EA046969B1 - TREATMENT OF HYDRADENITI SUPUPRATORY USING JAK INHIBITORS - Google Patents

Abstract

The invention provides methods for treating hidradenitis suppurativa in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound that inhibits JAK1 and/or JAK2, or a pharmaceutically acceptable salt thereof.

Description

This application claims priority to US Provisional Application No. 62/650600, filed March 30, 2018, which is incorporated herein by reference in its entirety.

Field of technology

This application provides methods of treating hidradenitis suppurativa (HS) using compounds that modulate the activity of Janus kinase (JAK) 1 and/or 2.

State of the art

Protein kinases (PKs) regulate various biological processes including, but not limited to, cell growth, survival, differentiation, organ formation, morphogenesis, neovascularization, tissue repair, and regeneration. Protein kinases also play specialized roles in a variety of human diseases, including cancer. Cytokines, small molecule polypeptides or glycoproteins, regulate many pathways involved in the host inflammatory response to sepsis. Cytokines influence cell differentiation, proliferation, and activation and can modulate both pro- and anti-inflammatory responses, allowing the host to respond appropriately to pathogens. Signaling of a wide range of cytokines includes the Janus kinase (JAK) family of protein tyrosine kinases and signal transducers and activators of transcription (STAT). There are four known mammalian JAKs: JAK1 (Janus kinase 1), JAK2, JAK3 (also known as Janus kinase, leukocyte; JAKL and L-JAK), and TYK2 (protein tyrosine kinase 2).

Immune and inflammatory responses stimulated by cytokines contribute to the pathogenesis of diseases: pathologies such as severe combined immunodeficiency (SCID) arise from suppression of the immune system, while an overactive or inappropriate immune/inflammatory response contributes to the pathology of autoimmune diseases (e.g. asthma, systemic lupus erythematosus, thyroiditis, myocarditis) and diseases such as scleroderma and osteoarthritis (Ortmann, R. A., T. Cheng, et al. (2000) Arthritis Res 2(1): 16-32).

Disturbances in JAK expression are associated with many disease conditions. For example, Jak1-/mice are small at birth, unable to nurse, and die during the perinatal period (Rodig, S. J., M. A. Meraz, et al. (1998) Cell 93(3): 373-83). Jak2 −/− mouse embryos are anemic and die at approximately 12.5 days after mating due to a lack of definitive erythropoiesis.

The JAK/STAT pathway and, in particular, all four JAKs are believed to play a role in the pathogenesis of the asthmatic response, chronic obstructive pulmonary disease, bronchitis and other associated inflammatory diseases of the lower respiratory tract.

Multiple cytokines that signal through JAK have been associated with inflammatory diseases/conditions of the upper respiratory tract, such as those affecting the nose and sinuses (eg, rhinitis and sinusitis), whether they are classic allergic reactions or not. The JAK/STAT pathway is also involved in inflammatory eye diseases/conditions and chronic allergic reactions.

Activation of JAK/STAT in cancer may occur by stimulation of cytokines (eg, IL-6 or GM-CSF) or by reduction of endogenous suppressors of JAK signaling, such as SOCS (suppressor or cytokine signaling) or PIAS (protein inhibitor of activated STAT) ( Boudny, V., and Kovarik, J., Neoplasm 49:349-355, 2002). Activation of STAT signaling, as well as other pathways downstream of JAK (eg, Akt), has been correlated with poor prognosis in many types of cancer (Bowman, T., et al. Oncogene 19:2474-2488, 2000). Elevated levels of circulating cytokines that signal through JAK/STAT play a causative role in cachexia and/or chronic fatigue. Thus, JAK inhibition may be beneficial for cancer patients for reasons beyond potential antitumor activity.

JAK2 tyrosine kinase may be beneficial for patients with myeloproliferative diseases, such as polycythemia vera (PV), essential thrombocythemia (ET), and myeloid metaplasia with myelofibrosis. (Levin, et al., Cancer Cell, vol. 7, 2005: 387-397). Inhibition of JAK2V617F kinase reduces hematopoietic cell proliferation, suggesting that JAK2 is a potential target for pharmacological inhibition in patients with PV, ET and MMM.

JAK inhibition may benefit patients suffering from cutaneous immune disorders such as psoriasis and skin sensitization. The maintenance of psoriasis is thought to depend on a number of inflammatory cytokines in addition to various chemokines and growth factors (JCI, 113: 1664–1675), many of which signal through JAK (Adv Pharmacol. 2000; 47:113–74).

Thus, new or improved agents that inhibit kinases such as JAK are continually needed to develop new and more effective pharmaceuticals aimed at enhancing or suppressing immune and inflammatory pathways, such as the treatment of hidradenitis suppurativa. This application is designed for this and other needs.

The essence of the invention

Provided herein are methods of treating hidradenitis suppurativa in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound that inhibits JAK1 and/or JAK2, or a pharmaceutically acceptable salt thereof.

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In some embodiments, the compound or salt is selective for JAK1 and JAK2, which are selective for JAK3 and TYK2.

In some embodiments, the compound or salt is selective for JAK1 over JAK2, JAK3, and TYK2.

In some embodiments, the compound is ruxolitinib or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is ruxolitinib or a pharmaceutically acceptable salt thereof in which one or more hydrogen atoms are replaced by deuterium atoms.

In some embodiments, the salt is a phosphate of ruxolitinib.

In some embodiments, the compound is {1-{1-[3-fluoro-2(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3D]pyrimidin-4-yl )-III-pyrazol1-yl]azetidin-3-yl}acetonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments, the salt is {1-{1-[3-fluoro-2(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3D]pyrimidin-4- yl)-III-pyrazol1-yl]azetidin-3-yl}acetonitrile

In some embodiments, the compound is 4-[3-(cyanomethyl)-3-(3',5'dimethyl-III,1H-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro- N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof.

In some embodiments, the salt is 4-[3-(cyanomethyl)-3-(3',5'dimethyl-III, 1'H-bipyrazol-1 -yl)azetidin-1 -yl]-2,5-phosphate difluoro-N-[( 1 S)-2,2,2-trifluoro-1-methylethyl]benzamide.

In some embodiments, the compound or salt is administered at a dosage of 15, 30, 60, or 90 mg, based on the free base.

In some embodiments, the compound is ((2R,5S)-5-{2-[(1R)-1hydroxyethyl]-III-imidazo[4,5D]thieno[3,2-b]pyridin-1-yl}tetrahydro -2H-pyran-2-yl)acetonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is ((2R,5S)-5-{2-[(1R)-1hydroxyethyl]-1H-imidazo[4,5D]thieno[3,2-b]pyridin-1-yl}tetrahydro -2H-pyran-2-yl)acetonitrile monohydrate.

In some embodiments, the methods further comprise administering an additional therapeutic agent (eg, an antibiotic, a retinoid, a corticosteroid, an anti-TNF-alpha agent, or an immunosuppressant).

In some embodiments, the administration of the compound or salt is topical. In some embodiments, the compound or salt is administered orally.

In some embodiments, the method results in a 10%, 20%, 30%, 40%, or 50% improvement in HiSCR (Hidradenitis Suppurativa Clinical Response).

This application also provides a compound that inhibits JAK1 and/or JAK2, or a pharmaceutically acceptable salt thereof, for use in the treatment of hidradenitis suppurativa.

This application further provides the use of a compound that inhibits JAK1 and/or JAK2, or a pharmaceutically acceptable salt thereof, for the preparation of a medicament for use in the treatment of hidradenitis suppurativa.

Brief description of graphic materials

In fig. Figure 1 shows individual gene expression (MFI) values for JAK1 for each experimental replication in keratinocytes modeled with TNFa and IFN-γ in the presence/absence of compounds A-D. Keratinocytes were stimulated with TNFa (25 ng/ml) and IFNy (25 ng/ml) in the presence/absence of increasing concentrations of JAK inhibitors. Data are presented as JAK1 expression levels for each group.

In fig. Figure 2 shows individual gene expression (MFI) values for JAK2 for each experimental replication in keratinocytes modeled with TNFa and IFN-γ in the presence/absence of compounds A-D. Keratinocytes were stimulated with TNFa (25 ng/ml) and IFNy (25 ng/ml) in the presence/absence of increasing concentrations of JAK inhibitors. Data are presented as JAK2 expression levels for each group.

In fig. Figure 3 shows individual gene expression (MFI) values for IL-1a for each experimental replicate in keratinocytes modeled with TNFa and IFN-γ in the presence/absence of compounds A-D. Keratinocytes were stimulated with TNFa (25 ng/ml) and IFNy (25 ng/ml) in the presence/absence of increasing concentrations of JAK inhibitors. Data are presented as IL-1a expression levels for each group.

In fig. Figure 4 shows individual gene expression (MFI) values for IL-6, for each experimental replication in keratinocytes modeled with TNFa and IFN-γ in the presence/absence of compounds A-D. Keratinocytes were stimulated with TNFa (25 ng/ml) and IFNy (25 ng/ml). ml) at

- 2 046969 presence/absence of increasing concentrations of JAK inhibitors. Data are presented as IL-6 expression levels for each group.

In fig. Figure 5 shows the individual protein concentrations (pg/ml) for IL-1a for each experimental replication in keratinocytes modeled with TNFa and IFN-γ in the presence/absence of compounds A-D. Keratinocytes were stimulated with TNFa (25 ng/ml) and IFNy (25 ng/ml) in the presence/absence of increasing concentrations of JAK inhibitors. Data are presented as IL-1a concentrations for each group.

In fig. 6 shows individual protein concentrations (pg/ml) for IL-6 for each experimental replicate in keratinocytes modeled with TNFa and IFN-γ in the presence/absence of compounds A-D. Keratinocytes were stimulated with TNFa (25 ng/ml) and IFNy (25 ng/ml) in the presence/absence of increasing concentrations of JAK inhibitors. Data are presented as IL-6 concentrations for each group.

In fig. 7 shows gene expression (MFI) of JAK1, JAK3 and TYK2 in the skin of healthy controls and subjects with hidradenitis suppurativa. Data are presented as JAK1, JAK3, or TYK2 gene expression levels for each healthy control (n=4) and hidradenitis suppurativa (n=41) subject.

In fig. 8 shows gene expression (MFI) of STAT1, STAT2 and STAT3 in the skin of healthy control subjects and subjects with hidradenitis suppurativa. Data are presented as STAT1, STAT2, or STAT3 gene expression levels for each healthy control subject (n=4) and subject with hidradenitis suppurativa (n=41).

In fig. 9 shows gene expression (MFI) of IRAKI, IRAK2, and IRAK4 in the skin of healthy control subjects and subjects with hidradenitis suppurativa Data are presented as the expression levels of the IRAKI, IRAK2, or IRAK4 genes for each healthy control subject (n=4) and subject with hidradenitis suppurativa. hidradenitis (n=41).

Detailed description

This application provides, among other things, a method of treating hidradenitis suppurativa in a patient in need thereof, comprising administering a therapeutically effective amount of a compound that inhibits JAK1 and/or JAK2, or a pharmaceutically acceptable salt thereof.

The method described herein uses a compound or salts that are inhibitors of JAK1 and/or JAK2. In some embodiments, the compound is: ruxolitinib;

ruxolitinib, in which one or more hydrogen atoms are replaced by deuterium atoms;

{1-{1-[3-Fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7Hpyrrolo[2,3-<1]pyrimidin-4-yl)-1H-pyrazole -1-yl]azetidin-3-yl (acetonitrile;

4-{3-(Cyanomethyl)-3-[4-(7H-pyrrolo[2,3-c1]pyrimidin-4-yl)-1H-pyrazol-1yl]azetide in-1-yl}-T4-[4 -fluoro-2-(trifluoromethyl)phenyl]piperide in-1-carboxamide;

[3 - [4-(7H-pyrrolo[2,3 -<1]pyrimidin-4-yl)- 1H-pyrazol-1 -yl]-1 -(1 - {[2(trifluoromethyl)pyrimidin-4-yl ]carbonyl}piperidin-4-yl)azetidin-3-yl]acetonitrile;

4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,GN-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5difluoro-T4-[( 18)-2,2,2-trifluoro-1-methylethyl]benzamide;

((2R, 5 S)- 5 - { 2- [(1 R)-1 -hydroxyethyl] -1 H-imidazo[4,5 -<1]thieno[3,2-b]pyridin-1 yl}tetrahydro -2H-pyran-2-yl)acetonitrile;

- [1 -(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3 - [4-(7H-pyrrolo[2,3 -<1]pyrimidin-4yl)-1H-pyrazol-1 -yl] propanenitrile;

3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3<1]pyrimidin-4-yl )-1H-pyrazol-1-yl]propanenitrile;

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4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-b]pyrimidin-4-yl)-1H-pyrazol-1yl]propyl (piperazin-1-yl)carbonyl] -3-fluorobenzonitrile;

4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-c1]pyrimidin-4-yl)-1H-pyrrol-1yl]propyl (piperazin-1-yl)carbonyl] -3-fluorobenzonitrile;

[trans-1 - [4-(7H-pyrrolo[2,3-b]pyrimidin-4-yl)- 1H-pyrazol-1 -yl]-3 -(4- {[2(trifluoromethyl)pyrimidin-4- yl]carbonyl(piperazin-1-yl)cyclobutyl]acetonitrile;

{trans-3-(4-{[4-[(3-hydroxyazetidin-1-yl)methyl]-6-(trifluoromethyl)pyridin-2yl]oxy(piperidin-1-yl)-1 -[4-(7H -pyrrolo[2,3-b]pyrimidin-4-yl)-1H-pyrazol-1 yl]cyclobutyl (acetonitrile;

{trans-3-(4-{[4-{[(28)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6(trifluoromethyl)pyride in-2-yl]oxy(piper idin-1- yl)-1-[4-(7H-pyrrolo[2,3-<1]pyrimide in4-yl)-1 H-pyrazol-1 -yl]cyclobutyl } acetonitrile;

{trans-3-(4-{[4- {[(2K)-2-(hydroxymethyl)pyrrolidin-1 -yl]methyl (-6(trifluoromethyl)pyride in-2-yl]oxy(piper idin-1 - yl)-1-[4-(7H-pyrrolo[2,3-b]pyrimide in4-yl)-1H-pyrazol-1 -yl]cyclobutyl}acetonitrile;

4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy(piperidin-1-yl)-3-[4-(7Hpyrrolo[2,3-c!]pyrimidin-4-yl)-1H- pyrazol-1-yl]butanenitrile;

5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1 yl]azetidin-1-yl(-L1-isopropylpyrazine -2-carboxamide;

4- {3 -(cyanomethyl)-3 - [4-(7H-pyrrolo[2,3 -d]pyrimidin-4-yl)-1 H-pyrazol-1 yl]azetidin-1-yl(-2,5 -difluoro-K-[(18)-2,2,2-trifluoro-1-methylethyl]benzamide;

5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1 yl]azetidin-1-yl(-L1-isopropylpyrazin- 2-carboxamide;

{1-(cis-4-{[6-(2-hydroxyethyl)-2-(trifluoromethyl)pyrimidin-4yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidine -4-yl)-1 H-pyrazol-1 yl]azetidin-3-yl(acetonitrile;

{1-(cis-4-{ [4-[(ethylamino)methyl]-6-(trifluoromethyl)pyridin-2yl]oxy } cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimide in-4-yl)-1 H-pyrazol-1 yl]azetidin-3-yl(acetonitrile;

{1-(cis-4-{[4-(1-hydroxy-1-methylethyl)-6-(trifluoromethyl)pyridin-2yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3 -d]pyrimide in-4-yl)-1 H-pyrazol-1 yl]azetidin-3-yl(acetonitrile;

{1 -(cis-4- {[4- {[(3R)-3-hydroxypyrrolidin-1 -yl]methyl (-6(trifluoromethyl)pyridin-2-yl]oxy(cyclohexyl)-3-[4-( 7H-pyrrolo[2,3-b]pyrimidin-4yl)-1H-pyrazol-1 -yl] azetidin-3 -yl (acetonitrile;

{1 -(cis-4- {[4- {[(3 S)-3 -hydroxypyrrolidin-1 -yl]methyl (-6(trifluoromethyl)pyridin-2-yl]oxy(cyclohexyl)-3-[4- (7H-pyrrolo[2,3-b]pyrimidin-4yl)-1H-pyrazol-1 -yl] azetidin-3 -yl (acetonitrile;

{trans-3-(4-{[4-({[(18)-2-hydroxy-1-methylethyl]amino(methyl)-6(trifluoromethyl)pyridin-2-yl]oxy(piperidin-1-yl) -1-[4-(7H-pyrrolo[2,3^]pyrimidin4-yl)-1H-pyrazol-1 -yl]cyclobutyl(acetonitrile;

[trans-3-(4-[[4-([[(2K)-2-hydroxypropyl]amino}methyl)-6(trifluoromethyl)pyridin-2-yl]oxy(piperidin-1-yl)-1-[ 4-(7n-pyrrolo[2,3-4]pyrimidin-4yl)-1H-pyrazol-1-yl]cyclobutyl(acetonitrile;trans-3-(4-{[4-({[(28)-2- hydroxy ipropyl]amino(methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy]piperidin-1-yl)-1[4-(7H-pyrrolo[2,3^]pyrimidin-4-yl)-1H -pyrazol-1-yl]cyclobutyl(acetonitrile;

{trans-3-(4-{[4-(2-hydroxyethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy(piperidin1 -yl)-1 - [4-(7H-pyrrolo[2,3^ ]pyrimidin-4-yl)-1H-pyrazol-1 yl]cyclobutyl (acetonitrile;

or a pharmaceutically acceptable salt of any of the above.

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In some embodiments, the compound or salt is selective for JAK1 and JAK2 over JAK3 and TYK2. In some embodiments, the compound is 3-cyclopentyl-3-[4-(7H-pyrrolo[2,3D]pyrimidin-4-yl)-N-pyrazol-1-yl]propanenitrile or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile (ruxolitinib) . or a pharmaceutically acceptable salt thereof. Ruxolitinib has an IC50 of less than 10 nM at 1 mM ATP (assay A) for JAK1 and JAK2. 3-Cyclopentyl-3-[4-(7H-pyrrolo[2,3D]pyrimidin-4-yl)-1Hpyrazol-1-yl]propanenitrile and ruxolitinib can be prepared according to the procedure described in US 7598257 (Example 67), filed December 12, 2006, which is incorporated herein by reference in its entirety. In some embodiments, the JAK1 and/or JAK2 inhibitor is the phosphate salt of (3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1yl ]propanenitrile. The phosphoric acid salt can be prepared as described in U.S. Pat. 8722693, which is incorporated herein by reference in its entirety.

In some embodiments, the compound or salt is a JAK1 inhibitor. In some embodiments, the compound or salt is selective for JAK1 over JAK2, JAK3, and TYK2. For example, some of the compounds described herein, or a pharmaceutically acceptable salt thereof, preferentially inhibit JAK1 over one or more of JAK2, JAK3 and TYK2. JAK1 plays a central role in a number of cytokine and growth factor signaling pathways that, when dysregulated, can lead to or contribute to disease states. For example, IL-6 is elevated in rheumatoid arthritis, a disease in which it is thought to have detrimental effects (Fonesca, et al., Autoimmunity Reviews, 8:538-42, 2009). Because IL-6 signals, at least in part, through JAK1, IL-6 may, indirectly through inhibition of JAK1, result in potential clinical benefit (Guschin, et al. Embo J 14:1421, 1995; Smolen, et al. Lancet 371:987, 2008) Moreover, in some forms of cancer, JAK1 is mutated, leading to undesirable growth and survival of tumor cells (Mullighan, Proc Natl Acad Sci USA. 106:9414-8, 2009; Flex, J Exp Med. 205 :751-8, 2008). In other autoimmune diseases and cancers, elevated systemic levels of inflammatory cytokines that activate JAK1 may also contribute to the disease and/or associated symptoms. Therefore, patients with such diseases may benefit from JAK1 inhibition. Selective JAK1 inhibitors may be effective while avoiding the unnecessary and potentially undesirable effects of inhibiting other JAK kinases.

Hidradenitis suppurativa is characterized by significant inflammation of the skin; however, there are a limited number of publications describing this inflammation (Hoffman et al., PLOS One, September 28, 2018, https://doi.org/10.1371/journal.pone.0203672). This paper provides examples that support the hypothesis that inflammation is largely caused by JAK/STAT-mediated pathways. Examples C, D and E illustrate increased levels of JAK/STAT gene expression in the skin of patients with HS compared to healthy skin. Additionally, Examples C, D and E demonstrate that pro-inflammatory cytokines known to be elevated in HS (TNFalpha and IFN-gamma) induce the JAK/STAT pathway in cultured keratinocytes and that this induction can be reduced by the addition of JAK inhibitors. Therefore, patients with HS may benefit from JAK1 inhibition. Selective JAK1 inhibitors may be effective while avoiding the unnecessary and potentially undesirable effects of inhibiting other JAK kinases.

In some embodiments, the compound or salt inhibits JAK1 preferentially over JAK2 (eg, has a JAK2/JAK1 IC ₅₀ ratio >1). In some embodiments, the compounds or salts are about 10 times more selective for JAK1 compared to JAK2. In some embodiments, the compounds or salts are about 3, about 5, about 10, about 15, or about 20 times more selective for JAK1 over JAK2, as calculated by measuring the IC50 at 1 mM ATP (see Example A).

In some embodiments, the JAK1 inhibitor is a compound from Table. 1 or a pharmaceutically acceptable salt thereof. Connections in the table 1 are selective inhibitors of JAK1 (selective over JAK2, JAK3 and TYK2). The _IC50 values obtained by the method of Example A at 1 mM ATP are presented in table. 1.

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Table 1

Soy d. No. Rev. Name Structure JAK1 1C ₅₀ (nM) JAK2/ JAK1

1 US 2011/0224190 (Example 1) {1-{ 1-[3-Fluoro-2(trifluoromethyl)isonico tinoyl]piperidin-4yl}-3-[4-(7Hpyrrolo[2,3- <1]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin3-yl(acetonitrile T T CF ₃ Q ^F NN Λ \ |Ι Λ / Η + >10 2 US 2011/0224190 (Example 154) 4-{3-(Cyanomethyl)-3[4-(7H-pyrrolo[2,3- <1]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin1-yl}-M-[4-fluoro-2(trifluoromethyl)phenyl]p iperidin-1carboxamide FΑ. Ο^ΝΗ Q Ν Ν-Ν At A NAG. Chg'i Fl + >10 3 US 2011/0224190 (Example 85) [3-[4-(7H-pyrrolo[2,3- <1]pyrimidin-4-yl)- 1Hpyrazol- Pyl] -1 -(1 - {[2(trifluoromethyl)pyrimidin-4- yl]carbonyl}piperidi n-4-yl)azetidin-3yl]acetonitrile Ο у=у > 4 Ν <Cl :, rN ^Z jy' ¹ .N Ν VV'< J 7 l LL > + >10 4 US 2014/03430 30 (Example 7) 4-[3-(cyanomethyl)-3(3',5'-dimethyl-1H,GH4,4'-bipyrazol-1 yl)azetidin-1 -yl]-2,5difluoro-X-[(18)-2 ,2,2- chlu Υ*F HNN +++ >10

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trifluoro-1methylethyl]benzamide 5 US 2014/01211 98 (Example 20) ((2R,5S)-5-{2-[(lR)-l- hydroxyethyl]-1Nimidazo[4,5- <1]thieno[3,2-b]pyridin1-yl}tetrahydro-2Hpyran-2yl)acetonitrile he ,' A % ί-Ν ^Ν ·>, S ¢0 ++ >10 6 US 2010/0298334 (Example 2) ^a 3 - [ 1 -(6-chloropyridin-2yl)pyrrole id in-3 - yl] - 3 [4-(7H-pyrrolo[2,3<1]pyrimidin-4-yl)- 1Hpyrazol-1yl]propanenitrile N Η ί> } Ν -4 C.I. Ν' ' Ν ' ’ ΝΗ + >10 7 US 2010/0298334 (Example 13c) 3-(1-[1,3]oxazolo[5,4b]pyridin-2ylpyrrolidin-3-yl)-3 [4-(7H-pyrrolo[2,3<1]pyrimidin-4-yl)-1Hpyrazol-1yl ]propanenitrile ^Ν χ ....../ Ν ^w Ν' Ν Η + >10 8 US 2011/0059951 (Example 12) 4-[(4-{3-cyano-2-[4- (7H-pyrrolo[2,3- <1]pyrimidin-4-yl)- 1Hpyrazol-1- yl]propyl } piperazine- 1 -yl)carbonyl]-3 fluorobenzonitrile \\ / \ , /“D CN \\ //“~F Ν -Ν' // Λ NC W ^Ν Η + >10

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9 US 2011/0059951 (Example 13) 4-[(4-{3-cyano-2-[3- (7H-pyrrolo[2,3- <1]pyrimidin-4-yl)- 1H- pyrrole-1- yl]propyl } piperazine- 1 -yl)carbonyl]-3 fluorobenzonitrile O >—· * ' · CN /—N ⁴ — O (y /CN O W + >10 10 US 2012/0149681 (Example 7b) [trans-1 - [4-(7Hpyrrolo[2,3- <1]pyrimidin-4-yl)-1Hpyrazol-1-yl]-3-(4-{ [2(trifluoromethyl)pyrimidin-4- yl]carbonyl } piperazine -1- yl)cycle butyl]acetony tril ^F ? ν' Ν o N NN ^Z μ JL 7 N ^{N N} H + >10 eleven US 2012/0149681 (Example 157) [trans-3-(A-{ [4-[(3hydroxyazetidin-1yl)methyl]-6(trifluoromethyl)pyridinium n-2- yl]oxy}piperidin-1 yl)-1-[4-(7Hpyrrolo[2,3- <1]pyrimidin-4-yl)- 1Hpyrazol-1- yl ]cyclobutyl}acetony tril _OH N^ o ^N f ^F 0 NN i.· ¹ i LL X n N H + >10

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12 US 2012/0149681 (Example 161) {trans-3-(D-{ [4-{ [(2S)2- (hydroxymethyl)pyrrole id in-1 - yl] methyl } -6(trifluoromethyl)pyridinium-2- yl]oxy}piperidin-1 yl)-1-[4-(7Hpyrrolo[2,3- <1]pyrimidin-4-yl)- 1Hpyrazol-1- yl ]cyclobutyl}acetony tril z ^{- Z} \A ^z o + >10 13 US 2012/0149681 (Example 162) {trans-3-(4-{ [4-{ [(2R)2- (hydroxymethyl)pyrrol ide in-1 - yl] methyl } -6(trifluoromethyl)pyridium n-2- yl]oxy}piperidine-1 - yl)-1-[4-(7H- pyrrolo[2,3- <1]pyrimidin-4-yl)- 1Hpyrazol-1- yl ]cyclobutyl}acetony tril π·π § + >10 14 US 2012/0149682 (Example 20) ^b 4-(4-{3- [(dimethylamino)methyl] -5- fluorophenoxy}piperidi n-1-yl)-3-[4-(7H- pyrrolo[2,3- <1]pyrimidin-4-yl)- 1Hpyrazol-1- silt]butanenitrile n A—iw N + >10

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15 US 2013/0018034 (Example 18) 5-{3-(cyanomethyl)-3-[4(7H-pyrrolo[2,3- <1]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin1-yl}-bGisopropylpyrazin-2carboxamide N=—\/\ ^N H-N X t/O Η + >10 16 US 2013/0018034 (Example 28) 4-{3-(cyanomethyl)-3-[4(7H-pyrrolo[2,3- <1]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin1-yl}-2,5-difluoro-I[(1S)-2,2,2-trifluoro-1 methylethyl]benzamide F ,____/ o N=—/=x ,. X .n-Ч XX Ν-Λ У ⁷ , ^ΝΗ // \ F |1Л7 F γ γ Υπ ^{F Ν} Η + >10 17 US 2013/0018034 (Example 34) 5-{3-(cyanomethyl)-3-[4(1H-pyrrolo[2,3- b] pyridin-4-yl)-1Hpyrazol-1 -yl] azetidin1-yl}-bGisopropylpyrazin-2carboxamide ί|Ι - φ + >10 18 US 2013/0045963 (Example 45) {1-(g/is-4-{[6-(2hydroxyethyl)-2(trifluoromethyl)pyrimidine-4- yl] oxy } cyclohexyl)3-[4-(7H-pyrrolo[2,3<1]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin3-yl(acetonitrile κι .--- ^- % Ο _C .X Φ A Γ J τ Η + >10 19 US 2013/0045963 (Example 65) {1-(g/is-4-{[4[(ethylamino)methyl]-6(trifluoromethyl)pyridinium-2- yl] oxy } cyclohexyl)- Λ 1 Γ ί X N-CT Ν-Μ _c X i \ F ψΈ 11 X / + >10

- 10 046969

3-[4-(7H-pyrrolo[2,3<1]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin3-yl(acetonitrile 20 US 2013/0045963 (Example 69) {1-(g/is-4-{[4-(1—hydroxy-1-methylethyl)6- (trifluoromethyl)pyridium n-2- yl]oxy(cyclohexyl)3-[4-(7H-pyrrolo[2,3<1]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin3-yl(acetonitrile Vzj'V Hz Ο T ^F + >10 21 US 2013/0045963 (Example 95) {l-(z/z/c-4-{[4-{[(3R)-3hydroxypyrrolidin-1 yl]methyl(-6(trifluoromethyl)pyridinium n-2- yl]oxy(cyclohexyl)3-[4-(7H-pyrrolo[2,3<1]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin3-yl(acetonitrile f 4/ ά)π F, _-iSN FA—Ч // . /HuT F ^M X)U / ^I U\| Ν Ν Η + >10 22 US 2013/0045963 (Example 95) {l-(z/z/c-4-{[4-{[(3S)-3hydroxypyrrolidin-1 yl]methyl(-6(trifluoromethyl)pyridinium n-2- yl]oxy(cyclohexyl)3-[4-(7H-pyrrolo[2,3<1]pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin3-yl(acetonitrile Γ' ^Ν Ο*ΟΗ ^Ν Η + >10

- 11 046969

23 US 2014/0005166 (Example 1) {trans-3-(A-{ [4-( {[(1S)2-hydroxy-1methylethyl] amino } methyl l)-6(trifluoromethyl)pyridinium n-2- yl]oxy}piperidin-1 yl)-1-[4-(7Hpyrrolo[2,3- <1]pyrimidin-4-yl)- 1Hpyrazol-1- yl ]cyclobutyl}acetony tril . OH ΝΗ o ^N F ^F 0 Λ / Ν-Ν ^Ν Ι^> Η + >10 24 US 2014/0005166 (Example 14) {trans-b-(D-{[A({[(2R)-2hydroxypropyl] amino }methyl)-6(trifluoromethyl)pyridinium n-2- yl]oxy}piperidin-1 yl)-1-[4-(7Hpyrrolo[2,3- <1]pyrimidin-4-yl)- 1Hpyrazol-1- yl ]cyclobutyl}acetony tril /OH ΝΗ Of ο ^Ν f ^F 0 Ν-Ν ν ΤΑ Η + >10

- 12 046969

25 US 2014/0005166 (Example 15) {trans-3-(4-{[4-({ [(2S)2- hydroxypropyl]amino}methyl)-6- (trifluoromethyl)pyridium n-2- yl]oxy}piperidine-1 - yl)-1-[4-(7H- pyrrolo[2,3- <1]pyrimidin-4-yl)- 1Hpyrazol-1- yl ]cyclobutyl}acetony tril OH NH f o ^N F ^F Ν ΝΉ ^Ν τΛ and Λ / Ν Ν Η + >10 26 US 2014/0005166 (Example 20) {trans-3-(4-{ [4-(2hydroxyethyl)-6(trifluoromethyl)pyridinium-2- yl]oxy}piperidine-1 - yl)-1-[4-(7H- pyrrolo[2,3- <1]pyrimidin-4-yl)- 1Hpyrazol-1- yl ]cyclobutyl}acetony tril BUT ο ^Ν f ^F 0 у/ ΝΉ ^Ν τΛ Ν Ν Η + >10

+ Means <10 nM (for assay conditions see example A), ++ means <100 nM (for assay conditions see example A), +++ means <300 nM (for assay conditions see example A).

^a Data for enantiomer 1.

^b Data for enantiomer 2.

In some embodiments, the JAK1 inhibitor is {1-{1-[3-fluoro-2(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3H]pyrimidin-4- yl)-1H-pyrazol1-yl]azetidin-3-yl}acetonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is the adipate salt of {1-{1[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)-1Hpyrazol-1-yl]azetidin-3-yl}acetonitrile.

Synthesis and preparation of {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H -pyrazol-1-yl]azetidin-3-yl}acetonitrile and its adipic acid salt can be found, for example, in US Patent Publication No. 2011/0224190, filed March 9, 2011, US Patent Publication No. 2013/0060026, filed 6 September 2012, and US Patent Publication No. 2014/0256941, filed March 5, 2014, each of which is incorporated herein by reference in its entirety.

In some embodiments, the JAK1 inhibitor is 4-[3-(cyanomethyl)-3-(3',5'dimethyl-1H,1Ή-4,4'-bipyrazol-1-yl)azetidin-1-yl]- 2,5-difluoro-\-[( 1 S)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is 4-[3-(cyanomethyl)3-(3',5'-dimethyl-1H,1H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-phosphate 2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.

In some embodiments, the JAK1 inhibitor is 4-[3(cyanomethyl)-3-(3',5'-dimethyl-1H,1Ή-4,4'-bipyrazol-1-yl)azetidin-1-yl]- 2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.

In some embodiments, the JAK1 inhibitor is 4-[3(cyanomethyl)-3-(3',5'-dimethyl-1H,1Ή-4,4'-bipyrazol-1-yl)azetidin-1-yl]- hydrobromide 2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide.

- 13 046969

In some embodiments, the JAK1 inhibitor is 4-[3('cyanomethyl)-3-('3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1 sulfate -yl|-2,5-difluoro-X-[('^)-2,2,2-trifluoro-1-methylethyl]benzamide.

Synthesis and preparation of 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]2,5- difluoro-X-[('^)-2,2,2-trifluoro-1-methylethyl|benzamide and its phosphoric acid salt can be found, for example, in US Patent Publ. No. US 2014/0343030, filed May 16, 2014, which is incorporated herein by reference in its entirety.

In some embodiments, the JAK1 inhibitor is ((2R,5S)-5-{2-[(1R)-1hydroxyethyl]-1H-imidazo[4,5^]thieno[3,2-b]pyridin-1-yl }tetrahydro-2H-pyran-2-yl)acetonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is ((2R,5S)-5-{2-[(1R)-1hydroxyethyl]-1H-imidazo[4,5^]thieno[3,2-b]pyridin-1-yl }tetrahydro-2H-pyran-2-yl)acetonitrile monohydrate.

Synthesis of ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H -pyran-2-yl)acetonitrile and the characteristics of its anhydrous and monohydrate forms are described in US Patent Publ. No. 2014/0121198, filed October 31, 2013, and US Patent Publ. No. 2015/0344497, filed April 29, 2015, each of which is incorporated herein by reference in its entirety.

In some embodiments of the connection table. 1 is produced by synthetic methods described in US Patent Publ. No. 2011/0224190, filed March 9, 2011, US Patent Publication No. 2014/0343030, filed May 16, 2014, US Patent Publication No. 2014/0121198, filed October 31, 2013, US Patent Publication No. 2010/0298334, filed May 21, 2010, US Patent Publication No. 2011/0059951, filed August 31, 2010, US Patent Publication No. 2012/0149681, filed November 18, 2011, US Patent Publication No. 2012/0149682, filed November 18, 2011 ., US Patent Publication 2013/0018034, filed June 19, 2012, US Patent Publication No. 2013/0045963, filed August 17, 2012, and US Patent Publication No. 2014/0005166, filed May 17, 2013, each is incorporated herein by reference in its entirety.

In some embodiments, the JAK1 inhibitor is selected from the compounds or pharmaceutically acceptable salts thereof set forth in US Patent Publ. No. 2011/0224190, filed March 9, 2011, US Patent Publication No. 2014/0343030, filed May 16, 2014, US Patent Publication No. 2014/0121198, filed October 31, 2013, US Patent Publication No. 2010/0298334, filed May 21, 2010, US Patent Publication No. 2011/0059951, filed August 31, 2010, US Patent Publication No. 2012/0149681, filed November 18, 2011, US Patent Publication No. 2012/0149682, filed November 18, 2011 ., US Patent Publication 2013/0018034, filed June 19, 2012, US Patent Publication No. 2013/0045963, filed August 17, 2012, and US Patent Publication No. 2014/0005166, filed May 17, 2013, each is incorporated herein by reference in its entirety.

In some embodiments, the JAK1 inhibitor is a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein X is N or CH;

L represents C(=O) or C(=O)NH;

A is phenyl, pyridinyl or pyrimidinyl, each of which is optionally substituted with 1 or 2 independently selected R ¹ groups; and each R ¹ is fluoro or trifluoromethyl.

In some embodiments, the compound of formula I is {1-{1-[3-fluoro-2

- 14 046969 (trifluoromethyl)isonicotinoyl] piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol1-yl]azetidin-3-yl} acetonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula I is 4-{3-(cyanomethyl)-3[4-(7H-pyrrolo[2,3D]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1- yl}-K-[4-fluoro-2-(trifluoromethyl)phenyl]piperidine-1-carboxamide or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of formula I is [3-[4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-1-(1-{[2-( Fluoromethyl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)azetidin-3-yl]acetonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is a compound of Formula II

R ^e R ⁷

HN-N

II or a pharmaceutically acceptable salt thereof, wherein R ² is C _1-6 alkyl, C _1-6 haloalkyl, C _3-6 cycloalkyl or C _3-6 cycloalkyl-C _1-3 alkyl, wherein said Cp is dlkyl. C _3-6 cycloalkyl and C _3-6 cycloalkyl-C _1-6 alkyl are each optionally substituted with 1, 2 or 3 substituents independently selected from fluorine, -CF3 and methyl;

R ³ represents H or methyl;

R ⁴ represents H, F or Cl;

R ⁵ represents H or F;

R ⁶ represents H or F;

R ⁷ represents H or F;

R ⁸ represents H or methyl;

R ⁹ represents H or methyl;

R ¹⁰ represents H or methyl and

R ¹¹ represents H or methyl.

In some embodiments, the compound of Formula II is 4-[3-(cyanomethyl)3-(3',5'-dimethyl-1H,GN-4,4'-bipyrazol-1-yl)azetidin-1-yl]- 2,5-difluoro-K-[(^)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is a compound of Formula III

III or a pharmaceutically acceptable salt thereof, wherein Cy is a tetrahydro-2H-pyran ring which is optionally substituted with 1 or 2 groups independently selected from CN, OH, F, Cl, C _1-3 alkyl, C _1-3 haloalkyl, CN -C _1-3 alkyl, HO-Ci.3 alkyl, amino, C _1-3 alkylamino and di(C _1-3 alkyl)amino, where said C _1-3 alkyl and di^.3 alkyl)amino is optionally substituted with 1 , 2 or 3 substituents independently selected from F, Cl, C _1-3 alkylaminosulfonyl and C _1-3 alkylsulfonyl; And

R ¹² represents -CH2-OH, -CH(CH ₃ )-OH, or -CH2-NHSO2CH3.

In some embodiments, the compound of Formula III is ((2R,5S)-5-{2[(III)-1-hydroxyethyl]-1H-imidazo[4,5D]thieno[3,2-b]pyridin-1- yl}tetrahydro-2H-pyran-2-yl)acetonitrile or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 and/or JAK2 inhibitor is barcitinib, tofacitinib, oclacitinib, filgotinib, gandotinib, lesturnitib, momelotinib, bacritinib, PF04965842, upadacitinib, pecitinib, fedbitacin-501, cucurtinib (ATI-501), cucurtinib (ATI) - 502 (Aclaris), JTE052 (Leo Pharma and Japan Tobacco) or CHZ868.

In some embodiments, the JAK1 and/or JAK2 inhibitor may be isotopically labeled with

- 15 046969 compound or a pharmaceutically acceptable salt thereof. An isotopically or radiolabeled compound is a compound according to the disclosure in which one or more atoms are replaced or replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number commonly found in nature (i.e., naturally occurring ). Suitable radionuclides that may be included in the compounds of this disclosure include, but are not limited to, H (also referred to as D for deuterium), ^3H (also referred to as T for tritium),

11C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ¹⁸ F, ³⁵ S, ³⁶ Cl, ⁸² Br, ⁷⁵ Br, ⁷⁶ Br, ⁷⁷ Br, ¹²³ I, ¹²⁴ I, ¹²⁵ I and ¹³¹ I. For example, one or more hydrogen atoms in a compound of this disclosure may be replaced by deuterium atoms, for example, -CD ₃ is replaced by -CH ₃ ).

One or more of the constituent atoms of the compounds described herein may be replaced or replaced by isotopes of natural or non-natural frequency atoms. In some embodiments, the compound includes at least one deuterium atom. In some embodiments, the compound includes two or more deuterium atoms. In some embodiments, the compound includes 1-2, 1-3, 1-4, 1-5, or 1-6 deuterium atoms. In some embodiments, all of the hydrogen atoms in the compound may be replaced or replaced by deuterium atoms.

Synthetic methods for incorporating isotopes into organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, N.Y., Appleton-CenturyCrofts, 1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labeling by James R. Hanson, 2011). NMR spectroscopy, metabolism experiments and/or analyses.

Substitution with heavier isotopes such as deuterium may provide certain therapeutic benefits due to greater metabolic stability, such as increased in vivo half-life or reduced dosage requirements, and may therefore be preferable in some circumstances (see, e.g., A. Kerekes et al. J. Chem. 2011, 54, 201-210; R. Xu et al. In particular, replacement at one or more metabolic sites may provide one or more therapeutic benefits.

Accordingly, in some embodiments, the JAK1 and/or JAK2 inhibitor is a compound in which one or more hydrogen atoms in the compound are replaced by deuterium atoms, or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 and/or JAK2 inhibitor is ruxolitinib in which one or more hydrogen atoms are replaced by deuterium atoms, or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK1 and/or JAK2 inhibitor is any of the compounds in US Pat. No. 9,249,149 (which is incorporated herein by reference in its entirety) or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK1 and/or JAK2 inhibitor is CTP-543 or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound is a compound of Formula I or a pharmaceutically acceptable salt thereof, wherein R ¹ is selected from H and D, each R ² is independently selected from H and D, with the proviso that each R ² attached to the total carbon is one and the same, each R ³ is independently selected from H and D, provided that each R ³ attached to the total carbon is the same,

R ⁴ is selected from H and D, each R ⁵ is the same and is selected from H and D, R ⁶ , R ⁷ , and R ⁸ each independently

- 16 046969 selected from H and D; provided that when R ¹ is H, each R ² and each R ³ is H, R ⁴ is H, and each of R ⁶ , R ⁷ , and R ⁸ is H, then each R ⁵ is D .

In some embodiments, the JAK1 and/or JAK2 inhibitor is a compound of formula I selected from the following compounds 100-130 in the table below (where R ⁶ , R ⁷ and R ⁸ each represent H) or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK1 and/or JAK2 inhibitor is a compound of formula I selected from the following compounds 200-231 in the table below (wherein R ⁶ , R ⁷ and R ⁸ each represent D) or a pharmaceutically acceptable salt thereof.

Compound R ¹ Each R ² Each R ³ R ⁴ Each R ⁵ 100 N N N D N 101 N N n N D 102 N n n D D 103 N n D N N

- 17 046969

104 Η Η D D Η 105 Η Η D Η D 106 Η Η D D D 107 Η D Η Η Η 108 Η D Η D Η 109 Η D Η Η D BY Η D Η D D 111 Η D D Η Η 112 Η D D D Η 113 Η D D Η D 114 Η D D D D 115 D Η Η Η Η 116 D Η Η D Η 117 D Η Η Η D 118 D Η Η D D 119 D Η D Η Η 120 D Η D D Η 121 D Η D Η D 122 D Η D D D 123 D D Η Η Η 124 D D Η D Η 125 D D Η Η D 126 D D Η D D 127 D D D Η Η 128 D D D D Η 129 D D D Η D 130 D D D D D 200 Η Η Η D Η 201 Η Η Η Η D

- 18 046969

202 N N N D D 203 n N D N N 204 n N D D N 205 n N D N D 206 n N D D D 207 n D N N N 208 n D N D N 209 n D N N D 210 n D N D D 211 n D D N N 212 n D D D N 213 n D D N D 214 n D D D D 215 D N N N N 216 D N N D N 217 D N N N D 218 D N N D D 219 D N D N N 220 D N D D N 221 D N D N D 222 D N D D D 223 D D N N N 224 D D N D N 225 D D N N D 226 D D N D D 227 D D D N N 228 D D D D N 229 D D D N D 230 D D D D D 231 N N N N N

In some embodiments, the JAK1 and/or JAK2 inhibitor is baricitinib in which one or more hydrogen atoms are replaced by deuterium atoms, or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK1 and/or JAK2 inhibitor is any of the compounds in US Pat. No. 9,540,367 (which is incorporated herein by reference in its entirety) or a pharmaceutically acceptable salt thereof.

As used herein, the phrase optionally means unsubstituted or substituted. As used herein, the term substituted means that a hydrogen atom has been removed and replaced by a substituent. It should be understood that substitution at a given atom is limited by valence.

As used herein, the term C _n -m alkyl, used alone or in combination

- 19 046969 tion with other terms, refers to a saturated hydrocarbon group, which may be linear or branched, having from n to m carbon atoms. In some embodiments, the alkyl group contains from 1 to 6 or from 1 to 3 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, 2-methyl-1butyl, 3 -pentyl, n-hexyl, 1,2,2-trimethylpropyl and the like.

As used herein, the term alkylene, used alone or in combination with other terms, refers to a divalent alkyl linking group, which may be branched or linear, where two substituents may be attached at any position of the alkylene linking group. Examples of alkylene groups include, but are not limited to, ethane1,2-diyl, propane-1,3-diyl, propane-1,2-diyl and the like.

As used herein, the term HO-Ci-3-alkyl refers to a group of the formula alkylene-OH, wherein said alkylene group has from 1 to 3 carbon atoms.

As used herein, the term C#Cl _-3 alkyl refers to C1. ₃ alkyl substituted with a cyano group.

As used herein, the term amino refers to a group of the formula -NH ₂ .

As used herein, the term di(C _1-3 -alkyl)amino refers to a group of the formula -N(alkyl)2, in which the two alkyl groups each have, independently, from 1 to 3 carbon atoms

As used herein, the term _C1-3 alkylamino refers to a group of the formula -^H(alkyl) in which the alkyl group has from 1 to 3 carbon atoms.

As used herein, the term di(C1 _-3 alkyl)aminosulfonyl refers to a group of the formula A(O) ₂ Malkyl) ₂ . in which each alkyl group independently has from 1 to 3 carbon atoms.

As used herein, the term _C1-3 alkylsulfonyl refers to a group of the formula 8(O)2-alkyl, wherein the alkyl group has from 1 to 3 carbon atoms.

As used herein, halo or halogen, used alone or in combination with other terms, includes fluorine, chlorine, bromine and iodine. In some embodiments, the halogen group is fluorine or chlorine.

As used herein, the term _Cnm haloalkyl, used alone or in combination with other terms, refers to a _Cnm alkyl group having up to {2(n to m)+1} halogen atoms, which may be the same or different. In some embodiments, the halogen atoms are fluorine atoms. In some embodiments, the alkyl group has 1-6 or 1-3 carbon atoms. Examples of haloalkyl groups include CF3, C2F5, CHF2, CC13, _CHCl2 , C2C15 and the like. In some embodiments, the haloalkyl group is a fluoroalkyl group.

As used herein, the term C1 _-3 fluoroalkyl refers to a C1 _-3 alkyl group that may be partially or completely replaced by fluorine atoms.

As used herein, the term C _3-6 cycloalkyl, used alone or in combination with other terms, refers to a non-aromatic monocyclic hydrocarbon group having 3-6 carbon atoms, which may optionally contain one or more alkenylene groups as part of the ring structure. One or more ring-forming carbon atoms of a cycloalkyl group can be oxidized to form carbonyl bonds. Exemplary C _3-6 cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, and the like. In some embodiments, the cycloalkyl group is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

As used herein, the term C _3-6 cycloalkyl-C 1 _-3 alkyl refers to a group of the formula -C 1 _-3 alkylene-C _3-6 cycloalkyl.

The compounds described herein may be asymmetric (eg, have one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended to be used unless otherwise noted. Compounds that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically inactive starting materials are known in the art, such as resolution of racemic mixtures or stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, etc. may also be present in the compounds described herein, and all such stable isomers are contemplated herein. Cis and trans geometric isomers of the compounds of this application are described and can be isolated as a mixture of isomers or as separate isomeric forms. In some embodiments, the compound has a (Reconfiguration. In some embodiments, the compound has a ^ configuration.

Separation of racemic mixtures of compounds can be accomplished by any of numerous methods known in the art. A typical method involves fractional recrystallization using a chiral resolving acid, which is an optically active salt-forming organic acid. Suitable separating agents for fractional recapitulation methods

- 20 046969 Stallizations are, for example, optically active acids such as the D- and L-forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or various optically active camphorsulfonic acids such as P-camphorsulfonic acid . Other resolving agents suitable for fractional crystallization methods include stereoisomerically pure forms of α-methylbenzylamine (e.g., S- and R-forms or diastereomerically pure forms), 2-phenylglycine, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2- diaminocyclohexane, and the like.

The separation of racemic mixtures can also be accomplished by elution on a column packed with an optically active resolving agent (eg dinitrobenzoylphenylglycine). The appropriate composition of the elution solvent can be determined by one skilled in the art.

The compounds described herein include tautomeric forms. Tautomeric forms arise from the exchange of a single bond with an adjacent double bond, together with concomitant migration of a proton. Tautomeric forms include prototropic tautomers, which are isomeric protonation states having the same empirical formula and net charge. Examples of prototropic tautomers include ketone-enol pairs, amide-imidic acid pairs, lactam-lactim pairs, enamine-imine pairs, and ring forms where the proton can occupy two or more positions in the heterocyclic system, for example, 1H- and 3H-imidazole, 1H- , 2H- and 4H-1,2,4-triazole, 1H and 2H-isoindole and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. For example, it will be appreciated that the following pyrazole ring can form two tautomers:

MVMV aww

ΗΝ-Ν Ν-ΝΗ

The claims are intended to cover both tautomers.

All compounds and their pharmaceutically acceptable salts can be found together with other substances, such as water and solvents (eg, hydrates and solvates), or can be isolated.

In some embodiments, the compounds described herein, or salts thereof, are substantially isolated. By substantially isolated is meant that the compound is at least partially or substantially isolated from the environment in which it was formed or detected. The partial separation may include, for example, a composition enriched with the compounds described herein. Substantial separation may include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97% or at least about 99% by weight of the compounds described herein, or a salt thereof. Methods for isolating compounds and their salts are well known in the art.

The phrase pharmaceutically acceptable is used herein to designate those compounds, materials, compositions and/or dosage forms that, within the bounds of sound medical judgment, are suitable for use in contact with tissues of humans and animals without excessive toxicity, irritation, allergic reaction or other problem. or complications commensurate with a reasonable benefit/risk ratio.

The expressions ambient temperature and room temperature or rt, as used herein, are understood in the art and generally refer to a temperature, for example a reaction temperature, that is close to the temperature of the room in which the reaction is carried out, for example a temperature from about 20°C to about 30°C.

This application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, pharmaceutically acceptable salts refer to derivatives of the described compounds in which the parent compound is modified by converting an existing acidic or basic moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids of basic moieties such as amines; salts of basic cations and organic salts of acidic residues such as carboxylic acids; etc. Pharmaceutically acceptable salts herein include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of this application can be synthesized from a starting compound that contains a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture of both; generally, non-aqueous media such as ether, ethyl acetate, alcohols (eg, methanol, ethanol, isopropanol or butanol) or acetonitrile (ACN) are preferred. Lists of suitable salts

- 21 046969 can be found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.

As used herein, the term contacting refers to the combination of these moieties in an in vitro system or in an in vivo system. For example, contacting a JAK with a compound of the invention involves administering a compound of this application to an individual or patient, such as a human, having a JAK, as well as, for example, administering a compound of the invention to a sample containing a cell or purified preparation containing a JAK.

As used herein, the term subject, individual or patient, used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, pigs, cattle, sheep, horses , or primates and most preferably humans. In some embodiments, the subject, individual, or patient is in need of said treatment.

In some embodiments, the inhibitors are administered in a therapeutically effective amount. As used herein, the phrase therapeutically effective amount refers to the amount of an active compound or pharmaceutical agent that produces a biological or drug response that is desired in a tissue, system, animal, individual or human by a researcher, veterinarian, family physician or other clinician.

As used herein, the term treatment or therapy refers to one or more of: (1) suppression of a disease; for example, suppressing a disease, condition or disorder in an individual who is experiencing or exhibiting the pathology or symptoms of the disease, condition or disorder (ie, stopping further development of the pathology and/or symptoms); (2) alleviation of disease; for example, alleviating a disease, condition or disorder in an individual who experiences or exhibits pathology or symptomatology of the disease, condition or disorder (ie, reversal of the pathology and/or symptomatology), such as reducing the severity of the disease; or (3) preventing a disease, condition or disorder in a person who may be susceptible to the disease, condition or disorder but is not yet experiencing or exhibiting the disease pathology or symptoms. In some embodiments, treatment relates to suppressing or alleviating a disease. In some embodiments, the treatment is the prevention of a disease.

Combined treatment methods.

The methods described herein may further include administering one or more additional therapeutic agents. One or more additional therapeutic agents can be administered to the patient simultaneously or sequentially.

In some embodiments, the additional therapeutic agent is an antibiotic. In some embodiments, the antibiotic is clindamycin, doxycycline, minocycline, trimethoprim-sulfamethoxazole, erythromycin, metronidazole, rifampin, moxifloxacin, dapsone, or a combination thereof. In some embodiments, the antibiotic is clindamycin, doxycycline, minocycline, trimethoprim-sulfamethoxazole, or erythromycin in combination with metronidazole. In some embodiments, the antibiotic is a combination of rifampin, moxifloxacin, and metronidazole. In some embodiments, the antibiotic is a combination of moxifloxacin and rifampin.

In some embodiments, the additional therapeutic agent is a retinoid. In some embodiments, the retinoid is etretinate, acitretin, or isotretinoin.

In some embodiments, the additional therapeutic agent is a steroid. In some embodiments, the additional therapeutic agent is a corticosteroid. In some embodiments, the steroid is triamcinolone, dexamethasone, fluocinolone, cortisone, prednisone, prednisolone, or flumetholone.

In some embodiments, the additional therapeutic agent is an anti-TNF-alpha agent. In some embodiments, the anti-TNF-alpha agent is an anti-TNF-alpha antibody. In some embodiments, the anti-TNF-alpha agent is infliximab or etanercept or adalimumab.

In some embodiments, the additional therapeutic agent is an immunosuppressant. In some embodiments, the immunosuppressant is methotrexate or cyclosporine A. In some embodiments, the immunosuppressant is mycophenolate mofetil or sodium mycophenolate.

In some embodiments, the additional therapeutic agent is finasteride, metformin, adapalene, or azelaic acid.

In some embodiments, the method further includes administering an additional therapeutic agent selected from IMiDs, an anti-IL-6 agent, a hypomethylation agent, and a biological response modifier (BRM).

- 22 046969

Generally, BRMs are substances obtained from living organisms to treat diseases that may occur naturally in the body or may be produced in a laboratory. Examples of BRMs include IL-2, interferon, various types of colony-stimulating factors (CSF, GM-CSF, G-CSF), monoclonal antibodies such as abciximab, etanercept, infliximab, rituximab, trasturzumab and high doses of ascorbate.

In some embodiments, the hypomethylation agent is a DNA methyltransferase inhibitor. In some embodiments, the DNA methyltransferase inhibitor is selected from 5azacytidine and decitabine.

Typically, IMiD is an immunomodulatory agent. In some embodiments, the IMiD is selected from thalidomide, lenalidomide, pomalidomide, CC-11006, and CC-10015.

In some embodiments, the method further includes administering an additional therapeutic agent selected from antithymocyte globulin, recombinant human granulocyte colony-stimulating factor (G CSF), granulocyte-monocyte CSF (GMCSF), an erythropoiesis-stimulating agent (ESA), and cyclosporine

In some embodiments, the method further includes administering an additional JAK inhibitor to the patient. In some embodiments, the additional JAK inhibitor is barcitinib, tofacitinib, oclacitinib, filgotinib, gandotinib, lesturtinib, momelotinib, bacritinib, PF-04965842, upadacitinib, pecitinib, fedratinib, cucurbitacin I, or CHZ868.

One or more additional pharmaceutical agents, such as, for example, anti-inflammatory agents, immunosuppressants, and PI3I<i5 kinase inhibitors. mTor, Bcr-Ab1, Flt-3, RAF and FAK, such as, for example, those described in WO 2006/056399, which is incorporated herein by reference in its entirety, or other agents may be used in combination with the compounds described herein, for the treatment of JAK-associated diseases, disorders or conditions. One or more additional pharmaceutical agents can be administered to the patient simultaneously or sequentially.

Examples of Bcr-Ab1 inhibitors include compounds and pharmaceutically acceptable salts thereof of the genera and species disclosed in U.S. Pat. Pat. No. 5521184, WO 04/005281 and U.S. Ser. No. 60/578491, all of which are incorporated herein by reference in their entirety.

Examples of suitable Flt-3 inhibitors include the compounds and their pharmaceutically acceptable salts as described in WO 03/037347, WO 03/099771 and WO 04/046120, all of which are incorporated herein by reference in their entirety.

Examples of suitable RAF inhibitors include the compounds and their pharmaceutically acceptable salts as described in WO 00/09495 and WO 05/028444, both of which are incorporated herein by reference in their entirety.

Examples of suitable FAK inhibitors include the compounds and their pharmaceutically acceptable salts as described in WO 04/080980, WO 04/056786, WO 03/024967, WO 01/064655, WO 00/053595 and WO 01/014402, all of which are fully included into this document by reference.

In some embodiments, one or more compounds of this invention can be used in combination with one or more other kinase inhibitors, including imatinib, particularly for the treatment of patients resistant to imatinib or other kinase inhibitors.

In some embodiments, the additional therapeutic agent is fluocinolone acetonide (Retisert®) or rimexolone (AL-2178, Vexol, Alcon).

In some embodiments, the additional therapeutic agent is cyclosporine (Restasis®).

In some embodiments, the additional therapeutic agent is selected from Dehydrex™ (Holies Labs), Civamide (Opko), sodium hyaluronate (Vismed, Lantibio/TRB Chemedia), cyclosporine (ST603, Sirion Therapeutics), ARG101 (T) (testosterone, Argentis), AGR1012 (P) (Argentis), ecabeta sodium (Senju-Ista), gefarnate (Santen), 15-^)-hydroxyeicosatetraenoic acid (15(S)-HETE), sevilemine, doxycycline (ALTY-0501, Alacrity), minocycline, iDestrin™ (NP50301, Nascent Pharmaceuticals), cyclosporine A (Nova22007, Novagali), oxytetracycline (duramycin, MOLI1901, Lantibio), CF101 (2S,3S,4R,5R)-3,4-dihydroxy-3,4-dihydroxy [6 -[(3-iodophenyl)methylamino]purin-9-yl]-N-methyloxolan-2-carbamyl, Cap-Fite Biopharma), voclosporin (LX212 or LX214, Lux Biosciences), ARG103 (Agentis), RX-10045 (synthetic resolvin analogue, Resolvyx), DYN15 (Dyanmis Therapeutics), rivoglitazone (DE011, Daiichi Sanko), TB4 (RegeneRx), OPH-01 (Ophtalmis Monaco), PCS101 (Pericor Science), REV1-31 (Evolutec), Lacritina (Senju) , rebamipide (Otsuka-Novart), OT-551 (Othera), PAI-2 (University of Pennsylvania and Temple University), pilocarpine, tacrolimus, pimecrolimus (AMS981, Novartis), loteprednol etabonate, rituximab, tetrasodium diquafosol (INS365, Inspire), KLS-0611 (Kissei Pharmaceuticals), dehydroepiandrosterone, anakinra, efalizumab, mycophenolate sodium, etanercept (Embrel®), hydroxychloroquine, NGX267 (TorreyPines Therapeutics), Actemra, gemcitabine, oxaliplatin, L-asparaginase, or thalidomide.

In some embodiments, the additional therapeutic agent is an

- 23 046969 thiangiogenic agent, cholinergic agonist, TRP-1 receptor modulator, calcium channel blocker, mucin secretagogue, MUC1 stimulator, calcineurin inhibitor, corticosteroid, P2Y2 receptor agonist, muscarinic receptor agonist, mTOR inhibitor, other JAK inhibitor, Bcr kinase inhibitor- Ab1, Flt-3 kinase inhibitor, RAF kinase inhibitor and FAK kinase inhibitor, such as, for example, those described in WO 2006/056399, which is incorporated herein by reference in its entirety. In some embodiments, the additional therapeutic agent is a tetracycline derivative (eg, minocycline or doxyline). In some embodiments, the additional therapeutic agent binds to FKBP12.

In some embodiments, the additional therapeutic agent is an alkylating agent or DNA cross-linking agent; antimetabolite/demethylating agent (eg, 5-fluorouracil, capecitabine, or azacitidine); antihormonal therapy (eg, hormone receptor antagonists, SERMs, or aromatase inhibitor); a mitotic inhibitor (eg, vincristine or paclitaxel); topoisomerase inhibitor (I or II) (eg, mitoxantrone and irinotecan); apoptosis inducers (for example, ABT-737); nucleic acid therapy (eg, antisense or RNAi); nuclear receptor ligands (eg agonists and/or antagonists: all-trans retinoic acid or bexarotene); epigenetic targeting agents such as histone deacetylase inhibitors (eg, vorinostat), hypomethylating agents (eg, decitabine); protein stability regulators such as Hsp90 inhibitors, ubiquitin and/or ubiquitin-like conjugating or deconjugating molecules; or an EGFR inhibitor (erlotinib).

In some embodiments, the additional therapeutic agent includes an antibiotic, antiviral, antifungal, anesthetic, anti-inflammatory agent, including steroidal and non-steroidal anti-inflammatory agents, and antiallergic agents. Examples of suitable drugs include aminoglycosides such as amikacin, gentamicin, tobramycin, streptomycin, netilmicin and kanamycin; fluoroquinolones such as ciprofloxacin, norfloxacin, ofloxacin, trovafloxacin, lomefloxacin, levofloxacin and enoxacin; naphthyridine; sulfonamides; polymyxin; chloramphenicol; neomycin; paramomycin; colistimethate; bacitracin; vancomycin; tetracyclines; rifampin and its derivatives (rifampins); cycloserine; beta-lactams; cephalosporins; amphotericins; fluconazole; flucytosine; natamycin; miconazole; ketoconazole; corticosteroids; diclofenac; flurbiprofen; ketorolac; suprofen; cromolyn; boatsamide; levocabastine; naphazoline; antazoline; pheniramine; or azalide antibiotic.

Pharmaceutical compounds and dosage forms.

When used as pharmaceuticals, the compounds of the invention can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the pharmaceutical art and can be administered in a variety of ways depending on whether local or systemic treatment is required and the area being treated. Administration may be topical (including transdermal, epidermal, ophthalmic and mucosal, including intranasal, vaginal and rectal), pulmonary (eg, by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal), orally or parenterally. Parenteral administration includes intravenous, intra-arterial, subcutaneous, intraperitoneal, intramuscular, or injection or infusion; or intracranial, for example, intrathecal or intraventricular administration. Parenteral administration may be in the form of a single bolus dose or may be administered, for example, using a continuous infusion pump. Pharmaceutical and topical compositions may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners, etc. may be necessary or desirable.

In some embodiments, the administration is a local administration. In some embodiments, administration is a topical application to the skin.

In some embodiments, administration is oral administration.

The present invention also includes pharmaceutical compositions that contain as an active ingredient a compound of the invention or a pharmaceutically acceptable salt thereof in combination with one or more pharmaceutically acceptable carriers (excipients). In some embodiments, the composition is suitable for topical use. In preparing the compositions of this invention, the active ingredient is typically mixed with an excipient, diluted with an excipient, or enclosed in such a carrier, for example, in the form of a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it may be a solid, semi-solid or liquid material that acts as a carrier medium, carrier or vehicle for the active ingredient. Thus, the compositions may be in the form of tablets, pills, powders, lozenges, sachets, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosols (solid or liquid), ointments, containing, for example, up to 10% by weight active compound, soft and hard gelatin capsules, suppositories, sterile injection solutions and powders in sterile packaging.

When forming the composition of the drug, the active compound can be crushed to provide

- 24 046969 making particles of suitable size before combining with other ingredients. If the active compound is substantially insoluble, it can be ground to a particle size of less than 200 mesh. If the active compound is substantially soluble in water, the particle size can be adjusted by grinding to ensure substantially uniform distribution in the formulation, for example, about 40 mesh.

The compounds of the invention can be crushed using known grinding procedures, such as wet grinding, to obtain a particle size suitable for the formation of tablets and other types of formulations. Fine (nanoparticle) preparations of the compounds of the invention can be prepared by methods known in the art, for example, see International Application No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum arabic, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. The compositions may further include: lubricants such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservatives such as methyl and propyl hydroxybenzoates; sweeteners; and flavors. The compositions of the invention can be formulated to provide rapid, sustained or delayed release of the active ingredient following administration to a patient using procedures known in the art.

In some embodiments, the pharmaceutical composition contains silicified microcrystalline cellulose (SMCC) and at least one compound described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the silicated microcrystalline cellulose contains about 98% microcrystalline cellulose and about 2% silica by weight.

In some embodiments, the composition is a sustained release composition containing at least one compound described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In some embodiments, the composition contains at least one compound described herein herein, or a pharmaceutically acceptable salt thereof, and at least one component selected from microcrystalline cellulose, lactose monohydrate, hydroxypropyl methylcellulose and polyethylene oxide. In some embodiments, the composition contains at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate, and hydroxypropyl methylcellulose. In some embodiments, the composition contains at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate, and polyethylene oxide. In some embodiments, the composition further comprises magnesium stearate or silica. In some embodiments, the microcrystalline cellulose is Avicel PHI02™. In some embodiments, the lactose monohydrate is Fast-flo 316™. In some embodiments, the hydroxypropyl methylcellulose is hydroxypropyl methylcellulose 2208 K4M (eg, Methocel K4 M Premier™) and/or hydroxypropyl methylcellulose 2208 K100LV (eg, Methocel K00LV™). In some embodiments, the polyethylene oxide is polyethylene oxide WSR 1105 (eg, Polyox WSR 1105™).

In some embodiments, a wet granulation process is used to produce the composition. In some embodiments, a dry granulation process is used to prepare the composition.

The compositions may be formulated in unit dosage form, with each dose containing from about 1 to about 1000 mg, from about 1 mg to about 100 mg, from 1 mg to about 50 mg, and from about 1 mg to 10 mg of active ingredient. Preferably, the dose is from about 1 mg to about 50 mg or from about 1 mg to about 10 mg of active ingredient. In some embodiments, each dose contains about 10 mg of active ingredient. In some embodiments, each dose contains about 50 mg of active ingredient. In some embodiments, each dose contains about 25 mg of active ingredient. The term unit dosage dosage forms refers to physically discrete units suitable as unitary dosages for humans and other mammals, each unit containing a predetermined amount of active material calculated to produce the desired therapeutic effect, in combination with a suitable pharmaceutical excipient.

In some embodiments, the compositions contain from about 1 to about 1000 mg, from about 1 mg to about 100 mg, from 1 mg to about 50 mg, and from about 1 mg to 10 mg of active ingredient. Preferably, the compositions contain from about 1 mg to about 50 mg, or from about 1 mg to about 10 mg of active ingredient. One skilled in the art will understand that this embodies compounds or compositions containing from about 1 mg to about 10 mg, from about 1 mg to about 20 mg, from about 1 mg to

- 25 046969 about 25 mg, from about 1 mg to about 50 mg of active ingredient.

In some embodiments, the dosage of the compound or a pharmaceutically acceptable salt thereof is 15, 30, 60, or 90 mg based on the free base. In some embodiments, the dosage is 15, 30, 60, or 90 mg, based on the free base of Compound 4 or a pharmaceutically acceptable salt thereof. In some embodiments, the dosage of the compound or a pharmaceutically acceptable salt thereof is 15 mg based on the free base. In some embodiments, the dosage of the compound or a pharmaceutically acceptable salt thereof is 30 mg based on the free base. In some embodiments, the dosage of the compound or a pharmaceutically acceptable salt thereof is 60 mg based on the free base. In some embodiments, the dosage of the compound or a pharmaceutically acceptable salt thereof is 90 mg based on the free base.

The active compound can be effective over a wide range of dosages and is typically administered in a pharmaceutically effective amount. However, it should be understood that the actual amount of compound administered is generally determined by the physician in accordance with the relevant circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, age, weight, response of the individual patient, severity of the patient's symptoms, and the like.

To prepare solid compositions, such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation of a composition containing a homogeneous mixture of the compound of this application. When referring to these preformulated compositions as homogeneous, the active ingredient is typically uniformly dispersed throughout the composition so that the composition can be readily divided into equally effective unit dosage forms such as tablets, pills and capsules. These solid preformulations are divided into unit dosage forms of the type described above, containing, for example, from about 0.1 to about 1000 mg of the active ingredient of this application.

The tablets or pills of this invention may be coated or otherwise formulated to provide a dosage form that provides the benefit of a sustained release. For example, a tablet or pill may contain an internal dose component and an external dose component, the latter being in the form of a coating covering the former. The two components may be separated by an enteric layer, which serves to prevent disintegration in the stomach and allows the inner component to pass into the duodenum intact or with delayed release. A variety of materials can be used for such enteric layers or coatings, including a number of polymer acids and mixtures of polymer acids with materials such as shellac, cetyl alcohol and cellulose acetate.

Liquid forms in which the compounds and compositions of this application may be formulated for oral or injectable administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil or peanut butter, as well as elixirs and similar pharmaceutical carrier vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof and powders. Liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. The compositions can be nebulized using inert gases. Nebulized solutions can be inhaled directly from the nebulization device, or the nebulization device can be attached to the cover of a face mask or intermittent positive pressure breathing apparatus. The compositions, in the form of a solution, suspension or powder, can be administered orally or nasally using devices that deliver the composition in a suitable manner.

Formulations for topical use may contain one or more conventional carriers. In some embodiments, ointments may contain water and one or more hydrophobic carriers selected, for example, from liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white petrolatum, and the like. Cream carrier compositions may be water based in combination with glycerin and one or more other components, for example glycerol monostearate, PEG-glycerol monostearate and cetyl stearyl alcohol. Gels can be formulated using isopropyl alcohol and water, suitably in combination with other components such as, for example, glycerin, hydroxyethylcellulose and the like. In some embodiments, the topical formulations contain at least about 0.1, at least about 0.25, at least about 0.5, at least about 1, at least about 2, or at least about 5 % wt. compounds according to the invention. Formulations for topical use may be suitably packaged in tubes, for example, 100 g, which are optionally associated with instructions for the treatment of the selected indication, for example, psoriasis or other skin condition.

The amount of compound or composition administered to a patient will vary depending

- 26 046969 from what is administered, the purpose of administration, such as prophylaxis or therapy, the condition of the patient, the route of administration, etc. For therapeutic purposes, the compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially relieve the symptoms of the disease and its complications. Effective dosages will depend on the disease state being treated as well as the judgment of the attending physician depending on factors such as the severity of the disease, the age, weight and general condition of the patient, and the like.

Compositions administered to a patient may be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization methods or can be sterilized by filtration. Aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile aqueous vehicle before administration. The pH of the formulations of the compounds is typically from 3 to 11, more preferably from 5 to 9 and most preferably from 7 to 8. It will be understood that the use of some of the above excipients, carriers or stabilizers will result in the formation of pharmaceutical salts.

The therapeutic dosage of a compound herein may vary depending, for example, on the specific application being treated, the route of administration of the compound, the health and condition of the patient, and the prescribing judgment of the attending physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition may vary depending on a number of factors, including dosage, chemical characteristics (eg, hydrophobicity), and route of administration. For example, the compounds of this invention can be presented in an aqueous physiological buffer solution containing from about 0.1 to about 10% w/v. compounds for parenteral administration. Some typical dosage ranges are from about 1 mcg/kg to about 1 g/kg body weight per day. In some embodiments, the dosage range is from about 0.01 mg/kg to about 100 mg/kg body weight per day. Dosage will likely depend on variables such as the type and degree of progression of the disease or disorder, the general health of the individual patient, the relative biological effectiveness of the selected compound, the composition of the excipient, and the route of administration. Effective doses can be extrapolated from dose-response curves obtained from in vitro test systems or animal models.

The compositions of the invention may further include one or more additional pharmaceutical agents, examples of which are listed above.

Sets.

This application also includes pharmaceutical kits that can be used, for example, for the treatment and/or prevention of cytokine-related diseases or disorders, such as CRS, which include one or more containers containing a pharmaceutical composition containing a therapeutically effective amount of a compound described herein . Such kits may further include, if desired, one or more of various conventional pharmaceutical kit components, such as, for example, containers containing one or more pharmaceutically acceptable carriers, additional containers, and the like, as will be apparent to those skilled in the art. . The kit may also include instructions in the form of inserts or labels indicating the quantities to be administered, instructions for use and/or instructions for mixing the components.

Examples

The invention will be described in more detail using specific examples. The following examples are offered for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize many non-critical parameters that can be changed or modified to obtain substantially the same results.

Example A: In vitro JAK kinase assay.

JAK1 inhibitors that can be used to treat diseases or disorders associated with cytokines are tested for the inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains of human JAK1 (aa 837-1142), JAK2 (aa 828-1132) and JAK3 (aa 781-1124) with an N-terminal His tag are expressed using baculovirus in insect cells and purified . The catalytic activity of JAK1, JAK2, or JAK3 was analyzed by measuring the phosphorylation of the biotinylated peptide. Phosphorylated peptide was detected using homogeneous time-resolved fluorescence (HTRF). The IC ₅₀ of compounds is measured for each kinase in 40 μl reactions that contain enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT and 0.1 mg/ml (0 .01%). B.S.A. For 1 mM IC ₅₀ measurements, the ATP concentration in the reactions is 1 mM. Reactions are carried out at room temperature for 1 hour and then stopped with 20 μl of 45 mM EDTA, 300 nM SA-APC, 6 nM Ei.i-Pv20 in assay buffer (Perkin Elmer, Boston, MA) Antibody binding. labeled with europium occurs within 40 min, and the HTRF signal is measured on a Fusion plate reader (Perkin Elmer, Boston, MA). Connections in the table 1 were tested in this assay and were shown to have the IC ₅₀ values in Table. 1.

Example B: Study of the Safety and Efficacy of JAK1 and/or JAK2 Inhibitors in Subjects

- 27 046969 with moderate to severe hidradenitis suppurativa.

A randomized, double-blind, placebo-controlled, multicenter trial is being conducted in men and women aged 18–75 years with moderate (Hurley stage II) to severe (Hurley stage III) hidradenitis suppurativa for at least 6 months. Stage I Hurley is associated with the formation of an abscess (single or multiple) without sinus passages and scarring. Hurley stage II is associated with recurrent abscesses with tract formation and scarring; single or multiple, widely separated lesions. Hurley stage III is associated with diffuse or near-diffuse disease or multiple interconnected tracts and abscesses throughout the area. Study participants were randomized into 5 groups (approximately 50 participants per group) and received 15, 30, 60, or 90 mg of a JAK1 and/or JAK2 inhibitor (eg, ruxolitinib, Compound 4 or Compound 5, or a pharmaceutically acceptable salt thereof) or placebo. At week 16 (the primary endpoint), participants in the placebo group are re-randomized equally to active treatment for 8 weeks. Double blind method supported. The primary endpoint is the proportion of subjects achieving HiSCR (Hidradenitis Suppurativa Clinical Response) at week 16.

Secondary endpoints include: (1) proportion of subjects with HiSCR compared to baseline at each visit; (2) the proportion of subjects with abscess and inflammatory nodule (AN) counts between 0 and 2 at each visit; (3) mean deviation from baseline on the HS1 pain rating scale at each visit; (4) change in modified Sartorius score at 16 and 24 weeks; (5) change in the number of draining fistulas at each visit; (6) the proportion of subjects requiring reserve treatment of lesions before week 24; (7) number of lesion repair episodes up to week 24; (8) population PK of the JAK1 and/or JAK2 inhibitor (eg, apparent clearance, apparent volume of distribution); (9) safety and tolerability assessed by monitoring the frequency, duration, and severity of AEs, physical examination, vital signs, and laboratory data for hematology, serum chemistry, and urinalysis; (10) assessment of changes in the dermatology quality of life index (DLQI Dermatology Quality of Life Index); (11) change from baseline in IHS43 disease severity at each visit; (12) change in hidradenitis suppurativa quality of life (HiSQOL) score at each visit compared to baseline; and (13) assessing dose/exposure-response relationships for percentage change from baseline in terms of efficacy and safety endpoints across treatment periods.

HiSCR is defined as a reduction of at least 50% in the number of abscess and inflammatory nodule (AN - abscess and inflammatory nodule) without an increase in the number of abscesses and no increase in the number of draining fistulas at week 16 compared to baseline). A numerical pain rating scale is used to evaluate worst cutaneous and average cutaneous pain due to HS. Scores for the two items range from 0 (no skin pain) to 10 (skin pain as bad as you can imagine). Ratings are recorded by participants in a daily diary before bed and are based on the recall period over the past 24 hours. The Modified Sartorius Scale is used to quantify the severity of HS. Points are awarded for 12 areas of the body (left and right axilla, left and right sub/inframammary areas, intermammary area, left and right buttocks, left and right inguinal-femoral folds, perianal area, perineal area and others): points are awarded for the nodule (2 points each); abscesses (4 points); fistulas (4 points); scar (1 point); and the greatest distance between two damages (2-6 points, 0 if there are no damages); and whether the lesions are separated by normal skin (yes - 0 points; no - 6 points). The overall Sartorius scale is the sum of 12 regional ratings. Life-saving therapy for the lesion: In case the acutely painful lesion requires immediate intervention, doctors can perform life-saving interventions. Only two types of interventions are permitted: (1) intralesional injection of triamcinolone acetonide suspension (up to a total of 30 mg per visit) and/or (2) incision and drainage. The intervention can be performed on a maximum of two different lesions at one visit or on the same lesion at two different study visits. The same lesion cannot be treated twice in one visit. If a subject requires more than two interventions before week 16, they are excluded from the study. International Hidradenitis Suppurativa Severity Scoring System (IHS4): IH4 (score) = (number of nodules x 1) + (number of abscesses x 2) + (number of drainage tunnels [fistulas/sinuses] x 4). Mild HS: < 3 points; Moderate HS: 4-10 points; Severe HS: > 11 points.

Study treatment 1 (active) includes an oral tablet containing 15 mg 4-[3(cyanomethyl)-3-(3',5'-dimethyl-1H,1Ή-4,4'-bipyrazol-1-yl)azetidin-1 -yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide. Dosage levels include 15 mg (1 tablet), 30 mg (2 tablets), 60 mg (4 tablets), and 90 mg (6 tablets). Study treatment 2 (placebo) includes oral placebo tablets.

Blood samples to measure plasma concentrations of JAK1 and/or JAK2 inhibitor are collected at the

- 28 046969 time points at least 2, 12, 16, 20 and 24 weeks before and after administration of the study drug before dosing, 1 hour after dosing, and 2-5 hours after dosing. At the early withdrawal visit, if subjects discontinue treatment before week 8, collect a PK sample if possible. The date/time of the last previous dose is also recorded.

Tests for superiority of JAK1 and/or JAK2 inhibitor doses of 90, 60, 30 and 15 mg compared to placebo were performed using the Hochberg procedure with an overall two-sided α=0.05. Comparisons between each active group and placebo at week 16 are performed using logistic regression. At all dose levels, the superiority tests are significant (e.g., 10%, 20%, 30%, 40%, or 50% improvement in HiSCR (Hidradenitis Suppurativa Clinical Response)) and demonstrate the effectiveness of a JAK1 and/or JAK2 inhibitor for the treatment of HS. Tests show a reduction in the number of nodules and non-inferiority/superiority compared to placebo.

All secondary and exploratory effectiveness measures are assessed using descriptive statistics. Clinical safety data (vital signs, routine laboratory tests, and AEs) were analyzed using descriptive statistics. Exposure-response relationships (E-R) between PK exposures of plasma JAK1 and/or JAK2 inhibitor and efficacy/safety data are determined. An interim analysis to assess response to treatment and facilitate planning of future studies is conducted when at least half of the randomized subjects reach week 16.

Example C. Janus kinase expression in keratinocytes induced by interferon-gamma and tumor necrosis factor-alpha and subsequent production of inflammatory mediators.

Transformed human keratinocyte cells (HaCaT) were purchased from AddexBio (cat. no. T0020001) and cultured in Dulbecco's modified Eagle's medium (AddexBio, cat. no. C0003-02) supplemented with 10% fetal bovine serum (Hyclone, cat. no. 16140 -071) and 1x Penicillin/Streptomycin (Gibco, cat. no. 15140-122). When cells reached 80-90% confluency, they were washed with 1x DPBS, then separated from tissue culture flasks by incubation with 0.25% trypsin (Gibco, cat. no. 25200-056) for 3-5 min at 37°C/ 5%CO2. Cell culture medium was added to the trypsinized cells, and the cell suspension was then transferred to a sterile 15 mL centrifuge tube for centrifugation for 10 min at 1300 rpm. The trypsin-containing medium was aspirated from the cell pellet, and the pellet was then resuspended in 10 ml of cell culture medium. Cells were counted using a Countess II automated cell counter, then seeded into 24-well tissue culture-treated plates at a concentration of 4 x 104 cells/ml and incubated for 48 hours at 37°C/5% CO ₂ . After 48 hours, the medium was removed and replaced with 500 μl of cell culture or combination stimulation medium with recombinant human interferon gamma (R&D Systems, catalog no. 285-IF-100) and recombinant human tumor necrosis factor alpha (R&D Systems, catalog no. 210-TA- 020). HaCaT cells treated with combinatorial cytokine stimulation were treated with final concentrations of 10 ng/ml, 25 ng/ml, 50 ng/ml, or 100 ng/ml of each cytokine. The treated plates were mixed by gentle shaking for 30 s, then incubated for 24 h at 37°C/5% CO ₂ . At the end of the 24-hour incubation, the medium was immediately removed from each plate.

RNA was isolated from HaCaT cells using QuantiGene Plex Assay reagents and protocols (Affymetrix, cat. no. QGP-232-M18042302). Cells were washed with 1x DPBS, then lysed by incubation with the provided QuantiGene lysis buffer for 30 min at 50-55°C. Cell lysates were incubated for 18–24 h at 55°C with capture beads and a probe set designed to specifically hybridize to mRNA from targets of interest. A panel of 32 targets of interest included housekeeping genes used to normalize results. After 18-24 hours of incubation, the signal was amplified using branched DNA techniques according to the manufacturer's procedures (Affymetrix, catalog no. QGP-232M18042302). After the hybridization and washing steps, the assay plate was read on a Luminex 200 and data were expressed as net average fluorescence intensity. The data were then normalized to the net mean fluorescence intensity of the housekeeping gene HPRT1 (Table 2).

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table 2

Stimulation of human keratinocytes with TNFa and IFNy induces the JAK/STAT pathway and proinflammatory cytokines

Gene Treatment MFI ^a p-value JAK1 Carrier medium 126.7 ±6.55 - 10 ng/ml TNFa/IFNy 178.19 ±3.41 <0.0001 25 ng/ml TNFa/IFNy 195.02 ±3.47 <0.0001 50 ng/ml TNFa/IFNy 198.23 ±2.52 <0.0001 100 ng/ml TNFa/IFNy 207.34 ±3.91 <0.0001 JAK2 Carrier medium 21.7 ±0.53 - 10 ng/ml TNFa/IFNy 154.13 ± 11.65 <0.0001 25 ng/ml TNFa/IFNy 174.07 ± 12.34 <0.0001 50 ng/ml TNFa/IFNy 180.71 ± 13.63 <0.0001 100 ng/ml TNFa/IFNy 187.94 ± 13.12 <0.0001 JAK3 Carrier medium 0.1 ±0.02 - 10 ng/ml TNFa/IFNy 0.16 ±0.05 0.8111 25 ng/ml TNFa/IFNy 0.18 ±0.05 0.596 50 ng/ml TNFa/IFNy 0.33 ± 0.06 0.0082

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100 ng/ml TNFa/ΙΡΝγ 0.28 ± 0.06 0.0532 TYK2 Carrier medium 167.84 ±2.25 - 10 ng/ml TNFa/ΙΡΝγ 240.49 ± 4.4 <0.0001 25 ng/ml TNFa/ΙΡΝγ 250.15 ±3.41 <0.0001 50 ng/ml TNFa/ΙΡΝγ 257.24 ±3.55 <0.0001 100 ng/ml TNFa/ΙΡΝγ 265.37 ±3.1 <0.0001 STAT1 Carrier medium 484.33 ±4.52 - 10 ng/ml TNFa/ΙΡΝγ 3834.09 ±65.62 <0.0001 25 ng/ml TNFa/ΙΡΝγ 3935.51 ±66.15 <0.0001 50 ng/ml TNFa/ΙΡΝγ 3943.03 ± 63.05 <0.0001 100 ng/ml TNFa/ΙΡΝγ 4136.09 ±67.06 <0.0001 STAT3 Carrier medium 606.76 ± 11.51 - 10 ng/ml TNFa/ΙΡΝγ 1561.14 ±40.35 <0.0001 25 ng/ml TNFa/ΙΡΝγ 1652.97 ±39.53 <0.0001 50 ng/ml TNFa/ΙΡΝγ 1666.52 ± 52.15 <0.0001 100 ng/ml TNFa/ΙΡΝγ 1742.81 ±38.26 <0.0001 STAT4 Carrier medium 2.27 ±0.12 - 10 ng/ml TNFa/ΙΡΝγ 3.78 ± 0.22 <0.0001 25 ng/ml TNFa/ΙΡΝγ 3.84 ±0.23 <0.0001 50 ng/ml TNFa/ΙΡΝγ 3.72 ±0.25 <0.0001 100 ng/ml TNFa/ΙΡΝγ 3.61 ±0.28 0.0003 STAT5A Carrier medium 1.03 ±0.1 - 10 ng/ml TNFa/ΙΡΝγ 26.06 ±3.1 <0.0001 25 ng/ml TNFa/ΙΡΝγ 28.58 ±3.23 <0.0001 50 ng/ml TNFa/ΙΡΝγ 31.01 ±3.37 <0.0001 100 ng/ml TNFa/ΙΡΝγ 29.61 ±2.91 <0.0001 STAT6 Carrier medium 626.95 ± 22 - 10 ng/ml TNFa/ΙΡΝγ 1010.38 ± 14.28 <0.0001 25 ng/ml TNFa/ΙΡΝγ 1044.97 ± 12.71 <0.0001 50 ng/ml TNFa/ΙΡΝγ 1039.59 ± 10.5 <0.0001 100 ng/ml TNFa/ΙΡΝγ 1059.01 ± 13.45 <0.0001 ILIA Carrier medium 156.9 ± 1.89 - 10 ng/ml TNFa/ΙΡΝγ 1786.44 ±31.13 <0.0001 25 ng/ml TNFa/ΙΡΝγ 2135.03 ±66.58 <0.0001

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50 ng/ml TNFa/ΙΕΝγ 2256.89 ±90.79 <0.0001 100 ng/ml TNFa/ΙΕΝγ 2459.6 ± 106.2 <0.0001 IL6 Carrier medium 5.89 ±0.19 - 10 ng/ml TNFa/IFNy 311.31 ±38.81 0.0002 25 ng/ml TNFa/IFNy 410.93 ± 52.93 <0.0001 50 ng/ml TNFa/IFNy 464.27 ±61.46 <0.0001 100 ng/ml TNFa/IFNy 519.31 ±68.04 <0.0001

^a Data are presented as mean ± standard error (SEM)

Target proteins of interest in the media were detected and quantified using ProCarta multiplex immunoassay reagents and protocols (Invitrogen, catalog number EPX450-12171-901). The medium was incubated with beads conjugated with antibodies designed to bind to epitopes of specific target proteins and identify the bound protein by the characteristic spectral pattern of the beads. Biotinylated detection antibodies, designed to bind to different epitopes of the same target proteins, and streptavidin-PE are added to assay plates to quantify the amount of target proteins. Assay plates were read on a Luminex 200 and data were expressed as net mean fluorescence intensity. Net median fluorescence intensity values for an antigen standard curve generated according to the manufacturer's procedures (Invitrogen, Cat. No. EPX450-12171-901) were plotted against expected concentrations for each standard. The concentration of each protein was extrapolated from the antigen standard curve, and concentrations were expressed in pg/ml (Table 3).

Table 3

Stimulation of human keratinocytes with TNFa and IFNy induces the production of proinflammatory cytokines

Protein Treatment pg/ml ^a R- magnitude IL-la Carrier medium 0.37 ± 0.05 - 10 ng/ml TNFa/IFNy 13.22 ± 1.24 <0.0001 25 ng/ml TNFa/IFNy 15.12 ± 1.48 <0.0001 50 ng/ml TNFa/IFNy 14.74 ± 1.45 <0.0001 100 ng/ml TNFa/IFNy 13.64 ± 1.29 <0.0001 IL-6 Carrier medium 72.86 ± 9.77 - 10 ng/ml TNFa/IFNy 2012.1 ±337.23 0.0001 25 ng/ml TNFa/IFNy 2329.01 ± 384.78 <0.0001 50 ng/ml TNFa/IFNy 2208.6 ±370.81 <0.0001

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100 ng/ml TNFa/ΙΡΝγ 1889.75 ±298.39 0.0004 IP-10 Carrier medium 16.61 ± 1.6 - 10 ng/ml TNFa/ΙΡΝγ 3275.51 ± 174.48 <0.0001 25 ng/ml TNFa/ΙΡΝγ 3243.28 ± 178.41 <0.0001 50 ng/ml TNFa/ΙΡΝγ 3209.56 ±211.43 <0.0001 100 ng/ml TNFa/ΙΡΝγ 2978.45 ± 167.27 <0.0001 ΜΙΡΙα Carrier medium 7.47 ± 1.13 - 10 ng/ml TNFa/ΙΡΝγ 525.75 ± 87.5 <0.0001 25 ng/ml TNFa/ΙΡΝγ 546.69 ±92.35 <0.0001 50 ng/ml TNFa/ΙΡΝγ 531.55 ±91.88 <0.0001 100 ng/ml TNFa/ΙΡΝγ 409.14 ±60.62 0.0012 RANTES Carrier medium 11.78 ± 1.41 - 10 ng/ml TNFa/ΙΡΝγ 126.13 ± 5.15 <0.0001 25 ng/ml TNFa/ΙΡΝγ 127.73 ± 2.8 <0.0001 50 ng/ml TNFa/ΙΡΝγ 119.95 ±4.67 <0.0001 100 ng/ml TNFa/ΙΡΝγ 103.48 ± 7.09 <0.0001 ^a Data are presented as mean ± standard error (SEM)

Example D: Janus kinase inhibitors interfere with interferon-gamma and tumor necrosis-alpha-mediated inflammation in keratinocytes.

Transformed human keratinocyte cells (HaCaT) were purchased from AddexBio (cat. no. T0020001) and cultured as described in Example C. Four compounds A-D (A: ruxolitinib, B: itacitinib ({1-{1-[3-fluoro-2 -(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile) , C: 4-[3-(cyanomethyl)-3-(3',5'-dimethylIII,1'H-4,4'bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro- N--[(^)-2,2,2-trifluoro-1-methylethyl]benzamide, D:

((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H- pyran2-yl)acetonitrile) were reconstituted in DMSO, then each compound was serially diluted with cell culture medium to concentrations of 400 nM, 200 nM, 100 nM and 50 nM. After 48 h, cell culture medium was removed from the 24-well plates and replaced with 250 μl of medium containing serially diluted drug, then incubated for 15 min at 37°C/5% CO2. After drug incubation, 250 μl of combinatorial stimulation containing recombinant human interferon gamma (R&D Systems, cat. no. 285-IF-100) and recombinant human tumor necrosis factor alpha (R&D Systems, cat. no. 210-TA-020) was added to the plates. . The final concentration of recombinant human interferon gamma and recombinant human tumor necrosis factor alpha was 25 ng/ml of each cytokine. Cytokine stimulation added to drug-containing wells brought the final concentrations for each drug treatment to 25 nM, 50 nM, 100 nM, and 200 nM. The treated plates were mixed by gentle shaking for 30 s, then incubated for 24 h at 37°C/5% CO2. At the end of the 24-hour incubation, the medium was immediately removed from each plate.

RNA was isolated from HaCaT cells using QuantiGene Plex Assay reagents and protocols (Affymetrix, cat. no. QGP-232-M18042302) according to the manufacturer's instructions. Cells were washed with 1x DPBS, then lysed by incubation with the provided QuantiGene lysis buffer for 30 min at 50–55°C. Cell lysates were incubated for 18–24 h at 55°C with capture beads and a probe set designed to specifically hybridize to mRNA from targets of interest. Genes included housekeeping genes (eg, HPRT1, GAPDH) used to normalize results. After 18-24 hours of incubation, the signal was amplified using branched DNA techniques according to the manufacturer's procedures (Affymetrix, catalog no. QGP-232-M18O423O2). After the hybridization and washing steps, the assay plate was read on a Luminex 200 and data were expressed as net mean fluorescence intensity. The data were then normalized to the net mean fluorescence intensity of the housekeeping gene HPRT1 (Table 4).

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Table 4

Normalized expression of target genes in human keratinocyte cells stimulated with TNFa and IFNy in the presence/absence of JAK inhibitors

Compound A Compound IN Connection C eD connections Gene ^Stimulation Concentration of the drug MFI ^b rvel ichi na ^s MFI ^b rvel ichi na ^s MFI ^b ranks were torn from MFI ^b tear it ^up JA K1 - - 183.21 ±7.55 25 ng/ml - 213.93 ± 5.55 ^e - 200 nM 159.13 ± 7.08 - 171.53 ±9.49 - 177.67 ± 11.84 - 177.9 7± 14.91 - 25 ng/ml 25 nM 206.18 ± 7.99 0.89 4 216.23 ±6.41 0.99 93 206.29 ± 6.84 0.783 4 200.4 ±9.84 0.5 65

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4 25 ng/ml 50 nM 195.48 ± 9.54 0.29 25 210.42 ± 10.89 0.99 65 194.2 ± 8.24 0.085 2 210.5 2± 7.73 0.9 94 2 25 ng/ml 100 nM 186.97 ± 7.49 0.06 21 205.03 ± 11.49 0.90 26 193.28 ± 4.55 0.066 9 200.2 5 ± 8.15 0.5 56 2 25 ng/ml 200 nM 180.99 ± 8.58 0.01 91 195.97 ± 10.45 0.45 97 182.86 ± 4.07 0.002 6 190.5 3± 7.68 0.1 28 6 - - 25.35 ±0.95 25 ng/ml - ¥126.63± ^4.89 200 23.67 ± 25.21 ± 25.25 ± 25.67 nM 0.92 1.12 1.04 ± 1.03 JA 25 ng/ml 25 nM 89.4 ± 2.21 <0.0 001 109.39 ±2.8 0.00 21 114.94 ± 2.16 0.041 9 108.8 9± 3.25 0.0 16 5 K2 25 ng/ml 50 nM 69.7 ± 1.78 <0.0 001 101 ± 2.26 <0.0 001 107.16 ± 2.86 0.000 3 106.8 3 ± 5.94 0.0 06 3 25 ng/ml 100 nM 54.4 ± 1.8 <0.0 001 94.5 ± 2.65 <0.0 001 95.51 ± 3.13 <0.00 01 102.6 4± 3.52 0.0 00 7 25 ng/ml 200 nM 40.25 ± 1.3 <0.0 001 89.16 ± 3.43 <0.0 001 91.17 ± 2.15 <0.00 01 92.21 ±2.9 <0.00 01 - - 0.66 ±0.14 JA short circuit 25 ng/ml - 0.52 ±0.16 200 0.53 ± 0.63 ± 0.68 ± 0.71 ± nM 0.09 0.10 0.17 0.15

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25 ng/ml 25 nM 0.81 ± 0.15 0.52 84 0.84 ± 0.12 0.32 47 1.02 ± 0.19 0.102 2 0.97 ± 0.18ъ 0.2 18 7 25 ng/ml 50 nM 1.01 ± 0.23 0.12 84 0.83 ± 0.15 0.34 97 0.99 ± 0.16 0.149 3 0.99 ± 0.20 0.1 85 4 25 ng/ml 100 nM 0.84 ± 0.13 0.44 73 0.92 ± 0.13 0.16 08 0.99 ± 0.15 0.149 1 1.01 ± 0.17 0.1 53 1 25 ng/ml 200 nM 0.68 ± 0.13 0.91 33 0.79 ± 0.15 0.48 76 0.85 ± 0.15 0.432 3 0.86 ± 0.16 0.4 44 2 τ Υ K2 - - 217.40 ± 8.13 25 ng/ml - 296.98 ± 6.92 ^¥ - 200 nM 205.57 ± 10.87 - 217.28 ± 10.09 - 217.28 ± 14.28 - 220.7 8± 12.01 - 25 ng/ml 25 nM 298.27 ± 10.83 0.99 9 292.92 ±7.99 0.99 29 283.97 ± 8.59 0.501 5 283.9 3 ± 8.16 0.7 98 1 25 ng/ml 50 nM 287.93 ± 16.28 0.93 05 287.31 ± 11.08 0.86 03 273.68 ± 7.44 0.082 3 307.3 6± 14.87 0.8 95 8 25 ng/ml 100 nM 260.21 ± 7.05 0.05 46 284.15 ±9.62 0.70 43 266 ± 6.82 0.012 3 280.6 3 ± 10.46 0.6 5 25 ng/ml 200 nM 264.75 ± 8.44 0.12 04 277.52 ± 8.67 0.35 78 263.49 ± 5.05 0.006 1 283.2 8± 10.88 0.7 70 7 ST A - - 545.83 ± 15.37 25 - 3106.13 ±217.15 ^¥

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ΤΙ ng/ml - 200 nM 526.90 ± 13.46 - 535.07 ±22.13 - 554.39 ± 11.80 - 554.6 4± 11.36 - 25 ng/ml 25 nM 2907.12 ±206.85 0.86 32 2868.6 9± 202.69 0.78 33 3111.17± 182.20 >0.99 99 3164.74 ± 242.3 5 0.9 98 6 25 ng/ml 50 nM 2902.82 ± 173.71 0.85 44 2862.5 8± 163.98 0.76 85 3058.64 ± 154.86 0.998 9 3017.08 ± 167.9 6 0.9 92 8 25 ng/ml 100 nM 2712.93 ± 182.91 0.37 89 2790.3 2± 176.4 0.58 07 3035.33 ± 122.14 0.995 2999.87 ± 197.8 6 0.9 86 2 25 ng/ml 200 nM 2475.58 ± 134.64 0.07 34 2857.2 ± 174.57 0.75 53 2984.14 ± 163.4 0.963 4 3161, 66 ± 135.8 0.9 98 8 ST A TK - - 751.20 ± 14.97 25 ng/ml - 1608.39 ± 70.09 ^¥ - 200 nM 728.97 ± 20.48 - 732.19 ±23.03 - 746.17 ± 16.73 - 750.9 0± 27.68 - 25 ng/ml 25 nM 1434.08 ± 43.26 0.07 4 1466.7 3 ± 66.75 0.32 06 1557.84 ± 58.15 0.939 9 1572.76 ± 65.5 0.9 88 25 ng/ml 50 nM 1301.55 ± 51.7 0.00 05 1437.2 8± 60.69 0.17 62 1519.61 ± 69.92 0.704 4 1543.4± 58.65 0.9 04 2 25 ng/ml 100 nM 1150.46 ±52.66 <0.0 001 1373.3 4± 0.03 52 1457.24 ± 54.48 0.26 1549, 17 ± 0.9 28

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55.51 89.41 8 25 ng/ml 200 nM 1082.84 ±39.32 <0.0 001 1400.7 7± 58.44 0.07 38 1483.1 ± 51.73 0.420 1 1570, 19± 51.51 0.9 84 5 ST A T4 - - 4.52 ±0.64 25 ng/ml - 6.19 ± 0.53 ^€ - 200 nM 3.75 ± 0.33 - 4.01 ± 0.45 - 4.28 ± 0.61 - 4.32 ± 0.53 - 25 ng/ml 25 nM 6.15 ± 0.47 >0.9 99 6.00 ± 0.46 0.99 67 5.65 ± 0.44 0.798 1 5.4 ± 0.45 0.5 46 2 25 ng/ml 50 nM 5.57 ± 0.53 0.77 12 6.22 ± 0.42 >0.9 99 5.41 ± 0.33 0.515 1 6.1 ± 0.36 0.9 99 7 25 ng/ml 100 nM 5.63 ± 0.39 0.82 69 6.21 ± 0.48 >0.9 99 5.32 ± 0.46 0.415 7 5.83 ± 0.34 0.9 44 8 25 ng/ml 200 nM 5.25 ± 0.45 0.46 53 6.27 ± 0.56 0.99 99 5.04 ± 0.36 0.183 3 5.42 ± 0.52 0.5 69 1 ST A T5 A - - 2.17 ±0.54 25 ng/ml - 26.41 ±2.26 ^¥ - 200 nM 1.12 ± 0.19 - 1.44 ± 0.41 - 1.75 ± 0.44 - 1.99 ± 0.51 - 25 ng/ml 25 nM 19.04 ± 1.94 0.01 eleven 23.69± 1.63 0.74 71 22.82 ± 1.77 0.452 0 20.12 ± 1.29 0.0 42 8 25 ng/ml 50 nM 16.18 ± 1.66 0.00 03 22.32 ± 2.16 0.42 25 20.71 ± 1.77 0.111 7 22.69 ± 1.71 0.3 62 9

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25 ng/ml 100 nM 12.94 ± 1.27 <0.0 001 20.87 ± 2.1 0.17 84 18.44 ± 1.85 0.013 8 19.54 ± 1.34 0.0 23 3 25 ng/ml 200 nM 9.48 ± 0.86 <0.0 001 19.2 ± 1.94 0.05 05 17.64 ± 1.46 0.005 9 18.33 ± 1.83 0.0 05 9 ST A T6 - - 749.34 ± 20.85 25 ng/ml - 1045.99 ± 26.73 ^¥ - 200 nM 723.56 ± 20.76 - 740.11 ±34.98 - 762.04 ± 9.44 - 777.0 3 ± 29.31 - 25 ng/ml 25 nM 1043.96 ± 20.37 >0.9 99 1004.8 2± 23.76 0.55 57 1020.89 ± 23.57 0.823 8 1042, 76 ± 29.23 >0.99 9 25 ng/ml 50 nM 1016.85 ±25.68 0.80 28 990.05 ±21.06 0.28 95 982.62 ± 14.34 0.138 9 1046, 46 ± 29.12 >0.99 9 25 ng/ml 100 nM 966.76 ± 28.58 0.07 39 987.64 ± 15.75 0.25 57 943.66 ± 25.99 0.005 9 985.1 ± 39.79 0.3 95 5 25 ng/ml 200 nM 976.22 ± 14.93 0.14 87 985.17 ±29.31 0.22 4 966.51 ± 12.3 0.042 9 1013, 25 ± 17.15 0.8 45 3 IL la - - 95.72 ± 5.84 25 ng/ml - 1405.01 ±27.93 ^¥ - 200 nM 84.51 ± 7.04 - 85.16 ± 6.50 - 88.72 ± 5.90 - 92.67 ±5.54 - 25 ng/ml 25 nM 1115.1 ± 18.96 <0.0 001 1288.0 2 ±20 0.00 47 1370.52 ± 35.28 0.837 9 1269, 66 ± 50.59 0.0 74 4

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25 ng/ml 50 nM 962.51 ± 23 <0.0 001 1258.7 6± 23.63 0.00 03 1308.7 ± 45.12 0.099 5 1336, 95 ± 50.97 0.5 87 1 25 ng/ml 100 nM 839.16 ± 21.04 <0.0 001 1162.3 5 ± 23.34 <0.0 001 1194.29 ± 12.27 <0.00 01 1244, 96 ± 41.03 0.0 26 4 25 ng/ml 200 nM 755.65 ± 16.88 <0.0 001 1126.9 4± 26.22 <0.0 001 1151.31 ± 20.01 <0.00 01 1163, 14 ± 26.71 0.0 00 4 IL -6 - - 5.86 ±0.38 25 ng/ml - 170.83 ± 5.28 ^¥ - 200 nM 4.70 ± 0.32 - 4.97 ± 0.36 - 4.98 ± 0.28 - 5.15 ± 0.31 - 25 ng/ml 25 nM 93.79 ± 4.03 <0.0 001 130.24 ±3.84 <0.0 001 135.32 ± 3.36 <0.00 01 132.2 8± 7.41 <0.00 01 25 ng/ml 50 nM 69.7 ± 2.81 <0.0 001 122.69 ±4.36 <0.0 001 128.14 ± 6.83 <0.00 01 137.6 1 ± 5.87 0.0 00 6 25 ng/ml 100 nM 51.01 ± 1.57 <0.0 001 111.07 ±4.74 <0.0 001 112.13 ± 3.37 <0.00 01 122.4 6± 5.35 <0.00 01 25 ng/ml 200 nM 40.39 ± 2.19 <0.0 001 93.03 ± 3.25 <0.0 001 101.17 ± 2.91 <0.00 01 119.4 9± 4.42 <0.00 01 ^a Stimulation with TNFa (25 ng/ml) and ΙΡΝγ (25 ng/ml) ^b Data are presented as mean ± standard error ^c Statistically significant differences compared to stimulation with TNFa and FFNy alone ^¥ Indicates a significant difference p < 0.0001 from carrier medium (without stimulation and without drug concentration) ^€ Indicates a significant difference p <0.1 from the carrier medium

Fig. 1-4 illustrate individual gene expression (MFI) values for JAK1, JAK2, IL-Ια and IL-6, respectively, for each experimental replicate in keratinocytes modeled with TNFa and IFN-γ in the presence/absence of JAK inhibitors.

Target proteins of interest in the media were detected and quantified using ProCarta multiplex immunoassay reagents and protocols (Invitrogen, catalog number EPX450-12171-901). The medium was incubated with beads conjugated to antibodies designed to bind to epitopes of specific target proteins and identify the bound protein by the characteristic spectral pattern of the beads. Biotinylated detection antibodies, designed to bind to different epitopes of the same target proteins, and streptavidin-PE are added to assay plates to quantify the amount of target proteins. Assay plates were read on a Luminex 200 and data were expressed as net mean fluorescence intensity. Net median fluorescence values for standard curve

- 40 046969 antigen, constructed according to the manufacturer's procedures (Invitrogen, cat. no. EPX450-12171901), was plotted against the expected concentrations for each standard. The concentration of each protein was extrapolated from the antigen standard curve, and concentrations were expressed in pg/ml (Table 5).

Table 5

Concentrations of inflammatory mediators produced by human keratinocyte cells stimulated with TNFa and IFNy in the presence/absence of JAK inhibitors

Compound A Connection B Connection C Compound D Protein ^Stimulation Concentration of the drug pg/ ^ml grow on ^s pg/ ^ml grow on ^s pg/ ^ml grow on ^s pg/ml ^b rvel ichi na ^s IL- la - - 0.29 ± 0.03 25 ng/ml - ¥7.82± ^0.18 - 200 nM 0.26 ± 0.05 - 0.29 ± 0.03 - 0.30 ±0.05 - 0.31 ± 0.04 - 25 ng/ml 25 nM 5.93 ± 0.29 <0.000 1 7.34 ±0.31 0.7043 7.74 ±0.36 0.9994 6.8 ± 0.39 0.14 98 25 ng/ml 50 nM 4.9 ±0.3 <0.000 1 7.06 ±0.37 0.3281 7.01 ±0.39 0.3537 6.76 ± 0.4 0.12 49 25 100 4.12 ± <0.000 7 ±0.41 0.2631 7.27 ± 0.47 0.6747 6.92 ± 0.22

- 41 046969

ng/ml nM 0.26 1 0.4 81 25 ng/ml 200 nM 3.45 ± 0.23 <0.000 1 6.16 ±0.35 0.0034 6.45 ± 0.38 0.0358 6.3 ± 0.35 0.01 21 IL-6 - - 30.57 ±2.89 25 ng/ml - 862.33 ± 17.95 ^¥ - 200 nM 26.86 ± 2.62 - 28.49 ± 2.89 - 28.79 ± 2.91 - 28.84 ± 1.89 - 25 ng/ml 25 nM 594.5 ± 25.17 <0.000 1 749.64 ± 32.94 0.0158 774.87 ± 31.09 0.1794 743.07 ± 36.3 0.04 76 25 ng/ml 50 nM 446.35 ± 19.73 <0.000 1 674.21 ± 27.15 <0.000 1 710.89 ± 36.7 0.006 698.04 ± 29.79 0.00 37 25 ng/ml 100 nM 362.14 ± 18.73 <0.000 1 643.8 ± 27.14 <0.000 1 690.4 ± 35.25 0.0016 703.99 ± 42.22 0.00 54 25 ng/ml 200 nM 295.21 ± 15.22 <0.000 1 568.73 ± 24.74 <0.000 1 621.79 ± 33.44 <0.000 1 646.2 ± 32.46 <0, OOO 1 IP 10/ CXC LIO - - 20.14 ±0.36 25 ng/ml - 3935.46 ± 375.68 ^¥ - 200 nM 19.75 ± 0.42 - 19.83 ± 0.40 - 20.23 ± 0.48 - 20.39 ± 0.57 - 25 ng/ml 25 nM 3497.56 ± 194.81 0.6232 4068.98 ± 507.12 0.9982 3999.39 ± 370.53 0.9998 3903.67 ± 366.97 >0.999 25 ng/ml 50 nM 3599.04 ± 402.58 0.7995 3872.74 ± 295.01 0.9999 3665.2 ± 277.11 0.9431 3998.62 ±456.34 0.99 99 25 ng/ml 100 nM 3158.24 ± 189.25 0.1574 4050.7 ± 471.31 0.999 3860.41 ± 323.05 0.9995 4100.26 ±502.48 0.99 78 25 ng/ml 200 nM 2662.18 ± 89.27 0.0059 4071.78 ± 411.22 0.9979 3835.78 ± 304.58 0.9984 4407.56 ± 645.63 0.89 45 MIP - - 3.14 ±0.24

- 42 046969

1α 25 ng/ml - 105.63 ± 3.74 ^¥ - 200 nM 2.63 ± 0.35 - 2.75 ± 0.26 - 2.90 ±0.21 - 3.11 ± 0.28 - 25 ng/ml 25 nM 82.56 ± 3.1 <0.000 1 103.81 ± 3.29 0.9925 101.71 ± 3.84 0.931 102.06 ± 4.18 0.93 03 25 ng/ml 50 nM 70.57 ± 3.32 <0.000 1 100.64 ± 4.66 0.7866 104.54 ± 6.56 0.9994 96.35 ± 3.57 0.33 35 25 ng/ml 100 nM 50.91 ± 1.6 <0.000 1 91.52 ± 5.05 0.0532 96.4 ±4.18 0.4229 96.22 ± 3.58 0.32 15 25 ng/ml 200 nM 40.36 ± 0.88 <0.000 1 83.1 ±2.77 0.0007 98.72 ± 3.87 0.6469 88.49 ± 5.06 0.01 6 RAN TES - - 9.56 ±0.56 25 ng/ml - ¥230.17± ^9.43 - 200 nM 10.17 ± 0.54 - 8.42 ±0.51 - 8.61 ±0.52 - 9.51 ± 0.56 - 25 ng/ml 25 nM 192 ± 12.74 0.0311 203.77 ± 12.55 0.4195 216.88 ± 13.45 0.9096 237.57 ± 17.46 0.99 67 25 ng/ml 50 nM 165.12± 11.76 0.0001 198.35 ± 15.1 0.262 201.93 ± 15.44 0.439 237.55 ± 21.78 0.99 67 25 ng/ml 100 nM 136.24 ± 7.8 <0.000 1 194.21 ± 12.67 0.1736 207.79 ± 17.38 0.6354 241.39 ± 22.79 0.98 41 25 ng/ml 200 nM 111.94 ± 6.48 <0.000 1 183.18± 13.92 0.0416 189.62 ± 13.78 0.1403 238.51 ± 23.12 0.99 42 ^a Stimulation with TNFa (25 ng/ml) and ΙΡΝγ (25 ng/ml) ^b Data are presented as mean ± standard error ^c Statistically significant differences compared to stimulation with TNFa and ΙΡΝγ alone ^¥ Indicates a significant difference p < 0.0001 from one vehicle (without stimulation and without drug concentration)

Fig. 5 and 6 illustrate individual protein concentrations (pg/ml) for IL-Ια and IL-6, respectively, for each experimental replicate in keratinocytes modeled with TNFa and IFN-γ in the presence/absence of JAK inhibitors.

Example E: Skin biopsy from hidradenitis suppurativa is characterized by increased expression of Janus kinase.

Total RNA of Healthy Control skin from 3 separate donors was purchased from Amsbio (Cat. Nos. HR101 and R1234218-50). Total RNA of Healthy Control skin from the donor pool was purchased from Life Technologies Corporation (Cat. No. QS0639). Hidradenitis suppurativa skin biopsies (41 donors) were purchased from Discovery Life Sciences in formaldehyde-embedded paraffin (FFPE) blocks from which total RNA was purified.

Gene expression from the Healthy Control (n=4) and hidradenitis suppurativa (n=41) skin total RNA samples was measured for the genes listed in Table 1. 6 using QuantiGene Plex Assay reagents and protocols (Life Technologies Corporation, Cat. No. QGP-277-M19012402). Purified RNAs were used in the recommended

-

Claims (8)

assay range from 50 ng to 500 ng and incubated overnight with capture beads designed for specific hybridization with mRNA from selected genes (Table 6). This panel of targets included several housekeeping genes that were used to normalize the results. After overnight incubation, the signal was amplified using branched DNA techniques according to the manufacturer's procedures (Life Technologies Corporation). The assay plate was read on a Luminex 200 and data were expressed as net mean fluorescence intensity (net MFI). Data were normalized to the geometric mean net MFI for the housekeeping genes ACTB and GAPDH. Fig. 7-9 illustrate the expression of the JAK1, JAK3, TYK2, STAT1, STAT2, STAT3, IRAKI, IRAK2 and IRAK4 genes in the skin of healthy controls and subjects with hidradenitis suppurativa. Table 6 Target genes Gene ID Name Gena JAK1 Janus kinase 1 JAK2 Janus kinase 2 JAK3 Janus kinase 3 IRAKI interleukin 1 receptor-associated kinase 1 IRAK2 interleukin 1 receptor-associated kinase 2 IRAK4 Interleukin 1 receptor-associated kinase 4 STAT1 signal transducer and transcription activator 1 STAT3 signal transducer and transcription activator 3 STAT4 signal transducer and transcription activator 4 STAT5A signal transducer and transcription activator 5A STAT6 signal transducer and transcription activator 6 STAT2 signal transducer and transcription activator 2 STAT5B signal converter and transcription activator 5V TYK2 tyrosine kinase 2 SYK spleen-associated tyrosine kinase GAPDH glyceraldehyde-3-phosphate dehydrogenase ASTV actin beta Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also included within the scope of the appended claims. Each reference cited in this application, including all patents, patent applications and publications, is incorporated herein by reference in its entirety. CLAIM 1. A method of treating hidradenitis suppurativa in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a compound that inhibits JAK1, or a pharmaceutically acceptable salt thereof, wherein the compound or salt is selective for JAK1 over JAK2, JAK3 and TYK2, where the compound is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3^]pyrimidin-4-yl) -1 H-pyrazol-1 -yl] azetidin-3 -yl } acetonitrile; 4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3^]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-U-[4fluoro -2-(trifluoromethyl)phenyl]piperidine-1-carboxamide; [3-[4-(7H-pyrrolo[2,3^]pyrimidin-4-yl)-1H-pyrazol-1-yl]-1-(1-{ [2-(trifluoromethyl)pyrimidin-4yl]carbonyl } piperidin-4-yl)azetidin-3-yl] acetonitrile; 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H,1'H-4,4'-bipyrazol-1-yl)azetidin-1-yl]-2,5-difluoro- Y-[(^)2,2,2-trifluoro-1-methylethyl]benzamide; ((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]-1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2Hpyran- 2-yl)acetonitrile; 3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3^]pyrimidin-4-yl)-III-pyrazol1 -yl]propanenitrile ; - 44 046969 3-(1-[1,3|oxazo.to[5,4-b|pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrho.to[2,3d|pyrimidin-4 -yl)1H-pyrazol-1-yl]propanenitrile; 4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2.3d|pyrimidin-4-yl)-1H-pyrazol-1-yl|propyl;piperazin-1yl)carbonyl] -3 - fluorobenzonitrile; 4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1yl)carbonyl| -3-fluorobenzonitrile; [trans-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-3-(4-{[2-(trifluoromethyl)pyrimidin-4 -yl]carbonyl}piperazin-1-yl)cyclobutyl]acetonitrile; {trans-3 -(4-{[4-[(3-hydroxyazetidin-1-yl)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4- (7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; {trans-3 -(4-{[4-{[(2S)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2yl]oxy}piperidin-1-yl)- 1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; {trans-3 -(4-{[4-{[(2R)-2-(hydroxymethyl)pyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2yl]oxy}piperidin-1-yl)- 1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; 4-(4-{3-[(dimethylamino)methyl|-5-fluorophenoxy}piperidin-1-yl)-3 -[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H -pyrazol-1-yl| butanenitrile; 5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-Nisopropylpyrazin-2 -carboxamide; 4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5difluoro -N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide; 5-{3-(cyanomethyl)-3-[4-(1H-pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-isopropylpyrazine -2-carboxamide; {1-(cis-4-{[6-(2-hydroxyethyl)-2-(trifluoromethyl)pyrimidin-4-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; {1-(cis-4-{[4-[(ethylamino)methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d ]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; {1 -(cis-4-{[4-(1-hydroxy-1-methylethyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3 -[4(7H-pyrrolo[2,3 -d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; {1-(cis-4-{ [4-{ [(3R)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4- (7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; {1-(cis-4-{[4-{[(3S)-3-hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4- (7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile; {trans-3 -(4-{ [4-({ [(1 S)-2-hydroxy-1-methylethyl]amino }methyl)-6-(Ίriforomethyl)pyridin-2-yl]oxy}piperidin-1- yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}aceΊΌnicΊril; {trans-3-(4-{[4-({[(2R)-2-hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1- [4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile; trans-3-(4-{[4-({[(2S)-2-hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[ 4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile or {trans-3-(4-{[4-(2-hydroxyethyl)- 6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl }acetonitrile. 2. The method according to claim 1, characterized in that the compound is {1-{1-[3-fluoro-2(Ίriforomethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2 ,3-d]pyrimidin-4-yl)-1H-pyrazol1-yl|azetidin-3-yl}acetonitrile or a pharmaceutically acceptable salt thereof. 3. The method according to claim 2, characterized in that the salt is a salt of adipic acid {1-{1-[3fluoro-2-(Ίriforomethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo [2,3-d]pyrimidin-4-yl)-1Hpyrazol-1 -yl|azetidin-3-yl}acetonitrile. 4. Method according to claim 1, characterized in that the compound is 4-[3-(cyanomethyl)-3-(3',5'dimethyl-1H,1Ή-4,4'-bipyrazol-1-yl)azetidine -1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide or a pharmaceutically acceptable salt thereof. 5. The method according to claim 4, characterized in that the salt is a salt of phosphoric acid 4-[3(cyanomethyl)-3-(3',5'-dimethyl-1H,1Ή-4,4'-bipyrazole-1- yl)azetidin-1-yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl|benzamide. 6. The method according to claim 1, characterized in that the compound is ((2R,5S)-5-{2-[(1R)-1hydroxyethyl|-1H-imidazo[4,5D|thieno[3,2-b |pyridin-1-yl}tetrahydro-2H-pyran-2-yl)acetonitrile or a pharmaceutically acceptable salt thereof. 7. The method according to claim 1, characterized in that the compound is ((2R,5S)-5-{2-[(1R)-1hydroxyethyl|-1H-imidazo[4,5D|thieno[3,2-b |pyridin-1 -yl}tetrahydro-2H-pyran-2-yl)acetonitrile monohydrate. 8. Method according to any one of claims 1 to 7, characterized in that the compound or salt is administered in a dosage of 15, 30, 60 or 90 mg, calculated as the free base. -