Methods of treating alopecia disorders with TYK2 inhibitors

Abstract

The invention relates to a method of treating an alopecia disorder with a TYK2 inhibitor, and discloses a method of preventing or treating an immune-mediated alopecia disorder, such as alopecia areata, in a mammalian subject comprising administering to the mammalian subject an inhibitor of TYK2. TYK2 inhibitors useful in such methods include compounds having the structure of formula (I) as described herein and compounds having the structure of formula (II) as described herein.

Description

[0001] This application is a divisional application of the invention application with an application date of September 22, 2022, Chinese application number 202280075809.3, and invention name “Method for treating hair loss disorders with TYK2 inhibitors”. Technical Field

[0002] The present invention generally relates to methods of preventing or treating hair loss disorders using tyrosine kinase 2 (TYK2) inhibitors. Background Art

[0003] Alopecia areata (AA) is an immune-mediated disease of the hair follicles that results in non-scarring hair loss. Individuals with alopecia areata often experience chronic or recurring disease with severe psychological consequences. This is due in part to an incomplete understanding of the pathogenesis of AA and the limited treatment options available to AA patients despite a clinical need for therapy. The present invention addresses this need by providing novel methods for treating and managing hair loss disorders, including alopecia areata. Summary of the invention

[0004] Described herein are methods of treating a hair loss disorder in a subject, the methods comprising administering to the subject a TYK2 inhibitor. In some embodiments, the TYK2 inhibitor is a compound having a structure of Formula (I):

[0005]

[0006] This compound is also known as deucravacitinib. In certain embodiments, the TYK2 inhibitor is a compound having the structure of formula (II):

[0007]

[0008] In certain embodiments, the TYK2 inhibitor is a pharmaceutically acceptable salt of a compound having the structure of Formula (I), or a pharmaceutically acceptable salt of a compound having the structure of Formula (II).

[0009] In certain embodiments, the hair loss disorder is alopecia areata (AA). For example, in some embodiments, the method comprises administering a TYK2 inhibitor to a subject suffering from alopecia areata. Alopecia areata includes various phenotypic subtypes, such as localized patchy alopecia areata, alopecia totalis, and alopecia universalis. The TYK2 inhibitor can be administered orally, topically (such as by topical application or local injection to the affected one or more skin areas), or both orally and topically.

[0010] In some embodiments, the subject suffers from alopecia totalis or alopecia universalis.For example, certain embodiments of the invention relate to a method of treating alopecia totalis in a subject, the method comprising administering a TYK2 inhibitor to the subject.

[0011] Also described herein are methods of preventing hair loss in a subject previously suffering from a hair loss disorder (e.g., such as alopecia areata), the methods comprising administering a TYK2 inhibitor to the subject. In some embodiments, the TYK2 inhibitor is a compound having a structure of formula (I). In certain embodiments, the TYK2 inhibitor is a compound having a structure of formula (II). The TYK2 inhibitor can be administered orally, topically (e.g., by topical application or local injection to one or more previously affected skin areas), or both orally and topically. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is a graph showing the relative expression of MHC class I (MHC-I) in the proximal outer root sheath of hair follicles treated with vehicle, IL-12 and IL-18 or IFNγ.

[0013] Figure 2 is a graph showing the relative expression of MHC-I in the dermal cups of hair follicles treated with vehicle, IL-12 and IL-18 or IFNγ.

[0014] Figure 3 is a graph showing the relative number of MHC class II positive (MHC-II+) cells in the proximal outer root sheath of hair follicles treated with vehicle, IL-12 and IL-18 or IFNγ.

[0015] Figure 4 is a graph showing the relative number of MHC-II+ cells in the bulbar connective tissue sheath of hair follicles treated with vehicle, IL-12 and IL-18 or IFNγ.

[0016] Figure 5 is a graph showing the relative expression of MICA / B in the proximal outer root sheath of hair follicles treated with vehicle, IL-12 and IL-18 or IFNγ.

[0017] Figure 6 is a graph showing the relative expression of MICA / B in the dermal cups of hair follicles treated with vehicle, IL-12 and IL-18 or IFNγ.

[0018] Figure 7 Images of hair follicles immunostained for MHC-I are shown. The left image shows hair follicles treated with vehicle; the middle image shows hair follicles treated with IL-12 and IL-18; and the right image shows hair follicles treated with IFNγ.

[0019] Figure 8 Images of hair follicles immunostained for MHC-II are shown. The left image shows hair follicles treated with vehicle; the middle image shows hair follicles treated with IL-12 and IL-18; and the right image shows hair follicles treated with IFNγ.

[0020] Fig. 9 Images of hair follicles immunostained for MICA / B are shown. The left image shows hair follicles treated with vehicle; the middle image shows hair follicles treated with IL-12 and IL-18; and the right image shows hair follicles treated with IFNγ.

[0021] Fig.10 is a graph showing the relative number of CD3+ T cells per follicle as measured in the mesenchyme and epithelium of follicles treated with vehicle, IL-12 and IL-18 or IFNγ.

[0022] Fig.11 is a graph showing the relative number of CD56+ NK cells per follicle as measured in the mesenchyme and epithelium of follicles treated with vehicle, IL-12 and IL-18 or IFNγ.

[0023] Fig.12 Images of hair follicles immunostained for CD3 and CD56 are shown. The top image shows hair follicles treated with vehicle; the middle image shows hair follicles treated with IL-12 and IL-18; and the bottom image shows hair follicles treated with IFNγ. The bright areas in the top and middle images are areas stained with CD3 or CD56. The bright areas in the bottom image are areas stained with CD3. Some stained areas are indicated with arrows.

[0024] Fig.13 Shown are the results of differential gene expression analysis comparing gene expression in follicles treated with IL-12 and IL-18 to that in follicles treated with vehicle.

[0025] Fig.14 is a graph showing the number of genes upregulated in follicles treated with IL-12 and IL-18 compared to vehicle-treated follicles when the genes are grouped by specific pathways or functions. For example, for IFNγ-inducible genes, 35 of 78 such genes were upregulated in follicles treated with IL-12 and IL-18 compared to vehicle-treated follicles; for genes related to antigen presentation machinery, 27 of 123 such genes were upregulated in follicles treated with IL-12 and IL-18 compared to vehicle-treated follicles.

[0026] Fig.15Shown are the results of differential gene expression analysis comparing gene expression in follicles treated with IFNγ to that in follicles treated with vehicle.

[0027] Fig.16 Shown are the results of differential gene expression analysis comparing gene expression in follicles treated with IL-12 and IL-18 to that in follicles treated with IFNγ.

[0028] Fig.17 is a graph showing the relative expression of MHC-I in the proximal outer root sheath of hair follicles treated with vehicle, compound of formula (II) (which is represented as "BMS" in the figure), or tofacitinib for 5-6 days, with vehicle or cytokines IL-12+IL-18 added on day 2 of culture.

[0029] Fig.18 is a graph showing the relative expression of MHC-I in the dermal cup of hair follicles treated with vehicle, compound of Formula (II), or tofacitinib for 5-6 days, with vehicle or cytokines IL-12+IL-18 added on day 2 of culture.

[0030] Fig.19 is a graph showing the relative number of MHC-II+ cells in the proximal outer root sheath of hair follicles treated with vehicle, compound of Formula (II), or tofacitinib for 5-6 days, with vehicle or cytokines IL-12+IL-18 added on day 2 of culture.

[0031] Fig. 20 is a graph showing the relative number of MHC-II+ cells in the bulbar connective tissue sheath of hair follicles treated with vehicle, compound of formula (II), or tofacitinib for 5-6 days, with vehicle or cytokines IL-12+IL-18 added on day 2 of culture.

[0032] Fig.21 Images of hair follicles immunostained for MHC-I are shown. The left image shows hair follicles treated with vehicle for 5-6 days; the middle image shows hair follicles treated with vehicle for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; and the right image shows hair follicles treated with a compound of formula (II) for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture.

[0033] Fig. 22Images of hair follicles immunostained for MHC-II are shown. The left image shows hair follicles treated with vehicle for 5-6 days; the middle image shows hair follicles treated with vehicle for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; and the right image shows hair follicles treated with a compound of formula (II) for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture.

[0034] Fig.23 is a graph showing the relative number of CD3+ T cells per hair follicle as measured in the mesenchyme of hair follicles treated as follows: treated with vehicle for 5-6 days; treated with vehicle for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; treated with a compound of Formula (II) for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; or treated with tofacitinib for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture.

[0035] Fig.24 is a graph showing the relative number of CD3+ T cells per follicle as measured in the epithelium of follicles treated as follows: treated with vehicle for 5-6 days; treated with vehicle for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; treated with a compound of Formula (II) for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; or treated with tofacitinib for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture.

[0036] Fig.25 is a graph showing the relative number of CD56+ NK cells per hair follicle as measured in the mesenchyme of hair follicles treated as follows: treated with vehicle for 5-6 days; treated with vehicle for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; treated with a compound of formula (II) for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; or treated with tofacitinib for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture.

[0037] Fig.26 is a graph showing the relative number of CD56+ NK cells per follicle as measured in the epithelium of follicles treated as follows: treated with vehicle for 5-6 days; treated with vehicle for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; treated with a compound of formula (II) for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; or treated with tofacitinib for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture.

[0038] Fig. 27 Images of hair follicles immunostained for CD3 and CD56 are shown. The left image shows hair follicles treated with vehicle for 5-6 days; the middle image shows hair follicles treated with vehicle for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture; and the right image shows hair follicles treated with a compound of formula (II) for 5-6 days and treated with IL-12 and IL-18 added on day 2 of culture. Bright areas are areas stained with CD3 or CD56. Some stained areas are indicated with arrows.

[0039] Fig.28 is a graph showing the relative expression of MHC-I in the proximal outer root sheath of hair follicles treated as follows: with vehicle for 5-6 days; with IL-12 and IL-18 for 5-6 days; with IL-12 and IL-18 for 5-6 days and with a compound of Formula (II) added on day 2 of culture; or with IL-12 and IL-18 for 5-6 days and with tofacitinib added on day 2 of culture.

[0040] Fig.29 is a graph showing the relative expression of MHC-I in the dermal cup of hair follicles treated as follows: with vehicle for 5-6 days; with IL-12 and IL-18 for 5-6 days; with IL-12 and IL-18 for 5-6 days and with a compound of Formula (II) added on day 2 of culture; or with IL-12 and IL-18 for 5-6 days and with tofacitinib added on day 2 of culture.

[0041] Fig.30 is a graph showing the relative number of MHC-II+ cells in the proximal outer root sheath of hair follicles treated as follows: treated with vehicle for 5-6 days; treated with IL-12 and IL-18 for 5-6 days; treated with IL-12 and IL-18 for 5-6 days and treated with a compound of formula (II) added on day 2 of culture; or treated with IL-12 and IL-18 for 5-6 days and treated with tofacitinib added on day 2 of culture.

[0042] Fig.31 is a graph showing the relative number of MHC-II+ cells in the bulbous connective tissue sheath of hair follicles treated as follows: treated with vehicle for 5-6 days; treated with IL-12 and IL-18 for 5-6 days; treated with IL-12 and IL-18 for 5-6 days and treated with a compound of formula (II) added on day 2 of culture; or treated with IL-12 and IL-18 for 5-6 days and treated with tofacitinib added on day 2 of culture.

[0043] Fig.32Images of hair follicles immunostained for MHC-I are shown. The left image shows hair follicles treated with vehicle for 5-6 days; the middle image shows hair follicles treated with IL-12 and IL-18 for 5-6 days; the right image shows hair follicles treated with IL-12 and IL-18 for 5-6 days and treated with a compound of formula (II) added on day 2 of culture.

[0044] Fig.33 Images of hair follicles immunostained for MHC-II are shown. The left image shows hair follicles treated with vehicle for 5-6 days; the middle image shows hair follicles treated with IL-12 and IL-18 for 5-6 days; the right image shows hair follicles treated with IL-12 and IL-18 for 5-6 days and treated with a compound of formula (II) added on day 2 of culture.

[0045] Fig.34 is a graph showing the relative number of CD3+ T cells per hair follicle as measured in the mesenchyme of hair follicles treated as follows: treated with vehicle for 5-6 days; treated with IL-12 and IL-18 for 5-6 days; treated with IL-12 and IL-18 for 5-6 days and treated with a compound of Formula (II) added on day 2 of culture; or treated with IL-12 and IL-18 for 5-6 days and treated with tofacitinib added on day 2 of culture.

[0046] Fig.35 is a graph showing the relative number of CD3+ T cells per follicle as measured in the epithelium of follicles treated as follows: treated with vehicle for 5-6 days; treated with IL-12 and IL-18 for 5-6 days; treated with IL-12 and IL-18 for 5-6 days and treated with a compound of Formula (II) added on day 2 of culture; or treated with IL-12 and IL-18 for 5-6 days and treated with tofacitinib added on day 2 of culture.

[0047] Fig.36 is a graph showing the relative number of CD56+ NK cells per hair follicle as measured in the mesenchyme of hair follicles treated as follows: treated with vehicle for 5-6 days; treated with IL-12 and IL-18 for 5-6 days; treated with IL-12 and IL-18 for 5-6 days and treated with a compound of Formula (II) added on day 2 of culture; or treated with IL-12 and IL-18 for 5-6 days and treated with tofacitinib added on day 2 of culture.

[0048] Fig.37is a graph showing the relative number of CD56+ NK cells per follicle as measured in the epithelium of follicles treated as follows: treated with vehicle for 5-6 days; treated with IL-12 and IL-18 for 5-6 days; treated with IL-12 and IL-18 for 5-6 days and treated with a compound of Formula (II) added on day 2 of culture; or treated with IL-12 and IL-18 for 5-6 days and treated with tofacitinib added on day 2 of culture.

[0049] Fig.38 Images of hair follicle immunostaining for CD3 and CD56 are shown. The left image shows hair follicles treated with vehicle for 5-6 days; the middle image shows hair follicles treated with IL-12 and IL-18 for 5-6 days; the right image shows hair follicles treated with IL-12 and IL-18 for 5-6 days and treated with a compound of formula (II) added on day 2 of culture. Bright areas are areas stained with CD3 or CD56. Some stained areas are indicated with arrows.

[0050] Fig.39 is a graph showing the relative production of IFNγ from hair follicles treated as follows: treated with vehicle; pre-treated with vehicle followed by treatment with IL-12 and IL-18 added to the culture medium; pre-treated with a compound of Formula (II) followed by treatment with IL-12 and IL-18 added to the culture medium; or pre-treated with tofacitinib followed by treatment with IL-12 and IL-18 added to the culture medium.

[0051] Fig.40 is a graph showing the relative production of IFNγ from hair follicles treated as follows: treated with vehicle; treated with IL-12 and IL-18; pre-treated with IL-12 and IL-18 followed by treatment with a compound of Formula (II) added to the culture medium; pre-treated with IL-12 and IL-18 followed by treatment with tofacitinib added to the culture medium.

[0052] Fig.41 is a graph showing the relative number of IL-12RB2-positive cells in hair follicles obtained from healthy donors, patients with acute AA, and patients with chronic AA.

[0053] Fig.42 Images of hair follicle immunostaining for IL-12RB2 are shown. DETAILED DESCRIPTION

[0054] The present disclosure relates in part to the discovery that local IL-12 is important in the pathogenesis of AA. As shown in the Examples, IL-12 is sufficient to induce IFNγ secretion and collapse of hair follicle immune privilege with the support of IL-18. In addition, IL-12 receptor-positive immune cells are present around the affected hair bulbs of AA patients. Based on these findings, the present disclosure further relates to the discovery that inhibition of tyrosine kinase 2 (TYK2) can prevent IL-12-induced collapse of hair follicle immune privilege. TYK2 inhibition can also restore the immune privilege of hair follicles after the collapse of immune privilege is induced by IL-12 and IL-18. The ability to restore the immune privilege of hair follicles via inhibition of TYK2 demonstrates that TYK2 inhibition is a viable therapeutic strategy in AA patients.

[0055] TYK2 is a member of the Janus kinase (JAK) family of non-receptor tyrosine kinases and has been shown to be involved in both cytokine signaling in vivo and IL-12 / Th1 / Th17 axes in vivo in mice (Ishizaki, M. et al., “Involvement of tyrosine kinase-2 in both the IL-12 / Th1 and IL-23 / Th17 axes in vivo,” J. Immunol., 187:181-189 (2011); Prachal-Murphy, M. et al., “TYK2 kinase activity is required for functional type I interferon responses in vivo,” PLoS One, 7:e39141 (2012)) and humans (Minegishi, Y. et al., “Human tyrosine kinase 2 deficiency reveals its requisite roles in multiple cytokine signals involved in innate and acquired TYK2 is critical in regulating signal transduction cascades downstream of the receptors for IL-12, IL-23, and type I interferons in both immunity,” Immunity, 25:745-755 (2006)). TYK2 mediates receptor-induced phosphorylation of members of the STAT family of transcription factors, which is an essential signal leading to STAT protein dimerization and STAT-dependent pro-inflammatory gene transcription. TYK2-deficient mice are resistant to experimental models of colitis, psoriasis, and multiple sclerosis, demonstrating the importance of TYK2-mediated signaling in autoimmune and related disorders (Ishizaki, M. et al., “Involvement of tyrosine kinase-2in both the IL-12 / Th1 andIL-23 / Th17 axes in vivo,” J. Immunol., 187:181-189 (2011); Oyamada, A. et al., “Tyrosine kinase 2plays critical roles in the pathogenic CD4 T cell responses for the development of experimental autoimmune encephalomyelitis,” J. Immunol., 183:7539-7546 (2009)).

[0056] In humans, individuals expressing an inactive variant of TYK2 are protected from multiple sclerosis and possibly other autoimmune disorders (Couturier, N. et al., “Tyrosine kinase 2 variant influences T lymphocyte polarization and multiple sclerosis susceptibility”, Brain, 134:693-703 (2011)). Genome-wide association studies have shown that other variants of TYK2 are associated with autoimmune disorders such as Crohn's disease, psoriasis, systemic lupus erythematosus, and rheumatoid arthritis, which further proves the importance of TYK2 in autoimmunity (Ellinghaus, D. et al., "Combined Analysis of Genome-wide Association Studies for Crohn Disease and Psoriasis Identifies Seven Shared Susceptibility Loci", Am. J. Hum. Genet., 90: 636-647 (2012); Graham, D. et al., "Association of polymorphisms across the tyrosine kinase gene, TYK2 in UK SLE families", Rheumatology (Oxford), 46: 927-930 (2007); Eyre, S. et al., "High-density genetic mapping identifies new susceptibility loci for rheumatoid arthritis". arthritis", Nat. Genet., 44: 1336-1340 (2012)).

[0057] The present disclosure generally relates to the use of inhibitors of TYK2 to treat immune-mediated hair loss disorders, such as alopecia areata. Alopecia areata is a disease characterized by infiltration of diseased hair follicles by inflammatory T cells and NK cells. Healthy hair follicle epithelium is an immune privileged site, thereby protecting hair follicles from autoinflammatory immune responses; this immune privilege is due to a relatively immunosuppressive environment achieved by, for example, downregulation or loss of MHC class I and MHC class II expression. In contrast to healthy hair follicles, hair follicles in AA have higher MHC-I and MHC-II expression. This collapse of immune privilege of hair follicles in AA is considered to be the cause of hair loss. Bertolini et al., "Hair follicle immune privilege and its collapse in alopeciaareata," Experimental Dermatology, 29: 1-23 (2020). Although IFNγ is considered a key cytokine driving AA, the early stages and events of disease onset are not fully understood.

[0058] The examples provided herein show that TYK2 inhibition can prevent immune privilege collapse and, in addition, restore immune privilege to hair follicles. As shown herein, local IL-12 signaling may be critical in the early stages and maintenance of AA pathogenesis: it promotes resident IL-12RB2+ immune cells to produce IFNγ, leading to hair follicle immune privilege collapse. Inhibition of TYK2-dependent, IL-12-mediated signaling prevents hair follicle immune privilege collapse, and treatment with selective TYK2 inhibitors can restore immune privilege (after collapse), as demonstrated herein. These findings support targeting TYK2 for pharmacological AA therapy.

[0059] TYK2 inhibitors that can be used in the methods described herein include compounds disclosed in U.S. Pat. No. RE47,929E, the contents of which are hereby incorporated herein by reference in their entirety. For example, in certain embodiments, the TYK2 inhibitor is deuterated lexitinib. Deuterated lexitinib is also known as 6-(cyclopropanecarboxamido)-4-((2-methoxy-3-(1-methyl-1H-1,2,4-triazol-3-yl)phenyl)amino)-N-(methyl-d3)pyridazine-3-carboxamide, which has a structure of formula (I):

[0060]

[0061] Deuterated lexitinib allosterically inhibits TYK2 by binding to the regulatory domain of TYK2 rather than the catalytic domain of the enzyme. Deuterated lexitinib is highly selective for TYK2.

[0062] Other TYK2 inhibitors that can be used in the methods described herein include compounds disclosed in U.S. Patent No. 9,663,467, the contents of which are hereby incorporated by reference in their entirety. For example, in certain embodiments, the TYK2 inhibitor used in the methods described herein is a compound having a structure of Formula (II):

[0063]

[0064] TYK2 inhibitors can be administered or formulated in the form of pharmaceutically acceptable salts, such as pharmaceutically acceptable salts of compounds having the structure of formula (I), or pharmaceutically acceptable salts of compounds having the structure of formula (II). For example, TYK2 inhibitors can be formulated as hydrochlorides, mesylate salts, or sulfate salts of compounds having the structure of formula (I). See, for example, International Application Nos. PCT / US2019 / 034534 and PCT / US2020 / 036727 (published as WO 2019 / 223138 and WO 2020 / 2251911, respectively), the entire contents of each of which are incorporated herein by reference.

[0065] Administration of a TYK2 inhibitor as described herein to treat a hair loss disorder may include administering the TYK2 inhibitor systemically (e.g., orally) or topically (e.g., by topical application or by local injection) to one or more affected parts of the skin. In some embodiments, the TYK2 inhibitor is administered orally, topically, or both orally and topically. Oral dosage forms include, for example, dosage forms described in International Application No. PCT / US2020 / 051342 (disclosed as WO 2021 / 055652), the contents of which are hereby incorporated herein by reference in their entirety. Topical dosage forms include gels, creams, ointments, foams, solutions, and the like. Administration of the TYK2 inhibitor may include administration once a day, twice a day, or three times a day. In addition, the TYK2 inhibitor may be administered over the course of weeks (e.g., at least two weeks) or months (e.g., one month, two months, three months, or longer).

[0066] For any embodiment described herein, the dosage of the TYK2 inhibitor that can be administered (e.g., orally) to the subject can be in the range of about 1 mg to about 100 mg per day or about 1 mg to about 40 mg per day. For example, in some embodiments, a dosage of 3 mg, 6 mg, 12 mg, 15 mg, or 36 mg of the TYK2 inhibitor per day is administered to the subject in the method as described herein. Such a daily dose can be administered once a day, or can be administered in two or more divided doses (e.g., for a total daily dose of 12 mg, 12 mg can be administered once a day, or can be administered as two 6 mg doses, or can be administered as three 4 mg doses). In certain embodiments, the TYK2 inhibitor is deuterated lexitinib. In other embodiments, the TYK2 inhibitor is a compound having a structure of formula (II).

[0067] Certain embodiments of the present invention relate to methods for preventing recurrence of hair loss. For example, in some embodiments, the method comprises administering a TYK2 inhibitor to a subject who previously suffered from a hair loss disorder or has been diagnosed with a hair loss disorder (e.g., alopecia areata). In certain such embodiments, the subject may not currently be experiencing hair loss but has previously suffered from hair loss. In other embodiments, the TYK2 inhibitor is topically administered, for example, to certain areas of the subject's scalp.

[0068] In addition, embodiments of the present invention relate to methods of treating hair loss in a subject. For example, in some embodiments, the method comprises administering a TYK2 inhibitor to a subject suffering from hair loss. Such a subject may be a patient experiencing hair loss associated with alopecia areata. Administering a TYK2 inhibitor to such a subject may promote hair regrowth in the affected area.

[0069] There are several forms of autoimmune diseases known as alopecia areata. Subjects suffering from alopecia areata may suffer from localized patchy hair loss in the location where hair normally grows on the body (e.g., scalp). In some cases, the hair loss develops to the entire scalp (this is called alopecia totalis) or to the whole body (this is called alopecia universalis). All types of alopecia areata fall within the scope of the embodiments described herein.

[0070] In any of the embodiments described herein, a TYK2 inhibitor may be administered to a subject in combination with one or more additional agents.

[0071] In the context of the present invention, a subject, in particular a human subject, may also be referred to as a patient.

[0072] Example

[0073] The present invention will be further described by the following examples. The examples are only used to illustrate the present invention and its practice. The examples should not be interpreted as limiting the scope or spirit of the present invention.

[0074] In the following examples and figures, the following abbreviations apply: AA = alopecia areata; HF = hair follicle; IFNγ = interferon gamma; IL = interleukin; IR = immune reactivity; MHC = major histocompatibility complex; NK = natural killer cell; ORS = outer root sheath; DC = dermal cup; CTS = connective tissue sheath; Tofa = tofacitinib; FC = fold change. The compound having the structure of formula (II) is represented as "BMS" in the figures.

[0075] To elucidate the role of IL-12 in AA pathobiology and its potential as a therapeutic target, microdissected hair follicles from human scalp were cultured in the presence of IL-12 (3 ng / mL) and IL-18 (20 ng / mL) and in the presence or absence of a selective allosteric TYK2 inhibitor, a compound of formula (II) (300 nM). IFNγ (75 UI / mL) was used as a positive control to induce immune privilege (IP) collapse, and tofacitinib was used as a positive control to block IFNγ signaling. Hair follicle immune privilege (HF-IP) was assessed by quantitative immunohistomorphometry of MHC class I, MHC class II, and MHC class chain-associated protein A and MHC class chain-associated protein B (MICA / B). Resident immune cell populations were assessed by quantification of CD3-positive cells and CD56-positive cells. Whole transcriptome analysis was used to assess gene expression of treated hair follicles. IFNγ production was quantified by enzyme-linked immunosorbent assay (R&D systems). IL-12RB2 expressing cells were evaluated in healthy donors, acute AA patients, and chronic AA patients using immunohistomorphometry.

[0076] Example 1: Evaluation of the role of IL-12 in the pathogenesis of AA and the potential of IL-12 as a therapeutic target in AA

[0077] An ex vivo model of hair follicle immune privilege collapse was established. Microdissected hair follicles from human scalp were cultured ex vivo for 5-6 days in the presence of vehicle; IL-12 (3 ng / mL) and IL-18 (20 ng / mL); or IFNγ (75 UI / mL). Each group included 19-48 hair follicles obtained from 4-9 independent healthy donors. Hair follicle immune privilege collapse was assessed by quantitative immunohistomorphometry for MHC class I and MHC class II and MICA / B. See Figure 1-Figure 9 . Figure 1-Figure 6 The graphs in show mean ± SEM (standard error of the mean) and the results of Dunn's multiple comparison test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Figure 7 , Figure 8and Fig. 9 Immunostaining for MHC-I, MHC-II and MICA / B are shown, respectively (scale bar = 100 μm).

[0078] Treatment with IL-12 and IL-18 resulted in a significant increase in antigen presenting molecules. Treatment with IL-12 and IL-18 increased MHC class I expression in the outer root sheath of hair follicles compared to vehicle, and similarly in the dermal cup. Treatment with IL-12 and IL-18 also increased the number of MHC class II expressing cells in the bulbar connective tissue sheath compared to vehicle, and increased ectopic expression of MHC class II in the outer root sheath. This ectopic expression of MHC class II is the most prominent feature of HF-IP collapse.

[0079] The effects of IL-12 and IL-18 treatment on T cell and NK cell expansion were also examined. Microdissected hair follicles were again treated with vehicle, with IL-12 (3 ng / mL) and IL-18 (20 ng / mL), or with IFNγ (75 UI / mL) for 5-6 days. Each group included 9-29 follicles obtained from 2-6 independent healthy donors. Resident immune cell populations were assessed by quantification of CD3-positive cells and CD56-positive cells. See Figure 10-12 . Fig.10 and Fig.11 The graphs in show the mean ± SEM and the results of Dunn's multiple comparison test (*p<0.05; ***p<0.001). Fig.12 Immunostaining of CD3 (T cells) and CD56 (NK cells) in hair follicles is shown (scale bar = 50 μm).

[0080] IL-12+IL-18 treatment of hair follicles resulted in an increase in the number of CD3+T cells and CD56+NK cells in the epithelium and mesenchyme of the hair follicles. A small number of T cells and NK cells were observed in vehicle-treated hair follicles. However, treatment of hair follicles with IL-12+IL-18 increased the number of T cells and NK cells. T cells and NK cells are key effector cells in AA, and these results show that treatment with IL-12 and IL-18 increases the number of these cells in healthy scalp hair follicles.

[0081] Whole transcriptome analysis was used to assess gene expression of treated hair follicles. For this analysis, 6-15 hair follicles obtained from 2-3 independent healthy donors were cultured with vehicle, IL-12 (3, 4.5 or 6 ng / mL) and IL-18 (20, 30 or 40 ng / mL) or with IFNγ (75 IU / mL) for 24 hours and then subjected to whole transcriptome analysis. Fig.13Differentially expressed genes in IL-12+IL-18 treated follicles (from 3 donors) normalized to vehicle treated follicles (from 3 donors) are shown (dashed horizontal lines indicate adjusted p<0.05). Fig.14 The top ten significantly enriched pathways referring to the MetaCore database are shown ( Fig.14 The vertical lines in the figure indicate adjusted p<0.05). Fig.15 Differentially expressed genes in IFNγ-treated follicles (from 2 donors) normalized to vehicle-treated follicles are shown (dashed horizontal lines indicate adjusted p<0.05). Fig.16 Differentially expressed genes of IL-12+IL-18 treated follicles compared to IFNγ treated follicles are shown (dashed horizontal lines indicate adjusted p<0.05).

[0082] The above-described whole transcriptome and pathway analyses revealed that IL-12+IL-18 treatment of hair follicles selectively induced the expression of IFNγ and IFNγ-inducible genes, as well as the expression of genes associated with the antigen presentation pathway (e.g., MHC-II, consistent with the above-described protein expression results), and the expression of AA-related chemoattractants (e.g., CXCL-10). The differentially regulated genes detected in IFNγ-treated follicles compared to vehicle-treated follicles were similar. No significant gene expression differences were observed between IL-12+IL-18-treated follicles and IFNγ-treated follicles (see Fig.16 ). These results suggest that IL-12 induces IFNγ expression with the support of IL-18.

[0083] Example 2: Inhibition of TYK2 prevents IL-12+IL-18-induced collapse of hair follicle immune privilege

[0084] In the preventive assay, hair follicles were pretreated with a compound having the structure of formula (II) (denoted as "BMS" in the figure) and then treated with IL-12 and IL-18. Tofacitinib was used as a control for comparison. For this assay, each group included 19-40 hair follicles obtained from 3-5 independent healthy donors. Each group was treated as follows: with vehicle, with a compound of formula (II) (300 nM) or with tofacitinib (400 nM) for 5-6 days; on day 2 of culture, vehicle or IL-12 (3 ng / mL) and IL-18 (20 ng / mL) were added. The levels of MHC class I and MHC class II were assessed as described above, and Figure 17-Figure 22 Results are provided. Figure 17-Figure 20 The graphs in the figure show the mean ± SEM and the results of Dunn's multiple comparison test (*p<0.05; **p<0.01; ****p<0.0001). Fig.21 and Fig. 22 Staining of MHC-I and MHC-II in hair follicles is shown, respectively (scale bar = 100 μm).

[0085] Inhibition of TYK2 with compounds of formula (II) prevents IL-12+IL-18 mediated upregulation of MHC class I and class II in the outer root sheath. The TYK2 inhibition achieved by compounds of formula (II) is greater than that observed with tofacitinib. This effect is also observed in other hair follicle compartments. See, e.g., Fig.18 These results show that pretreatment with the compound of formula (II) prevents IL-12 and IL-18-induced collapse of hair follicle immune privilege. These results also confirm that IL-12 signaling is required for the induction of IL-12+IL-18-mediated collapse of immune privilege.

[0086] The effect of pretreatment with a compound of formula (II) on the expansion of T cells and NK cells after IL-12 and IL-18 stimulation was also examined. 22-40 hair follicles obtained from 4-5 independent healthy donors were incubated with vehicle, compound of formula (II) (300nM) or tofacitinib (400nM) for 5-6 days. On the second day of culture, vehicle or IL-12 (3ng / mL) and IL-18 (20ng / mL) were added. The resident immune cell population was evaluated by quantification of CD3 positive cells and CD56 positive cells. Figure 23-Figure 27 Results are provided. Figure 23-Figure 26 The graphs in show the mean ± SEM and the results of Dunn's multiple comparison test (*p<0.05; ***p<0.001). Fig. 27 Staining of CD3 and CD56 in hair follicles is shown (scale bar = 50 μm). Pretreatment with the compound of formula (II) prevented the increase in CD3+ T cell frequency and CD56+ NK cell frequency, which is consistent with the above results.

[0087] Example 3: Restoration of hair follicle immune privilege by inhibiting TYK2 with a compound of formula (II) after collapse of immune privilege induced by IL-12 and IL-18

[0088] The above assays were adapted to investigate whether TYK2 inhibition could inhibit the ongoing IL-12+IL-18-mediated collapse of hair follicle immune privilege, and thus whether TYK2 inhibitors are a viable therapeutic strategy for treating alopecia areata. In these assays, immune privilege collapse was first induced by treatment with IL-12+IL-18, and then the compound of formula (II) was added.

[0089] To evaluate whether TYK2 inhibition can restore hair follicle immune privilege, 21-31 hair follicles obtained from 3-4 independent healthy donors were cultured for 5-6 days with vehicle or with IL-12 (3 ng / mL) and IL-18 (20 ng / mL). On day 2 of culture, compound of formula (II) (300 nM) or tofacitinib (400 nM) were added until day 5-6. MHC class I expression and MHC class II expression were measured as previously done. See Figure 28-Figure 33 . Figure 28-Figure 31 Graphs in the table show mean ± SEM, with p values ​​from Dunn's multiple comparison test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Fig.32 and Fig.33 Staining of MHC-I and MHC-II in hair follicles is shown, respectively (scale bar = 100 μm). The compound of formula (II) and tofacitinib each significantly reduced the expression of MHC class I and MHC class II induced by IL-12 + IL-18. These results show that TYK2 inhibition can restore MHC class I and MHC class II levels to the levels observed in vehicle-treated hair follicles (which reflect the levels in healthy hair follicles). As discussed above, MHC class I and class II are markers of immune privilege collapse.

[0090] To evaluate whether TYK2 inhibition can block ongoing T cell and NK cell expansion, hair follicles were first incubated with vehicle or with IL-12+IL-18, and then the compound of formula (II) or tofacitinib was added. Specifically, 24-33 hair follicles obtained from 3-4 independent healthy donors were treated with vehicle or with IL-12 (3ng / mL) and IL-18 (20ng / mL) for 5-6 days. On the second day of culture, the compound of formula (II) (300nM) or tofacitinib (400nM) was added. T cell expansion and NK cell expansion were evaluated by immunostaining for CD3 (T cells) or CD56 (NK cells). Figure 34-Figure 37 Graphs in the table show mean ± SEM, with p values ​​from Dunn's multiple comparison test (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Fig.38 Images of staining for CD3 and CD56 in hair follicles are provided (scale bar = 50 μm).

[0091] Treatment with the compound of formula (II) rescued hair follicles from the increase of T cells and NK cells in the hair follicle epithelium and mesenchyme after IL-12+IL-18 stimulation. Figure 34-Figure 37As shown, inhibition of TYK2 with the compound of formula (II) achieved T cell and NK cell numbers similar to those observed in vehicle-treated follicles; the compound reversed T cell and NK cell expansion induced by IL-12 and IL-18.

[0092] The effect of TYK2 inhibition on IFNγ production by hair follicles was also evaluated. Specifically, two assays were performed to examine how TYK2 inhibition affects IFNγ secretion in the culture medium of ex vivo cultured hair follicles treated with IL-12+IL-18. In the preventive assay, hair follicles were pretreated with a compound of formula (II) or with tofacitinib and then cultured in the presence of IL-12+IL-18, after which IFNγ was measured in the culture medium. See Fig.39 In the therapeutic assay, hair follicles were initially treated with IL-12 + IL-18; then the compound of formula (II) or tofacitinib was added and culture continued, after which IFNγ was measured in the culture medium. Fig.40 . Fig.39 and Fig.40 The data in are based on the amount of IFNγ measured in pooled culture medium from n=8 hair follicles per group obtained from 1-2 healthy donors. The graphs show the mean±SEM of technical replicates.

[0093] Local stimulation with IL-12+IL-18 resulted in the release of IFNγ from isolated hair follicles, such as Fig.39 and Fig.40 TYK2 inhibition prevented IFNγ secretion into the culture medium and restored secretion to almost baseline levels, whereas this restoration was not observed with tofacitinib.

[0094] The above examples show that IL-12 is a key effector cytokine that promotes IFNγ secretion, immune cell expansion, and HF-IP collapse. Notably, tofacitinib treatment did not reduce the release of IFNγ into the culture medium. In contrast to tofacitinib, the compound of formula (II) is a TYK2 inhibitor that directly and selectively targets the TYK2 and IL-12 receptor signaling pathways, thereby inhibiting IFNγ release.

[0095] Example 4: IL-12 receptor expression in AA

[0096] To demonstrate that TYK2 inhibition is an attractive new target for alopecia areata management, IL-12 receptor expression in freshly obtained lesional skin from acute and chronic AA patients was compared with that in skin from healthy controls. IL-12RB2 expressing cells were evaluated in healthy donors, acute AA patients, and chronic AA patients using immunohistomorphometry. Fig.41Data from groups of 25-27 hair follicles obtained from scalp biopsies from 3-4 independent donors (healthy donors, patients with acute AA, or patients with chronic AA, as indicated) are presented. Fig.42 Immunostaining in , scale bar = 200 μm.

[0097] like Fig.41 As shown in , a higher number of IL-12RB2+ cells were observed around the hair bulbs in the skin from acute AA patients compared to the number of IL-12RB2+ cells in the skin from healthy individuals. This result further supports the role of IL-12 signaling in the pathogenesis of AA. IL-12RB2+ cells were also observed in chronic AA patients, suggesting that the remaining resident immune cells retain the ability to respond to IL-12 stimulation.

[0098] While the invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art from this disclosure that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the following claims.

[0099] The present disclosure relates to the following embodiments:

[0100] 1. A method of treating a hair loss disorder in a mammalian subject, the method comprising administering to the mammalian subject a TYK2 inhibitor.

[0101] 2. The method of claim 1, wherein the hair loss disorder is alopecia areata.

[0102] 3. The method of claim 1, wherein the hair loss disorder is alopecia totalis.

[0103] 4. The method of claim 1, wherein the hair loss disorder is alopecia universalis.

[0104] 5. The method according to any one of items 1-4, wherein the TYK2 inhibitor is deuterated lecitinib.

[0105] 6. The method according to any one of items 1-4, wherein the TYK2 inhibitor is a pharmaceutically acceptable salt of a compound having a structure of formula (I):

[0106]

[0107] 7. The method according to any one of items 1-4, wherein the TYK2 inhibitor is a compound having the structure of formula (II):

[0108]

[0109] 8. The method according to any one of items 1-4, wherein the TYK2 inhibitor is a pharmaceutically acceptable salt of a compound having a structure of formula (II):

[0110]

[0111] 9. The method according to any one of items 1-8, wherein the TYK2 inhibitor is administered orally.

[0112] 10. The method according to any one of items 1-8, wherein the TYK2 inhibitor is administered topically.

[0113] 11. A method of preventing hair loss in a mammalian subject, the method comprising administering to the mammalian subject a TYK2 inhibitor, wherein the mammalian subject previously suffered from alopecia areata.

[0114] 12. A method of preventing hair loss in a mammalian subject, the method comprising administering to the mammalian subject a TYK2 inhibitor, wherein the mammalian subject previously suffered from alopecia totalis.

[0115] 13. A method of preventing hair loss in a mammalian subject, the method comprising administering to the mammalian subject a TYK2 inhibitor, wherein the mammalian subject previously suffered from alopecia universalis.

[0116] 14. The method according to any one of items 11-13, wherein the TYK2 inhibitor is a compound having a structure of formula (I) or a pharmaceutically acceptable salt thereof:

[0117]

[0118] 15. The method according to any one of items 11-13, wherein the TYK2 inhibitor is a compound having a structure of formula (II) or a pharmaceutically acceptable salt thereof:

[0119]

[0120] 16. The method according to any one of items 11-15, wherein the TYK2 inhibitor is administered orally.

[0121] 17. The method according to any one of items 11-15, wherein the TYK2 inhibitor is administered topically.

[0122] 18. The method according to any one of items 1-10, wherein said administering said TYK2 inhibitor to said mammalian subject comprises administering said TYK2 inhibitor at least twice per week.

[0123] 19. The method of claim 18, wherein the TYK2 inhibitor is administered to the mammalian subject once daily.

[0124] 20. A method according to any one of items 18-19, wherein the method further comprises promoting hair regeneration in the mammalian subject.

[0125] 21. A method of regenerating hair in a human subject suffering from alopecia areata, the method comprising administering to the human subject a TYK2 inhibitor.

[0126] 22. The method according to item 21, wherein the TYK2 inhibitor is a compound having a structure of formula (I) or a pharmaceutically acceptable salt thereof:

[0127]

[0128] 23. The method according to item 21, wherein the TYK2 inhibitor is a compound having a structure of formula (II) or a pharmaceutically acceptable salt thereof:

[0129]

Claims

1. A method of treating a hair loss disorder in a mammalian subject, the method comprising administering to the mammalian subject a TYK2 inhibitor.

2. The method of claim 1, wherein the hair loss disorder is alopecia areata.

3. The method of claim 1, wherein the hair loss disorder is alopecia totalis.

4. The method of claim 1, wherein the hair loss disorder is alopecia universalis.

5. The method of any one of claims 1-4, wherein the TYK2 inhibitor is deuterated lexitinib.

6. The method according to any one of claims 1-4, wherein the TYK2 inhibitor is a pharmaceutically acceptable salt of a compound having the structure of formula (I):

7. The method according to any one of claims 1 to 4, wherein the TYK2 inhibitor is a compound having the structure of formula (II):

8. The method according to any one of claims 1-4, wherein the TYK2 inhibitor is a pharmaceutically acceptable salt of a compound having the structure of formula (II):

9. The method of any one of claims 1-8, wherein the TYK2 inhibitor is administered orally.

10. The method of any one of claims 1-8, wherein the TYK2 inhibitor is administered topically.

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