, elsamitrucin, eltrombopag olamine, enloplatin, enpromate, epipropidine, epirubicin hydrochloride, epoetin alfa, er bulozol, erlotinib hydrochloride, esorubicin hydrochloride, estramustine, estramustine phosphate sodium, etanidazole, etoposide, etoposide phosphate, etoprine, everolimus, exemestane, fadrozole hydrochloride, fazarabine, fenretinide, filgrastim, floxuridine, fludarabine phosphate, fluorouracil, fluorocitabine, fosquidone, fostriecin sodium, fulvestrant, gefitinib, gemcitabine, gemcitab ina hydrochloride, gemcitabine-cisplatin , gemtuzumab ozogamicin, goserelin acetate, histrelin acetate, hydroxyurea, idarubicin hydrochloride, ifosfamide, imifosin, ibritumomab tiuxetan, idarucibin, ifosfamide, imatinib mesylate, imiquimod, interleukin II (including recombinant interleukin II or rIL2 ), interferon alpha-2a, interferon alpha- 2b, interferon alpha-n1, interferon alpha-n3, interferon beta-1a, interferon gamma-1b, iproplatin, irinotecan hydrochloride, ixabepilone, lanreotide acetate, lapatinib, lenalidomide, letrozole, leuprolide acetate, leucovorin calcium, leuprolide acetate, levamisole, liposomal cytarabine, liarozole hydrochloride, lometrexol sodium, lomustine, losoxantrone hydrochloride, mazoprocol, maytansine, mechlorethamine hydrochloride, megestrol acetate, melengestrol acetate, melphalan, menogaryl, mercaptopurine, methotrexate, methotrexate sodium, methoxalen, metho prina, meturedepa, mitindomide, mitocarcin, mitocromine, mitogillin , mitomalcin, mitomycin C, mitosper, mitotane, mitoxantrone hydrochloride, mycophenolic acid, nandrolone fenpropionate, nelarbine, nilotinib, nocodazole, nofetumomab, nogalamycin, ofatumumab, oprelvekin, ormaplatin, oxaliplatin, oxysuran, paclitaxel , palifermin, palonosetron hydrochloride, pamidronate, pegfilgrastim, pemetrexed disodium, pentostatin, panitumumab, pazopanib hydrochloride, pemetrexed sodium, plerixafor, pralatrexate, pegaspargase, peliomycin, pentamustine, peplomycin sulfate, perfosfamide, pipobromane, piposulfan, piroxantrone hydrochloride, plicamycin, plomestan, pomalidomide , porfimer sodium, porfiromycin, prednimustine, procarbazine hydrochloride , pyromucin, pyromucin hydrochloride, pyrazofurin, quinacrine, raloxifene

- 43 044096 hydrochloride, rasburicase, recombinant bivalent vaccine against HPV, recombinant quadrivalent vaccine against HPV, riboprin, rogletimide, rituximab, romidepsin, romiplostim, safingol, safingol hydrochloride, sargramostim, semustine, simtrazene, sipuleucel -T, sorafenib, sodium sparphosate, sparsomycin , spirogermanium hydrochloride, spiromustine, spiroplatinum, streptonigrin, streptozocin, sulofenir, sunitinib malate, talisomycin, tamoxifen citrate, tecogalane sodium, tegafur, teloxantrone hydrochloride, temozolomide, temoporfin, temsirolimus, teniposide, teroxiron, testolactone, thalidomide, thiamiprin, thioguanine, thiotepa, thiazofurin, tirapamazine, topotecan hydrochloride, toremifene, tositumomab and I ¹³¹ iodine tositumomab, trastuzumab, trestolone acetate, tretinoin, triciribine phosphate, trimetrexate, trimetrexate glucuronate, triptorelin, tubulosole hydrochloride, uracil mustard, uredepa, valrubicin, vapreotide, verteporfin, vinblastine, vinblastine sulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidine sulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine tartrate, vinrosidine sulfate, vinzolidine sulfate, vorinostat, vorozole, zeniplatin, zinostatin, zoledronic acid or zorubicin hydrochloride.

Monoclonal antibodies include, but are not limited to, trastuzumab (Herceptin) and rituximab (Rituxan).

In some embodiments, the at least one additional chemotherapeutic agent is selected from, for example, alemtuzumab, arsenic trioxide, asparaginase (PEGylated or non-PEGylated), bevacizumab, cetuximab, platinum-based compounds such as cisplatin, cladribine, daunorubicin/doxorubicin/ idarubicin, irinotecan, fludarabine, 5-fluorouracil, gemtuzumab, methotrexate, taxol, temozolomide, thioguanine or classes of drugs including hormones (analogs of anti-estrogen, anti-androgen or gonadotropin-releasing hormones), interferons such as alpha-interferon, nitrogen mustards such such as busulfan or melphalan or mechlorethamine, retinoids such as tretinoin, topoisomerase inhibitors such as irinotecan or topotecan, tyrosine kinase inhibitors such as gefitinib or imatinib, or agents for the treatment of signs or symptoms caused by such therapy, including allopurinol, filgrastim, granisetron/ondansetron/polanosetron, dronabinol.

In one aspect, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered or formulated in combination with one or more anticancer agents. In accordance with some embodiments, the one or more anticancer agents are proapoptotic agents. Examples of anticancer agents include, but are not limited to, any of the following: gossypol, genesense, polyphenol E, chlorofusin, all-trans retinoic acid (ATRA), bryostatin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), 5-aza-2'-deoxycytidine, all-trans -retinoic acid, doxorubicin, vincristine, etoposide, gemcitabine, imatinib, geldanamycin, 17-K-allylamino-17demethoxygeldanamycin (17-AAG), flavopiridol, LY294002, bortezomib, carfilzomib, trastuzumab, BAY 11-7082, PKC412 or PD184352, paclitaxel and paclitaxel analogues. It has also been shown that compounds having a basic taxane skeleton as a common structural feature have the ability to arrest cells in G2-M phases due to stabilized microtubules and may be useful for the treatment of cancer in combination with compound I or a pharmaceutically acceptable salt or solvate thereof.

Additional examples of anticancer agents for use in combination with Compound I or a pharmaceutically acceptable salt or solvate thereof include inhibitors of the mitogen-activated protein kinase signaling pathway, for example, U0126, PD98059, PD184352, PD0325901, ARRY142886, SB239063, SP600125, BAY 43-9006, wortmannin or LY294002; Syk inhibitors; mTOR inhibitors; activin inhibitors, PKM2 inhibitors, c-fms inhibitors and histone deacetylase inhibitors. Additional examples of anticancer agents for use in combination with Compound I or a pharmaceutically acceptable salt or solvate thereof include aromatase inhibitors. Aromatase inhibitors include steroidal aromatase inhibitors and non-steroidal aromatase inhibitors. Steroidal aromatase inhibitors include, without limitation, exemestane. Non-steroidal aromatase inhibitors include, but are not limited to, anastrozole and letrozole.

Other anticancer agents for use in combination with Compound I or a pharmaceutically acceptable salt or solvate thereof include alkylating agents, antimetabolites, natural products or hormones, for example, nitrogen mustards (eg, mechlorethamine, cyclophosphamide, chlorambucil, etc.), alkyl sulfonates (eg busulfan), nitrosoureas (eg carmustine, lomustine, etc.) or triazines (decarbazine, etc.). Examples of antimetabolites include, but are not limited to, folic acid analogs (eg, methotrexate) or pyrimidine analogs (eg, cytarabine), purine analogs (eg, mercaptopurine, thioguanine, pentostatin).

Examples of natural products for use in combination with Compound I or a pharmaceutically acceptable salt or solvate thereof include, but are not limited to, vinca alkaloids (e.g., vinblastine, vincristine), epipodophyllotoxins (e.g., etoposide), antibiotics (e.g., daunorubicin, doxorubicin, bleomycin), enzymes ( for example, L-asparaginase) or biologo modifiers

- 44 044096 gical response (for example, interferon-alpha).

Examples of alkylating agents for use in combination with Compound I or a pharmaceutically acceptable salt or solvate thereof include, but are not limited to, nitrogen mustards (e.g., mechlorethamine, cyclophosphamide, chlorambucil, melphalan, etc.), ethyleneimine, and methylmelamines (e.g., hexamethylmelamine, thiotepa), alkylsulfonates (eg busulfan), nitrosoureas (eg carmustine, lomustine, semustine, streptozocin, etc.) or triazines (decarbazine, etc.).

In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used for the treatment of cancer in combination with a second antiestrogen (e.g., tamoxifen), antiandrogen (e.g., bicalutamide, flutamide), gonadotropin-releasing hormone (e.g., leuprolide).

Other agents that may be used in the methods and compositions described herein for the treatment or prevention of cancer include platinum coordination complexes (e.g., cisplatin, carboplatin), anthracenedione (e.g., mitoxantrone), substituted urea (e.g., hydroxyurea), derivative methylhydrazine (for example, procarbazine), substances that suppress the function of the adrenal cortex (for example, mitotane, aminoglutethimide).

Examples of anticancer agents that act by blocking cells in G2-M phases through stabilized microtubules include, but are not limited to, the following marketed and developmental drugs: erbulozole, dolastatin 10, mivobulin isethionate, vincristine, NSC-639829, discodermolide, ABT-751, altoryrtins (such as altorirtin A and altoryrtin C), spongistatins (such as spongistatin 1, spongistatin 2, spongistatin 3, spongistatin 4, spongistatin 5, spongistatin 6, spongistatin 7, spongistatin 8 and spongistatin 9), cemadotin hydrochloride, epothilones (such as epothilone A, epothilone B, epothilone C, epothilone D, epothilone E, epothilone F, epothilone B N-oxide, epothilone A N-oxide, 16azaepothilone B, 21-aminoepothilone B, 21-hydroxyepothilone D, 26 -fluoroepothilone, auristatin RE, soblidotin, vincristine sulfate, cryptophycin 52, vitilevuamide, tubulisin A, canadensol, centaureidine, oncocidin A1, fidzhianolide B, laulimalide, narcozine, nascapine, hemiasterin, vanadocene acetylacetonate, indanocin, eleutherobines (such as desmethyleleutherobin, desethyleleutherobine, isoeleutherobin A and Zeleutherobin), caribeoside, caribeolin, halichondrin B, diazonamide A, taccalonolide A, diosostatin, (-)phenilagistin, myoseverin B, resverastatin sodium phosphate.

In one aspect, Compound I or a pharmaceutically acceptable salt or solvate thereof is co-administered with a thrombolytic agent (eg, alteplase, anistreplase, streptokinase, urokinase, or tissue plasminogen activator), heparin, tinzaparin, warfarin, dabigatran (eg, dabigatran etexilate), factor inhibitors Xa (eg, fondaparinux, draparinux, rivaroxaban, DX-9065a, otamixaban, LY517717 or YM150), ticlopidine, clopidogrel, CS-747 (prasugrel, LY640315), ximelagatran, or BIBR 1048.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in combination with antiemetic agents to treat nausea or vomiting that may result from the use of Compound I or a pharmaceutically acceptable salt or solvate thereof, anticancer agent(s). and/or radiation therapy.

Antiemetics include, but are not limited to, neurokinin-1 receptor antagonists, 5HT3 receptor antagonists (such as ondansetron, granisetron, tropisetron, palonosetron, and zatisetron), GABA _B receptor agonists (such as baclofen), corticosteroids (such as dexamethasone, prednisone, prednisolone, and others). ), dopamine antagonists (such as but not limited to domperidone, droperidol, haloperidol, chlorpromazine, promethazine, prochlorperazine, metoclopramide), antihistamines (histamine H1 receptor antagonists such as but not limited to cyclizine, diphenhydramine, dimenhydrinate, meclizine, promethazine, hydroxyzine), cannabinoids (such as without limitation cannabis, marinol, dronabinol) and others (such as without limitation trimethobenzamide; ginger preparation, emetrol, propofol).

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in combination with an agent useful in the treatment of anemia. Such an anemia treatment agent is, for example, a continuous ER activator (such as epoetin-α).

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in combination with an agent useful in the treatment of neutropenia. Examples of agents useful for treating neutropenia include, but are not limited to, hematopoietic growth factors that regulate neutrophil production and function, such as human granulocyte colony-stimulating factor (G-CSF). Examples of G-CSF include filgrastim.

In one aspect, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered in combination with one or more immunosuppressive agents. Immunosuppressive therapy is used clinically to treat or prevent transplant rejection

- 45 044096 organs and tissues (for example, bone marrow, heart, kidney, liver), treatment of autoimmune diseases or diseases that are most likely to be of autoimmune origin (for example, rheumatoid arthritis, myasthenia gravis, systemic lupus erythematosus, Crohn's disease and ulcerative colitis ) and the treatment of certain other non-autoimmune inflammatory diseases (eg, long-term control of allergic asthma) and in the treatment of fibrotic conditions. Immunosuppressants include, without limitation, glucocorticoids, cytostatics, antibodies and drugs that act on immunophilins. Examples of glucocorticoids include cortisol, cortisone, prednisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone, deoxycorticosterone and aldosterone. Examples of cytostatics include alkylating agents (eg, nitrogen mustards such as cyclophosphamide, nitrosoureas, platinum compounds) and antimetabolites (eg, folic acid analogs such as methotrexate, purine analogs such as azathioprine and mercaptopurine, pyrimidine analogs such as fluorouracil, inhibitors protein synthesis). Examples of drugs for use in the described methods include cyclosporine, tacrolimus, sirolimus, interferons, opioids, TNF-binding proteins, mycophenolate and fingolimod. Examples of antibodies suitable for co-administration with Compound I or a pharmaceutically acceptable salt or solvate thereof according to the method described herein include antithymocyte globulin, 1D09C3, adalimumab/D2E7 (Humira; Trudexa), afelimomab, afutuzumab/GA101 (type II), alemtuzumab /campat-III (mabcampat), apolizumab/Hu1D10, aselizumab, atlizumab, basiliximab (simulect), bektumomab/IMMU-LL2, belimumab (benlysta, lymphostat-B), bertilimumab, BL22/CAT-3888, brentuximab/cAC10/SGN- 35, briakinumab/ABT-874, canakinumab/ACZ885 (ilaris), certolizumab pegol/CDP870 (cimzia), clenoliximab, dacetuzumab/SGN-40, daclizumab (zenapax), eculizumab/5G1.1 (soliris), efalizumab (raptiva, formerly xanelim), epratuzumab/hLL2/IMMU102 (Lymphocyde©), fontolizumab, fresolimumab/GC-1008, galiximab/IDEC-114, gavilimomab/ABXCBL, gemtuzumab, golimumab/CNTO148 (Simponi), HL2434P (IMMU-114), ibritumomab tiuxetan ( MXDPTA)/IDEC Y2B8 (zevalin), infliximab/chimeric A2 (cA2) (remicade), inolimomab/BT563, inotuzumab, keliximab/IDEC CE9.1, lerdelimumab/CAT-152, lintuzumab/HuM195 (zamil), LMB-2, lorvotuzumab mertansine, lumiliximab/IDEC-152, Lym-1 (oncolim), MDX-060, mepolizumab/SB-240563, metelimumab/CAT-192, mogamulizumab/KW-0761/AMG-761, moxetumomab pasudotox/CAT8015/HA22, muromonab -CD3 (orthoclone OKT3), natalizumab (tysabri, antegren), nerelimomab/CDP571, ocrelizumab/PRO70769 (type I), odulimomab, ofatumumab/2F2/HuMax-CD20 (arzerra) (type I), omalizumab (xolair), otelixizumab/ TRX4, pascolizumab/SB 240683, reslizumab/SCH 55700 (Zinquil), rituximab/chimeric 2B8 (IDEC-C2B8) (Rituxan, MabThera) (type I), ruplizumab (Antova), SAR-3419, secukinumab/AIN-457, SGN30 , siplizumab/MEDI-507, teplizumab/MGA031/hOKT3γ1(Ala-Ala), tocilizumab (actemra), tositumomab (type II), ustekinumab/CNTO 1275 (stelara), vedolizumab/MNL-0002, veltuzumab/IMMU-106/hA20 (type I), visilizumab (Nuvion), zanolimumab/HuMax-CD4, zolimomab aritox/H65, abatacept/CTLA4-Ig/BMS-188667 (orenzia), belatacept/LEA29Y, atacicept/BLyS/APRIL-Ig, etanercept/TNFR- Ig (Enbrel), pegsunercept/PEGylated TNFR-Ig, alefacept (Ameviv), and rilonacept (Arcalyst). Immunosuppressive antibodies include antibodies that target complement-dependent proteins and interleukins.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered with a corticosteroid. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered with a therapeutic agent selected from the following: calcineurin inhibitors (such as, but not limited to, cyclosporine, tacrolimus); mTOR inhibitors (such as without limitation sirolimus, everolimus); antiproliferative agents (such as, but not limited to, azathioprine, mycophenolic acid); corticosteroids (such as, without limitation, prednisone, cortisone acetate, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone, hydrocortisone); antibodies (such as, but not limited to, monoclonal antibodies to the IL-2Raa receptor (basiliximab, daclizumab), polyclonal antibodies to T cells (antithymocyte globulin (ATG), antilymphocyte globulin (ALG)), B cell antagonists, rituximab, natalizumab.

Other therapeutic agents suitable for combination with Compound I or a pharmaceutically acceptable salt or solvate thereof that are described herein include, without limitation, cyclophosphamide, penicillamine, cyclosporine, nitrosourea, cisplatin, carboplatin, oxaliplatin, methotrexate, azathioprine, mercaptopurine, pyrimidine analogs, protein synthesis inhibitors, dactinomycin, anthracyclines, mitomycin C, bleomycin, mithramycin, Atgam ^(R) , Thymoglobuline®, OKT3®, basiliximab, daclizumab, cyclosporine, tacrolimus, sirolimus, interferons (IFN-β, IFN-γ), opioids, TNF-binding proteins (infliximab, etanercept, adalimumab, golimumab), leflunomide, gold thioglucose, gold thiomalate, aurofin, sulfasalazine, hydroxychloroquinine, minocycline, rapamycin, mycophenolic acid, mycophenolate mofetil, FTY720, and those listed in the publication US 7060697.

- 46 044096

In accordance with one embodiment, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered in combination with cyclosporine A (CsA) or tacrolimus (FK506). In one embodiment, Compound I, or a pharmaceutically acceptable salt or solvate thereof, is administered to a mammal in combination with an anti-inflammatory agent, including, without limitation, nonsteroidal anti-inflammatory drugs (NSAIDs), phosphodiesterase-4 inhibitors, JNK kinase inhibitors, and corticosteroids (glucocorticoids).

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered with corticosteroids. Corticosteroids include, but are not limited to, betamethasone, prednisolone, aclomethasone, aldosterone, amcinonide, beclomethasone, betamethasone, budesonide, ciclesonide, clobetasol, clobetasone, clocortolone, cloprednol, cortisone, cortivasole, deflazacort, deoxycorticosterone, desonide, deoxymethasone, deoxycortone, dexamethasone, diflorazon, diflucortolone, difluprednate, fluclorolone, fludrocortisone, fludroxycortide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonides, fluocortin, fluocortolone, fluorometholone, fluperolone, fluprednidene, fluticasone, formocortal, galcinonide, halomethasone, hydrocortisone/cortisol, hydrocorti zon aceponate, hydrocortisone buteprate, hydrocortisone butyrate, loteprednol, medrisone, meprednisone, methylprednisolone, methylprednisolone aceponate, mometasone furoate, paramethasone, prednicarbate, prednisone/prednisolone, rimexolone, thixocortol, triamcinolone and ulobetazole.

In accordance with one embodiment, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered to a mammal in combination with a nonsteroidal anti-inflammatory drug (NSAID). NSAIDs include, but are not limited to, aspirin, salicylic acid, gentisic acid, choline magnesium salicylate, choline salicylate, choline magnesium salicylate, choline salicylate, magnesium salicylate, sodium salicylate, diflunisal, carprofen, fenoprofen, fenoprofen calcium, flurobiprofen, ibuprofen, ketoprofen, nabutone, ketorolac, ketorolac tromethamine, naproxen, oxaprozin, diclofenac, etodolac, indomethacin, sulindac, tolmetin, meclofenamate, meclofenamate sodium, mefenamic acid, piroxicam, meloxicam, specific COX-2 inhibitors (such as, but not limited to, celecoxib, rofecoxib, valdecoxib, parecoxib, this ricoxib , lumiracoxib, CS502, JTE-522, L-745,337 and NS398).

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is co-administered with an analgesic agent.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is used in combination with radiation therapy (or radiotherapy). Radiation therapy is a method of treating cancer and other diseases with ionizing radiation. Radiation therapy can be used to treat localized solid tumors, such as cancers of the skin, tongue, larynx, brain, breast, prostate, colon, liver, uterus, and/or cervix. It can also be used to treat leukemia and lymphoma (forms of cancer of the blood-forming cells and lymphatic system, respectively).

The technique for delivering radiation to cancer cells involves placing radioactive implants directly into the tumor or body cavity. This technique is called internal radiotherapy (brachytherapy, interstitial irradiation and endocavity irradiation are different types of internal radiotherapy). With internal radiotherapy, the radiation dose is concentrated in a small area and the patient remains in the hospital for only a few days. Internal radiotherapy is often used for cancers of the tongue, uterus, prostate, colon and cervix.

The term radiotherapy or ionizing radiation includes all forms of radiation, including, but not limited to, α, β, and γ radiation and ultraviolet radiation.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered with a glucose-lowering agent. In accordance with some embodiments, the glucose-lowering agent is selected from a peroxisome proliferator-activated receptor (PPAR) agonist (gamma, dual, or pan-agonist), dipeptidyl peptidase (IV) inhibitor, glucagon-like peptide-1 (GLP-I) analogue , insulin or insulin analogue, insulin secretagogue, sodium-dependent glucose transporter 2 (SGLT2) inhibitor, glucophage, human amylin analogue, biguanide, alpha-glucosidase inhibitor, meglitinide, thiazolidinedione and sulfonylurea. In some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered with metformin, sitagliptin, saxagliptin, repaglinide, nateglinide, exenatide, liraglutide, insulin lispro, insulin aspart, insulin glargine, insulin detemir, insulin isophane, and glucagon-like peptide 1 or any combination thereof. In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered with a lipid-lowering agent.

In accordance with some embodiments, Compound I or a pharmaceutically acceptable salt or solvate thereof is administered in combination with at least one additional therapeutic agent used to treat a cardiovascular disease. In accordance with

- 47 044096 In some embodiments, the therapeutic agent used to treat a cardiovascular disease is an angiotensin converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), a beta blocker, a diuretic, a calcium channel blocker, an inhibitor of the renin-angiotensin system ( RAS), anticoagulant, statin and fibrate and any combination thereof.

Sets and products.

Disclosed herein are kits for the treatment of a condition, disease or disorder associated with LOXL2 activity comprising administering Compound I or a pharmaceutically acceptable salt or solvate thereof to a specified individual.

For use in the therapeutic applications described herein, kits and articles are also described herein. In accordance with some embodiments, such kits include a carrier, package, or container that is divided into sections containing one or more containers, such as vials, tubes, and the like, each of these containers containing one of the individual elements that subject to use in the method described herein. Suitable containers include, for example, bottles, vials, syringes and test tubes. Containers can be made from various materials such as glass or plastic.

The products presented herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, inhalers, pumps, pouches, vials, containers, syringes, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment. A wide range of compound formulations and compositions are provided herein, as well as a variety of treatments for any disorder that will benefit from inhibition of LOXL2, or in which LOXL2 is a mediator or contributing factor to the symptoms or cause.

The container(s) optionally have a sterile access opening (eg, the container is an intravenous solution bag or a vial with a stopper pierced by a hypodermic needle). Such kits optionally contain the compound with an identifying description or label or instructions pertaining to its use in the methods described herein.

The kit will typically include one or more additional containers, each containing one or more different materials (such as reagents, optionally in concentrated form, and/or devices) required from a commercial and user point of view for the use of the compound described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, filters, needles, syringes; labels on the media, packaging, container, bottle and/or tube indicating the contents and/or instructions for use, as well as package inserts with instructions for use. A set of instructions will also usually be included.

In accordance with some embodiments, the label is on or attached to the container. A label may be located on a container if the letters, numbers or other symbols forming the label are affixed, cast or engraved into the container itself; a label may be associated with a container when it is present within a container or carrier that also contains the container, such as a package insert. The label may be used to indicate that the contents are to be used for a particular therapeutic use. The label may also indicate directions for use of the contents, for example, in the methods described herein.

In accordance with certain embodiments, a pharmaceutical composition containing Compound I or a pharmaceutically acceptable salt or solvate thereof is contained in a package or dispensing device, which may contain one or more unit dosage forms. The packaging may, for example, contain metal or plastic foil, for example in the case of a blister pack. The packaging or dispensing device may be accompanied by instructions for use. The package or dispenser may also be accompanied by a notice associated with the container in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceuticals, which notice reflects the agency's approval of the form of the drug for use in humans or animals. For example, such a notice could be an FDA-approved label for a prescription drug or an approved product insert. Compositions containing a compound provided herein admixed with a compatible pharmaceutical carrier may also be prepared, packaged and labeled for the treatment of the specified condition.

- 48 044096

Examples

The following examples are presented for purposes of illustration only and are not intended to limit the scope of the claims presented herein. Compound I was prepared as described in international patent application no. PCT/US2016/020732, filed March 3, 2016. The mesylate salt was prepared by treating compound I with methanesulfonic acid in acetonitrile.

Example A-1: preparation of compound 1 in capsule form.

Compound 1 was directly added to size 9 capsules (Torpac, Inc., New Jersey).

Example A-2: drugs in tablet form.

Two different preparations were obtained in the form of tablets with dosages of 50 mg and 250 mg (according to the amount of compound I). Tablets were prepared according to standard tableting procedures.

Table 1

Preparation A

Dose 250 mg (compound I) Mass. % Mass. per tablet (mg) Mass. per 50 g batch (g) Connection 2 35.27% 317.42 17,634 Prosolv HD90 55.73% 501.58 27,866 Ac-Di-Sol 5.00% 45.00 2,500 NPC Kluckel EXF 3.00% 27.00 1,500 Aerosil 200 0.50% 4.50 0.250 Magnesium stearate 0.50% 4.50 0.250 Total 100.00% 900.00 50,000

table 2

Preparation B

Dose 250 mg (compound I) Mass. % Mass. on tablet (mg) Mass. per 50 g batch ( ^g ) Connection 2 35.27% 317.42 17,634 Avicel RN 102 14.06% 126.52 7,029 Partek M200 (mannitol) 42.17% 379.56 21,087 Explotab 5.00% 45.00 2,500 PVP VA 64 3.00% 27.00 1,500 PRUV 0.50% 4.50 0.250 Total 100.00% 900.00 50,000

Two different preparations were obtained in the form of tablets with different dosages: 50 mg and 250 mg (according to the amount of compound I). Tablets were prepared according to standard tableting procedures and stored at 20-25°C. The tableting formulation was prepared by direct mixing and compressed into tablets in the form of 900 mg capsules.

Table 3

Composition of tablets with compound 2.50 mg (compound I)

Component Amount per tablet (wt.%) Connection 2 62.46 mg (6.94%) Siliconized microcrystalline cellulose 756.5 mg (84.1%) Croscarmellose sodium 45.00 mg (5.0%) Hydroxypropyl cellulose 27.00 mg (3.0%) Colloidal silicon dioxide 4.50 mg (0.5%) Magnesium stearate 4.50 mg (0.5%) Total 900 mg

- 49 044096

Table 4

Composition of tablets with compound 2.250 mg (compound I)

Component Amount per tablet (wt.%) Connection 2 312.3 mg(34.7%) Prosolv HD90 506.7 mg (56.3%) Ac-Di-Sol® 45.00 mg (5.0%) NPC Kluckel EXF 27.00 mg (3.0%) Aerosil 200 4.50 mg (0.5%) Magnesium stearate 4.50 mg (0.5%) Total 900 mg

Example A-3: oral solution.

Oral solutions of Compound 2 were prepared that had a concentration of Compound 2 ranging from 5 mg/mL to 50 mg/mL in an aqueous vehicle containing sodium citrate dehydrate, anhydrous citric acid, FONA bittering flavorings, and sucralose in an aqueous solution.

Solutions were prepared as follows. Add the required amount of water to the container (see Table 3 for quantities). The required amount of sodium citrate and citric acid was weighed, added to the container and stirred until completely dissolved. Weigh out the required amount of flavoring (FONA Bitter Masking Flavors), add it to the solution and mix until smooth. Weighed the required amount of sucralose, added it to the solution and stirred until completely dissolved. Appearance (colorless to slightly yellow) and pH (pH range 3 to 5) were evaluated to ensure that the diluent met specifications. Weigh the required amount of compound 2 and slowly add it to the diluent. Stir until compound 2 is completely dissolved (sonicate, heat or stir if necessary). The pH had to be between 3 and 5 and the appearance colorless or slightly yellow. Measure up to 80 ml of bulk dosage solution into a glass container.

When stored at 2 to 8°C or 25°C/60% RH for up to 7 days in glass containers, the oral solution showed no significant changes in appearance, potency, purity or pH. The recommended storage of the oral solution is either at a temperature of 2 to 8°C, or at a temperature of 20 to 25°C in a glass container for up to 7 days.

Table 5

Composition of Compound 2 Oral Solution

Component Amount per unit Concentration (mg/ml) Connection 2 6.3 mg/ml - 63 mg/ml Sodium citrate dihydrate 9.50 Anhydrous citric acid 10.50 Sucralose 2.00 Flavoring for masking bitterness - liquid (936.0504 units) 0.50 Flavor for masking bitterness - solid (936.0592 units) 0.50 Purified water up to 1 ml as needed

Example A-4: Pharmacokinetic study of compound 1 or 2 in rat.

Compound 1 or 2 was administered orally in solution at 30 mg/kg or 15 mg/kg of Compound 1 capsule [Compound 1 directly added to a size 9 capsule (Torpac, Inc., NJ)].

Blood samples were collected from each rat (approximately 0.3 ml of total blood per time point) at predose, 5 or 15 min, and then at various time points up to 24 h postdose. Samples were collected on wet ice into tubes containing potassium EDTA (w/v in normal saline; BD Biosciences, Franklin Lakes, NJ). Plasma samples obtained by centrifugation of whole blood were stored frozen (−80°C) until analysis. All

- 50 044096 other reagents were of analytical quality.

Analysis was performed on LC-MS/MS systems consisting of a Sciex API-4000Qt tandem mass spectrometer (AB Sciex, Foster City, California) coupled to an HPLC system consisting of a single Agilent 1200 Series Quaternary system pump (Santa Clara , California) and the LEAP PAL autoinjector (Greenville, South Carolina). Analyzes were performed using an Agilent Zorbax SB-C8 column (2.1 x 50 mm; 5 μm) for chromatographic separation at room temperature. The data is described in table. 6.

Table 6

Pharmacokinetics of compound 1 or 2 in rats using different dosage forms

View Rat Compound 1 1 2 1 Path IV RO RO RO Dosage form filler Saline solution 0.5% MS 0.5% MS Capsule Dose (mg/kg) 2 thirty thirty 15 On an empty stomach/after meals After food On an empty stomach On an empty stomach On an empty stomach Floor M M M M AUC(o-t h) (µg"h/ml) 0.14 1.4 2.0 0.74 AISocch/dose 0.07 0.05 0.07 0.05 Stach (mcg/ml) 0.63 1.7 2.6 1.2 Ttah (h) 0.083 0.25 0.42 0.6 F(%) 100 71 95 69

IV - intravenous; RO - oral; MS - methylcellulose; M = male; AUC is the area under the plasma concentration versus time curve; C _max - maximum concentration in plasma; T _max - time until maximum plasma concentration is reached; %F - bioavailability calculated by AUC ₀ . _tPO /AUC ₀ . _tIV .

Example A-5: Pharmacokinetic study of compound 1 or 2 in a dog.

Compound 1 or 2 was administered PO in solution at a dose of 100 or 300 mg/kg. Compound 2 was administered PO in a capsule and also in two different tablet formulations (Formulation A or B). The data is described in table. 5. Vomiting was common in all dose groups, but clinical observations concluded that the oral capsule and possibly the tablet were better tolerated than oral solutions.

Blood samples were collected from each dog (approximately 1 mL of total blood per time point) at predose, 5 or 15 min, and then at various time points up to 24 h postdose. Samples were collected on wet ice into tubes containing potassium EDTA (w/v in normal saline; BD Biosciences, Franklin Lakes, NJ). Plasma samples obtained by centrifugation of whole blood were stored frozen (−80°C) until analysis. All other reagents were of analytical quality.

Analysis was performed on LC-MS/MS systems consisting of a Sciex API-4000Qt tandem mass spectrometer (AB Sciex, Foster City, California) coupled to an HPLC system consisting of a single Agilent 1200 Series Quaternary system pump (Santa Clara , California) and the LEAP PAL autoinjector (Greenville, South Carolina). Analyzes were performed using an Agilent Zorbax SB-C8 column (2.1 x 50 mm; 5 µm) for chromatographic separation at room temperature.

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Table 7

Pharmacokinetics of compound 1 or 2 in dogs using _______________different dosage forms_______________

View Dog Compound 1 1 2 2 2 2 Path IV RO RO RO RO RO Dosage form filler Saline solution 0.5% MS Citrate Capsule The drug is in the form of tablets A The drug is in the form of tablets B Dose (mg/kg) 5 100 300 300 100 100 On an empty stomach/after meals After food On an empty stomach On an empty stomach On an empty stomach On an empty stomach On an empty stomach Floor M M M M M M AUC(o-t h) (µg"h/ml) 1.4 85 286 100 75.6 63.9 AISocch/dose 0.28 0.85 0.95 0.33 0.76 0.64 Stach (MCG/ML) 2.5 27 143 80 33.7 25.1 Ttah (h) 0.083 1.3 o, s 0.5 0.4 0.8 F(%) 100 283 339 100 253 210

IV - intravenous; RO - oral; MS - methylcellulose; M = male beagle; AUC is the area under the plasma concentration versus time curve; Cmax - maximum plasma concentration; T _max - time until maximum plasma concentration is reached; %F bioavailability calculated from AUC ₀ -t PO/AUC ₀ -t IV.

Example A-6: Pharmaceutical composition for parenteral administration.

To prepare a pharmaceutical composition for parenteral administration suitable for administration by injection (subcutaneous, intravenous), 1-1000 mg of compound (I), or a pharmaceutically acceptable salt or solvate thereof, was dissolved in sterile water and then mixed with 10 ml of 0.9 % sterile saline solution. Optionally, a suitable buffer and an optional acid or base are added to adjust the pH. The mixture was formulated into a unit dosage form suitable for injection.

Example A-7: Gel composition for topical use.

To prepare a pharmaceutical gel composition for topical use, compound (I) or a pharmaceutically acceptable salt thereof was mixed with hydroxypropylcellulose, propylene glycol, isopropyl myristate and USP purified alcohol. The resulting gel mixture was then introduced into containers, such as tubes, that were suitable for topical application.

Example B-1: preparation of concentrated conditioned media (CCM).

Stable human LOXL2/CHO and human LOX/HEK cell lines were cultured under normal growth conditions in 15 cm tissue culture plates until the cells reached ∼80% confluence. The cells were then washed with PBS before adding 2530 ml of serum-free medium (DMEM/F12 without phenol red with glutamax containing penicillin/streptomycin, 10-100 µM CuCl ₂ ±0.1% BSA). Cells were incubated at 37°C, 5% CO ₂ in serum-free medium for 40-48 hours, after which the conditioned medium was removed and centrifuged at 2000 rpm for 5 minutes at 4°C to pellet cells/debris. The medium was concentrated 10- to 20-fold using 10 to 30 MWCO preparative centrifugal chromatography columns according to the manufacturer's instructions (EMD Millipore, Billerica, MA) before aliquoting and storing at −80°C.

Example B-2: CFM analysis of human LOXL2.

LOXL2 amine oxidase activity was assessed by measuring Amplex Red fluorescence using 10- to 20-fold concentrated conditioned medium (free of BSA) from CHO cells stably expressing human LOXL2. To assay amine oxidase activity, 10 μL of concentrated conditioned medium was incubated with 2 μL of test compound in DMSO and 73 μL of assay buffer (50 mM borate buffer, pH 8) for 2 h at 37°C. After a 2-hour incubation, 5 μl of 10 mM 1,5-diaminopentane (DAP) was diluted in assay buffer and 10 μl of Amplex Red Mix (8.5 μl of assay buffer + 0.5 μl of 10 mM Amplex Red + 1 μl of horseradish peroxidase) was added at a concentration of 500 units/ml) and the contents of the plate were mixed and the plate was immediately placed on the FlexStation for fluorescence measurement. Fluorescence was read in kinetic mode every 2 minutes for 0.5-1 hour with excitation wavelength = 544 and emission = 590. Amine oxidase activity was calculated from the slope of the linear part of the curve.

- 52 044096

Wells containing dosage form excipient (DMSO) showed maximum activity and were taken as 0% inhibition, and wells containing 100 μM eAPN (3-aminopropionitrile) showed no activity and were taken as 100% inhibition.

A is <300 nM.

Example B-3: SFM analysis for human LOX.

The amine oxidase activity of human LOXL was assessed by measuring Amplex Red fluorescence using 10- to 20-fold concentrated conditioned medium (containing BSA) from HEK cells stably expressing human LOXL. To assay amine oxidase activity, 10 μL of concentrated conditioned medium was incubated with 2 μL of test compound in DMSO and 73 μL of assay buffer (50 mM borate buffer, pH 8) for 2 h at 37°C. After a 2-hour incubation, 5 μl of 10 mM 1,5-diaminopentane (DAP) was diluted in assay buffer and 10 μl of Amplex Red Mix (8.5 μl of assay buffer + 0.5 μl of 10 mM Amplex Red + 1 μl of horseradish peroxidase) was added at a concentration of 500 units/ml) and the contents of the plate were mixed and the plate was immediately placed on the FlexStation for fluorescence measurement. Fluorescence was read in kinetic mode every 2 minutes for 1 hour with excitation wavelength = 544 and emission = 590. Amine oxidase activity was calculated from the slope of the linear part of the curve. Wells containing dosage form excipient (DMSO) showed maximum activity and were taken as 0% inhibition, and wells containing 100 μM eAPN (3-aminopropionitrile) showed no activity and were taken as 100% inhibition.

Example B-4: Analysis of purified recombinant human LOXL2 protein.

Amine oxidase activity was assessed by measuring Amplex Red fluorescence using commercially available purified recombinant human LOXL2 (Sino Biologicals, Beijing, China). To analyze amine oxidase activity, 10 μl of purified recombinant LOXL2 at a concentration of 0.025 μg/μl, diluted in assay buffer (50 mM borate buffer, pH 8), was incubated with 2 μl of the test compound in DMSO and 73 μl of assay buffer for 2 h at 37°C. WITH. After a 2-hour incubation, 5 μl of 10 mM 1,5-diaminopentane (DAP) was diluted in assay buffer and 10 μl of Amplex Red Mix (8.5 μl of assay buffer + 0.5 μl of 10 mM Amplex Red + 1 μl of horseradish peroxidase) was added at a concentration of 500 units/ml) and the contents of the plate were mixed and the plate was immediately placed on the FlexStation for fluorescence measurement. Fluorescence was read in kinetic mode every 2 minutes for 0.5-1 hour with excitation wavelength = 544 and emission = 590. Amine oxidase activity was calculated from the slope of the linear part of the curve. Wells containing dosage form excipient (DMSO) showed maximum activity and were taken as 0% inhibition, and wells containing 100 μM eAPN (3-aminopropionitrile) showed no activity and were taken as 100% inhibition.

Example B-5: Analysis of purified recombinant human LOXL3 protein.

Amine oxidase activity was assessed by measuring Amplex Red fluorescence using commercially available purified recombinant human LOXL3 (R&D Systems, Minneapolis, MN). To analyze amino oxidase activity, 10 μl of purified recombinant LOXL3 at a concentration of 0.075 μg/μl, diluted in assay buffer (50 mM borate buffer, pH 8), was incubated with 2 μl of the test compound in DMSO and 73 μl of assay buffer for 2 h at 37°C. WITH. After a 2-hour incubation, 5 μl of 10 mM 1,5-diaminopentane (DAP) was diluted in assay buffer and 10 μl of Amplex Red Mix (8.5 μl of assay buffer + 0.5 μl of 10 mM Amplex Red + 1 μl of horseradish peroxidase) was added at a concentration of 500 units/ml) and the contents of the plate were mixed and the plate was immediately placed on the FlexStation for fluorescence measurement. Fluorescence was read in kinetic mode every 2 minutes for 0.5-1 hour with excitation wavelength = 544 and emission = 590. Amine oxidase activity was calculated from the slope of the linear part of the curve. Wells containing dosage form excipient (DMSO) showed maximum activity and were taken as 0% inhibition, and wells containing 100 µM BAPN (3-aminopropionitrile) showed no activity and were taken as 100% inhibition.

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Table 9

IC50 values for compound 1 in LOX, LOXL2 and LOXL3 assays

Activity Analysis IC50 (µM) LOXL2 (ССМ) 0.0751 LOXL2 (CCM containing BSA) 0.116 LOXL2 (purified, recombinant) 0.209 LOX (CCM containing BSA) 47.0 LOXL3 (purified, recombinant) 1.21

Example B-6: human blood test for LOXL2.

The amine oxidase activity of human LOXL2 in the context of human whole blood was measured using the Amplex Red assay. Purified recombinant human LOXL2 (Sino Biologicals, Beijing, China) was resuspended to 0.25 μg/ml using sterile water, then 16 μl of LOXL2 was added to 182 μl of fresh human blood collected in heparin-containing vacuum tubes. Add 2 μl of test compound in DMSO (or DMSO alone) and incubate at 37°C for 2 hours. After 2 hours of incubation, the blood is centrifuged at 2000xg for 15 minutes at room temperature to isolate blood plasma. 50 μl of plasma was collected and mixed with 25 μl of 40 mM DAP (diluted in water) and 25 μl of Amplex Red Mix (23.5 μl of 50 mM borate buffer, pH 8 + 0.5 μl of 10 mM Amplex Red + 1 μl of horseradish peroxidase with a concentration of 500 units/ml). Samples were mixed and immediately placed on the FlexStation for fluorescence measurement. Fluorescence was read in kinetic mode every 2 minutes for 1 hour with excitation wavelength = 544 and emission = 590. Amine oxidase activity was calculated from the slope of the linear part of the curve. Wells containing dosage form excipient (DMSO) exhibited maximum activity and were scored as 0% inhibition, and wells containing blood without added LOXL2 showed no activity and were scored as 100% inhibition.

Example B-7: Mouse model of pulmonary fibrosis induced by oropharyngeal administration of bleomycin.

Pulmonary fibrosis was induced in male C57BL/6 mice by administering bleomycin (0.1-4 U/kg) via oropharyngeal instillation. Mice were pretreated with formulation excipient or test compound orally, intraperitoneally, intravenously, or subcutaneously either prophylactically (1 day to 1 hour before bleomycin instillation) or therapeutically (7 to 14 days after bleomycin instillation). The dosing route and frequency were based on previously determined pharmacokinetic properties of the LOXL2 inhibitor in mice. Following bleomycin instillation, animals were monitored daily for weight loss and clinical signs for 14–28 days until sacrifice. Animals were sacrificed at the end of the study and weighed. Blood (for blood plasma isolation) and bronchoalveolar lavage fluid were collected and frozen for subsequent analyses. The lungs were removed, weighed, and then either inflated and fixed with 10% formalin instillation and prepared for histological examination or homogenized in 1 ml PBS to determine collagen content using the hydroxyproline assay. For histological examination, lung sections were stained with Masson's trichrome or picrosirius red to measure fibrillar collagen as an indicator of fibrosis, and the Ashcroft score for pulmonary fibrosis and inflammatory damage was determined. To determine the hydroxyproline content in the lungs, 0.5 ml of lung homogenate was taken and added to 0.5 ml of 12 N. HCl and samples were heated to 120°C and left overnight. After hydrolysis in an acidic medium, 25-100 μl of the supernatant was dried, resuspended in 25 μl of water and the hydroxyproline content was determined by adding 0.5 ml of chloramine T solution (140 mg of chloramine T in 6.5 ml of ddH ₂ O + 1 ml of n-propanol + 2.5 ml of 1 M sodium acetate) and kept at room temperature for 20 min. After incubation, 0.5 ml of Ehrlich solution (1.48 g of 4(dimethylamino(benzaldehyde) in 7 ml of n-propanol + 2.88 ml of 60% perchloric acid and 0.12 ml of ddH ₂ O) was added and incubated at 65°C for 15 min before reading absorbance at 550 nm.

Example B-8: dose-dependent effectiveness in pulmonary fibrosis.

Compound Rac-1 was orally administered prophylactically to mice instilled with bleomycin at 3, 10, 30, or 60 mg/kg/day with lungs collected for histological evaluation on day 14. In fig. Figure 1 shows the histopathological Ashcroft score reflecting pulmonary fibrosis in a prophylactic 14-day Rac-1 dose-response study in a mouse model of bleomycin-induced pulmonary fibrosis (*p < 0.05; **p < 0.01 ; ***p <0.001). Rac-1 reduced fibrosis in a dose-dependent manner, suggesting that 30 mg/kg is the minimum dose to achieve maximum antifibrotic efficacy.

Example B-9: Prophylactic Efficacy Versus Therapeutic Efficacy for Pulmonary Fibrosis.

Compound 1, Compound Rac-1, and Compound Ent-1 were administered orally at a dose of 60 mg/kg/day to bleomycin-instilled mice in two regimens: prophylactic (dose from day 1) and therapeutic ( dose from the 7th day). In fig. Figure 2 shows the Ashcroft scale based on the results of histopathological analysis, reflecting pulmonary fibrosis, the comparative effectiveness of compounds 1,

Rac-1 and Ent-1 in both preventive (profil.) and therapeutic (therapeutic) regimens in a mouse model of bleomycin-induced pulmonary fibrosis (*p <0.05, **p <0.01, *** *p<0.0001).

Fibrotic status was assessed histologically at day 21 using the Ashcroft score as the primary indicator of pulmonary fibrosis. All three compounds significantly reduced fibrosis in a mouse model of pulmonary fibrosis with similar efficacy regardless of whether the compound was administered prophylactically or therapeutically.

Example B-10: relief of developed pulmonary fibrosis.

In a mouse model of bleomycin-induced pulmonary fibrosis, fibrosis is known to gradually develop over the first 14–21 days, but then spontaneously resolve in younger mice over the next few weeks (Hecker et al., 2014). To determine whether LOXL2 inhibitors could accelerate resolution of established fibrosis, compound 1 was administered orally starting on day 14 after bleomycin, with lungs collected on day 28 for histological analysis. The degree of fibrosis remained constant from days 14 to 28 in mice treated with the formulation vehicle. Compound 1 reduced fibrosis with 73% normalization of Ashcroft score (Figure 3). In fig. 3 shows the Ashcroft histopathological score reflecting pulmonary fibrosis in a 28-day recovery study in which Compound 1 was administered at a dose of 60 mg/kg starting on day 14.

Example B-11: Effect of Dosing Frequency in Pulmonary Fibrosis.

Compound Rac-1 was administered prophylactically orally at a dose of 60 mg/kg to bleomycin-induced mice using different dosing schedules. The efficacy of daily (QD) dosing was compared with that of every two days (Q2D) and every three days (Q3D) for lungs collected for histological evaluation on day 14. Regardless of dosing frequency, Rac-1 reduced pulmonary fibrosis, with QD being slightly more effective than Q2D or Q3D (Figure 4).

In fig. Figure 4 shows the Ashcroft histopathological score reflecting pulmonary fibrosis in a 14-day prophylactic study comparing QD, Q2D and Q3D doses of Rac-1 in a mouse model of bleomycin-induced pulmonary fibrosis (**p<0 .01; ****p<0.0001).

Example B-12: Comparison of efficacy with anti-LOXL2 antibody in pulmonary fibrosis.

The efficacy of Compound 1 was compared in a head-to-head comparison study with rAB0023. rAB0023 is a recombinant mouse fusion antibody that has the heavy chain variable region of the anti-LOXL2 antibody, AB0023, cloned into the mouse IgG2a backbone, and the light chain variable region, AB0023, cloned into the mouse IgG2 backbone. rAB0023 binds to LOXL2 with an affinity equal to AB0023, an antibody that has demonstrated efficacy in various in vivo models, including a mouse model of bleomycin-induced pulmonary fibrosis (Barry-Hamilton et al., Nat Med. 2010 Sep;16(9) ): 1009-17). Compound 1 was administered orally prophylactically at a dose of 60 mg/kg/day starting on day -1; rAB0023 was administered intraperitoneally (IP) at a dose of 30 mg/kg on days -4, -1, 1, 4, 8 and 11 (twice a week), with On day 1, the lungs were collected for histological analysis. Compound 1 significantly reduced pulmonary fibrosis in a bleomycin mouse model, as evidenced by a decrease in Ashcroft score from a mean of 3.1 to 0.8 (Figure 5). Antibody rAB0023 showed a trend towards a decrease in Ashcroft score (from 3.1 to 1.7), but these trends were not statistically significant. The results show that compound 1 has better efficacy than rAB0023 in a mouse model of bleomycin-induced pulmonary fibrosis.

In fig. 5 shows the histopathological Ashcroft score reflecting pulmonary fibrosis in a 14-day prophylaxis study comparing 60 mg/kg Compound 1 with 30 mg/kg rAB0023, an anti-LOXL2 antibody.

Example B-13: combination with other antifibrotic agents for pulmonary fibrosis.

LOXL2 inhibitors can be used in combination with other antifibrotic drugs for pulmonary fibrosis. Pirfenidone and nintedanib are currently approved for the treatment of pulmonary fibrosis in patients with IPF. LOXL2 inhibitors were tested alone and in combination with pirfenidone at prophylactic or therapeutic dosages for 14–28 days. LOXL2 inhibitors have also been tested alone and in combination with nintedanib at prophylactic or therapeutic dosages for 14–28 days. Fibrosis was measured using the Ashcroft score or hydroxyproline concentration as described above.

Combination therapy is advantageous when its effectiveness is greater than that of a single agent or when the dose required for either drug is reduced, thereby improving the side effect profile.

Example B-14: Mouse model of renal fibrosis in Alport syndrome.

Mice with mutations in one of the glomerular basement membrane collagen IV genes, collagen

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IV-a3/a4/a5, have defects in glomerular function with the development of kidney fibrosis. These mice develop renal dysfunction and are susceptible to premature death from renal failure, with the exact time point at which this occurs depending on the lineage background in which the mutation is present. Compound 1 was administered orally to Col4A3-deficient mice on a 129/Sv background either prophylactically (about 2-3 weeks old) or therapeutically (about 4-6 weeks old). Mice were either sacrificed at a predetermined time (7–9 weeks) or continuously dosed until they lost >15% of body weight, which preceded death 1–3 days later. If the study was specifically terminated, mice were transcardially perfused with PBS and the renal artery was clamped in one kidney while the other was perfused with Dynabeads microcarriers to magnetically isolate the glomeruli. The other kidney was bisected and a small sample of renal cortex was fixed for transmission electron microscopy (TEM) analysis, and a second sample of renal cortex was used for RNA isolation. The second half of the bisected kidney was embedded in OCT for immunohistochemical analysis. RNA from glomeruli and renal cortex was analyzed by real-time RT-PCR for genes of interest, including MMP-10, MMP-12, IL6, MCP-1, TGF-b1, CTGF, MMP-2, and MMR-9. Immunohistochemical analysis included staining for collagen 1, CD45, fibronectin, smooth muscle actin, WT-1, and integrin alpha 8/laminin α5. Collagen 1 staining was blinded to assess fibrosis, and fibronectin staining was blinded to assess glomerulosclerosis. In all studies, albuminuria was assessed weekly and BUN was assessed at the time of tissue collection.

Compound 1 reduced both glomerular and interstitial fibrosis in Col4A3-deficient mice when administered orally at a dose of 30 mg/kg/day starting at 2 weeks of age (Fig. 6a), i.e. a time when the kidneys are still fairly normal in this model. Fibrosis was assessed at 7 weeks of age using blinded immunohistochemistry for collagen and fibronectin. Compound 1 also reduced fibrosis when dosing was started at 5 weeks of age, which could be considered a therapeutic intervention.

In fig. Figure 6a shows glomerulosclerosis (left) and interstitial fibrosis (right) scores reflecting renal fibrosis in a Col4A3-deficient mouse model of Alport syndrome and chronic kidney disease. Samples were collected at 7 weeks of age following oral administration of Compound 1 at a 30 mg/kg dose starting at 2 or 5 weeks of age (**p < 0.01).

Example B-15: combination with other antifibrotic agents for kidney fibrosis.

LOXL2 inhibitors can be used in combination with other drugs for chronic kidney disease, including Alport syndrome. Angiotensin P-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) are often used in patients with kidney disease. LOXL2 inhibitors were tested alone and in combination with ramipril (ACE inhibitor) or candesartan (ARB) prophylactically starting at 2–3 weeks of age. If efficacy occurs after prophylactic dosing, combination studies will be conducted in a therapeutic dosing regimen (starting at 4–6 weeks of age). Fibrosis was measured by histology as described above, and renal function was measured by proteinuria and/or serum BUN. The effects of combination therapy on survival were also measured as described above. Combination therapy is advantageous when its effectiveness is greater than that of a single agent or when the dose required for either drug is reduced, thereby improving the side effect profile.

Example B-16: Mouse orthotopic model of breast cancer.

Compound 1 was evaluated in an orthotopic mouse model of human breast cancer. Tumor masses were prepared by subcutaneous (sc) injection of MDA-MB-435-GFP cells at a concentration of 5x106 cells/100 μl into the flank of nude mice. When subcutaneous tumors reached 50-100 mm ³ , the tumors were collected and cut into 1 mm ³ fragments. Two tumor fragments were transplanted into the mammary fat pad of each mouse. Compound 1 was administered orally at a dose of 60 mg/kg/day after the average primary tumor volume reached 100 mm ³ . Docetaxel was administered intravenously at a dose of 10 mg/kg once a week for 4 weeks as a positive control. Tumor size was calculated from the results of measuring the minor perpendicular side (W) and the major perpendicular side (L) using the formula (W2xL)XV ₂ , while W and L were measured using a caliper with a digital display. Mice were sacrificed after 4 weeks. Compound 1 attenuated tumor growth with a statistically significant 35% volume reduction after 4 weeks.

In fig. 6b shows tumor volume in an orthotopic human breast cancer model with MDA-MB-435-GFP cells implanted into the mammary fat pads of nude mice. Tumor volumes were measured weekly during the 4-week study (***p < 0.001, ****p < 0.0001).

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Example B-17: Mouse model of skin and lung fibrosis induced by subcutaneous administration of bleomycin.

Skin and lung fibrosis were induced in female C57B1/6 mice by administering bleomycin via subcutaneous injection into one (50-100 μg bleomycin/site) or two sites (50 μg bleomycin/site) on the backs of mice. For the two-site model, animals were anesthetized with isoflurane and injected with bleomycin (100 μl or control PBS) at the same site daily for 28 days to induce skin and lung fibrosis. In the single-site model, mice were restrained and injected into the same site marked with a permanent marker. Mice were pretreated with either the dosage form excipient or test compound (1 day to 1 hour) orally, intravenously or subcutaneously before injection of bleomycin (prophylactic dosing) or 7-14 days after injection of bleomycin (therapeutic dosing). At the end of the study, animals were sacrificed and weighed, and blood (for plasma isolation) and bronchoalveolar lavage fluid were collected and frozen for subsequent analyses. The lungs were either removed, weighed, then homogenized in PBS to determine collagen content using a hydroxyproline assay, or inflated and fixed by instillation of 10% formaldehyde and prepared for histological examination by trichrome or picrosirius red staining. Skin biopsies were taken from each injection site using a 6-mm dermal biopsy (Acuderm). One puncture biopsy specimen was covered with a sponge on both sides, placed in formalin and prepared for histological examination by histological staining with hematoxylin and eosin, trichrome and/or picrosirius red. Another core biopsy was placed in 0.5 ml PBS and minced using fine scissors. Then 500 μl of 12 N was added. HCl and samples were heated at 120°C overnight. After hydrolysis in an acidic medium, 25-100 μl of the supernatant was dried, resuspended in 25 μl of water and the hydroxyproline content was determined by adding 0.5 ml of chloramine T solution (140 mg of chloramine T in 6.5 ml of ddH ₂ O + 1 ml of n-propanol + 2.5 ml of 1 M sodium acetate) and kept at room temperature for 20 min. After incubation, 0.5 ml of Ehrlich solution (1.48 g of 4-(dimethylamino(benzaldehyde) in 7 ml of n-propanol + 2.88 ml of 60% perchloric acid and 0.12 ml of ddH ₂ O) was added and incubated at 65° C for 15 min before absorbance reading at 550 nm.The hydroxyproline concentration in each skin biopsy was determined using a hydroxyproline standard curve (purchased from Sigma).

Example B-18: Rat/Mouse CCl ₄ Model of Liver Fibrosis.

Liver fibrosis in mice (Balb/c or C57B1/6) was induced by intraperitoneal administration of CCl4 (0.5-2 ml/kg body weight) diluted in corn oil twice a week for 4-8 weeks or by oral administration two to three times a week according to a protocol of increasing doses (Popov et al. 2011 Gastroenetrology; 140(5): 1642-1652.). Liver fibrosis in rats was induced either by intraperitoneal administration (1-2.5 ml/kg) or orally in oil (mineral, olive or corn) twice a week for 6-12 weeks. LOXL2 inhibitors were given orally, intraperitoneally, intravenously, or subcutaneously 1 day to 1 hour before the initial dose of CCl4 (prophylactic dosing) or 1 to 4 weeks after the initial dose of _CCl4 (therapeutic dosing). At the end of the study, mice were sacrificed by opening the chest cavity under isoflurane, blood was collected via cardiac puncture into EDTA tubes, and livers were collected. A portion of the liver was fixed in 10% neutral buffered formalin for subsequent histopathological analysis of inflammation and fibrosis by hematoxylin and eosin staining and picrosirius red staining. The remaining tissue was quickly frozen at -80°C for subsequent analysis of total collagen content by hydroxyproline.

Example B-19: Mdr2 knockout mouse model of biliary fibrosis.

The BALB/c.Mdr2-/- mouse model develops liver disease with bridging fibrosis/early cirrhosis between 8 and 12 weeks of age (Ikenaga et al. 2015 Am J Pathology, 185: 325-334). Compound 1 was given orally at 30 and 60 mg/kg/day in BALB/c.Mdr2-/- mice once daily for 6 weeks starting at 6 weeks postnatal. At the end of the study, mice were anesthetized with isoflurane (1.5% v/v) using a precision vaporizer. After laparotomy, portal pressure was measured directly by inserting a high-precision pressure catheter into the portal vein and measuring pressure-related signals for 5 min. Serum was collected for analysis of biochemical parameters in the liver (ALT, AST, ALP and bilirubin) and kidneys (creatinine). A portion of the liver was fixed in 10% neutral buffered formalin for histopathological analysis of inflammation, necrosis and fibrosis by hematoxylin and eosin staining and picrosirius red staining. Collagen area fraction was measured on picrosirius red-stained images using an algorithm that separates red-stained collagen from nonspecific staining by color subtraction and determining threshold values for the processed image. Sections were analyzed in random order using the same threshold value for each set. Data were reconstructed from convolution after analysis to determine group assignment. Collagen content was determined from portions of liver tissue using the hydroxyproline assay.

Compound 1 reduced liver fibrosis as measured by changes in picrosirium staining

- 57 044096 som red, when administered at a dose of 30 or 60 mg/kg, starting from the age of 6 weeks and up to the age of 12 weeks. A similar reduction in liver fibrous area was observed for both dose levels. Treatment with rAB0023, while tending to reduce fibrosis, did not result in a statistically significant improvement. In fig. Figure 7 shows the results of quantification of the area of fibrosis stained with picrosirius red (**p < 0.01 using one-way ANOVA followed by Dunnett's test in comparison with mice that received the vehicle formulation).

Example B-20: Thioacetamide (TAA) mouse model of liver fibrosis.

Liver fibrosis was induced in male C57B1/6 mice by intraperitoneal injection of thioacetamide (TAA) at a dose of 200 mg/kg 3 times a week. Compound 1 was administered orally at a dose of 30 mg/kg QD, with treatment starting 3 or 6 weeks after the start of TAA administration. Liver fibrosis was examined 12 weeks after initiation of TAA. At the end of the study, mice were sacrificed by laparotomy under isoflurane, blood was collected via cardiac puncture into EDTA tubes, and livers were collected. A portion of the liver was fixed in 10% neutral buffered formalin for subsequent histopathological analysis of inflammation and fibrosis by hematoxylin and eosin staining and picrosirius red staining. The remaining tissue was quickly frozen at -80°C for subsequent analysis of total collagen content by hydroxyproline or mRNA analyses. Serum was collected for analysis of liver biochemical parameters (ALT, AST, ALP, and bilirubin) as an indicator of liver function.

Compound 1 reduced liver fibrosis, as measured by changes in picrosirius red staining, when administered at a dose of 30 mg/kg/day starting 3 or 6 weeks after the onset of TAA. The reduction in fibrosis after 12 weeks of treatment with TAA was greater when dosing began at 3 weeks, suggesting that fibrosis may be better treated at an earlier stage. In fig. Figure 8 shows the results of a quantitative assessment of the area of fibrosis stained with picrosirius red. (**, ***p < 0.01, p < 0.001, respectively, using one-way ANOVA followed by Dunnett's test compared with TAA- and vehicle-treated mice).

Example B-21: Diet-induced model of non-alcoholic steatohepatitis (NASH).

The non-alcoholic steatohepatitis (NASH) phenotype was induced by feeding male C57B1/6J mice an AMLN diet (D09100301, Research Diet, USA) (40% fat (18% trans fat), 40% carbohydrate (20% fructose) and 2% cholesterol) for 26 years. 35 weeks before the start of the study and during the study period. Mice underwent liver biopsy under anesthesia, and mice with marked steatosis and fibrosis were recruited for the efficacy study. LOXL2 inhibitors were administered orally, intraperitoneally, intravenously, or subcutaneously at a dose of 30–100 mg/kg/day for 12 weeks. At or before the end of the study, blood was drawn via the tail vein to measure blood ALT, triglycerides, total cholesterol, glucose, and insulin as an indicator of metabolic status. At the end of the study, mice were sacrificed by opening the chest cavity under isoflurane, blood was collected via cardiac puncture into EDTA tubes, and livers were collected. A portion of the liver was fixed in 10% neutral buffered formalin for subsequent histopathological analysis of inflammation and fibrosis by staining with hematoxylin and eosin, trichrome or picrosirius red. The remaining tissue was quickly frozen at -80°C for subsequent analysis of total collagen content by hydroxyproline or analyzes of total cholesterol, liver triglycerides and mRNA.

Example B-22: Mouse model of NASH induced by a choline-deficient diet with defined amino acids (CDAAs) supplemented with high fat.

Liver fibrosis was induced by feeding C57B1/6 mice a high-fat, choline-deficient, L-amino acid-deficient diet (CDAAHFD) containing 60% kcal% fat and 0.1% methionine (Research Diets C/N A06071302), starting at 6 weeks of age. After 4-6 weeks of this diet, mice were screened for mice with impaired liver function and mice with elevated bilirubin levels were excluded. The remaining mice were assigned to groups and dosing was started. LOXL2 inhibitors were administered orally, intraperitoneally, intravenously, or subcutaneously at a dose of 30–100 mg/kg/day for an additional 8–12 weeks. At the end of the study, mice were sacrificed by opening the chest cavity under isoflurane, blood was collected via cardiac puncture into EDTA tubes, and livers were collected. Parts of the liver were fixed in 10% neutral buffered formalin for subsequent histopathological analysis of inflammation and fibrosis by staining with hematoxylin and eosin, trichrome and/or picrosirius red. The remaining tissue was quickly frozen at -80°C for subsequent analysis of total collagen content by hydroxyproline or analyzes of total cholesterol, liver triglycerides and/or mRNA.

Example B-23: Combined Liver Fibrosis and NASH Studies.

LOXL2 inhibitors can be used in combination with other drugs to treat liver fibrosis and NASH. ASK1 inhibitors are currently in clinical trials in multiple fibrotic indications and have demonstrated efficacy in rodent models of liver fibrosis. LOXL2 inhibitors have been tested alone and in combination with ASK1 inhibitors for

- 58 044096 models of liver fibrosis described above, including the TAA and CDAA-HFD models. JNK1 is a profibrotic kinase downstream of ASK1 with inhibitors that exhibit antifibrotic efficacy in rodent models. JNK1 inhibitors are being tested in clinical trials for IPF. LOXL2 inhibitors were tested alone and in combination with JNK1 inhibitors in the liver fibrosis models described above, including the TAA and CDAA-HFD models. Additional combinations included LOXL2 inhibitors with FXR agonists (OCA), PPARa/δ/γ agonists and antagonists (GFT505); dual CCR2/5 antagonist (cenicriviroc); drugs that target galectin-3 (GR-MD-02), ACC inhibitors (NDI-010976). Efficacy was assessed using either a prophylactic dosing regimen or a therapeutic dosing regimen (starting at 4-6 weeks of age). Fibrosis was measured using histology as described above, and liver function was measured using liver enzymes. Combination therapy is advantageous when its effectiveness is greater than that of a single agent or when the dose required for either drug is reduced, thereby improving the side effect profile.

Example B-24: Laser-induced choroidal neovascularization in mouse eyes.

Laser-induced choroidal neovascularization (CNV) is a semi-acute model of fibrosis associated with age-related macular degeneration. C57B1/6 mice were anesthetized with a ketamine/xylazine cocktail and their pupils dilated with tropicamide. Laser burns were induced at multiple locations around the optical disk using a green laser in a slit lamp lasing system. Lubricating eye drops were applied between each spot and animals were monitored for signs of pain and distress. One eye of each animal was subjected to laser treatment, and the opposite eye served as an uninjured control. At the end of the study (14-42 days after laser treatment), mice were subjected to CO ₂ asphyxiation and cervical dislocation. The eyes were removed and fixed overnight in 1% paraformaldehyde or 10% formaldehyde and embedded in paraffin. Histological sections were stained for several parameters, including standard hematoxylin and eosin stains, trichrome or picrosirus red staining, and various immunohistochemical stains: i.e., anti-CD31 in the case of blood vessels, anti-fibrillary acidic protein in the case of glial cells, anti-CD45 in the case of infiltrate inflammatory cells. In some studies, angiogenesis and vascular integrity were assessed using retrobulbar perfusion with FITC-labeled dextran for 2 min; RPE-choroid/sclera complexes were dissected and placed on a glass slide for analysis of fluorescence and vessel area. LOXL2 inhibitors were administered orally, intraperitoneally, intravenously, subcutaneously at a dose of 30-100 mg/kg/day, or administered by intravitreal injection or topical solution to the eye.

Example B-25: Effect of LOXL2 inhibitor on fibroblast-like synoviocyte (FLS) invasion.

The effects of a LOXL2 inhibitory compound were examined in an FLS invasion assay. The FLS invasion assay was a two-chamber model in which cells invaded through Matrigel. The results of this in vitro assay correlate with joint damage in vivo (Tolboom TC, et al. Invasiveness of fibroblast-like synoviocytes is an individual patient characteristic associated with the rate of joint destruction in patients with rheumatoid arthritis. Arthritis Rheum 52: 1999-2002 ( 2005)).

Briefly, FLS cell lines (rodent RA and FLS cell lines) were placed in the upper chamber. Over a period of 24 hours, the cells penetrated the Matrigel layer, then passed through the pores at the base of the upper chamber. The bottom of the upper chamber was stained and cells were counted.

The experiment was repeated, but now the FLS cell lines were pretreated with increasing concentrations of the LOXL2 inhibitory compound. For example, cell lines were pretreated with a LOXL2 inhibitory compound for approximately 1-2 hours to ensure effective binding of the compound and then placed in an upper chamber over a layer of Matrigel.

Example B-26: Collagen-induced arthritis (CIA) model system.

A non-limiting example of the action of LOXL2 inhibitory compounds is described below.

In this example, the effect of LOXL2 inhibitors on the development of collagen-induced arthritis in a mouse model system was assessed. The DBA/1 mouse strain was used because it is highly susceptible to the development of CIA. On day 0 and day 21, all animals received an intradermal tail injection of 200 μg collagen in 0.1 ml type II collagen/complete Freund's adjuvant (CFA) emulsion. The injection site was located on the tail at a distance of approximately 1 cm from the base of the tail.

Male DBA/1 mice, 6-7 weeks old, all within +20% of mean weight, were randomly assigned to one of three treatment groups. Group 1 was the dosage form excipient control group. Animals in this group received 10 ml/kg of formulation vehicle (0.5% methylcellulose), orally (PO), once daily starting on day 16. Animals in group 2, the positive control group, received 0.05 mg/kg dexamethasone at a dose of 10 ml/kg, PO once daily starting on day 16. Animals in group 3 received 0.5-60 mg/kg LOXL2 inhibitory compound once or twice daily starting on day 16. The study was completed on the 35th day and blood was taken from all remaining animals for examination under isofluorane, after which the cervical vertebrae were displaced.

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Mice were examined for signs of arthritogenic reactions in peripheral joints on days 0 and 16, and then daily until the end of the study.

Arthritis reactions were graded for each paw on an increasing scale of severity as follows:

Grade 0: no reaction, normal.

Grade 1: two posterior or anterior joints affected or mild diffuse erythema and swelling.

Grade 2: three posterior or anterior joints affected or moderate diffuse erythema and swelling.

Grade 3: The four posterior or anterior joints are affected or there is marked diffuse erythema and swelling.

Grade 4: The entire paw is affected, with severe diffuse erythema and severe swelling, and inability to flex the toes.

Clinical examinations were performed on day 0, day 16, and daily thereafter. Observations included changes in skin, fur, eyes, mucous membranes, secretions and discharge (eg, diarrhea), and autonomic activity (eg, lacrimation, salivation, piloerection, pupil size, and unusual breathing patterns). Changes in gait, posture, and response to manipulation were also noted, as well as bizarre behavior, tremors, seizures, sleep, and coma.

Animals were weighed shortly before tail injection on day 0, again on day 16, and then three times a week until the end of the study.

As an indicator of experimental arthritis, the thickness of both hind paws was measured on day 0, day 16, and daily thereafter. The left and right paws were measured dorsoventrally just above the toes and below the calcaneus using a caliper (Kroeplin, Munich, Germany).

At the end of the study on day 35, the paws of all remaining animals were removed, skinned, and fixed in 10% pH-neutral buffered formaldehyde. Thin sections were analyzed histologically using hematoxylin and eosin staining for inflammation, pannus formation, cartilage damage, and bone resorption.

Evaluation of the data to determine the significance of any observed effects is based primarily on comparisons of group means for arthritis scores, body weight, and paw thickness measurements (all as described above) using ANOVA followed by Tukey's post hoc analysis (Winsat 2005.1 for Excel ).

Example B-27: Clinical trials in pulmonary fibrosis.

A non-limiting example of a human clinical trial for pulmonary fibrosis is described below.

Objective: The objectives of this study were to evaluate the effectiveness of Compound I or its pharmaceutically acceptable salt or solvate alone or in combination in the treatment of patients with pulmonary fibrosis, collecting information on any side effects that the compound may cause alone or in combination. , and evaluating the pharmacokinetic properties of the compound as a single agent or in combination.

Intervention: Patients were administered 100-2000 mg of Compound I or a pharmaceutically acceptable salt or solvate thereof per day, either alone or in combination.

Detailed Description: Patients were given Compound I or a pharmaceutically acceptable salt or solvate thereof orally once or twice daily, either alone or in combination. Before each dosing cycle, a physical examination, blood work, and assessment of any side effects were performed.

Primary outcome measures: progression-free survival, defined as no deaths or a decrease in FVC from baseline of at least 10%.

Secondary outcome measures: number of acute exacerbations of IPF; health-related quality of life; PO ₂ at rest and during physical activity relative to baseline; Р(Аа)О ₂ at rest and during physical activity relative to the initial level; predicted FEV1 relative to baseline; forced expiratory volume in one second (FEV1) to FVC relative to baseline; plethysmographic lung volumes relative to baseline; diffusivity for carbon monoxide (DLco) relative to the initial level; six-minute walk test relative to baseline: rest and 6-minute SpO2, presence or absence of desaturation to 88% or lower at the end of the six-minute walk, distance walked; Borg dyspnea severity index before and after walking; HRCT grade of pulmonary fibrosis from two independent chest radiologists relative to baseline; number and severity of side effects.

Suitability: Male and female subjects between 40 and 80 years of age.

Inclusion criteria: clinical symptoms of IPF for at least 3 months; forced vital capacity (FVC) from 50 to 90% of the predicted value; DLco at least

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35% of the predicted value; PaO2 > 55 mm Hg. Art. when breathing ambient air at rest; High-resolution computed tomography (HRCT) scan showing definite or probable IPF criteria.

Exclusion criteria: clinically significant exposure to known fibrogenic agents (birds, molds, asbestos, radiation, and drugs known to cause pulmonary fibrosis (amiodarone, nitrofurantoin, bleomycin, etc.)); a history of neurofibromatosis, Hermann-Pudlak syndrome, metabolic disorders, etc.; a history of fever, weight loss, myalgia, arthralgia, skin rash, arthritis; active infection within one week before enrollment; alternative cause of interstitial lung disease; the ratio of forced expiratory volume in one second (VEF1) to FVC is less than 0.6 after using a bronchodilator; residual volume more than 120% of the predicted value (if available); more than 20% lymphocytes or eosinophils in bronchoalveolar lavage (BAL) (if present); granulomas, infection or malignancy in transbronchial or surgical biopsy (if present); previous therapy with azathioprine, prednisolone (>0.5 mg/kg/day or more for at least 3 months), cyclophosphamide, or new biotechnological drugs; unstable cardiovascular or neurological disease; uncontrolled diabetes mellitus; pregnancy; lactation; the probability of death, according to the researcher's forecast, over the next year; the number of leukocytes in the blood <4000/mm ³ ; platelet count <100,000/mm ³ ; hematocrit <30% or >59%; liver enzymes more than 3 times the upper limit of the normal range; creatinine level >1.5 mg/dl; albumin level <3 g/dL; refusal to sign the informed consent of the patient or guardian.

Example B-28: Clinical trials in liver fibrosis.

A non-limiting example of a human clinical trial for liver fibrosis is described below.

Objective: The objectives of this study were to evaluate the effectiveness of Compound I or its pharmaceutically acceptable salt or solvate alone or in combination in the treatment of patients with liver fibrosis, collecting information on any side effects that the compound may cause alone or in combination. , and evaluating the pharmacokinetic properties of the compound as a single agent or in combination.

Intervention: Patients were administered 100-2000 mg of Compound I or a pharmaceutically acceptable salt or solvate thereof per day, either alone or in combination.

Detailed Description: Patients were given Compound I or a pharmaceutically acceptable salt or solvate thereof orally once or twice daily, either alone or in combination. Before each dosing cycle, a physical examination, blood work, and assessment of any side effects were performed.

Primary outcome measures: liver enzymes (ALT, AST, ALP), liver biopsy.

Secondary outcome measures: Pharmacodynamic markers could include: tissue PD markers by mRNA expression, autotaxin, LOXL2, LOX, other LOXL proteins, aSMA, collagen 1A1, NFκB1, caspase 1, SMAD and NOD; Serum and plasma PD markers included: AST to platelet ratio (APRI), autotoxin activity, concentrations of LOXL2, osteopontin, hyaluronic acid, CXCL 9, 10 and 11, MMP1, MMP3, MMP9, TIMP1, CD40L, TGF-p1, ET -1, VEGF, GAL3, IL-6 / IL-8 / TNFa / IFNy, a2-macroglobulin, apolipoprotein A1, PINP, PIIINP, PVCP-1230, PDGF; assessing the effect of chronic dosing on liver structure and fibrotic markers; the incidence of adverse events resulting from multiple doses of a compound.

Suitability: Male and female subjects aged 18 to 60 years.

Inclusion criteria: stage 1-3 fibrosis according to the Metavir scale based on liver biopsy results; body mass index <36 kg/ ^m2 .

Exclusion criteria: any signs of liver decompensation in the past or present; subjects currently abusing amphetamines, cocaine, opiates, or alcohol; clinically significant heart disease; history of cancer other than nonmelanoma skin cancer within 5 years before screening; systemic fungal, bacterial, viral or other infection that cannot be controlled; use of systemic immunosuppressants within 28 days before the pretreatment phase; use of an approved therapeutic agent for hepatitis C virus or hepatitis B virus within 28 days prior to the pretreatment phase; pregnancy or lactation; a history of hemorrhagic diathesis within the last 6 months before the 1st day of the study.

Example B-29: Clinical Trial for Fatty Liver Steatosis (NAFLD, NASH).

A non-limiting example of a human clinical trial for fatty liver/steatosis is described below.

Objective: The objectives of this study were to evaluate the effectiveness of Compound I or its pharmaceutically acceptable salt or solvate as a single agent or in combination in the treatment of patients with fatty liver/steatosis, collecting information on any adverse effects.

- 61 044096 effects that the compound may cause as a single agent or in combination, and evaluation of the pharmacokinetic properties of the compound as a single agent or in combination.

Intervention: Patients were administered 10-2000 mg of Compound I or a pharmaceutically acceptable salt or solvate thereof per day, either alone or in combination.

Detailed Description: Patients were given Compound I or a pharmaceutically acceptable salt or solvate thereof orally once or twice daily, either alone or in combination. Before each dosing cycle, a physical examination, blood work, and assessment of any side effects were performed.

Suitability: Male and female subjects aged 21 to 80 years.

Inclusion criteria: patients with a clinically confirmed diagnosis of non-alcoholic fatty liver or non-alcoholic steatohepatitis; histological evidence of definite or probable non-alcoholic steatohepatitis (NASH) based on a liver biopsy taken no more than 90 days before randomization and a non-alcoholic fatty liver (NASH) activity score of 4 or higher.

Exclusion criteria: current or history of significant alcohol consumption, use of medications historically associated with non-alcoholic fatty liver disease (NAFLD) (amiodarone, methotrexate, systemic glucocorticoids, tetracyclines, tamoxifen, estrogens in doses higher than those used for hormone replacement, anabolic steroids, valproic acid and other known hepatotoxins) for more than 2 weeks in the year prior to randomization, previous or planned (during the study period) bariatric surgery (eg, gastroplasty, gastric bypass with Roux-en-Y gastrojejunostomy), uncontrolled diabetes mellitus, defined as hemoglobin Alc 9.5% or higher within 60 days before study entry, presence of liver cirrhosis on liver biopsy, platelet count below 100,000/mm ³ ; clinical signs of liver decompensation, defined by the presence of any of the following abnormalities: serum albumin less than 3.2 g/deciliter (g/dL), INR (international normalized ratio) more than 1.3, direct bilirubin more than 1.3 mg per deciliter (mg/ dl), a history of esophageal varices, ascites or hepatic encephalopathy; signs of other forms of chronic liver disease: hepatitis B, determined by the presence of hepatitis B surface antigen (HBsAg), hepatitis C, determined by the presence of hepatitis C virus (HCV) ribonucleic acid (RNA), or a positive reaction to hepatitis C antibodies (HCV antibodies) ), evidence of ongoing autoimmune liver disease as determined by comparative liver histology, primary biliary cirrhosis, primary sclerosing cholangitis, Wilson's disease, alpha-1 antitrypsin (A1AT) deficiency, history of hemochromatosis or iron overload, drug-induced liver disease as determined by based on typical presentation and history, known bile duct obstruction, suspected or proven liver cancer, any other type of liver disease other than non-alcoholic steatohepatitis (NASH); serum alanine aminotransferase (ALT) more than 300 units per liter (U/L); serum creatinine 2.0 mg/dL or higher; use of ursodeoxycholic acid (ursodiol, urso) within 90 days before trial entry; inability to safely obtain a liver biopsy, history of bile duct drainage abnormality, known positivity for human immunodeficiency virus (HIV) infection; pregnancy, planned pregnancy, possibility of pregnancy and unwillingness to use effective birth control during the trial, breastfeeding.

Primary outcome measures: liver function tests, liver biopsy, NAS score.

Secondary outcome measures: fibrosis biomarkers, liver imaging (ultrasound, MRI), insulin resistance as measured by HOMA-IR, lipid profile.

Example B-30: Clinical trial for pancreatic cancer.

A non-limiting example of a human clinical trial for pancreatic cancer is described below.

Objective: The objectives of this study were to evaluate the effectiveness of Compound I or its pharmaceutically acceptable salt or solvate alone or in combination in the treatment of patients with pancreatic cancer, collecting information on any side effects that the compound may cause alone or in combination. combination, and evaluating the pharmacokinetic properties of the compound as a single agent or in combination.

Intervention: Patients were administered 100-2000 mg of Compound I or a pharmaceutically acceptable salt or solvate thereof per day, either alone or in combination.

Detailed Description: Patients were given Compound I or a pharmaceutically acceptable salt or solvate thereof orally once or twice daily, either alone or in combination. Before each dosing cycle, a physical examination, blood work, and assessment of any side effects were performed.

Eligibility: Male and female subjects aged 21 to 80 years with advanced pancreatic cancer.

Inclusion criteria: radiographic or clinical evidence of measurable advanced pancreatic carcinoma (stage II, III, IV). Subjects must have measurable disease with a diameter of at least 2 cm.

ECOG performance status score of 0 or 1.

Exclusion criteria: previous history of malignancy (excluding basal cell or squamous cell carcinoma or carcinoma of the breast in situ), unless the subject had been disease free for 1 year or more. Moderate or severe heart disease; active infection; not pregnant or breastfeeding; negative pregnancy test; Fertile patients should use effective contraception during and for >3 months after completion of study treatment; able to swallow oral medications; there must be no other malignant neoplasms in the last 5 years, with the exception of local forms of cancer or basal cell or squamous cell carcinoma of the skin; absence of hypersensitivity or intolerance to statins; there is no other non-malignant systemic disease that would preclude the use of rosuvastatin or long-term follow-up.

Primary outcome measures: progression-free survival, overall survival, worsening pain, new pain.

Secondary outcome measures: tumor size/response (RECIST).

Example B-31: Clinical trial for hepatocellular carcinoma (HCC).

A non-limiting example of a human clinical trial in hepatocellular carcinoma is described below.

Objective: The objectives of this study were to evaluate the effectiveness of Compound I or its pharmaceutically acceptable salt or solvate alone or in combination in the treatment of patients with hepatocellular carcinoma, collecting information on any side effects that the compound may cause alone or in combination. , and evaluating the pharmacokinetic properties of the compound as a single agent or in combination.

Intervention: Patients were administered 100-2000 mg of Compound I or a pharmaceutically acceptable salt or solvate thereof per day, either alone or in combination.

Detailed Description: Patients were given Compound I or a pharmaceutically acceptable salt or solvate thereof orally once or twice daily, either alone or in combination. Before each dosing cycle, a physical examination, blood work, and assessment of any side effects were performed.

Suitability: Male and female subjects aged 21 to 80 years.

Inclusion criteria: patients with a histopathologically or clinically confirmed diagnosis of hepatocellular carcinoma; who do not respond to standard therapy, or for whom standard therapy is intolerable, or for which there is no suitable therapy; ECOG general condition score of 0-2.

Exclusion criteria: patients with a primary malignant tumor; a history of liver transplantation; brain metastases; a mental disorder that may cause difficulties in obtaining informed consent or in conducting the trial; not pregnant or breastfeeding; Fertile patients should use effective contraception during and for >3 months after completion of study treatment; there must be no other malignant neoplasms in the last 5 years, with the exception of local forms of cancer or basal cell or squamous cell carcinoma of the skin; absence of hypersensitivity or intolerance to statins; there is no other non-malignant systemic disease that would preclude the use of rosuvastatin or long-term follow-up.

Primary outcome measures: time to progression, progression-free survival, overall response (RECIST).

Secondary outcome measures: liver function tests, tumor biomarkers.

The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes suggested to those skilled in the art are intended to be within the spirit and scope of the present application and the scope of the appended claims.

Claims (6)

1. Use of a compound for the treatment of a disease or condition in a mammal that will benefit from inhibiting or reducing the activity of LOXL2, wherein the compound is a small molecule inhibitor of LOXL2 that is at least 100 times more selective for inhibiting or binding to LOXL2 than regarding LOX; where the small molecule LOXL2 inhibitor is: R,R-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4hydroxypyrrolidin-1-yl)methanone or a pharmaceutically acceptable salt thereof; and where the disease or condition is primary myelofibrosis or secondary myelofibrosis. 2. Use according to claim 1, where (R,R)-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin- 1-yl)methanone is administered to the mammal as the mesylate salt. 3. Use according to claim 1, wherein the myelofibrosis is primary myelofibrosis. 4. Use according to claim 1, wherein the myelofibrosis is secondary myelofibrosis. 5. Use according to claim 1, where the myelofibrosis is a primary myelofibrosis that occurs after polycythemia vera or after essential thrombocythemia. 6. Use according to any one of claims 1 to 5, wherein the mammal is a human.