JAK1 pathway inhibitors for the treatment of cytokine-related disorders

Abstract

This disclosure relates to JAK1 pathway inhibitors and the use thereof in treating cytokine-related diseases or disorders such as cytokine release syndrome (CRS), hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), and 5 CAR-T-cell-related encephalopathy syndrome (CRES). 20 24 20 48 91 16 J ul 2 02 4 A B S T R A C T 2 0 2 4 2 0 4 8 9 1 1 6 J u l 2 0 2 4 5

Description

25 related to the selective JAKI pathway inhibitor and doses of the same, the embodiments related to any salt forms of the compounds disclosed herein, the embodiments related to the individual types of cytokine related diseases or disorders, and the embodiments related to composition and / or administration can be combined in any combination). For example, provided herein is a method for treating a cytokine related disease or 30 disorder selected from the group consisting of cytokine release syndrome (CRS), hemophagocyiic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), or CAR-T-cell-related encephalopathy syndrome (CRES), in a subject, the method comprising administering to the subject a once-daily dose of about 200 mg on a free base basis of {1 -{1-[3-fhioro-2-(trifIuoromethyI)isonicolinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin- 2024204891   16 Jul 2024 4-yi)-lH-pyrazol-l-yl]azetidin-3-yl}acetonitrile, or a pharmaceutically acceptable salt thereof, wherein the dose comprises one or more sustained-release dosage forms each comprising the {l-{I-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-IH-pyrazol-l-yl]azetidin-3-yl}acetonitrile, or a 5 pharmaceutically acceptable salt thereof. Sustained-release dosage forms of {l-{l-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl} -3 [4-(7H-pyrrolo[2,3 -d]pyrimidin-4-yl)-1H-pyrazol-l-yl]azetidin-3-yl} acetonitrile, or a pharmaceutically acceptable salt thereof (Table 1, Compound 1) can be found in US Publ. No. 2015 / 0065484, fded August 6, 2014, which is 10 hereby incorporated by reference in its entirety. All possible combinations are not separately listed herein merely for the sake of brevity. The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless 15 otherwise indicated. Compounds that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how7 to prepare optically active forms from optically inactive starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all 20 such stable isomers are contemplated in the present invention. Cis and trans geometric isomers of the compounds of the present invention are described and may be isolated as a mixture of isomers or as separated isomeric forms. In some embodiments, the compound has the (R)-configuration. In some embodiments, the compound has the (^-configuration. 25          Resolution of racemic mixtures of compounds can be carried out by any of numerous methods know n in the art. An example method includes fractional recrystallizaion using a chiral resolving acid which 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, 30 mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids such as p-camphorsulfonic acid. Other resolving agents suitable for fractional crystallization methods include stereoisomerically pure forms of a-methylbenzylamine (e.g., S and R forms, or diastereomerically pure forms), 2-phenylglycinol, norephedrine, ephedrine, N-methylephedrine, cyclohexylethylamine, 1,2-diaminocyclohexane, and the like. 2024204891   16 Jul 2024 Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art. Compounds described herein also include tautomeric forms. Tautomeric forms result 5 from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more 10 positions of a heterocyclic system, for example, 1H- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, 1H- and 2H- isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution. Compounds described herein can also include isotopically-labeled compounds of the disclosure. An “isotopically” or “radio-labeled” compound is a compound of the disclosure 15 where one or more atoms are replaced or substituted by an atom having an atomic mass or mass number different from the atomic mass or mass number typically found in nature (i.e., naturally occurring). Suitable radionuclides that may be incorporated in compounds of the present disclosure include but are not limited to 2H (also written as D for deuterium), 3H (also written as T for tritium), !1C, 13C, 14C, i3N, i5N, 15O, i7O, 18O, 18F, 35S, 36C1, S2Br, 75Br, 76Br, 20 "Br, !23I, 124I, I25I and !3iI. For example, one or more hydrogen atoms in a compound of the present disclosure can be replaced by deuterium atoms (e.g., one or more hydrogen atoms of a Ci-6 alkyl group of Formulae (I), (II), or (III) or a compound of Table 1 can be optionally substituted with deuterium atoms, such as -CDs being substituted for -CH3).The term, “compound,” as used herein is meant to include all stereoisomers, geometric isomers, 25 tautomers, and isotopes of the structures depicted, unless the name indicates a specific stereoisomer. Compounds herein identified by name or structure as one particular tautomeric form are intended to include other tautomeric forms unless otherwise specified. All compounds, and pharmaceutically acceptable salts thereof, can be found together with other substances such as water and solvents (e.g. hydrates and solvates) or can be 30 isolated. In some embodiments, the compounds described herein, or salts thereof, are substantially isolated. By “substantially isolated” is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compounds 2024204891   16 Jul 2024 described herein. Substantial separation can 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 salt thereof. Methods for isolating compounds and their salts are routine in the art. 5          The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. 10          The expressions, “ambient temperature” and “room temperature” or “rt” as used herein, are understood in the art, and refer generally to a temperature, e.g. a reaction temperature, that is about the temperature of the room in which the reaction is carried out, for example, a temperature from about 20 °C to about 30 °C. The present invention also includes pharmaceutically acceptable salts of the 15 compounds described herein. As used herein, “pharmaceutically acceptable salts” refers to derivatives of the disclosed compounds wherein 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; alkali or organic salts of acidic residues such as carboxylic acids; and the 20 like. The pharmaceutically acceptable salts of the present invention include the 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 invention can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free 25 acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixtur e of the two; generally, non-aqueous media like ether, ethyl acetate, alcohols (e.g., methanol, ethanol, iso-propanol, or butanol) or acetonitrile (ACN) are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 30 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety. As used herein, the term “subject”, “individual,” or “patient,” used interchangeably, refers to any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, 2024204891   16 Jul 2024 cats, swine, cattle, sheep, horses, or primates, and most preferably humans. In some embodiments, the “subject,” “individual,” or “patient” is in need of said treatment. In some embodiments, the inhibitors are administered in a therapeutically effective amount. As used herein, the phrase “therapeutically effective amount” refers to the amount of 5 active compound or pharmaceutical agent that elicits the biological or medicinal response that is being sought in a tissue, system, animal, individual or human by a researcher, veterinarian, medical doctor or other clinician. In some embodiments, the dosage of the compound, or a pharmaceutically acceptable salt thereof, administered to a patient or individual is about 1 mg to about 2 g, about I mg to about 1000 mg, about 1 mg to about 500 mg, about 1 mg to about 10   200 mg, about 1 mg to about 100 mg, about 1 mg to 50 mg, or about 50 mg to about 500 mg. In some embodiments, the dosage of the compound, or a pharmaceutically acceptable salt thereof, is about 200 mg. As used herein, the term “treating” or “treatment” refers to one or more of (1) inhibiting the disease; for example, inhibiting a disease, condition or disorder in an individual 15 who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., arresting further development of the pathology and / or symptomatology); (2) ameliorating the disease; for example, ameliorating a disease, condition or disorder in an individual who is experiencing or displaying the pathology or symptomatology of the disease, condition or disorder (i.e., reversing the pathology and / or symptomatology) such as 20 decreasing the severity of disease; or (3) preventing the disease, condition or disorder in an individual who may be predisposed to the disease, condition or disorder but does not yet experience or display the pathology or symptomatology of the disease. In some embodiments, treating refers to inhibiting or ameliorating the disease. In some embodiments, treating is preventing the disease. 25 Combination Therapies The methods described herein can further comprise administering one or more additional therapeutic agents. The one or more additional therapeutic agents can be administered to a patient simultaneously or sequentially. 30          In some embodiments, the additional therapeutic agent is an IL-6 antagonist or receptor antagonist. In some embodiments, the IL-6 receptor antagonist is tocilizumab. In some embodiments, the additional therapeutic agent is an inhibitor of MCP-1. In some embodiments, the additional therapeutic agent is an inhibitor of MIP1B. In some embodiments, the additional therapeutic agent is an inhibitor of IL-2R. In some 2024204891   16 Jul 2024 embodiments, the additional therapeutic agent is an inhibitor of IL-1R. In some embodiments, the additional therapeutic agent is an inhibitor of TNF-a. In some embodiments, the additional therapeutic agent is an anti-CD25 antibody. In some embodiments, the anti-CD25 antibody is daclizumab. 5          In some embodiments, the additional therapeutic agent is an antagonist of IL-1 p. In some embodiments, the additional therapeutic agent is an IL1 receptor antagonist (ILIRa). In some embodiments, the IL1 receptor antagonist (ILIRa) is anakinra. In some embodiments, the additional therapeutic agent is a corticosteroid. In some embodiments, the corticosteroid is prednisone. 10          In some embodiments, any of the preceding additional therapeutic agents is used in further combination with a corticosteroid (e.g., prednisone). In some embodiments, the additional therapeutic agents comprise tocilizumab and a corticosteroid. In some embodiments, the additional therapeutic agents comprise tocilizumab and prednisone. 15 Pharmaceutical Formulations and Dosage Forms When employed as pharmaceuticals, the JAKI pathway inhibitors or pharmaceutically acceptable salts thereof, can be administered in the form of pharmaceutical compositions. These compositions can be prepared in a manner well known in the 20 pharmaceutical art, and can be administered by a variety of routes, depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including transdermal, epidermal, ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery'), pulmonary (e.g., by inhalation or insufflation of pow'ders or aerosols, including by nebulizer; intratracheal or intranasal), oral or parenteral. 25 Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal intramuscular or injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, 30 drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary' or desirable. This invention also includes pharmaceutical compositions which contain, as the active ingredient, the JAKI pathway' inhibitor described herein, or a pharmaceutically acceptable salt thereof, in combination with one or more pharmaceutically acceptable carriers 2024204891   16 Jul 2024 (excipients). In some embodiments, the composition is suitable for topical administration. In making the compositions, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, 5 semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, 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, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and 10 sterile packaged powders. In preparing a formulation, the active compound can be milled to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it can be milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size can be adjusted by milling to 15 provide a substantially uniform distribution in the formulation, e.g. about 40 mesh. The JAKI pathway inhibitors may be milled using known milling procedures such as wet milling to obtain a particle size appropriate for tablet formation and for other formulation types. Finely divided (nanoparticulate) preparations of the JAKI selective inhibitors can be prepared by processes known in the art, e.g., see International App. No. WO 2002 / 000196. 20           Some examples of suitable excipients include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; 25 preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art. The compositions can be formulated in a unit dosage form, each dosage containing 30 from about 5 to about 1000 mg (1 g), more usually about 100 to about 500 mg, of the active ingredient. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. 2024204891   16 Jul 2024 In some embodiments, the compositions of the invention contain from about 5 to about 50 mg of the active ingredient. One having ordinary skill in the art will appreciate that this embodies compositions containing about 5 to about 10, about 10 to about 15, about 15 to about 20. about 20 to about 25, about 25 to about 30, about 30 to about 35, about 35 to about 5   40, about 40 to about 45, or about 45 to about 50 mg of the active ingredient. In some embodiments, the compositions of the invention contain from about 50 to about 500 mg of the active ingredient. One having ordinary’ skill in the art will appreciate that this embodies compositions containing about 50 to about 100, about 100 to about 150, about 150 to about 200, about 200 to about 250, about 250 to about 300, about 350 to about 400, or 10 about 450 to about 500 mg of the active ingredient. In some embodiments, the compositions of the invention contain from about 500 to about 1000 mg of the active ingredient. One having ordinary skill in the art will appreciate that this embodies compositions containing about 500 to about 550, about 550 to about 600, about 600 to about 650, about 650 to about 700, about 700 to about 750, about 750 to about 15   800, about 800 to about 850, about 850 to about 900, about 900 to about 950, or about 950 to about 1000 mg of the active ingredient. Similar dosages may be used of the compounds described herein in the methods and uses of the invention. The active compound can be effective over a wide dosage range and is generally 20 administered in a pharmaceutically effective amount. It will be understood, however, that the amount of the compound actually administered will usually be determined by a physician, according to the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like. 25           For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical excipient to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, the active ingredient is typically dispersed evenly throughout the composition so that the composition can be readily subdivided into 30 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 from, for example, about 0.1 to about 1000 mg of the active ingredient of the present invention. 2024204891   16 Jul 2024 The tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer 5 which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate. 10          The liquid forms in which the compounds and compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles. 15           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 supra. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions can be nebulized by use of 20 inert gases. Nebulized solutions may be breathed directly from the nebulizing device or the nebulizing device can be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can be administered orally or nasally from devices which deliver the formulation in an appropriate manner. Topical formulations can contain one or more conventional carriers. In some 25 embodiments, ointments can contain water and one or more hydrophobic carriers selected from, for example, liquid paraffin, polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and the like. Carrier compositions of creams can be based on water in combination with glycerol and one or more other components, e.g. glycerinemonostearate, PEG-glycerinemonostearate and cetylstearyl alcohol. Gels can be formulated using isopropyl 30 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 % of the compound described herein. The topical formulations can be 2024204891   16 Jul 2024 suitably packaged in tubes of, for example, 100 g which are optionally associated with instructions for the treatment of the select indication, e.g., psoriasis or other skin condition. The amount of compound or composition administered to a patient will vary depending upon what is being administered, the purpose of the administration, such as 5 prophylaxis or therapy, the state of the patient, the maimer of administration, and the like. In therapeutic applications, compositions can be administered to a patient already suffering from a disease in an amount sufficient to cure or at least partially arrest the symptoms of the disease and its complications. Effective doses will depend on the disease condition being treated as well as by the judgment of the attending clinician depending upon factors such as 10 the severity of the disease, the age, weight and general condition of the patient, and the like. The compositions administered to a patient can be in the form of pharmaceutical compositions described above. These compositions can be sterilized by conventional sterilization techniques, or may be sterile filtered. Aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier 15 prior to administration. The pH of the compound preparations typically will be between 3 and 11, more preferably from 5 to 9 and most preferably from 7 to 8. Il will be understood that use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of pharmaceutical salts. The therapeutic dosage of a compound of the present invention can vary according to, 20 for example, the particular use for which the treatment is made, the manner of administration of the compound, the health and condition of the patient, and the judgment of the prescribing physician. The proportion or concentration of a compound described herein in a pharmaceutical composition can vary depending upon a number of factors including dosage, chemical characteristics (e.g., hydrophobicity), and the route of administration. For example, 25 the compounds described herein can be provided in an aqueous physiological buffer solution containing about 0.1 to about 10% w / v of the compound for parenteral administration. Some typical dose ranges are from about 1 pg / kg to about 1 g / kg of body weight per day. In some embodiments, the dose range is from about 0.01 mg''kg to about 100 mg / kg of body weight per day. The dosage is likely to depend on such variables as the type and extent of 30   progression of the disease or disorder, the overall health sta tus of the particular patient, the relative biological efficacy of the compound selected, formulation of the excipient, and its route of administration. Effective doses can be ext rapolated from dose-response curves derived from in vitro or animal model test systems. 2024204891   16 Jul 2024 The compositions of the invention can further include one or more additional pharmaceutical agents such as a chemotherapeutic, steroid, anti-inflammatory compound, or immunosuppressant, examples of which are listed herein. 5 Kits The present invention 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. Such kits can further 10   include, if desired, one or more of various 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. Instructions, either as inserts or as labels, indicating quantities of the components to be administered, guidelines for administration, and / or guidelines for mixing the components, can also be included in the kit. 15 EXAMPLES The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes, and are not intended to limit the invention in any maimer. Those of skill in the art will readily recognize a variety of non- 20 critical parameters which can be changed or modified to yield essentially the same results. The compounds of the Examples have been found to be JAK inhibitors according to a t least one assay described herein. Example A: In vitro JAK Kinase Assay 25          JAKI pathway inhibitors that can be used for the treatment of cytokine-related diseases or disorders are tested for inhibitory activity of JAK targets according to the following in vitro assay described in Park et al., Analytical Biochemistry 1999,269, 94-104. The catalytic domains of human JAKI (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3 (a.a. 781-1124) with an N-terminal His tag are expressed using baculovirus in insect cells and 30 purified. The catalytic activity of JAKI, JAK2 or JAK3 was assayed by measuring the phosphorylation of a biotinylated peptide. The phosphorylated peptide was detected by homogenous time resolved fluorescence (HTRF). ICsos of compounds are measured for each kinase in the 40 microL reactions that contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8) buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg / mL (0.01%) BSA. For 2024204891   16 Jul 2024 the 1 mM ICso measurements, ATP concentration in the reactions is 1 mM. Reactions are carried out at room temperature for 1 hour and then stopped with 20 pL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assay buffer (Perkin Elmer, Boston, MA). Binding to the Europium labeled antibody takes place for 40 minutes and HTRF signal was measured on a 5 Fusion plate reader (Perkin Elmer, Boston, MA). The compounds in Table 1 were tested in this assay and shown to have the IC50 values also found in Table 1. Example B: Anti-CD3 Antibody-Induced Cytokine Release Syndrome in BALB / c Mice 10          JAKI pathway inhibitors can be tested for efficacy against CRS according to an in vivo assay described in Ferran, C. et al. Clin. Exp. Immunol. 1991, 86, 537-543. Specifically, this study can test the ability of a compound to reduce or ameliorate anti-CD3 antibody-induced cytokine release syndrome (CRS) in BALB / c mice. The antibody, clone 145-2C11, is an immunoglobin G (IgG) hamster MoAb that is specific for the e chain of the CD3 murine 15 molecule (Leo, O. et al., Proc. Natl. Acad. Sei. USA, 1987, 34, 1374). Treatment with 145-2C11 induces high affinity IL-2 receptors at the surface of spleen T-cells and results in a release of some cytokines such as tumor necrosis factor (TNF-a), IL-2, IL-3, IL-6, and interferon-gamma (IFN-y) (Ferran, et al. Eur. J. Immunol. 1990, 20, 509-515 and Algre, M. et al., Eur. J. Immunol., 1990, 707). Release of these cytokines results in behavioral changes 20    (e.g., inactivity, piloerection, etc) of the animals. A, Materials and Methods Species / strain: Mice: Male BALB / c Physiological state: Normal Age / weight range al start of study. 6-8 weeks old Animal supplier: Charles River Laboratories Number / sex of animals: 32 total Male mice Randomization: Mice will be randomized into four (4) groups of eight (8) mice prior to the commencement of the study. Justification: Injection ofanti-CD3 antibody (clone I45-2C11) has been shown in the literature to induce cytokine release syndrome and serves as a model with which to test the efficacy of potential therapies. Replacement Animals will not be replaced during the course of the study. 2024204891   16 Jul 2024 Anti-CD3e Identity and lot number: Source: Storage conditions: Vehicle: Dose: Dosina Route / Volume Anti-CD3e Clone 145-2C11 BioXCell 4°C Sterile saline lOpig IV, WOpL per animal Compound: Storage conditions: Vehicle: Dose(s): Dosing Route / Volume Frequency and duration of dosing: Compound 1 (Jaki inhibitor)A RT (formulation RT on tube rotator) 0.5% Methylcellulose 60 mg / kg and 120 mg / kg PO, 0.1mL / 20g (5mL / kg) QD on day 0 A The synthesis and preparation of Compound I of Table 1 or {l-{l-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl} -3 [4-(7H-pyrrolo[2,3-d]pyrimidin-4-yI)-1H-pyrazol-I-yl]azetidin-3-yl}acetonitrile and the adipic acid salt of the same can be found, e.g., in US Patent Publ. No. 2011 / 0224190, filed March 9, 2011, US Patent Publ. No. 2013 / 0060026, filed September 6, 2012, and US Patent Publ. No. 2014 / 0256941, filed March 5, 2014, each of which is incorporated herein by reference in its entirety. B. Experimental Design The main objective of this study was io test the ability of a JAKI pathway inhibitor (e.g., Compound 1) to reduce or ameliorate anti-CD3 antibody-induced cytokine release syndrome (CRS) in BALB / c mice. A total of thirty-two (32) BALB / c mice were used for this 15 one day study. Animals were weighed prior to test article dosing, and monitored for the duration of the experiment. On day 0, one hour (1) prior to anti-CD3 antibody administration, vehicle (0.5% methylcellulose) or Compound I were given in a single dose via oral gavage (PO) to animals in groups 2-4 as is detailed in Table 1 A. Group 1 served as naive controls and were not treated. Following the 1 hour pre-treatment with vehicle or Compound I, 20 animals in Groups 2-4 were administered 10 pig of an anti-CD3s antibody (clone I45-2C11) via intravenous injection (IV) in order to induce CRS. All animals were euthanized via CO2 inhalation 1.5 hours after anti-CD3 administration. Whole blood was collected via cardiac puncture into K2EDTA tubes and stored on ice until plasma processing occurred. Plasma was collected and stored at -80 °C until cytokine multiplex was performed. 2024204891   16 Jul 2024 Table 1A. Study Design Group No. Animals TA PreTreatment (PO) Dosing schedule Anti-CD3 (io pg, (IV) Sacrifice Schedule / Collecti on Endpoints 1 8 / males Naive 60 minutes before anti-CD3 - 1.5 hr after anti- CD3 administration Whole blood Ga cardiac puncture (K2EDTA tubes) Plasma collection for multiplex cytokine analysis 2 8 / males Vehicle + 3 8 / males Compound 1 (60 mg / kg) + 4 8 / males Compound 1 (120 mg / kg) + C. Experimental Procedures I. Test Article Pre-Treatment 5          On day 0, animals were dosed with vehicle or test articles or Compound 1 as shown in Table 1 A. Group 2 received a single dose of vehicle (0.5% methylcellulose) via PO at 0.1 mL / 20g. Group 3 received a single dose of 60 mg / kg Compound 1 via PO at 0.1 mL / 20g. Group 4 received a single dose of 120 mg / kg Compound 1 via PO at 0.1 mL / 20g. Group 1 served as the naive controls and were not treated. 10 II. Anti-CD3s Antibody Administration One (1) hour after test article administration, an anti-CD3s antibody (clone 145-2C11) was administered via IV injection to Groups 2-4. Each animal in Groups 2-4 received 10 pg of anti-CD3s antibody in 0.1 mL. 15 III. In-Life monitoring After the administration of the anti-CD3 antibody, animals were closely monitored for signs of distress due to the resulting systemic inflammatory response. Animals that were unable to right themselves, cold to the touch, or moribund were euthanized. Moribund 20 animals were euthanized by CO2 inhalation, and blood was collected via cardiac puncture and plasma retained. 2024204891   16 Jul 2024 IV. Sacrifice One and a half (1.5) hours after anti-CD3 antibody administration all animals were euthanized by CO2 inhalation. 5          V. Collection of Samples Upon sacrifice, whole blood was collected from each animal via cardiac puncture into K2EDTA tubes. The blood was centrifuged and the plasma collected in cryovials. The plasma was frozen and stored at -80°C for the downstream cytokine multiplex assay. 10          VI. Cytokine Multiplex Analysis Plasma samples are thawed on ice and used for a cytokine multiplex according to the manufacturer’s protocol (ThermoFisher). D. Results 15          Compound 1 dose-dependently inhibited IL-6 concentrations within the blood compartment (FIG. 1). This serves as confirmation of the biological activity observed in the Con A preclinical model described below in Example C. An unpaired one-way analysis of variance (ANOVA) incorporating Sidak’s multiple test comparison was performed using GraphPad Prism (version 4.00; GraphPad Software, San Diego California, USA). A value of 20 p < 0.05 was considered significant. Example C: Concanavalin A Induced Cytokine Release Syndrome Concanavalin A (Con A) is a selective T lymphocyte mitogen resulting in broad inflammatory cytokine release and proliferation of CD4 and CD8 T-cells. Injection of Con-A 25 has been shown in the literature to induce cytokine release syndrome and serves as a model with which to test the efficacy of cytokine release syndrome therapies (Gantner, F. at al. Hepatology, 1995, 21, 190-198). The mitogen response is dependent on expression of the T-cell receptor. Animals exhibit behavioral changes such as fever, malaise, hypotension, hypoxia, capillary leak, and potential multi-organ toxicity. 30 A, Materials and Methods Species / strain:                        Mice: Female BALB / c Physiological state:                    Normal Age / weight range at start of study:      6-8 weeks old 2024204891   16 Jul 2024 Animal supplier: Number / sex of animals: Taconic 40 total mice Randomization: Mice were randomized into five (5) groups of eight (8 ) mice prior to the commencement of the study. Justification: Injection of Con-A has been shown in the. literature to induce cytokine release syndrome and serves as a model with which to test the efficacy of potential therapies. B, Experimental Design In particular, this study tests the ability of a selective JAKI inhibitor (e.g., Compound 1, Table 1) to reduce or ameliorate Con A-induced cytokine release syndrome (CRS) in BALB / c mice. A total of forty (40) BALB / c mice were used for this one day study. Animals 5 were weighed prior to test article dosing, and monitored for the duration of the experiment. On day 0, Sixty (60) minutes prior to Con A administration, vehicle (0.5% methylcellulose) or Compound 1 (60 and 120 mg / kg) was given in a single dose via oral gavage (PO) to animals in groups 2-4 as detailed in Table 2A. Group 1 served as naive controls and were not treated. Following the 45 minutes pre-treatment with vehicle or Compound I, animals in 10 Groups 2-4 were administered 20 mg / kg of Con A via intravenous injection (IV) in order to induce CRS. All animals were euthanized via CO2 inhalation two hours after Con A administration. Whole blood was collected via cardiac puncture into K2EDTA tubes and stored on ice until plasma processing occurs. Plasma was collected and stored at -80 °C until cytokine multiplex was performed. Table 2A. Study Design Group No. of Animals PreTreatment (PO) Dosing schedule Con-A (IV) Sacrifice Schedule / Collection Endpoints 1 10 Naive 60 minutes before Con A - 2 h after Con A administration Whole blood via cardiac puncture Plasma collection for multiplex cytokine analysis 2 10 Vehicle + 3 10 Compound 1 (60 mg / kg) + 4 10 Compound 1 (120 mg / kg) + 2024204891   16 Jul 2024 C. Experimental Procedures Day-1 Animals were weighed and Con-A dose (20 mg / kg) calculated. 5 Vehicle and Compound 1 were prepared at corresponding doses. Day 0 I. Test Article Pre-Treatment On day 0, animals were dosed with vehicle or Compound 1, as in Table 2A. Group 1 10 served as the naive controls and were not treated. Group 2 received a single dose of vehicle (0.5% methylcellulose) via PO at 0.1 mL / 20g. Group 3 received a single dose of 60 mg / kg Compound 1 via PO at 0.1 mL / 20g. Group 4 received a single dose of 120 mg / kg Compound i Ga PO at 0.1 mL / 20g. 15          II. Con-A Administration Sixty (60) minutes after test article administration, Con-A was administered via IV injection to Groups 2-4. Each animal in Groups 2-4 received 20 mg / kg of Con-A in 0.2 mL. III. In-Life monitoring 20          After the administration of Con-A, animals were closely monitored for signs of distress due to the resulting systemic inflammatory response. IV. Sacrifice Two hours after Con-A administration all animals were euthanized by CO2 inhalation. 25 V. Collection of Samples Upon sacrifice, whole blood was collected from each animal via cardiac puncture into K2EDTA tubes. The blood was centrifuged and the plasma collected in cryovials. The plasma was frozen and stored at -80°C for the downstream cytokine multiplex assay. 30 VI. Cytokine Multiplex Analysis Plasma samples are thawed on ice and used for a cytokine multiplex according to the manufacturer’s protocol (ThermoFisher). 2024204891   16 Jul 2024 D. Results Compound 1 dose-dependently inhibited IL-6 concentrations within the blood compartment (FIG. 2A). This cytokine is a key mediated of CRS pathophysiology. The T-cell derived IFNy and GM-CSF cytokines were also significantly inhibited suggesting that 5 Compound 1 has therapeutic potential beyond tocilizumab’s restricted mechanism of action (anti-IL-6R only) (FIGs. 2B and 2C). Monocytes and / or macrophage derived cytokines were also reduced. Statistically significant dose-dependent IL-12 reduction (FIG. 3A) was observed as well as trends for treatment effect with IL-Ip (FIG. 3B) and IL-18 (FIG. 3C) suggesting that JAKI specific 10 inhibition has therapeutic potential across immune cell types implicated in CRS pathology. Importantly, the cytokine IL-5 (FIG. 4) was unaffected by Compound 1 treatment, is JAKI independent and not implicated in CRS pathology. This data suggests that Compound 1 based efficacy is not mediated via broad, non-specific, immune suppression. An unpaired one-way analysis of variance (ANOVA) incorporating Sidak’s multiple 15 test comparison was performed using GraphPad Prism (version 4.00; Graph Pad Software, San Diego California, USA). A value of p < 0.05 was considered significant. Example D: Preparation of Sustained Release Formulations of Compound 1 Sustained release tablets comprising Compound 1 were prepared with the excipients 20 being in the amounts shown in the tables below. Protocol A was used for the SRI tablets, Protocol B was used for the SR2 tablets, Protocol C was used for the SR3 tablets and the 25 mg SR tablets, and Protocol D was used for the SR4 tablets. These procedures are disclosed in US Patent Publ. No. 2015 / 0065484, which is directed to sustained release dosage forms of Compound 1. 25 Protocol A: Step 1. Individually screen the adipic acid salt of Compound 1, microcrystalline cellulose, hypromelloses (Methocel KI00 LV and Methocel K4M), and lactose monohydrate. 30          Step 2. Transfer the screened material from Step 1 to a suitable blender and mix. Step 3. Transfer the blend from Step 2 to a suitable granulator and mix. Step 4. Add purified water while mixing. 2024204891   16 Jul 2024 Step 5.        Transfer the granules from Step 4 into a suitable dryer and dry until LOD is less than 3%. Step 6.       Screen the granules from Step 5. Step 7.       Mix screened Magnesium Stearate with granules in Step 6 in a suitable 5 blender. Step 8.        Compress the final blend in Step 7 on a suitable rotary tablet press. Protocol B: Step 1.        Indi vidually screen the adipic acid salt of Compound 1, 10 15 microcrystalline cellulose, hypromellose and pregelatinized starch. Step 2.        Transfer the screened material from Step 1 to a suitable blender and mix. Step 3.       Transfer the blend from Step 2 to a suitable granulator and mix. Step 4.       Add purified water while mixing. Step 5.        Transfer the granules from Step 4 into a suitable dryer and dry until LOD is less than 3%. Step 6.        Screen the granules from Step 5. Step 7.       Individually screened polyox, butylated hydroxytoluene and colloidal silicone dioxide. 20 Step 8.       Transfer the granules from Step 6 and material from Step 7 into a suitable blender and mix. Step 9.       Add screened Magnesium Stearate to the material in Step 8 and continue blending. Step 10.      Compress the final blend in Step 9 on a suitable rotaiy tablet press. 25 Protocol C: Step 1.        Individually screen lactose monohydrate, the adipic acid salt of Compound 1, microcrystalline cellulose and hypromelloses through a suitable screen. Step 2.        Transfer the screened material from Step 1 to a suitable blender and 30 mix. Step 3.       Transfer the blend from Step 2 to a suitable granulator and mix. Step 4.       Add purified water while mixing. Step 5.        Screen wet granules through a suitable screen. 2024204891   16 Jul 2024 Step 6. Transfer the granules from Step 5 into a suitable dryer and dry until LOD is less than 3%. Step 7. Mill the granules from Step 6. Step 8. Mix screened magnesium stearate with granules in Step 7 in a suitable 5 blender. Step 9. Compress the final blend in Step 8 on a suitable rotary tablet press. Protocol D: Step i. Individually screen pregelatinized starch, the adipic acid salt of 10 Compound i, hypromellose, and a portion of required microcrystalline cellulose through a suitable screen. Step 2. Transfer the screened material from Step 1 to a suitable blender and mix. Step 3. Transfer the blend from Step 2 to a suitable granulator and mix. Step 4. Add purified water while mixing. Step 5. Screen wet granules through a suitable screen. Step 6. Transfer the granules from Step 5 into a suitable dryer and dry until LOD is less than 3%. Step 7. Mill the granules from Step 6. 20          Step 8. Screen the remaining portion of microcrystalline cellulose and half of the sodium bicarbonate. Step 9. Transfer the milled granules from Step 7 and screened materials from Step 8 into a suitable blender and mix. Step i 0. Screen the remaining portion of sodium bicarbonate and mix with 25 blend in Step 9. Step 11.      Screen magnesium stearate and mix with blend in Step 10. Step 12.      Compress the final blend in Step 11 on a suitable rotary tablet press. SRI: Composition of 100 mg Sustained Release Tablets Component Function Weight (mg / tablet) Composition (wt%) Adipic acid salt of Compound 1 a Active 126.42a 21.1 Microcrystalline Cellulose Filler 60.0 10.0 2024204891   16 Jul 2024 Component Function Weight (mg / tablet) Composition (wt%) Hypromellose (Methocel K100LV) Release Control 60.0 10.0 Hypromellose (Methocel K4M) Release Control 60.0 10.0 Lactose Monohydrate Filler 290.58 48.4 Magnesium Stearate b Lubricant 3.0 0.5 Purified Waterc Granulating Liquid q.s. — Total 600.0 100 a Conversion factor for adipate salt to free base is 0.791 i b Added after granulation Removed during processing SR2: Composition of 100 mg Sustained Release Tablets Component Function Weight (mg / tablet) Composition (wt%) Adipic acid salt of Compound la Active 126.4 a 21.1 Microcrystalline Cellulose Filler 180.0 30.0 Hypromellose (Methocel K100LV) Binder 6.0 1.0 Polyethylene Oxide (Polyox WRS 1105)b Release Control 180.0 30.0 Pregelatinized Starch Filler 101.6 16.9 Colloidal Silicon Dioxide b Glidant 3.0 0.5 Butylated Hydroxytoluene b Antioxidant 0.012 0.002 Magnesium Stearate b Lubricant 3.0 0.5 Purified Waterc Granulating Liquid q.s. __ Total 600.0 100.0 a Conversion factor for adipate salt to free base is 0.7911 D Added after granulation c Removed during processing 2024204891   16 Jul 2024 SR3 (100 mg): Composition of 100 mg Sustained Release Tablets Component Function Weight (mg / tablet) Composition (wt%) Adipic acid salt of Compound 1a Active 126.4a 21.1 Microcrystalline Cellulose Filler 108.0 18.0 Hypromellose (Methocel K100LV) Release Control 42.0 7.0 Hypromellose (Methocel K4M) Release Control 30.0 5.0 Lactose Monohydrate Filler 290.6 48.4 Magnesium Stearate b Lubricant 3.0 0.5 Purified Walerc Granulating Liquid q.s. __ Total 600.0 100.0 a Conversion factor for adipate salt to free base is 0.7911 b Added after granulation c Removed during processing SR4: Composition of 100 mg Sustained Release Tablets Excipient Function Weight (mg / tablet) Composition (wt%) Adipic acid salt of Compound la Active 126.4a 21.1 Microcrystalline Cellulose d Filler 104.6 17.4 Hypromellose (Methocel K100LV) Release Control 210.0 35.0 Pregelatinized Starch Filler 60.0 10.0 Sodium Bicarbonateb Gastric Floating Aid 96.0 16.0 Magnesium Stearate b Lubricant 3.0 0.5 Purified Waterc Granulation Liquid q.s. — Total 600.0 100.0 a Conversion factor for adipate salt to free base is 0.7911 D Added after granulation 2024204891   16 Jul 2024 c Removed during processing " Partial added before and partial added after granulation 25mg SR: Composition of 25 mg Sustained Release Tablets Component Function Weight (mg / tablet) Composition (wt%) Adipic acid salt of Compound la Active 31.6a 12.6 Microcrystalline Cellulose Filler 105.0 42.0 Hypromellose, (Methocel K100LV) Release Control 25.0 10.0 Hypromellose, (Methocel K4M) Release Control 25.0 10.0 Lactose Monohydrate Filler 62.15 24.9 Magnesium Stearate b Lubricant 1.25 0.5 Purified Water c Granulating Liquid q.s. __ Total 250 100.0 a Conversion factor for adipate salt to free base is 0.7911 D Added after granulation c Removed during processing Various modifications of the invention, in addition to those described herein, will be 10 apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference, including all patent, patent applications, and publications, cited in the present application is incorporated herein by reference in its entirety.

Claims

1. A method for treating a cytokine-related disease or disorder in a subject, said method comprising administering to the subject a JAKI pathway inhibitor which is 4-[3-(cyanomethyl)-3 -(3 ',5 '-dimethyl-1H, 1 'H-4,4'-bipyrazol-1 -yljazetidin-1 -yl] -2,5-difluoro-N-[(lS)-2,2,2-trifhioro-l-methylethyl]benzamide, or a pharmaceutically acceptable salt thereof, wherein the cytokine-related disease or disorder is cytokine release syndrome (CRS), hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), or CAR-T-cell-related encephalopathy syndrome (CRES).

2. The method of claim 1, wherein the cytokine-related disease or disorder is cytokine release syndrome (CRS).

3. The method of claim 1, wherein the cytokine-related disease or disorder is hemophagocytic lymphohistiocytosis (HLH).

4. The method of claim 1, wherein the cytokine-related disease or disorder is macrophage activation syndrome (MAS).

5. The method of claim 4, wherein the macrophage activation syndrome (MAS) is associated with systemic juvenile idiopathic arthritis.

6. The method of claim 1, wherein the cytokine-related disease or disorder is CAR-T-cell-related encephalopathy syndrome (CRES).

7. The method of any one of claims 1-6, wherein the JAKI pathway inhibitor is 4-[3-(cyanomethyl)-3 -(3 ',5 '-dimethyl-1H, 1 'H-4,4'-bipyrazol-1 -yljazetidin-1 -yl] -2,5-difluoro-N-[(lS)-2,2,2-trifluoro-l-methylethyl]benzamide phosphoric acid salt.

8. The method of any one of claims 1-7, further comprising administering tocilizumab to said subject.2024204891   16 Jul 20249. The method of any one of claims 1-7, further comprising administering a corticosteroid to said subject.

10. The method of any one of claims 1-7, further comprising administering prednisone to said subject.

11. The method of any one of claims 1-7, further comprising administering tocilizumab and a corticosteroid to said subject.

12. The method of any one of claims 1-7, wherein the JAKI pathway inhibitor, or a pharmaceutically acceptable salt thereof, is administered as a monotherapy.

13. The method of any one of claims 1-12, wherein the treating comprises ameliorating or inhibiting the cytokine-related disease or disorder in the subject.

14. The method of any one of claims 1-12, wherein the treating comprises preventing.

15. Use of a JAKI pathway inhibitor which is 4-[3-(cyanomethyl)-3-(3',5'-dimethyl-1H, 1 'H-4,4'-bipyrazol-1 -yljazetidin-1 -yl]-2,5-difluoro-N-[( 1 S)-2,2,2-trifluoro-1 -methylethyl]benzamide, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for treating a cytokine-related disease or disorder in a subject, wherein the cytokine-related disease or disorder is cytokine release syndrome (CRS), hemophagocytic lymphohistiocytosis (HLH), macrophage activation syndrome (MAS), or CAR-T-cell-related encephalopathy syndrome (CRES).

16. The use of claim 15, wherein the cytokine-related disease or disorder is cytokine release syndrome (CRS).

17. The use of claim 15, wherein the cytokine-related disease or disorder is hemophagocytic lymphohistiocytosis (HLH).

18. The use of claim 15, wherein the cytokine-related disease or disorder is macrophage activation syndrome (MAS).2024204891   16 Jul 202419. The use of claim 18, wherein the macrophage activation syndrome (MAS) is associated with systemic juvenile idiopathic arthritis.

20. The use of claim 15, wherein the cytokine-related disease or disorder is CAR-T-cell-related encephalopathy syndrome (CRES).

21. The use of any one of claims 15-20, wherein the JAKI pathway inhibitor is 4-[3-(cyanomethyl)-3 -(3 ',5 '-dimethyl-1H, 1 'H-4,4'-bipyrazol-1 -yljazetidin-1 -yl] -2,5-difluoro-N-[(lS)-2,2,2-trifluoro-l-methylethyl]benzamide phosphoric acid salt.

22. The use of any one of claims 15-21, further comprising administering tocilizumab to said subject.

23. The use of any one of claims 15-21, further comprising administering a corticosteroid to said subject.

24. The use of any one of claims 15-21, further comprising administering prednisone to said subject.

25. The use of any one of claims 15-21, further comprising administering tocilizumab and a corticosteroid to said subject.

26. The use of any one of claims 15-21, wherein the medicament comprising the JAKI pathway inhibitor, or a pharmaceutically acceptable salt thereof, is for administration as a monotherapy.

27. The use of any one of claims 15-26, wherein the treating comprises ameliorating or inhibiting the cytokine-related disease or disorder in the subject.

28. The use of any one of claims 15-26, wherein the treating comprises preventing.

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