CN119816304A - Dosing regimen of NLRP3 inhibitors - Google Patents

Abstract

The present disclosure relates to the field of pharmacy, and in particular to NLRP3 inhibitors for use in the treatment of autoinflammatory syndromes. The present disclosure also relates to NLRP3 inhibitors for use in the treatment of autoinflammatory syndromes or a drug combination comprising an NLRP3 inhibitor or a pharmaceutically acceptable salt thereof and at least one other therapeutic agent; to a method for treating autoinflammatory syndromes, the method involving the administration of an NLRP3 inhibitor or the combination; and to the use of an NLRP3 inhibitor or the combination for the manufacture of a medicament for the treatment of autoinflammatory syndromes. In particular, N'-((1,2,3,5,6,7-hexahydro-symmetric indacene-4-yl)carbamoyl)-2-(2-hydroxypropyl-2-yl)thiazole-5-sulfonylimide amide and its enantiomers are used to treat autoinflammatory syndromes, particularly cryptopyrin-associated periodic syndromes (CAPS), familial cold autoinflammatory syndromes (FCAS), Muckler-Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease/chronic infantile neurocutaneous and joint syndrome (NOMID/CINCA) or familial Mediterranean fever (FMF).

Description

Dosing regimen for NLRP3 inhibitors

Technical Field

The present disclosure relates to the field of pharmacy, in particular to NLRP3 inhibitors for use in the treatment of autoinflammatory syndrome. The disclosure also relates to an NLRP3 inhibitor or a pharmaceutical combination comprising an NLRP3 inhibitor or a pharmaceutically acceptable salt thereof and at least one additional therapeutic agent for use in the treatment of autoinflammatory syndrome, to a method for treating autoinflammatory syndrome, which method involves administering an NLRP3 inhibitor or the combination, and to the use of an NLRP3 inhibitor or the combination for the manufacture of a medicament for the treatment of autoinflammatory syndrome.

Background

Cryptothermal protein-related periodic syndrome (CAPS) is a group of rare diseases characterized by cutaneous, musculoskeletal, ocular and nervous system symptoms and chronic systemic inflammation that can lead to organ damage and/or amyloidosis and is caused by mutations obtained by heterozygous functions of the thermo-protein (pyrin) domain 3 (NLRP 3) gene in the nucleotide-binding oligomerization domain-like receptor family. CAPS is classified on the basis of disease severity Cheng Dupu into three clinical phenotypes, familial cold auto-inflammatory syndrome (FCAS), mu Kele-Weiles syndrome (MWS), and Neonatal Onset Multisystem Inflammatory Disease (NOMID) (also known as chronic infant nerve skin and joint syndrome (CINCA)) (Hoffman et al, (2019) Cryopyrin-Associated Periodic Syndromes (CAPS) [ Cryptothermal protein-related periodic syndrome (CAPS) ] which is described in: hashkes P, laxer R, simon A (eds. Textbook of Autoinflammation. [ auto-inflammatory textbook ] Springer, cham.) pages 347-365.

FCAS represents the mildest CAPS clinical phenotype and symptoms are generally limited to low fever, extensive rash, conjunctivitis and polyarthritis, triggering within 1-2 hours after cold exposure and regressing within 24 hours after warmth. These clinical manifestations, attacks or attacks typically begin in infancy and extend throughout life. Many patients with FCAS also show evidence of chronic inflammation between episodes, particularly skin rash every afternoon, which can be associated with headache, myalgia and fatigue occurring at night, but chronic inflammation rarely leads to amyloidosis (about 2%) in this patient population.

Familial Mediterranean Fever (FMF) is caused by mutations in the MEFV gene, and is most common among all known auto-inflammatory diseases. Mutations in the MEFV gene reduce the activity of the thermoprotein, similar to the NLRP3 gene in MWS, FCAS and NOMID/CINCA, thereby disrupting the control of the inflammatory process. FMF is characterized by recurrent painful inflammation in the abdomen, chest, or joints. These attacks are often accompanied by fever and sometimes rashes or headaches. Inflammation may occasionally occur in other parts of the body, such as the heart, membranes around the brain and spinal cord, and in men, in the testes. Typically, the onset lasts 12 to 72 hours and the severity may vary. The length of time between incidences is also variable and may vary between days to years. During these periods, the affected individual is generally free of signs or symptoms associated with the condition. However, if no treatment is performed to help prevent morbidity and complications, accumulation of protein deposits (amyloidosis) can occur in organs and tissues of the body, especially in the kidneys, which can lead to renal failure.

Chronic inflammation and abnormal immune activity are the basis and driving factors for many serious human diseases, ranging from rare and acute inflammatory diseases, rheumatic indications, cardiovascular and metabolic diseases, neurodegenerative diseases and cancer. In these cases, molecular hazard signals generated by dead cells, metabolic disorders, environmental toxins, or diets can act as stimulators to activate NLRP3 inflammatory corpuscles (Mangan MSJ et al, TARGETING THE NLRP3 inflammasome in inflammatory diseases [ targeting NLRP3 inflammatory corpuscles in inflammatory diseases ] Nat Rev Drug Discov [ natural review: drug discovery ];2018;17 (8): 588-606). Once activated, NLRP3 performs nuclear assembly on the inflammatory small complex that coordinates both innate and adaptive immune responses to drive a strong inflammatory response (Evavold et al, how Inflammasomes Inform Adaptive Immunity [ how the inflammatory small body tells adaptive immunity ] J.mol. Biol [ journal of molecular biology ];2018;430 (2): 217-237). Inflammatory corpuscles are large cytoplasmic multimeric protein complexes assembled in response to danger signals, and activation of inflammatory corpuscles causes caspase-1 mediated production of interleukin-1 beta (IL-1 beta) and interleukin-18 (IL-18) and apoptosis of cells (inflammation mediated cell death) (Dinarello, A CLINICAL PERSPECTIVE of IL-1 beta AS THE GATEKEEPER of inflammation [ clinical view of IL-1 beta as an inflammatory gatekeeper ] Eur J Immunol [ J.European Immunol ];2011;41 (5): 1203-17). By producing IL-1 beta and IL-18, nlrp3 inflammatory bodies have been considered as the primary drivers of inflammation associated with autoinflammatory, acute and chronic inflammatory diseases (e.g., CAPS). In these and other cases, the use of NLRP3 inhibitors to inhibit NLRP3 and thereby block IL-1 beta, IL-18 and cell apoptosis potentially provides treatment for conditions where sustained inflammatory body activation leads to pathology (Ridker PM et al ,Modulation of the interleukin-6signaling pathway and incidence rates of atherosclerotic events and all-cause mortality:analyses from the Canakinumab Anti-Inflammatory Thrombosis Outcomes Study(CANTOS).[ regulate the interleukin-6 signaling pathway and the incidence of and total mortality of atherosclerotic events: analysis from the anti-inflammatory thrombosis results study of kanagab (CANTOS) ] Eur. Heart J ];2018;39 (38): 3499-3507.).

In autoinflammatory syndrome, NLRP3 inhibitors can address the underlying etiology of the disease by directly inhibiting the NLRP3 inflammatory bodies. MCC950 is a selective small molecule inhibitor of NLRP3, shows activity in inhibiting activation of NLRP3 inflammatory corpuscles in various NLRP3 dependent mouse models, and shows activity in inhibiting IL-1β release in isolated samples of Peripheral Blood Mononuclear Cells (PBMC) of individuals with CAPS (Coll et al, A small-molecule inhibitor of the NLRP-inflammasome for THE TREATMENT of inflammatory diseases. [ small molecule inhibitor of NLRP3 inflammatory corpuscles for the treatment of inflammatory diseases ] Nat.Med [ Nature ];2015;21 (3): 248-55). However, administration of the NLRP3 inhibitor MCC950/CRID3 failed to rescue CAPS mouse models carrying the L351P mutation in NLRP3, which corresponds to the L353P mutation of human NLRP3 (Vande et al ,MCC950/CRID3potently targets the NACHT domain of wild-type NLRP3 but not disease-associated mutants for inflammasome inhibition.[MCC950/CRID3 effectively targeted the NACHT domain of wild-type NLRP3, but failed to target disease-related mutants for inflammatory platelet inhibition ] PLoS Biol [ public science library biology ];2019;17 (9): e 3000354).

While supportive therapy is widely available for lighter symptoms such as fever, rash, and pain, people with auto-inflammatory syndromes may require corticosteroids and, in some cases, biological IL-1 blockers to treat or prevent more severe inflammatory activities. Compared to biological agents, NLRP3 inhibitors may provide additional benefits to patients as well as patients whose disease is not adequately treated by currently available therapies.

Provided herein are NLRP3 inhibitors that can be used to prevent or reduce NLRP3 inflammatory small body reactions, thereby addressing unmet medical needs, including the treatment of autoinflammatory syndromes including CAPS, MWS, FCAS, NOMID/CINCA and FMF.

Disclosure of Invention

Described herein are methods of treating a subject with an NLRP3 inhibitor, particularly compound I, for use in treating autoinflammatory syndrome. Also described herein are methods of treating autoinflammatory syndrome by administering a therapeutically effective amount of an NLRP3 inhibitor, particularly compound I, to a subject in need thereof.

Further provided herein are specific dosage regimens for the methods or uses of the NLRP3 inhibitors described herein, particularly compound I.

In addition, described herein are pharmaceutical combinations for use in the treatment of auto-inflammatory syndrome, comprising a) compound I and b) at least one further therapeutic agent (optionally in the presence of a pharmaceutically acceptable carrier), and pharmaceutical compositions comprising the same. Preferably, wherein compound I is compound IA.

Other features and advantages of the described methods and uses will become apparent from the following detailed description.

Drawings

Fig. 1 is a summary view of the treatment regimen detailed in example 1.

FIG. 2 is a schematic overview of the study design of the first human (FIH) study detailed in example 2.

Detailed Description

Described herein are methods of treating auto-inflammatory syndrome by administering to a subject in need thereof an effective amount of compound I or a pharmaceutically acceptable salt thereof. Accordingly, in one aspect, there is provided a method of treating auto-inflammatory syndrome, the method comprising administering to a subject in need thereof an effective amount of compound I or a pharmaceutically acceptable salt thereof. Also provided are compound I for use in the treatment of auto-inflammatory syndrome. In one embodiment of any of the methods or uses described herein, the compound I is compound IA.

Definition of the definition

In order that this document may be more readily understood, certain terms are first defined. Other definitions are set forth throughout this document.

All patents, published patent applications, publications, references, and other materials mentioned herein are incorporated by reference in their entirety for all purposes described.

As used herein, the term "comprising" encompasses "including" and "consisting of". The composition (consisting of) ", for example, a composition that" comprises "X may consist of X alone, or may include some additional, such as x+y.

As used herein, the article "a" or "an" refers to one or more than one (e.g., at least one) grammatical object of the article.

The term "or" is used herein to mean and be used interchangeably with the term "and/or" unless the context clearly indicates otherwise.

The term "about" in connection with a reference value and grammatical equivalents thereof as used herein may include the value itself as well as a range of values that add or subtract 10% from the value. For example, an amount "about 10" includes 10 and any amount from 9 to 11. For example, the term "about" in relation to a reference value may also include a series of values from which 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% are added or subtracted. In some instances, a numerical value described throughout may be "about" the numerical value even though the term "about" is not specifically mentioned.

As used herein, the term "baseline" refers to the degree of a state or condition (e.g., disease) of a subject, or one or more parameters related to the state of a patient, observed prior to treatment according to the methods and uses described (e.g., prior to administration of a compound, e.g., prior to administration of compound I optionally in combination with at least one additional therapeutic agent).

As used herein, the term "administration" in relation to a compound (e.g., compound I optionally in combination with at least one additional therapeutic agent) is used to refer to delivery of the compound by any delivery route. Such delivery may be, for example, intravenous administration or oral administration. Such delivery may also be, for example, subcutaneous administration.

As used herein, the word "substantially" does not exclude "complete", e.g., a composition that is "substantially free" of Y may be completely free of Y. The word "substantially" may be omitted from the definition, if desired.

As used herein, the term "pharmaceutically acceptable" means a non-toxic material that does not substantially interfere with the effectiveness of the biological activity of one or more active ingredients.

As used herein, the term "patient" is used interchangeably with the term "subject" and includes any human or non-human animal. The term "non-human animal" includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dogs, cats, horses, cows, chickens, amphibians, reptiles, and the like. In particular embodiments, the compositions, methods and uses described herein relate to a human patient or human subject.

As used herein, a subject "needs" such treatment if the subject has a condition of interest (i.e., disease, disorder, or syndrome) and will receive a biological, medical, or quality of life benefit from treatment.

As used herein, the term "autoinflammatory syndrome" is a form of inflammatory response syndrome that may be initiated or manifested by a variety of factors, such as fatigue, stress or physical (e.g., FMF), and cold exposure (e.g., CAPS, MWS, FCAS, NOMID/CINCA).

The terms "treatment (treat, treating, treatment)", "prevention (prevent, preventing, prevention)" include therapeutic treatment, prophylactic treatment, and use, wherein the risk of a subject developing a disorder or other risk factor is reduced. Treatment does not require complete cure of the disorder and encompasses alleviation of symptoms or potential risk factors.

The term "treatment" includes administration of a compound (e.g., compound I), optionally in combination with at least one additional therapeutic agent, to prevent, to alleviate or to delay the onset of symptoms, complications or biochemical indicators of a disease, condition, disorder or syndrome (e.g., cryptopyrene related periodic syndrome (CAPS), familial Cold Autoinflammatory Syndrome (FCAS), mu Kele-wils syndrome (MWS), neonatal onset multisystem inflammatory disease/chronic infant nerve skin and joint syndrome (NOMID/CINCA) or Familial Mediterranean Fever (FMF)), thereby preventing outbreaks of a disease, condition, disorder or syndrome, alleviating its symptoms or preventing or inhibiting its further development or manifestation.

As used herein, the term "prevention (prevent, preventing or presenting)" in connection with a disease, condition, disorder or syndrome (e.g., cryptopyrene protein-related periodic syndrome (CAPS), familial Cold Autoinflammatory Syndrome (FCAS), mu Kele-wils syndrome (MWS), neonatal onset multisystem inflammatory disease/chronic infant nerve skin and joint syndrome (NOMID/CINCA) or Familial Mediterranean Fever (FMF)) refers to prophylactic treatment of a subject at risk of developing a condition such that the subject has a reduced probability of developing the condition (e.g., a particular disease or disorder associated with CAPS, FCAS, MWS, NOMID/CINCA or FMF or clinical symptoms thereof, such as skin disease, joint pain, myalgia, headache/migraine, conjunctivitis, fatigue/discomfort, and organ or tissue damage).

For example, "treating familial cold-type autoinflammatory syndrome (FCAS)" may refer to alleviating, moderating, or modulating at least one symptom or pathological feature associated with familial cold-type autoinflammatory syndrome (FCAS), such as, for example, low fever, generalized rash, conjunctivitis, polyarthritis, headache, myalgia, and fatigue, for example, may refer to slowing progression, thereby reducing or stopping at least one symptom or pathological feature associated with familial cold-type autoinflammatory syndrome (FCAS), such as, for example, low fever, generalized rash, conjunctivitis, polyarthritis, headache, myalgia, and fatigue. It may also refer to preventing or delaying the progression of one or more of the described symptoms, such as slowing the progression of a disease, condition, disorder, manifestation or syndrome, stopping or reversing the progression of a disease, condition, disorder, manifestation or syndrome, and improving clinical outcome.

"Treating" may also refer to slowing the progression of a disease, condition, disorder, performance or syndrome progression, stopping or reversing the progression of a disease, condition, disorder, performance or syndrome, and improving clinical outcome, e.g., moving from a higher number to a lower number on the 5-point disease-related clinical signs and symptoms scale:

As used herein, the term "excipient" or "pharmaceutically acceptable excipient" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, carrier, solvent, or encapsulating material. In one embodiment, each component is "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical formulation and suitable for contact with tissues or organs of humans and animals without undue toxicity, irritation, allergic response, immunogenicity, or other problems or complications commensurate with a reasonable benefit/risk ratio. See, e.g., remington: THE SCIENCE AND PRACTICE of Pharmacy [ Lemington: pharmaceutical science and practice ], 21 st edition, lippincott Williams & Wilkins [ LiPink Williams and Wilkins publishing company ]: philadelphia, pa., 2005;Handbook of Pharmaceutical Excipients [ handbook of pharmaceutical excipients ], 6 th edition, rowe et al, editions, the Pharmaceutical PRESS AND THE AMERICAN Pharmaceutical Association [ British medical publishing company and American society of medicine ] 2009;Handbook ofPharmaceutical Additives [ handbook of pharmaceutical additives ], 3 rd edition, ash and Ash editions, gower Publishing Company [ Golgi publishing company ]:2007;Pharmaceutical Preformulation and Formulation [ pharmaceutical pre-formulation and formulation ], 2 nd edition, gibson editions, CRC PRESS LLC [ CRC publishing company ]: boca Raton, FL) in Florida.

As used herein, the term "NLRP3 inhibitor" is a compound that inhibits the ability of NLRP3 to induce IL-1 β and/or IL-18 by binding to NLRP3 directly or by inactivating, destabilizing, altering the distribution of, or otherwise. Typically, in the hTHP-1 assay defined herein containing 2% fetal bovine serum, hTHP-1IC ₅₀ <1 μm for NLRP3 inhibitors.

Preferably, the NLRP3 inhibitor is a compound of compound I, compound IA or compound IB. More preferably, the NLRP3 inhibitor is compound IA.

As used herein, "compound of formula I" or "compound I" are used interchangeably and mean a compound having the structure shown below, and can be synthesized using procedures known in the art and described in WO 2019/023754 (incorporated by reference in its entirety).

Compound I, compound IA or compound IB may be used in crystalline or amorphous form, as a solvate (e.g., hydrate) or unsolvated form.

Tautomers:

the scope of the compounds disclosed herein includes tautomeric forms of the compounds. Thus, by way of example, is represented as a compound containing the following moieties

Also intended to include tautomeric forms containing

Stereoisomers of:

Non-limiting exemplary compounds of the formulae described herein include a steric sulfur atom. The present disclosure provides examples of mixtures of stereoisomers (e.g., racemic mixtures of enantiomers; mixtures of diastereomers). The present disclosure also describes and exemplifies methods for separating individual components of the stereoisomer mixture (e.g., resolving enantiomers of a racemic mixture). For example, compound I represents a non-racemic mixture of compound IA and compound IB, a racemic mixture of compound IA and compound IB, compound IA in enantiomerically pure form, or compound IB in enantiomerically pure form. As used herein, "compound I" is also intended to include an enantiomeric excess of compound IA or compound IB. For example, compound IA may be present in an enantiomeric excess of about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5%. Alternatively, compound IB may be present in about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or about 99.5% enantiomeric excess.

Any formulae given herein are also intended to represent unlabeled as well as isotopically-labeled forms of the compounds. Isotopically-labeled compounds have structures depicted by the formulae given herein, but one or more atoms are replaced by an atom having a selected atomic mass or mass number. Isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, and oxygen, such as ³H、¹¹C、¹³C、¹⁴ C and ¹⁵ N, for example. Thus, it should be appreciated that the methods of the invention can or may involve compounds incorporating one or more of any of the above isotopes, including, for example, radioisotopes such as ³ H and ¹⁴ C, or compounds in which non-radioactive isotopes such as ² H and ¹³ C are present. Such isotopically-labeled compounds are useful in metabolic studies (with ¹⁴ C), in kinetic studies (e.g., with ² H or ³ H), detection or imaging techniques, such as Positron Emission Tomography (PET) or Single Photon Emission Computed Tomography (SPECT), including drug or substrate tissue distribution assays, or in radiotherapy of patients. Isotopically-labeled compounds can generally be prepared by conventional techniques known to those skilled in the art, for example, by using an appropriate isotopically-labeled reagent in place of the previously employed unlabeled reagent.

The present invention encompasses the following examples, which include all pharmaceutically acceptable salts of the compounds provided herein that are useful according to the present invention. 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, inorganic or organic acid salts of basic residues such as amines, alkali metal or organic salts of acidic residues such as carboxylic acids, and the like. Pharmaceutically acceptable salts include, for example, conventional non-toxic salts of the parent compound formed from non-toxic inorganic or organic acids. Pharmaceutically acceptable salts can be synthesized from the parent compound containing a basic or acidic moiety by conventional chemical methods. In general, such salts can be prepared by reacting the free 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 mixture of both, with non-aqueous media (like ether, ethyl acetate, ethanol, isopropanol or acetonitrile) being generally preferred. A list of suitable salts is found in Remington's Pharmaceutical Sciences [ Lemington pharmaceutical science ], 17 th edition, mack Publishing Company [ Mark publication Co ], iston, pa., 1985, p.1418, and Journal ofPharmaceutical Science [ journal of pharmaceutical science ],66,2 (1977), each of which is incorporated herein by reference in its entirety. For example, preferred pharmaceutically acceptable salts include, but are not limited to, inorganic or organic acid salts of basic residues (e.g., amines). For example, the salt may be a hydrochloride salt.

The phrase "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for 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.

As used herein, unless otherwise indicated, a "dose" or amount of an NLRP3 inhibitor (e.g., compound I) refers to the amount of the free base or free acid form of the compound. For the salt form of the NLRP3 inhibitor, the actual amount will be adjusted based on the salt form used.

An "effective amount" refers to an amount sufficient to achieve a beneficial or desired result. For example, a therapeutic amount is an amount that achieves a desired therapeutic effect. This amount may be the same as or different from a prophylactically effective amount, which is the amount required to prevent the onset of a disease, condition, disorder or syndrome or related symptom. An effective amount may be administered in one or more administrations, applications, or one or more doses. The "therapeutically effective amount" (i.e., effective dose) of a therapeutic compound depends on the therapeutic compound selected. The composition may be administered one or more times per day to one or more times per week and also includes less frequent administration, e.g., as described herein. The skilled artisan will appreciate that certain factors may influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease, condition, disorder or syndrome, previous treatments, the general health and/or age of the subject, and other diseases, conditions, disorders or syndromes that are concurrently present. Furthermore, treatment of a subject with a therapeutically effective amount of a therapeutic compound described herein may include a single treatment or a series of treatments.

As used herein, the term "therapeutically effective amount" of a compound as described herein refers to the amount of the compound that will elicit a biological or medical response in a subject, such as alleviation of symptoms, alleviation of conditions, slowing or delaying the progression of a disease, or prevention of a disease, condition, disorder, manifestation, syndrome, or the like. In one non-limiting embodiment, the term "therapeutically effective amount" refers to an amount of a compound described herein that is effective, when administered to a subject, to at least partially alleviate, inhibit, prevent, and/or ameliorate autoinflammatory syndrome (e.g., cryptopyrene Associated Periodic Syndrome (CAPS), familial Cold Autoinflammatory Syndrome (FCAS), mu Kele-wils syndrome (MWS), neonatal onset multisystem inflammatory disease/chronic infant nerve skin and joint syndrome (NOMID/CINCA), or Familial Mediterranean Fever (FMF)).

As defined herein, "combination" refers to a fixed combination of unit dosage forms (e.g., capsules, tablets, sachets, or vials), a free (i.e., non-fixed) combination, or a kit of parts (kit of parts) for combined administration, wherein compound I and one or more additional therapeutic agents may be administered independently at the same time or separately within time intervals, particularly where these time intervals allow the combination partners to exhibit a synergistic effect (e.g., a synergistic effect).

As used herein, the terms "co-administration" or "combined administration" and the like are intended to encompass administration of an additional therapeutic agent to a single subject (e.g., subject) in need thereof, and the additional therapeutic agent is intended to include treatment regimens that do not require administration of compound I and the additional therapeutic agent by the same route of administration and/or at the same time. Each component of the combination described herein may be administered simultaneously or sequentially in any order. Co-administration includes simultaneous, sequential, overlapping, spaced, and/or sequential administration, and any combination thereof.

As used herein, the term "pharmaceutical combination" means a pharmaceutical composition resulting from the combination (e.g., mixing) of more than one active ingredient, and includes both fixed and free combinations of active ingredients.

The term "fixed combination" means that the active ingredients are administered to a subject simultaneously in the form of a single entity or dose.

The term "free combination" (non-fixed combination) means that the active ingredients as defined herein are administered to a subject simultaneously, concurrently or sequentially with no specific time limitation, as well as in any order, as distinct entities, wherein such administration provides therapeutically effective levels of the compound in the subject. In particular, reference to a combination comprising a) compound I, and b) at least one additional therapeutic agent as used herein (e.g., in any embodiment or any claim herein) refers to a "non-fixed combination" and may be administered independently at the same time or separately over a time interval.

By "simultaneous administration" is meant administration of the active ingredients as defined herein on the same day. The active ingredients may be administered at the same time (for fixed or free combination) or one at a time (for free combination).

The term "sequentially administered" may mean that only one of the active ingredients as defined herein is administered on any given day during a continuous co-administration period of two or more days.

By "overlapping administration" is meant that during a continuous co-administration period of two or more days, simultaneous administration is performed for at least one day and administration of only one of the active ingredients as defined herein is performed for at least one day.

By "continuous administration" is meant a period of co-administration without any blank day. As noted above, sequential administration may be simultaneous, sequential or overlapping.

The term "dose" refers to a specified amount of a drug administered at one time. For example, the dose may be declared on the product package or in the product information sheet.

As used herein, the term "NLRP3" is intended to include, but is not limited to, nucleic acids, polynucleotides, oligonucleotides, sense and antisense polynucleotide strands, complementary sequences, peptides, polypeptides, proteins, homologous and/or orthologous NLRP3 molecules, isoforms, precursors, mutants, variants, derivatives, splice variants, alleles, different species, and active fragments thereof.

Examples listed (examples 1.1 to 1.32):

1.1 an NLRP3 inhibitor for use in treating auto-inflammatory syndrome in a subject in need thereof.

1.2 The NLRP3 inhibitor for use as described in example 1.1 wherein the NLRP3 inhibitor is administered to a subject in a single dose or in divided doses in a total daily dose of about 50mg to about 500mg, preferably about 50mg to about 200 mg.

1.3 The NLRP3 inhibitor for use as described in example 1.2 wherein the NLRP3 inhibitor is administered to the subject in a single dose or in divided doses at a total daily dose of about 100mg or about 200 mg.

1.4 The NLRP3 inhibitor for use as described in example 1.3 wherein the NLRP3 inhibitor is administered to the subject at a dose of about 100mg twice daily for three consecutive days and once in the morning of the fourth day for about 100mg.

1.5 The NLRP3 inhibitor for use of any one of claims 1.1-1.4 wherein the autoinflammatory syndrome is CAPS, FCAS, MWS, NOMID/CINCA or FMF.

1.6 The NLRP3 inhibitor for use of any one of claims 1.1-1.5 wherein the autoinflammatory syndrome is FCAS.

1.7 The NLRP3 inhibitor for use of example 1.6 wherein the patient's white blood cell count (WCC) is not increased by more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% after administration of the NLRP3 inhibitor.

1.8 The NLRP3 inhibitor for use as in example 1.6 wherein after administration of the NLRP3 inhibitor, the patient exhibits a lower score of at least 1, at least 2, at least 3 on a scale of 1-10 on a physician general assessment scale after cold exposure.

1.9 The NLRP3 inhibitor for use as in example 1.6 wherein after administration of the NLRP3 inhibitor, the patient exhibits a lower score of at least 10%, at least 20%, at least 30% on a scale of 1-100 on the physician general assessment scale after cold exposure.

1.10 The NLRP3 inhibitor for use of any one of embodiments 1.1-1.9 wherein the patient's C-reactive protein is not increased by more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% after administration of the NLRP3 inhibitor.

1.11 The NLRP3 inhibitor for use of any one of embodiments 1.1-1.10 wherein the patient exhibits no more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% increase in IL-1 β or IL-18 after administration of the NLRP3 inhibitor.

1.12 The NLRP3 inhibitor for use of any of the previously enumerated embodiments wherein the NLRP3 inhibitor is orally administered to the subject.

1.13 The NLRP3 inhibitor for use of any of the previously listed embodiments wherein the NLRP3 inhibitor is present in a tablet formulation.

1.14 The NLRP3 inhibitor for use of any one of enumerated embodiments 1.1-1.6, wherein the NLRP3 inhibitor is compound I or a pharmaceutically acceptable salt thereof:

1.15 NLRP3 inhibitor for use as in example 1.14 wherein compound I comprises compound IA enantiomer or a pharmaceutically acceptable salt thereof:

1.16 NLRP3 inhibitor for use as in example 1.15 wherein compound I comprises a compound IB enantiomer or a pharmaceutically acceptable salt thereof:

1.17 NLRP3 inhibitor for use as described in example 1.15, wherein compound IA has an enantiomeric excess of at least 90%.

1.18 NLRP3 inhibitor for use as described in example 1.16, wherein compound IB has an enantiomeric excess of at least 90%.

1.19 A pharmaceutical composition comprising an NLRP3 inhibitor as described in examples 1.12-1.18 for use as described in any of the preceding listed examples.

1.20 A pharmaceutical combination comprising an NLRP3 inhibitor as described in examples 1.12-1.18 and at least one additional therapeutic agent for use as described in any of the preceding listed examples, the pharmaceutical combination further comprising.

Use and method

The following and elsewhere in this document include various embodiments of the methods and uses described herein. It should be appreciated that the features specified in each embodiment may be combined with other specified features to provide further embodiments:

The following examples are taught herein to relate to the use of any NLRP3 inhibitor and are not limited to compound I. Preferably, compound I of the following examples is compound IA (i.e., the R enantiomer) with an enantiomeric excess of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5%. Preferably, compound IA has an enantiomeric excess of at least 90%. More preferably, compound IA has an enantiomeric excess of at least 95%.

Example 1

In one embodiment, provided herein is a method of treating or alleviating a symptom of autoinflammatory syndrome in a subject in need thereof, the method comprising administering an effective amount of compound I or a pharmaceutically acceptable salt thereof. In one embodiment, provided herein is compound I or a pharmaceutically acceptable salt thereof for use in treating auto-inflammatory syndrome in a subject in need thereof. In some embodiments, provided herein is the use of compound I for the manufacture of a medicament for the treatment of auto-inflammatory syndrome.

Example 2

In one embodiment, provided herein is a method of treating or alleviating symptoms of cryptomelane-related periodic syndrome (CAPS) in a subject in need thereof, the method comprising administering an effective amount of compound I or a pharmaceutically acceptable salt thereof. In one embodiment, provided herein is compound I or a pharmaceutically acceptable salt thereof for use in treating cryptomelane-associated periodic syndrome (CAPS) in a subject in need thereof. In some embodiments, provided herein is the use of compound I for the manufacture of a medicament for the treatment of cryptomelane-related periodic syndrome (CAPS).

Example 3

In one embodiment, provided herein is a method of treating or alleviating symptoms of familial cold-type autoinflammatory syndrome (FCAS) in a subject in need thereof, the method comprising administering an effective amount of compound I or a pharmaceutically acceptable salt thereof. In one embodiment, provided herein is compound I or a pharmaceutically acceptable salt thereof for use in treating familial cold-type autoinflammatory syndrome (FCAS) in a subject in need thereof. In some embodiments, provided herein is the use of compound I for the manufacture of a medicament for the treatment of familial cold-type autoinflammatory syndrome (FCAS).

Example 4

In one embodiment, provided herein is a method of treating Mu Kele-wilt syndrome (MWS) or alleviating a symptom thereof in a subject in need thereof, the method comprising administering an effective amount of compound I or a pharmaceutically acceptable salt thereof. In one embodiment, provided herein is compound I or a pharmaceutically acceptable salt thereof for use in treating Mu Kele-wils syndrome (MWS) in a subject in need thereof. In some embodiments, provided herein is the use of compound I for the manufacture of a medicament for the treatment of Mu Kele-wils syndrome (MWS).

Example 5

In one embodiment, provided herein is a method of treating or alleviating symptoms of multisystem inflammatory disease/chronic infant nerve skin and joint syndrome (NOMID/CINCA) in a neonatal onset in a subject in need thereof, the method comprising administering an effective amount of compound I or a pharmaceutically acceptable salt thereof. In one embodiment, provided herein is compound I or a pharmaceutically acceptable salt thereof for use in treating a multisystem inflammatory disease/chronic infant nerve skin and joint syndrome (NOMID/CINCA) in neonatal onset in a subject in need thereof. In some embodiments, provided herein is the use of compound I for the manufacture of a medicament for the treatment of neonatal onset multisystem inflammatory disease/chronic infant neurodermatitis and joint syndrome (NOMID/CINCA).

Example 6

In one embodiment, provided herein is a method of treating Familial Mediterranean Fever (FMF) or alleviating symptoms thereof in a subject in need thereof, the method comprising administering an effective amount of compound I or a pharmaceutically acceptable salt thereof. In one embodiment, provided herein is compound I or a pharmaceutically acceptable salt thereof for use in treating Familial Mediterranean Fever (FMF) in a subject in need thereof. In some embodiments, provided herein is the use of compound I for the manufacture of a medicament for the treatment of Familial Mediterranean Fever (FMF).

In any of the embodiments described herein, the patient's white blood cell count (WCC) does not increase by more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% after administration of the NLRP3 inhibitor.

In any of the embodiments described herein, the patient's C-reactive protein is not increased by more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% after administration of the NLRP3 inhibitor.

In any of the embodiments described herein, the patient exhibits no more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% increase in IL-1 β or IL-18 after administration of the NLRP3 inhibitor.

In any of the embodiments described herein for treating or alleviating symptoms of FCAS, after administration of the NLRP3 inhibitor, the patient exhibits a lower score of at least 1, at least 2, at least 3 on a scale of 1-10 on a physician general assessment scale after cold exposure. In any of the embodiments described herein for treating or alleviating symptoms of FCAS, after administration of the NLRP3 inhibitor, the patient exhibits a lower score of at least 10%, at least 20%, at least 30% on a scale of 1-100 on the physician general assessment scale after cold exposure.

In any of the embodiments described herein, compound I or a pharmaceutically acceptable salt thereof can be administered to a subject in a single dose or in divided doses at a total daily dose (as measured by the non-salt equivalent) of about 50mg to about 200 mg. In particular embodiments, compound I is administered to a subject in a single dose or in divided doses at a total daily dose of about 200 mg. In yet a specific embodiment, compound I is administered to the subject at a dose of about 100mg twice daily for three consecutive days, and once in the morning on day four for about 100mg.

In some embodiments, provided herein is a pharmaceutical composition comprising compound I or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable excipient. In a particular embodiment, the pharmaceutical composition is a tablet. In yet a further specific embodiment, the pharmaceutical composition is administered in a monolithic or crushed tablet. In some embodiments, the pharmaceutical composition comprises about 5mg, about 10mg, about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 60mg, about 70mg, about 80mg, about 90mg, or about 100mg per unit dose.

Provided herein is a pharmaceutical composition comprising compound I or a pharmaceutically acceptable salt thereof for use in any of the embodiments described herein.

In any of the embodiments described herein, compound I or a pharmaceutically acceptable salt thereof is administered orally to a subject in need thereof. In some embodiments, compound I is in the form of a tablet, which is administered in whole or in sub-divided form (i.e., crushed prior to administration). In certain embodiments, compound I may be administered via a nasogastric tube, for example, when the patient is unable to swallow.

For all embodiments defined herein, the preferred NLRP3 inhibitor is compound IA. In particular embodiments, compound IA is in an enantiomeric excess of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%.

A subject

As described herein, a subject receiving the presently described NLRP3 inhibitors may develop symptoms of or risk for Familial Cold Autoinflammatory Syndrome (FCAS), e.g., as described above.

Combination therapy

In practicing some of the therapeutic methods or uses described herein, a therapeutically effective amount of compound I is administered to a patient, such as a mammal (e.g., a human). In addition, for less severe FCAS cases, the patient may receive treatment in a supportive manner to control symptoms such as fever, muscle pain, or fatigue. For more severe FCAS cases, immunosuppressants such as corticosteroids may be required, but a judgment must be made to avoid negating the effect of the drug intended to activate the immune system.

Pharmaceutical composition

When combined with a pharmaceutically acceptable carrier, compound I can be used as a pharmaceutical composition. Such compositions may contain, in addition to compound I, carriers, various diluents, fillers, salts, buffers, stabilizers, solubilizers and other known materials. The characteristics of the carrier will depend on the route of administration. The pharmaceutical compositions for use in the compositions, uses and methods described herein may also contain at least one or more additional therapeutic agents for treating a particular targeted disorder, disease, condition or syndrome. Such additional factors and/or agents may be included in the pharmaceutical composition to produce a synergistic effect with compound I.

In particular embodiments, compound I may be administered in combination with one or more conventional pharmaceutical excipients. Pharmaceutically acceptable excipients include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) (such as d-alpha-tocopheryl polyethylene glycol 1000 succinate), surfactants used in pharmaceutical dosage forms (such as tween, poloxamer or other similar polymer delivery matrices), serum proteins (such as human serum albumin), buffer substances (such as phosphates, tris, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts)), colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycols, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, and lanolin. Cyclodextrins such as α -, β -and γ -cyclodextrins, or chemically modified derivatives such as hydroxyalkyl cyclodextrins (including 2-and 3-hydroxypropyl- β -cyclodextrins), or other solubilized derivatives, may also be used to enhance delivery of the compounds described herein. Dosage forms or compositions containing chemical entities as described herein in the range of 0.005% to 100% can be prepared with the remainder being complemented by non-toxic excipients. Contemplated compositions may contain from 0.001% to 100%, in one embodiment from 0.1% to 95%, in another embodiment from 75% to 85%, and in still other embodiments from 20% to 80% of the chemical entities provided herein. Practical methods of preparing such dosage forms are known to, or will be apparent to, those skilled in the art, see, for example, remington: THE SCIENCE AND PRACTICE of Pharmacy [ Lemington: pharmaceutical science and practice ], 22 nd edition (Pharmaceutical Press [ medical Press ], london, UK.2012).

Routes of administration and composition Components

In some embodiments, the chemical entities described herein or pharmaceutical compositions thereof may be administered to a subject in need thereof by any accepted route of administration. Acceptable routes of administration include, but are not limited to, buccal, cutaneous, endocervical, intracardial (endosinusial), intratracheal, enteral, epidural, interstitial, intra-abdominal, intra-arterial, intrabronchial, intracapsular, intra-cerebral, intracisternal, intra-coronary, intradermal, intraductal, intraduodenal, intra-dural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intra-meningeal, intramuscular, intra-ovarian, intraperitoneal, intraprostatic, intrapulmonary, intracavitary (intrasinal), intraspinal, intrasynovial, intrathecal, intrauterine, intravascular, intravenous, nasal feeding, oral, parenteral, transdermal, epidural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transtracheal, ureteral, urethral and vaginal administration. In certain embodiments, the preferred route of administration is parenteral (e.g., intratumoral).

The compositions may be formulated for parenteral administration, for example, for injection via intravenous, intramuscular, subcutaneous, or even intraperitoneal routes. Typically, such compositions may be prepared as injectables, either as liquid solutions or suspensions, as solid forms suitable for preparing solutions or suspensions upon addition of liquids prior to injection, and as formulations which are also emulsified. The preparation of such formulations will be known to those skilled in the art in light of the present disclosure.

Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions, formulations including sesame oil, peanut oil, or aqueous propylene glycol, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It should also be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.

The carrier may also be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), suitable mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, sorbic acid, thimerosal (thimerosal), and the like). In many cases, it is preferable to include isotonic agents, for example, sugars or sodium chloride. The absorption of the injectable composition may be prolonged by the use of agents delaying absorption in the composition, such as aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvents with various other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains an alkaline dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Intratumoral injection is discussed in, for example Lammers et al ,"Effect ofIntratumoral Injection on the Biodistribution and the Therapeutic Potential of HPMACopolymer-Based Drug Delivery Systems[ influence of intratumoral injection on the biodistribution and therapeutic potential of HPMA copolymer-based drug delivery systems ] "Neoplasia [ neoplasia ]2006,10,788-795.

In certain embodiments, the chemical entities described herein or pharmaceutical compositions thereof are suitable for topical, topical administration to the digestive tract or Gastrointestinal (GI) tract, e.g., rectal administration. Rectal compositions include, but are not limited to, enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly-like suppositories, and enemas (e.g., retention enemas).

Pharmacologically acceptable excipients that may be used in the rectal composition as a gel, cream, enema, or rectal suppository include, but are not limited to, any one or more of cocoa butter glycerides, synthetic polymers such as polyvinylpyrrolidone, PEG (e.g., PEG ointments), glycerol, glycerinated gelatin, hydrogenated vegetable oils, poloxamers, mixtures of polyethylene glycols and fatty acid esters of polyethylene glycols of various molecular weights, petrolatum, anhydrous lanolin, shark liver oil, sodium saccharinate, menthol, sweet almond oil, sorbitol, sodium benzoate, anoxid SBN, vanilla essential oil, aerosols, parabens in phenoxyethanol, sodium methyl paraoxybenzoate, sodium propyl paraoxybenzoate, diethylamine, carbomer, carbopol, methoxybenzoate, polyethylene glycol stearyl ether, cocoyl caprylyl caprate, isopropyl alcohol, propylene glycol, liquid paraffin, xanthan gum, carboxy-metabisulfite, sodium edetate, sodium benzoate, metabisulfite, grape seed extract, methylsulfonylmethane (MSM), vitamins such as potassium acetate and potassium acetate.

In certain embodiments, suppositories may be prepared by mixing the chemical entities described herein with suitable non-irritating excipients or carriers (such as cocoa butter, polyethylene glycol or a suppository wax) which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum and release the active compound. In other embodiments, the composition for rectal administration is in the form of an enema.

In other embodiments, the compounds described herein or pharmaceutical compositions thereof are suitable for topical delivery to the digestive tract or Gastrointestinal (GI) tract by oral administration (e.g., solid or liquid dosage forms).

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the chemical entity is admixed with one or more pharmaceutically acceptable excipients, such as sodium citrate or dicalcium phosphate and/or a) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and silicic acid, b) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia, c) humectants, such as glycerin, d) disintegrants, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate, e) solution retarding agents, such as paraffin, f) absorption accelerators, such as quaternary ammonium compounds, g) wetting agents, such as, for example, cetyl alcohol and glyceryl monostearate, h) absorbents, such as kaolin and bentonite clays, and i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents. Excipients such as lactose or milk sugar and high molecular weight polyethylene glycols can also be used, as can solid compositions of similar type for use as fillers in soft and hard-filled gelatin capsules.

In one embodiment, the composition is in the form of a unit dosage form, such as a pill or tablet, and thus the composition may contain diluents, such as lactose, sucrose, dicalcium phosphate, and the like, lubricants, such as magnesium stearate, and the like, and binders, such as starches, acacia, polyvinylpyrrolidone, gelatin, cellulose derivatives, and the like, along with the chemical entities provided herein. In another solid dosage form, a powder, pellet (marume), solution, or suspension (e.g., in propylene carbonate, vegetable oil, PEG, poloxamer 124, or triglycerides) is encapsulated in a capsule (gelatin or cellulose-based capsule). It is also contemplated that the one or more chemical entities or additional active agents provided herein are physically separate unit dosage forms, e.g., capsules (or tablets in capsules) containing particles of each drug, bilayer tablets, dual compartment gel caps, and the like. Enteric coatings or delayed release oral dosage forms are also contemplated.

Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives particularly useful for preventing the growth or action of microorganisms. Various preservatives are well known and include, for example, phenol and ascorbic acid.

In certain embodiments, the excipient is sterile and generally free of undesirable substances. These compositions may be sterilized by conventional well-known sterilization techniques. For various oral dosage excipients such as tablets and capsules, sterility is not required. The USP/NF standard is generally adequate.

In certain embodiments, the solid oral dosage form may further include one or more components that chemically and/or structurally facilitate the composition in delivering the chemical entity to the stomach or lower GI, e.g., the ascending and/or transverse colon and/or distal colon and/or small intestine. Exemplary formulation techniques are described, for example, in Filipski, K.J., et al, current diagnostics IN MEDICINAL CHEMISTRY [ Current Topics of medicinal chemistry ],2013,13,776-802, which is incorporated herein by reference in its entirety.

Examples include superior-GI targeting techniques, such as Accordion Pill (actodion hill) (intel pharmaceutical company (INTEC PHARMA)), floating capsules, and materials that can adhere to mucosal walls.

Other examples include inferior-GI targeting techniques. Several enteric/pH-reactive coatings and excipients are available for targeting various regions in the gut. These materials are typically polymers designed to dissolve or erode at a particular pH range selected based on the GI region of the desired drug release. These materials are also used to protect acid labile drugs from gastric juice or limit exposure where the active ingredient may stimulate the upper GI (e.g., hydroxypropyl methylcellulose phthalate series, coateric (polyvinyl acetate phthalate), cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate, eudragit series (methacrylic acid-methyl methacrylate copolymer), and Marcoat). Other techniques include dosage forms that are responsive to localized flora in the Gastrointestinal (GI) tract, pressure-controlled colon delivery capsules, and Pulsincap.

The ophthalmic composition may include, but is not limited to, any one or more of a thickening agent (viscogen) (e.g., carboxymethyl cellulose, glycerol, polyvinylpyrrolidone, polyethylene glycol), a stabilizing agent (e.g., pluronic (triblock copolymer), cyclodextrin), a preservative (e.g., benzalkonium chloride, ETDA, softzia (boric acid, propylene glycol, sorbitol, and zinc chloride; ai Kang Gongsi (Alcon Laboratories, inc.)), purite (stabilized oxy-chloro complex; ai-building company (Allergan, inc.)).

Topical compositions may include ointments and creams. Ointments are semisolid preparations, typically based on petrolatum or other petroleum derivatives. Creams containing the selected active agent are typically viscous liquid or semisolid emulsions, usually oil-in-water or water-in-oil. The cream base is typically water-washable and contains an oil phase, an emulsifier, and an aqueous phase. The oil phase (sometimes also referred to as the "internal" phase) is typically composed of petrolatum and fatty alcohols (such as cetyl or stearyl alcohol), and the aqueous phase is typically (although not necessarily) more voluminous than the oil phase and typically contains a humectant. The emulsifier in the cream formulation is typically a nonionic, anionic, cationic or amphoteric surfactant. With respect to other carriers or vehicles, the ointment base should be inert, stable, non-irritating and non-sensitizing.

In any of the foregoing embodiments, the pharmaceutical compositions described herein may include one or more of lipids, multilamellar vesicles crosslinked between bilayers, nanoparticles or microparticles based on biodegradable poly (D, L-lactic-co-glycolic acid) [ PLGA ] or polyanhydride, and nanoporous particle-supported lipid bilayers.

Dosing regimen and mode of administration

The dosage regimen is adjusted to provide the best desired response (e.g., therapeutic response). Depending on the compound used, the targeted disease, condition, disorder or syndrome and their associated stages, the dosing regimen (i.e., the dosage and/or frequency of administration of the pharmaceutical composition comprising compound I) may vary. Depending on the compound used, the disease, condition, disorder or syndrome and their associated stages, the dosing regimen (i.e., the dosage and/or frequency of administration of the pharmaceutical combination comprising a) compound I and b) at least one additional therapeutic agent may vary.

For administration of compound I in a method for treating autoinflammatory syndrome, the dose is in the range of about 0.0001 to about 100mg/kg of subject body weight, and more typically in the range of about 0.01 to about 30mg/kg of subject body weight. In particular embodiments, compound I is administered in a daily dose of about 50mg to about 500mg, about 50mg to about 200mg, about 50mg to about 150mg, about 50mg to about 100mg, about 50 mg. In particular embodiments, compound I is administered at a daily dose of about 50mg, about 100mg, about 150mg, or about 200 mg. In certain embodiments, compound I is administered once a day. In other embodiments, compound I is administered twice, three times, or four times daily. In a preferred embodiment, compound I is administered at once, or in two divided doses, at a total daily dose of about 200 mg. Preferably, compound I is compound IA. Preferably, compound IA is administered in a total daily dose of about 50mg to 500 mg. More preferably, compound 1A is administered at a daily dose of 200 mg.

In some embodiments, compound IA is administered at a daily dose of 200 mg. In some embodiments, 100mg of compound IA is administered twice daily.

In some embodiments, the period of time for which the compounds described herein are administered lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. In another embodiment, the period of discontinuation of administration lasts for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, or longer. The dose is repeated as necessary and may range from about once every week up to about once every 10 weeks, for example once every 4 weeks or once every 8 weeks.

Kit for detecting a substance in a sample

Also included herein are kits for use in methods for treating or preventing cytokine release syndrome or cytokine storm syndrome, which kits may include compound I in liquid or lyophilized form or a pharmaceutical composition comprising compound I. Further, such kits may include means (e.g., syringes and vials, drug-loaded syringes, drug-loaded pens) and instructions for use for administering compound I. These kits may contain, for example, additional therapeutic agents (described elsewhere herein) for delivery in combination with compound I.

The phrase "means for administering" is used to indicate any available instrument for systemic administration of a drug to a patient, including but not limited to droppers, drug delivery syringes, vials and syringes, injection pens, auto-injectors, intravenous (i.v.) drip chambers and bags, pumps, and the like. With such articles, the patient may self-administer the drug (i.e., administer the drug by themselves), the caregiver may administer the drug to the patient, or a doctor or other medical professional may administer the drug.

Each component of the kit is typically enclosed in a separate container, and all of the various containers are in a single package, with instructions for use.

It is to be understood that each embodiment may be combined with one or more other embodiments to the extent that such combinations are consistent with the description of the embodiments. It should also be understood that the embodiments provided above should be understood to include all embodiments, including such embodiments resulting from combinations of embodiments.

Other features, objects, and advantages of the described methods and uses will be apparent from the description and drawings, and from the claims.

Synthesis of Compound I

Compounds I, IA and IB were synthesized according to the syntheses defined in WO 2019/023754 for examples 4, 5 and 6 and described in detail below. However, the compounds may be assembled in various ways, using related reaction procedures to build up the final molecule in a modular manner allowing for different reaction sequences and/or different reagents.

The progress of the reaction is typically monitored by TLC or LC-MS. The identity of the product is usually confirmed by LC-MS. LC-MS was recorded using the following method:

Method A, shim-pack XR-ODS, C18,3X 50mm,2.5um column, 1.0uL injection, 1.5mL/min flow rate, 90-900amu scan range, 190-400nm UV range, 5% -100% (1.1 min), 100% (0.6 min) gradient with ACN (0.05% TFA) and water (0.05% TFA), 2 min total run time.

The final target was purified by preparative HPLC. Preparative HPLC was performed using the following procedure:

Method B preparative HPLC: column, XBridge Shield RP OBD (19X 250mm,10 um), mobile phase, water (10 mmol/L NH ₄HCO₃) and ACN, UV detection 254/210nm.

NMR was recorded on BRUKER NMR 300.03MHz, DUL-C-H, ULTRASHIELD ^TM300,AVANCE II 300B-ACS^TM or BRUKER NMR 400.13MHz, BBFO, ULTRASHIELD ^TM400,AVANCE III 400,B-ACS^TM or BRUKER AC250NMR instrument with TMS as reference in ppm (parts per million) as measurement unit.

Scheme 1:

Compound I:

compound I N' - (1, 2,3,5,6, 7-hexahydro-symmetrical indacen-4-ylcarbamoyl) -2- (2-hydroxy-propyl)

-2-Yl) thiazole-5-sulfonylimido amide

Step 1N- (tert-Butyldimethylsilyl) -N' - (1, 2,3,5,6, 7-hexahydro-symmetrical indacen-4-ylcarbamoyl) -2- (2-hydroxypropan-2-yl) thiazole-5-sulfonylimido amide

A50 mL round bottom flask was charged with a solution of N' - (tert-butyldimethylsilyl) -2- (2-hydroxy-prop-2-yl) thiazole-5-sulfonylimide amide (intermediate I) (336 mg,1.0 mmol) in THF (10 mL). To this solution was added NaH (60% wt,80mg,2.0 mmol) in portions at 0 ℃. The solution was stirred at 0 ℃ for 15 min, and then a solution of 4-isocyanato-1, 2,3,5,6, 7-hexahydro-symmetrical indacene (209 mg,1.1 mmol) in THF (5 mL) was added dropwise with stirring at RT. The resulting solution was stirred at RT for 12h. The reaction was then quenched by addition of 10mL NH ₄ Cl (saturated). The resulting solution was extracted with 3x 10ml DCM and the combined organic layers were concentrated in vacuo. This gives 535mg (crude material) of the title compound as a brown oil. MS-ESI 535.0 (M+1).

Step 2N' - (1, 2,3,5,6, 7-hexahydro-symmetrical indacen-4-ylcarbamoyl) -2- (2-hydroxypropyl-2-yl) thiazole-5-sulfonylimido amide

A50-mL round bottom flask was charged with a solution of N- (tert-butyldimethylsilyl) -N' - (1, 2,3,5,6, 7-hexahydro-symmetrical indacen-4-ylcarbamoyl) -2- (2-hydroxypropan-2-yl) thiazole-5-sulfonylimid-mide (535 mg, crude material, 1.0 mmol) in THF (10 mL). To this solution was added HF/Py (70% wt,143mg,5.0 mmol) dropwise at 0 ℃. The solution was stirred at RT for 4h. The reaction was then quenched by the addition of 10mL of water. The resulting solution was extracted with 3x 10ml ethyl acetate and the combined organic layers were concentrated in vacuo. The crude product was purified by preparative HPLC using method B with ACN/water (20% to 60% in 10 min). This gives 189mg (45%, 2 steps) of compound I as a white solid.

Compounds of formula (I) I：MS-ESI:421.0(M+1).¹H NMR(400MHz,DMSO-d₆)δ8.46(br s,1H),8.04(s,1H),7.80(br s,2H),6.86(s,1H)6.28(s,1H),2.88-2.71(m,4H),2.71-2.56(m,4H),2.02-1.80(m,4H),1.49(s,6H).

Compound IA and compound IB:

Compounds IA and IB (R) and (S) -N' - (1, 2,3,5,6, 7-hexahydro-symmetrical indacen-4-ylcarbamoyl) -2- (2-hydroxypropan-2-yl) thiazole-5-sulfonylimido amide

And 3, chiral separation.

The compound I product (189 mg) obtained as described in the previous step was resolved by chiral preparative HPLC using a column, CHIRAL Cellulose-SB,2 x 25cm,5um, mobile phase, hex (0.1% DEA) and EtOH (20% EtOH maintained over 16 min), flow rate, 20mL/min, detector, UV 254/220nm. This gave 70mg of Compound IB as a white solid (front peak, 99% ee) and 65mg of Compound IA as a white solid (second peak, 97.5% ee).

Compounds of formula (I) IB：MS-ESI:421.0(M+1).¹H NMR(400MHz,DMSO-d₆)δ8.43(br s,1H),8.05(s,1H),7.83(br s,2H),6.87(s,1H)6.29(s,1H),2.82-2.71(m,4H),2.71-2.56(m,4H),2.02-1.80(m,4H),1.50(s,6H).

Compounds of formula (I) IA：MS-ESI:421.0(M+1).¹H NMR(400MHz,DMSO-d₆)δ8.41(br s,1H),8.05(s,1H),7.83(s,2H),6.87(s,1H)6.27(s,1H),2.82-2.71(m,4H),2.71-2.56(m,4H),2.02-1.80(m,4H),1.50(s,6H).

Intermediate I of scheme 1 was synthesized according to the synthesis shown in WO 2019/023754 and provided in scheme 2 below.

Scheme 2:

intermediate I

N' - (tert-butyldimethylsilyl) -2- (2-hydroxypropyl-2-yl) thiazole-5-sulfonylimid amide

Step 1 2- (2-methyl-1, 3-dioxolan-2-yl) thiazole

A500-mL round-bottom flask was charged with a solution of 1- (thiazol-2-yl) ethanone (20 g,157.0 mmol) in toluene (300 mL) and ethane-1, 2-diol (19.5 g,314 mmol). TsOH (2.7 g,15.7 mmol) was added to the solution. The resulting solution was refluxed overnight and water was separated from the solution during the reflux. The resulting solution was diluted with 200mL of water and extracted with 2x 100mL of ethyl acetate. The organic layers were combined, dried over anhydrous Na ₂SO₄, and then concentrated in vacuo. This gave 26.6g (99%) of the title compound as a pale yellow oil. MS-ESI 172.0 (M+1).

Step 2- (2-methyl-1, 3-dioxolan-2-yl) thiazole-5-sulfonamide

A500-mL 3-neck round bottom flask purged with nitrogen and maintained under nitrogen was charged with a solution of 2- (2-methyl-1, 3-dioxolan-2-yl) thiazole (14 g,81.6 mmol) in THF (200 mL). n-BuLi (2.5M in THF, 35.2mL,88.0 mmol) was then added dropwise with stirring at-78 ℃. The resulting solution was stirred at-78 ℃ for 0.5h, and then SO ₂ was introduced into the above reaction mixture. The reaction was slowly warmed to RT and NCS (12.8 g,95.86 mmol) was then added. The resulting solution was stirred at RT for 1h. The solid was filtered off. The resulting filtrate was concentrated in vacuo, and then diluted in DCM (160 mL). To the above dilution was added a saturated solution of ammonia in DCM (300 mL). The resulting solution was stirred at RT for 3h and then concentrated in vacuo. The residue was applied to a silica gel column and eluted with a gradient of ethyl acetate/petroleum ether (1:20 to 1:5). This gives 12.5g (61%) of the title compound as a yellow solid. MS-ESI:251.0 (M+1).

Step3 2-acetylthiazole-5-sulfonamide

A250-mL round bottom flask was charged with a solution of 2- (2-methyl-1, 3-dioxolan-2-yl) thiazole-5-sulfonamide (12.5 g,50.0 mmol) in THF (125 mL). To the above solution was added aqueous HCl (4 n,50.0 ml). The resulting solution was stirred at 70 ℃ for 6h. The resulting solution was diluted with 100mL of water and extracted with 2x 200mL of ethyl acetate. The organic layers were combined, dried over anhydrous Na ₂SO₄, and then concentrated in vacuo. The residue was applied to a silica gel column and eluted with a gradient of ethyl acetate/petroleum ether (1:2 to 1:1). This gives 9.3g (90%) of the title compound as a yellow solid. MS-ESI:207.0 (M+1).

Steps 4-6 use the same procedure as indicated for the conversion of compound Z to compound Y shown in scheme 3 to afford intermediate I from compounds I-d. MS-ESI 336.1 (M+1).

Scheme 3:

Compound Y

N' - (tert-butyldimethylsilyl) -5- (2-hydroxypropyl-2-yl) thiazole-2-sulfonylimid amide

Step1 methyl 2-mercaptothiazole-5-carboxylate

Into a 2-L round bottom flask was placed methyl 2-bromothiazole-5-carboxylate (100 g,450 mmol), etOH (1000 mL), sodium hydrogen sulfide (50 g, 890mmol). The resulting solution was stirred at 80 ℃ for 2h and then cooled to 0 ℃ under a water/ice bath. The pH of the solution was adjusted to 3 with hydrogen chloride (1N). The solid was collected by filtration. This gave 63.2g (80%) of the title compound as a pale yellow solid. MS-ESI 176.0 (M+1).

Step 2 methyl 2- (chlorosulfonyl) thiazole-5-carboxylate

Into a 1-L round bottom flask was placed methyl 2-mercaptothiazole-5-carboxylate (30 g,170 mmol) and acetic acid (300 mL). Sodium hypochlorite (300 ml,8% -10% wt.) was then added in portions at 0 ℃. The resulting solution was stirred at RT for 2h and then diluted with 500mL of water. The solution was extracted with 3x 300ml DCM and the combined organic layers were washed with 2x 300ml brine and dried over anhydrous Na ₂SO₄. The crude product was used in the next step as a yellow solution in DCM.

Step 3 2-sulfamoylthiazole-5-carboxylic acid methyl ester

To a 2-L round bottom flask was placed methyl 2- (chlorosulfonyl) thiazole-5-carboxylate as a crude solution in DCM (900 mL). NH ₃ (g) was introduced into the solution at below 0℃for 20 minutes. The resulting solution was stirred at RT for 1h and then concentrated in vacuo. The residue was applied to a silica gel column and eluted with ethyl acetate/petroleum ether (1:5 to 1:3). This gave 23g (75%, 2 steps) of the title compound as a white solid. MS-ESI 223.0 (M+1).

Step 4 5- (2-hydroxy-prop-2-yl) thiazole-2-sulfonamide

A500-mL round-bottom flask purged with nitrogen and maintained under nitrogen was charged with a solution of methyl 2-sulfamoylthiazole-5-carboxylate (15 g,67.5 mmol) in THF (150 mL). MeMgBr/THF (3M, 90 mL) was then added dropwise with stirring at 0 ℃. The resulting solution was stirred at RT for 14h and then quenched by addition of 100mL NH ₄ Cl (saturated). The resulting solution was extracted with 3x 150ml DCM. The organics were laminated and dried over anhydrous Na ₂SO₄ then concentrated in vacuo. The residue was applied to a silica gel column and eluted with ethyl acetate/petroleum ether (1:5 to 1:3). This gives 11.5g (78%) of the title compound as a white solid. MS-ESI 223.0 (M+1), 221.0 (M-1) in positive and negative ion modes, respectively.

Step 5N- (tert-Butyldimethylsilyl) -5- (2-hydroxy-prop-2-yl) thiazole-2-sulfonamide

A250-mL 3-neck round bottom flask purged with nitrogen and maintained under nitrogen was charged with a solution of 5- (2-hydroxypropan-2-yl) thiazole-2-sulfonamide (5 g,22.5 mmol) in THF (100 mL). NaH (60% wt,1.8g,45.0 mmol) was then added in portions to the above solution in an ice/water bath. After stirring in a water/ice bath for 20 minutes, a solution of TBSCl (4.1 g,27.2 mmol) in THF (10 mL) was then added dropwise with stirring at 0 ℃. The resulting solution was stirred at RT for 4h. The reaction was quenched with saturated NH ₄ Cl (100 mL). The resulting solution was extracted with 3x100mL ethyl acetate and the combined organic layers were dried over Na ₂SO₄ and concentrated in vacuo. The crude solid was washed with ethyl acetate/hexane (1:5) (2X 100 mL). This gave 6.81g (90%) of the title compound as a yellow solid. MS-ESI 337.1 (M+1), 335.1 (M-1) in positive and negative ion modes, respectively.

Step 6:N' - (tert-butyldimethylsilyl) -5- (2-hydroxypropyl-2-yl) thiazole-2-sulfonylimid amide

A solution of PPh ₃Cl₂ (3 g,9.0 mmol) in CHCl ₃ (100 mL) was placed in a 100-mL 3-neck round bottom flask purged with nitrogen and maintained under nitrogen. DIEA (1.54 g,11.9 mmol) was then added dropwise with stirring at RT. The resulting solution was stirred at RT for 10min. A solution of N- (tert-butyldimethylsilyl) -5- (2-hydroxy-prop-2-yl) thiazole-2-sulfonamide (2.0 g,5.9 mmol) in CHCl ₃ (30 mL) was then added dropwise with stirring in an ice/water bath. The resulting solution was stirred in an ice/water bath for 30min. NH ₃ (g) was introduced into the above solution at below 0℃for 15 minutes. The resulting solution was stirred at RT for 20 min. The solid was filtered off, and the filtrate was concentrated and the residue was dissolved in 300mL ethyl acetate. The solution was washed with brine (2 x 100 ml), dried over Na ₂SO₄ and concentrated in vacuo. The crude solid was washed with CHCl ₃ (100 mL). The filtrate was then concentrated under vacuum and the residue was further purified by passing through a silica gel column with ethyl acetate/petroleum ether (1:10 to 1:3). The initially washed solids and the solids from the silica gel purification were combined. This gives 1.2g (60%) of the title compound as a white solid .MS-ESI:336.1(M+1).¹H-NMR(300MHz,DMSO-d₆)δ7.66(s,1H),7.12(s,2H),5.78(s,1H),1.51(s,6H),0.86(s,9H),0.02(s,3H),0.01(s,3H).

The following abbreviations have the indicated meanings:

acn=acetonitrile

Btc=trichloromethyl chloroformate

Boc=tert-butoxycarbonyl group

Davephos = 2-dicyclohexylphosphino-2' - (N, N-dimethylamino) biphenyl

Dcm=dichloromethane

Dea=diethylamine

Dmf=n, N-dimethylformamide

DMSO = dimethyl sulfoxide

Diea=n, N-diisopropylethylamine

Dppa=diphenyl azide phosphate

Dppf=1, 1' -bis (diphenylphosphino) ferrocene

Etoh=ethanol

HATU = 1- [ bis (dimethylamino) methylene ] -1H-1,2, 3-triazolo [4,5-b ] pyridinium 3-oxide hexafluorophosphate

Hex=hexane

HPLC = high performance liquid chromatography

LC-ms=liquid chromatography-mass spectrometry

LiHMDS = lithium bis (trimethylsilyl) amide

LDA = lithium diisopropylamide

M=mol/L

Me=methyl group

Meoh=methanol

MSA = methanesulfonic acid

NBS = N-bromosuccinimide

NCS = N-chloro succinimides

Nmr=nuclear magnetic resonance

Pd (dppf) Cl ₂ = dichloro [1,1' -bis (diphenylphosphino) ferrocene ] palladium

Ph=phenyl

PPh ₃Cl₂ = triphenylphosphine dichloride

Py = pyridine

Rt=room temperature

Rt=retention time

R _f =blocking coefficient

Sat=saturated

Tbaf=tetrabutylammonium fluoride

Tbs=tert-butyldimethylsilyl group

TBSCl = tert-butyldimethylchlorosilane

TBDPSCl = tert-butyldiphenylchlorosilane

Tea=triethylamine

TFA = trifluoroacetic acid

THF = tetrahydrofuran

TLC = thin layer chromatography

Tsoh=4-methylbenzenesulfonic acid

UV = ultraviolet light

B.i.d=twice daily

Wcc=white blood cell count

Ep=endpoint

Y = year old

Y/n=yes/no

Examples

The following examples illustrate the methods and uses described herein. However, they are not intended to limit the scope of the methods and uses described in any way. Other variations of the embodiments will be apparent to those of ordinary skill in the art and are encompassed by the appended claims.

EXAMPLE 1 clinical study with Compound IA

An open-label, single arm phase II study on compound IA was used to assess safety, tolerability, and efficacy in participants with Familial Cold Autoinflammatory Syndrome (FCAS) who developed evidence of inflammatory activity following cold challenge during screening. Fig. 1 is a summary of a treatment regimen with compound IA (i.e., the R enantiomer of compound I). Compound IA 100mg will be administered twice daily for 3 days, and 100mg will be administered on day 4 in the morning.

The main objective of this study was to evaluate the efficacy of compound IA in reducing cold-induced inflammation in participants with FCAS.

The Endpoint (EP) of the primary objective is the change in white blood cell count (WCC) from pre-challenge to post-challenge highest value.

Secondary objectives of the study are as follows:

1. Evaluation of safety and tolerability of Compound IA

2. Assessing the efficacy of Compound IA in improving signs and symptoms of FCAS

3. Assessing the Effect of Compound IA on patient reporting outcome

The Endpoint (EP) of the secondary target is as follows:

1. Safety endpoints (including vital signs, ECG parameters, safety laboratory assessment and adverse events)

2. Doctor general assessment of autoinflammatory disease activity and doctor severity assessment of autoinflammatory disease signs and symptoms, change in terms of pre-challenge to post-challenge

3. General assessment of patients with disease activity varies from pre-challenge to post-challenge

Study design

This is an open-label, single-arm, multi-dose study with approximately 6 participants with FCAS who confirmed that NLRP3 function was mutated, showing evidence of inflammatory activity following cold challenges during screening.

Cold challenge regimens were formulated to study the acute inflammatory mechanisms of FCAS patients following general cold exposure and to study the effect of pretreatment with IL-1 blocking therapy. Participants with a short history of self-limiting rashes, fever and/or joint pain, who will receive a controlled cold challenge at most twice to elicit a short inflammatory response while residing in the clinical setting under medical supervision, exhibit symptoms characteristic of FCAS after cold exposure and resolve after warming. After 45 minutes of cold challenge, the participants will return to ambient temperature of approximately 25 ℃ for at least 24 hours at the clinical site to ensure resolution of the inflammatory response.

Typical symptoms and signs caused by cold challenges in participants with FCAS include an increase in systemic inflammatory markers (e.g., WCC, neutrophils), low fever, rash, and joint pain, which peak at 4-8 hours starting from 1 hour after challenge and diminish the following day, and there is no report on long-term sequelae (Hoffman et al ,Prevention of cold-associated acute inflammation in familial cold autoinflammatory syndrome by interleukin-1receptor antagonist.[ prevention of cold-related acute inflammation in familial cold-type autoinflammatory syndrome by interleukin-1 receptor antagonists ] Lancet; (2004) (9447): 1779-85.).

The study involved three phases:

Screening period, including screening for cold challenges (up to three months)

Treatment period, including the second cold challenge (duration for a total of 5 days)

Follow-up period, with study completion visit as end (10 days after dose administration)

Screening period to confirm compliance with study inclusion and exclusion criteria, and clinical observations and biological sampling were performed:

Screening visit-informed consent will be collected and the qualification of the participants assessed. For those participants meeting the screening cold challenge, the use of the disabling agent will cease. Before a screening cold challenge is scheduled, the results of laboratory samples collected at the screening visit, as well as active disease reports of the participants, must be obtained.

Screening cold challenge the participants were resident for three days on site in connection with the screening cold challenge. On the first day (day-10), the participants will be admitted in the morning, subjected to a safety assessment, and then stay in the room at ambient temperature (preferably above 25 ℃) to ensure that the conditions are stable before the cold challenge. On the next day (day-9), after the pre-challenge evaluation and breakfast, the participants will receive a 45 minute cold challenge and then monitor for the next 23 hours. On the third day (day-8), the participants will be discharged after the last evaluation, or at a later time at the discretion of the researcher. Participants should recover from the cold challenge for at least one week prior to recruitment and initiation of treatment.

Treatment period

Treatment initiation visit participants who met all inclusion and non-exclusion criteria (including evidence of inflammatory activity after cold challenge during screening) will recruit and initiate treatment on day 1.

The first dose of compound IA will be administered at the clinic and the participants will be assigned study treatment in order to continue treatment at home. The participants and researchers may decide themselves to stay in the clinic during treatment, for convenience in terms of participants and/or organization.

Cold challenge the participants will visit the clinic in the morning of day 3 (i.e., the day prior to the cold challenge), and if required by the researcher or local regulations, the participants will live for a total of three days or more. On day 3, the participants will receive a safety assessment and then stay in the room at ambient temperature (preferably above 25 ℃) to ensure that the conditions are stable before the cold challenge. In the morning of day 4, a pre-challenge assessment will be made and breakfast provided. The pre-dose PK samples will be collected immediately after breakfast and the last dose of study treatment will be administered. One hour after study treatment administration, another PK sample will be collected and cold challenges will begin. The cold challenge lasted 45 minutes. After the cold challenge ends, the participants will be monitored until the next morning (day 5). At the discretion of the investigator, the participants will be discharged after the last evaluation on or after day 5.

A follow-up period to ensure the safety of the participants after discontinuation of treatment and recovery from cold challenges.

At the end of study visit, i.e., approximately 10 days after the last dose, participants will be followed for end of study evaluation.

Critical inclusion criteria

1. Written informed consent must be obtained prior to any study-specific evaluation

2. Male and female participants between 18-80 years of age

3. The body mass index is in the range of 18-35kg/m ²

4. Gene diagnosis of participants with FCAS

5. Participants with clinical history and findings consistent with FCAS without history or diagnosis of amyloidosis and/or organ damage (e.g., deafness, periorbital edema, lymphadenopathy, and serositis)

6. Participants with evidence of inflammatory activity after screening for cold challenge

7. The minimal autoinflammatory disease activity > was assessed based on physician general assessment, with a clinical history of active disease occurring in the environment in response to cold exposure during screening in at least one scenario

Critical exclusion criteria

1. Participants carrying non-responsive NLRP3 mutations to NLRP3 inhibition, including but not limited to L353P carriers (based on primary researcher/registration data, published evidence and/or internal research by North Co., ltd.)

2. Participants currently undergoing anti-rejection and/or immunomodulatory drug treatment cannot discontinue treatment or change other agents until they are screened for cold challenge and during the duration of the study, within 28 days or 5 half-lives (if local regulations require longer) or until the expected pharmacodynamic effects return to baseline for the immunomodulatory therapeutic antibody

The exception is that anakinra, kanamab and/or other investigational IL-1/NLRP3 binding or blocking therapies must be discontinued at the time of screening. Once the evidence criteria for active disease are met, participants may proceed with screening for cold challenges

3. Clinically significant, suspected active or chronic bacterial (including mycobacterium tuberculosis), viral or fungal infections occur within 30 days prior to administration

4. Participants with congenital (e.g., TLR immunodeficiency, IFN- γ signaling) or acquired immunodeficiency (e.g., AIDS)

5. Human Immunodeficiency Virus (HIV) infection, hepatitis B surface antigen (HBsAg) or hepatitis B core antibody (anti-HBc) or hepatitis C antibody is present at the time of screening

6. Live vaccine vaccination within 4 weeks of day 1 (i.e., the first dose of compound IA)

7. The absolute peripheral blood neutrophil count is less than or equal to 1000/mm ³

8. Estimating glomerular filtration rate (eGFR). Ltoreq.90 mL/min/1.73m2 (based on the formula of chronic kidney disease epidemiological cooperation (CKD-EPI))

A medical history or current diagnosis of ecg abnormalities indicates that the enrolled participants present a significant safety risk

10. Pregnant or lactating (lactation) women, wherein pregnancy is defined as the state of the female after conception until termination of pregnancy, as determined by positive human serum chorionic gonadotrophin (hCG) laboratory tests

11. Women with fertility potential are defined as all women that are physiologically pregnant unless they use a high-efficiency contraceptive method during dosing and within 10 days after cessation of study treatment

12. Researchers believe that any significant concurrent medical condition may affect the ability of the participants to tolerate or complete the study

Efficacy evaluation:

white blood cell count (WCC)

Doctor general assessment of autoinflammatory disease Activity

Doctor severity assessment of signs and symptoms of auto-inflammatory disease

General assessment of patients with disease Activity

Critical security assessment

Physical examination

Vital signs

ECG parameters

Pregnancy and fertility assessment

Laboratory marker monitoring in blood and urine

Adverse event and serious adverse event monitoring

Other evaluation

Inflammatory markers (including but not limited to Absolute Neutrophil Count (ANC), hypersensitive C-reactive protein (hsCRP) and Serum Amyloid A (SAA))

Plasma concentration and pharmacokinetic parameters of Compound IA

Pharmacodynamics in serum, inflammatory small pathways and inflammation-related biomarkers, such as IL-1 beta, IL-6, IL-18 binding protein (IL-18 BP), CXCL10, caspase-1 and proteomics

Data analysis

If the estimated difference in total WCC between treatment and screening period relative to the change before challenge satisfies the following conditions, the main objective will be achieved and compound IA will be considered as effective in treating inflammation caused by cold in participants with FCAS:

1. Has statistical significance (p < 0.10) and

2. Less than-4 x 109 cells/L

The change in the highest WCC from pre-challenge to post-challenge will be analyzed by a linear model, including pre-challenge values as covariates and modeling cycle effects, while taking into account intra-participant correlations.

Basic principle of dosage/regimen, duration of treatment

Based on preliminary PK results and preliminary PK/PD relationships, the study selected a dose of 100mg administered as a tablet twice daily. Compound IA tablets will be administered soon after completion with a meal, and all doses will be administered approximately 12h (+/-1 h) apart.

100Mg tablets had a positive food effect on PK (peak plasma levels (Cmax) increased 2.05-fold and AUClast increased 1.49-fold). Compound IA tablets had an apparent terminal elimination half-life of about 10 hours under fed conditions, and thus were expected to reach steady state on day 4 after multiple dosing.

NLRP3 has no defined placeholder marker. The effective dose has been estimated using ex vivo whole blood, LPS stimulated IL-1 beta secretion data from FIH studies. Based on the results of this assay, the average total compound IA plasma trough concentration (IC 90) required to inhibit 90% of stimulated IL-1 β release in healthy participants was 3.17 μm on average, corresponding to about 1.3 μg/mL.

Based on the in vitro IC50 values of inhibition of IL-1 β secretion measured in PBMCs of patients with certain FCAS-related mutations being 3-5 times greater than healthy participants, participants with FCAS may require higher concentrations. This is probably due to the fact that mutations in the NLRP3 protein affect the binding KD of the compounds. Assuming that a 5-fold higher plasma level than the IC90 value obtained by the preliminary PK/PD study was required to maintain full targeted occupancy of participants with FCAS over a 24-hour period, administration of a dose of 100mg twice daily shortly after completion of the meal would be used to ensure complete inhibition of NLRP3 on day 4.

In one human study against healthy volunteers, a = compound 1A administered once daily at a dose of 200mg for up to 14 days, with average AUC0-24h and Cmax at steady state of 182 μg h/mL and 26.4 μg/mL, respectively. For a twice daily administration of 100mg tablets, the predicted steady state mean AUC0-24h (225 μg h/mL) was 1.2-fold higher than the corresponding PK parameters for healthy volunteers administered 200mg Compound 1A, and Cmax (12.1 μg/mL) was 2.2-fold lower.

Good laboratory specification (GLP) toxicology studies performed in rats and cynomolgus monkeys for 13 weeks support the safety of the treatment period.

TABLE 1

^a Based on the total exposure. For unbound exposure (correction of plasma protein binding), the safety margin was higher for rats (2-fold) and lower for monkeys (0.8-fold).

^b The NOAEL Cmax (μg/mL) and AUC0-24h (μg h/mL) for the 13-week toxicology study for rats (30 mg/kg) were 52.9 and 400, and 258 and 3540 for monkeys (150 mg/kg), respectively.

The administration of compound IA in this study was considered safe given the short treatment duration of 4 days and sufficient safety margin.

Drug-drug interaction problem:

Evaluation and recommendation of clinical studies of drug-drug interactions of cytochrome P450 (CYP) substrates/modulators and compound IA are based on in vitro/preclinical data and physiological-based PK simulation. Compound IA is expected to be primarily responsible for the elimination via hepatic CYP mediated metabolism, CYP2C9 (68%) and CYP3A4 (29%). Participants who were poor metabolites of CYP2C9 will be excluded from this study.

Administration of compound IA is considered safe even under conditions of elevated exposure to compound IA, given the duration of treatment and sufficient safety margin.

Disabling drugs and herbs

Anti-rejection/immunomodulatory therapies (e.g., anakinra, kanamab or other investigational IL-1/NLRP3 binding or blocking therapies)

Live vaccine

Strong or moderate inducers of CYP2C9 or CYP3A, including carbamazepine, enzalutamide, lu Maka Torr, phenobarbital, phenytoin, rifabutin, mitotane and St.John's grass (Hypericum perforatum (Hypericum perforatum))

Strong inhibitors of CYP2C9, including miconazole, berberine (herbal product), sulfafenazole, fluconazole, resveratrol (herbal product)

Other research products

Cautious herbs

Drugs metabolized by CYP3A

In vitro metabolic studies have shown that compound IA may have the potential to induce metabolism of drug substrates metabolized by the isozymes CYP 3A. Thus, researchers may decide to administer concomitant agents known to be metabolized by CYP3A 4/5. Patients receiving such agents may require dose adjustments or increases in concomitant medications. In particular, caution is recommended when compound IA is co-administered with drugs that are sensitive substrates for CYP3A and/or have a narrow therapeutic index.

Drugs as strong or moderate inhibitors of CYP3A

Compound IA was identified as a substrate for CYP3A in vitro and thus an increase in systemic exposure of compound IA cannot be excluded when co-administered with strong CYP3A inhibitors such as antiviral drugs (e.g., ritonavir), antifungal agents (e.g., itraconazole, ketoconazole) and antibiotics (e.g., erythromycin, clarithromycin). The researcher may decide on his own to co-administer known inhibitors of CYP3A, but its duration should be kept as short as possible and the patient must be closely monitored.

Example 2 clinical first human (FIH) study of Compound IA:

Study design

The study design consisted of 4 parts, a single ascending dose (SAD; part A), relative bioavailability of the tablet formulation (part B), multiple ascending doses (MAD; part C) and relative bioavailability and food effect (part D) (FIG. 2). In each of the groups of parts a and C, 8 subjects were randomized at a 3:1 ratio to receive compound IA (6 subjects) or to match placebo (2 subjects).

For part a, a total of 8 groups of 8 eligible subjects were recruited. Each subject received a single oral dose of compound IA (3, 10, 30, 100, 300mg crystalline suspension and 100, 300, 600mg SDD under fasted conditions). In group 1 of part a, 2 sentinel subjects were dosed at least 24 hours before the rest of the subjects in the group to ensure maximum safety.

Part B was skipped because the data collected in part a provided adequate comparison of the crystalline formulation to the SDD formulation.

In part C, eligible subjects were recruited into 6 different groups. Each subject received multiple doses of compound IA (10, 30mg crystalline suspension and 100, 200mg SDD suspension for 14 days) once daily (QD) in a fasted state, or multiple doses of compound IA (25, 50mg encapsulated crystalline tablet for 13 days and single dose or placebo on day 14) twice daily (BID) under fed conditions. After review of available safety, tolerability and PK data from the previous group in part a, subjects in part C were dosed.

Part D has an open label, randomized, 3-time period crossover design, consisting of 1 group with 6 subjects. The PK profile of the evaluated crystalline sheet formulation supported by compound IA non-clinical safety pharmacology and toxicology program was compared between fed and fasted conditions and compared to that of the crystalline suspension of compound IA under fasted conditions. Subjects received 3 doses of compound IA with a washout period of 7-14 days between each dose (dose 1: 100mg oral suspension under fasted conditions; dose 2: 100mg oral tablet under fasted conditions; dose 3: 100mg oral tablet under fed conditions). Based on these doses, subjects were randomly assigned to 1 treatment sequence (Wang B-S、Wang X-J、Gong L-K.The Construction of a Williams Design and Randomization in Cross-Over Clinical Trials Using SAS.[ of 6 treatment sequences prepared using Williams design (1 subject per sequence) using SAS construction cross-clinical trial Williams design and randomization [ Journal of Statistical Software [ journal of statistical software ]2009; 29).

A subject

The eligible subjects are healthy volunteers between 18 and 64 years of age and having a Body Mass Index (BMI) of 18.5 and 30.0kg/m ² or less. No subjects participated in >1 fraction or group. Written informed consent was obtained prior to any study procedure. The subjects involved in part D must be willing and able to eat the entire high fat breakfast within the specified time frame. Subjects were excluded if they had a history of severe mental disorders, diagnosis of mental disability, clinically significant vital sign abnormalities, or had tobacco product usage within 90 days prior to (first) drug administration until follow-up.

Blind setting

In part a and part C, the active and placebo treatments were not distinguishable based on the label, they were identical in appearance and similar in taste and odor. To maintain blindness, each subject in the respective group is given the same number of tablets or suspensions. The investigator and subject remained blind throughout the relevant portion of the study, and remained blind without disruption throughout the course of the study. The sponsor remains non-blind to all study data and obtains a copy of the randomized code to support decisions regarding the study. According to the williams design, part D is open-labeled, and compound IA is administered only to subjects of 1 of the 6 treatment sequences (1 subject per sequence).

Target object

The main objective of the study was to evaluate the safety and tolerability of SAD and MAD oral doses of compound IA in healthy subjects throughout the part of the study. A key secondary objective was to characterize PK profile after single and multiple doses of compound IA, and to evaluate the effect of food on PK profile of compound IA.

Evaluation of

Security assessment

Safety assessment in all parts of the study included Adverse Events (AEs) reported using the regulatory active medical dictionary (Medical Dictionary for Regulatory Activities) (version 22.1), clinical laboratory tests (biochemistry, hematology and urinalysis), vital signs, electrocardiography (ECG), physical examination and skin biopsies, if applicable.

Pharmacokinetic (PK) assessment

In the single dose portion, blood samples were collected at the following time points relative to the dosing on day 1 to determine the concentration of compound IA at 0.25, 0.5, 0.75, 1, 1.5, 2,3, 4,6, 8, 12, 24, 36 and 48 hours prior to and after dosing, and at follow-up. In the multi-dose portion, blood samples were collected at time points of 0.25, 0.5, 0.75, 1, 1.5, 2,3, 4,6, 8 and 12 hours before and after dosing, at 2,4, 7, 9 and 11 days before dosing, at 24 and 36 hours (15 th) and 48 hours (16 th) after the last dosing on 14 days, and at follow-up, relative to dosing on days 1 and 14.

In the multiple increasing dose (MAD) fraction, pools were collected over 24 hours for QD doses (0-12 h and 12-24 h) and over 12 hours for BID doses relative to day 1 and day 14 dosing. Plasma samples were analyzed by fully validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). The measurement accuracy of compound IA concentration >1ng/mL (lower limit of quantification, LLOQ) is 4.4% or less, and the relative error in accuracy is-2.0% to 1.5%. Urine samples were analyzed by LC-MS/MS, with LLOQ of 1ng/mL. The concentration below LLOQ is set to 0.

The PK parameters were estimated using non-compartmental analysis, maximum concentration in plasma (C _max), time to maximum concentration (t _max), concentration 24h post-dosing (C _24h) (part a), lag time, observation time before first quantifiable concentration (t _lag), time to last quantifiable concentration (t _last), area under the concentration-time curve from time 0 to last quantifiable concentration (AUC _0-last), area under the plasma concentration-time curve from time 0 to infinity (AUC _0-inf), area under the plasma concentration-time curve from time 0 to 24 hours post-dosing (AUC _0-24), end-stage rate constant (K _el), end-stage half-life (t _1/2), apparent total clearance (CL/F), and end-stage apparent distribution volume (V _z/F), furthermore, for part C only, apparent clearance under steady state (CLss/F) over the dosing interval from time 0 to 12 hours post-dosing (auc3474/F), apparent clearance under steady state (AUC _0-tau), and ratio (R3742) based on cumulative ratio (R35).

Pharmacodynamic (PD) evaluation

To determine the PD response to the NLRP3 inhibition pathway, whole blood samples were collected for exploratory PD analysis (groups 1-3 of parts a and C). After ex vivo stimulation of whole blood samples with Lipopolysaccharide (LPS), the inflammatory marker IL-1β was analyzed. Whole blood stimulation by 1 μg/mL LPS was successful and analysis of IL-1β concentration in plasma samples performed using validated electrochemiluminescent assays was found to be effective and scientifically acceptable. The LLOQ after dilution was 64.6ng/L.

Statistical analysis

All data are summarized using descriptive statistics and listed and summarized in tabular and/or graphical form. Descriptive statistics for all relevant PK parameters include n, arithmetic mean, standard Deviation (SD), coefficient of variation (CV%), minimum, median, maximum, geometric mean, and geometric CV%. For t _max, only the median, minimum and maximum values are presented. PK parameters were calculated using non-compartmental methods using Phoenix version 8.1 software. Concentrations below the lower limit of quantitation were treated as zero in summary statistics of individual concentration data. The linear trapezoidal rule is used for AUC calculation. The regression analysis for determining the final plasma elimination period of t _1/2 included at least 3 data points after C _max. The parameters for which the adjusted r ² was below 0.80 were marked but included in the descriptive statistics. The parameters AUC _0-inf、％AUC_extra, CL/F and% AUC _extra were excluded from descriptive statistics for VZ/F above 20%.

In section a, the regression power model that relates log-transformed C _max、AUC_0-last and AUC _0-inf to log-transformed dose levels was used to explore dose proportionality. The point estimates for intercept and slope are calculated, along with the corresponding 90% Confidence Interval (CI) for slope. Dose proportionality was not explored for part C. In part D, the analysis of variance (ANOVA) model of PK data was used to explore the relative bioavailability of crystalline tablets relative to crystalline suspensions, as well as the food effect. ANOVA models include fixed effects of treatment, cycles, and sequences, as well as random effects of subjects within the sequences.

The least squares geometric mean ratio is presented with 90% CI for 100mg of Compound IA tablet versus 100mg of Compound IA suspension in fasted versus 100mg of Compound IA tablet in fasted for the following treatments.

Individual versus mean graphs of combinations of IL-1 beta concentration versus time are presented per treatment.

To characterize the inhibitory potency of compound IA on IL-1β release, a bayesian Emax PK/PD model was applied. The model was developed in the free software environment R (version 4.0.5) using the rstan library. Convergence problems (which may be related to the fitting of baseline values and inter-individual variability) are the ultimate logical basis for selecting bayesian rather than frequency-based methods, which were also initially evaluated-again for reasons of including only one parameter with inter-individual variability. Two results are presented, one is to estimate the maximum inhibition and the other is the ability to assume complete inhibition (E _max = 1). The in vitro LPS stimulation results were corrected for the unstimulated results of the same sample. The concentration below LLOQ was set to half LLOQ (IL-1β) and 0 (Compound IA).

Results

Subject treatment and demographics

The study recruited a total of 122 subjects. All 122 subjects were included in the safety and PD analysis set, and 94 subjects receiving active agent treatment (compound IA) were included in the PK analysis set. Overall, 58 (48%) men and 64 (52%) women between the ages of 18 and 64 and BMI between 18.9 and 29.4kg/m ² participated in the study. Most (105 [86% ]) subjects (part a, n=57, part c, n=42, part d, n=6) are caucasians.

Of the enrolled subjects, 107 (88%) subjects completed the study on schedule, and 15 (12%) subjects discontinued the study prematurely. Early discontinuation included 1 of 64 subjects in part a (2%), 13 of 52 subjects in part C (25%), and 1 of 6 subjects in part D (17%). Reasons for study discontinuation included 12 (10%) subjects were withdrawn from AE, with 1 (1%) subjects each discontinuing the study by withdrawal of consent, loss of follow-up, or suspension of the study by COVID-19 pandemic (blocking access; independent of compound IA safety). A total of 4 discontinued subjects were replaced in part C.

Safety of

Tolerability of single and multiple doses of compound IA was generally good. Death or Severe AE (SAE) were not reported during the study. Overall, 87 of 122 subjects (71%) reported AE (TEAE) present in treatment, 66 of 94 subjects (70%) in compound IA group and 21 of 28 subjects (75%) in placebo group reported AE (TEAE) present in treatment. Most TEAEs reported by 84 (69%) subjects were of mild intensity, while 15 subjects (12%) reported moderate TEAE. Frequently reported Systemic Organ Category (SOC) events in >20% of subjects are neurological disorders (34%), general disorders and administration site conditions (29%) and gastrointestinal disorders (27%).

In general, 46 relevant TEAEs reported by 24 of 122 subjects (20%) were considered relevant to study drug, including 21 of 94 subjects receiving compound IA (22%) and 3 of 28 subjects receiving placebo (11%). For 12 out of 122 subjects (10%), TEAE with 20 maculopapules rash and/or itching was considered particularly interesting AEs as they were considered to be relevant to study drug. All 12 subjects received compound IA either in a single dose (100 mg cs [ n=1 ] or 600mg sdd [ n=1 ]) or in multiple doses (30 mg QD cs [ n=2 ], 100mg QD sdd [ n=3 ], 200mg QD sdd [ n=2 ] or 50mg BID ect [ n=3 ]). These TEAEs have mild to moderate intensity, generally starting within 1-17 days after the onset of treatment with compound IA and regressing within 1-18 days after onset, all cases regressing without combination therapy. For 10 subjects, these TEAEs resulted in discontinuation of treatment. Two other subjects were discontinued prematurely due to TEAE independent of study drug.

The mild decrease in neutrophil and leukocyte counts was considered non-clinically significant and was only occasionally noted, which may be consistent with the PD effect of compound IA caused by inhibition of IL-1β signaling downstream of NLRP 3. One subject was considered to have secondary atrioventricular block independent of the study drug. No other clinically relevant findings were reported for vital signs, 12-lead ECG, 24-hour Holter (Holter) monitoring or physical examination.

Pharmacokinetics of

As shown by dose normalized Cmax and AUC, when compound IA is administered as a crystalline suspension, the plasma exposure of single dose of compound IA increases in a less proportional manner to the dose (3-300 mg, slope [90% CI ] 0.518[0.460;0.577] for Cmax, and 0.701[0.614;0.788] for AUC0 last). Although at lower dose levels (3 mg to 30 mg) the average AUC _0-last appears to increase in proportion to the dose, at 100mg and 300mg dose levels the average AUC _0-last is similar (79500 (CV 54.8%) and 64400ng h/mL (CV 23.5%), respectively). When applied as an SDD suspension, exposure increased in proportion to the dose (100-600 mg, slope [90% CI ] 0.913[0.802;0.1.024] for Cmax, and 1.121[0.977;0.1.265] for AUC _0-last).

Only about 1.1-1.3 times the limited drug accumulation was observed at steady state after QD administration for 2 weeks at compound IA in the 30-200mg dose range. This is consistent with an average t _1/2 over the range of 9.83-16.2 hours at QD and BID dose levels. At steady state, compound IA exhibited very low CLss/F (about 0.83 to 1.11L/h) and Vss/F (about 12.6 to 23.3L), with low to moderate inter-subject variability at QD and BID dose levels of compound IA.

As the multi-dose level increases, the total cumulative amount of compound IA excreted in the urine increases linearly. Most of compound IA was expelled within 12 hours. On days 1 and 14, the mean dose fractions expelled were in the range of 0.3% -0.4% and 0.7% -1.1% respectively, and the renal clearance was in the range of 3.6-5.0mL/h and 6.8-9.1mL/h respectively.

Administration of a single dose of 100mg of compound IA as a crystalline suspension under fed conditions resulted in a 2.05-fold increase in C _max and a 1.49-fold increase in AUC _0-last of compound IA compared to fasted conditions. For crystalline tablets (100 mg of compound IA under fasted conditions), median t _max of compound IA was delayed from 2 hours to 5 hours, C _max was 78% lower, AUC was similar (104%) and t _1/2 was comparable between crystalline tablet and suspension. Encapsulated crystalline tablets (25 and 50mg BID under fed conditions) are characterized by median lag times of 0.75 and 0.25 hours, respectively, and a median t _max on day 1 of 4 hours. The average t _1/2 of compound IA was comparable between the tablet (18.6 hours) and suspension (17.7 hours) formulations.

Pharmacodynamics of medicine

A dose-dependent decrease in ex vivo stimulated IL-1 beta concentration was observed (average nadir concentration was about 5% -20% of baseline) with increasing single and multiple oral doses of compound IA. Inhibition of IL-1β release was observed at most dose levels of compound IA, from 1 hour after dosing up to the last sampling time point, single (day 3 or up to 6 hours for the lowest +.10mg dose level) and multiple (day 15) oral doses of compound IA.

Typical baseline (E ₀) (+ -SD) for IL-1β stimulation was 1820 (+ -102) ng/L, E _max for IL-1β was-0.985 (+ -0.00277), and Hill coefficient was 0.758 (+ -0.0351) based on the fractional maximum stimulation (E _max) model tested with Hill coefficient. The effective concentration of the estimated maximal effect against compound IA caused by ex vivo stimulated IL-1 beta release was EC ₅₀：59ng/mL(90％ CI：48,72),EC₉₀:1080 ng/mL (90% CI:942,1240). When considering complete inhibition (fixed E _max = 1), compound IA inhibited 50% and 90% of the ex vivo stimulated IL-1β release of LPS at plasma concentrations (IC ₅₀ and IC ₉₀) of 61ng/mL (90% CI:50; 70) and 1340ng/mL (90% CI:1190; 1490), respectively, with a hill coefficient of 0.715 (±0.0333). The similarity between the models shows that replacing values below LOQ with LOQ/2 has limited impact.

Discussion of the invention

For patients with inflammatory, small-body NLRP3 mediated inflammatory, metabolic, and neurodegenerative diseases, the medical need to expand the therapeutic modality to provide an effective and predictable treatment option without increasing AE risk has not been met. In this study, NLRP3 antagonist compound IA was administered orally to human subjects for the first time, exploring its safety, tolerability, PK and PD properties. The initial dose is selected based on the predicted human PK and the expected effective dose, as well as the non-clinical safety of animal and in vitro data.

Safety of

Single and multiple doses of compound IA or placebo were generally well tolerated. No mortality or SAE was reported during the study. Similar TEAE rates were observed between subjects receiving compound IA (70%) and placebo (75%). Most TEAEs reported by subjects were mild (69%) or moderate (12%). Subcutaneous tissue and gastrointestinal disorder TEAE were reported only by subjects receiving compound IA, while subjects receiving placebo were not reported. The maculopapules and/or pruritic rashes are most frequently reported at higher multi-dose levels of compound IA, indicating a relationship to exposure to compound IA, independent of the formulation used. Safety and tolerability data for other NLRP3 inhibitors such as ZYIL1 or dapasuntrile tested in clinical trials (Parmar DV,Kansagra KA,Momin T,Patel HB,Jansari GA,Bhavsar J,Shah C,Patel JM,Ghoghari A,Barot A,Sharma B,Viswanathan K,Patel HV,Jain MR.Safety,Tolerability,Pharmacokinetics,and Pharmacodynamics of the Oral NLRP3 Inflammasome Inhibitor ZYIL1:First-in-Human Phase 1Studies(Single Ascending Dose and Multiple Ascending Dose).[ oral administration of NLRP3 inflammatory small inhibitor ZYIL1 safety, tolerability, pharmacokinetics and pharmacodynamics: first human phase 1 study (single increment dose and multiple increment dose) ] Clin Pharmacol Drug Dev [ clinical pharmacology in drug development ]2023;12:202-211;Marchetti C,Swartzwelter B,Gamboni F,Neff CP,Richter K,Azam T,Carta S,Tengesdal I,Nemkov T,D'Alessandro A,Henry C,Jones GS,Goodrich SA,Laurent JP,Jones TM,Scribner CL,Barrow RB,Altman RD,Skouras DB,Gattorno M,Grau V,Janciauskiene S,Rubartelli A,Joosten LAB,Dinarello CA.OLT1177,aβ-sulfonyl nitrile compound,safe in humans,inhibits the NLRP3 inflammasome and reverses the metabolic cost of inflammation.[OLT1177 is a β -sulfonyl nitrile compound which is safe to humans, inhibits NLRP3 inflammatory small and reverses the metabolic cost of inflammation ] Proc NATL ACAD SCI USA [ national academy of sciences of the united states of america ]2018;115:E1530-E1539;Klück V,Jansen TLTA,Janssen M,Comarniceanu A,EfdéM,Tengesdal IW,Schraa K,Cleophas MCP,Scribner CL,Skouras DB,Marchetti C,Dinarello CA,Joosten LAB.Dapansutrile,an oral selective NLRP3 inflammasome inhibitor,for treatment of gout flares:an open-label,dose-adaptive,proof-of-concept,phase 2a trial.[Dapansutrile is an oral selective NLRP3 inflammatory small inhibitor for treating gout flares: an open label, dose adaptation, proof of concept, phase 2a trial ] Lancet Rheumatol [ salix ]2020;2:e270-e 280) does not include any drug-related skin reaction directed to specific compound IA-related effects, but not due to mode of action.

Pharmacokinetics of

Following a single oral dose of compound IA (3-300 mg cs,100-600mg SDD) in suspension under fasted conditions, compound IA is generally rapidly absorbed, with a median t _max at the dose level in the range of 0.76-3.00 hours. However, the median t _max was slightly delayed (1.5-3.0 hours) at the higher dose range of 30-600mg compared to the lower doses (3 and 10mg: 0.76 and 1.00 hours, respectively). The increase in drug exposure is not much proportional to the dose of the crystalline suspension (especially 100 and 300 mg), whereas in the case of the SDD suspension (100-600 mg) a dose-proportional increase in exposure is observed, indicating that the absorption of crystalline material is limited by the solubility at doses > 100 mg.

Multiple doses and formulations of compound IA showed no deviation from dose-proportional drug exposure after 2 weeks, indicating that the multiple dose PK parameters were linear and not limited by solubility. After an oral dose of compound IA on day 1, a slight delay in absorption was observed in the case of encapsulated crystalline tablets under fed conditions. This slower absorption is consistent with the bioavailability results, where no significant effect of food on t _max was observed. These findings indicate that the lag absorption time is due to encapsulation. Renal clearance was determined to be about 0.004L/h (day 1) or 0.008L/h (day 14), approaching < 0.8% of the oral dose. This suggests that direct secretion of compound IA into urine is not expected to be the primary elimination pathway of this drug in humans.

Compound IA in the form of a 100mg crystalline tablet showed a positive food effect (greater exposure in the presence of food) with a 2.05-fold and 1.49-fold increase in C _max and AUC, respectively, in the fed (high fat, high calorie diet) state compared to the fasted state. The median T _max for 100mg crystalline tablets was 5 hours, while the suspension reported a shorter T _max value (0.76-3.0 hours). Compound IA has very low apparent oral clearance (CLss/F of about 1.0L/h), which involves < 2% human liver blood flow and low apparent volume of distribution (Vss/F) of about 12.6-23.3L. A slight drug accumulation of about 1.2 times was observed after QD dosing reached steady state and a slight drug accumulation of 2 times was observed after BID dosing reached steady state, consistent with an effective t _1/2 of about 10 hours determined for crystalline tablets upon food dosing.

Pharmacodynamics of medicine

Non-clinical studies have shown that compound IA blocks IL-1 β release using a range of NLRP3 dependent activators. This phenomenon is also observed, for example, in ZYIL1 compounds that selectively inhibit MCC950(Tapia-Abellan A,Angosto-Bazarra D,Martinez-Banaclocha H,de Torre-Minguela C,Ceron-Carrasco JP,Perez-Sanchez H,Arostegui JI,Pelegrin P.MCC950 closes the active conformation of NLRP3 to an inactive state.[MCC950 of NLRP3 activation to block the active conformation of NLRP3 to an inactive state ] Nat Chem Biol [ Nat chemical biology ]2019; 15:560-64) or that exhibit >90% IL-1b inhibition in healthy subjects. In contrast, another NLRP3 inhibitor dapansutrile (OLT 1177) only partially reduced the release of IL-1b in healthy subjects and gout flares.

In this study, a dose-dependent decrease in IL-1β concentration was observed with increasing single and multiple oral doses of compound IA. IL-1β production may be mediated by other inflammatory or inflammatory-body independent pathways (Gaidt MM,Hornung V.Alternative inflammasome activation enables IL-1beta release from living cells.[ alternative inflammatory-body activation enabling IL-1β to be released from living cells Curr Opin Immunol [ current point of immunology ]2017; 44:7-13), and thus inhibitors against IL-1β may lead to unintended immunosuppression. Thus, pharmacological inhibitors that specifically target NLRP3 inflammatory corpuscles alone may be a better choice for the treatment of NLRP 3-related diseases (Zahid A, li B, kombe AJK, jin T, tao J.pharmaceutical Inhibitors of the NLRP3 Inflammasome. [ pharmacological inhibitors of NLRP3 inflammatory corpuscles ] Front Immunol [ immunoprospective ]2019; 10:2538). Safety laboratory findings in 27 subjects were a mild non-clinically significant decrease in neutrophil and leukocyte counts. This is likely consistent with the PD effect of compound IA (similar to the known effect of the anti-IL-1 β monoclonal antibody kanamab) caused by inhibition of signaling downstream of NLRP3 (Dhimolea e.canakinumab. [ kanamab ] MAbs [ monoclonal antibody ]2010; 2:3-13). Compound IA rapidly acts on IL-1b inhibition after both single and multiple doses, with a clear dose response over the entire study dose range, with no apparent delay in onset, indicating a direct PK/PD relationship. To maintain IL-1b inhibition of about 90% over 24h, a dose of 25mg in crystalline tablet form was selected twice daily in a phase 2a study in knee osteoarthritis.

The latest report from the early phase 2a clinical trial of compound IA, which involved patients with the associated pneumonia and impaired respiratory function caused by COVID-19 infection, also showed that compound IA 50mg BID tablets were well tolerated in this group of patients and no new safety signal was discerned. The results indicate that there is a trend towards improved patient response to compound IA on an SoC basis compared to SoC alone. Sub-analysis of patients with more severe inflammation (CRP high) but lower corticosteroid doses showed a faster reduction and normalization of inflammatory markers in compound ia+soc group (Madurka I,Vishnevsky A,Soriano JB,Gans SJ,Ore DJS,Rendon A,Ulrik CS,Bhatnagar S,Krishnamurthy S,Mc Harry K,Welte T,Fernandez AA,Mehes B,Meiser K,Gatlik E,Sommer U,Junge G,Rezende E.COMPOUND IA:a new oral NLRP3 inhibitor-tested in an early phase 2a randomised clinical trial in patients with COVID-19pneumonia and impaired respiratory function.Infection[ infection ] 2022:1-14.

In summary, single and multiple oral doses of compound IA were well tolerated in healthy subjects for up to 14 days without safety or tolerability issues. The PK profile of compound IA was compatible with BID dosing regimen, and PK/PD data supported dose and formulation selection for further development. Safety and tolerability, PK and PD results indicate that compound IA has the potential to be an effective oral first-in-class innate immune modulator, requiring further clinical evaluation.

Example 3

The following procedure is suitable for testing the activity of NLRP3 inhibitors, according to those disclosed herein.

Procedure 1 IL-1. Beta. Production in THP-1 cells differentiated with PMA stimulated with gramicidin.

THP-1 cells were purchased from american type culture collection (AMERICAN TYPE Culture Collection) and subcultured according to instructions from the supplier. Prior to the experiment, cells were cultured in complete RPMI 1640 (containing 10% heat-inactivated FBS, penicillin (100 units/ml) and streptomycin (100 μg/ml)) and maintained in the log phase prior to the experimental setup. THP-1 was treated with PMA (phorbol 12-myristate 13-acetate) (20 ng/ml) for 16-18 hours prior to the experiment. The compound was dissolved in Dimethylsulfoxide (DMSO) to produce a 30mM stock solution. On the day of the experiment, the medium was removed and the adherent cells were isolated with trypsin for 5 minutes. Cells were then harvested, washed with complete RPMI 1640, spun down, suspended in RPMI 1640 (containing 2% heat inactivated FBS, penicillin (100 units/ml) and streptomycin (100. Mu.g/ml)). Cells were plated in 384-well plates at a density of 50,000 cells/well (final assay volume 50 μl). The compounds were first dissolved in assay medium to obtain a 5x maximum concentration of 500 μm. 10 dilutions were then performed in assay medium containing 1.67% DMSO (1:3). The 5x compound solution was added to the medium to achieve the desired final concentration (e.g., 100, 33, 11, 3.7, 1.2, 0.41, 0.14, 0.046, 0.015, 0.0051, 0.0017 μm). The final DMSO concentration was 0.37%. Cells were incubated with the compound for 1 hour and then stimulated with gramicidin (5 μm) (enzocine) for 2 hours. The plate was then centrifuged at 340g for 5min. Cell-free supernatants (40 μl) were collected using 96-channel PLATEMASTER (gilsen) and evaluated for IL-1β production by HTRF (xisi biosystems). For each experiment, vehicle-only control and CRID3 dose titration (100-0.0017 μm) were run simultaneously. Data were normalized to vehicle treated samples (equivalent to 0% inhibition) and CRID3 at 100 μm (equivalent to 100% inhibition). The compounds exhibit concentration-dependent inhibition of IL-1β production in THP-1 cells differentiated from PMA.

Procedure 2

1. Experimental procedure

1.1 Cell culture

1) THP-1 cells were cultured in complete RPMI-1640 medium with 10% FBS at 37℃under 5% CO ₂.

2) Cells were passaged every 3 days by seeding 3x 10 ⁵ cells/ml.

1.2 Preparation of Compounds

A 3-fold serial dilution of compounds was prepared with DMSO in 384-well LDV microplates using a TECAN EVO system to produce 10 concentrations of compound source plates. The highest concentration was 30mM. Fig. 3 depicts a layout of a microplate.

1.3 Cell preparation

1) THP-1 cells were centrifuged at 350g for 5min.

2) Cells were resuspended in complete RMPI-1640 medium and counted.

3) Seed cells in T225 flasks (approximately 2.5x10 ⁷/flask) were treated with 20ng/ml PMA (final DMSO concentration < 1%).

4) Incubate overnight.

1.4THP-1 stimulation

1) The adherent THP-1 cells were washed with PBS and the cells were isolated with 4ml trypsin for use in T225 flasks.

2) Cells were centrifuged at 350g for 5min, resuspended in RPMI-1640 containing 2% FBS, and counted with trypan blue.

3) Serial dilutions of 50 nl/well of test compound were transferred to 384 well plates by Echo, 165nl for the high control and first spot of CRID3 (MCC 950), then backfilled to make the DMSO concentrations uniform in all wells, and the plate layout was as follows.

4) 50K cells per well were seeded in 40ul RPMI-1640 with 2% FBS in 384 well plates.

5) Incubate at 37 ℃, 5% CO ₂ h.

6) 5 Xbacitracin was prepared, added at 10 μl/well and final concentration of 5 μM, and incubated at 37℃for 2 hours at 5% CO ₂.

7) Centrifuge at 350g for 1min.

8) Mu.l of supernatant was pipetted through apricot and transferred to white 384 proxiplate. FIG. 3 depicts the plate layout HC: 100. Mu.M CRID3 (MCC 950) +5. Mu.M bacitracin LC: 5. Mu.M bacitracin.

1.5IL-1 beta detection

1) The 5x diluent #5 was homogenized with vortexing and 1 volume of stock solution was added to 4 volumes of distilled water.

2) A 20x stock solution of anti-IL 1 beta-cryptand antibody and anti-IL 1 beta XL antibody was thawed. Both antibodies were diluted to 1x with detection buffer # 3.

3) The two ready-to-use antibody solutions were pre-mixed just prior to use.

4) 4Ul of pre-mixed anti-IL 1. Beta. Antibody working solution was dispensed into all wells.

5) Plates were sealed and incubated overnight at 4 ℃.

6) Cell plates were read using EnVison and the readings plotted against the concentration of test compound to calculate IC ₅₀.

2. Data analysis:

1. the IC of a compound can be calculated using the following formula ₅₀

Formulas of IC ₅₀

Inhibition% = 100-100x [ HC _{Average of} -reading/(HC _{Average of} -LC _{Average of} ) ]

2. The normalized data were fitted in a dose-response manner using XLfit and the compound concentrations were calculated.

Table 2 shows the biological activity of the compounds in hTHP-1 assay containing 2% fetal bovine serum: <0.008 μm= "+++++++"; not less than 0.008 and less than 0.04 mu m= "+++"; mu m= "+" ++ ".

Compounds of formula (I)	hTHP-1IC50
		I	++++
IA	+++++
		IB	+++

All publications and patent documents cited herein are incorporated by reference as if each such publication or document were specifically and individually indicated to be incorporated by reference. The present invention and its embodiments have been described in detail. However, the scope of the present invention is not intended to be limited to the particular embodiments of any process, article of manufacture, composition of matter, compounds, means, methods and/or steps described in the specification. Various modifications, substitutions, and alterations are possible to the disclosed materials without departing from the spirit and/or essential characteristics of the present invention. Accordingly, one of ordinary skill in the art will readily appreciate from the disclosure that subsequent modifications, substitutions, and/or variations may be utilized in accordance with such related embodiments of the present invention to perform substantially the same functions or achieve substantially the same results as the embodiments described herein. Accordingly, the following claims are intended to cover within their scope modifications, substitutions, and variations to the processes, articles of manufacture, compositions of matter, compounds, means, methods, and/or steps disclosed herein. The claims should not be read as limited to the described order or elements unless stated to that effect. It should be understood that various changes in form and details may be made therein without departing from the scope of the appended claims.

Claims (20)

1. A method of treating auto-inflammatory syndrome in a patient in need thereof, the method comprising administering a therapeutically effective amount of an NLRP3 inhibitor.

2. A method of alleviating a symptom of autoinflammatory syndrome in a patient in need thereof, the method comprising administering a therapeutically effective amount of an NLRP3 inhibitor.

3. The method of claim 1 or 2, wherein the NLRP3 inhibitor is administered to the subject in a single dose or in divided doses in a total daily dose of about 50mg to about 500mg, optionally about 50mg to about 200 mg.

4. The method of claim 3, wherein the NLRP3 inhibitor is administered to the subject in a single dose or in divided doses at a total daily dose of about 100 mg.

5. The method of claim 4, wherein the NLRP3 inhibitor is administered to the subject in a dose of about 100mg twice daily for three consecutive days and once in the morning of the fourth day for about 100mg.

6. The method of any one of claims 1 to 5, wherein the autoinflammatory syndrome is cryptopyrene related periodic syndrome (CAPS), familial Cold Autoinflammatory Syndrome (FCAS), mu Kele-wils syndrome (MWS), neonatal onset multisystem inflammatory disease/chronic infant nerve skin and joint syndrome (NOMID/CINCA), or Familial Mediterranean Fever (FMF).

7. The method of any one of claims 1 to 6, wherein the autoinflammatory syndrome is Familial Cold Autoinflammatory Syndrome (FCAS).

8. The method of any one of claims 1 to 7, wherein the patient's white blood cell count (WCC) is not increased by more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% after administration of the NLRP3 inhibitor.

9. The method of any one of claims 1 to 7, wherein after administration of the NLRP3 inhibitor, the patient exhibits a lower score of at least 1, at least 2, at least 3 on a scale of 1-10 on a physician general assessment scale after cold exposure.

10. The method of any one of claims 1 to 7, wherein after administration of the NLRP3 inhibitor, the patient exhibits a lower score of at least 10%, at least 20%, at least 30% on a scale of 1-100 on the doctor general assessment scale after cold exposure.

11. The method of any one of claims 1 to 10, wherein the patient's C-reactive protein is not increased by more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% after administration of the NLRP3 inhibitor.

12. The method of any one of claims 1 to 11, wherein the patient exhibits no more than about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100% increase in IL-1 β or IL-18 after administration of the NLRP3 inhibitor.

13. The method of any one of the preceding claims, wherein the NLRP3 inhibitor is administered orally to the subject.

14. The method of any one of the preceding claims, wherein the NLRP3 inhibitor is present in a tablet formulation.

15. The method of any one of the preceding claims, comprising administering at least one additional therapeutic agent.

16. The method of any one of the preceding claims, wherein the NLRP3 inhibitor is compound I or a pharmaceutically acceptable salt thereof:

17. The method of claim 16, wherein compound I is an enantiomer of compound IA or a pharmaceutically acceptable salt thereof:

18. the method of claim 17, wherein compound IA has an enantiomeric excess of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%.

19. The method of claim 16, wherein compound I is a compound IB enantiomer or a pharmaceutically acceptable salt thereof:

20. The method of claim 19, wherein compound IB has an enantiomeric excess of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%.