Methods of treating pde iv mediated diseases or conditions

Abstract

The present disclosure provides methods and compositions for treating PDE IV-mediated diseases or disorders, including inflammatory diseases or disorders, using compounds of Formula (I), or pharmaceutically acceptable salts thereof:

Description

[0001] Cross-references to related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 076,774, filed September 10, 2020, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This disclosure relates to methods and compositions for treating PDE IV-mediated diseases or conditions. Background Technology

[0004] Phosphodiesterase IV (PDEIV) is a family of cAMP-specific phosphodiesterases composed of four different gene products, AD. See DP Rotella, Phosphodiesterases, Comprehensive Medicinal Chemistry II, 2007, pp. 919-957. PDE4 enzymes are expressed in the CNS and other nervous system tissues, smooth muscle, inflammatory and endothelial cells, and the heart.

[0005] PDEIV is responsible for cAMP catabolism and inflammation regulation in many cell types. Several small-molecule inhibitors of PDEIV have been shown to possess anti-inflammatory properties. See, for example, Sekut, L. et al., 1995, Anti-inflammatory activity of phosphodiesterase (PDE)-IV inhibitors in acute and chronic models of inflammation, Clinical & Experimental Immunology, Vol. 100: pp. 126-132. Inflammatory diseases or disorders, as well as other diseases and disorders with inflammatory components, represent a significant portion of the ailments plaguing modern populations.

[0006] Given the large number of cases where inflammation is an undesirable aspect, there is a need for methods to treat patients with inflammation-related disorders. Summary of the Invention

[0007] This disclosure relates in particular to methods for treating patients with phosphodiesterase IV (PDEIV)-mediated diseases or conditions, the methods comprising administering to said patient an amount of a compound of formula (I) effective in treating the disease or condition:

[0008]

[0009] Or its pharmaceutically acceptable salt.

[0010] In other aspects, the present disclosure relates to methods of inhibiting the release of inflammatory cytokines from a mammalian inflammatory cell by contacting the mammalian inflammatory cell with an amount of a compound of Formula (I):

[0011]

[0012] or a pharmaceutically acceptable salt thereof effective to inhibit PDE IV activity in a mammalian inflammatory cell.

[0013] In other aspects, the present disclosure relates to methods of inhibiting the release of inflammatory cytokines from a mammalian inflammatory cell by contacting the mammalian inflammatory cell with an amount of a compound of Formula (I):

[0014]

[0015] or a pharmaceutically acceptable salt thereof effective to inhibit PDE IV activity in a mammalian inflammatory cell. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Body weight of each group at 6 hours in Example 1 is shown.

[0017] Figure 2 Body weight of each group at 6 hours in Example 1 is shown.

[0018] Figure 3 Body weight of each group at 24 hours in Example 1 is shown.

[0019] Figure 4 Right ear thickness of each group at 6 hours in Example 1 is shown.

[0020] Figure 5 Left ear thickness of each group at 6 hours in Example 1 is shown.

[0021] Figure 6 Right ear thickness of each group at 24 hours in Example 1 is shown.

[0022] Figure 7 Left ear thickness of each group at 24 hours in Example 1 is shown.

[0023] Figure 8 Change in right ear thickness of each group from 0 hours to 6 hours in Example 1 is shown.

[0024] Figure 9 Change in left ear thickness of each group from 0 hours to 6 hours in Example 1 is shown.

[0025] Figure 10 Change in right ear thickness of each group from 0 hours to 24 hours in Example 1 is shown.

[0026] Figure 11Changes in right ear weight at 6 hours for each group in Example 1 are shown.

[0027] Figure 12 Changes in right ear weight at 6 hours for each group in Example 1 are shown.

[0028] Figure 13 Changes in right ear weight at 6 hours for each group in Example 1 are shown.

[0029] Figure 14 Changes in right ear weight at 24 hours for each group in Example 1 are shown.

[0030] Figure 15 Changes in right ear weight at 24 hours for each group in Example 1 are shown.

[0031] Figure 16 Ear tissue MPO at 6 hours for each group in Example 1 is shown.

[0032] Figure 17 Ear tissue MPO at 24 hours for each group in Example 1 is shown.

[0033] Figure 18 Ear tissue total protein at 6 hours for each group in Example 1 is shown.

[0034] Figure 19 Ear tissue total protein at 24 hours for each group in Example 1 is shown.

[0035] Figure 20 G-CSF and Eotaxin in ear tissue homogenate for each group in Example 1 is shown.

[0036] Figure 21 GM-CSF and IL-1 beta in ear tissue homogenate for each group in Example 1 is shown.

[0037] Figure 22 IL-2 and IL-5 in ear tissue homogenate for each group in Example 1 is shown.

[0038] Figure 23 IL-6 and IL-10 in ear tissue homogenate for each group in Example 1 is shown.

[0039] Figure 24 KC (Keratinocyte Chemoattractant-like) and MCP-1 (Monocyte Chemoattractant Protein-1) in ear tissue homogenate for each group in Example 1 is shown.

[0040] Figure 25 MIP-1 beta and TNF-alpha in ear tissue homogenate for each group in Example 1 is shown.

[0041] Figure 26Plasma and ear tissue concentrations of the compound of formula (I) in Example 1 are shown.

[0042] Figure 27 Body weights of the groups in Example 2 are shown.

[0043] Figure 28 Percent change in body weight of the groups in Example 2 is shown.

[0044] Figure 29 Ear thickness of the groups in Example 2 is shown.

[0045] Figure 30 Dorsal skin thickness of the groups in Example 2 is shown.

[0046] Figure 31 Erythema of the groups in Example 2 is shown.

[0047] Figure 32 Ratios of the groups in Example 2 are shown.

[0048] Figure 33 Skin thickness of the groups in Example 2 is shown.

[0049] Figure 34 Cumulative clinical scores of the groups in Example 2 are shown.

[0050] Figure 35 Spleen weights of the groups in Example 2 are shown.

[0051] Figure 36 Normalized spleen weights of the groups in Example 2 are shown.

[0052] Figure 37 MPO of the groups in Example 2 is shown.

[0053] Figure 38 Cytokine levels in ear homogenates of Example 2 are shown.

[0054] Figure 39 Cytokine levels in ear homogenates of Example 2 are shown.

[0055] Figure 40 Cytokine levels in ear homogenates of Example 2 are shown.

[0056] Figure 41 Plasma and ear tissue concentrations of the compound of formula (I) in Example 2 are shown.

[0057] Figure 42 Ear histopathology scores in Example 2 are shown.

[0058] Figure 43 Dorsal skin histopathology scores in Example 2 are shown. DETAILED DESCRIPTION

[0059] The present application can be understood with reference to the following detailed description in conjunction with the drawings in which the same reference notations are used to refer to like elements throughout. The application is not limited to the specific methods, conditions or parameters described and / or shown herein, and the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application as claimed.

[0060] Unless otherwise defined, scientific and non-scientific terms used in connection with the present patent application have the meanings that are commonly understood by one of ordinary skill in the art.

[0061] In some aspects, the present disclosure relates to methods for treating a patient having a phosphodiesterase IV (PDE IV)-mediated disease or disorder, comprising administering to the patient an amount of a compound of Formula (I)

[0062]

[0063] or a pharmaceutically acceptable salt thereof.

[0064] As used herein, the term "treatment" refers to any indicia of success in the treatment or amelioration of a PDE IV-mediated disease or disorder, e.g., an inflammatory disease or disorder, including any objective or subjective parameter such as alleviation; -remission; -diminishment of symptoms or making the disease or disorder more tolerable to the patient; -slowing in rate of degeneration or decline; -lessening of final point of degeneration; -improvement in the patient's physical or mental well-being. Treatment or amelioration can be based on objective or subjective parameters including, but not limited to, physical examination, neurologic examination, and / or psychiatric evaluation. The term "treatment" and variations thereof, include prophylaxis.

[0065] As used herein, the term "patient" or "subject" are used interchangeably and refer to a living organism suffering from or susceptible to a disease or disorder that can be treated by administration of a compound of Formula (I). Non-limiting examples include humans, other mammals, bovine, rats, mice, dogs, monkeys, goats, sheep, cattle, deer, and other non-mammalian animals.

[0066] In some embodiments, the patient is a human.

[0067] As used herein, the phrase "phosphodiesterase IV (PDE IV)-mediated disease or disorder" refers to any disease or disorder characterized by or caused by PDE IV activity. PDE IV-mediated diseases or disorders include, for example, various inflammatory disorders, allergic disorders, immune disorders, CNS disorders, atherosclerosis, and vascular inflammation. These disorders include asthma, chronic obstructive pulmonary disease (COPD) (e.g., chronic bronchitis and / or emphysema), atopic dermatitis, urticaria, allergic rhinitis, allergic conjunctivitis, vernal conjunctivitis, eosinophilic granuloma, psoriasis, rheumatoid arthritis, septic shock, ulcerative colitis, Crohn's disease, reperfusion injury of the myocardium and brain, chronic glomerulonephritis, endotoxic shock, adult respiratory distress syndrome, multiple sclerosis, cognitive impairment (e.g., in neurological disorders), depression, or pain. See, e.g., WO 2004 / 024728 A2. The use of PDE IV inhibitors in the treatment of inflammatory diseases is generally described in, e.g., Press, Neil J. and Katharine H. Banner. "2 PDE4 Inhibitors A Review of the Current Field." Progress in Medicinal Chemistry 2009): 37; Dastidar, Sunanda G., Deepa Rajagopal, and Abijit Ray. "Therapeutic Benefits of PDE4 Inhibitors in Inflammatory Diseases." Current Opinion in Investigational Drugs 8.5 (2007): 364; Schafer, P. H., et al. "Apilimod Is a Selective PDE4 Inhibitor with Modulatory Effects on Innate Immunity." Cell Signaling 26.9 (2014): 2016-2029; Li, Heng, Jianping Zuo, and Wei Tang. "Phosphodiesterase-4 Inhibitors for the Treatment of Inflammatory Diseases." Frontiers in pharmacology 9 (2018): 1048.

[0068] The use of PDE IV inhibitors in the treatment of inflammatory skin diseases is described, for example, in Makins, Caitlyn, Ravina Sanghera, and Parbeer S. Grewal. “Off-Label Treatment Potential of Crisaborole.” Journal of Cutaneous Medicine and Surgery 2020): 1203475420909794; Kitzen, Jan M., et al. “Crisaborole and Amlexanox: PDE4 Inhibitors with Similar Mechanisms of Action for Different Indications in the Management of Inflammatory Skin Disorders” (2018) Dastidar, Sunanda G., Deepa Rajagopal, and Abijit Ray. “Therapeutic Benefits of PDE4 Inhibitors in Inflammatory Diseases.” Current Opinion in Investigational Drugs 8.5 (2007): 364; J. M. Hanifin et al., “Type 4 phosphodiesterase inhibitors having clinical and in vitro antiinflammatory effects in atopic dermatitis,” J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M. Hanifin et al., J. M.wherein 201-209); A. M. Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466-473 (atopic dermatitis; see, for example, p. 470); H. J. Dyke et al. Expert Opinion Invest. Drugs 2002, 11(1), 1-13 (atopic dermatitis; see, for example, page 7 and references. Cited therein as 74, 75 and 76); W. et al., European [J. Pharmacol., 2002, 446, 195-200 and et al., J. Pharmacy Pharmacol., 2003, 55, 1107-1114 (allergic dermatitis).

[0069] The use of PDE IV inhibitors in COPD is described in S. L. Wolda, Emerging Drugs, 2000, 5(3), 309-319; Z. H. Huang et al., Current Opinion in Chemical Biology, 2001, 5:432-438; H. J. Dyke et al., Expert Opinion on Investigational Drugs, January 2002, 11(1), 1-13; C. Burnouf et al., Current Pharmaceutical Design, 2002, 8(14), 1255-1296; A. M. Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466-473; A. M. Vignola, Respiration, 2004, 98, 495-503; D. Spina, Drugs, 2003, 63(23), 2575-2594; and references cited in those publications; G. Krishna et al., Expert Opinion on Investigational Drugs, 2004, 13(3), 255-267 (see especially pages 259-261 and references. 102-111 and 201 therein); C. H. Compton et al., The Lancet, 2001, vol. 358, 265-270), E. Gamble et al., Am. J. Respir. Crit. Care Med., 2003, 168, 976-982); R. D. Border et al., Chest, 2003, vol. 124 Suppl. 4, p. 170S (abstract) and J. D. Eddleston et al., Am. J. Respir. Crit. Care Med., 2001, 163, A277 (abstract); B. J. Lipworth, The Lancet, 2005, 365, 167-175 and references 49-50 therein); S. L. Wolda, Emerging Drugs, 2000, 5(3), 309-319).

[0070] The use of PDE IV inhibitors in asthma is described in M. A. Giembycz, Drugs, Feb 2000, 59(2), 193-212; Z. M. A. Giembycz, 59(2), 59(1), 59(1), 59(1), 59(1), 59(1), 59(1), 59(Huang et al., Current Opinion in Chemical Biology, 2001, 5:432-438; H. J. Dyke et al., Expert Opinion on Investigational Drugs, January 2002, 11(1), 1-13; C. Burnouf et al., Current Pharmaceutical Design, 2002, 8(14), 1255-1296; A. M. Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466-473; P. J. Barnes, Nature Reviews Drug Discovery, October 2004, 831-844; and references cited in these publications).

[0071] The use of PDE IV inhibitors in allergic rhinitis is described in B. M. Schmidt et al. J. Allergy and Clinical Immunology 108(4), 2001, 530-536).

[0072] The use of PDE IV inhibitors in rheumatoid arthritis and multiple sclerosis is described in H. J. Dyke et al., Expert Opinion on Investigational Drugs, January 2002, 11(1), 1-13; C. Burnouf et al., Current Pharmaceutical Design, 2002, 8(14), 1255-1296; and A. M. Doherty, Current Opinion Chem. Biol., 1999, 3(4), 466-473; and references cited in these publications.

[0073] The use of PDE IV inhibitors in the treatment of pain is described in A. Kumar et al. Indian J. Exp. Biol., 2000, 38(1), 26-30).

[0074] The use of PDE IV inhibitors in the treatment of cognitive impairment (e.g. cognitive impairment in neurological disorders such as Alzheimer's disease) is described in H. T. Zhang et al: Psychopharmacology, June 2000, 150(3), 311-316; Neuropsychopharmacology, 2000, 23(2), 198-204; and T. Kawashima et al. Japanese J. Pharmacol., 1997, 75(3), 275-81.

[0075] The use of PDE IV inhibitors as antidepressants is described in J. Zhu et al., CNS Drug Reviews, 2001, 7(4), 387-398; O'Donnell, Expert Opinion on Investigational Drugs, 2000, 9(3), 621-625; H. T. Zhang et al., Neuropsychopharmacology, October 2002, 27(4), 587-595; J. M. O'Donnell and H.-T. Zhang, Trends Pharmacol. Sci, March 2004, 25(3), 158-163; and T. E. Renau, Curr. Opinion Investig. Drugs, 2004, 5(1), 34-39.

[0076] The use of PDE IV inhibitors in the treatment of inflammatory bowel disease (e.g. ulcerative colitis and / or Crohn's disease) has been described in K. H. Banner and M. A. Trevethick Trends Pharmacol. 2004, 25, 8, 430, 436.

[0077] In some embodiments, the phosphodiesterase IV (PDE IV)-mediated disease or disorder is an inflammatory disease or disorder. Such diseases and disorders include inflammatory diseases and disorders, as well as diseases and disorders having an inflammatory component. These disorders are known to those skilled in the art and include rheumatoid arthritis, Crohn's disease, inflammatory bowel disease (IBD), colitis, cachexia, adult respiratory distress syndrome, asthma, hyperoxic alveolar injury, allergic rhinitis, chronic pulmonary inflammatory disease (including COPD), depression, psoriatic arthropathies such as psoriatic arthritis, systemic lupus erythematosus, arthritis and related arthritic conditions (e.g., osteoarthritis and rheumatoid arthritis), rheumatoid spondylitis, inflammatory diseases associated with infection (e.g., HIV, hepatitis, etc.), cystic fibrosis, sepsis and sepsis syndrome, endotoxic shock, septic shock, toxic shock, endotoxic and hemodynamic shock, post ischemic reperfusion injury, meningitis, fibrotic diseases, transplant rejection, osteoporosis, multiple sclerosis, ENL in leprosy, radiation injury, bone resorption diseases, periodontitis, psoriasis, atopic dermatitis, contact dermatitis, Behcet's syndrome, lupus, alopecia, frontal fibrosing alopecia, vitiligo, acne, lichen planus, uveitis, nodular prurigo, and discoid lupus erythematosus.

[0078] In some embodiments, the PDE IV-mediated disease or disorder is an inflammatory skin disease or disorder.

[0079] In some embodiments, the inflammatory skin disease is psoriasis, plaque psoriasis, atopic dermatitis, contact dermatitis, seborrheic dermatitis, stasis dermatitis, scleroderma, Behcet's syndrome, lupus, alopecia, frontal fibrosing alopecia, vitiligo, acne, lichen planus, uveitis, nodular prurigo, or discoid lupus erythematosus.

[0080] In some embodiments, the inflammatory skin disease is psoriasis.

[0081] In some embodiments, the inflammatory skin disease is plaque psoriasis.

[0082] In other embodiments, the inflammatory skin disease is atopic dermatitis.

[0083] In other embodiments, the inflammatory skin disease is contact dermatitis.

[0084] In other embodiments, the inflammatory skin disease is seborrheic dermatitis.

[0085] In other embodiments, the inflammatory skin disease is stasis dermatitis.

[0086] In other embodiments, the inflammatory skin disease is scleroderma.

[0087] In some embodiments, the inflammatory disease or disorder is psoriatic arthritis, plaque psoriasis, atopic dermatitis, or COPD.

[0088] In some aspects, the phosphodiesterase IV (PDE IV)-mediated disease or disorder is an immune disorder.

[0089] In some embodiments, the immune disorder is a chronic inflammatory disorder, asthma, rheumatoid arthritis, or Behcet's syndrome.

[0090] In some aspects, the phosphodiesterase IV (PDE IV)-mediated disease or disorder is a disease mediated by cytokines.

[0091] In some implementations, cytokine-mediated diseases include periodontitis, dry eye disease, rheumatoid arthritis, osteoarthritis, Crohn's disease, ulcerative colitis, psoriatic arthritis, traumatic arthritis, rubella arthritis, inflammatory bowel disease, multiple sclerosis, psoriasis, graft-versus-host disease, systemic lupus erythematosus, toxic shock syndrome, irritable bowel syndrome, muscle degeneration, allogeneic transplant rejection, pancreatitis, islet inflammation, glomerulonephritis, diabetic nephropathy, renal fibrosis, chronic renal failure, gout, leprosy, acute synovitis, Reiter's syndrome, gouty arthritis, Becton's disease, spondylitis, endometriosis, and non-articular conditions (e.g., intervertebral disc syndrome, chondritis, vascular pain, visceral pain, labor pains, or fibromyalgia). Pain syndromes, acute or chronic pain (including but not limited to neuropathic pain, neuropathy, polyneuropathy, diabetes-associated polyneuropathy, trauma, migraine, fibromyalgia, back pain, spinal pain, postoperative pain, sciatica induced by herniated disc, cancer-related pain, vascular pain, visceral pain, childbirth or HIV-related pain, allergies, metabolic diseases, myofibromyalgia), non-articular conditions (e.g., disc syndromes, chondritis, keratoconjunctivitis, neuralgia), type 1 diabetes, type 2 diabetes, liver disease, polyneuropathy, diabetes-associated polyneuropathy, trauma, migraine, fibromyalgia, back pain, spinal pain, postoperative pain, lung compliance, vasculitis, myocardial ischemia, restenosis, thrombosis, angiogenesis, etc. Coronary artery disease, coronary artery disease, acute coronary syndrome, spondyloarthritis, fibromyalgia; diseases or conditions related to blood clotting or fibrinolysis, such as acute Karoch disease. Acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, acute Karoch disease, cluster headache, phantom limb pain, gastrointestinal diseases, Sjögren's syndrome, cerebral malaria, leprosy, endotoxemia, arteritis, aortic stenosis, myocarditis, venous thromboembolism, disseminated intravascular coagulation syndrome, tenosynovitis, high-intensity venous thromboembolism, severe arterial obstruction, venous occlusive disease, leukopenia, blood flow resistance, coronary artery bypass grafting, lying on the operating table, arteriosclerosis, uveitis, glaucoma,neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis and other demyelinating diseases, viral and bacterial meningitis, CNS trauma, spinal cord injury, seizures, convulsions, olivopontocerebellar atrophy, AIDS dementia complex, MERRF and MELAS syndromes, Leber's disease, Wemicke's encephalopathy, Rett's disease, hyperhomocystinuria, hyperprolinemia, hyperhomocysteinuria, nonketotic hyperglycinemia, hydroxybutyric acidaemia, sulfite oxidase deficiency, inborn errors of the urea cycle, plumbism, Tourette's syndrome, hepatic encephalopathy, drug intoxication, drug tolerance, drug dependence, depression, anxiety and schizophrenia, aneurysm, epilepsy, bone resorption diseases, osteopetrosis, osteoporosis or osteoarthritis, diabetes mellitus, cachexia secondary to infection or malignancy, cachexia secondary to acquired immune deficiency syndrome (AIDS), obesity, anorexia or bulimia nervosa, sepsis, HIV, HCV, malaria, infectious arthritis, leishmaniasis, lyme disease, cancer, including but not limited to, breast cancer, colon cancer, lung cancer, prostate cancer, multiple myeloma, acute myelogenous leukemia, serum cancer, non-Hodgkin's lymphoma, Castleman's disease, or drug resistance.

[0092] In some embodiments, the phosphodiesterase IV (PDE IV)-mediated disease or disorder is allergic conjunctivitis.

[0093] In some embodiments, the present disclosure relates to a method of treating a patient having allergic conjunctivitis, the method comprising administering to the patient an amount of a compound of Formula (I), or a pharmaceutically acceptable salt thereof, effective to treat allergic conjunctivitis.

[0094] In some embodiments, the amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is effective to reduce a sign or symptom of allergic conjunctivitis.

[0095] In some embodiments, the sign or symptom of allergic conjunctivitis is ocular itching, conjunctivital redness, ciliary redness, supraciliary redness, conjunctival edema, swelling of the eyelids, lacrimation, rhinorrhea, nasal itching, itching of the ears or palate, or nasal congestion. In other embodiments, the sign or symptom of allergic conjunctivitis is ocular itching. In other embodiments, the sign or symptom of allergic conjunctivitis is conjunctivital redness.

[0096] One of ordinary skill in the art will understand how to design a method for determining whether a reduction (i.e., improvement) in signs or symptoms of allergic conjunctivitis has occurred. Indeed, such methods are known to those of skill in the art and include, for example, objective methods and subjective measures. Signs of allergic conjunctivitis, and their severity, can be assessed by an ophthalmic examination and can include the use of instruments such as a slit lamp microscope. Patient self-assessment scales, such as visual analog scales, numerical scales, and severity indices, can be used for subjective assessment of symptoms and their severity. See, e.g., A. Leonardi Diagnosing Tools for Ocular Allergies Allergy. 2017, 72-1485 (2008) (Phillips et al., Cancer Res., 68:9280-9290, 2008).

[0097] In some embodiments, the reduction in signs or symptoms of allergic conjunctivitis is measured using a numerical scale ranging from, at one extreme, no signs or symptoms to, at the other extreme, severe signs or symptoms. For example, a numerical scale for assessing ocular itch can range from 0 to 5, or 0 to 10, or 0 to 20, where 0 indicates no itch and the highest number indicates severe itch. Similar scales can be used to measure objective signs of allergy, such as conjunctival redness.

[0098] In some aspects, the phosphodiesterase IV (PDE IV)-mediated disease or disorder is a neutrophil-mediated disease.

[0099] In some embodiments, the neutrophil-mediated disease is bronchial asthma, rhinitis, influenza, stroke, myocardial infarction, thermal injury, adult respiratory distress syndrome (ARDS), multiple organ injury secondary to trauma, acute glomerulonephritis, skin diseases with acute inflammatory components, acute purulent meningitis, hemodialysis, leukopheresis, granulocyte transfusion associated syndromes, or necrotizing enterocolitis.

[0100] In some aspects, the phosphodiesterase IV (PDE IV)-mediated disease or disorder is a neurodegenerative disorder.

[0101] In some embodiments, the neurodegenerative disorder is Alzheimer’s disease, Parkinson’s disease, an inflammatory bowel disease selected from Crohn’s disease and ulcerative colitis, diarrhea, a liver disease selected from autoimmune hepatitis, hepatitis C, primary biliary cirrhosis, primary sclerosing cholangitis, or severe liver function failure, a gastrointestinal disease selected from celiac disease and non-specific colitis, a bone disease such as osteoporosis, a pulmonary disease selected from allergic rhinitis, asthma, chronic obstructive pulmonary disease, chronic granulomatous inflammation, cystic fibrosis, and sarcoidosis, a cardiovascular disease selected from atherosclerotic heart disease, congestive heart failure, and restenosis, and a renal disease selected from glomerulonephritis and vasculitis.

[0102] In some aspects, the phosphodiesterase IV (PDE IV) mediated disease or disorder is an acute and chronic (in particular inflammatory and allergen-induced) respiratory disorder of different origin (bronchitis, allergic bronchitis, bronchial asthma); a skin disorder (in particular of the proliferative, inflammatory and allergic type), such as psoriasis (psoriasis vulgaris), toxic and allergic contact eczema, atopic eczema, seborrhoeic eczema, simple lichen planus, sunburn, pruritus in the anogenital region, alopecia areata, hypertrophic scarring, discoid lupus erythematosus, follicular and extensive pyodermias, endogenous and exogenous pain, alcohol acne and other proliferative, inflammatory and allergic skin disorders; - a disorder based on the excessive release of TNF and leukotrienes, such as disorders of the arthritic type (rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and other arthritic disorders), disorders of the immune system (AIDS), shock types [septic shock, endotoxic shock, gram-negative sepsis, toxic shock syndrome and adult respiratory distress syndrome (ARDS) and systemic inflammation in the gastrointestinal region (Crohn's disease and ulcerative colitis); - disorders based on allergic and / or chronic immune pseudo-reactions in the upper airway region (pharynx, nose) and adjacent regions (paranasal sinuses, eyes), such as allergic rhinitis / sinusitis, chronic rhinitis / sinusitis, allergic conjunctivitis and nasal polyps; and cardiac disorders which can be treated with PDE inhibitors, such as cardiac insufficiency, or disorders which can be treated due to the tissue relaxing action of PDE inhibitors, such as renal and ureteral colic associated with kidney stones.

[0103] The method of the present application comprises administering a compound of formula (I) or a pharmaceutically acceptable salt thereof. The compound of formula (I) is 2 - methyl-N 4 -phenyl-6-(2233-tetrafluoropropoxy)-135-triazine-2,4-diamine has been described, for example, in WO2017112951. See also S. Lee et al., J. Med. Chem. 2017, 60 3, (1210).

[0104] In some embodiments of the method of the present application, a compound of formula (I) is administered.

[0105] In other embodiments of the methods of the application, pharmaceutically acceptable salts of the compounds of Formula (I) are administered. The term "pharmaceutically acceptable salt" means an acid addition salt of a compound of Formula (I) prepared with relatively non-toxic acids. The acid addition salts can be prepared by contacting the neutral form of a compound of Formula (I) with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids such as hydrochloric, hydrobromic, nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids, and the like, as well as salts derived from relatively nontoxic organic acids such as acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, oxalic, methanesulfonic, etc. Salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like, also see, e.g., Berge et al, "Pharmaceutical Salts", Journal of Pharmaceutical Science, 1977, 66, 1-19. Accordingly, the compounds of Formula (I) can exist as salts, including hydrochloride, hydrobromide, sulfate, methanesulfonate, nitrate, maleate, acetate, citrate, fumarate, tartrate (e.g., (+)-tartrate, (-)-tartrate, or mixtures thereof, including racemic mixtures), succinate, benzoate, and salts with amino acids such as glutamic acid. These salts can be prepared by methods known to those skilled in the art.

[0106] In some embodiments, the compounds of Formula (I) can also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. For example, the compounds of Formula (I) can be radiolabeled with isotopes such as deuterium ( 2 H), tritium ( 3 H), fluorine- 18 ( 18 F), iodine- 125 ( 125 I), or carbon- 14 ( 14 C). All isotopic variations of the compounds of Formula (I), whether radioactive or not, are encompassed within the scope of the present application.

[0107] In the methods of the present disclosure, the amount of the compound of Formula (I), or a pharmaceutically acceptable salt thereof, is effective to treat a PDE IV-mediated disease or disorder.

[0108] As used herein, the phrase "therapeutically effective amount" refers to an amount that is sufficient to achieve the stated purpose (e.g., to achieve the effect for which it is administered, to treat a disease, to reduce one or more symptoms of a disease or condition). An example of a "therapeutically effective amount" is an amount that is sufficient to help treat, prevent, or reduce a sign or symptom of a disease or condition. "Reduction" of a sign or symptom (and grammatical equivalents of that phrase) refers to a decrease in severity, or frequency of the sign or symptom, or elimination of the sign or symptom. Such reduction can be determined using the numerical scales of signs and / or symptoms outlined above.

[0109] In some embodiments, a "therapeutically effective amount" is an amount that has a prophylactic effect, e.g., prevents or delays the onset (or recurrence) of a disease or condition, or reduces the likelihood of the onset (or recurrence) of a disease or condition or symptoms thereof.

[0110] Administration of a "therapeutically effective amount" can include administration of the amount in a single dose or multiple doses. The exact amount depends on the purpose of the treatment and can be determined by one of skill in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms, Vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); Remington: The Science and Practice of Pharmacy, 20th Ed., 2003, Gennaro, Ed., Lippincott, Williams Wilkins).

[0111] A "therapeutically effective amount" can be estimated from tests and animal models, e.g., those described herein. Such information can be used by one of skill in the art to determine a dose that is useful in humans.

[0112] For systemic administration, the dosage and interval can be adjusted individually to provide plasma levels of the active moiety that are sufficient to achieve the desired effects. In some embodiments, the dose for systemic administration ranges from about 0.1 mg / day to about 1000 mg / day, e.g., 1-500 mg / day, 10-200 mg / day, or 100-200 mg / day.

[0113] In embodiments where a compound of Formula (I) is administered topically, a "therapeutically effective amount" can be from about 0.01 mg / cm 2 to about 10 mg / cm 2 , e.g., about 0.1 mg / cm 2 to about 10 mg / cm 2 , or about 0.1 mg / cm 2 to about 2 mg / cm 2 .

[0114] The dosage and frequency (single or multiple doses) of administration of the compounds of the present disclosure to a mammal will vary according to a variety of factors, including, for example, whether the mammal is suffering from another disease and its route of administration; the size, age, sex, health, body weight, body mass index, and diet of the recipient; the nature and extent of the disease treated, the kind of concurrent treatment, the condition of the disease being treated, or other health-related problems. Adjustments and manipulations of dosages (e.g., frequency and duration) are within the ability of those skilled in the art.

[0115] In some aspects, the present disclosure relates to a method of inhibiting the release of an inflammatory cytokine from a mammalian inflammatory cell by contacting the mammalian inflammatory cell with an amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof effective to inhibit the release of the inflammatory cytokine from the mammalian inflammatory cell.

[0116] In some embodiments of these methods, the inflammatory cytokine is tumor necrosis factor alpha (TNFa); an interleukin (IL), including IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, and IL-12; an interferon, including alpha-interferon, beta-interferon, or gamma-interferon.

[0117] As used herein, the term "inhibiting release" means preventing or reducing the amount of an inflammatory cytokine released by an inflammatory cell relative to the amount released by the cell in the absence of a compound of Formula (I). Methods of measuring cytokine levels are known to those skilled in the art.

[0118] As used herein, the term "contacting" means bringing a compound of Formula (I) into proximity with a mammalian inflammatory cell such that they are able to make physical contact.

[0119] In some aspects, the present disclosure relates to a method of inhibiting PDE IV activity in a mammalian inflammatory cell, the method comprising contacting the mammalian inflammatory cell with an amount of a compound of Formula (I) or a pharmaceutically acceptable salt thereof effective to inhibit the PDE IV activity.

[0120] As used herein, the term "inhibiting PDE IV activity" means preventing or reducing the enzymatic activity of a PDE IV enzyme relative to the PDE IV enzyme activity in the absence of a compound of Formula (I). Methods of measuring PDE IV activity are known to those skilled in the art.

[0121] In the methods of the present disclosure, the compound of Formula (I) or a pharmaceutically acceptable salt thereof can be administered in a pharmaceutical composition comprising the compound of Formula (I) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0122] A "pharmaceutically acceptable excipient" refers to a substance that aids administration of an active agent to and absorption by a subject and that can be included in the compositions of the present application without significant adverse toxicological effects on the patient. Non-limiting examples of pharmaceutically acceptable excipients include water, NaCl, saline solution, lactated Ringer's, sucrose, glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (e.g., Ringer's solution), alcohols, oils, gelatins, carbohydrates such as lactose, straight or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and coloring agents, and the like. Such formulations can be sterilized and, if desired, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, coloring agents, and / or aromatic substances and the like that do not deleteriously react with the compounds of the present application. Those of skill in the art will recognize other pharmaceutical excipients that can be used in the present application.

[0123] Pharmaceutical compositions suitable for use in the methods of the present application include any dosage form suitable for administration to a patient. Such dosage forms include, but are not limited to, tablets, powders, capsules, pills, cachets, troches, applicator-mouthing sticks, solutions, suspensions, emulsions, gels, creams, ointments, pastes, jellies, paints, powders, aerosols, dragees, liquids, syrups, slurries, suspensions, suppositories, microspheres, liposomes, and dispersable granules.

[0124] As used herein, the term "administering" means applying a compound of Formula (I) or a pharmaceutically acceptable salt thereof (including as a pharmaceutical composition) to the body of a patient in a manner that results in the presence of the compound of Formula (I) at the site of inflammation. Administration includes oral administration, administration as a suppository, topical contact, transdermal, intravenous, parenteral, intraperitoneal, intramuscular, intralesional, intrathecal, intranasal, or subcutaneous administration, implanting a slow-release device, e.g., a microosmotic pump, into a subject, transmucosal (e.g., buccal, sublingual, palatine, gingival, nasal, vaginal, rectal, or transdermal), parenteral (e.g., intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraventricular, and intracranial). Other modes of delivery include, but are not limited to, the use of liposomal formulations, intravenous infusion, transdermal patches, and the like.

[0125] In some embodiments of the disclosed methods, a compound of Formula (I) or a pharmaceutically acceptable salt thereof is administered topically, orally, perorally, as a suppository, intravenously, parenterally, intraperitoneally, intramuscularly, intralesionally, intrathecally, intranasally, or subcutaneously.

[0126] In other embodiments of the disclosed methods, a compound of Formula (I) or a pharmaceutically acceptable salt thereof is administered topically.

[0127] In some embodiments of the disclosed methods, a compound of Formula (I) or a pharmaceutically acceptable salt thereof is administered orally.

[0128] In the methods of the present disclosure, the compound of Formula (I), or a pharmaceutically acceptable salt thereof, can be administered in combination with other active pharmaceuticals known to be useful in the treatment of a given disease or condition, or in combination with ancillary agents that can not be effective by themselves but can contribute to the efficacy of the compound of Formula (I). In such embodiments, the compound of Formula (I) is administered simultaneously, before, or after administration of one or more additional agents.

[0129] The following examples further illustrate aspects of the methods of the present disclosure and are not intended to be limiting.

[0130] Example

[0131] Example 1 :

[0132] Animals

[0133] The animals were housed individually in IVC cages and autoclaved corn cob was used as bedding material. The animals were maintained in a controlled environment with temperature of 22 ± 3 °C, humidity of 50 ± 20 %, light / dark cycle of 12 hours each and 15-20 fresh air changes per hour. The animals were fed with certified irradiated laboratory rodent feed Nutrilab brand, Tetragon Chemie Pvt. Ltd., Bangaalore.

[0134] Acclimation

[0135] The animals were kept under climatization for a period of about 5-7 days before starting the study. On the day of the study, the animals were randomized based on body weight.

[0136] Randomization

[0137] The inter-group randomization was done based on body weight so that the animal variation was less than 10 %. Immediately after randomization, the animals were assigned a permanent number through ear notching. The cages were identified by cage cards indicating study code, group number, gender, dose, cage number, animal number and animal number details.

[0138] Test System

[0139] Species / Strain BALB / c Gender Male Age at start of experiment 7-8 weeks Weight range ～19-23g Source Taconic or other vendor Total number of animals 67 Temperature (23)±(2)℃ Humidity Produce: 60 ± 20% Food Ad libitum Water Ad libitum Light cycle Normal 7 AM - 7 PM light cycle

[0140] Test article details

[0141] 1. Dexamethasone Catalogue Number or Batch Number: BCBM4557V (Sigma-Aldrich)

[0142] Vehicle for Dexamethasone (Veh. 1): Acetone: Ethanol (1 : 1), 20 μL / ear

[0143] Dose: 0.1 mg / ear

[0144] Dose volume: 20 μL / ear

[0145] 2. Crisaborole PZ0037 (Sigma)

[0146] Vehicle (Veh. 1): Acetone: Ethanol (1 : 1), 20 μL / ear

[0147] Dose: 0.1 mg / ear

[0148] Dose volume: 20 μL / ear

[0149] 3. Compound of formula (I)

[0150] Vehicle (Veh. 1): Acetone: Ethanol (1 : 1), 20 μL / ear

[0151] Dose: 0.1 mg / ear

[0152] Dose volume: 20 μL / ear

[0153] Study groups

[0154] Dexamethasone and test article preparation and administration:

[0155] Concentration of working solution = 5 mg / ml

[0156] Group 1 was topically administered with vehicle alone (ethanol:acetone = 1 : 1) on day 0.

[0157] Animals in groups 2, 3 and 4 received dexamethasone, Crisaborole and compound of formula (I) respectively (as a solution in 1 : 1 ratio of acetone and ethanol) on both ears 30 minutes prior to PMA challenge.

[0158] Study procedure

[0159] Timeline :

[0160] -24 hr Randomization (based on body weight)

[0161] -30 min Treatment topically

[0162] 0 min - Disease induction PMA application

[0163] 15 min Treatment topically

[0164] 6 hr Skin thickness, skin collection (Group 1)

[0165] 24 hr Skin thickness, skin collection (Group 2)

[0166]

[0167] Disease induction :

[0168] 5 μg / 20 μl / right ear and 5 μg / 20 μl / left ear (2.5 μg / 10 μl to the anterior and posterior surfaces of both ears)

[0169] Phorbol 12-myristate 13-acetate (PMA) was prepared in acetone at a concentration of 250 μg / ml. This working solution was applied topically to the anterior and posterior surfaces of both ears (10 μl per side).

[0170] Observation results :

[0171] Ear thickness (right and left ear): at -1 hour (prior to PMA challenge), 6 hours and 24 hours post PMA challenge.

[0172] Ear collection :

[0173] At 6 hours and 24 hours, the animals of each group were euthanized and the ear tissue was collected. The right ear was collected, snap-frozen in liquid nitrogen, and stored at -80°C until homogenized for cytokine estimation (3 sham control animals were used for comparison). The left ear was collected and stored in 10% NBF for histopathology. Half of the left ear was used for drug concentration measurement and the other half of the left ear was used for histopathology.

[0174] Blood collection :

[0175] Blood was collected into K2-EDTA tubes by retro-orbital bleeds prior to euthanasia; plasma was isolated and stored at -80°C for further analysis (for test article concentration).

[0176] Results

[0177] As shown in Figure 4 , Figure 5 , Figure 8 , Figure 9 , Figure 12 and Figure 13 , the compounds of Formula (I) prevent or reduce PMA-induced ear thickening and ear weight gain (i.e., a marker of hyperplasia) at 6 hours to an extent comparable to that of dexamethasone and crisaborole.

[0178] Example 2 :

[0179] Housing and feeding of animals

[0180] The animals were housed individually in IVC cages and autoclaved corn cob was used as bedding material. The animals were maintained in a controlled environment with temperature of 22 ± 3 °C, humidity of 50 ± 20 %, light / dark cycle of 12 hours each and fresh air changed 15-20 times per hour. The animals were fed ad libitum with certified irradiated Laboratory Rodent Diet (Nutrilab brand, Tetragon Chemie Pvt. Ltd., Bangaalore).

[0181] Acclimation

[0182] On receipt, the animals were assigned a temporary number at the base of the tail using non-erasable marker pen. All animals were acclimatized for about 5-7 days before the start of the study. On the day of the study, the animals were randomized based on body weight.

[0183] Randomization

[0184] The groups were randomized based on body weight so that the animal variation was less than 10 %. Immediately after randomization, the animals were assigned a permanent number through ear punch. The cages were identified by cage cards indicating the study code, group number, sex, dose, cage number, animal number and animal number details.

[0185] Test System

[0186]

[0187]

[0188] Test article details

[0189] 1. Dexamethasone Catalogue Number or Batch Number: BCBM4557V (Sigma-Aldrich)

[0190] Vehicle for Dexamethasone (Veh. 1): Acetone: Ethanol (1 : 1)

[0191] Dose: 0.1 mg / ear and 0.25 mg / back skin

[0192] Dose Volume: 20 μΐ / ear and 50 μΐ back skin

[0193] 2. Crisaborole PZ0037 (Sigma-Aldrich)

[0194] Vehicle (Veh. 1): Acetone: Ethanol (1 : 1)

[0195] ​​​Dose: 0.1 mg / ear and 0.25 mg / back skin

[0196] Dose volume: 20 μL / ear, 50 μL back skin

[0197] 3. Compound of formula (I)

[0198] Vehicle (Veh. 1): Acetone: Ethanol (1 : 1)

[0199] Dose: 0.1 mg / ear and 0.25 mg / back skin

[0200] Dose volume: 20 μL / ear, 50 μL back skin

[0201] Dexamethasone and test article preparation and administration:

[0202] Strength = 5 mg / ml

[0203] Administration = 100 μg / 20 μl per ear + 250 μg / 50 μl back G2 to G4 received Dexamethasone, Crisaborole or test article: Daily topical treatment (D0-D6): 0.1 mg in 20 μl per ear x two ears + 0.25 mg in 50 μl (back) 6-8 hours after IMQ administration.

[0204] Study procedure

[0205] Timeline :

[0206] Disease induction: 5% IMQ cream 40 mg (back) and 5 mg (inner surface of each ear) administered daily from Day 0-6.

[0207] Treatment: 0.5% Dexamethasone solution: 0.25 mg (back) and 0.1 mg (per ear) 8 hours after IMQ.

[0208] Day 2 - Hair loss on back area

[0209] Day 0 - Observations (body weight; ear thickness using digital micrometer)

[0210] Day 2 - Observations (body weight; ear thickness using digital micrometer)

[0211] Day 4 - Observations (body weight; ear thickness using digital micrometer)

[0212] Day 6 - Observations (body weight; ear thickness using digital micrometer); Blood collection (PK) and termination (2 hours after last dose) • Ear collection: PK, cytokines, MPO, thickness • Back skin collection, scoring and thickness measurement • Histopathology (optional)

[0213] Study groups

[0214]

[0215] Animals in groups 2-4: as described in the table above.

[0216] Animals in group 1 received the vehicle (acetone and ethanol 1 :1 ) topically once a day.

[0217] Disease induction :

[0218] Disease induction: Imiquimod 5% cream

[0219] Animals were shaved on the dorsal back area (2 cm x 2 cm) using a hair clipper. Imiquimod 5% cream: 50 mg / animal (40 mg on the shaved back + 5 mg each in the inner part of the right and left ear) was applied QD from day 0 to 5 (groups 1-4).

[0220] Vaseline: 50 mg / animal (40 mg on the shaved back + 5 mg each in the inner part of the right and left ear) was applied QD from day 0 to 5 to sham control mice. They did not receive any additional treatment (group 5).

[0221] Observation :

[0222] Body weight of all animals was recorded at the time of inclusion, on the day of randomization, before treatment and once a day for two days after treatment. Mortality of animals was observed at regular intervals.

[0223] Blood collection :

[0224] On day 6, blood was collected by retro-orbital bleeds under isoflurane anesthesia before euthanasia. Blood was collected in K2-EDTA tubes, plasma was isolated and stored at -80°C for further analysis (for test item concentration).

[0225] Ear collection :

[0226] On day 6, 2 hours after compound treatment, all animals were euthanized and ear tissue was collected. The right ear was snap-frozen in liquid nitrogen and stored at -80°C until homogenized for MPO and cytokine estimation.

[0227] Half of the left ear was used for drug concentration determination. The other half of the left ear tissue was used for histopathology. Back skin was collected for visual assessment (scoring) and histopathology.

[0228] MPO analysis: Rapidly frozen ear tissue samples were homogenized in IX assay buffer provided with the kit. The homogenate was centrifuged in a refrigerated centrifuge at 8000 g for 10 minutes. The supernatant was discarded and the pellet was resuspended in lysis buffer (IX assay buffer containing 0.5% HTaBr). The sample was homogenized and subjected to three rounds of freeze-thawing followed by brief sonication for 10 seconds. The sample was centrifuged in a refrigerated centrifuge at 12000 g for 10 minutes and the resulting supernatant was analyzed for MPO levels based on a fluorescent detection kit. The kit utilizes a non-fluorescent detection reagent which is oxidized in the presence of hydrogen peroxide and MPO to produce its fluorescent analog. (Cayman Chemical; ADI-907-029.

[0229] Histopathology: At the end of the study, dorsal back skin and ear skin samples were collected. Skin samples were processed using routine histopathology methods and 4-5 μιη paraffin sections were prepared. Tissue sections (3 consecutive sections / animal) were stained with hematoxylin and eosin (H&E) to evaluate acanthosis, hyperkeratosis / parakeratosis and inflammatory cell infiltration in the dermis. The severity of the lesions was semi-quantitatively assessed on a scale of 0-4 by two independent pathologists in a blinded fashion. The percentage of the area involved in the lesion was taken into account when evaluating the score. The scores for each group (for each parameter) were averaged. The cumulative lesion score was calculated by adding the scores for acanthosis, hyperkeratosis, parakeratosis and cellular infiltration (thus maximum score = 16). All treatment groups were compared to the pathology control group.

[0230] Results

[0231] See van der Fits et al. Imiquimod-induced psoriasiform skin inflammation in mice is mediated via the IL-23 / IL-17 axis. The Journal of Immunology, 2009, 182: 5836-5845.

[0232] Sham control animals exhibited a significant increase in body weight compared to IMQ control animals. Animals treated with 0.5% dexamethasone showed a significant decrease in body weight when compared to IMQ control animals. Animals treated topically with the compound of formula (I) showed significantly less decrease in body weight (0.5%) when compared to IMQ control animals. See Figure 27 and 28 .

[0233] IMQ administration increased ear thickness in the pathological (IMQ control) animals. Dexamethasone (0.5%) administration resulted in a significant decrease in ear thickness on day 4 and day 6, respectively (p < 0.001). Topical treatment with Crisaborole (0.5%) and the compound of formula (I) (0.5%) showed a significant decrease in ear thickness on day 6. See Figure 29 .

[0234] To score the severity of the back skin inflammation, an objective scoring system was developed. The erythema, scale formation and skin thickness formation were scored independently on a scale of 0 to 4 in all animals. The cumulative clinical score was calculated by adding the scores of erythema, skin thickness and scale formation. Thus, the maximum score per animal was 12. The scoring system was based on the clinical Psoriasis Area and Severity Index (PASI) as follows: (van der Fits et al; J Immunol 2009) (0 - normal 1 - mild 2 - moderate 3 - marked 4 - very marked). Signs of erythema, skin thickness and scale formation were observed on the back skin of the animals 2-3 days after imiquimod administration. All individual scores and cumulative clinical scores of the IMQ control animals were significantly increased compared to the sham control animals.

[0235] The IMQ control group showed a significant increase in the cumulative clinical score including erythema, skin thickness and scale. Dexamethasone treatment showed a significant decrease in the degree of erythema, skin thickness and scale and the overall cumulative clinical score. Animals treated with Crisaborole (0.5%) showed a significant decrease in the cumulative clinical score on day 6 (p < 0.05) when compared to the IMQ control animals. Treatment with the compound of formula (I) did not show any significant decrease in the overall cumulative score. See Figure 34 .

[0236] Splenomegaly was observed in this model due to the differentiation of Th17 cells in the spleen. The IMQ control animals showed a significant increase in spleen weight. Dexamethasone treated animals exhibited a significant decrease in spleen weight (p < 0.0001). Animals treated with Crisaborole and the compound of formula (I) did not show a significant decrease in spleen cell weight or normalized spleen cell weight when compared to the IMQ control animals. See Figure 35 and 36 .

[0237] On day 6, at termination, the back skin thickness of each animal was measured. A significant increase in back skin thickness was observed in the IMQ control animals after imiquimod administration compared to the sham control. Dexamethasone treated animals exhibited a significant decrease in back skin thickness (p < 0.0001). Animals treated with Crisaborole and the compound of formula (I) did not exhibit a significant decrease in back skin thickness. See Figure 30 .

[0238] On day 6, plasma and ear skin concentrations were estimated for animals treated with the compound of formula (I) after 2 hours of treatment. Significant concentrations of the test compound, compound of formula (I), were observed in plasma and ear skin. Concentrations in ear skin were nearly 976-fold higher than plasma levels. See Table 6. Figure 41 .

[0239] Myeloperoxidase (MPO) is an oxidative enzyme present in neutrophils. During inflammation, there is infiltration of neutrophils and other immune mediators in the ear tissue. An increase in MPO activity was observed in ear tissue homogenates of the IMQ control group. Dexamethasone-treated animals exhibited a significant inhibition of MPO activity (p 0.001). Animals treated with Crisaborole and the compound of formula (I) did not show a significant inhibition of MPO activity. See Table 7. Figure 37 .

[0240] Histopathology (HP) scores for each grading feature were given taking into account the percentage of involvement of each grade. Grading criteria for skin and ear are shown below.

[0241] Histological grading criteria for IMQ-induced psoriasis Skin

[0242]

[0243] The percentage of involvement score for each grading feature was as follows:

[0244] 1 1-25％ 2 26-50％ 3 51-75％ 4 76-100％

[0245] Histological grading criteria for IMQ-induced psoriasis Ear

[0246]

[0247] The percentage of involvement score for each grading feature was as follows:

[0248] 1 1-25％ 2 26-50％ 3 51-75％ 4 76-100％

[0249] Dorsal and ear skin sections from IMQ control group showed acanthosis, hyperkeratosis, parakeratosis and inflammatory cell infiltration in the dermis and epidermis. Dexamethasone treatment resulted in a significant reduction in the degree of acanthosis, infiltration and total cumulative HP score of ear and dorsal skin. Animals treated with Crisaborole (0.5%) showed a significant reduction in total cumulative HP score of ear skin. Animals treated with the compound of formula (I) (0.5%) exhibited a significant reduction in the degree of acanthosis, cell infiltration in the ear and total cumulative HP score of ear skin. Treatment also showed a reduction in the degree of acanthosis of dorsal skin. A significant reduction in the cumulative HP score of dorsal skin was observed. In summary, animals treated with the compound of formula (I) (0.5%) exhibited a significant reduction in ear thickness and exhibited a significant reduction in ear and dorsal skin histopathology scores. See Figure 42 and Figure 43 .

[0250] When compared to normal control animals, a significant increase in cytokine levels was observed in ear tissue homogenates following IMQ administration in IMQ control animals. A significant increase in various pro-inflammatory cytokines was observed in IMQ control animals when compared to sham control animals. Local treatment of animals with dexamethasone showed a significant reduction in most cytokines elevated due to IMQ administration in the ear when compared to IMQ control animals. Local treatment with Crisaborole and the compound of formula (I) showed a significant reduction in several pro-inflammatory cytokines. See Figure 38 to 40 .

[0251] Example 3 :

[0252] The activity of the compound of formula (I) in phosphodiesterase IV subtypes was evaluated.

[0253] The methodology employed in this study was adapted from the scientific literature to maximize reliability and reproducibility.

[0254] Reference standards were run as part of each assay to ensure the validity of the results obtained. Reference compounds:

[0255] Phosphodiesterase PDE10A2 Papaverine

[0256] Phosphodiesterase PDE11A4 Dipyridamole

[0257] Phosphodiesterase PDE1A MMPX

[0258] Phosphodiesterase PDE2A EHNA

[0259] Phosphodiesterase PDE3A Cilostamide

[0260] Phosphodiesterase PDE4A1A Rolipram

[0261] Phosphodiesterase PDE4B1 Rolipram

[0262] Phosphodiesterase PDE5A Zardaverine

[0263] Phosphodiesterase PDE6 Zardaverine

[0264] Phosphodiesterase PDE7A IBMX

[0265] Phosphodiesterase PDE7B IBMX

[0266] Phosphodiesterase PDE8A1 Dipyridamole

[0267] Phosphodiesterase PDE9A2 Zardaverine

[0268] PDE4 assay was performed using a method adapted from the literature. See Richter W et al. (2001) Identification of the inhibitor binding site of cAMP-specific phosphodiesterase 4. Cell Signal. 13(4):287-297; Wang H et al. (2005) Multiple elements jointly determine inhibitor selectivity of cyclic nucleotide phosphodiesterases 4 and 7. J Biol Chem. 280 35 30949 30955

[0269] Phosphodiesterase PDE4A1A; Phosphodiesterase PDE4B1; Phosphodiesterase PDE10A2; Phosphodiesterase PDE11A4; Phosphodiesterase PDE2A; Phosphodiesterase PDE3A; Phosphodiesterase PDE7A; Phosphodiesterase PDE7B; and Phosphodiesterase PDE8A1:

[0270] Source: Human recombinant insect Sf9 cells

[0271] Substrate: 0.10 μΜ FAM-cAMP

[0272] Vehicle: 1.00% DMSO

[0273] Significance Analysis: ≥ 50% of maximum stimulation or inhibition

[0274] Incubation Buffer: 10 mM Tris-HCl, pH 7.2, 10 mM MgCl2

[0275] 0.05% NaN3, 0.1% non-phosphated BSA

[0276] Quantitative Method: Quantification of fluorescein-AMP-IMAP

[0277] Pre-Incub. Time / Temperature: 15 min @ 25°C

[0278] Incubation time / temperature: 30 min @ 25°C

[0279] Phosphodiesterase PDE1A; Phosphodiesterase PDE5A; Phosphodiesterase PDE9A2:

[0280] Source: Human recombinant insect Sf9 cells

[0281] Substrate: 0.10 μM FAM-cAMP

[0282] Carrier 1.00% DMSO

[0283] Significance analysis: ≥ 50% of maximum stimulation or inhibition

[0284] Incubation buffer: 10 mM Tris-HCl, pH 7.2, 10 mM MgCl2

[0285] 0.05% NaN3, 0.1% BSA without phosphate

[0286] Quantification method: Quantification of Fluorine-GMP-IMAP

[0287] Pre-Incub. time / temperature: 15 min @ 25°C

[0288] Incubation time / temperature: 30 min @ 25°C

[0289] Phosphodiesterase PDE6:

[0290] Source: Bovine retinal outer segments

[0291] Substrate: 100 μM[ 3 H]cGMP + cGMP

[0292] Carrier 1.00% DMSO

[0293] Significance analysis: ≥ 50% of maximum stimulation or inhibition

[0294] Incubation buffer: 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2

[0295] Quantification method: [ 3 Quantification of [H]Guanosine

[0296] Pre-Incub. time / temperature: 15 min @ 25°C

[0297] Incubation time / temperature: 20 min @ 25°C

[0298] IC50values were determined using MathIQ™ (ID Business Solutions Ltd., UK) by non-linear least squares regression analysis. When inhibition constants (Ki) are given, Ki values were calculated using the equation of Cheng and Prusoff (Cheng, Y., Prusoff, W. H., Biochem. Pharmacol. 22:3099-3108, 1973) using the observed IC50of the test compound, the concentration of the radioligand used in the assay, and the historical value of the ligand KD. When presented, the Hill coefficient (nH) defining the slope of the competitive binding curve was calculated using MathIQ™. Hill coefficients significantly different from 1.0 can indicate that the binding displacement does not follow the law of mass action for a single binding site. When IC50, Ki and / or nH data are presented without standard error of the mean (SEM), the data are not sufficient for quantitation, and the presented values (Ki, IC50, nH) should be interpreted with caution.

[0299]

[0300]

[0301]

[0302]

[0303] Example 4 :

[0304] Compounds of formula (I) were evaluated for activity in the phosphodiesterase IV isoform.

[0305] The methods employed in this study were adapted from the scientific literature to maximize reliability and reproducibility.

[0306] Reference standards were run as part of each assay to ensure the validity of the results obtained. Cilomilast was used for the PDE4 assay.

[0307] The PDE4 assay was performed using a method adapted from the literature. See Richter W et al. (2001) Identification of the inhibitor binding site of the cAMP-specific phosphodiesterase 4. Cell Signal. 13(4):287-297; Wang H et al. (2005) Multiple elements jointly determine inhibitor selectivity of cyclic nucleotide phosphodiesterases 4 and 7. J Biol Chem. 280 35 30949 30955

[0308] Phosphodiesterase PDE4A1A:

[0309] Source: Human recombinant insect Sf9 cells

[0310] Substrate: 0.10 μΜ FAM-cAMP

[0311] Carrier 1.00% DMSO

[0312] Significance Analysis: > 50% of maximum stimulation or inhibition

[0313] Incubation Buffer: 10 mM Tris-HCl, pH 7.2, 10 mM MgCl2

[0314] 0.05% NaN3, 0.1% phosphate free BSA

[0315] Quantification Method Fluor-AMP-IMAP quantification

[0316] Pre-Incub. Time / Temp: 15 min @ 25°C

[0317] Incubation Time / Temp: 30 min at 25°C

[0318] Phosphodiesterase PDE4B1:

[0319] Source: Human recombinant insect Sf9 cells

[0320] Substrate: 0.10 μΜ FAM-cAMP

[0321] Carrier 1.00% DMSO

[0322] Significance Analysis: > 50% of maximum stimulation or inhibition

[0323] Incubation Buffer: 10 mM Tris-HCl, pH 7.2, 10 mM MgCl2

[0324] 0.05% NaN3, 0.1% phosphate free BSA

[0325] Quantification Method Fluor-AMP-IMAP quantification

[0326] Pre-Incub. Time / Temp: 15 min @ 25°C

[0327] Incubation Time / Temp: 30 min at 25°C

[0328] IC determined by non-linear least square regression analysis using MathIQ™ (ID Business Solutions Ltd., UK) 50Values. When inhibition constants (Ki) are given, the equation of Cheng and Prusoff (Cheng, Y., Prusoff, W. H., Biochem. Pharmacol. 22:3099-3108, 1973) was used to calculate Ki values using the observed IC 50 , concentration of radioligand used in the assay, and historical values of the ligand KD. When presented, the Hill coefficient (nH) defining the slope of the competitive binding curve was calculated using MathIQ™. A Hill coefficient significantly different from 1.0 can indicate that the binding displacement does not follow the law of mass action for a single binding site. When IC 50 , Ki and / or nH data are presented without standard error of the mean (SEM), the data are insufficient for quantitation, and the presented values (Ki, IC 50 , nH) should be interpreted with caution.

[0329] Assay Species N Concentration % Inh. IC50 Phosphodiesterase PDE4A1A hum 2 10 μM 96 0.063 μM hum 2 3 μM 93 hum 2 1 μM 87 hum 2 0.3 μM 75 hum 2 0.1 μM 58 hum 2 0.03 μM 37 hum 2 10 nM 23

[0330] Assay Species N Concentration % Inh. IC50 Phosphodiesterase PDE4B1 hum 2 10 μM 94 0.094 μM hum 2 3 μM 91 hum 2 1 μM 83 hum 2 0.3 μM 70 hum 2 0.1 μM 49 hum 2 0.03 μM 34 hum 2 10 nM 17

[0331] Example 5 :

[0332] The activity of the compounds of formula (I) in PDE4D2 was evaluated.

[0333] The methods employed in this study were adapted from the scientific literature to maximize reliability and reproducibility.

[0334] Cincopafylline was used as a reference standard.

[0335] PDE4 assays were performed using methods adapted from the literature. See Houslay MD (2005) The long and short of phosphodiesterase-4 as putative therapeutic targets. Mol Pharmacol. 68(3):563-567; MacKenzie SJ and Houslay MD (2000) The effects of cincopafylline on specific PDE4 cAMP phosphodiesterase isotypes and on cAMP-response element-binding protein (CREB) and p38 mitogen-activated protein (MAP) kinase phosphorylation in U937 monocytes. Biochem J. 347(Pt 2):571-578.

[0336] Phosphodiesterase PDE4D2:

[0337] Source: Human recombinant insect Sf9 cells

[0338] Substrate: 0.10 μΜ FAM-cAMP

[0339] Vehicle 1.00% DMSO

[0340] Significance analysis: ≥50% of the maximum stimulus or inhibition

[0341] Culture buffer: 10mM Tris-HCl, pH 7.2, 10mM MgCl2

[0342] 0.05% NaN3, 0.1% phosphate-free BSA

[0343] Quantitative methods for quantifying fluorescein-AMP-IMAP

[0344] Pre-Incub. Time / Temperature: 15 minutes @ 25℃

[0345] Incubation time / temperature: 15 minutes @ 25℃

[0346] IC was determined using MathIQ™ (ID Business Solutions Ltd., UK) via nonlinear least squares regression analysis. 50 When the inhibition constant (Ki) is given, the Ki value is calculated using the equation of Cheng and Prusoff (Cheng, Y., Prusoff, WH, Biochem. Pharmacol. 22:3099-3108, 1973). The IC50 of the tested compound is used. 50 The concentrations of the radioligands used in the assays and historical values ​​of the ligand KD (obtained experimentally at Eurofins Panlabs, Inc.) were used. When presented, the Hill coefficient (nH) defining the slope of the competitive binding curve was calculated using MathIQ™. A Hill coefficient significantly different from 1.0 may indicate that binding substitution does not follow the mass effect law of individual binding sites. When IC 50 When Ki and / or nH data are presented without mean standard error (SEM), the data are insufficient for quantification, and the presented values ​​(Ki, IC) are... 50 (nH) should be interpreted with caution.

[0347] Assay Species N Concentration % Inh. IC 50 <!-- 24 -->]]> Phosphodiesterase PDE4D2 hum 2 10 μM 99 6.19 nM hum 2 3 μM 97 hum 2 1 μM 96 hum 2 0.3 μM 91 hum 2 0.1 μM 83 hum 2 0.03 μM 70 hum 2 10 nM 56

[0348] Example 6 :

[0349] In preclinical trials, compounds of formula (I) did not induce mutagenesis or biologically relevant harmful effects in vitro or in vivo. Ocular instillation studies demonstrated that compounds of formula (I) were well-tolerated.

[0350] The 0.034% ophthalmic solution of compound (I) has the following composition:

[0351] Ingredient % w / v Formula I 0.034 Macrogol tetraoleate 5.00 PEG-400 1.00 Propylene glycol 1.00 Sodium chloride 0.05 CMC sodium (Cekol 150) 0.30 Sodium thiosulfate pentahydrate 0.20 Disodium EDTA dihydrate 0.10 Monosodium phosphate monohydrate 0.0262 Disodium phosphate anhydrous 0.115 Water for injection QS to 100 mL

[0352] A 0.034% ophthalmic solution of the compound of formula (I) can be prepared using conventional techniques. For example, a solution of the compound of formula I in PEG-400 and propylene glycol is prepared by adding formula I to a mixture of PEG-400 and propylene glycol with stirring. The resulting solution is mixed with a solution of macrogol tetra cetyl stearate and a portion of the water used in the composition. The resulting solution is mixed with water, sodium phosphate monobasic monohydrate, sodium phosphate dibasic anhydrous, sodium CMC, sodium chloride, sodium thiosulfate pentahydrate, and disodium EDTA dihydrate solution to give the final solution, which is sterilized by filtration (0.2 pm PES filter). The filtered solution is aseptically filled into vials and sealed under sterile conditions. The composition is packaged in 5-mL, 20 mm USP Type I clear glass vials (silica-coated) with 20 mm gray stoppers and 20 mm Flip-off Seals.

[0353] The clinical model of allergic conjunctivitis used for this study was a conjunctival allergen challenge. See, e.g., Abelson MB, Changelian ML, Smith LM. Conjunctival allergen challenge. Arch Ophthalmol 1990, 108:84; Abelson MB, Loeffler O. Conjunctival allergen challenge: a model in ocular allergy studies. Curr Allergy Asthma Rep. 2003;3:363, 368 (Matteucci et al., J. Am. Chem. Soc, Vol. 103, p. 3185, 1981).

[0354] Diagnosis: Allergic conjunctivitis

[0355] Test Product: Compound of formula (I) 0.034%.

[0356] If each eligible subject has a positive reaction to their skin test, they are subjected to a bilateral conjunctival allergen challenge titration using allergens. Subjects who elicit a positive reaction to the postconjunctival allergen challenge are subjected to a confirmatory conjunctival allergen challenge using the same allergens.

[0357] Randomization is used to avoid bias in the assignment of subjects to study products, to increase the likelihood that known and unknown subject attributes (e.g., demographics and baseline characteristics) are balanced evenly across treatment groups, and to enhance the validity of statistical comparisons across treatment groups. Finally, blinded treatment is used to reduce potential bias during data collection and evaluation of clinical endpoints.

[0358] Treatment begins after subjects are randomized. Subjects receive in-clinic doses of treatment, which they are randomized to receive. Approximately 14-18 hours after instillation of the test drug, subjects undergo a conjunctival allergen challenge to assess the 16-hour duration of action.

[0359] In a subsequent clinic visit, the subject receives an in-clinic dose of the same test drug. Approximately 8 hours after the infusion of the test drug, the subject undergoes a conjunctival allergen challenge to assess the 8-hour duration of action.

[0360] In a subsequent clinic visit, the subject receives an in-clinic dose of the same test drug approximately 15 minutes prior to the conjunctival allergen challenge to assess the 15-minute duration of action.

[0361] Efficacy measurements

[0362] This clinical study evaluates the efficacy of the compound of formula (I) 0.034% compared to vehicle in treating signs and symptoms of allergic conjunctivitis.

[0363] Eye itch is assessed by the subject under up to 10 minutes of postconjunctival allergen provocation.

[0364] Eye itch is assessed using a numerical scale ranging from no itch at one extreme to severe itch at the other extreme.

[0365] The investigator assesses conjunctival redness at up to 25 minutes postconjunctival allergen provocation.

[0366] Conjunctival redness is assessed using a numerical scale ranging from no redness at one extreme to severe redness at the other extreme.

[0367] Additional efficacy measures include, for example, assessment of redness, overlying redness, conjunctival edema, eyelid swelling, tearing, rhinorrhea, nasal itching, ear or palate itching, and nasal congestion using a numerical scale ranging from no sign / symptom at one extreme to severe sign / symptom at the other extreme. Eyelid swelling is assessed using a numerical scale ranging from no swelling at one extreme to severe swelling at the other extreme.​

Claims

1. The use of a compound of formula (I) or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of a phosphodiesterase IV (PDEIV)-mediated disease or condition, said disease or condition being an inflammatory skin disease: 。 2. The use according to claim 1, wherein the PDEIV-mediated disease or condition is psoriasis, atopic dermatitis, contact dermatitis, seborrheic dermatitis, stasis dermatitis, nodular prurigo, or discoid lupus erythematosus.

3. The use according to claim 1, wherein the PDEIV-mediated disease or condition is psoriasis.

4. The use according to claim 3, wherein the psoriasis is plaque psoriasis.

5. The use according to claim 2, wherein the PDEIV-mediated disease or condition is atopic dermatitis.

6. The use according to claim 2, wherein the PDEIV-mediated disease or condition is contact dermatitis.

7. The use according to claim 2, wherein the PDEIV-mediated disease or condition is seborrheic dermatitis.

8. The use according to claim 2, wherein the PDEIV-mediated disease or condition is stasis dermatitis.

9. The use according to claim 2, wherein the PDEIV-mediated disease or condition is nodular prurigo.

10. The use according to claim 2, wherein the PDEIV-mediated disease or condition is discoid lupus erythematosus.

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