MX2022012285A

Treatment of hidradenitis suppurativa using jak inhibitors.

Abstract

The present application 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.

MX2022012285A, drawing sheet 1 — Sheet 1 of 15

Term

12.5 yearsleft in the term

Expires 29 March 2039.

13 claims: 7 independent, 6 dependent

1
Uso de un compuesto, o una sal farmacéuticamente aceptable de este, en la elaboración de un medicamento para el tratamiento de hidradenitis supurativa, en donde el compuesto es:ruxolitinib;ruxolitinib, en donde uno o más átomos de hidrógeno se reemplazan con átomos de deuterio;o una sal farmacéuticamente aceptable de cualquiera de los anteriores.
2
El uso de conformidad con la reivindicación 1, en donde el compuesto o sal es selectivo para JAK1 y JAK2 sobre JAK3 y TYK2.
3
El uso de conformidad con la reivindicación 2, en donde el compuesto es ruxolitinib, o una sal farmacéuticamente aceptable de este.
4
El uso de conformidad con la reivindicación 3, en donde el compuesto es ruxolitinib, o una sal farmacéuticamente aceptable de este, en donde uno o más átomos de hidrógeno se reemplazan con átomos de deuterio.
5
El uso de conformidad con la reivindicación 3, 107 en donde la sal es fosfato de ruxolitinib.
6
El uso de conformidad con cualquiera de las reivindicaciones 1-5, en donde además un aqente terapéutico está adaptado para ser adicionalmente administrable.
7
El uso de conformidad con la reivindicación 6, en donde el aqente terapéutico adicional es un corticosteroide.
8
El uso de conformidad con la reivindicación 7, en donde el corticosteroide es triamcinolona, dexametasona, fluocinolona, cortisona, prednisona, prednisolona o flumetolona.
9
El uso de conformidad con cualquiera de las reivindicaciones 1-8, en donde el compuesto o sal está adaptado para ser administrable de manera tópica.
10
El uso de conformidad con cualquiera de las reivindicaciones 1-8, en donde el compuesto o sal está adaptado para ser administrable de manera oral.
11
El uso de conformidad con cualquiera de las reivindicaciones 1-10, en donde el uso da como resultado una mejora de 10%, 20%, 30%, 40%, o 50% en HiSCR (respuesta clínica de hidradenitis supurativa).
12
El uso de conformidad con cualquiera de las reivindicaciones 1-10, en donde el compuesto o sal está adaptado para ser administrable como una composición farmacéutica adecuada para administración tópica.
13
El uso de conformidad con cualquiera de las reivindicaciones 1-10, en donde el compuesto es base libre de CP77 ίη/77Π7/Ε/ΥΙΛΙ 108 ruxolitinib.

Independent claims13

690 paragraphs in 6 sections, as filed

TREATMENT OF HIDRADENITIS SUPPURATIVA THROUGH THE USE OF JANUS KINASE (JAK) ACTIVITY INHIBITORS

FIELD OF THE INVENTION

The present application provides methods for the treatment of hidradenitis suppurativa (HS) through the use of compounds that modulate the activity of Janus kinase (JAK) 1 and/or 2.

BACKGROUND OF THE INVENTION

Protein kinases (PK) regulate several biological processes including growth, survival, cellular differentiation, organ formation, morphogenesis, neovascularization, tissue repair and regeneration, among others. Likewise, protein kinases also serve specialized functions in a host of human diseases including cancer. Cytokines, glycoproteins or low molecular weight polypeptides, regulate many pathways involved in the host inflammatory response to sepsis. Cytokines affect cell differentiation, proliferation, and activation and can modulate pro- and anti-inflammatory responses to allow the host to react appropriately to pathogens. Signaling by a wide variety of cytokines involves the Janus kinase (JAK) family of protein tyrosine kinases and signal transducers and activators of transcription (STAT,

Ref. 311897). There are four known JAKs in mammals: JAK1 (Janus kinase-1), JAK2, JAK3 (also known as leukocyte Janus kinase; JAKL; and L-JAK), and TYK2 (protein tyrosine kinase 2).

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

Deficiencies in JAK expression are associated with various disease states. For example, Jakl/- mice are the smallest of the litter at birth, fail to feed and die in the perinatal stage (Rodig, SJ, MA Meraz, et al. (1998) Cell 93(3): 373-83 ). Embryos from Jak2 −/− mice are anemic and die around day 12.5 after coitus due to the absence of definitive erythropoiesis.

The JAK/STAT pathway and, in particular, the four JAKs are believed to play a role in the pathogenesis of asthmatic response, chronic obstructive pulmonary disease, bronchitis

CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ and other related inflammatory diseases of the lower respiratory tract. Multiple cytokines signaling through JAK have been linked to inflammatory diseases/conditions of the upper respiratory tract, such as those affecting the nose and sinuses (e.g., rhinitis and sinusitis), whether classic allergic reactions or not. The JAK/STAT pathway has also been implicated in ocular inflammatory diseases/conditions and chronic allergic responses.

JAK/STAT activation in cancers may occur due to stimulation by cytokines (e.g., IL-6 or GM-CSF) or due to reduction of endogenous suppressors of JAK signaling such as SOCS (suppressors of JAK signaling). cytokines) or PIAS (inhibitor of proteins activated by STAT) (Boudny, V., and Kovarik, J., Neoplasm. 49:349-355, 2002). Activation of STAT signaling, as well as other pathways 3' to JAKs (e.g., Akt), has been linked to poor prognoses in many cancer types (Bowman, T., et al. Oncogene 19:2474- 2488, 2000). Elevated levels of circulating cytokines signaling through JAK/STAT play a causal role in cachexia and/or chronic fatigue. As such, JAK inhibition may be beneficial to cancer patients for reasons beyond potential antitumor activity.

JAK2 tyrosine kinase may be beneficial for

CQ77 ίη/ΖΖΠΖ/Ε/ΥΙΛΙ patients with myeloproliferative disorders, e.g., polycythemia vera (PV), essential thrombocythemia (ET), myelofibrosis with myeloid metaplasia (MMM) (Levin, et al., Cancer Cell, vol. 7, 2005 : 387-397). JAK2V617F kinase inhibition decreases 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 immunological skin disorders such as psoriasis and skin sensitization. The maintenance of psoriasis is thought to depend on a number of inflammatory cytokines, in addition to several chemokines and growth factors (JCI, 113:1664-1675), many of which are signaled through JAKs (Adv Pharmacol. 2000 ;47:113-74).

Therefore, new or improved agents that inhibit kinases such as JAK are continually needed to develop new and more effective pharmaceutical products that aim to increase or suppress immune and inflammatory pathways, such as the treatment of hidradenitis suppurativa. This request refers to that need and others.

SUMMARY OF THE INVENTION

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

In some embodiments, the compound or salt is selective with respect to 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, wherein one or more hydrogen atoms are replaced with deuterium atoms.

In some embodiments, the salt is ruxolitinib phosphate.

In some embodiments, the compound is {1-{1-[3-fluoro2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl) -lH-pyrazol-l-yl]azetidin-3yl}acetonitrile, or a pharmaceutically acceptable salt thereof.

In some embodiments, the salt is adipic acid salt {1—{1—[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4yl}-3-[4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)-IH-pyrazol-lyl]azetidin-3-yl}acetonitrile.

In some embodiments, the compound is 4—[3—(cyanomethyl)-3-(3',5'-dimethyl-lH,1Ή-4,4'-bipyrazol-1yl)azetidin-l-yl]-2,5 -difluoro-N-[(lS)-2,2,2-trifluoro-lmethylethyl]benzamide, or a pharmaceutically acceptable salt of

CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ this .

In some embodiments, the salt is 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-lH,1'H-4,4'-bipyrazol-1yl)azetidin-l-phosphoric acid salt. yl]-2,5-difluoro-N-[ (lS)-2,2,2-trifluoro-lmethylethyl]benzamide.

In some embodiments, the compound or salt is administered at a dose of 15, 30, 60 or 90 mg relative to the free base.

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

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

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

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

In some embodiments, the method results in a 10%, 20%, 30%, 40%, or 50% improvement in HiSCR (response

CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ hidradenitis suppurativa clinic).

The present 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.

The present application further provides 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 THE FIGURES

FIGURE 1 illustrates the individual gene expression values (MFI) for JAK1 for each experimental replicate in simulated keratinocytes with TNFα and IFN-γ in the presence/absence of Compounds AD. Keratinocytes were stimulated with TNFα (25 nq/mL) and IFNy (25 nq/mL) in the presence/absence of increasing concentrations of JAK inhibitors. Data are presented as JAK1 expression levels for each group.

FIGURE 2 illustrates the individual gene expression values (MFI) for JAK2 for each experimental replicate in simulated keratinocytes with TNFα and IFN-γ in the presence/absence of Compounds AD. Keratinocytes were stimulated with TNFα (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.

FIGURE 3 illustrates the individual gene expression values (MFI) for IL-Ια for each experimental replicate in simulated keratinocytes with TNFα and IFN-γ in the presence/absence of Compounds AD. Keratinocytes were stimulated with TNFα (25 ng/mL) and ΙΕΝγ (25 ng/mL) in the presence/absence of increasing concentrations of JAK inhibitors. Data are presented as IL-Ια expression levels for each group.

FIGURE 4 illustrates the individual gene expression values (MFI) for IL-6 for each experimental replicate in simulated keratinocytes with TNFα and IFN-γ in the presence/absence of Compounds AD. Keratinocytes were stimulated with TNFα (25 ng/mL) and IFNy (25 ng/mL) in the presence/absence of increasing concentrations of JAK inhibitors. Data are presented as IL-6 expression levels for each group.

FIGURE 5 illustrates the individual protein concentrations (pg/mL) for IL-Ια, for each experimental replicate in gueratinocytes simulated with TNFα and IFN-γ in the presence/absence of Compounds AD. Keratinocytes were stimulated with TNFα (25 ng/mL) and ΙΕΝγ (25 ng/mL) in the presence/absence of increasing concentrations of JAK inhibitors. Data are presented as IL-Ια concentrations for each group.

FIGURE 6 illustrates the individual protein concentrations (pg/mL) for IL-6 for each experimental replicate in keratinocytes simulated with TNFα and IFN-γ in the presence/absence of Compounds AD. Keratinocytes were stimulated with TNFα (25 ng/mL) and ΙΕΝγ (25 ng/mL) in the presence/absence of

CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ increasing concentrations of JAK inhibitors. Data are presented as IL-6 concentrations for each group.

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

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

FIGURE 9 illustrates the gene expression (MFI) of IRAK1, IRAK2, and IRAK4 in the skin of healthy controls and subjects with hidradenitis suppurativa. Data are presented as gene expression levels of IRAK1, IRAK2, or IRAK4 for each healthy control (n=4) and hidradenitis suppurativa subject (n=41).

DETAILED DESCRIPTION OF THE INVENTION

The present application provides, among others, 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 compounds or salts that are inhibitors of JAK1 and/or JAK2. In some modalities,

CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ the compound is: ruxolitinib;

ruxolitinib, wherein one or more hydrogen atoms are replaced with deuterium atoms;

{1—{1—[3-fluoro-2(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-i1)-lH-pyrazole-l -yl]azetidin-3yljacetonitrile;

4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4yl)-lH-pyrazol-1-yl]azetidin-l-yl]-N-[4- fluoro-2(trifluoromethyl)phenyl]piperidine-l-carboxamide;

[3-[4 -(7H-pyrrolo[ 2,3-d]pyrimidin-4-i1)-lH-pyrazol-1yl]-1-(1-{[2-(trifluoromethyl)pyrimidin-4yl]carbonyl}piperidin -4-yl)azetidin-3-yl]acetonitrile;

4-[3-(cyanomethyl)-3-(3',5'-dimethyl-ΙΗ,l'H-4,4'bipyrazol-l-yl)azetidin-l-yl]-2,5-difluoro-N -[(1S)-2,2,2trifluoro-l-methylethyl]benzamide;

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

3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-i1)-IH-pyrazol-l-yl] propanenitrile;

3- (1- [ 1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl) -3-

[4 - (7H-pyrrolo[2,3-d]pyrimidin-4-yl)-IH-pyrazol-lyl]propanonitrile;

CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ

4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-i1)IH-pyrazol-l-yl]propyl}piperazin-l-yl)carbonyl ]-3fluorobenzonitrile;

4-[ ( 4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-i1)IH-pyrrol-l-yl]propyl}piperazin-l-yl)carbonyl ]-3fluorobenzonitrile;

[ trans-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1Hpyrazol-l-yl]-3-(4-{[2-(trifluoromethyl)pyrimidin-4yl]carbonyl }piperazin-l-yl)cyclobutyl]acetonitrile;

{trans-3-(4-{ [4- [ (3-hydroxyazetidin-l-yl)methyl]-6(trifluoromethyl)pyridin-2-yl]oxy}piperidin-l-yl)-1-[4-( 7Hpyrrolo[2,3-d]pyrimidin-4-i1)-IH-pyrazol-lyl]cyclobutyl}acetonitrile;

{trans-3-(4-{[4-{[(2S)-2-(hydroxymethyl)pyrrolidin-1yl]methyl}-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-l-yl) 1 - [4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-IH-pyrazol-lyl]cyclobutyl}acetonitrile;

{trans-3-(4-{ [4-{ [ (2R)-2-(hydroxymethyl)pyrrolidin-1yl]methyl]-6-(trifluoromethyl)pyridin-2-yl]oxy}piperidin-l-yl) 1 -[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-IH-pyrazol-lyl]cyclobutyl}acetonitrile;

4-(4-{ 3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidinl-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-lH-pyrazole -1yl]butanenitrile;

5-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4CQ77 ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

11)-IH-pyrazol-l-yl]azetidin-l-yl}-N-isopropyIpyrazin-2carboxamide;

4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4yl)-IH-pyrazol-l-yl]azetidin-l-yl}-2,5-difluoro -N-[(1S)2,2,2-trifluoro-l-methylethyl]benzamide;

5-{3-(cyanomethyl)-3-[4-(IH-pyrrolo[2,3-b]pyridin-4-yl)IH-pyrazol-l-yl]azetidin-l-yl}-N-isopropiipyrazin- 2carboxamide;

{l-(cis-4-{[6-(2-hydroxyethyl)-2(trifluoromethyl)pyrimidin-4-yl]oxy}cyclohexyl)-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4 -yl)-lH-pyrazol-l-yl]azetidin-3yl}acetonitrile;

{l-(cis-4-{ [4 —[ (ethylamino)methyl]-6(trifluoromethyl)pyridin-2-yl]oxy]cyclohexyl)-3-[4-(7Hpyrrolo[2,3-d]pyrimidin- 4-yl)-lH-pyrazol-l-yl]azetidin-3yljacetonitrile;

{1-(cis-4-{[4-(1-hydroxy-l-methylethyl)-6(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-(7Hpyrrolo[2,3-d ]pyrimidin-4-yl)-lH-pyrazol-l-yl]azetidin-3yljacetonitrile;

{l-(cis-4-{ [4 —{ [(3R)-3-hydroxypyrrolidin-l-yl]methyl}-6(trifluoromethyl)pyridin-2-yl]oxy}cyclohexyl)-3-[4-( 7Hpyrrolo[2,3-d]pyrimidin-4-yl)-lH-pyrazol-l-yl]azetidin-3yl}acetonitrile;

{l-(cis-4-{ [4-{ [ (33)-3-hydroxypyrrolidin-l-yl]methyl}-6CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ (trifluoromethyl)pyridin-2-yl]oxy]cyclohexyl) -3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-i1)-lH-pyrazol-l-yl]azetidin-3yljacetonitrile;

{trans-3-(4-{[4-({[(lS)-2-hydroxy-lmethylethyl]aminojmethyl)-6-(trifluoromethyl)pyridin-2yl]oxy}piperidin-l-yl)-1-[4 -(7H-pyrrolo[2,3-d]pyrimidin-4yl)-lH-pyrazol-l-yl]cyclobutyl}acetonitrile;

{trans-3-(4-{[4-({[(2R)-2-hydroxypropyl]amino}methyl)-6(trifluoromethyl)pyridin-2-yl]oxy}piperidin-l-yl)—1—[ 4—(7H—pyrrolo[2,3-d]pyrimidin-4-i1)-lH-pyrazol-1yl]cyclobutyl}acetonitrile;

{trans-3- (4-{[4 - ( { [ (2 S)-2-hydroxypropyl]amino}methyl)-6(trifluoromethyl)pyridin-2-yl]oxy}piperidin-l-yl)-1- [4-(7Hpyrrolo[2,3-d]pyrimidin-4-i1)-lH-pyrazol-1yl]cyclobutyl}acetonitrile;

{trans-3-(4 —{ [4 - (2-hydroxyethyl)-6(trifluoromethyl)pyridin-2-yl]oxy}piperidin-l-yl)-1-[4-(7Hpyrrolo[2,3-d ]pyrimidin-4-i1)-IH-pyrazol-lyl]cyclobutyl}acetonitrile;

or a pharmaceutically acceptable salt of any of the above.

In some embodiments, the compound or salt is selective with respect to JAK1 and JAK2 over JAK3 and TYK2. In some embodiments, the compound is 3-cyclopentyl-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-i1)-IH-pyrazol-l-yl]propanonitrile,

CQ77 ίη/ΖΖΠΖ/Ε/ΥΙΛΙ or a pharmaceutically acceptable salt thereof. In some embodiments, the compound is (3R)-3-cyclopentyl-3-[4-(7Hpyrrolo[2,3-d]pyrimidin-4-yl)-IH-pyrazol-l-yl]propanenitrile (ruxolitinib), or a pharmaceutically acceptable salt thereof. Ruxolitinib has an IC50 less than 10 nM for 1 mM ATP (test A) in JAK1 and JAK2. 3-cyclopentyl-3-[4-(7H-pyrrolo[2,3d]pyrimidin-4-yl)-IH-pyrazol-l-yl]propanenitrile and ruxolitinib can be made by the procedure described in US 7,598,257 (Example 67 ), filed on December 12, 2006, which is incorporated herein in its entirety by reference. In some embodiments, the JAK1 and/or JAK2 inhibitor is a phosphoric acid salt (3R)3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1Hpyrazole- l-yl]propanenitrile. The phosphoric acid salt can be made as described in US Patent 8,722,693, which is incorporated herein in its entirety by reference.

In some embodiments, the compound or salt is a JAK1 inhibitor. In some embodiments, the compound or salt is selective with respect to 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 critical role in a number of cytokine and growth factor signaling pathways

CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ that, when dysregulated, can cause or contribute to disease states. For example, IL-6 levels are elevated in rheumatoid arthritis, a disease in which it has been suggested 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 be indirectly through JAK1 inhibition, resulting in potential clinical benefit (Guschin, et al. Embo J 14:1421, 1995; Emolen, et al. Lancet 371:987, 2008). Furthermore, in some cancers, JAK1 is mutated resulting in undesirable constitutive tumor cell growth and survival (Mullighan, Proc Nati Acad Sel US A. 106: 9414-8, 2009; Flex, J Exp Med. 205:7518, 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 inhibitors of JAK1 may be effective while avoiding unnecessary and potentially undesirable effects of inhibition of other JAK kinases.

Hydradenitis suppurativa is characterized by significant inflammation of the skin; however, there are only limited publications describing inflammation (Hoffman et al., PLOS One, September 28, 2018,

CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ https://doi.org/10.1371/journal.pone .0203672). Examples are presented here that support the hypothesis that inflammation is caused, in large part, by mediated pathways. by JAK/STAT.Examples C, D and E illustrate elevated levels of JAK/STAT gene expression in the skin of patients with HS compared to healthy skin.In addition, Examples C, D and E show that proinflammatory cytokines known to be elevated in HS (TNF-alpha and IFN-gamma) induce the JAK/STAT pathway in cultured keratinocytes and that this induction can be reduced by the addition of JAK inhibitors.By Therefore, patients with HS may benefit from JAK1 inhibition. Selective JAK1 inhibitors may be effective while avoiding unnecessary and potentially undesirable effects of inhibition of other JAK kinases.

In some embodiments, the compound or salt inhibits JAK1, preferably, over JAK2 (e.g., has a JAK2/JAK1 IC50 ratio >1). In some embodiments, the compounds or salts are about 10 times more selective for JAK1 than for JAK2. In some embodiments, the compounds or salts are about 3 times, about 5 times, about 10 times, about 15 times, or about 20 times more selective for JAK1 than for JAK2, as calculated by measuring IC50. in 1 mM ATP (see Example A).

In some embodiments, the JAK1 inhibitor is a

CQ77 ίη/ΖΖΠΖ/Ε/ΥΙΛΙ compound of Table 1, or a pharmaceutically acceptable salt thereof. The compounds in Table 1 are selective JAK1 inhibitors (selective over JAK2, JAK3 and TYK2). The IC50 values obtained by the method of Example A for 1 mM ATP are shown in Table 1.

Table 1

<td>Comp. No .</td><td>Prep.</td><td>Name</td><td>Structure</td><td>JAK1 I.C.b0(nM)</td><td>JAK 2/ JAK 1</td>

<td> 1</td><td>US 2011/0224190 (Example 1)</td><td>{l-{l-[3- fluoro-2- (trifluoromethyl)isonicotinoyl]piperidin-4- il}—3—[4 —(7Hpyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazol1-yl]azetidin3- iljacetonitrile</td><td> 1 1 C.F.3 QFN NN Á Á Η 1 > NNh</td><td> +</td><td> >10</td>

<td> 2</td><td>US 2011/0224190 (Example 154)</td><td>4-{ 3(cyanomethyl)-3[4-(7Hpyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazol1-yl]azetidinl-yl}-N-[4fluoro-2- (trifluoromethyl)phenyl]piperidi nl-carboxamide</td><td>F exC.F.3 O^NH QN N“N N^p</td><td> +</td><td> >10</td>

<td> 3</td><td>US 2011/0224190 (Example 85)</td><td>[3-[4-(7Hpyrrolo[2,3d]pyrimidin-4yl)-lH-pyrazol1-yl]-1-(l-{[2(trifluoromethyl )pyrimidin-4i 1 ]carbon11}pip eridin-4- il)azetidin-3yl]acetonitrile</td><td>EITHER N^NpurNA/N NN 1 L JO ^'N^'NIN h</td><td> +</td><td> >10</td>

<td> 4</td><td>US 2014/03430 30 (Example 7)</td><td>4- [3- (cyanomethyl)-3(3',5'-dimethyl1Η,1Ή-4,4'bipyrazol-1yl)azetidin-1yl]-2,5-difluoroN-[ (13)-2,2,2trifluoro-1methylethyl]benz ami gives</td><td>F, nn^N Mhn/FANDFF' HN-N</td><td> +++</td><td> >10</td>

CQ77 ίη/77Π7/Ε/ΥΙΛΙ

<td> 5</td><td>US 2014/01211 98 (Example 20)</td><td>((2R,5S)-5-{2[(IR)-1hydroxyethyl]IH-imidazo[4,5d]thieno[3,2— b]pyridin-1yl}tetrahydro2H-pyran-2- 11)acetonitrile</td><td>ooN</td><td> ++</td><td> >10</td>

<td> 6</td><td>US 2010/0298334 (F, example 2)to</td><td>3-[l-(6chloropyridin-2i Ί ) pyrro 1 idin 3-11]-3-[4-(7Hpyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazol1-yl ]propanonitrile</td><td>N N νΛ ' ' I í // i' and N -1 And ci n || ....7 'N M.H.</td><td> +</td><td> >10</td>

<td> 7</td><td>US 2010/0298334 (Example 13c)</td><td>3- (1- [1,3]oxazolo[5, 4-b]pyridin-21Ipyrro11din-311)-3-[4-(7Hpyrrolo[2,3— d]pyrimidin-4yl)-IH-pyrazol1-11]propanonitrile</td><td>_ II /-NN- NN or— N.N.</td><td> +</td><td> >10</td>

<td> 8</td><td>US 2011/0059951 (Example 12)</td><td>4- [ (4-{3-cyano2-[4-(7Hpyrrolo[2,3— d]pyrimidin-4- 11)-IH-pyrazole- 1- il]propyl}piperazin-1-</td><td> ° /---\ _N\ ~yn Ά #~F N.N. // TO NC</td><td> +</td><td> >10</td>

CQ77 ίη/77Π7/Ε/ΥΙΛΙ

<td></td><td></td><td>11)carbonyl]-3fluorobenzonitrile</td><td></td><td></td><td></td>

<td> 9</td><td>US 2011/0059951 (Example 13)</td><td>4- [ (4 —{3-cyano2-[3-(7Hpyrrolo[2,3d]pyrimidin-411)-IH-pyrrole1- 11]propyl}piperazin-1- i1)carbon)1]-3fluorobenzonitrile</td><td>F 0C.N.either N—' _yN q h 2 / H</td><td> +</td><td> >10</td>

<td> 10</td><td>US 2012/0149681 (Example 7b)</td><td>[trans-1-[4(7Hpyrrolo[2,3d]pyrimidin-411)-IH-pyrazoll-yl]-3-(4-{[2(trifluoromethyl )pyrimidin-411]carbonyl}piperazin-1yl)cyclobutyl]acetonitrile</td><td>or N N.N.Z/n already</td><td> +</td><td> >10</td>

<td> 11</td><td>US 2012/0149681 (Example 157)</td><td>{trans-3-(4- { [4-[ (3- hydroxyazetidin -1-yl)methyl]- 6(trifluoromethyl)pyridin-2yl]oxy}piperidin-l-yl)-1-[4(7H- pyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazole1- 11]cyclobutyl}acetonitrile</td><td>ZOH AF Λ Γ 7\ /N N.N. ΛA N τΛ it Λ > NNH</td><td> +</td><td> >10</td>

<td> 12</td><td>US 2012/0149681 (Example 161)</td><td>{trans-3-(4- { [ 4-{ [ (2S)-2(hydroxymethyl)pyrrolidin-111]methyl}- 6(trifluoromethyl)pyridin-211]oxy Jpiperidin-l-yl)-1-[4(7H- pyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazole1- yl]cyclobutyl}acetonitrile</td><td>\\ //j '\z/z-\ TZ Λ - J, \ UZ TI -A 1 ti -n O</td><td> +</td><td> >10</td>

CQ77 ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

<td> 13</td><td>US 2012/0149681 (Example 162)</td><td>{trans-3-(4- {[4-{[(2R)-2(hydroxymethyl)pyrrolidin-111]methyl}- 6(trifluoromethyl)pyridin-211]oxy}piperidin-l-yl)-1-[4(7H- pyrrolo[2,3d]pyrimidin-411)-IH-pyrazole1- yl]cyclobutyl}acetonitrile</td><td>N/'/ í '-oh AF 0>N FF0 ,N qj' NN ΧχΧ N.N.</td><td> +</td><td> >10</td>

<td> 14</td><td>US 2012/0149682 (Example 20)b</td><td>4- (4-{3- [(dimethylamino)methyl]-5- fluorophenoxy} piperidin-l-yl)- 3- [4-(7Hpyrrolo[2,3— d]pyrimidin-4- il)-IH-pyrazol-lil]butanonitri-lo</td><td>N Τι TI 11 ψ F O-™ N</td><td> +</td><td> >10</td>

<td> 15</td><td>US 2013/0018034 (Example 18)</td><td>5-{ 3(cyanomethyl)-3[4-(7Hpyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazol1-yl]azetidinl-yl}-N- isopropylpyrazin-2carboxamide</td><td>N=—\ ~ N=\ O NN '—N HN—( // \ ' lNh</td><td> +</td><td> >10</td>

CQ77 ίη/77Π7/Ε/ΥΙΛΙ

<td> 16</td><td>US 2013/0018034 (Example 28)</td><td>4-{ 3(cyanomethyl)-3[4-(7Hpyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazol1-yl]azetidinl-yl}-2,5- difluoro-N- [(1S)-2,2,2trifluoro-1methylethyl]benzamide</td><td>either ........ F h X / Nh</td><td> +</td><td> >10</td>

<td> 17</td><td>US 2013/0018034 (Example 34)</td><td>5-{ 3(cyanomethyl)-3[4-(1Hpyrrolo[2,3b]pyridin-4- il)-IH-pyrazol1-yl]azetidinl-yl}-N- isopropylpyrazin-2carboxamide</td><td>v-OL óo</td><td> +</td><td> >10</td>

<td> 18</td><td>US 2013/0045963 (Example 45)</td><td>(1-(cis-4-{[6(2-hydroxyethyl)- 2- (trifluoromethyl)pyrimidin-4- yl]oxy}cyclohexy 1)-3-[4-(7Hpyrrolo[2, 3d]pyrimidin-4yl)-IH-pyrazol1-yl]azetidin-3yljacetonitrile</td><td>NJ.J. \nn 03</td><td> +</td><td> >10</td>

CQ77 ίη/77Π7/Ε/ΥΙΛΙ

<td> 19</td><td>US 2013/0045963 (Example 65)</td><td>{1-(cis-4-{[4[(ethylamino)methyl]-6- (trifluoromethyl)pyridine-2- yl]oxy}cyclohex il)-3-[4-(7Hpyrrolo[2,3d]pyrimidin-411)-lH-pyrazol1-yl ]azetidin- 3- il}acetonitrile</td><td>N h</td><td> +</td><td> >10</td>

<td> 20</td><td>US 2013/0045963 (Example 69)</td><td>{1-(cis-4-{[4(1-hydroxy-l- methylethyl)-6- (trifluoromethyl)pyrimidin-2- 11]oxy}cyclohexyl)-3-[4-(7Hpyrrolo[2,3d]pyrimidin-4yl)-lH-pyrazol1-yl]azetidin- 3- il}acetonitrile</td><td>O—A LL or to</td><td> +</td><td> >10</td>

<td> 21</td><td>US 2013/0045963 (Example 95)</td><td>{1-(cis-4-{[4- {[(3R)—3—hydroxypyrrolidin-1yl]methyl}-6(trifluoromethyl)pyridin-2yl]oxy}cyclohexyl)-3-[4(7H-</td><td>y^N\L0H' nA J Λr o—\ JN_Nx</td><td> +</td><td> >10</td>

CQ77 ίη/77Π7/Ε/ΥΙΛΙ

<td></td><td></td><td>pyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazole1 — i 1 ]azetidin3- il}acetonitrile</td><td></td><td></td><td></td>

<td> 22</td><td>US 2013/0045963 (Example 95)</td><td>{1-(cls-4-{[4- {[(3S)-3hydroxypyrrolidin-1- 11]methyl}- 6- (tri f1uorometi 1 )pyridin-2- 11]oxy}cyclohexyl)-3-[4-(7Hpyrrolo[2,3d]pyrimidin-4yl)-lH-pyrazol1-yl]azetidin- 3- iljacetonitrile</td><td>r\ λΓν/NA/NAr OA/NN 1,IN h</td><td> +</td><td> >10</td>

<td> 23</td><td>US 2014/0005166 (Example 1)</td><td>{trans-3-(4-{[4({[(1S))-2hydroxy-1methylethyl]amino; methyl)-6- (trifluoromethyl)pyridin-2- yl]oxy Jpiperidin-l-yl)-1-[4(7H-pyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazol1-yl]cyclobutyl; acetonitrile</td><td>-~^OH NH EITHER>N U 0 N.N. U.A. óaO N.N.</td><td> +</td><td> >10</td>

CQ77 ίη/77Π7/Ε/ΥΙΛΙ

<td> 24</td><td>US 2014/0005166 (Example 14)</td><td>{trans-3-(4- { [ 4- ({ [ (2R) -2hydroxypropyl]aminoJmethyl)-6(trifluoromethyl )pyridin-2yl]oxy}piperidin-l-yl)-1-[4(7H- pyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazole1- 11]cyclobutyl}acetonitrile</td><td>^OH NH Af O>N F 0 NN (/A N.N.</td><td> +</td><td> >10</td>

<td> 25</td><td>US 2014/0005166 (Example 15)</td><td>{trans-3-(4- { [4- ({ [ (2S)-2hydroxypropyl]amino Jmethyl)-6(trifluoromethyl )pyridin-2yl]oxyJpiperidin-l-yl)-1-[4(7H- pyrrolo[2,3d]pyrimidin-4yl)-IH-pyrazole1- yl]cyclobutyl}acetonitrile</td><td>^oh NH EITHER>N Γ 0 X ,N N.N. N.N.</td><td> +</td><td> >10</td>

CQ77 ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

+ means < 10 nM (see Example A for test conditions) ++ means < 100 nM (see Example A for test conditions) +++ means d 300 nM (see Example A for test conditions) test conditions)toEnantiomer 1 databEnantiomer 2 data

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

In some embodiments, the JAK1 inhibitor is adipic acid salt {1-{1-[3-fluoro-2(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7H pyrrolo[2,3-d ]pyrimidin-4-yl)-lH-pyrazol-l-yl]azetidin-3yljacetonitrile.

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

In some embodiments, the JAK1 inhibitor is 4-[3(cyanomethyl)-3-(3',5'-dimethyl-ΙΗ,1Ή-4,4'-bipyrazol-1yl)azetidin-l-yl]-2, 5-difluoro-N-[(lS)-2,2,2-trifluoro-lmethylethyl]benzamide, or a pharmaceutically acceptable salt thereof.

In some embodiments, the JAK1 inhibitor is 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-ΙΗ,1'H4,4'-bipyrazol-l-yl)azetidin-phosphoric acid salt. l-yl]-2,5-difluoro-N-[(1S)2,2,2-trifluoro-l-methylethyl]benzamide.

In some embodiments, the JAK1 inhibitor is 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-ΙΗ,1'H4,4'-bipyrazol-l-yl)azetidin-hydrochloric acid salt. l-yl]-2,5-difluoro-N-[(1S)CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ

2,2,2-trifluoro-l-methylethyl]benzamide.

In some embodiments, the JAK1 inhibitor is 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-lH,1'H4,4'-bipyrazol-l-yl)azetidin-hydrobromic acid salt. l-yl]-2,5-difluoro-N-[(1S)2,2,2-trifluoro-l-methylethyl]benzamide.

In some embodiments, the JAK1 inhibitor is 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-lH,1'H4,4'-bipyrazol-l-yl)azetidin-sulfuric acid salt. l-yl]-2,5-difluoro-N-[(1S)2,2,2-trifluoro-l-methylethyl]benzamide.

The synthesis and preparation of 4-[3-(cyanomethyl)-3-(3',5'dimethyl-lH,1Ή-4,4'-bipyrazol-l-yl)azetidin-l-yl]-2,5difluoro- N-[(1S)-2,2,2-trifluoro-l-methylethyl]benzamide and the phosphoric acid salt thereof can be found, for example, in US Patent Publication No. US 2014/0,343,030, filed on 16 May 2014, which is incorporated herein in its entirety by reference.

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

In some embodiments, the JAK1 inhibitor is ((2R,5S)-5-{2-[(IR)-1-hydroxyethyl ]-1Himidazo[4,5-d]thieno[3,2-b]pyridine monohydrate. -l-yl]tetrahydro-2H-pyran-2yl)acetonitrile.

The synthesis of ((2R, 5S)-5-{2-[(IR)-1-hydroxyethyl]-1HCQ77 ίη/77Π7/Ε/ΥΙΛΙ imidazo[4,5-d]thieno[3,2-b]pyridin -l-yl}tetrahydro-2H-pyran-2yl)acetonitrile and the characterization of the anhydrous and monohydrate forms thereof are described in US Patent Publication No. 2014/0121198, filed October 31, 2013, the publication of US Patent No. 2015/0344497, filed on Wednesday, April 29, 2015, each of which is incorporated herein in its entirety by reference.

In some embodiments, the compounds of Table 1 are prepared by the synthetic procedures described in US Patent Publication No. 2011/0224190, filed March 9, 2011, US Patent Publication No. 2014/0343030, filed on May 16, 2014, US Patent Publication No. 2014/0121198, filed October 31, 2013, US Patent Publication No. 2 010/0298334, filed on May 21, 2010, US Patent Publication No. 2011/0059951, filed on August 31, 2010, US Patent Publication No. 2012/0149681, filed on November 18, 2011, US Patent Publication No. 2012/0149682, filed on November 18, 2011, US Patent Publication 2013/0018034, filed on June 19, 2012, US Patent Publication No. 2013/0045963,

CQ77 ίη/77Π7/Ε/ΥΙΛΙ filed on August 17, 2012, US Patent Publication No. 2014/0005166, filed on May 17, 2013, each of which is incorporated herein in its entirety by reference .

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

In some embodiments, the JAK1 inhibitor is a

CQ77 ίη/77Π7/Ε/ΥΙΛΙ compound of Formula I

CQ77 ίη/ΖΖΠΖ/Ε/ΥΙΛΙ

I or a pharmaceutically acceptable salt thereof, wherein: X is N or CH;

L is C(=0) or C(=0)NH;

A is phenyl, pyridinyl, or pyrimidinyl, each of which is optionally substituted with 1 or 2 R groups1 independently selected; and each R1 It is, independently, fluoro or trifluoromethyl.

In some embodiments, the compound of Formula I is {1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin4-yl}-3-[4-(7H-pyrrolo[2,3-d] pyrimidin-4-yl)-lH-pyrazol-1yl]azetidin-3-yl}acetonitrile, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula I is 4-{3-(cyanonornetyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-i1) lH-pyrazol-l-yl] azetidin-l-yl}-N-[4-fluoro-2(trifluoromethi1)phenyl]piperidine-1-carboxamide, or a pharmaceutically acceptable salt thereof.

In some embodiments, the compound of Formula I is [3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-IH-pyrazol-l-yl]-1(1-{[ 2-(trifluoromethyl)pyrimidin-4-yl]carboni1}piperidin-4yl)azetidin-3-yl]acetonitrile, or a pharmaceutically acceptable salt thereof.

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

CP77 ίη/77Π7/Ε/ΥΙΛΙ

II or a pharmaceutically acceptable salt thereof, wherein:

R2 is Ci-6 alkyl, Ci-6 haloalkyl, C3-6 cycloalkyl, or C3-6 cycloalkyl-C1-3 alkyl, wherein C1-6 alkyl, C3-6 cycloalkyl, and C3-6 cycloalkyl-C1-3 alkyl , is each substituted with 1, 2, or 3 substituents independently selected from fluoro, -CF3, and methyl;

R3 is H or methyl;

R4 is H, F or Cl;

R5 is H or F;

R6 is H or F;

R7 is H or F;

R8 is H or methyl;

R9 is H or methyl;

R10 is H or methyl; and

R11 It is H or methyl.

In some embodiments, the compound of Formula II is 4-[3-(cyanomethi1)-3-(3',5'-dimethyl-1H,1'H-4,4'bipyrazol-l-yl)azetidin-l -yl]-2,5-difluoro-N-[(1S)-2,2,2trifluoro-l-methylethyl]benzamide, or a pharmaceutically acceptable salt thereof.

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

CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ

III, or a pharmaceutically acceptable salt thereof, wherein:

Cy4 is a tetrahydro-2H-pyran ring, which is optionally substituted with 1 or 2 groups selected, independently, from CN, OH, F, Cl, C1-3 alkyl, C1-3 haloalkyl, CN-C1-3 alkyl, HO- C1-3 alkyl, amino, C1-3 alkylamino, and di(C1-3 alkyl)amino, where I alkyl them

C1-3 and di (alkyl

C1-3) are optionally replaced with 1,

2, or 3 substituents selected, independently, from F, Cl, C1-3 alkylaminosulfonyl, and C1-3 alkylsulfonyl; and

R12 is -CH2-OH, —CH(CH3)—OH, or -CH2-NHSO2CH3.

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

In some embodiments, the JAK1 and/or JAK2 inhibitor is barcitinib, tofacitinib, oclacitinib, filgotinib, gandotinib, lestaurtinib, momelotinib, bacritinib, PF04965842, upadacitinib, peficitinib, fedratinib, cucurbitacin I, ATI-501 (Aclaris), ATI-502 (Aclaris), JTE052 (Leo Pharma and Japan Tobacco), or CHZ868.

In some embodiments, the JAK1 and/or JAK2 inhibitor may be an isotopically labeled compound, or a pharmaceutically acceptable salt thereof. An “isotopically labeled” or “radiolabeled” compound is a compound of the description where one or more atoms have been replaced or substituted with an atom with an atomic mass or mass number different from the atomic mass or mass number it usually has. in nature (that is, of natural origin). Suitable radionuclides that can be incorporated into the compounds of the present disclosure include, but are not limited to,2H (also written as D for deuterium),3h

CP77 ίη/77Π7/Ε/ΥΙΛΙ (also written as T for tritium),13C,14C,13N,15N,15EITHER,17EITHER,18EITHER,18F,35Yes,36C1,82Br,75Br,76Br,77Br,123YO,124YO,125I and131I. For example, one or more hydrogen atoms in a compound of the present disclosure may be replaced with deuterium atoms, such as the substitution of -CD3 for CH3) .

One or more constituent atoms of the compounds described herein may be replaced or substituted with isotopes of the atoms in natural or unnatural abundance. 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 hydrogen atoms in a compound can be replaced or substituted by deuterium atoms.

Synthetic methods for including isotopes in organic compounds are known in the art (Deuterium Labeling in Organic Chemistry by Alan F. Thomas (New York, NY, Appleton-Century-Crofts, 1971; The Renaissance of H/D Exchange by Jens Atzrodt, Volker Derdau, Thorsten Fey and Jochen Zimmermann, Angew. Chem. Int. Ed. 2007, 7744-7765; The Organic Chemistry of Isotopic Labeling by James R. Hanson,

CQ77 ίΠ/ΖΖηΖ/Ε/ΥΙΛΙ

Royal Society of Chemistry, 2011). Isotopically labeled compounds can be used in various studies such as NMR spectroscopy, assays and/or metabolism experiments.

Substitution with heavier isotopes such as deuterium may provide certain therapeutic benefits arising from greater metabolic stability, for example, longer in vivo half-life or fewer dosage requirements, and may therefore be preferred in some circumstances (see e.g. A. Kerekes et al. J. Med. Chem. 2011, 54, 201-210; R. Xu et al. J. Label Compd. Radiopharm. 2015, 58, 308-312). In particular, substitution at one or more sites of metabolism may provide one or more therapeutic advantages.

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

In some embodiments, the JAK1 and/or JAK2 inhibitor is ruxolitinib, wherein one or more hydrogen atoms in the compound are replaced with deuterium atoms or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK1 and/or JAK2 inhibitor is any of the compounds of US Patent 9249149 (which is incorporated herein in its entirety by reference), or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK1 and/or JAK2 inhibitor is CTP-543, or a salt

CQ77 ίη/77Π7/Ε/ΥΙΛΙ pharmaceutically acceptable from this.

CQ77 ίη/77Π7/Ε/ΥΙΛΙ

In some embodiments, the compound is a compound of Formula I:

or a pharmaceutically acceptable salt thereof, wherein:

R1 is selected from H and D;

each R2 is selected, independently, from H and D, provided that each R2 attached to a common carbon is the same;

each R3 is selected, independently, from H and D, provided that each R3 attached to a common carbon is the same;

R4 is selected from H and D;

each R5 is the same and is selected from H and D; and each of R6, R.7, and R8is selected, independently, from H and D; provided that, when R2 is H, each R2 and each R3 is H, R4 is H, and each of R6, R.7, and R8 is H, so each R5 It's 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 (wherein each of Ryes, R.7, and R8 is H), or a pharmaceutical salt of these. 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 each of R5, R.7, and R8 es D), or a pharmaceutically acceptable salt thereof.

<td>Compound</td><td>R1</td><td>Each R2</td><td>Each R3</td><td>R4</td><td>Each R5</td>

CQ77 ίη/77Π7/Ε/ΥΙΛΙ

In some embodiments, the JAK1 and/or JAK2 inhibitor is baricitinib, wherein one or more hydrogen atoms in the compound are replaced with deuterium atoms or a pharmaceutically acceptable salt thereof. In some embodiments, the JAK1 and/or JAK2 inhibitor is any of the compounds of US Patent 9540367 (which is incorporated herein in its entirety by reference), or a salt

CP77 ίη/77Π7/Ε/ΥΙΛΙ pharmaceutically acceptable from this.

As used herein, the term "optionally substituted" means unsubstituted or substituted. As used herein, the term "substituted" means that a hydrogen atom is removed and replaced with a substituent. It will be understood that substitution on a given atom is limited by valence.

As used herein, the term "C alkyln-m» used alone or in combination with other terms refers to a saturated hydrocarbon group that can be straight-chain or branched, having nam carbon atoms. In some embodiments, the alkyl group contains 6 to 3 carbon atoms. Examples of alkyl moieties include, but are not limited to, chemical groups such as methyl, ethyl, npropyl, isopropyl, n-butyl, isobutyl, sec-butyl, terebutyl, n-pentyl, 2-methyl-l-butyl, 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 straight chain or branched, where the two substituents may be attached. at any position of the alkylene linking group. Examples of alkylene groups include, but are not limited to, ethane-1,2-diyl, propane-1,3-diyl, propane-1,2-diyl, and the like.

CQ77 ίη/77Π7/Ε/ΥΙΛΙ

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

As used herein, "CN-C1-3 alkyl" refers to a C1-3 alkyl substituted with a cyano group.

As used herein, the term "amino" refers to a group of the formula -NH2.

As used herein, the term "di(C1-3alkyl)amino" refers to a group of the formula -N(alkyl)2, wherein the two alkyl groups each independently have 1 to 3 carbon atoms.

As used herein, the term "C1-3 alkylamino" refers to a group of the formula -NH (alkyl), wherein the alkyl group has 1 to 3 carbon atoms.

As used herein, the term "di(Ci3alkyl)aminosulfonyl" refers to a group of the formula S(O)2N(alkyl)2, where each alkyl group independently has 1 to 3 atoms. carbon.

As used herein, the term "C1-3 alkylsulfonyl" refers to a group of the formula S(0)2-alkyl, wherein the alkyl group has 1 to 3 carbon atoms.

As used herein, "halo" or "halogen", used alone or in combination with other terms, includes fluoro, chlorine, bromine and iodine. In some modalities, the group

CP77 ίη/77Π7/Ε/ΥΙΛΙ halo is fluoro or chloro.

As used herein, the term "Cn-m haloalkyl", used alone or in combination with other terms, refers to a C alkyl groupn-m which has up to {2(nam)+l} halogen atoms that can be the same or different. In some embodiments, the halogen atoms are fluoro atoms. In some embodiments, the alkyl group has 1-6 or 1-3 carbon atoms. Examples of haloalkyl groups include CF3, C?Fs, CHF?, CC13, CHC1?, CpCls, and the like. In some embodiments, the haloalkyl group is a fluoroalkyl group.

As used herein, the term "Ci-fluoroalkyl"3» refers to a Ci-alkyl group3 which may be partially or completely substituted with fluoro atoms.

As used herein, the term "C cycloalkyl3-6", used alone or in combination with other terms, refers to a non-aromatic monocyclic hydrocarbon moiety having 3-6 carbon atoms, which may optionally contain one or more alkenylene groups as part of the ring structure. One or more carbon atoms that form the ring of a cycloalkyl group can be oxidized to form carbonyl bonds. Examples of cycloalkyl groups C3-g include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, and the like. In some embodiments, the cycloalkyl group is cyclopropyl,

CP77 ίη/77Π7/Ε/ΥΙΛΙ cyclobutyl, cyclopentyl or cyclohexyl.

As used herein, the term "C3-6 cycloalkyl-C1-3 alkyl" refers to a group of the formula -C3-6 alkylene-C3-6 cycloalkyl.

The compounds described herein may be asymmetric (e.g., have one or more stereocenters). Unless otherwise indicated, it is intended to consider all stereoisomers, such as enantiomers and diastereomers. Compounds containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods are known in the art on how to prepare optically active forms from optically inactive starting materials, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of deffins, C=N double bonds, and the like may also be present in the compounds described herein, and all of these stable isomers are contemplated in the present application. The cis and trans geometric isomers of the compounds of the present application are described and can be isolated as a mixture of isomers or as separate isomeric forms. In some embodiments, the compound has the (R) configuration. In some embodiments, the compound has the (S) configuration.

The resolution of racemic mixtures of compounds can be carried out by any of the various methods known in the art. An exemplary method includes fractional recrystallization by using a chiral resolving acid that is an optically active salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods 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 the various optically camphorsulfonic acids. actives such as β-camphorsulfonic acid. Other resolving agents suitable for fractional recrystallization methods include stereoisomerically pure forms of amethylbenzylamine (e.g., the S and R forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2diaminocyclohexane , and the like.

Resolution of racemic mixtures can also be carried out by elution on a column with an optically active resolving agent (for example, dinitrobenzoylphenylglycine). One skilled in the art can determine the appropriate composition of an elution solvent.

The compounds described herein include tautomeric forms. Tautomeric forms result from the exchange of a single bond for an adjacent double bond along with the concomitant migration of a proton.

Tautomeric forms include prototropic tautomers which are isomeric protonation states with the same empirical formula and total charge. Illustrative prototropic tautomers include ketone-enol pairs, imidic acid amide pairs, lactam-lactime pairs, enamine imine pairs, and ring forms where a proton can occupy two or more positions of a heterocyclic system, e.g., 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H-isoindole and 1H- and 2H-pyrazole. Tautomeric forms may be in equilibrium or spherically locked into one form by appropriate substitution. For example, it will be recognized that the following pyrazole ring can form two tautomers:

CP77 ίη/77Π7/Ε/ΥΙΛΙ

The claims are intended to encompass both tautomers.

All compounds and pharmaceutically acceptable salts thereof may be found together with other substances such as water and solvents (for example, hydrates and solvates) or may be isolated.

In some embodiments, the compounds described herein, or their salts, are substantially isolated. "Substantially isolated" means that the compound is at least partially or substantially separated from the environment in which it was formed or detected. The partial separation may include, for example, a composition enriched in 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 salts thereof. Methods for isolating compounds and their salts are common in the art.

The term "pharmaceutically acceptable" is used herein to refer to compounds, materials, compositions and/or dosage forms suitable within the scope of good medical judgment, for use in contact with human tissues. and animals without causing excessive toxicity, irritation, allergic response or other problem or complication, based on a reasonable benefit/risk ratio.

The terms "room temperature" or "ta", as used herein, are understood in the art and generally refer to a temperature, for example, a reaction temperature, which is about the temperature of the environment at which at which the reaction is carried out, for example, a temperature of about 20 °C to about 30 °C.

The present application also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the described compounds, where the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkaline or organic salts of acid residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts of the present application include conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present application can be synthesized from the parent compound containing an acidic or basic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the basic or free acid forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of both; In general, non-aqueous media such as ether, ethyl acetate, alcohols (for example, methanol, ethanol, iso-propanol or butanol) or acetonitrile (ACN) are preferred. Lists of suitable salts can be found at Remington's

CQ77 ίη/77Π7/Ε/ΥΙΛΙ that the researcher, veterinarian, doctor or other health professional seeks in a tissue, system, animal, individual or human being.

As used herein, the term "treat" or "treatment" refers to (1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual who suffers from or exhibits the pathology or symptomatology of the disease, condition or disorder (i.e., stopping further development of the pathology and/or symptomatology); (2) improve the disease; for example, improving a disease, condition or disorder in an individual who suffers from or exhibits the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and/or symptomatology) such as decreasing the severity of the illness; or (3) prevent the disease, condition, or disorder in an individual who may be predisposed to the disease, condition, or disorder, but does not yet have or exhibit the pathology or symptomatology of the disease. In some embodiments, treat refers to inhibiting or ameliorating the disease. In some embodiments, to treat is to prevent a disease.

Combination therapies

The methods described herein may further comprise administering one or more additional therapeutic agents. The one or more additional therapeutic agents may

CQ77 ίη/77Π7/Ε/ΥΙΛΙ be administered to a patient simultaneously or sequentially.

In some embodiments, the additional therapeutic agent is an antibiotic. In some embodiments, the antibiotic is clindamycin, doxycycline, minocycline, trimethoprimsulfamethoxazole, erythromycin, metronidazole, rifampicin, moxifloxacin, dapsone, or a combination of these. 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 spheroid. In some embodiments, the additional therapeutic agent is a corticosteroid. In some embodiments, the spheroid is, for example, 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

CQ77 ίη/77Π7/Ε/ΥΙΛΙ modalities, 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 cyclosporin A. In some embodiments, the immunosuppressant is mycophenolate mofetil or mycophenolate sodium.

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

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

In general, a BRM is a substance prepared from living organisms to treat diseases, which can occur naturally in the body or can be prepared in the laboratory. Some examples of BRMs include IL-2, interferon, various types of colony-stimulating factors (CSF, GMCSF, G-CSF), monoclonal antibodies such as abciximab, etanercept, infliximab, rituximab, trasturzumab, and high-dose ascorbate.

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

CP77 ίη/77Π7/Ε/ΥΙΛΙ

In general, IMiDs are immunomodulatory agents. In some embodiments, the IMiD is selected from thalidomide, lenalidomide, pomalidomide, CC-11006 and CC-10015.

In some embodiments, the method further comprises administering an additional therapeutic agent selected from anti-thymocyte globulin, recombinant human granulocyte colony-stimulating factor (G CSF), granulocyte-monocyte CSF (GM-CSF), an agent erythropoiesis stimulant (ESA) and cyclosporine.

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

One or more additional pharmaceutical agents may be used such as, for example, anti-inflammatory agents, immunosuppressants, as well as inhibitors of ΡΙ3Κδ, mTor, Bcr-Abl, Flt-3, RAF and FAK kinases, such as, for example, those described in WO 2006/056399, which is incorporated herein in its entirety by reference, or other agents, in combination with the compounds described herein for the treatment of diseases, disorders or conditions

CP77 ίη/77Π7/Ε/ΥΙΛΙ associated with JAK. The one or more additional pharmaceutical agents may be administered to a patient simultaneously or sequentially.

Examples of Bcr-Abl inhibitors include the compounds and pharmaceutically acceptable salts thereof, of the genera and species described in US Patent No. 5,521,184, WO 04/005281, and US Serial No. 60/578,491, all of which are incorporated herein by reference.

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

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

Examples of suitable FAK inhibitors include compounds and pharmaceutically acceptable salts thereof, 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 incorporated herein by reference.

In some embodiments, one or more of the compounds of the invention may be used in combination with one or more

CQ77 ίη/77Π7/Ε/ΥΙΛΙ additional kinase inhibitors including imatinib, in particular, for the treatment of patients with resistance 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™ (Bolles Labs), Civamide (Opko), sodium hyaluronate (Vismed, Lantibio/TRB Chemedia), cyclosporine (ST-603, Sirion Therapeutics), ARG101 (T) ( testosterone, Argentis), AGR1012(P) (Argentis), ecabet sodium (Senju-Ista), gefarnate (Santen), 15-(s)hydroxyeicosatetraenoic acid (15(S)-HETE), cevilemine, 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-5-[6-[(3iodophenyl)methylamino]purin-9-yl ]-N-methyl-oxolan-2-carbamyl, Can-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), Lacritin (Senju), rebamipide (Otsuka-Novartis),

OT-551 (Othera), ΡΑΙ-2 (University of Pennsylvania and Temple University), pilocarpine, tacrolimus, pimecrolimus (AMS981, Novartis), loteprednol etabonate, rituximab, diquafosol tetrasodium (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 antiangiogenic agent, a cholinergic agonist, a TRP-1 receptor modulator, a calcium channel blocker, a mucin secretagogue, a MUGI stimulant, a calcineurin inhibitor, a corticosteroid. , a P2Y2 receptor agonist, a muscarinic receptor agonist, an mTOR inhibitor, another JAK inhibitor, a Bcr-Abl kinase inhibitor, a Flt-3 kinase inhibitor, a RAF kinase inhibitor, a FAK kinase inhibitor such as, for example, those described in WO 2006/056399, which is incorporated herein in its entirety by reference. In some embodiments, the additional therapeutic agent is a tetracycline derivative (e.g., minocycline or doxycycline). 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; an anti-metabolite/demethylating agent (eg, 5-fluorouracil, capecitabine or azacitidine); an antihormonal therapy (for example, hormone receptor antagonists, SERMs, or aromatase inhibitor); a mitotic inhibitor (for example, vincristine or paclitaxel); a topoisomerase inhibitor (I or II) (for example, mitoxantrone and irinotecan); an apoptotic inducer (eg, ABT-737); a nucleic acid therapy (e.g., antisense or RNAi); nuclear receptor ligands (for example, 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 conjugation or deconjugation molecules; or an EGFR inhibitor (erlotinib).

In some embodiments, the additional therapeutic agent includes an antibiotic, antiviral, antifungal, anesthetic, anti-inflammatory agent including spheroid and non-spheroid anti-inflammatories, and anti-allergy agents. Examples of suitable medications 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; chloramphelicol; neomycin; paramomycin; colistimethate; bacitracin; vancomycin; tetracyclines; rifampin and its derivatives (“rifampins”); cycloserine; beta-lactams; caphalosporins; amphotericins; fluconazole; flucytosine; natamycin; miconazole; ketoconazole; corticosteroids; diclofenac; flurbiprofen; ketorolac; suprofen; Cromolin; lodoxamide; levocabastine; naphazoline; antazoline; pheniramine or azalide antibiotic.

Pharmaceutical formulations 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 known in the pharmaceutical art and can be administered by various routes, depending on whether local or systemic treatment is desired and the area to be treated. Administration may be topical (including transdermal, epidermal, ophthalmic and mucosal membranes, including intranasal, vaginal and rectal administration), pulmonary (for example, by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal or intranasal) , oral or parenteral. Parenteral administration includes administration, intra-arterial, subcutaneous, intraperitoneal, intramuscular or injection or infusion; or intracranial, for example, intrathecal or intraventricular. Parenteral administration may be in the form of a single dose

CP77 ίη/77Π7/Ε/ΥΙΛΙ individually in bolus or can be, for example, through a continuous infusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, aerosols, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oil bases, thickeners, and the like, may be necessary or desirable.

In some embodiments, administration is topical. In some embodiments, administration is topical to the skin.

In some embodiments, administration is oral.

The invention also includes pharmaceutical compositions containing, as an active ingredient, the 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 administration. In preparing the compositions of the invention, the active ingredient is typically mixed with an excipient, diluted by means of an excipient or surrounded within the carrier, for example, in the form of a capsule, sachet, paper or other container. When the excipient functions as a diluent, it can be a solid, semi-solid or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Therefore, the compositions may be in the form of tablets, pills, powders, dragees, sachets, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example , up to 10% by weight of the active compound, hard and soft gelatin capsules, suppositories, sterile injectable solutions and sterile packaged powders.

When preparing a formulation, the active compound can be ground to provide the appropriate particle size before combining it with the other ingredients. If the active compound is considerably insoluble, it can be ground to a particle size less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by grinding to provide a substantially uniform distribution in the formulation, for example, of about 40 mesh.

The compounds of the invention can be milled by using known milling procedures, such as wet milling, to obtain a particle size suitable for tableting and for other types of formulation. Finely divided preparations (nanoparticles) of the compounds of the invention can be prepared through processes 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, acacia gum, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup and methylcellulose. The formulations may additionally include: lubricating agents such as talc, magnesium stearate and mineral oil; wetting agents; emulsifying and suspending agents; preservative agents such as methyl- and propylhydroxy-benzoates; sweetening agents and flavoring agents. The compositions of the invention can be formulated to provide rapid, sustained or delayed release of the active ingredient after administration to the patient using methods known in the art.

In some embodiments, the pharmaceutical composition comprises silicified microcrystalline cellulose (SMCC) and at least one compound described herein, or a pharmaceutically acceptable salt thereof. In some embodiments, the silicified microcrystalline cellulose comprises about 98% microcrystalline cellulose and about 2% w/w silicone dioxide.

In some embodiments, the composition is a sustained release composition comprising at least one compound described herein, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable carrier. In some embodiments, the composition comprises at least one

CQ77 ίη/77Π7/Ε/ΥΙΛΙ compound described herein, or a pharmaceutically acceptable salt thereof, and at least one component selected from microcrystalline cellulose, lactose monohydrate, hydroxypropylmethylcellulose and polyethylene oxide. In some embodiments, the composition comprises at least one compound described herein, or a pharmaceutically acceptable salt thereof, and microcrystalline cellulose, lactose monohydrate, and hydroxypropylmethylcellulose. In some embodiments, the composition comprises 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 silicon dioxide. In some embodiments, the microcrystalline cellulose is Avicel PH102™. In some embodiments, the lactose monohydrate is Fast-flo 316™. In some embodiments, the hydroxypropylmethylcellulose is hydroxypropylmethylcellulose 2208 K4M (e.g., Methocel K4 M Premier™) and/or hydroxypropylmethylcellulose 2208 K100LV (e.g., Methocel K00LV™). In some embodiments, the polyethylene oxide is WSR 1105 polyethylene oxide (e.g., 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 produce the composition.

CP77 ίη/77Π7/Ε/ΥΙΛΙ

The compositions may be formulated in a unit dosage form, each dosage 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 dosage is about 1 mg to about 50 mg or about 1 mg to about 10 mg of active ingredient. In some embodiments, each dosage contains about 10 mg of the active ingredient. In some embodiments, each dosage contains about 50 mg of the active ingredient. In some embodiments, each dosage contains about 25 mg of the active ingredient. The term "unit dosage forms" refers to physically separate units suitable as individual doses for human subjects and other mammals, in which each unit contains a predetermined amount of active material that is calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient.

In some embodiments, the compositions comprise 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 comprise from about 1 mg to about 50 mg or from about 1 mg to about 10 mg of active ingredient. An expert in the technique

CP77 ίη/77Π7/Ε/ΥΙΛΙ You will appreciate that this covers 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 about 25 mg, from about 1 mg to about 50 mg of the 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 or 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 may be effective over a wide dosage range and is generally administered in a pharmaceutically effective amount. However, it will be understood that the amount actually administered of the compound will normally be determined by a physician, in accordance with the

CP77 ίη/77Π7/Ε/ΥΙΛΙ relevant circumstances, including the condition being treated, the route of administration chosen, the compound actually administered, the age, weight and response of the individual patient, the severity of the patient's symptoms, and Similar.

To prepare solid compositions such as tablets, the main active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present application. When these preformulation compositions are referred to as homogeneous, the active ingredient is typically dispersed uniformly throughout the composition so that the composition can be easily subdivided into equally effective unit dosage forms, such as tablets, pills and capsules. This solid preformulation is then subdivided 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 the present application.

The tablets or pills of the present application may be coated or otherwise composed to provide a dosage form that achieves the advantage of prolonged action. For example, the tablet or pill may comprise an internal dosage component and an external dosage component, the latter being located

CP77 ίη/77Π7/Ε/ΥΙΛΙ in the form of an envelope over the first. The two components may be separated by an enteric layer that serves to resist disintegration in the stomach and allow the internal component to pass intact into the duodenum or delay release. A variety of materials can be used for enteric layers or coatings, materials include a variety of polymeric acids and mixtures of polymeric acids with materials such as shellac, cephalic alcohol and cellulose acetate.

Liquid forms into which the compounds and compositions of the present application may be incorporated for oral or injection administration include aqueous solutions, syrups with a suitable flavor, aqueous or oily suspensions and emulsions flavored with edible oils such as seed oil. cotton, sesame oil, coconut oil or peanut oil, as well as elixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable aqueous or organic solvents or mixtures thereof, and powders. The liquid or solid compositions may contain suitable pharmaceutically acceptable excipients, as described above. In some embodiments, the compositions are administered via the nasal or oral respiratory route for a local or systemic effect. The compositions can be nebulized through the use of inert gases. Nebulized solutions can be inhaled directly from the nebulizer device or the nebulizer device can be attached to a protective cover of a face mask or intermittent positive pressure respirator. Compositions in the form of solutions, suspensions or powders can be administered orally or nasally by devices that appropriately administer the formulation.

Topical formulations may contain one or more conventional carriers. In some embodiments, the ointments may contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white petroleum jelly, and the like. Cream carrier compositions may be water-based in combination with glycerol and one or more additional components, for example, glycerin monostearate, glycerin-PEG monostearate and cetostearyl alcohol. The gels may be formulated using isopropyl alcohol and water, suitably in combination with other components such as, for example, glycerol, hydroxyethyl cellulose, and the like. In some embodiments, 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. invention. Topical formulations may suitably be packaged in tubes of, 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 the patient will vary depending on what is administered, the purpose of administration, such as prophylaxis or therapy, the condition of the patient, the mode of administration, and the like. In therapeutic applications, the compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. The effective amounts will depend on the state of the disease to be treated, as well as the judgment of the treating physician, depending on factors such as the severity of the disease, the age, weight and general condition of the patient, and the like.

The compositions administered to a patient may be in the form of the pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques or can be filtered under sterile conditions. Aqueous solutions can be packaged for use as is or lyophilized, where the lyophilized preparation is

CQ77 ίη/77Π7/Ε/ΥΙΛΙ combine with a sterile aqueous carrier before administration. The pH of the compound preparations will typically be between 3 and 11, more preferably, between 5 and 9, and even more preferably, between 7 and 8. It will be understood that the use of some of the above excipients, vehicles or stabilizers will result in the formation of pharmaceutical salts.

The therapeutic dosage of the compounds of the present application may vary, for example, according to the particular use for which the treatment is designed, the method of administration of the compound, the health and condition of the patient, as well as the criterion of the treating physician. The proportion or concentration of a compound of the invention in a pharmaceutical composition can vary depending on a number of factors including dosage, chemical characteristics (for example, hydrophobicity) and the route of administration. For example, the compounds of the invention can be provided in an aqueous physiological buffer containing from about 0.1 to about 10% w/v of the compound for parenteral administration. Some typical dosage ranges are from about 1 μρ/kp to about 1 p/kp body weight per day. In some embodiments, the dosage range is from about 0.01 mp/kp to about 100 mp/kp body weight per day. The dosage is likely to depend on variables such as the type and extent of progression of the disease or disorder, the general health status of the particular patient, the relative biological efficacy of the selected compound, the formulation of the excipient, and its route of administration. Effective doses can be extrapolated from dose response curves derived 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 hereinabove.

Kits

The present application also includes pharmaceutical kits useful, for example, in the treatment and/or prevention of cytokine-related diseases or disorders, such as CRS, which include one or more containers containing a pharmaceutical composition comprising a therapeutically effective amount of a compound described herein. The kits may further include, if desired, one or more of several conventional pharmaceutical kit components such as, for example, containers with one or more pharmaceutically acceptable carriers, additional containers, etc., as will be readily apparent to those skilled in the art. in the technique. The kit may also include instructions, either as package inserts or as labels, indicating the quantities of the components to be administered, administration guidelines and/or

CP77 ίη/77Π7/Ε/ΥΙΛΙ guidelines for mixing components.

EXAMPLES

The invention will be described in more detail by means of specific examples. The examples below are provided for illustrative purposes and are not intended to limit the invention in any way. Those skilled in the art will readily recognize various non-critical parameters that can be changed or modified to obtain essentially the same results.

Example A: In Vitro JAK Kinase Assay

JAK1 inhibitors that can be used for the treatment of cytokine-related diseases or disorders are evaluated for JAK target inhibitory activity according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999, 269, 94-104 . The catalytic domains of JAK1 (aa 837-1142), JAK2 (aa 8281132), and JAK3 (aa 781-1124) with a His tag at the N terminus are expressed using baculovirus in insect cells and purified. The catalytic activity of JAK1, JAK2, or JAK3 was analyzed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogeneous time-resolved fluorescence (HTRF). The IC50 of the compounds for each kinase is measured in the 40 microL reactions containing the enzyme, ATP and 500 nM peptide in buffer.

CQ77 ίη/77Π7/Ε/ΥΙΛΙ

50 mM Tris (pH 7.8) with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL BSA (0.01%). For ICso 1 mM 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 μΡ of 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, MA). Binding with the europium-labeled antibody occurred for 40 min, and the HTRF signal was measured in a Fusion plate reader (Perkin Elmer, Boston, MA). In this assay, the compounds in Table 1 were evaluated and shown to have the IC50 values in Table 1.

Example B: Safety and efficacy study of JAK1 and/or JAK2 inhibitors in subjects with moderate to severe hidradenitis suppurativa

A randomized, double-blind, placebo-controlled, multicenter study is conducted in men and women aged 1875 years with moderate (Hurley stage II) to severe hidradenitis suppurativa (Hurley stage III) for at least 6 months. Hurley stage I is associated with the formation of abscesses (single or multiple) without sinus tracts or healing. 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 involvement or with multiple interconnected tracts and abscesses throughout the area. Study participants are randomly assigned into 5 groups (about 50 participants per group) and treated with 15, 30, 60, or 90 mg of a JAK1 and/or JAK2 inhibitor (e.g., ruxolitinib, Compound 4 or Compound 5, or a pharmaceutically acceptable salt thereof), or placebo. At week 16 (primary endpoint), participants in the placebo group are randomly and equally reassigned to the active treatment groups for 8 weeks. The blind remains. The primary endpoint is the proportion of subjects who are able to achieve hidradenitis suppurativa clinical response (HiSCR) at week 16.

Secondary endpoints include (1) Proportion of subjects with HiSCR from baseline at each visit; (2) Proportion of subjects achieving an abscess and inflammatory nodule (AN) count of 0 to 2 at each visit; (3) Mean change from baseline on the HS Numerical Pain Rating Scale 1 at each visit; (4) Change in modified Sartorius scale at week 16 and week 24; (5) Change in the number of fistulas draining at each visit; (6) Proportion of subjects requiring injury recovery treatments through week 24; (7) Number of episodes of injury recovery treatments up to week 24; (8) PK of the JAK1 and/or JAK2 inhibitor population (e.g., apparent clearance, apparent volume of distribution); (9) Safety and tolerability assessed by monitoring AE frequency, duration, and severity, physical examination, vital signs, and laboratory data for hematology, serum chemistry, and urinalysis; (10) Change in dermatological life quality index (DLQI) assessment; (11) Change in disease severity from baseline, as assessed by IHS43 score at each visit; (12) Change in hidradenitis suppurativa quality of life (HiSQOL) assessment at home visit compared to baseline; and (13) Dose/exposure-response assessment of percentage change from baseline in terms of efficacy and safety endpoints during treatment periods.

HiSCR is defined as at least a 50% reduction in abscess and inflammatory nodule (AN) count with no increase in abscess count and no increase in draining fistula count at week 16 relative to baseline). The numerical pain rating scale is used to evaluate the worst skin pain and the average skin pain due to HS. Ratings for the 2 items range from 0 (no skin pain) to 10 (skin pain is as bad as you can imagine). Participants record assessments in a diary every day before going to bed and are based on a memory period of the “last 24 hours.” The scale of

Modified Sartorius 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 inguinocrural folds, perianal area, perineal area and others): points awarded per nodule ( 2 points for each one); abscesses (4 points); fistulas (4 points); scar (1 point); and the greatest distance between two injuries (2-6 points, 0 if there are no injuries); and if the lesions are separated, by normal skin (yes-0 point; no-6 points). The total Sartorius scale is the sum of the 12 regional scores. Injury recovery treatment: In the event that a very painful injury requires immediate intervention, doctors have the option of performing recovery interventions. Only two types of interventions are allowed: (1) injection with intralesional triamcinolone acetonide suspension (up to 30 mg total at the same visit) and/or (2) incision and drainage. An intervention can be performed on a maximum of two different lesions at the same visit or on the same lesion at two different study visits. The same injury cannot be treated twice in the same visit. If a subject requires more than two interventions before week 16, he or she is withdrawn from the study. International Hidradenitis Suppurativa Severity Scoring System (IHS4): IH4 (points) = (number of nodules x 1) + (number of abscesses x 2) + (number of tunnels

CQ77 ίη/77Π7/Ε/ΥΙΛΙ drainage [fistulas/sinuses] * 4). mild HS: 3 points; Moderate HS: 4-10 points; Severe HS: h 11 points.

Study treatment 1 (active) includes one oral tablet containing 15 mg of 4-[3-(cyanomethyl)-3(3',5'-dimethyl-lH,l'H-4,4'-bipyrazol-l -yl)azetidin-l-yl]-2,5difluoro-N-[(1S)-2,2,2-trifluoro-l-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 an oral placebo tablet.

Blood samples are taken to measure plasma concentrations of the JAK1 and/or JAK2 inhibitor, at least at weeks 2, 12, 16, 20, and 24 before and after administration of the study drug at pre-dose times. , 1 hour after dose, 2-5 hours after dose. At the premature discontinuation visit, if subjects withdraw before week 8, a minimal PK sample is taken, if possible. The date/time of the last previous dose administration is also recorded.

Superiority testing of JAK1 and/or JAK2 inhibitor 90, 60, 30 and 15 mg compared to placebo is carried out using the Hochberg procedure at a two-sided total level of α = 0.05. Comparisons between each active group and placebo at week 16 are made with logistic regression. At all dose levels, the evidence

CP77 ίη/77Π7/Ε/ΥΙΛΙ of superiority are significant (e.g., with a 10%, 20%, 30%, 40%, or 50% improvement in HiSCR (Hidradenitis Suppurativa Clinical Response)) and demonstrate the efficacy of the JAK1 and/or JAK2 inhibitor to treat HS. Testing shows a reduction in nodules and no inferiority/superiority compared to placebo.

All secondary and exploratory efficacy measurements are evaluated using descriptive statistics. Clinical safety data (vital signs, routine laboratory tests, and AEs) are analyzed using descriptive statistics. One or more exposure-response relationships (ER) are determined between plasma JAK1 and/or JAK2 inhibitor PK exposures and efficacy/safety data. An interim analysis is performed to estimate treatment response and facilitate planning for future studies when at least half of the randomized subjects reach week 16.

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

Transformed human keratinocyte (HaCaT) cells were obtained from AddexBio (Catalog # T0020001) and cultured in optimized Dulbecco's modified Eagle's medium (AddexBio, Catalog # C0003-02) supplemented with 10% fetal bovine serum (Hyclone, Catalog # 16140-071) and lx penicillin/streptomycin (Gibco, Catalog # 15140-122).

When the cells reached 80-90% confluency, they were washed with lx DPBS, then separated from the tissue culture flasks by incubation with 0.25% T trypsin (Gibco, Catalog # 25200-056) for 3-5 minutes at 37 °C/5% CO2. Cell culture medium was added to the cells with trypsin, then the cell suspension was transferred to a sterile 15 mL centrifuge tube to be centrifuged for 10 minutes at 1300 rpm. Trypsin-containing medium was aspirated from the cell pellet and then the pellet was resuspended in 10 mL of cell culture medium. Cells were counted using a Countess II automated cell counter, then plated into tissue culture-treated 24-well plates at a concentration of 4xl0.4 cells/mL and incubated for 48 hours at 37 °C/5% CO2. After 48 hours, the medium was removed and replaced with 500 uL of cell culture medium or a combinatorial stimulation of recombinant human interferon gamma (R&D Systems, Catalog #285-IF-100) and recombinant human tumor necrosis factor alpha ( R&D Systems, Catalog # 210-TA-020). HaCaT cells treated with combinatorial cytokine stimulation were treated at 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 shaking for 30 seconds, then incubated for 24 hours at 37 °C/5% CO2. At the end of the 24-h incubation, the medium was immediately removed from each plate.

RNA was isolated from HaCaT cells using QuantiGene Plex assay protocols and reagents (Affymetrix, Catalog # QGP-232-M18042302). Cells were washed with Ix DPBS, then used by incubation with the provided QuantiGene lysis buffer for 30 minutes at 50-55°C. Cell lysates were incubated for 18-24 hours at 55°C with capture beads and a set of probes designed to hybridize specifically to the mRNA of the targets of interest. The panel of 32 targets of interest included housekeeping genes used for normalization of the results. After incubation for 18 to 24 hours, the signal was amplified by using branched DNA methodologies, according to the manufacturer's procedures (Affymetrix, Catalog # QGP-232-M18042302). After the hybridization and washing steps, the assay plate was read on the Luminex 200 and the data were expressed as net average fluorescence intensity. The data were then normalized to the net average fluorescence intensity of the housekeeping gene HPRT1 (Table 2).

CP77 ίη/77Π7/Ε/ΥΙΛΙ

Table 2. Stimulation of human keratinocytes with

CO77 ίη/77Π7/Ε/ΥΙΛΙ

TNFa and IFNy induce the JAK/STAT pathway and proinflammatory cytokines

<td>gene</td><td>Treatment</td><td>MFIto</td><td>worth Q</td>

<td rowspan="5">JAK1</td><td>Vehicle</td><td> 126.7 ± 6.55</td><td></td>

<td rowspan="5">JAK2</td><td>Vehicle</td><td> 21.7 ± 0.53</td><td> —</td>

<td rowspan="5">JAK3</td><td>Vehicle</td><td> 0.1 + 0.02</td><td> -</td>

<td rowspan="5">ΤΥΚ2</td><td>Vehicle</td><td> 167.84 ± 2.25</td><td></td>

<td rowspan="3">STAT1</td><td>Vehicle</td><td> 484.33 ± 4.52</td><td></td>

CP77 ίη/77Π7/Ε/ΥΙΛΙ

<td rowspan="5">STAT3</td><td>Vehicle</td><td> 606.76 + 11.51</td><td></td>

<td rowspan="5">STAT4</td><td>Vehicle</td><td> 2.27 ± 0.12</td><td> —</td>

CP77 ίη/77Π7/Ε/ΥΙΛΙ

<td rowspan="5">STAT5A</td><td>Vehicle</td><td> 1.03 + 0.1</td><td> -</td>

<td rowspan="5">STAT 6</td><td>Vehicle</td><td> 626.95 ± 22</td><td> —</td>

<td rowspan="3">IL1A</td><td>Vehicle</td><td> 156.9 ± 1.89</td><td></td>

CP77 ίη/77Π7/Ε/ΥΙΛΙ

<td rowspan="5">IL6</td><td>Vehicle</td><td> 5.89 ± 0.19</td><td> -</td>

<td colspan="4">toData are presented as the mean + standard error (SEM)</td>

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Target proteins of interest were detected and quantified in the media using ProCarta Multiplex immunoassay protocols and reagents (Invitrogen, Catalog # EPX450-12171-901). The medium was incubated with antibody-conjugated microspheres designed to bind to the epitopes of specific target proteins and identify the bound protein through the distinctive spectral pattern of the microsphere. Biotinylated detection antibodies, designed to bind to different epitopes of the same target proteins, and streptavidin-PE were added to the assay plates to quantify the amount of target protein. Assay plates were read on the Luminex 200 and data were expressed as net average fluorescence intensity. Net average fluorescence intensity values for the antigen standard curve, prepared according to the manufacturer's procedures (Invitrogen, Catalog # EPX45012171-901), were plotted against the expected concentrations for each standard. The concentration of each protein was extrapolated from the antigen standard curve and the concentrations were expressed as pg/mL (Table 3).

Table 3. Stimulation of human keratinocytes with TNFa and IFNy induces the production of proinflammatory cytokines.

CP77 ίη/77Π7/Ε/ΥΙΛΙ

<td>Protein</td><td>Treatment</td><td>pg/mLto</td><td>worth Q</td>

<td rowspan="5">IL-la</td><td>Vehicle</td><td> 0.37 ± 0.05</td><td> -</td>

<td rowspan="2">IL-6</td><td>Vehicle</td><td> 72.86 ± 9.77</td><td> -</td>

<td rowspan="5">ΙΡ-10</td><td>Vehicle</td><td> 16.61 ± 1.6</td><td> -</td>

<td rowspan="5">ΜΙΡΙα</td><td>Vehicle</td><td> 7.47 + 1.13</td><td> -</td>

<td rowspan="4">RANTES</td><td>Vehicle</td><td> 11.78 ± 1.41</td><td> -</td>

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<td colspan="4">toData are presented as the mean ± standard error (SEM)</td>

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

Transformed human keratinocyte cells (HaCaT) were obtained from AddexBio (Catalog #T0020001) and cultured as described in Example C. Four AD compounds were reconstructed (A: ruxolitinib, B: itacitinib ({1-{1-[3-fluoro-2(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3[4-(7Hpyrrolo[2,3-d ]pyrimidin-4-yl)-lH-pyrazol-l-yl]azetidin-3yl}acetonitrile), C: 4-[3-(cyanomethyl)-3-(3',5'-dimethyl1H,1'H-4 ,4'-bipyrazol-l-yl)azetidin-l-yl]-2,5-difluoro-N[ (1S)-2,2,2-trifluoro-l-methylethyl] benzamide, D: ((2R,5S )-5{2-[(IR)-1-hydroxyethyl]-lH-imidazo[4,5-d]thieno[3,2b]pyridin-l-yl}tetrahydro-2H-pyran-2-yl)acetonitrile) in

DMSO, then each compound was serially diluted with culture medium to concentrations of 400 nM, 200 nM, 100 nM, and 50 nM. After 48 hours, the cell culture medium was removed from the 24-well plates and replaced with 250 uL of serially diluted drug-containing medium, then incubated for 15 minutes at 37°C/5% CO2. After incubation with drug, 250 uL of combinatorial stimulation containing recombinant human gamma interferon (R&D Systems, Catalog #285-IF-100) and recombinant human tumor necrosis factor alpha (R&D Systems, Catalog #210) was added to the plates. -TA-020). 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 the 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 shaking for 30 seconds, then incubated for 24 hours at 37 °C/5% CO2. At the end of the 24-h incubation, the medium was immediately removed from each plate.

RNA was isolated from HaCaT cells using QuantiGene Plex assay protocols and reagents (Affymetrix, Catalog # QGP-232-M18042302) according to the manufacturer's guidelines. Cells were washed with lx DPBS, then used by incubation with the provided QuantiGene lysis buffer for 30 minutes at 50-55°C. Cell samples were incubated for 18-24 hours at 55°C with capture beads and a set of probes designed to hybridize specifically to the mRNA of the targets of interest. Genes included housekeeping genes (e.g., HPRT1, GAPDH) used for normalization of results. After incubation for 18 to 24 hours, the signal was amplified by using branched DNA methodologies, according to the manufacturer's procedures (Affymetrix, Catalog # QGP-232-M18042302). After the hybridization and washing steps, the assay plate was read on the Luminex 200 and the data were expressed as net average fluorescence intensity. The data were then normalized to the net average fluorescence intensity of the housekeeping gene HPRT1 (Table 4).

CP77 ίη/77Π7/Ε/ΥΙΛΙ

Table 4. Normalized expression of target genes in human keratinocyte cells > archhcrroo stimulated with TNFα and IFNy in the presence/absence of JAK inhibitors.

<td></td><td></td><td></td><td colspan="2">Compound A</td><td colspan="2">Compound B</td><td colspan="2">Compound C</td><td colspan="2">Compound D</td>

<td>gene</td><td>stimulationto</td><td>Drug concentration</td><td>MFIb</td><td>worth Qc</td><td>MFIb</td><td>worth Qc</td><td>MFIb</td><td>p valuec</td><td>MFIb</td><td>worth P°</td>

Q l\

C h

Q l\ C hhc

roc

Q l\ C hhc

roc

Q l\

C h

Q l\

C h

archhc

roc

to Stimulation with TNFa (25 ng/mL) and IFNy (25 ng/mL)b Data are presented as mean + standard errorc Significant differences compared to stimulation with TNFα and IFNγ aloneANDIndicates a significant difference of p<0.3001 from vehicle (no stimulation and no drug concentration) alone€ Indicates a significant difference of p<0.1 from the vehicle

FIGURES 1-4 illustrate the individual gene expression values (MFI) for JAK1, JAK2, IL-Ία, and IL-6, respectively, for each experimental replicate in keratinocytes simulated with TNFα and IFN-γ in the presence/absence of inhibitors by JAK.

Target proteins of interest were detected and quantified in the media using ProCarta Multiplex immunoassay protocols and reagents (Invitrogen, Catalog # EPX450-12171-901). The medium was incubated with antibody-conjugated microspheres designed to bind to the epitopes of specific target proteins and identify the bound protein through the distinctive spectral pattern of the microsphere. Biotinylated detection antibodies, designed to bind to different epitopes of the same target proteins, and streptavidin-PE were added to the assay plates to quantify the amount of target proteins. Test plates were read on the Luminex 200 and data were expressed as net average fluorescence intensity. Net average fluorescence values for the antigen standard curve, prepared according to the manufacturer's procedures (Invitrogen, Catalog #EPX450-12171-901), were plotted against the expected concentrations for each standard. The concentration of each protein was extrapolated from the antigen standard curve and the concentrations were expressed as 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.

<td></td><td></td><td></td><td colspan="2">Compound A</td><td colspan="2">Compound B</td><td colspan="2">Compound C</td><td colspan="2">Compound D</td>

<td>Protein</td><td>stimulation3</td><td>Drug concentration</td><td>pg/mLb</td><td>P valuec</td><td>pg/mLb</td><td>P valuec</td><td>pg/mLb</td><td>P valuec</td><td>pg/mLb</td><td>worth Qc</td>

archhc

rroo

Q l\ C hh

C rroc

<td rowspan="3">RANTES</td><td></td><td> -</td><td colspan="8"> 9.56 ±0.56</td>

Q l\ C hh

C rroc

<td colspan="11">to Stimulation with TNFa (25 ng/mL) and IFNy (25 ng/mL)b Data are presented as mean+standard errorc Significant differences compared to stimulation with TNFα and IFNγ alone¥ Indicates a significant difference of p<0.0001 from vehicle (no stimulation and no drug concentration) alone</td>

Q l\ C hhc

roc

103

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

Example E: Skin biopsies from hidradenitis suppurativa are characterized by increased expression of Janus kinase

Total RNA from healthy control skin from 3 individual donors was purchased from Amsbio (Catalog # HR101 and R1234218-50). Total RNA from healthy control skin from a group of donors was purchased from Life Technologies Corporation (Catalog #QS0639). Hidradenitis suppurativa skin biopsies (41 donors) were purchased from Discovery Life Sciences as formalin-fixed paraffin-embedded (FFPE) blocks from which total RNA was purified.

Gene expression of total RNA samples from healthy control (n=4) and hidradenitis suppurativa (n=41) skin was measured for the genes listed in Table 6 using QuantiGene Plex assay protocols and reagents. (Life Technologies Corporation, Catalog # QGP-277-M19012402). Purified RNAs were used in the recommended assay range of 50 ng to 500 ng and incubated overnight with capture beads designed to hybridize

CP77 ίη/77Π7/Ε/ΥΙΛΙ

104 specifically with the mRNA of the selected genes (Table 6). This panel of targets included several housekeeping genes that were used for normalization of the results. After overnight incubation, the signal was amplified using branched DNA methodologies, according to the manufacturer's procedures (Life Technologies Corporation). The assay plate was read on a Luminex 200 and data were expressed as net average fluorescence intensity (net MFI). Data were normalized to the geometric mean of the net MFI for the housekeeping genes ACTB and GAPDH. FIGURES 7-9 illustrate the gene expression of JAK1, JAK3, TYK2, STAT1, STAT2, STAT3, IRAK1, IRAK2, and IRAK 4 in the skin of healthy controls and subjects with hidradenitis suppurativa.

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Table 6. Targeted genes

<td>Gene identifier</td><td>Gene name</td>

<td>JAK1</td><td>Janus Kinase 1</td>

<td>JAK 2</td><td>Janus Kinase 2</td>

<td>JAK 3</td><td>Janus 3 Kinase</td>

<td>IRAQ1</td><td>Interleukin 1 receptor-associated kinase 1</td>

<td>IRAQ 2</td><td>Interleukin 1 receptor-associated kinase 2</td>

<td>IRAQ 4</td><td>Interleukin 1 receptor-associated kinase 4</td>

<td>STAT1</td><td>Signal transducer and transcription activator 1</td>

<td>STAT3</td><td>Signal transducer and transcription activator 3</td>

105

<td>STAT4</td><td>Signal transducer and transcription activator 4</td>

<td>STAT5A</td><td>Signal transducer and transcription activator 5A</td>

<td>STAT6</td><td>Signal transducer and transcription activator 6</td>

<td>STAT2</td><td>Signal transducer and transcription activator 2</td>

<td>STAT5B</td><td>Signal transducer and transcription activator 5B</td>

<td>TYK2</td><td>Tyrosine kinase 2</td>

<td>SYK</td><td>Spleen-associated tyrosine kinase</td>

<td>GAPDH</td><td>Glyceraldehyde-3-phos fat dehydrogenase</td>

<td>ACTB</td><td>beta actin</td>

Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. It is also intended that the modifications be within the scope of the attached claims. Each reference cited in this application, including all patents, patent applications and publications, is incorporated herein in its entirety by reference.

It is stated that in relation to this date, the best method known to the applicant to put the aforementioned invention into practice is the one that is clear from the present description of the invention.

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Treatment of hidradenitis suppurativa using jak inhibitors.

Abstract

Term

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

SUMMARY OF THE INVENTION

BRIEF DESCRIPTION OF THE FIGURES

DETAILED DESCRIPTION OF THE INVENTION

EXAMPLES

Priority claims3

Members58

Numbers

Titles2

Classification