WO2021002887A1

WO2021002887A1 - Gut-targeted nlrp3 antagonists and their use in therapy

Info

Publication number: WO2021002887A1
Application number: PCT/US2020/012721
Authority: WO
Inventors: Luigi Franchi; Shomir Ghosh; Gary Glick; Jason Katz; Anthony William OPIPARI; William Roush; Hans Martin Seidel; Dong-Ming Shen; Shankar Venkatraman; David Guenther WINKLER
Original assignee: Novartis Inflammasome Research, Inc.
Priority date: 2019-07-02
Filing date: 2020-01-08
Publication date: 2021-01-07

Abstract

Provided herein are gut-targeted NLRP3 antagonists for use in the treatment or the prevention of a condition mediated by TNF-α, in a subject in need thereof; in particular a gut condition, disease or disorder. Further provided herein are gut-targeted NLRP3 antagonists for use in the treatment or the prevention of a condition mediated by TNF-α, in a subject having resistance to an anti- TNFα agent.

Description

GUT-TARGETED NLRP3 ANTAGONISTS AND THEIR USE IN THERAPY

RELATED APPLICATIONS

This application claims priority to International Application No. PCT/US2019/040357 filed July 2, 2019, the content of which is incorporated herein by reference in its entirety.

TECHNICAL FIELD

The invention relates to methods of treating a subject having a condition mediated by TNF- a (for example an inflammatory bowel disease), that include administration of a gut-targeted NLRP3 antagonist to said subject. The present disclosure also relates, in part, to said methods wherein the subject is anti-TNFa resistance. Said methods include administration of a gut- targeted NLRP3 antagonist, a gut-targeted NLRP3 antagonist and an anti-TNFa agent combination, or a composition encompassing a gut-targeted NLRP3 antagonist and optionally an anti-TNFa agent.

BACKGROUND

Alterations in NLRP3 and/or TNFa protein levels have been associated with the pathogenesis of a number of complex diseases, including intestinal or gut diseases, e.g., Crohn’s disease (CD), ulcerative colitis (UC) which are termed as types of inflammatory bowel disease (IBD).

Several patients having inflammatory (or autoimmune) bowel diseases are treated with anti-TNFa agents. Most if not all anti-TNFa agents on the market are systemic agents (i.e. agents that do not target the gut), and due to their systemic exposure (and activity) result in an increased risk of infection due to systemic immunosuppression by the anti-TNFa agent. Therefore, it is desireable to develop therapies that target the gut and thus, minimise the risk of such side effects associated with systemic therapeutic agents. Additionally, a subpopulation of patients develop resistance to treatment with anti-TNFa agents. Therefore, it is also desirable to develop methods/treatments for reducing a patient’s resistance to anti-TNFa agents, or provide an alternative therapy that circumnavigates or does not trigger this resistance.

In light of the above, it is desirable to provide alternative therapies for treating inflammatory (or autoimmune) bowel diseases to avoid or minimise the use of anti-TNFa agents, as detailed further below.

Inflammatory bowel disease (IBD) is an example of an inflammatory gut disease, and encompasses Ulcerative Colitis (UC) and Crohn’s disease (CD) that are chronic diseases characterized by barrier dysfunction and uncontrolled inflammation and mucosal immune reactions in the gut. A number of inflammatory pathways have been implicated in the progression of IBD, and anti-inflammatory therapy such as tumor necrosis factor-alpha (TNF-a) blockade has l shown efficacy in the clinic ( Rutgeerts P et al N Engl J Med 2005; 353:2462-76). Anti-TNFa therapies, however, do not show complete efficacy, however, other cytokines such as I L-1 b, IL- 6, IL-12, IL-18, IL-21 , and IL-23 have been shown to drive inflammatory disease pathology in IBD (Neurath MF Nat Rev Immunol 2014; 14; 329-42). I L-1 b and IL-18 are produced by the NLRP3 inflammasome in response to pathogenic danger signals, and have been shown to play a role in IBD. Anti-IL-1 b therapy is efficacious in patients with IBD driven by genetic mutations in CARD8 or IL-10R ( Mao L et al, J Clin Invest 2018;238:1793-1806, Shouval DS et al, Gastroenterology 2016; 151:1100-1104), IL-18 genetic polymorphisms have been linked to UC ( Kanai T et al, Curr Drug Targets 2013;14:1392-9), and NLRP3 inflammasome inhibitors have been shown to be efficacious in murine models of IBD ( Perera AP et al , Sci Rep 2018;8:8618). Resident gut immune cells isolated from the lamina propria of IBD patients can produce I L- 1 b , either spontaneously or when stimulated by LPS, and this I L-1 b production can be blocked by the ex vivo addition of an NLRP3 antagonist. Based on strong clinical and preclinical evidence showing that inflammasome- driven IL-1 b and IL-18 play a role in IBD pathology, it is clear that NLRP3 inflammasome inhibitors could be an efficacious treatment option for UC, Crohn’s disease, or subsets of IBD patients. These subsets of patients could be defined by their peripheral or gut levels of inflammasome related cytokines including I L- 1 b , IL-6, and IL-18, by genetic factors that pre-dispose IBD patients to having NLRP3 inflammasome activation such as mutations in genes including ATG16L1 , CARD8, I L-1 OR, or PTPN2 ( Saitoh T et al, Nature 2008,456:264, Spalinger MR, Cell Rep 2018,22:1835), or by other clinical rationale such as non-response to TNF therapy.

Though anti-TNF therapy is an effective treatment option for Crohn’s disease, about 40% to 50% of patients fail to respond (Leal RF et al Gut 2015;64:233-42), i.e. are resistant to anti- TNFa agents. One-third of non-responsive CD patients fail to respond to anti-TNF therapy at the onset of treatment, while another third lose response to treatment over time (secondary non response). Secondary non-response can be due to the generation of anti-drug antibodies, or a change in the immune compartment that desensitizes the patient to anti-TNF ( Ben-Horin S et al, Autoimmun Rev 2014; 13:24-30, Steenholdt C et al Gut 2014;63:919-27). Anti-TNF reduces inflammation in IBD by causing pathogenic T cell apoptosis in the intestine, therefore eliminating the T cell mediated inflammatory response ( Van den Brande et al Gut 2007:56:509-17). There is increased production of IL-1 b in the gut of TNF-non-responsive CD patients (Leal RF et al Gut 2015;64:233-42) compared to TNF-responsive patients. Furthermore, there is increased expression of TNF-receptor 2 (TNF-R2), which allows for TNF-mediated proliferation of T cells (Schmitt H et al Gut 2018;0:1-15). IL-1 b signaling in the gut promotes T cell differentiation toward Th1/17 cells which can escape anti-TNFa mediated apoptosis. It is therefore hypothesised herein that NLRP3 inflammasome activation, which triggers the release of I L- 1 b , can cause non responsiveness in CD patients to anti-TNF-a therapy by sensitizing pathogenic T cells in the gut to anti-TNF-a mediated apoptosis. Experimental data from immune cells isolated from the gut of TNF-resistant Crohn’s patients show that these cells spontaneously release I L- 1 b , which can be inhibited by the addition of an NLRP3 antagonist. It is hypothesised herein that NLRP3 inflammasome antagonists - in part by blocking I L-1 b secretion - would inhibit the mechanism leading to anti-TNF non-responsiveness, re-sensitizing the patient to anti-TNF therapy. Therefore, in IBD patients who are naive to anti-TNF therapy, treatment with an NLRP3 antagonist would be expected to prevent primary- and secondary-non responsiveness by blocking the mechanism leading to non-response. Therefore, NLRP3 antagonists that are efficacious locally in the gut can be efficacious drugs to treat IBD; in particular in the treatment of TNF-resistant CD alone or in combination with anti-TNF therapy.

Furthermore, systemic inhibition of both I L-1 b and TNF-a has been shown to increase the risk of opportunistic infections (Genovese MC et al, Arthritis Rheum 2004; 50: 1412), therefore, selectively blocking the NLRP3 inflammasome at the site of inflammation would reduce the infection risk inherent in neutralizing both IL-1 b and TNF-a. It is presented herein that NLRP3 antagonists that are i) potent in NLRP3-inflammasome driven cytokine secretion assays in cells and ii) have low permeability in vitro in a permeability assay (such as an MDCK assay), result in poor systemic bioavailability in a rat or mouse pharmacokinetic experiment and high levels of compound in the colon and/or small intestine. Such NLRP3 antagonists target the gut, and thus would be useful therapeutic alternative for gut restricted purposes (e.g. gut TNFa and NLRP3 mediated diseases), reducing the risk of infection.

The present invention provides alternative therapies for the treatment of inflammatory or autoimmune diseases, including IBD, that solves the above problems associated with anti-TNFa agents.

SUMMARY OF THE INVENTION

The present invention is based on the discovery of mutations and mRNA/protein expression profiles that correlate with a subject’s sensitivity to treatment with an NLRP3 antagonist.

The present invention also relates to the inventor’s discovery that inhibition of NLRP3 inflammasomes can increase a subject’s sensitivity to an anti-TNFa agent or can overcome resistance to an anti-TNFa agent in a subject, or provide an alternative therapy to anti-TNFa agents.

As mentioned above, a subset of patients with IBD can be effectively treated with anti- TNF therapy, however about half of patients either don’t respond or lose responsiveness over time. Patients that fail to respond to anti-TNF agents have hyper- activation of the innate immune system that involves in part increased IL-1 b production (Gut. 2015 Feb;64(2):233-42; J Clin Gastroenterol. 2019 Mar;53(3):210-215). NLRP3, however, underlies increased IL-1 b production by the innate immune system. An NLRP3 antagonist blocks release of I L-1 b by cells of the innate immune system, thereby removing a primary mechanisms of anti-TNF resistance. Additionally, the basis for non-responsiveness to anti-TNFa therapy in Crohn’s is resistance of pathogenic, autoreactive CD4+ T cells to anti-TNF-triggered, activation-induced cell death (AICD), which allows disease causing CD4+ T cells to survive (Nat. Med. 2000;6:583; Nat. Med. 2014;20:313). It is hypothesised herein that NLRP3 activation drives multiple signaling pathways that increase the resistance of CD4+ T cells to anti-TNF-induced AICD; and thus, inhibition of NLRP3 is an attractive therapeutic approach to reverse resistance to anti-TNFa therapies for Crohn’s disease, as well as other conditions mediated by TNFa, in particular gut related conditions, whilst reducing the risk of infection caused by systemic therapies.

Therefore, NLRP3 antagonists provide treatment of IBD patients who are known to be anti-TNF-non-responsive, by inhibiting the non-response mechanism and allowing anti-TNF therapy to be effective.

Addionally, NLRP3 antagonist treatment of IBD patients who are naive to anti-TNF therapy at the time of NLRP3 antagonist treatment is expected to prevent primary non-responsiveness by blocking the mechanism that leads to non-responsivess, in part by blocking release of I L- 1 b .

Furthermore, NLRP3 antagonist treatment of IBD patients who are treated with anti-TNF therapy reduces the dose of anti-TNFa agents required for therapeutic effect by blocking the mechanism that leads to resistance to anti-TNF (in part, by blocking release of I L- 1 b) , and thus increasing sensitivity to anti-TNFa thereby.

Therefore, gut-targeted NLRP3 antagonists provide:

i. alternative therapeutic agents to anti-TNFa agents for the treatment of TNFa mediated conditions, in particular gut conditions (for example inflammatory bowel conditions and diseases), whilst also reducing the risk of infection,

ii. therapeutic agents for anti-TNFa resistant subjects, in particular for gut conditions mediated by TNFa (for example inflammatory bowel conditions and diseases), iii. restoration benefit of anti-TNFa agents by increasing a subject’s sensitivity towards such agents, and

iv. reduce resistance to anti-TNFa agents in a subject.

Accordingly, the present invention relates to a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, for use in the treatment or the prevention of a condition mediated by TNF-a, in a subject in need thereof, wherein the NLRP3 antagonist is administered to said subject at a therapeutically effective amount. The subject may be resistant to treatment with an anti-TNFa agent. Preferably, the condition is a gut disease or disorder; more preferably the condition is an Inflammatory Bowel Disease (eg. Crohn’s Disease, or Ulcerative Colitis).

The present invention also relates to a method of treatment or prevention of a condition mediated by TNF- a, comprising administering to a subject in need thereof a therapeutically effective amount of a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof. The subject may be resistant to treatment with an anti-TNF a agent. Preferably, the condition is a gut disease or disorder; more preferably the condition is an Inflammatory Bowel Disease (eg. Crohn’s Disease, or Ulcerative Colitis).

The present invention also relates to the manufacture of a medicatment for use in the treatment or prevention of a condition mediated by TNFa, comprising administering to a subject in need thereof, a therapeutically effective amount of a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof. The subject may be resistant to treatment with an anti- TNFa agent. Preferably, the condition is a gut disease or disorder; more preferably the condition is an Inflammatory Bowel Disease (eg. Crohn’s Disease, or Ulcerative Colitis).

The present invention also relates to a pharmaceutical composition comprising a gut- targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient. Preferably, a pharmaceutical composition comprises an anti-TNFa agent; optionally the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab; preferably the anti-TNFa agent is Adalimumab.

In one embodiment, the invention relates to a method of treatment or prevention of a condition mediated by TNF-a, comprising administering to a subject in need thereof a therapeutically effective amount of a pharmaceutical composition comprising a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient. Preferably, the pharmaceutical composition comprises an anti-TNFa agent; optionally the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab; preferably the anti-TNFa agent is Adalimumab.

In one embodiment, the invention relates to a combination comprising a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, and at least one other therapeutically active agent. Preferably, the combination comprises an anti-TNFa agent; optionally the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab; preferably the anti-TNFa agent is Adalimumab.

In one embodiment, the invention relates to a method of treatment or prevention of a condition mediated by TNF-a, comprising administering to a subject in need thereof, a therapeutically effective amount of a combination comprising a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, and at least one other therapeutically active agent. Preferably, the combination comprises an anti-TNF a agent; optionally the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab; preferably the anti-TNFa agent is Adalimumab.

DEFINITIONS:

To facilitate understanding of the disclosure set forth herein, a number of terms are defined below. Generally, the nomenclature used herein and the laboratory procedures in organic chemistry, medicinal chemistry, and pharmacology described herein are those well-known and commonly employed in the art. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Each of the patents, applications, published applications, and other publications that are mentioned throughout the specification and the attached appendices are incorporated herein by reference in their entireties.

As used herein, the term“antagonist of NLRP3” or“NLRP3 antagonist” is an agent, a genetic mutation, or altered signaling pathways in a mammalian cell that results in a decrease in one or both of (i) the activity of an NLRP3 inflammasome (e.g., any of the exemplary activities of an NLRP3 inflammasome described herein) (e.g., as compared to the level of NLRP3 inflammasome activity in the absence of the agent) and (ii) the expression level of NLRP3 inflammasomes in a mammalian cell (e.g., using any of the exemplary methods of detection described herein) (e.g., as compared to the expression level of NLRP3 inflammasomes in a mammalian cell not contacted with the agent). Typically, an NRLP3 antagonist has a <1 micromolar activity in cellular assay systems, asassessed for example using a nigericin- stimulated I L-1 b secretion assay in THP-1 cells. Non-limiting examples of NLRP3 antagonists are described herein.

As used herein, the term“NLRP3” is meant to include, without limitation, nucleic acids, polynucleotides, oligonucleotides, sense and antisense polynucleotide strands, complementary sequences, peptides, polypeptides, proteins, homologous and/or orthologous NLRP molecules, isoforms, precursors, mutants, variants, derivatives, splice variants, alleles, different species, and active fragments thereof.

The term“NLRP3 inflammasome expression” means the level of one or more of NLRP3 protein, ASC protein, procaspase-1 protein, caspase-1 protein, NLRP3 mRNA, ASC mRNA, and procaspase-1 mRNA in a mammalian cell (e.g., a mammalian cell obtained from a subject).

The term“acceptable” with respect to a formulation, composition, or ingredient, as used herein, means having no persistent detrimental effect on the general health of the subject being treated.

“API” refers to an active pharmaceutical ingredient.

The terms“effective amount” or“therapeutically effective amount,” as used herein, refer to a sufficient amount of a gut-targeted NLRP3 antagonist being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result includes reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an“effective amount” for therapeutic uses is the amount of the composition comprising a gut-targeted NLRP3 antagonist disclosed herein required to provide a clinically significant decrease in disease symptoms. An appropriate “effective” amount in any individual case is determined using any suitable technique, such as a dose escalation study. 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 a pharmaceutical formulation, and suitable for use in contact with the tissue or organ of humans and animals without excessive 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, 21st ed.; Lippincott Williams & Wilkins: Philadelphia, PA, 2005; Handbook of Pharmaceutical Excipients, 6th ed.; Rowe et al. , Eds.; The Pharmaceutical Press and the American Pharmaceutical Association: 2009; Handbook of Pharmaceutical Additives, 3rd ed.; Ash and Ash Eds.; Gower Publishing Company: 2007; Pharmaceutical Preformulation and Formulation, 2nd ed.; Gibson Ed.; CRC Press LLC: Boca Raton, FL, 2009.

The term“pharmaceutically acceptable salt” may refer to pharmaceutically acceptable addition salts prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. In certain instances, pharmaceutically acceptable salts are obtained by reacting a compound described herein, with acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like. The term “pharmaceutically acceptable salt” may also refer to pharmaceutically acceptable addition salts prepared by reacting a compound having an acidic group with a base to form a salt such as an ammonium salt, an alkali metal salt, such as a sodium or a potassium salt, an alkaline earth metal salt, such as a calcium or a magnesium salt, a salt of organic bases such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, and salts with amino acids such as arginine, lysine, and the like, or by other methods previously determined. The pharmacologically acceptable salts not specifically limited as far as it can be used in medicaments. Examples of a salt that the compounds described herein from with a base include the following: salts thereof with inorganic bases such as sodium, potassium, magnesium, calcium, and aluminum; salts thereof with organic bases such as methylamine, ethylamine and ethanolamine; salts thereof with basic amino acids such as lysine and ornithine; and ammonium salt. The salts may be acid addition salts, which are specifically exemplified by acid addition salts with the following: mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, and phosphoric acid:organic acids such as formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid, and ethanesulfonic acid; acidic amino acids such as aspartic acid and glutamic acid.

The term“pharmaceutical composition” refers to a mixture of an NLRP3 antagonistor other compound described herein with other chemical components (referred to collectively herein as “excipients”), such as carriers, stabilizers, diluents, dispersing agents, suspending agents, and/or thickening agents. The pharmaceutical composition facilitates administration of the NLRP3 antagonist or other compound to an organism. Multiple techniques of administering a compound exist in the art including, but not limited to: rectal, oral, intravenous, aerosol, parenteral, ophthalmic, pulmonary, and topical administration.

The term “subject” refers to an animal, including, but not limited to, a primate (e.g., human), monkey, cow, pig, sheep, goat, horse, dog, cat, rabbit, rat, or mouse. The terms“subject” and“patient” are used interchangeably herein in reference, for example, to a mammalian subject, such as a human. In some embodiments of any of the methods described herein

In some embodiments of any of the methods described herein, the subject has been previously diagnosed or identified as having a disease associated with NLRP3 inflammasome activity (e.g., any of the types of NLRP3 inflammasome activity associated-diseases described herein or known in the art, e.g., an inflammatory disease or an autoimmune disease). In some embodiments of any of the methods described herein, the subject is suspected of having a NLRP3 inflammasome activity-associated disease (e.g., any of the types of NLRP3 inflammasome activity -associated diseases described herein or known in the art, e.g., an inflammatory disease or an autoimmune disease). In some embodiments of any of the methods described herein, the subject is presenting with one or more (e.g., two, three, four, or five) symptoms of a NLRP3 inflammasome activity -associated disease (e.g., any of the NLRP3 inflammasome activity- associated disease described herein or known in the art).

In some embodiments of any of the methods described herein, the subject has been previously diagnosed or identified as having a disease associated with an elevated level of TNFa activity and/or expression (e.g., any of the types of TNFa associated-diseases described herein or known in the art). In some embodiments of any of the methods described herein, the subject has been previously diagnosed or identified as having a disease associated with resistance to an anti-TNFa agent (e.g., any of the anti-TNFa agent described herein or known in the art).

In some embodiments of any of the methods described herein, the subject is a participant in a clinical trial. In some embodiments of any of the methods described herein, the subject has been previously administered a pharmaceutical composition and the different pharmaceutical composition was determined not to be therapeutically effective. In some embodiments of any of the methods described herein, the subject has been previously administered an anti-TNFa agent and the anti-TNFa agent was determined not to be therapeutically effective

The term“administration” or“administering” refers to a method of providing a dosage of a pharmaceutical composition or a compound to an invertebrate or a vertebrate, including a fish, a bird and a mammal (e.g., a human). In some aspects, administration is performed, e.g., orally, intravenously, subcutaneously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonarilly, intralymphatic, topically, intraocularly, vaginally, rectally, intrathecally, or intracystically. The method of administration can depend on various factors, e.g., the site of the disease, the severity of the disease, and the components of the pharmaceutical composition.

The terms “treat,” “treating,” and “treatment,” in the context of treating a disease or disorder, are meant to include alleviating or abrogating a disorder, disease, or condition, or one or more of the symptoms associated with the disorder, disease, or condition; or to slowing the progression, spread or worsening of a disease, disorder or condition or of one or more symptoms thereof.

The phrase“an elevated level” or“an increased level” as used herein can be an increase of 1.1x to 100x. In some aspects,“an elevated level” or“an increased level” can be an increase of between 1 % and 1000 (as compared to a reference level, e.g., any of the exemplary reference levels described herein).

The term “NLRP3 inflammasome activity” means direct activity of an NLRP3 inflammasome in a mammalian cell (e.g., caspase-1 cleavage activity, secretion of IL-18, and secretion of I L- 1 b) ; an upstream activity or mutation (e.g., any of the exemplary mutations or single nucleotide polymorphisms described herein) in a mammalian cell that results in increased NLRP3 inflammasome activity in the mammalian cell (e.g., increased expression of one or more of lipocalin-2 protein, lipocalin-2 mRNA, S100A8 protein, S100A8 mRNA, S100A9 protein, and S100A9 mRNA, e.g., as compared to any of the exemplary reference levels described herein; detection of any of the exemplary types of gain-of-function or loss-of-function mutations, or single nucleotide polymorphisms described herein); and/or an increased downstream activity of an NLRP3 inflammasome activity in a mammalian cell (e.g., increased expression of one or more of increased expression of one or more of CRP protein, CRP mRNA, SAA protein, SAA mRNA, HP protein, HP mRNA, ceruloplasmin protein, ceruloplasmin mRNA, IL-6 protein, IP-6 mRNA, calprotectin (S100A8) protein, calprotectin (S100A8) mRNA, IL-8 protein, IL-8 mRNA, leukotriene B4 protein, leukotriene B4 mRNA, myeloperoxidase protein, and myeloperoxidase mRNA, e.g., as compared to any of the exemplary reference levels described herein). Non-limiting examples of human protein and human cDNA sequences for CRP protein, CRP mRNA, SAA protein, SAA mRNA, HP protein, HP mRNA, ceruloplasmin protein, ceruloplasmin mRNA, IL-6 protein, IP-6 mRNA, calprotectin (S100A8) protein, calprotectin (S100A8) mRNA, IL-8 protein, IL-8 mRNA, leukotriene B4 protein, leukotriene B4 mRNA, myeloperoxidase protein, and myeloperoxidase mRNA are shown below.

NLRP3 inflammasome activity can be detected, e.g., by determining the level of expression of one or more of NLRP3, ASC, CASP1 , LCN2, IL-18, I L- 1 b , S100A8, and S100A9 in a mammalian cell; detection of a gain-of-function mutation in a NLRP3 gene (e.g., a NLRP3 protein having a Q705K amino acid substitution, a T350M amino acid substitution, a R262M amino acid substitution, a A441V amino acid substitution, a V200M amino acid substitution, an E629G amino acid substitution, a L355P amino acid substitution, a R260W amino acid substitution, a G571 R amino acid substitution, a A354V amino acid substitution, a D305N amino acid substitution, a F31 1S amino acid substitution, a R920Q amino acid substitution, or a D21 H amino acid substitution), each numbered according to the mature NLRP3 protein sequence of SEQ I D NO: 1 ; detection of one or more of a loss-of-function mutation in one or more of a CARD8 gene (e.g., a C allele at rs204321 1); detection of a T allele at rs3024505 flanking IL10 gene; detection of a R620W amino acid substitution in PTPN22; detection of a C allele at rs478582 in the PTPN2 gene; detection of a G allele at rs713875 in the MTMR3 gene; detection of a C allele at rs1042058 in the TPL2 gene; and detection of a ATG16L1 gene that encodes a ATG16L1 protein having a T300A amino acid substitution. Methods of detecting a level of each of these exemplary types of NLRP3 inflammasome activity are described herein. Additional examples of NLRP3 inflammasome activities are known in the art, as well as methods for detecting a level of the same.

As used herein,“gain-of-function mutation” refers to one or more nucleotide substitutions, deletions, and/or insertions in a gene that results in: an increase in the level of expression of the encoded protein as compared to the level of the expression by the corresponding wildtype gene, and/or the expression of a protein encoded by the gene that has one or more increased activities in a mammalian cell as compared to the version of the protein encoded by the corresponding wildtype gene.

As used herein,“loss-of-function mutation” refers to one or more nucleotide substitutions, deletions, and/or insertions in a gene that results in: a decrease in the level of expression of the encoded protein as compared to the level of the expression by the corresponding wildtype gene, and/or the expression of a protein encoded by the gene that has one or more decreased activities in a mammalian cell as compared to the version of the protein encoded by the corresponding wildtype gene.

As used herein, the phrase“resistance to an anti-TNFa agent” refers to a reduced or decreased level of sensitivity to treatment with an anti-TNFa agent in a subject (e.g., as compared to a similar subject or as compared to the level of sensitivity to the anti-TNFa agent at an earlier time point). For example, resistance to an anti-TNFa in a subject can be observed by a physician, e.g., by observing the requirement of a increasing dosage amounts of an anti-TNFa agent over time in order to achieve the same therapeutic effect in a subject, observing the requirement for an increased number of doses and/or an increased frequency of doses of an anti-TNFa agent over time in order to achieve the same therapeutic effect in a subject, a decrease in the observed therapeutic response to treatment with the same dosage of an anti-TNFa agent over time, or an observed progression of disease or disease relapse in a subject administered an anti-TNFa agent.

As used herein, the phrase“beneficial response” refers to a therapeutic benefit and/or an improved clinical outcome to a subject suffering from a TN Fa-associated disease from or as a result of the treatment with a NLRP3 antagonist. In some embodiments, a beneficial response is a cellular response. The term“condition mediated by TNF-a“ refers to a human state that is negatively affected by overexpression of TNF-a, for example an illnesses, injury, impairment, disease, disorder.

The term“resistant to treatment with an anti-TNFa agent” refers to a subject that does not obtain a beneficial response (e.g. relief of symptoms) of a condition mediated by TNFa with anti- TNA-a therapy (e.g treatment with anti-TNA-a agents).

The term “gut-targeted NLRP3 antagonist” or“gut restricted NLRP3 antagonist” is an NLRP3 antagonist that has a colomplasma exposure ratio of at least (i.e. =/>) 10:1. In other words, a compound which has a high (> 90%) exposure in the colon (or gut) compared to plasma (i.e. systemic exposure). The higher the colomratio exposure ratio, the better, to minimise systemic exposure (and corresponding systemic side effect) and optimise gut exposure. Preferably, the colon:plasma ratio is =/> 50: 1. Ideally, the colomplasma ratio is =/> 100: 1. The =/> 100: 1 colon: plasma relative exposure level was derived in order to obtain an IC₉₀ in colon, and what is considered no inhibition (e.g., no more than ICio) in plasma (or systemic exposure). The activity ratio of ICgo to ICio is approximately 100, and thus, selective gut exposure over systemic exposure as measured by the relative colomplasma exposure is expected to result at colomplasma exposure ration of =/> 100: 1. Therefore, at =/> 100:1 a compound will not only target the gut but be selective over systemic exposure, and no/minimal systemic side effects are expected.

As a result of the above NLRP3 antagonist characteristics a gut-targeted NLRP3 antagonist has relatively low systemic bioavailability (as assessed in a rat or mouse PK experiment) compared to relatively high exposure levels in the colon (as assessed using tissue distribution models), i.e. =/> 10: 1 relative exposure, preferably =/> 50: 1 , ideally, =/> 100: 1. Hence, gut-targeted exposure of these gut-targeted compounds minimises/avoids systemic exposure and associated side effects. It also allows for the targeting of gut related diseases, and thus, increased potency for gut diseases. Gut-targeted NLRP3 antagonists can be assess in accordance with the examples defined herein with Balb/c Mouse (Example 1). Corresponding relative exposure levels are expected in human. The colomplasma gut-targeted levels can be obtained at any time after dosing, and depends on the pharmacokinetics of the NLRP3 antagonist, however, ideally gut- target colon: plasma levels are achieved at, at least 12 hours after dosing; preferably, at 6 hours after dosing; preferably at 3 hours after dosing; preferably, at 1 hour after dosing.

As used herein, the terms “patient” or “subject” refer to a mammalian organism, preferably a human being, who is diseased with the condition (i.e. disease or disorder) of interest and who would benefit from the treatment.

As used herein, the term“prevent”,“preventing” or "prevention" in connection to a disease or disorder refers to the prophylactic treatment of a subject who is at risk of developing a condition (e.g., specific disease or disorder or clinical symptom thereof) resulting in a decrease in the probability that the subject will develop the condition.

As used herein, the term“treat”,“treating" or "treatment" of any disease or disorder refers in one embodiment to ameliorating the disease or disorder (i.e. slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms or pathological features thereof). In another embodiment “treat”, "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter or pathological features of the disease, e.g. including those, which may not be discernible by the subject. In yet another embodiment,“treat”, "treating" or "treatment" refers to modulating the disease or disorder, either physically, (e.g. stabilization of at least one discernible or non-discernible symptom), physiologically (e.g. stabilization of a physical parameter) or both. In yet another embodiment,“treat”, "treating" or "treatment" refers to preventing or delaying the onset or development or progression of the disease or disorder, or of at least one symptoms or pathological features associated thereof. In yet another embodiment, “treat”, "treating" or "treatment" refers to preventing or delaying progression of the disease to a more advanced stage or a more serious condition.

As used herein, the term "therapeutically effective amount" refers to an amount of the compound of the invention, e.g. tropifexor (as herein defined, e.g. in free form or as a stereoisomer, an enantiomer, a pharmaceutically acceptable salt, solvate, prodrug, ester thereof and/or an amino acid conjugate thereof), or cenicriviroc (in free form or as a pharmaceutically acceptable salt, solvate, prodrug, and/or ester thereof, e.g. in free form or as a pharmaceutically acceptable salt thereof), which is sufficient to achieve the stated effect. Accordingly, a therapeutically effective amount used for the treatment or prevention of a liver disease or disorder as hereinabove defined is an amount sufficient for the treatment or prevention of such a disease or disorder.

The terms“hydrogen” and“H” are used interchangeably herein.

The term "halo" refers to fluoro (F), chloro (Cl), bromo (Br), or iodo (I).

The term "alkyl" refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, CM_O (or C1-10) indicates that the group may have from 1 to 10 (inclusive) carbon atoms in it. Non-limiting examples include methyl, ethyl, iso-propyl, tert-butyl, n-hexyl.

The term "haloalkyl" refers to an alkyl, in which one or more hydrogen atoms is/are replaced with an independently selected halo.

The term "alkoxy" refers to an -O-alkyl radical (e.g., -OCH₃).

The term "carbocyclic ring" as used herein includes an aromatic or nonaromatic cyclic hydrocarbon group having 3 to 10 carbons, such as 3 to 8 carbons, such as 3 to 7 carbons, which may be optionally substituted. Examples of carbocyclic rings include five-membered, six membered, and seven-membered carbocyclic rings.

The term “heterocyclic ring” refers to an aromatic or nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1 , 2 or 3 atoms of each ring may be substituted by a substituent. Examples of heterocyclic rings include five-membered, six membered, and seven-membered heterocyclic rings.

The term "cycloalkyl" as used herein includes an aromatic or nonaromatic cyclic hydrocarbon radical having 3 to 10 carbons, such as 3 to 8 carbons, such as 3 to 7 carbons, wherein the cycloalkyl group which may be optionally substituted. Examples of cycloalkyls include five membered, six-membered, and seven-membered rings. Examples include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl,.

The term “heterocycloalkyl” refers to an aromatic or nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system radical having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein 0, 1 , 2 or 3 atoms of each ring may be substituted by a substituent. Examples of heterocycloalkyls include five-membered, sixmembered, and seven-membered heterocyclic rings. Examples include piperazinyl, pyrrolidinyl, dioxanyl, morpholinyl, tetrahydrofuranyl, and the like.

The term“hydroxy” refers to an OH group.

The term“amino” refers to an NH2 group.

The term“oxo” refers to O. By way of example, substitution of a CH2 a group with oxo gives a C=0 group.

The term "aryl" is intended to mean an aromatic ring radical containing 6 to 10 ring carbons. Examples include phenyl and naphthyl.

The term "heteroaryl" is intended to mean an aromatic ring system containing 5 to 14 aromatic ring atoms that may be a single ring, two fused rings or three fused rings wherein at least one aromatic ring atom is a heteroatom selected from, but not limited to, the group consisting of O, S and N. Examples include furanyl, thienyl, pyrrolyl, imidazolyl, oxazolyl, thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, oxadiazolyl, triazolyl, thiadiazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, triazinyl and the like. Examples also include carbazolyl, quinolizinyl, quinolinyl, isoquinolinyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, triazinyl, indolyl, isoindolyl, indazolyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl. phenazinyl, phenothiazinyl, phenoxazinyl, benzoxazolyl, benzothiazolyl, 1 H-benzimidazolyl, imidazopyridinyl, benzothienyl, benzofuranyl, isobenzofuran and the like.

As used herein, the terms “the ring A” or “A” are used interchangeably to denote

formulas AA, AB and AC, wherein the bond that is shown as being broken by the wavy line connects A to the S(0)(NHR³)=N moiety of Formula AA etc.. As used herein, the terms “the ring B” or “B” are used interchangeably to denote

in formula AA wherein the bond that is shown as being broken by the wavy line

connects B to the NH(CO) group of Formula AA, etc.

As used herein, the term “the optionally substituted ring A” is used to denote

formula AA, wherein the bond that is shown as being broken by the wavy line connects A to the S(0)(NHR³)=N moiety of Formula AA, etc..

As used herein, the term“the substituted ring B” is used to denote

formulas AA, AB and AC, wherein the bond that is shown as being broken by the wavy line connects B to the NH(CO) group of Formula AA, etc..

As used herein, the recitation“S(C>2)”, alone or as part of a larger recitation, refers to the

group

In addition, atoms making up the compounds of the present embodiments are intended to include all isotopic forms of such atoms. Isotopes, as used herein, include those atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium, and isotopes of carbon include ¹³C and ¹⁴C.

The scope of the compounds disclosed herein includes tautomeric form of the compounds. Thus, by way of example, a compound that is represented as containing the moiety

is also intended to include the tautomeric form containing the moiety

In addition, by way of example, a compound that is represented as containing the moiety

is also intended to include the tautomeric form containing the moiety

Non-limiting exemplified compounds of the formulae described herein include a stereogenic sulfur atom and optionally one or more stereogenic carbon atoms. This disclosure provides examples of stereoisomer mixtures (e.g., racemic mixture of enantiomers; mixture of diastereomers). This disclosure also describes and exemplifies methods for separating individual components of said stereoisomer mixtures (e.g., resolving the enantiomers of a racemic mixture). In cases of compounds containing only a stereogenic sulfur atom, resolved enantiomers are graphically depicted using one of the two following formats: formulas A/B (hashed and solid wedge three-dimensional representation); and formula C (“flat structures with *-labelled stereogenic sulfur).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art to which this disclosure belongs. All patents, applications, published applications, and other publications are incorporated by reference in their entirety. In the event that there is a plurality of definitions for a term herein, those in this section prevail unless stated otherwise.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF THE DRAWINGS

Figure 1 : Expression levels of RNA encoding NLRP3 in Crohn’s Disease patients who are responsive and non-responsive to infliximab.

Figure 2: Expression levels of RNA encoding I L-1 b in Crohn’s Disease patients who are responsive and non-responsive to infliximab.

Figure 3: Expression levels of RNA encoding NLRP3 in Ulcerative Colitis (UC) patients who are responsive and non-responsive to infliximab.

Figure 4: Expression levels of RNA encoding I L-1 b in Ulcerative Colitis (UC) patients who are responsive and non-responsive to infliximab.

Figure 5: Schematic Summary of In Vivo Procedures of Example 3

Figure 6: LPS-induced Serum IL-1 b and I L-18 Levels 6 Hours after Treatment with IFM-000491 1 or IFM-0000514.

Figure 7: Total Levels of IFM-0004911 and IFM-0000514 in Plasma and Colon at the End of the Study.

Figure 8: Schematic Summary of In Vivo Procedures of Example 4.

Figure 9: Inhibition of Release of Active Caspase-1 in Feces by IFM-0004911 in the Mouse DSS- Induced Colitis Model.

Figure 10: Inhibition of Release of Active Caspase-1 in Feces by IFM-0004911 in the Mouse DSS- Induced Colitis Model.

Figure 1 1 : Inhibition of Processing of I L-1 b in Colon Interstitial Extract by IFM-0004911 in the Mouse DSS-lnduced Colitis Model.

Figure 12: Schematic Summary of In Vivo Procedures of Example 5.

Figure 13: Inhibition of DSS-induced Body Weight Loss by IFM-0004911 in the Mouse Acute DSS Colitis Model.

Figure 14: Inhibition of DSS-induced Increase in Colon Weight/Length Ratio (mg/cm) by IFM- 0004911 in the Mouse Acute DSS Colitis Model.

Figure 15: Inhibition of Histology Score by IFM-0004911 in the Mouse DSS-lnduced Colitis Model Figure 16: Inhibition of Mucosal and Submucosal Inflammation Score by IFM-0004911 in the Mouse DSS-lnduced Colitis Model Figure 17: Inhibition of Mucosal Ulceration and Erosion Score by IFM-0004911 in the Mouse DSS- Induced Colitis Model

Figure 18: Total Levels of IFM-000491 1 in Plasma and Colon at End of Study

Figure 19: Schematic Summary of In Vivo Procedures of example 6

Figure 20: Total Levels of IFM-0003764 and IFM-0000514 in Plasma and Colon at the End of the Study

Figure 21 : LPS-induced Serum I L- 1 b , IL-18 and IL-6 Levels 6 Hours after Treatment with IFM- 0003764 or IFM-0000514

Figure 22: Inhibition of NLRP3 Pathway Activation Biomarkers (Caspase-1 and I L- 1 b) by IFM- 0003764 in Colon Tissue Extract from the Mouse DSS-lnduced Colitis Model

Figure 23: Plasma concentration vs time profile for IFM-0004911 after 30 mg/kg PO of Example 1

Figure 24: Topological Polar Surface Area (TPSA) of gut-targeted compounds compared to systemic compounds

Figure 25: Fraction % absorbtion graphs of gut-targeted compounds compared to systemic compounds

DETAILED DESCRIPTION

The present inventions are based on the discovery that specific genetic mutations and/or protein/ RNA expression profiles correlate with increased NLRP3 inflammasome activity and expression, and can be used to identify subjects who are more likely to have a therapeutic response to treatment with an NLRP3 antagonist.

Some aspects of the present invention is based on the discovery that a NLRP3 antagonist (e.g., any of the NLRP3 antagonists described herein) can reduce resistance to an anti-TNFa agent (e.g., any of the exemplary resistances to an anti-TNFa agent described herein or known in the art) in a subject.

In one embodiment, the invention relates to a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, for use in the treatment or the prevention of a condition mediated by TNF-a, in a subject in need thereof, wherein the NLRP3 antagonist is administered to said subject at a therapeutically effective amount. Preferably, the subject is resistant to treatment with an anti-TNFa agent. Preferably, the condition is a gut disease or disorder; more preferably the condition is Inflammatory Bowel Disease (eg. Crohn’s Disease, or Ulcerative Colitis).

In one embodiment, the invention relates to a method of treatment or prevention of a condition mediated by TNF-a, comprising administering to a subject in need thereof a therapeutically effective amount of a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof. Preferably, the subject is resistant to treatment with an anti-TNFa agent. Preferably, the condition is a gut disease or disorder; more preferably the condition is Inflammatory Bowel Disease (eg. Crohn’s Disease, or Ulcerative Colitis).

In one embodiment, the invention relates to the manufacture of a medicatment for use in the treatment or prevention of a condition mediated by TNF-a, comprising administering to a subject in need thereof, a therapeutically effective amount of a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof. Preferably, the subject is resistant to treatment with an anti-TNFa agent. Preferably, the condition is a gut disease or disorder; more preferably the condition is Inflammatory Bowel Disease (eg. Crohn’s Disease, or Ulcerative Colitis).

In one embodiment, the invention relates to a pharmaceutical composition comprising a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient. Preferably, a pharmaceutical composition comprises an anti-TNFa agent; optionally wherein the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab.

In one embodiment, the invention relates to a method of treatment or prevention of a condition mediated by TNF-a, comprising administering to a subject in need thereof, a therapeutically effective amount of a pharmaceutical composition comprising a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable excipient. Preferably, the pharmaceutical composition comprises an anti-TNFa agent; optionally wherein the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab.

In one embodiment, the invention relates to a combination comprising a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, and at least one other therapeutically active agent. Preferably, the combination comprises an anti-TNFa agent; optionally wherein the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab.

In one embodiment, the invention relates to a method of treatment or prevention of a condition mediated by TNF-a, comprising administering to a subject in need thereof, a therapeutically effective amount of a combination comprising a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, and at least one other therapeutically active agent. Preferably, the combination comprises an anti-TNFa agent; optionally wherein the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab.

In one embodiment, the present invention relates to a method for the treatment or the prevention of a condition mediated by TNF-a, in particular a gut disease or disorder, in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a gut-targeted NLRP3 antagonist.

In one embodiment, the present invention relates to a method for the treatment or the prevention of a condition, in particular a gut disease or disorder, in a patient in need thereof, comprising administering to said patient a therapeutically effective amount of a gut-targeted

NLRP3 antagonist.

In one embodiment, the present invention relates to a method for the treatment, stabilization or lessening the severity or progression of gut disease or disorder, in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a gut-targeted NLRP3 antagonist.

In one embodiment, the present invention relates to a method for slowing, arresting, or reducing the development of a gut disease or disorder, in a patient in need thereof comprising administering to said patient a therapeutically effective amount of a gut-targeted NLRP3 antagonist.

In one embodiment, the present invention relates to a method according to any of the above embodiments, wherein the gut disease is IBD.

In one embodiment, the present invention relates to a method according to any of the above embodiments, wherein the gut disease is UC or CD.

Enumerated Embodiments A of the invention:

Embodiment 1A: A gut-targeted NLRP3 antagonist for use in the treatment or the prevention of a condition mediated by TNF-a, in a subject in need thereof, wherein the NLRP3 antagonist is administered to said subject at a therapeutically effective amount.

Advantageously, gut-targeted NLRP3 antagonists (substantially) restrict the exposure of the NLRP3 antagonist to the gut, rather than systemic expsoure. This enables selective therapuetic targeting of the gut for gut related diseases (e.g. IBD), and thus minimises the risk of systemic infection that are known to result with the treatment of autoinflam matory immune therapies (e.g. anti-TNFa agents, systemic I L-1 b or IL-18 antagonists, etc.).

Embodiment 2A: The gut-targeted NLRP3 antagonist for use according to embodiment 1 A, wherein said subject is resistant to treatment with an anti-TNFa agent.

Increased levels of NLRP3 have been found for anti-TNFa agent resistant CD and UC patients. Use of an gut-targeted NLRP3 antagonist blocks the NLRP3 pathway which causes anti- TNFa resistance, whilst also treating TNFa related gut disease. Therefore, NLRP3 antagonists provide: i) an alternative therapy to anti-TNFa agents for patients with TNFa related gut disease, and ii) an effective therapy for anti-TNFa resistant populations of said patients.

Embodiment 3A: The gut-targeted NLRP3 antagonist for use according to any preceeding embodiment, wherein the condition is a gut disease or disorder. Embodiment 4A: The gut-targeted NLRP3 antagonist for use according to any preceeding embodiment, wherein the condition is Inflammatory Bowel Disease.

Embodiment 5A: The gut-targeted NLRP3 antagonist for use according to any preceeding embodiment, wherein the condition is Crohn’s Disease, or Ulcerative Colitis.

Embodiment 6A: A pharmaceutical composition comprising a gut-targeted NLRP3 antagonist and at least one pharmaceutically acceptable excipient, for use according to any preceding embodiment.

Embodiment 7A: A combination comprising a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, and at least one other therapeutically active agent, for use according to any one of embodiments 1A to 5A.

Embodiment 8A: The pharmaceutical composition of embodiment 6A, or combination of embodiment 7A, comprising an anti-TNFa agent.

Treatment with NLRP3 antagonists blocks the involvement of the NLRP3 protein in effecting resistance to anti-TNFa agents. Consequently, an anti-TNFa resistant patient will become resensitised to TNFa agents, such that, the combination of an NLRP3 antagonist and an anti-TNFa agent provide a synergistic therapeutic effect in the treatment of TNFa related diseases, for example IBD. Additionally, use of the combination of NLRP3 antagonist with an anti- TNFa agent, i) provides a synergistic effect which enables reduced dosing of both therapeutic agents, and ii) prevents NLRP3 driven resistance to anti-TNFa agents. This combination, synergistically, further reduces anti-TNFa agent resistance in patients that develop such resistance over time, as reduced concentration of anti-TNFa agents are reqiured for therapeutic efficacy.

Embodiment 9A: The pharmaceutical composition or the combination of embodiment 8A, wherein the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab.

Embodiment 10A: The pharmaceutical composition or the combinatoin of embodiment 9, wherein the anti-TNFa agent is Adalimumab.

Enumerated Embodiments B of the invention:

Embodiment 1 B: A method of treatment or prevention of a condition mediated by TNF-a, comprising administering to a subject in need thereof a therapeutically effective amount of a gut- targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof.

Embodiment 2B: The method of treatment or prevention of a condition mediated by TNF- a according to embodiment 1 B, wherein said subject is resistant to treatment with an anti-TNFa agent.

Embodiment 3B: The method of treatment or prevention of a condition mediated by TNF- a according to any preceeding embodiment, wherein the condition is a gut disease or disorder. Embodiment 4B: The method of treatment or prevention of a condition mediated by TNF- a according to any preceeding embodiment, wherein the condition is Inflammatory Bowel Disease.

Embodiment 5B: The method of treatment or prevention of a condition mediated by TNF- a according to any preceeding embodiment, wherein the condition is Crohn’s Disease, or Ulcerative Colitis.

Further Embodiments:

Also provided are methods of selecting a treatment for a subject that include selecting a treatment comprising a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof for a subject identified as resistant to an anti-TNFa agents.

Also provided herein are methods of selecting a subject for treatment that include: (a) identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity and/or expression as compared to a reference level; and (b) selecting an identified subject for treatment with a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof. Also provided are methods selecting a subject for treatment that include selecting a subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity and/or expression as compared to a reference level, for treatment with a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof.

Also provided herein are methods of selecting a subject for participation in a clinical trial that include selecting a subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity and/or expression as compared to a reference level for participation in a clinical trial that comprises administration of a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof.

Some embodiments of the invention are based on the discovery that a NLRP3 antagonist (e.g., any of the NLRP3 antagonists described herein) can reduce resistance to an anti-TNFa agent (e.g., any of the exemplary resistances to an anti-TNFa agent described herein or known in the art) in a subject. In view of these discoveries, provided herein are methods of treating a subject in need thereof that include (a) identifying a subject having resistance to an anti-TNFa agent (e.g., any of the exemplary resistances to an anti-TNFa agent described herein or known in the art) as compared to a reference level (e.g., any of the exemplary reference levels of NLRP3 inflammasome activity described herein or known in the art); and (b) administering a treatment comprising a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof to the identified subject. Also provided herein are methods of treating a subject identified as having resistance to an anti-TNFa agent (e.g., any of the exemplary resistances to an anti-TNFa agent described herein or known in the art) with a gut-targeted NLRP3 antagonist (e.g., any of the exemplary NLRP3 antagonists described herein) or a pharmaceutically acceptable salt, solvate, or co-crystal thereof

Non-liming aspects of these methods are described below, and can be used in any combination without limitation. Additional aspects of these methods are known in the art.

Provided herein are methods of treating a subject having a condition mediated by TNFa inflammatory disease that include: (a) identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity and/or expression as compared to a reference level; and (b) administering to the identified subject a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof.

Provided herein are methods of treating a subject that include: administering a therapeutically effective amount of a gut-targeted NLPR3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof to a subject having an inflammatory or autoimmune gut disease and identified as having a cell that has an elevated level of NLRP3 inflammasome activity and/or expression as compared to a reference level.

In some embodiments of any of the methods described herein, the subject has or is suspected of having an autoimmune disease selected from the group consisting of: an inflammatory bowel disease (IBD). In some embodiments, the IBD is selected from the group consisting of: Crohn’s disease, ulcerative colitis, autoimmune colitis, iatrogenic autoimmune colitis, ulcerative colitis, colitis induced by one or more chemotherapeutic agents, colitis induced by treatment with adoptive cell therapy, colitis associated with one or more alloimmune diseases such as GVHD, radiation enteritis, collagenous colitis, lymphocytic colitis, microscopic colitis, and radiation enteritis, celiac disease, and inflammatory bowel syndrome.

Provided herein are methods of treating a subject having an inflammatory or autoimmune gut disease in need thereof, that include: (a) identifying a subject having resistance to an anti- TNFa agent; and (b) administering a treatment comprising a therapeutically effective amount of a gut-targeted (i.e. gut restricted) NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof to the identified subject.

In some embodiments of any of the methods described herein, step (b) can further include identifying the subject as also having an elevated level of NLRP3 inflammasome activity and/or expression in a cell obtained from the subject, as compared to a reference level.

In some embodiments, the identifying the subject having an inflammatory or autoimmune gut disease and also having a cell that has an elevated level of NLRP3 inflammasome expression includes detecting the level of one or more of NLRP3 protein, ASC protein, procaspase-1 protein, and caspase-1 protein.

In some embodiments, the identifying the subject having an inflammatory or autoimmune gut disease and also having a cell that has an elevated level of NLRP3 inflammasome expression includes detecting the level of one or more of NLRP3 mRNA, ASC mRNA, and procaspase-1 mRNA.

In some embodiments of any of the methods described herein, the treatment further includes a therapeutically effective amount of an anti-TNFa agent, in addition to the gut-targeted NLRP3 antagonist.

Provided herein are methods of treating a subject having an inflammatory or autoimmune gut disease, that include: (a) administering one or more doses of an anti-TNFa agent to the subject; (b) detecting an elevated level of NLRP3 inflammasome activity and/or expression in a cell obtained from the subject after step (a) as compared to a reference level; and (c) administering a treatment comprising a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof to a subject determined to have an elevated level of NLRP3 inflammasome activity and/or expression as compared to the reference level in step (b).

Provided herein are methods of treating a subject in need thereof, that include: (a) detecting an elevated level of NLRP3 inflammasome activity and/or expression as compared to a reference level in a cell obtained from a subject previously administered one or more doses of an anti-TNFa agent; and (b) administering a treatment comprising a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof to a subject determined to have an elevated level of NLRP3 inflammasome activity and/or expression as compared to the reference level in step (a).

Provided herein are methods of treating a subject in need thereof, that include administering a treatment comprising a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof to a subject determined to have an elevated level of NLRP3 inflammasome activity and/or expression as compared to a reference level in a cell obtained from the subject after previous administration with one or more doses of an anti-TNFa agent.

In some embodiments of any of the methods described herein, the treatment further includes a therapeutically effective amount of an anti-TNFa agent, in addition to the NLRP3 antagonist.

Provided herein are methods of treating a subject in need thereof, that include: (a) administering one or more doses of an anti-TNFa agent to the subject; (b) after step (a), detecting resistance to the anti-TNFa agent in the subject; and (c) administering a treatment comprising a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof to a subject determined to have resistance to the anti-TNFa agent in step (b).

Provided herein are methods of treating a subject in need thereof, that include: (a) detecting resistance to an anti-TNFa agent in a subject previously administered one or more doses of the anti-TNFa agent; and (b) administering a treatment comprising a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof to a subject determined to have resistance to the anti-TNFa agent in step (a).

Provided herein are methods of treating a subject in need thereof, that include: administering a treatment comprising a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof to a subject previously administered one or more doses of an anti-TNFa agent and determined to have resistance to the anti-TNFa agent.

Provided herein are methods of reducing the risk of developing resistance to an anti-TNFa agent in a subject in need thereof, that include: administering to a subject in need thereof a therapeutically effective amount of an anti-TNFa agent and a therapeutically effective amount of a gut-targeted NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof.

In some embodiments, the anti-TNFa agent and the NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof are administered at substantially the same time.

In some embodiments, the anti-TNFa agent and the NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof are formulated into a single dosage form.

In some embodiments, the NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co-crystal thereof is administered to the subject prior to administration of the anti-TNFa agent.

In some embodiments, the anti-TNFa agent is administered to the subject prior to administration of the NLRP3 antagonist or a pharmaceutically acceptable salt, solvate, or co crystal thereof.

Gut-Taraeted NLRP3 Antagonist:

The gut-targeted NLRP3 antagonist is any NLRP3 antagonist as defined above. The gut- targeted NLRP3 antagonist may be any one compound defined herein, or a pharmaceutically acceptable salt, solvate, or co-crystal thereof. The gut-targeted NLRP3 antagonist may also be an inhibitory nucleic acid (e.g., a short interfering RNA, an antisense nucleic acid, or a ribozyme).

Screening of Gut-targeted NLRP3 Compounds: A screending method was developed to identify gut-tartgeted NLRP3 antagonists, that is, compounds with a colomplasma exposure ratio of =/>100: 1. Said screening method is applicable to any anti-NLRP3 antagonist compounds, and comprises identifying compounds with: i. Topological Polar Surface Area (TPSA) of greater than 125 angstroms squared, and ii. permeability of Papp < 2 x 10 ⁶ cm/sec, as assessed in an MDCK assay (an intestinal permeability assay).

Compounds that satisfied the above criteria were further assessed for having a colomplasma exposure ratio of =/>10:1 , but ideally =/>100: 1 in Balb/c Mice (see example 1 , below), which is expected to have a corresponding colomplasma exposure ratio in humans. Consequently, these gut-targeted NLRP3 antagonists will have a relatively low systemic exposure and relatively high gut exposure (as measured by colon exposure). The advantage of an NLRP3 antagonist which targets the gut (i.e. a“gut-targeted NLRP3 antagonist”), is that comparably low systemic exposure is obtained, and consequently: i) minimises systemic side effects, such as increased risk of infection, which are known to be associated with systemic anti-TNFa agents, and systemic I L-1 b agents; and ii) increased exposure and efficacy at the desired site which enables reduced dosing to obtain the same thereapeutic effect, and further reduction of side effects associated with dosing concentrations.

Topological Polar Surface Area (TPSA):

Literature highlights the importance of TPSA for gut-targeting (lower fa_*fgas†TPSA) ( Pharm . Res., 1997;4:568 J. Med. Chem., 2010;5:1098). Of the various compounds screening to be gut- targeted. Figure 24 shows the screening results of gut and systemic compounds which are differentiated by their TPSA. The y-axis relates to the number of compounds (and ranges from 0 to 220), the x-axis relates to the TPSA value (and ranges from 0 to 170 angstroms squared). As shown in the graph, systemic compounds have a TPSA approximately less than 125 angstroms squared, whereas, gut-targeted compounds have a TPSA of at least 125 angstroms squared. Figure 25 depicts the % fraction absorbed relative to TPSA. Compounds of TPSA of at least 125 angstroms squared provided gut-targeted compounds (i.e. compounds with low fraction % absorbtion).

COMPOUND TABLE:

EXAMPLES OF GUT-TARGETED NLRP3 COMPOUNDS:

In one embodiment, provided herein is a combination of a compound of any preceding embodiment, for use in the treatment or the prevention of a condition mediated by TNF-a, in a patient in need thereof, wherein the compound is administered to said patient at a therapeutically effective amount. Preferably, the subject is resistant to treatment with an anti-TNF a agent. Preferably, the condition is a gut disease or disorder.

In one embodiment, provided herein is a pharmaceutical composition of comprising a compound of any preceding embodiment, and an anti-TNFa agent disclosed herein. Preferably wherein the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab, more preferably wherein the anti-TNFa agent is Adalimumab.

In one embodiment, provided herein is a pharmaceutical combination of a compound of any preceding embodiment, and an anti-TNFa agent Preferably wherein the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab, more preferably wherein the anti-TNFa agent is Adalimumab.

Mature Human NLRP3 Protein (SEQ ID NO: 1 )

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leylsrisic kmkkdyrkky rkyvrsrfqc iedrnarlge svslnkrytr Irlikehrsq

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kilfeesdlr nhglqkadvs aflrmnlfqk evdcekfysf ihmtfqeffa amyylleeek

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glsheccfdi slvlssnqkl veldlsdnal gdfgirllcv glkhllcnlk klwlvscclt

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csalssvlst nqnlthlylr gntlgdkgik llcegllhpd cklqvleldn cnltshccwd

Istlltssqs Irklslgnnd Igdlgvmmfc evlkqqscll qnlglsemyf nyetksalet

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Methods of Treating Provided herein are methods of treating a subject having an inflammatory or autoimmune gut disease (e.g., any of the exemplary subjects described herein) that include: (a) identifying a subject having a cell that has an elevated level (e.g., an increase of 1.1x to 100x or higher, or any of the subranges of this range described herein) of NLRP3 inflammasome activity and/or expression as compared to a reference level; and (b) administering to the identified subject a therapeutically effective amount of a gut-targeted NLRP3 antagonist (e.g., any of the exemplary NLRP3 antagonists described herein) or a pharmaceutically acceptable salt, solvate, or co-crystal thereof.

Also provided herein are methods of treating a subject having an inflammatory or autoimmune gut disease (e.g., any of the exemplary subjects described herein) that include: administering a therapeutically effective amount of a gut-targeted NLPR3 antagonist (e.g., any of the exemplary NLRP3 antagonists described herein) or a pharmaceutically acceptable salt, solvate, or co-crystal thereof to a subject identified as having a cell that has an elevated level (e.g., an increase of 1.1x to 100x or higher, or any of the subranges of this range described herein) of NLRP3 inflammasome activity and/or expression as compared to a reference level.

In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is secretion of IL-18. In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is secretion of I L- 1 b . In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is caspase-1 activity. In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is the level of lipocalin- 2 (LCN2). In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is the level of S100A8. In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is the level of S100A9.

In some embodiments of any of the methods described herein, the identifying a subject having an inflammatory or autoimmune gut disease and having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a gain-of-function mutation in an NLRP3 gene (e.g., a NLRP3 protein having a Q705K amino acid substitution, a T350M amino acid substitution, a R262M amino acid substitution, a A441V amino acid substitution, a V200M amino acid substitution, an E629G amino acid substitution, a L355P amino acid substitution, a R260W amino acid substitution, a G571 R amino acid substitution, a A354V amino acid substitution, a D305N amino acid substitution, a F311 S amino acid substitution, a R920Q amino acid substitution, or a D21 H amino acid substitution, each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1) in a cell from the subject.

In some embodiments of any of the methods described herein, the subject having an inflammatory or autoimmune gut disease and identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having a gain-of-function mutation in an NLRP3 gene (e.g., a NLRP3 protein having a Q705K amino acid substitution, a T350M amino acid substitution, a R262M amino acid substitution, a A441V amino acid substitution, a V200M amino acid substitution, an E629G amino acid substitution, a L355P amino acid substitution, a R260W amino acid substitution, a G571 R amino acid substitution, a A354V amino acid substitution, a D305N amino acid substitution, a F31 1 S amino acid substitution, a R920Q amino acid substitution, or a D21 H amino acid substitution, each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1).

In some embodiments of any of the methods described herein, the identifying a subject having an inflammatory or autoimmune gut disease and having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a loss-of-function mutation in a CARD8 gene (e.g., a C allele at rs204321 1) in a cell from the subject.

In some embodiments of any of the methods described herein, the subject identified as having an inflammatory or autoimmune gut disease and having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having a loss-of-function mutation in a CARD8 gene (e.g., a C allele at rs2043211).

In some embodiments of any of the methods described herein, the identifying a subject having an inflammatory or autoimmune gut disease and having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a gain-of-function mutation in an NLRP3 gene (e.g., a NLRP3 protein having a Q705K amino acid substitution, a T350M amino acid substitution, a R262M amino acid substitution, a A441V amino acid substitution, a V200M amino acid substitution, an E629G amino acid substitution, a L355P amino acid substitution, a R260W amino acid substitution, a G571 R amino acid substitution, a A354V amino acid substitution, a D305N amino acid substitution, a F311 S amino acid substitution, a R920Q amino acid substitution, or a D21 H amino acid substitution, each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1) and a loss-of-function mutation in a CARD8 gene (e.g., a C allele at rs2043211) in a cell from the subject.

In some embodiments of any of the methods described herein, the subject having an inflammatory or autoimmune gut disease and identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having a gain-of-function mutation in an NLRP3 gene (e.g., a NLRP3 protein having a Q705K amino acid substitution, a T350M amino acid substitution, a R262M amino acid substitution, a A441V amino acid substitution, a V200M amino acid substitution, an E629G amino acid substitution, a L355P amino acid substitution, a R260W amino acid substitution, a G571 R amino acid substitution, a A354V amino acid substitution, a D305N amino acid substitution, a F31 1 S amino acid substitution, a R920Q amino acid substitution, or a D21 H amino acid substitution, each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1) and a loss-of-function mutation in a CARD8 gene (e.g., a C allele at rs204321 1).

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a T allele at rs3024505 flanking an IL10 gene in a cell from the subject. In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having a T allele at rs3024505 flanking IL10 gene in a cell.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a PTPN22 gene that encodes a PTPN22 protein having a R620W amino acid substitution in a cell from the subject.

In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having a PTPN22 gene that encodes a PTPN22 protein having a R620W amino acid substitution.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a C allele at rs478582 in the PTPN2 gene in a cell from the subject.

In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having a C allele at rs478582 in the PTPN2 gene.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a G allele at rs713875 in the MTMR3 gene in a cell from the subject.

In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having a G allele at rs713875 in the MTMR3 gene.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a C allele at rs1042058 in the TPL2 gene in a cell from the subject.

In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having a C allele at rs1042058 in the TPL2 gene.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a ATG16L1 gene that encodes a ATG16L1 protein having a T300A amino acid substitution in a cell from the subject.

In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having a ATG16L1 gene that encodes a ATG16L1 protein having a T300A amino acid substitution. In some embodiments of any of the methods described herein, the gain-of-function mutation in an NLRP3 gene results in the expression of a NLRP3 protein having a Q705K amino acid substitution. In some embodiments of any of the methods described herein, the loss-of- function mutation in a CARD8 gene is a C allele at rs2043211.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome expression comprises detecting the level of one or more of NLRP3 protein, ASC protein, procaspase-1 protein, caspase-1 protein, IL-18 protein (e.g., mature or pro-IL-18 protein), I L-1 b protein (e.g., mature or pro-l L-1 b protein), LCN2 protein, S100A8 protein, and S100A9 protein.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome expression comprises detecting the level of one or more of NLRP3 mRNA, ASC mRNA, and procaspase-1 mRNA, pro-IL-18 mRNA, pro-l L-1 b mRNA, LCN2 mRNA, S100A8 mRNA, and S100A9 mRNA.

In some embodiments of any of the methods described herein the subject identified as having a cell that has an elevated level of NLRP3 inflammasome expression has been determined to have a cell having an elevated level of one or more of NLRP3 protein, ASC protein, procaspase- 1 protein, caspase-1 protein, IL-18 protein (e.g., mature or pro-IL-18 protein), I L-1 b protein (e.g., mature or pro-IL-1 b protein), LCN2 protein, S100A8 protein, and S100A9 protein.

In some embodiments of any of the methods described herein the subject identified as having a cell that has an elevated level of NLRP3 inflammasome expression has been determined to have a cell having an elevated level of one or more of NLRP3 mRNA, ASC mRNA, and procaspase-1 mRNA.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome expression comprises detecting the level of one or more of CRP protein, SAA protein, HP protein, ceruloplasmin protein, IL-6 protein (e.g., mature or pro-IL-6 protein), calprotectin (S100A8) protein, IL-8 protein (e.g., mature or pro-IL-8 protein), leukotriene B4 protein, and myeloperoxidase protein.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome expression comprises detecting the level of one or more of CRP mRNA, SAA mRNA, HP mRNA, ceruloplasmin mRNA, pro-IL-6 mRNA, calprotectin (S100A8) mRNA, pro-IL-8 mRNA, leukotriene B4 mRNA, and myeloperoxidase mRNA.

In some embodiments of any of the methods described herein the subject identified as having a cell that has an elevated level of NLRP3 inflammasome expression has been determined to have a cell having an elevated level of one or more of CRP protein, SAA protein, HP protein, ceruloplasmin protein, IL-6 protein (e.g., mature or pro-IL-6 protein), calprotectin (S100A8) protein, IL-8 protein (e.g., mature or pro-IL-8 protein), leukotriene B4 protein, and myeloperoxidase protein. In some embodiments of any of the methods described herein the subject identified as having a cell that has an elevated level of NLRP3 inflammasome expression has been determined to have a cell having an elevated level of one or more of CRP mRNA, SAA mRNA, HP mRNA, ceruloplasmin mRNA, pro-1 L-6 mRNA, calprotectin (S100A8) mRNA, pro-IL-8 mRNA, leukotriene B4 mRNA, and myeloperoxidase mRNA.

In some embodiments of any of the methods described herein the subject has or is suspected of having Crohn’s disease, inflammatory bowel disease (IBD), or other gastrointestinal, autoimmune, or autoinflammatory disorders.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a mutation in an NLRP3 gene that results in the expression of a NLRP3 protein having one or both of a T350M and a R262M amino acid substitution (each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1) in a cell from the subject. In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having an NLRP3 gene that results in the expression of a NLRP3 protein having one or both of a T350M and a R262M amino acid substitution (each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1). In some embodiments of these methods, the subject has or is suspected of having hereditary periodic fever.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a mutation in an NLRP3 gene that results in the expression of an NLRP3 protein having one or more of a A441V, a V200M, a E629G, and a L355P amino acid substitution (each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1) in a cell from the subject. In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having an NLRP3 gene that results in the expression of an NLRP3 protein having one or more of a A441 V, a V200M, a E629G, and a L355P amino acid substitution (each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1). In some embodiments of these methods, the subject has or is suspected of having familial cold autoinflammatory syndrome (FCAS).

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a mutation in an NLRP3 gene that results in the expression of an NLRP3 protein having one or more of a R260W, a G571 R, and a A354V amino acid substitution (each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1) in a cell from the subject. In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having an NLRP3 gene that results in the expression of an NLRP3 protein having one or more of a R260W, a G571 R, and a A354V amino acid substitution (each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1). In some of embodiments of these methods, the subject has or is suspected of having Muckle-Wells syndrome (MWS).

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a mutation in an NLRP3 gene that results in the expression of an NLRP3 protein having one or both of a D305N and a F31 1S amino acid substitution (each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1) in a cell from the subject. In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having an NLRP3 gene that results in the expression of an NLRP3 protein having one or both of a D305N and a F31 1S amino acid substitution (each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1). In some embodiments of these methods, the subject has or is suspected of having Cinca syndrome.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a mutation in an NLRP3 gene that results in the expression of an NLRP3 protein having one or both of a R920Q amino acid substitution (each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1) in a cell from the subject. In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having a mutation in an NLRP3 gene that results in the expression of an NLRP3 protein having a R920Q amino acid substitution (each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1). In some embodiments of these methods, the subject has or is suspected of having deafness with or without inflammation.

In some embodiments of any of the methods described herein, the identifying a subject having a cell that has an elevated level of NLRP3 inflammasome activity comprises detecting a mutation in an NLRP3 gene that results in the expression of an NLRP3 protein having a D21 H amino acid substitution (numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1) in a cell from the subject. In some embodiments of any of the methods described herein, the subject identified as having a cell that has an elevated level of NLRP3 inflammasome activity has been determined to have a cell having an NLRP3 gene that results in the expression of an NLRP3 protein having a D21 H amino acid substitution (numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1). In some embodiments of these methods, the subject has or is suspected of having keratoendotheliitis fugax hereditaria.

In some embodiments of any of the methods described herein, the subject has or is suspected of having an inappropriate host response to infectious diseases where active infection exists at any body site. In some embodiments of these methods, the inappropriate host response to infectious disease where active infection exists at any body site is selected from the group of: septic shock, disseminated intravascular coagulation, and adult respiratory distress syndrome.

In some embodiments of any of the methods of treatment described herein, the method can result in a decreased risk (e.g., a 1 % to a 99% decrease) of developing a comorbidity in the subject (e.g., as compared to the risk of developing a comorbidity in a subject having a similar elevated level of NLRP3 inflammasome activity and/or expression in a cell and/or a similar level of anti-TNFa resistance, but administered a different treatment or a placebo).

In some embodiments of any of the methods described herein, where the subject has inflammatory bowel disease, such as ulcerative colitis or Crohn’s, the methods can result in a decrease (e.g., a 1 % to 99% decrease, or any of the subranges of this range described herein) in the disease activity index (DAI) for Crohn’s disease or ulcerative colitis in the subject (e.g., as compared to the DAI in the same subject prior to treatment).

In some embodiments of any of the methods described herein, wherein the subject has inflammatory bowel syndrome, such as Crohn’s disease or ulcerative colitis, the methods can result in an improvement in stool consistency in the subject (e.g., as compared to the stool consistency in the subject prior to treatment).

Methods of Predicting a Subject’s Responsiveness to an anti-TNFa agent

Also provided herein are methods of predicting a subject’s responsiveness (e.g., any of the exemplary subjects desecribed herein) to an anti-TNFa agent (e.g., any of the exemplary anti- TNFa agents described herein or known in the art), that include: (a) determining that a subject has an elevated level (e.g., an increase of 1 % to 1000%, or any of the subranges of this range described herein) of NLRP3 inflammasome activity and/or expression in a cell obtained from the subject, as compared to a reference level (e.g., any of the exemplary reference levels of NLRP3 inflammasome activity described herein or known in the art); and (b) identifying that the subject determined to have an elevated level of NLRP3 inflammasome activity and/or expression in step (a) has an increased likelihood of being resistant to treatment with an anti-TNFa agent.

Also provided herein are methods of predicting a subject’s responsiveness (e.g., any of the exemplary subjects desecribed herein) to an anti-TNFa agent (e.g., any of the exemplary anti- TNFa agents described herein or known in the art), that include: identifying a subject determined to have an elevated level (e.g., an increase of 1 % to 1000%, or any of the subranges of this range described herein) of NLRP3 inflammasome activity and/or expression in a cell obtained from the subject, as having an increased likelihood of being resistant to treatment with an anti-TNFa agent.

In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is secretion of IL-18 (processed IL-18). In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is secretion of I L-1 b (processed I L-1 b). In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is caspase-1 activity. In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is the level of lipocalin-2. In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is the level of S100A8. In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is the level of S100A9.

In some embodiments of any of the methods described herein, the determining that a subject has an elevated level of NLRP3 inflammasome activity and/or expression includes detecting the level of one or more (e.g., 1 , 2, 3, or 4) of NLRP3 protein, ASC protein, procaspase- 1 protein, and caspase-1 protein. In some embodiments of any of the methods described herein, the determining that a subject has an elevated level of NLRP3 inflammasome expression includes detecting the level of one or more (e.g., 1 , 2, 3, or 4) of NLRP3 mRNA, ASC mRNA, and procaspase-1 mRNA.

In some embodiments of any of the methods described herein, the identifying of the subject as having a cell that has an elevated level of NLRP3 inflammasome activity includes detecting the level of one or more of CRP protein, SAA protein, HP protein, ceruloplasmin protein, IL-6 protein (e.g., mature or pro-IL-6 protein), calprotectin (S100A8) protein, IL-8 protein (e.g., mature or pro-IL-8 protein), leukotriene B4 protein, and myeloperoxidase protein. In some embodiments of any of the methods described herein, the identifying of the subject as having a cell that has an elevated level of one or more of CRP mRNA, SAA mRNA, HP mRNA, ceruloplasmin mRNA, pro-IL-6 mRNA, calprotectin (S100A8) mRNA, pro-IL-8 mRNA, leukotriene B4 mRNA, and myeloperoxidase mRNA.

In some embodiments of any of the methods described herein, the subject determined to have an elevated level of NLRP3 inflammasome expression has been determined to have a cell having an elevated level of one or more of NLRP3 protein, ASC protein, procaspase-1 protein, and capsase-1 protein. In some embodiments of any of the methods described herein, the subject determined to have an elevated level of NLRP3 inflammasome expression has been determined to have a cell having an elevated level of one or more of NLRP3 mRNA, ASC mRNA, and procaspase-1 mRNA.

In some embodiments of any of the methods described herein, the subject has not previously been administered a dose of an anti-TNFa antagonist.

Some embodiments of any of the methods described herein can further include: administering to the subject identified as having an increased likelihood of being resistant to treatment with an anti-TNFa agent, a treatment including (i) a therapeutically effective amount of an anti-TNFa agent (e.g., any of the exemplary anti-TNFa agent described herein) and (ii) a therapeutically effective amount of a gut-targeted NLRP3 antagonist (e.g., any of the exemplary gut-targeted NLRP3 antagonsits described herein or known in the art).

Some embodiments of any of the methods described herein can further include: recording the identification of the subject as having an increased likelihood of being resistant to treatment with an anti-TNFa agent in the subject’s clinical record (e.g., a computer readable medium). Some embodiments of any of the methods described herein can further include recording in the clinical record for the subject identified as having an increased likelihood of being resistant to treatment with an anti-TNFa agent, that the subject should be administered an NLRP3 antagonist (e.g., alone or in combination with an anti-TNFa agent). Additional exemplary aspects that can be used or incorporated in these methods are described herein.

Some embodiments of any of the methods described herein can further include recording the selected treatment in the subject’s clinical record (e.g., a computer readable medium). Some embodiments of any of the methods described herein can further include administering one or more doses (e.g., at least two, at least four, at least six, at least eight, at least ten doses) of the selected treatment to the identified subject.

Additional exemplary aspects that can be used or incorporated in these methods are described herein.

Methods of Determining Resistance of an Anti-TNFa Agent

Resistance to an anti-TNFa agent (e.g., primary resistance) is a reduced or decreased level of sensitivity to treatment with an anti-TNFa agent in a subject (e.g., as compared to a similar subject or as compared to the level of sensitivity to the anti-TNFa agent at an earlier time point). For example, resistance to an anti-TNFa in a subject can be observed by a physician or trained medical professional, e.g., by observing the requirement of increasing dosage amounts of an anti- TNFa agent (e.g., any of the exemplary anti-TNFa agents described herein or known in the art) over time in order to achieve the same therapeutic effect in a subject (e.g., any of the exemplary subjects described herein), observing the requirement for an increased number of doses and/or an increased frequency of doses of an anti-TNFa agent (e.g., any of the exemplary anti-TNFa agents described herein or known in the art) over time in order to achieve the same therapeutic effect in a subject (e.g., any of the exemplary subjects described herein), a decrease in the observed therapeutic response to treatment with the same dosage of an anti-TNFa agent (e.g., any of the exemplary anti-TNFa agents described herein or known in the art) in a subject (e.g., any of the exemplary subjects described herein) over time, or an observed progression of disease or disease relapse (e.g., any of the inflammatory diseases or autoimmune diseases described herein) in a subject (e.g., any of the exemplary subjects described herein) administered an anti- TNFa agent (e.g., any of the exemplary anti-TNFa agents described herein or known in the art). Additional metrics and assessments of resistance to an anti-TNFa agent are known in the art.

Anti-TNFa Agents

The term“anti-TNFa agent” refers to an agent which directly or indirectly blocks, down- regulates, impairs, inhibits, impairs, or reduces TNFa activity and/or expression. In some embodiments, an anti-TNFa agent is an antibody or an antigen-binding fragment thereof, a fusion protein, a soluble TNFa receptor (a soluble tumor necrosis factor receptor superfamily member 1A (TNFR1) or a soluble tumor necrosis factor receptor superfamily 1 B (TNFR2)), an inhibitory nucleic acid, or a small molecule TNFa antagonist. In some embodiments, the inhibitory nucleic acid is a ribozyme, small hairpin RNA, a small interfering RNA, an antisense nucleic acid, or an aptamer.

Exemplary anti-TNFa agents that directly block, down-regulate, impair, inhibit, or reduce TNFa activity and/or expression can, e.g., inhibit or decrease the expression level of TNFa or a receptor of TNFa (TNFR1 or TNFR2) in a cell (e.g., a cell obtained from a subject, a mammalian cell), or inhibit or reduce binding of TNFa to its receptor (TNFR1 and/or TNFR2). Non-limiting examples of anti-TNFa agents that directly block, down-regulate, impair, inhibit, or reduce TNFa activity and/or expression include an antibody or fragment thereof, a fusion protein, a soluble TNFa receptor (e.g., a soluble TNFR1 or soluble TNFR2), inhibitory nucleic acids (e.g., any of the examples of inhibitory nucleic acids described herein), and a small molecule TNFa antagonist.

Exemplary anti-TNFa agents that can indirectly block, down-regulate, impair, inhibitreduce TNFa activity and/or expression can, e.g., inhibit or decrease the level of downstream signaling of a TNFa receptor (e.g., TNFR1 or TNFR2) in a mammalian cell (e.g., decrease the level and/or activity of one or more of the following signaling proteins: AP-1 , mitogen-activated protein kinase kinase kinase 5 (ASK1), inhibitor of nuclear factor kappa B (IKK), mitogen-activated protein kinase 8 (JNK), mitogen-activated protein kinase (MAPK), MEKK 1/4, MEKK 4/7, MEKK 3/6, nuclear factor kappa B (NF-KB), mitogen-activated protein kinase kinase kinase 14 (NIK), receptor interacting serine/threonine kinase 1 (RIP), TNFRSF1A associated via death domain (TRADD), and TNF receptor associated factor 2 (TRAF2), in a cell), and/or decrease the level of TNFa- induced gene expression in a mammalian cell (e.g., decrease the transcription of genes regulated by, e.g., one or more transcription factors selected from the group of activating transcription factor 2 (ATF2), c-Jun, and NF-KB). A description of downstream signaling of a TNFa receptor is provided in Wajant et al., Cell Death Differentiation 10:45-65, 2003 (incorporated herein by reference). For example, such indirect anti-TNFa agents can be an inhibitory nucleic acid that targets (decreases the expression) a signaling component downstream of a TN Fa-induced gene (e.g., any TNFa-induced gene known in the art), a TNFa receptor (e.g., any one or more of the signaling components downstream of a TNFa receptor described herein or known in the art), or a transcription factor selected from the group of NF-KB, c-Jun, and ATF2.

In other examples, such indirect anti-TNFa agents can be a small molecule inhibitor of a protein encoded by a TNFa-induced gene (e.g., any protein encoded by a TNFa-induced gene known in the art), a small molecule inhibitor of a signaling component downstream of a TNFa receptor (e.g., any of the signaling components downstream of a TNFa receptor described herein or known in the art), and a small molecule inhibitor of a transcription factor selected from the group of ATF2, c-Jun, and N F-KB. In other embodiments, anti-TNFa agents that can indirectly block, down-regulate, impair, or reduce one or more components in a cell (e.g., acell obtained from a subject, a mammalian cell) that are involved in the signaling pathway that results in TNFa mRNA transcription, TNFa mRNA stabilization, and TNFa mRNA translation (e.g., one or more components selected from the group of CD14, c-Jun, ERK1/2, IKK, I KB, interleukin 1 receptor associated kinase 1 (IRAK), JNK, lipopolysaccharide binding protein (LBP), MEK1/2, MEK3/6, MEK4/7, MK2, MyD88, N F-KB, NIK, PKR, p38, AKT serine/threonine kinase 1 (rac), raf kinase (raf), ras, TRAF6, TTP). For example, such indirect anti-TNFa agents can be an inhibitory nucleic acid that targets (decreases the expression) of a component in a mammalian cell that is involved in the signaling pathway that results in TNFa mRNA transcription, TNFa mRNA stabilization, and TNFa mRNA translation (e.g., a component selected from the group of CD14, c-Jun, ERK1/2, IKK, IKB, IRAK, JNK, LBP, MEK1/2, MEK3/6, MEK4/7, MK2, MyD88, N F-KB, NIK, IRAK, lipopolysaccharide binding protein (LBP), PKR, p38, rac, raf, ras, TRAF6, TTP). In other examples, an indirect anti-TNFa agents is a small molecule inhibitor of a component in a mammalian cell that is involved in the signaling pathway that results in TNFa mRNA transcription, TNFa mRNA stabilization, and TNFa mRNA translation (e.g., a component selected from the group of CD14, c-Jun, ERK1/2, IKK, I KB, IRAK, JNK, lipopolysaccharide binding protein (LBP), MEK1/2, MEK3/6, MEK4/7, MK2, MyD88, N F-KB, NIK, IRAK, lipopolysaccharide binding protein (LBP), PKR, p38, rac, raf, ras, TRAF6, TTP).

Antibodies

In some embodiments, the anti-TNFa agent is an antibody or an antigen-binding fragment thereof (e.g., a Fab or a scFv). In some embodiments, an antibody or antigen-binding fragment of an antibody described herein can bind specifically to TNFa. In some embodiments, an antibody or antigen-binding fragment described herein binds specifically to any one of TNFa, TNFR1 , or TNFR2. In some embodiments, an antibody or antigen-binding fragment of an antibody described herein can bind specifically to a TNFa receptor (TNFR1 or TNFR2).

In some embodiments, the antibody can be a humanized antibody, a chimeric antibody, a multivalent antibody, or a fragment thereof. In some embodiments, an antibody can be a scFv- Fc, a VHH domain, a VNAR domain, a (scFv)2, a minibody, or a BiTE.

In some embodiments, an antibody can be a crossmab, a diabody, a scDiabody, a scDiabody-CH3, a Diabody-CH3, a DutaMab, a DT-lgG, a diabody-Fc, a scDiabody-HAS, a charge pair antibody, a Fab-arm exchange antibody, a SEEDbody, a Triomab, a LUZ-Y, a Fcab, a kA-body, an orthogonal Fab, a DVD-lgG, an lgG(H)-scFv, a scFv-(H)lgG, an lgG(L)-scFv, a scFv-(L)-lgG, an IgG (L,H)-Fc, an lgG(H)-V, a V(H)-lgG, an lgG(L)-V, a V(L)-lgG, an KIH IgG- scFab, a 2scFv-lgG, an lgG-2scFv, a scFv4-lg, a Zybody, a DVI-lgG, a nanobody, a nanobody- HSA, a DVD-lg, a dual-affinity re-targeting antibody (DART), a triomab, a kih IgG with a common LC, an ortho-Fab IgG, a 2-in-1 -IgG, IgG-ScFv, scFv2-Fc, a bi-nanobody, tanden antibody, a DART-Fc, a scFv-HAS-scFv, a DAF (two-in-one or four-in-one), a DNL-Fab3, knobs-in-holes common LC, knobs-in-holes assembly, a TandAb, a Triple Body, a miniantibody, a minibody, a TriBi minibody, a scFv-CH3 KIH, a Fab-scFv, a scFv-CH-CL-scFv, a F(ab')2-scFV2, a scFv-KIH, a Fab-scFv-Fc, a tetravalent HCAb, a scDiabody-Fc, a tandem scFv-Fc, an intrabody, a dock and lock bispecific antibody, an ImmTAC, a HSAbody, a tandem scFv, an IgG-lgG, a Cov-X-Body, and a scFv1-PEG-scFv2.

Non-limiting examples of an antigen-binding fragment of an antibody include an Fv fragment, a Fab fragment, a F(ab')2 fragment, and a Fab' fragment. Additional examples of an antigen-binding fragment of an antibody is an antigen-binding fragment of an antigen-binding fragment of an IgA (e.g., an antigen-binding fragment of lgA1 or lgA2) (e.g., an antigen-binding fragment of a human or humanized IgA, e.g., a human or humanized lgA1 or lgA2); an antigen binding fragment of an IgD (e.g., an antigen-binding fragment of a human or humanized IgD); an antigen-binding fragment of an IgE (e.g., an antigen-binding fragment of a human or humanized IgE); an IgG (e.g., an antigen-binding fragment of lgG1 , lgG2, lgG3, or lgG4) (e.g., an antigen binding fragment of a human or humanized IgG, e.g., human or humanized lgG1 , lgG2, lgG3, or lgG4); or an antigen-binding fragment of an IgM (e.g., an antigen-binding fragment of a human or humanized IgM).

Non-limiting examples of anti-TNFa agents that are antibodies that specifically bind to TNFa are described in Ben-Horin et al. , Autoimmunity Rev. 13(1 ):24-30, 2014; Bongartz et al., JAMA 295(19):2275-2285, 2006; Butler et al., Eur. Cytokine Network 6(4)-.225-230, 1994; Cohen et al., Canadian J. Gastroenterol. Hepatol. 15(6):376-384, 2001 ; Elliott et al., Lancet 1994; 344: 1 125-1127, 1994; Feldmann et al. , Ann. Rev. Immunol. 19(1): 163-196, 2001 ; Rankin et al. , Br. J. Rheumatol. 2:334-342, 1995; Knight et al., Molecular Immunol. 30(16):1443-1453, 1993; Lorenz et al., J. Immunol. 156(4): 1646- 1653, 1996; Hinshaw et al., Circulatory Shock 30(3):279-292, 1990; Ordas et al., Clin. Pharmacol. Therapeutics 91 (4): 635-646, 2012; Feldman, Nature Reviews Immunol. 2(5):364-371 , 2002; Taylor et al., Nature Reviews Rheumatol. 5(10):578-582, 2009; Garces et al., Annals Rheumatic Dis. 72(12): 1947-1955, 2013; Palladino et al., Nature Rev. Drug Discovery 2(9):736-746, 2003; Sandborn et al., Inflammatory Bowel Diseases 5(2): 1 19-133, 1999; Atzeni et al., Autoimmunity Reviews 12(7):703-708, 2013; Maini et al., Immunol. Rev. 144(1): 195-223, 1995; Wanner et al., Shock 1 1 (6):391-395, 1999; and U.S. Patent Nos. 6,090,382; 6,258,562; and 6,509,015).

In certain embodiments, the anti-TNFa agent can include or is golimumab (golimumabTM), adalimumab (Humira™), infliximab (Remicade™), CDP571 , CDP 870, or certolizumab pegol (Cimzia™). In certain embodiments, the anti-TNFa agent can be a TNFa inhibitor biosimilar. Examples of approved and late-phase TNFa inhibitor biosimilars include, but are not limited to, infliximab biosimilars such as Flixabi™ (SB2) from Samsung Bioepis, Inflectra® (CT-P13) from Celltrion/Pfizer, GS071 from Aprogen, Remsima™, PF-06438179 from Pfizer/Sandoz, NI-071 from Nichi-lko Pharmaceutical Co., and ABP 710 from Amgen; adalimumab biosimilars such as Amgevita® (ABP 501) from Amgen and Exemptia™ from Zydus Cadila, BMO-2 or MYL-1401-A from Biocon/Mylan, CHS-1420 from Coherus, FKB327 from Kyowa Kirin, and Bl 695501 from Boehringer lngelheim;Solymbic®, SB5 from Samsung Bioepis, GP-2017 from Sandoz, ONS-3010 from Oncobiologics, M923 from Momenta, PF-06410293 from Pfizer, and etanercept biosimilars such as Erelzi™ from Sandoz/Novartis, Brenzys™ (SB4) from Samsung Bioepis, GP2015 from Sandoz, TuNEX® from Mycenax, LBEC0101 from LG Life, and CHS-0214 from Coherus.

In some embodiments of any of the methods described herein, the anti-TNFa agent is selected from the group consisting of: adalimumab, certolizumab, etanercept, golimumab, infliximabm, CDP571 , and CDP 870.

Fusion Proteins

In some embodiments, the anti-TNFa agent is a fusion protein (e.g., an extracellular domain of a TNFR fused to a partner peptide, e.g., an Fc region of an immunoglobulin, e.g., human IgG) (see, e.g., Deeg et al., Leukemia 16(2): 162, 2002; Peppel et al., J. Exp. Med. 174(6): 1483-1489, 1991) or a soluble TNFR (e.g., TNFR1 or TNFR2) that binds specifically to TNFa. In some embodiments, the anti-TNFa agent includes or is a soluble TNFa receptor (e.g., Bjornberg et al., Lymphokine Cytokine Res. 13(3):203-211 , 1994; Kozak et al., Am. J. Physiol. Reg. Integrative Comparative Physiol. 269(1 ):R23-R29, 1995; Tsao et al., Eur Respir J. 14(3):490-495, 1999; Watt et al., J Leukoc Biol. 66(6): 1005-1013, 1999; Mohler et al., J. Immunol. 151 (3): 1548-1561 , 1993; Nophar et al., EMBO J. 9(10):3269, 1990; Piguet et al., Eur. Respiratory J. 7(3):515-518, 1994; and Gray et al., Proc. Natl. Acad. Sci. U.S.A. 87(19):7380-7384, 1990). In some embodiments, the anti-TNFa agent includes or is etanercept (Enbrel™) (see, e.g., WO 91/03553 and WO 09/406,476, incorporated by reference herein). In some embodiments, the anti-TNFa agent inhibitor includes or is r-TBP-l (e.g., Gradstein et al., J. Acquir. Immune Defic. Syndr. 26(2): 111-117, 2001).

Inhibitory Nucleic Acids

Inhibitory nucleic acids that can decrease the expression of AP-1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF-kB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP mRNA expression in a mammalian cell include antisense nucleic acid molecules, i.e. , nucleic acid molecules whose nucleotide sequence is complementary to all or part of a AP- 1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, N F-KB, NIK, p38, PKR, rac, ras, raf, RIP, TNFD, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP mRNA (e.g., complementary to all or a part of any one of the sequences presented in Table A). Table A.

An antisense nucleic acid molecule can be complementary to all or part of a non-coding region of the coding strand of a nucleotide sequence encoding an AP-1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF-kB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTPMEKK1 protein. Non-coding regions (5' and 3' untranslated regions) are the 5' and 3' sequences that flank the coding region in a gene and are not translated into amino acids.

Based upon the sequences disclosed herein, one of skill in the art can easily choose and synthesize any of a number of appropriate antisense nucleic acids to target a nucleic acid encoding an AP-1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF-KB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP protein described herein. Antisense nucleic acids targeting a nucleic acid encoding an AP-1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF-KB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTPMEKK1 protein can be designed using the software available at the Integrated DNA Technologies website.

An antisense nucleic acid can be, for example, about 5, 10, 15, 18, 20, 22, 24, 25, 26, 28, 30, 32, 35, 36, 38, 40, 42, 44, 45, 46, 48, or 50 nucleotides or more in length. An antisense oligonucleotide can be constructed using enzymatic ligation reactions and chemical synthesis using procedures known in the art. For example, an antisense nucleic acid can be chemically synthesized using variously modified nucleotides or naturally occurring nucleotides designed to increase the physical stability of the duplex formed between the antisense and sense nucleic acids, e.g., phosphorothioate derivatives and acridine substituted nucleotides or to increase the biological stability of the molecules.

Examples of modified nucleotides which can be used to generate an antisense nucleic acid include 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-fluorouracil, 5-bromouracil, 5- chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 5-methylcytosine, N6- adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 5-methyl-2- thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methylester, uracil-5- oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e. , RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest).

The antisense nucleic acid molecules described herein can be prepared in vitro and administered to a subject, e.g., a human subject. Alternatively, they can be generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding an AP-1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF-KB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP protein to thereby inhibit expression, e.g., by inhibiting transcription and/or translation. The hybridization can be by conventional nucleotide complementarities to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule that binds to DNA duplexes, through specific interactions in the major groove of the double helix. The antisense nucleic acid molecules can be delivered to a mammalian cell using a vector (e.g., an adenovirus vector, a lentivirus, or a retrovirus).

An antisense nucleic acid can be an a-anomeric nucleic acid molecule. An a-anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual, b-units, the strands run parallel to each other (Gaultier et al. , Nucleic Acids Res. 15:6625-6641 , 1987). The antisense nucleic acid can also comprise a chimeric RNA-DNA analog (Inoue et al., FEBS Lett. 215:327-330, 1987) or a 2'-0-methylribonucleotide (Inoue et al., Nucleic Acids Res. 15:6131-6148, 1987).

Another example of an inhibitory nucleic acid is a ribozyme that has specificity for a nucleic acid encoding an AP-1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF-KB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP mRNA, e.g., specificity for any one of Table A). Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes (e.g., hammerhead ribozymes (described in Haselhoff and Gerlach, Nature 334:585-591 , 1988)) can be used to catalytically cleave mRNA transcripts to thereby inhibit translation of the protein encoded by the mRNA. An AP-1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF-KB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel et al. , Science 261 :141 1-1418, 1993.

Alternatively, a ribozyme having specificity for an AP-1 , ASK1 , CD14, c-jun, ERK1/2, I KB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, N F-KB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP mRNA can be designed based upon the nucleotide sequence of any of the AP-1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF-kB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP mRNA sequences disclosed herein. For example, a derivative of a Tetrahymena L-19 I VS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an AP-1 , ASK1 , CD14, c-jun, ERK1/2, I KB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF- KB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP mRNA (see, e.g., U.S. Patent. Nos. 4,987,071 and 5, 116,742).

An inhibitory nucleic acid can also be a nucleic acid molecule that forms triple helical structures. For example, expression of an AP-1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF-KB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP polypeptide can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the gene encoding the AP-1 , ASK1 , CD14, c-jun, ERK1/2, IKB, IKK, IRAK, JNK, LBP, MAPK, MEK1/2, MEKK1/4, MEKK4/7, MEKK 3/6, MK2, MyD88, NF-KB, NIK, p38, PKR, rac, ras, raf, RIP, TNFa, TNFR1 , TNFR2, TRADD, TRAF2, TRAF6, or TTP polypeptide (e.g., the promoter and/or enhancer, e.g., a sequence that is at least 1 kb, 2 kb, 3 kb, 4 kb, or 5 kb upstream of the transcription initiation start state) to form triple helical structures that prevent transcription of the gene in target cells. See generally Maher, Bioassays 14(12):807-15, 1992; Helene, Anticancer Drug Des. 6(6):569-84, 1991 ; and Helene, Ann. N. Y. Acad. Sci. 660:27-36, 1992.

In various embodiments, inhibitory nucleic acids can be modified at the sugar moiety, the base moiety, or phosphate backbone to improve, e.g., the solubility, stability, or hybridization, of the molecule. For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (see, e.g., Hyrup et al., Bioorganic Medicinal Chem. 4(1 ):5-23, 1996). Peptide nucleic acids (PNAs) are nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs allows for specific hybridization to RNA and DNA under conditions of low ionic strength. PNA oligomers can be synthesized using standard solid phase peptide synthesis protocols (see, e.g., Perry-O'Keefe et al., Proc. Natl. Acad. Sci. U.S. A. 93: 14670-675, 1996). PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, e.g., inducing transcription or translation arrest or inhibiting replication. Small Molecules

In some embodiments, the anti-TNFa agent is a small molecule. In some embodiments, the small molecule is a tumor necrosis factor-converting enzyme (TACE) inhibitor (e.g., Moss et al. , Nature Clinical Practice Rheumatology 4: 300-309, 2008). In some embodiments, the anti- TNFa agent is C87 (Ma et al., J. Biol. Chem. 289(18):12457-66, 2014). In some embodiments, the small molecule is LMP-420 (e.g., Haraguchi et al., AIDS Res. Ther. 3:8, 2006). In some embodiments, the TACE inhibitor is TMI-005 and BMS- 561392. Additional examples of small molecule inhibitors are described in, e.g., He et al., Science 310(5750): 1022-1025, 2005.

In some examples, the anti-TNFa agent is a small molecule that inhibits the activity of one of AP-1 , ASK1 , IKK, JNK, MAPK, MEKK 1/4, MEKK4/7, MEKK 3/6, NIK, TRADD, RIP, NF-KB, and TRADD in a cell (e.g., in a cell obtained from a subject, a mammalian cell).

In some examples, the anti-TNFa agent is a small molecule that inhibits the activity of one of CD14, MyD88 (see, e.g., Olson et al. , Scientific Reports 5:14246, 2015), ras (e.g., Baker et al., Nature 497 :577-578, 2013), raf (e.g., vemurafenib (PLX4032, RG7204), sorafenib tosylate, PLX- 4720, dabrafenib (GSK2118436), GDC-0879, RAF265 (CHIR-265), AZ 628, NVP-BHG712, SB590885, ZM 336372, sorafenib, GW5074, TAK-632, CEP-32496, encorafenib (LGX818), CCT196969, LY3009120, R05126766 (CH5126766), PLX7904, and MLN2480).

In some examples, the anti-TNFa agent TNFa inhibitor is a small molecule that inhibits the activity of one of MK2 (PF 3644022 and PHA 767491), JNK (e.g., AEG 3482, Bl 78D3, CEP 1347, c-JUN peptide, IQ 1S, JIP-1 (153-163), SP600125, SU 3327, and TCS JNK6o), c-jun (e.g., AEG 3482, Bl 78D3, CEP 1347, c-JUN peptide, IQ 1S, JIP-1 (153-163), SP600125, SU 3327, and TCS JNK6o), MEK3/6 (e.g., Akinleye et al., J. Hematol. Oncol. 6:27, 2013), p38 (e.g., AL 8697, AMG 548, BIRB 796, CMPD-1 , DBM 1285 dihydrochloride, EO 1428, JX 401 , ML 3403, Org 48762-0, PH 797804, RWJ 67657, SB 202190, SB 203580, SB 239063, SB 706504, SCIO 469, SKF 86002, SX 011 , TA 01 , TA 02, TAK 715, VX 702, and VX 745), PKR (e.g., 2-aminopurine or CAS 608512-97-6), TTP (e.g., CAS 329907-28-0), MEK1/2 (e.g., Facciorusso et al., Expert Review Gastroentrol. Hepatol. 9:993-1003, 2015), ERK1/2 (e.g., Mandal et al., Oncogene 35:2547-2561 , 2016), NIK (e.g., Mortier et al., Bioorg. Med. Chem. Lett. 20:4515-4520, 2010), IKK (e.g., Reilly et al., Nature Med. 19:313-321 , 2013), IKB (e.g., Suzuki et al., Expert. Opin. Invest. Drugs 20:395-405, 201 1), NF-KB (e.g., Gupta et al., Biochim. Biophys. Acta 1799(10- 12):775-787, 2010), rac (e.g., U.S. Patent No. 9,278,956), MEK4/7, IRAK (Chaudhary et al., J. Med. Chem. 58(1):96-110, 2015), LBP (see, e.g., U.S. Patent No. 5,705,398), and TRAF6 (e.g., 3-[(2,5-Dimethylphenyl)amino]-1-phenyl-2-propen-1-one).

In some embodiments, the inhibitory nucleic acid can be formulated in a liposome, a micelle (e.g., a mixed micelle), a nanoemulsion, or a microemulsion, a solid nanoparticle, or a nanoparticle (e.g., a nanoparticle including one or more synthetic polymers). Additional exemplary structural features of inhibitory nucleic acids and formulations of inhibitory nucleic acids are described in US 2016/0090598.

In some embodiments, the inhibitory nucleic acid (e.g., any of the inhibitory nucleic acid described herein) can include a sterile saline solution (e.g., phosphate-buffered saline (PBS)). In some embodiments, the inhibitory nucleic acid (e.g., any of the inhibitory nucleic acid described herein) can include a tissue-specific delivery molecule (e.g., a tissue-specific antibody).

Indications

In some embodiments, methods for treating a subject having condition, disease or disorder in which a decrease or increase in NLRP3 activity (e.g., an increase, e.g., NLRP3 signaling) contributes to the pathology and/or symptoms and/or progression of the condition, disease or disorder are provided, comprising administering to a subject an effective amount of a chemical entity described herein (e.g., a compound described generically or specifically herein or a pharmaceutically acceptable salt thereof or compositions containing the same). In some embodiments of any of the methods described herein, the subject can have, or be diagnosed or identified as having, an inflammatory disease or an anutoimmune gut disease. In some embodiments of any of the methods described herein, the subject can have, or be identified or diagnosed as having, any of the conditions, diseases, or disorders in which a decrease or increase in NLRP3 activity contributes to the pathology and/or symptoms and/or progression of the condition, disease, or disorder. In some embodiments of any of the methods described herein, the subject can be suspected of having or present with one or more symptoms of any of the conditions, diseases, or disorders described herein.

In some embodiments, the condition, disease or disorder is an inflammatory bowel diseases (IBDs). In certain embodiments, the condition is Crohn disease (CD) or ulcerative colitis (UC). In certain embodiments, the condition is Crohn’s disease, autoimmune colitis, iatrogenic autoimmune colitis, ulcerative colitis, colitis induced by one or more chemotherapeutic agents, colitis induced by treatment with adoptive cell therapy, colitis associated by one or more alloimmune diseases (such as graft-vs-host disease, e.g., acute graft vs. host disease and chronic graft vs. host disease), radiation enteritis, collagenous colitis, lymphocytic colitis, microscopic colitis, and radiation enteritis. In certain of these embodiments, the condition is alloimmune disease (such as graft-vs-host disease, e.g., acute graft vs. host disease and chronic graft vs. host disease), celiac disease, inflammatory bowel syndrome, rheumatoid arthritis, lupus, scleroderma, psoriasis, cutaneous T-cell lymphoma, uveitis, and mucositis (e.g., oral mucositis, esophageal mucositis or intestinal mucositis).

In some embodiments, the condition, disease or disorder is IBD.

In some embodiments of any of the methods described herein, the subject has or is suspected of having Crohn’s disease, inflammatory bowel disease (IBD), or other gastrointestinal, autoimmune, or autoinflammatory disorders. In some embodiments of any of the methods described herein, the subject has or is suspected of having an intestinal disease, such as Crohn’s disease or ulcerative colitis.

In some embodiments, the IBD is selected from the group consisting of: Crohn’s disease, ulcerative colitis, autoimmune colitis, iatrogenic autoimmune colitis, ulcerative colitis, colitis induced by one or more chemotherapeutic agents, colitis induced by treatment with adoptive cell therapy, colitis associated with one or more alloimmune diseases such as GVHD, radiation enteritis, collagenous colitis, lymphocytic colitis, microscopic colitis, and radiation enteritis, celiac disease, and inflammatory bowel syndrome.

Combination Therapy

This disclosure contemplates both monotherapy regimens as well as combination therapy regimens.

In some embodiments, the methods described herein can further include administering one or more additional therapies (e.g., one or more additional therapeutic agents and/or one or more therapeutic regimens) in combination with administration of the gut-targeted NLRP3 antagonist (e.g., any gut-targeted NLRP3 antagonists described herein or known in the art).

In certain embodiments, the second therapeutic agent or regimen is administered to the subject prior to contacting with or administering the gut-targeted NLRP3 antagonist (e.g., about one hour prior, or about 6 hours prior, or about 12 hours prior, or about 24 hours prior, or about 48 hours prior, or about 1 week prior, or about 1 month prior).

In other embodiments, the second therapeutic agent or regimen is administered to the subject at about the same time as contacting with or administering the gut-targeted NLRP3 antagonist. By way of example, the second therapeutic agent or regimen and the NLRP3 antagonist are provided to the subject simultaneously in the same dosage form. As another example, the second therapeutic agent or regimen and the gut-targeted NLRP3 antagonist are provided to the subject concurrently in separate dosage forms.

In still other embodiments, the second therapeutic agent or regimen is administered to the subject after contacting with or administering the gut-targeted NLRP3 antagonist (e.g., about one hour after, or about 6 hours after, or about 12 hours after, or about 24 hours after, or about 48 hours after, or about 1 week after, or about 1 month after).

In preferred embodiments, the second therapeutic agent is an anti-TNFa agent.

Patient Selection

In some embodiments, the methods described herein include the step of identifying a subject having an inflammatory or autoimmune gut disease (e.g., a patient) in need of treatment for an indication related to NLRP3 activity, such as an indication related to NLRP3 polymorphism.

In some embodiments, the methods described herein further include the step of identifying a subject (e.g., a patient) in need of treatment for an indication related to NLRP3 activity, such as an indication related to NLRP3 where a polymorphism in a NLRP3 gene is a gain-of-function mutation (e.g., a NLRP3 protein having a Q705K amino acid substitution, a T350M amino acid substitution, a R262M amino acid substitution, a A441V amino acid substitution, a V200M amino acid substitution, an E629G amino acid substitution, a L355P amino acid substitution, a R260W amino acid substitution, a G571 R amino acid substitution, a A354V amino acid substitution, a D305N amino acid substitution, a F311 S amino acid substitution, a R920Q amino acid substitution, or a D21 H amino acid substitution, each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1).

In some embodiments, the methods described herein include the step of identifying a subject (e.g., a patient) in need of treatment for an indication related to NLRP3 activity, such as an indication related to NLRP3 polymorphism found in CAPS syndromes.

In some embodiments, the methods described herein include the step of identifying a subject (e.g., a patient) in need of treatment for an indication related to NLRP3 activity, such as an indication related NLRP3 polymorphism where the polymorphism is VAR_014104 (R262W, numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1).

In some embodiments, the methods described herein include the step of identifying a subject (e.g., a patient) in need of treatment for an indication related to NLRP3 activity, such as an indication related NLRP3 polymorphism where the polymorphism is a natural variant reported in www.uniprot.org/uniprot/Q96P20.

In some embodiments, the methods described herein further include the step of identifying a subject (e.g., a patient) in need of treatment for an indication related to NLRP3 activity, such as an indication related to a point mutation in a gene involved in NLRP3 signaling.

Methods of Detecting the Level of NLRP3 Inflammasome Activity and/or Expression

In some embodiments of any of the methods described herein, the NLRP3 inflammmasome activity is the secretion of IL-18. In some embodiments of any of the methods described herein, the NLPR3 inflammasome activity is the secretion of I L- 1 b . In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity is caspase-1 activity in a mammalian cell (e.g., a mammalian cell obtained from the subject). Non limiting examples of methods that can be used to detect the secretion of IL-18 and I L-1 b include immunohistochemistry, immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, immunoprecipitation, and immunofluorescent assay. Non-limiting examples of commercially available assays for determining caspase-1 activity include Caspase 1 Assay Kit (Fluormetric) (ab39412) (Abeam), FAM-FLICA® Caspase-1 Assay Kit (ImmunoChemistry), Caspase-1 Colorimetric Assay Kit (K1 11) (Biovision, Inc.), and Caspase-1/ICE Colorimetric Assay Kit (R&D Systems).

In some embodiments of any of the methods described herein, the NLRP3 inflammasome activity can be the level of expression of an upstream activator of NLRP3 inflammasomes (e.g., the level of one or more (e.g., two, three, four, five, or six) of lipocalin-2 protein, lipocalin-2 mRNA, S100A8 protein, S1008A8 RNA, S100A9 protein, or S100A9 mRNA) in a mammalian cell (e.g., a mammalian cell obtained from a subject). Non-limiting assays that can be used to determine NLRP3 activity include: Southern blot analysis, Norther blot analysis, polymerase chain reaction (PCR)-based methods, e.g., next generation sequencing, reverse transcription polymerase chain reaction (RT-PCR), TaqMan™, microarray analysis, immunohistochemistry, immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, immunoprecipitation, immunofluorescent assay, mass spectrometry, immunoblot (Western blot), RIA, and flow cytometry. In some embodiments of any of the methods described herein, a mammalian cell have an increased level of NLRP3 activity can be identified by detecting the presence of one of more of the following the mammalian cell: a gain-of-function mutation in a NLRP3 gene (e.g., a NLRP3 protein having a Q705K amino acid substitution, a T350M amino acid substitution, a R262M amino acid substitution, a A441V amino acid substitution, a V200M amino acid substitution, an E629G amino acid substitution, a L355P amino acid substitution, a R260W amino acid substitution, a G571 R amino acid substitution, a A354V amino acid substitution, a D305N amino acid substitution, a F311 S amino acid substitution, a R920Q amino acid substitution, or a D21 H amino acid substitution, each numbered according to the mature NLRP3 protein sequence of SEQ ID NO: 1); a loss-of-function mutations in one or more of a CARD8 gene (e.g., a C allele at rs2043211); a T allele at rs3024505 flanking IL10 gene; detection of a R620W amino acid substitution in PTPN22; detection of a C allele at rs478582 in the PTPN2 gene; detection of a G allele at rs713875 in the MTMR3 gene; detection of an C allele at rs1042058 in the TPL2 gene; and detection of a ATG16L1 gene that encodes a ATG16L1 protein having a T300A amino acid substitution. Non-limiting examples of assays that can be used to determine the level of the presence of any of these mutations (e.g., any of the mutations described herein) include Southern blot analysis, Norther blot analysis, polymerase chain reaction (PCR)-based methods, e.g., next generation sequencing, reverse transcription polymerase chain reaction (RT-PCR), TaqMan™, and microarray analysis.

In some embodiments of any of the methods described herein, the NLRP3 inflammasome expression can be determined by detecting the level of one or more (e.g., two, three, four, five, six, or seven) of: NLRP3 protein, ASC protein, procaspase-1 protein, caspase-1 protein, NLRP3 mRNA, ASC mRNA, and procaspase-1 mRNA, in a mammalian cell (e.g., in a mammalian cell obtained from the subject). Non-limiting examples of assays that can be used to determine the level of NLRP3 protein, ASC protein, procaspase-1 protein, and caspase-1 protein include immunohistochemistry, immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, immunoprecipitation, immunofluorescent assay, and flow cytometry. Non limiting examples of assays that can be used to determine the level of NLRP3 mRNA, ASC mRNA, and procaspase-1 mRNA include Southern blot analysis, Norther blot analysis, polymerase chain reaction (PCR)-based methods, e.g., next generation sequencing, reverse transcription polymerase chain reaction (RT-PCR), TaqMan™, and microarray analysis.

In some embodiments of any of the methods described herein, the NLRP3 inflammasome expression can be determined by detecting the level of one or more of CRP protein, SAA protein, HP protein, ceruloplasmin protein, IL-6 protein (e.g., mature or pro-IL-6 protein), calprotectin (S100A8) protein, IL-8 protein (e.g., mature or pro-IL-8 protein), leukotriene B4 protein, myeloperoxidase protein, CRP mRNA, SAA mRNA, HP mRNA, ceruloplasmin mRNA, pro-IL-6 mRNA, calprotectin (S100A8) mRNA, pro-IL-8 mRNA, leukotriene B4 mRNA, and myeloperoxidase mRNA. Non-limiting examples of assays that can be used to determine the level of CRP protein, SAA protein, HP protein, ceruloplasmin protein, IL-6 protein (e.g., mature or pro-IL-6 protein), calprotectin (S100A8) protein, IL-8 protein (e.g., mature or pro-IL-8 protein), leukotriene B4 protein, myeloperoxidase protein include immunohistochemistry, immunoassays, e.g., enzyme-linked immunosorbent assay (ELISA), sandwich ELISA, immunoprecipitation, immunofluorescent assay, and flow cytometry. Non-limiting examples of assays that can be used to determine the level of CRP mRNA, SAA mRNA, HP mRNA, ceruloplasmin mRNA, pro-IL-6 mRNA, calprotectin (S100A8) mRNA, pro-IL-8 mRNA, leukotriene B4 mRNA, and myeloperoxidase mRNA include Southern blot analysis, Norther blot analysis, polymerase chain reaction (PCR)-based methods, e.g., next generation sequencing, reverse transcription polymerase chain reaction (RT-PCR), TaqMan™, and microarray analysis.

In some embodiments of any of the methods described herein, the level of the protein or mRNA can be detected in a biological sample including blood, serum, exosomes, plasma, tissue, urine, feces, sputum, and cerebrospinal fluid.

In some embodiments of any of the methods described herein, the level of at least one (e.g., 2, 3, 4, 5, 6, 7 or 8) NLRP3 inflammasome activity and/or expression can be determined, e.g., in any combination.

In one aspect, the cell can be a cell isolated from a subject who has been screened for the presence of an inflammatory disease or indication that is associated with a mutation in a NLRP3 activity.

Reference Levels

In some embodiments of any of the methods described herein, the reference can be a corresponding level detected in a similar cell or sample obtained from a healthy subject (e.g., a subject that has not been diagnosed or identified as having an inflammatory disease or autoimmune disorder, or any disorder associated with aberrant NLRP3 inflammasome activity and/or expression) (e.g., a subject who is not suspected or is not at increased risk of developing an inflammatory disease or autoimmune disease, or any disorder associated with aberrant NLRP3 inflammasome activity and/or expression) (e.g., a subject that does not present with any symptom of an inflammatory disease or autoimmune disease, or any disorder associated with aberrant NLRP3 inflammasome activity and/or expression).

In some embodiments, a reference level can be a percentile value (e.g., mean value, 99% percentile, 95% percentile, 90% percentile, 85% percentile, 80% percentile, 75% percentile, 70% percentile, 65% percentile, 60% percentile, 55% percentile, or 50% percentile) of the corresponding levels detected in similar samples in a population of healthy subjects (e.g., a population of subjects that have not been diagnosed or identified as having an inflammatory disease or autoimmune disorder, or any disorder associated with aberrant NLRP3 inflammasome activity and/or expression) (e.g., a population of subjects who are not suspected or are not at increased risk of developing an inflammatory disease or autoimmune disease, or any disorder associated with aberrant NLRP3 inflammasome activity and/or expression) (e.g., a population of subjects that do not present with any symptom of an inflammatory disease or autoimmune disease, or any disorder associated with aberrant NLRP3 inflammasome activity and/or expression).

In some embodiments, a reference can be a corresponding level detected in a similar sample obtained from the subject at an earlier time point.

NLR Family Pyrin Domain Containing 3 (NLRP3) Antagonists

In any of the methods described herein, the NLRP3 antagonist can be any of the NLRP3 antagonists described herein (e.g., any of the compounds described in this section), or any NLRP3 antagonist that satisfies the gut-targeted or gut-restricted criteria defined above. In any of the methods described herein, the NLRP3 antagonist has an IC50 of between about 1 nM and about 10 mM for NLRP.

Preferably the gut-targeted NLRP3 antagonist is one of a sulfonimidamide, or a sulfonylurea; and wherein said antagonist has:

- a TPSA of at least 125 angstroms squared, and

- an MDCK Papp of < 2 x 10 ⁶ cm/sec.

Preferably, the gut-targeted NLRP3 antagonist is an NLRP3 antagonist disclosed in WO2019023147, PCT/US2019/060770, W02016131098, WO2017140778, WO2018215818, WO2019166621 , WO2019068772, WO2019211463, WO2018225018; W02019/043610 (which are incorporated herein by reference in their entiret); and

wherein said antagonist has:

- a TPSA of at least 125 angstroms squared, and

- an MDCK Papp of < 2 x 10 ⁶ cm/sec. These compounds defined above will accordingly have a gut-targeted distribution profile, i.e. a colon:plasma NLRP3 antagonist concentrations of >10:1 , and indeed colomplasma ratios of >100:1 for these compounds are expected.

In one embodiment, the gut-targeted NLRP3 antagonist is a compound of Formula AA, or a pharmaceutically acceptable salt thereof,

Formula AA

wherein

m = 0, 1 , or 2;

n = 0, 1 , or 2;

o ⁼ 1 or 2;

p = 0, 1 , 2, or 3,

wherein

A is a 5- to 10-membered monocyclic or bicyclic heteroaryl or a C6-C10 monocyclic or bicyclic aryl;

B is a 5-membered heteroaryl, a 7-10 membered monocyclic or bicyclic heteroaryl, or a C6-C10 monocyclic or bicyclic aryl;

wherein

at least one R⁶ is ortho to the bond connecting the B ring to the NH(CO) group of Formula AA;

R¹ and R² are each independently selected from C1-C6 alkyl, C1-C6 haloalkyl, C1-C6 alkoxy, Cr C₆ haloalkoxy, halo, CN, NO2, COC1-C6 alkyl, CO-C6-C10 aryl; CO(5- to 10-membered heteroaryl); C0₂CrC₆ alkyl, C0₂C₃-C₈ cycloalkyl, OCOCrCe alkyl, OCOC₆-Cio aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), C6-C10 aryl, 5- to 10- membered heteroaryl, NH₂, NHC1-C6 alkyl, N(C_I-C₆ alkyl)₂, NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), NHCOC₂-C₆ alkynyl, NHCOOCCrCe alkyl, NH-(C=NR¹³)NR¹¹ R¹², CONR⁸R⁹, SF₅, SCrCe alkyl, S(0₂)CrC₆ alkyl, S(0)Ci-C₆ alkyl, S(0₂)NR¹¹ R¹², C3-C7 cycloalkyl and 3- to 7-membered heterocycloalkyl, wherein the C₁-C₆ alkyl, C C₆ haloalkyl, C₃-C₇ cycloalkyl and 3- to 7-membered

heterocycloalkyl is optionally substituted with one or more substituents each independently selected from hydroxy, halo, CN, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, NR⁸R⁹, =NR¹⁰, COOC₁-C₆ alkyl, CONR⁸R⁹, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, OCOC₁-C₆ alkyl, OCOC₆-C₁₀ aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC₁-C₆ alkyl, NHCOC₆-C₁₀ aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), and NHCOC₂-C₆ alkynyl;

wherein each C₁-C₆ alkyl substituent and each C₁-C₆ alkoxy substituent of the R¹ or R² C₃-C₇ cycloalkyl or of the R¹ or R² 3- to 7-membered heterocycloalkyl is further optionally independently substituted with one to three hydroxy, halo, NR⁸R⁹, or oxo;

wherein the 3- to 7-membered heterocycloalkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl) and NHCO(3- to 7- membered heterocycloalkyl) are optionally substituted with one or more substituents independently selected from halo, C₁-C₆ alkyl, and OC₁-C₆ alkyl;

or at least one pair of R¹ and R² on adjacent atoms, taken together with the atoms connecting them, independently form at least one C4-C8 carbocyclic ring or at least one 5- to 8-membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from O, N, and S, wherein the carbocyclic ring or heterocyclic ring is optionally independently substituted with one or more substituents independently selected from hydroxy, halo, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, NR⁸R⁹, =NR¹⁰, COOCrCe alkyl, C₆-Cio aryl, and CONR⁸R⁹ wherein the CrC₆ alkyl and CrC₆ alkoxy are optionally substituted with hydroxy, halo, oxo, NR⁸R⁹, =NR¹⁰, COOC1-C6 alkyl, C6-C10 aryl, and CONR⁸R⁹;

R⁶ and R⁷ are each independently selected from C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, Cr Ce haloalkoxy, halo, CN, NO₂, COC Ce alkyl, CO₂C₁-C₆ alkyl, CO₂C₃-C₈ cycloalkyl, OCOCrCe alkyl, OCOC₆-C₁₀ aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered

heterocycloalkyl), C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, NH₂, NHC₁-C₆ alkyl, N(C_I-C₆ alkyl)₂, CONR⁸R⁹, SF₅, S(0₂)Ci-C₆ alkyl, C₃-C₁₀ cycloalkyl and 3- to 10-membered

heterocycloalkyl, and a C₂-C₆ alkenyl,

wherein R⁶ and R⁷ are each optionally substituted with one or more substituents independently selected from hydroxy, halo, CN, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, NR⁸R⁹, =NR¹⁰, COOC₁-C₆ alkyl, CONR⁸R⁹, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, OCOC₁-C₆ alkyl, OCOC₆-C₁₀ aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC₁-C₆ alkyl, NHCOC₆-C₁₀ aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), NHCOC₂-C₆ alkynyl, C₆-C₁₀ aryloxy, and S(C>₂)Cr C₆ alkyl; and wherein the C₁-C₆ alkyl or C₁-C₆ alkoxy that R⁶ or R⁷ is substituted with is optionally substituted with one or more hydroxyl, C₆-C₁₀ aryl or NR⁸R⁹, or wherein R⁶ or R⁷ is optionally fused to a five- to -seven-membered carbocyclic ring or heterocyclic ring containing one or two heteroatoms independently selected from oxygen, sulfur and nitrogen; wherein the 3- to 7-membered heterocycloalkyl, C6-C10 aryl, 5- to 10-membered heteroaryl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl) and NHCO(3- to 7- membered heterocycloalkyl) are optionally substituted with one or more substituents independently selected from halo, C1-C6 alkyl, and OC1-C6 alkyl;

or at least one pair of R⁶ and R⁷ on adjacent atoms, taken together with the atoms connecting them, independently form at least one C4-C8 carbocyclic ring or at least one 5- to 8-membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from O, N, and S, wherein the carbocyclic ring or heterocyclic ring is optionally independently substituted with one or more substituents independently selected from hydroxy, hydroxymethyl, halo, oxo, C1-C6 alkyl, CrC₆ alkoxy, NR⁸R⁹, CH₂NR⁸R⁹, =NR¹⁰, COOCrCe alkyl, C₆-Cio aryl, and CONR⁸R⁹; each of R⁴ and R⁵ is independently selected from hydrogen and C1-C6 alkyl;

R¹⁰ is CrCe alkyl;

each of R⁸ and R⁹ at each occurrence is independently selected from hydrogen, C1-C6 alkyl, (C=NR¹³)NR¹¹R¹², S(0₂)CrC₆ alkyl, S(0₂)NR¹¹ R¹², COR¹³, C0₂R¹³ and CONR¹¹R¹²; wherein the C1-C6 alkyl is optionally substituted with one or more hydroxy, halo, C1-C6 alkoxy, C6-C10 aryl, 5- to 10-membered heteroaryl, C3-C7 cycloalkyl or 3- to 7-membered heterocycloalkyl; or R⁸ and R⁹ taken together with the nitrogen they are attached to form a 3- to 7-membered ring optionally containing one or more heteroatoms in addition to the nitrogen they are attached to;

R¹³ is C1-C6 alkyl, C6-C10 aryl, or 5- to 10-membered heteroaryl;

each of R¹¹ and R¹² at each occurrence is independently selected from hydrogen and C1-C6 alkyl;

alkylene)

R³ is selected from hydrogen, cyano, hydroxy, CrC₆ alkoxy, CrC₆ alkyl,

wherein the C1-C2 alkylene group is optionally substituted by oxo; and

R¹⁴ is hydrogen, C1-C6 alkyl, 5- to 10-membered monocyclic or bicyclic heteroaryl or C6-C10 monocyclic or bicyclic aryl , wherein each C1-C6 alkyl, aryl or heteroaryl is optionally

independently substituted with 1 or 2 R⁶; and wherein the compound has:

- a TPSA of at least 125 angstroms squared, and

- an MDCK Papp of < 2 x 10 ⁶ cm/sec.

Synthesis of the compounds of formula AA are provided in WO2019023147, which is incorporated by reference in its entirety. In one embodiment, the gut-targeted NLRP3 antagonist is a compound of Formula AB, or a pharmaceutically acceptable salt thereof,

Formula AB

wherein

m = 0, 1 , or 2;

n = 0, 1 , or 2;

o ⁼ 1 or 2;

p = 0, 1 , 2, or 3; wherein the sum of o and p is from 1 to 4;

wherein

A is a 5- to 10-membered heteroaryl or a C6-C10 aryl;

B is a 6-membered heteroaromatic ring containing 1-3 N atoms, or an N-oxide thereof;

wherein at least one R⁶ is ortho to the bond connecting the B ring to the NHC(O) group of Formula

AB;

R¹ and R² are each independently selected from C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, halo, CN, N0₂, C0₂CrC₆ alkyl, C0₂C₃-C₈ cycloalkyl, OCOCrCe alkyl, OCOC₆-Cio aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, NR⁸R⁹, C(0)R¹³, CONR⁸R⁹, SF₅, SCrCe alkyl, S(0₂)CrC₆ alkyl, S(0₂)NR¹¹R¹², S(0)CI-C₆ alkyl, C₃-C₇ cycloalkyl and 3- to 7-membered heterocycloalkyl, wherein the C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkyl, C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, C₃-C₇ cycloalkyl, and 3- to 7-membered heterocycloalkyl are optionally substituted with one or more substituents each independently selected from hydroxy, halo, CN, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, R¹⁵, NR⁸R⁹, =NR¹⁰, COOC₁-C₆ alkyl, CONR⁸R⁹, 3- to 7-membered heterocycloalkyl, Ob- Cio aryl, 5- to 10-membered heteroaryl, OCOC₁-C₆ alkyl, OCOC₆-C₁₀ aryl, OCO(5- to 10- membered heteroaryl), and OCO(3- to 7-membered heterocycloalkyl);

wherein each C₁-C₆ alkyl substituent and each C₁-C₆ alkoxy substituent of the R¹ or R² C₃-C₇ cycloalkyl or of the R¹ or R² 3- to 7-membered heterocycloalkyl is further optionally independently substituted with one to three hydroxy, -0(Co-C₃ alkylene)C₆-Cio aryl, halo, NR⁸R⁹, or oxo;

wherein the 3- to 7-membered heterocycloalkyl, C6-C10 aryl, and 5- to 10-membered heteroaryl are each optionally substituted with one or more substituents independently selected from halo, CrCe alkyl, and OCrC₆ alkyl; or one pair of R¹ and R² on adjacent atoms, taken together with the atoms connecting them, independently form at least one monocyclic or bicyclic C₄-C₁₂ carbocyclic ring or at least one monocyclic or bicyclic 5- to-12-membered heterocyclic ring wherein:

a) when each of the adjacent atoms is a carbon atom, then the heterocyclic ring includes from 1- 3 heteroatoms and/or heteroatomic groups independently selected from O, NH, NR¹³, S, S(O), and S(0)2; and

b) when one or both of the adjacent atoms is/are a nitrogen atom(s), then the heterocyclic ring includes from 0-2 heteroatoms and/heteroatomic groups independently selected from O, NH, NR¹³, S, S(O), and S(0)₂ (in addition to the aforementioned nitrogen atom(s) attached to R¹ and/or R²), and

wherein the carbocyclic ring or heterocyclic ring is optionally independently substituted with one or more substituents each independently selected from hydroxy, halo, oxo, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy, OC₃-C₁₀ cycloalkyl, NR⁸R⁹, =NR¹⁰, CN, COOC₁-C₆ alkyl, OS(0₂)C₆-Cio aryl, S(0₂)C₆-Cio aryl, C₆-Ci₀ aryl, 5- to 10-membered heteroaryl, C₃-C₁₀ cycloalkyl, 3- to 10-membered heterocycloalkyl, and CONR⁸R⁹,

wherein the C₁-C₆ alkyl, C₁-C₆ alkoxy, S(C>₂)C₆-Cio aryl, C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, C₃-C₁₀ cycloalkyl, and 3- to 10-membered heterocycloalkyl are optionally substituted with one or more substituents each independently selected from hydroxy, halo, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₁₀ cycloalkyl, C₁-C₆ alkoxy, oxo, NR⁸R⁹, =NR¹⁰, COOC₁-C₆ alkyl, Ob- Cio aryl, and CONR⁸R⁹;

R⁶ and R⁷ are each independently selected from C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, C₁-C₆ haloalkoxy, halo, CN, NO₂, COCrCe alkyl, CO₂C₁-C₆ alkyl, CO₂C₃-C₈ cycloalkyl, OCOCrCe alkyl, OCOC₆-C₁₀ aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, NH₂, NHC₁-C₆ alkyl, N(CI-C₆ alkyl)₂, CONR⁸R⁹, SF₅, SC₁-C₆ alkyl, S(0₂)C C₆ alkyl, C₃-C₁₀ cycloalkyl and 3- to 10-membered heterocycloalkyl, and C₂-C₆ alkenyl,

wherein R⁶ and R⁷ are each optionally substituted with one or more substituents independently selected from hydroxy, halo, CN, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, NR⁸R⁹, =NR¹⁰, COOC₁-C₆ alkyl, CONR⁸R⁹, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, OCOC₁-C₆ alkyl, OCOC₆-C₁₀ aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), C₆-C₁₀ aryloxy, and S(0₂)CrC₆ alkyl; and wherein the C₁-C₆ alkyl or C₁-C₆ alkoxy that R⁶ or R⁷ is substituted with is optionally substituted with one or more hydroxyl, C₆-C₁₀ aryl or NR⁸R⁹, or wherein R⁶ or R⁷ is optionally fused to a five- to -seven-membered carbocyclic ring or heterocyclic ring containing one or two heteroatoms independently selected from oxygen, sulfur and nitrogen;

wherein the 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, NHCOC₆-C₁₀ aryl, NHCO(5- to 10-membered heteroaryl) and NHCO(3- to 7-membered heterocycloalkyl) are optionally substituted with one or more substituents independently selected from halo, C₁-C₆ alkyl, and OC₁-C₆ alkyl;

or at least one pair of R⁶ and R⁷ on adjacent atoms, taken together with the atoms connecting them, independently form at least one C₄-C₈ carbocyclic ring or at least one 5- to 8-membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from O, NH, NR¹³, and S, wherein the carbocyclic ring or heterocyclic ring is optionally independently substituted with one or more substituents independently selected from hydroxy, hydroxymethyl, halo, oxo, C₁-C₆ alkyl, CrCe haloalkyl, Ci-C₆ alkoxy, NR⁸R⁹, CH₂NR⁸R⁹, =NR¹⁰, COOH, COOCrCe alkyl, C₆-Cio aryl, and CONR⁸R⁹;

R¹⁰ is CrCe alkyl;

each of R⁸ and R⁹ at each occurrence is independently selected from hydrogen, C₁-C₆ alkyl, C₂- C₆ alkenyl, C₃-C₇ cycloalkyl, Ci-C₆ haloalkyl, (C=NR¹³)NR¹¹ R¹², S(0₂)Ci-C₆ alkyl, S(0₂)NR¹¹ R¹², COR¹³, CO₂R¹³ and CONR¹¹R¹²; wherein the C₁-C₆ alkyl is optionally substituted with one or more hydroxy, halo, C₁-C₆ alkoxy, C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, C₃-C₇ cycloalkyl, 3- to 7- membered heterocycloalkyl, or NR¹¹ R¹²;

or R⁸ and R⁹ taken together with the nitrogen they are attached to form a 3- to 10-membered monocyclic or bicyclic ring optionally containing one or more heteroatoms in addition to the nitrogen they are attached to, wherein the ring is optionally substituted with one or more substituents independently selected from halo, C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, oxo, N(C C₆ alkyl)₂, NH₂, NH(CI-C₆ alkyl), and hydroxy;

R¹³ is CrCe alkyl, Ci-C₆ haloalkyl, or -(Z¹-Z²)_ai-Z³;

each of R¹¹ and R¹² at each occurrence is independently selected from hydrogen, C₁-C₆ alkyl, and -(Z¹-Z²)ai-Z³;

a1 is an integer selected from 0-10 (e.g., 0-5);

each Z¹ is independently C₁-C₆ alkylene optionally substituted with one or more substituents independently selected from oxo, halo, and hydroxy;

each Z² is independently a bond, NH, N(CI-C₆ alkyl), -0-, -S-, or 5-10 membered heteroarylene; Z³ is independently C₆-C₁₀ aryl, C₂-C₆ alkyenyl, C₂-C₆ alkynyl, C₃-C₁₀ cycloalkyl, 5- to 10- membered heteroaryl, or 3- to 10-membered heterocycloalkyl, each of which is optionally substituted with one or more substituents independently selected from halo, C₁-C₆ alkyl, Ci-e haloalkyl, C₁-C₆ alkoxy, oxo, N(CrC₆ alkyl)₂, NH₂, NH(CrC₆ alkyl), and hydroxy;

R³ is selected from hydrogen, cyano, hydroxy, CO₂C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ alkyl, and alkylene)

, wherein the C₁-C₂ alkylene group is optionally substituted with oxo;

R¹⁴ is hydrogen, C₁-C₆ alkyl, 5- to 10-membered monocyclic or bicyclic heteroaryl or C₆-C₁₀ monocyclic or bicyclic aryl, wherein each C₁-C₆ alkyl, aryl or heteroaryl is optionally independently substituted with 1 or 2 R⁶; R¹⁵ is -(Z⁴-Z⁵)a2-Z⁶;

a2 is an integer selected from 1-10 (e.g., 1-5 (e.g., 2-5));

each Z⁴ is independently selected from -0-, -S-, -NH-, and -N(C_I-C3 alkyl)-;

provided that the Z⁴ group directly attached to R¹ or R² is -O- or -S-;

each Z⁵ is independently C1-C6 alkylene optionally substituted with one or more substituents independently selected from oxo, halo, and hydroxy; and

Z⁶ is OH, OCrCe alkyl, NH₂, NH(CI-C₆ alkyl), N(CI-C₆ alkyl)₂, NHC(0)(Ci-C₆ alkyl), NHC(0)(Cr Ce alkoxy), or an optionally substituted group selected from the group consisting of:

C6-C10 aryl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C3-C10 cycloalkyl, 5- to 10-membered heteroaryl, or 3- to 10-membered heterocycloalkyl, each of which is optionally substituted with one or more substituents independently selected from halo, C1-C6 alkyl, Ci-₆ haloalkyl, C1-C6 alkoxy, oxo, N(Cr C₆ alkyl)₂, NH₂, NH(C_I-C₆ alkyl), and hydroxy;

and wherein the compound has:

- a TPSA of at least 125 angstroms squared, and

- an MDCK Papp of < 2 x 10⁶ cm/sec.

Syntheses of the compounds of formula AB are provided in PCT/US2019/060770, which is incorporated by reference in its entirety

In one embodiment, the gut-targeted NLRP3 antagonist is a compound of Formula AC, or a pharmaceutically acceptable salt thereof,

Formula AC wherein

m = 0, 1 , or 2;

n = 0, 1 , or 2;

o ⁼ 1 or 2;

p = 0, 1 , 2, or 3;

wherein

A is a 5-10-membered monocyclic or bicyclic heteroaryl or a C6-C10 monocyclic or bicyclic aryl; B is a 5-10-membered monocyclic or bicyclic heteroaryl or a C6-C10 monocyclic or bicyclic aryl; wherein

at least one R⁶ is ortho to the bond connecting the B ring to the C(R⁴R⁵) group of Formula AC;

R¹ and R² are each independently selected from C C₆ alkyl, C C₆ haloalkyl, C₁-C₆ alkoxy, Cr Ce haloalkoxy, halo, CN, NO₂, COCrCe alkyl, CO₂C₁-C₆ alkyl, CO-Ce-Cio aryl, CO-5- to 10- membered heteroaryl, CO₂C₃-C₈ cycloalkyl, OCOC₁-C₆ alkyl, OCOC₆-C₁₀ aryl, OCO(5- to 10- membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), C₆-C₁₀ aryl, 5- to 10- membered heteroaryl, NH2, NHC1-C6 alkyl, N(CI-C6 alkyl)2, NHCOC1-C6 alkyl, NHCOC6-C10 aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), NHCOC₂-C₆ alkynyl, NHCOOCCrCe alkyl, NH-(C=NR¹³)NR¹¹R¹², CONR⁸R⁹, SF₅, SCrCe alkyl, S(0₂)Ci-C₆ alkyl, S(0)Ci-C₆ alkyl, S(0₂)NR¹¹R¹², C₃-C₇ cycloalkyl and 3- to 7-membered heterocycloalkyl, wherein the C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₃-C₇ cycloalkyl and 3- to 7-membered

wherein the 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, NHCOC₆-C₁₀ aryl, NHCO(5- to 10-membered heteroaryl) and NHCO(3- to 7- membered heterocycloalkyl) are optionally substituted with one or more substituents independently selected from halo, C₁-C₆ alkyl, and OC₁-C₆ alkyl;

or at least one pair of R¹ and R² on adjacent atoms, taken together with the atoms connecting them, independently form at least one C₄-C₈ carbocyclic ring or at least one 5-to-8-membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from O, N, and S, wherein the carbocyclic ring or heterocyclic ring is optionally independently substituted with one or more substituents independently selected from hydroxy, halo, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, NR⁸R⁹, =NR¹⁰, COOC₁-C₆ alkyl, C₆-C₁₀ aryl, and CONR⁸R⁹· wherein the C₁-C₆ alkyl and C₁-C₆ alkoxy are optionally substituted with hydroxy, halo, oxo, NR⁸R⁹, =NR¹⁰, COOC₁-C₆ alkyl, C₆-C₁₀ aryl, and CONR⁸R⁹;

R⁶ and R⁷ are each independently selected from C₁-C₆ alkyl, C₁-C₆ haloalkyl, C₁-C₆ alkoxy, Cr C₆ haloalkoxy, halo, CN, N0₂, COCrCe alkyl, C0₂CrC₆ alkyl, C0₂C₃-C₈ cycloalkyl, OCOCrCe alkyl, OCOC₆-C₁₀ aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered

heterocycloalkyl), C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, NH₂, NHC₁-C₆ alkyl, N(C C₆ alkyl)₂, CONR⁸R⁹, SF₅, S(0₂)C C₆ alkyl, C₃-C₁₀ cycloalkyl and 3- to 10-membered

heterocycloalkyl, and a C₂-C₆ alkenyl, wherein R⁶ and R⁷ are each optionally substituted with one or more substituents independently selected from hydroxy, halo, CN, oxo, C₁-C₆ alkyl, C₁-C₆ alkoxy, NR⁸R⁹, =NR¹⁰, COOC₁-C₆ alkyl, CONR⁸R⁹, 3- to 7-membered heterocycloalkyl, C₆-C₁₀ aryl, 5- to 10-membered heteroaryl, OCOC₁-C₆ alkyl, OCOC₆-C₁₀ aryl, OCO(5- to 10-membered heteroaryl), OCO(3- to 7-membered heterocycloalkyl), NHCOC₁-C₆ alkyl, NHCOC₆-C₁₀ aryl, NHCO(5- to 10-membered heteroaryl), NHCO(3- to 7-membered heterocycloalkyl), NHCOC₂-C₆ alkynyl, C₆-C₁₀ aryloxy, and S(C>₂)Ci- C₆ alkyl; and wherein the C₁-C₆ alkyl or CrCe alkoxy that R⁶ or R⁷ is substituted with is optionally substituted with one or more hydroxyl, C₆-C₁₀ aryl, or NR⁸R⁹, or wherein R⁶ or R⁷ is optionally fused to a five- to -seven-membered carbocyclic ring or heterocyclic ring containing one or two heteroatoms independently selected from oxygen, sulfur and nitrogen;

or at least one pair of R⁶ and R⁷ on adjacent atoms, taken together with the atoms connecting them, independently form at least one C₄-C₈ carbocyclic ring or at least one 5-to-8-membered heterocyclic ring containing 1 or 2 heteroatoms independently selected from O, N, and S, wherein the carbocyclic ring or heterocyclic ring is optionally independently substituted with one or more substituents independently selected from hydroxy, hydroxymethyl, halo, oxo, C₁-C₆ alkyl, CrC₆ alkoxy, NR⁸R⁹, CH₂NR⁸R⁹, =NR¹⁰, COOCrCe alkyl, C₆-Cio aryl, and CONR⁸R⁹; each of R⁴ and R⁵ is independently selected from hydrogen and C₁-C₆ alkyl;

R¹⁰ is CrCe alkyl;

each of R⁸ and R⁹ at each occurrence is independently selected from hydrogen, C₁-C₆ alkyl, (C=NR¹³)NR¹¹R¹², S(0₂)Ci-C₆ alkyl, S(0₂)NR¹¹ R¹², COR¹³, CO₂R¹³ and CONR¹¹R¹²; wherein the C₁-C₆ alkyl is optionally substituted with one or more hydroxy, halo, C₁-C₆ alkoxy, C₆-C₁₀ aryl, 5- to 1 0-membered heteroaryl, C₃-C₇ cycloalkyl or 3- to 7-membered heterocycloalkyl; or R⁸ and R⁹ taken together with the nitrogen they are attached to form a 3- to 7-membered ring optionally containing one or more heteroatoms in addition to the nitrogen they are attached to;

R¹³ is C₁-C₆ alkyl, C₆-C₁₀ aryl, or 5- to 1 0-membered heteroaryl;

each of R^{1 1} and R¹² at each occurrence is independently selected from hydrogen and C₁-C₆ alkyl; alkylene)

R³ is selected from hydrogen, cyano, hydroxy, CrCe alkoxy, C₁-C₆ alkyl, and

wherein the C₁-C₂ alkylene group is optionally substituted by oxo; R¹⁴ is hydrogen, C1-C6 alkyl, 5-10-membered monocyclic or bicyclic heteroaryl or C6-C10 monocyclic or bicyclic aryl , wherein each C1-C6 alkyl, aryl or heteroaryl is optionally

independently substituted with 1 or 2 R⁶;

and wherein the compound has:

- a TPSA of at least 125 angstroms squared, and

- an MDCK Papp of < 2 x 10 ⁶ cm/sec.

Syntheses of the compounds of formula AC are provided in WO 2019/023145, which is incorporated by reference in its entirety.

Pharmaceutical Compositions

In some embodiments, a gut-targeted NLRP3 antagonist (e.g., any of the gut-targeted NLRP3 antagonists described herein or known in the art) is administered as a pharmaceutical composition that includes the chemical entity and one or more pharmaceutically acceptable excipients, and optionally one or more additional therapeutic agents as described herein.

In some embodiments, the pharmaceutical composition includes a gut-targeted NLRP3 antagonist and an anti-TNFa agent.

In some embodiments, the gut-targeted NLRP3 antagonist, optionally with a anti-TNFa agent, can 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-a-tocopherol polyethylene glycol 1000 succinate, surfactants used in pharmaceutical dosage forms such as Tweens, poloxamers or other similar polymeric 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, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, and wool fat. Cyclodextrins such as a-, b, and g-cyclodextrin, or chemically modified derivatives such as hydroxyalkylcyclodextrins, including 2- and 3-hydroxypropyl^-cyclodextrins, or other solubilized derivatives can also be used to enhance delivery of the NLRP3 antagonists described herein. Dosage forms or compositions containing an NLRP3 antagonist as described herein in the range of 0.005% to 100% with the balance made up from non-toxic excipient may be prepared. The contemplated compositions may contain 0.001 %-100% of an NLRP3 antagonist, in one embodiment 0.1-95%, in another embodiment 75-85%, in a further embodiment 20-80%. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 22^nd Edition (Pharmaceutical Press, London, UK. 2012).

Routes of Administration and Composition Components

In some embodiments, the gut-targeted NLRP3 antagonist (e.g., any of the exemplary NLRP3 antagonists described herein or known in the art) or a pharmaceutical composition thereof (optionally with a anti-TNFa agent) can be administered to subject in need thereof by any accepted route of administration. Acceptable routes of administration include, but are not limited to, buccal, cutaneous, endocervical, endosinusial, endotracheal, enteral, epidural, interstitial, intra-abdominal, intra-arterial, intrabronchial, intrabursal, intracerebral, intracisternal, intracoronary, intradermal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraovarian, intraperitoneal, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratesticular, intrathecal, intratubular, intratumoral, intrauterine, intravascular, intravenous, nasal, nasogastric, oral, parenteral, percutaneous, peridural, rectal, respiratory (inhalation), subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transtracheal, ureteral, urethral and vaginal. In certain embodiments, a preferred route of administration is parenteral (e.g., intratumoral).

Compositions can be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, or even intraperitoneal routes. Typically, such compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for use to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified. The preparation of such formulations will be known to those of skill in the art in light of the present disclosure.

The 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 it may be easily injected. It also should 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 also can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The 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 dispersion, and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds (i.e. the gut-targeted NLRP3 antagonist, and optionally the anti-TNFa agent) in the required amount in the appropriate solvent with various of the 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 the basic 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 injections are discussed, e.g., in Lammers, et al. , “Effect of Intratumoral Injection on the Biodistribution and the Therapeutic Potential of HPMA Copolymer-Based Drug Delivery Systems’’ Neoplasia. 2006, 10, 788-795.

In certain embodiments, the chemical entities described herein (i.e. the gut-targeted NLRP3 antagonist and optionally the anti-TNFa agent) or a pharmaceutical composition thereof are suitable for local, topical administration to the digestive or Gl tract, e.g., rectal administration. Rectal compositions include, without limitation, enemas, rectal gels, rectal foams, rectal aerosols, suppositories, jelly suppositories, and enemas (e.g., retention enemas).

Pharmacologically acceptable excipients usable in the rectal composition as a gel, cream, enema, or rectal suppository, include, without limitation, any one or more of cocoa butter glycerides, synthetic polymers such as polyvinylpyrrolidone, PEG (like PEG ointments), glycerine, glycerinated gelatin, hydrogenated vegetable oils, poloxamers, mixtures of polyethylene glycols of various molecular weights and fatty acid esters of polyethylene glycol Vaseline, anhydrous lanolin, shark liver oil, sodium saccharinate, menthol, sweet almond oil, sorbitol, sodium benzoate, anoxid SBN, vanilla essential oil, aerosol, parabens in phenoxyethanol, sodium methyl p- oxybenzoate, sodium propyl p-oxybenzoate, diethylamine, carbomers, carbopol, methyloxybenzoate, macrogol cetostearyl ether, cocoyl caprylocaprate, isopropyl alcohol, propylene glycol, liquid paraffin, xanthan gum, carboxy-metabisulfite, sodium edetate, sodium benzoate, potassium metabisulfite, grapefruit seed extract, methyl sulfonyl methane (MSM) , lactic acid, glycine, vitamins, such as vitamin A and E and potassium acetate.

In certain embodiments, suppositories can be prepared by mixing the chemical entities (i.e. the NLRP3 antagonist and optionally the anti-TNFa agent) 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, compositions for rectal administration are in the form of an enema. In other embodiments, the gut-targeted NLRP3 antagonist, and optionally the anti-TNFa agent described herein, or a pharmaceutical composition thereof, are suitable for local delivery to the digestive or Gl tract by way of 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 gut-targeted NLRP3 antagonist, and optionally the anti- TNFa agent, is mixed 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, polyvinylpyrrolidinone, sucrose, and acacia, c) humectants such as glycerol, d) disintegrating agents 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 glycerol monostearate, h) absorbents such as kaolin and bentonite clay, 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 form may also comprise buffering agents. Solid compositions of a similar type may also be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.

In one embodiment, the compositions will take the form of a unit dosage form such as a pill or tablet and thus the composition may contain, along with a gut-targeted NLRP3 antagonist, and optionally the anti-TNFa agent provided herein, a diluent such as lactose, sucrose, dicalcium phosphate, or the like; a lubricant such as magnesium stearate or the like; and a binder such as starch, gum acacia, polyvinylpyrrolidine, gelatin, cellulose, cellulose derivatives or the like. In another solid dosage form, a powder, marume, solution or suspension (e.g., in propylene carbonate, vegetable oils, PEG’S, poloxamer 124 or triglycerides) is encapsulated in a capsule (gelatin or cellulose base capsule). Unit dosage forms in which one or more chemical entites (i.e. NLRP3 antagonists, and optionally the anti-TNFa agents) or additional active agents are physically separated are also contemplated; e.g., capsules with granules (or tablets in a capsule) of each drug; two-layer tablets; two-compartment gel caps, etc. Enteric coated or delayed release oral dosage forms are also contemplated.

Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives that are 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 excipients are sterile and generally free of undesirable matter. These compositions can be sterilized by conventional, well-known sterilization techniques. For various oral dosage form excipients such as tablets and capsules sterility is not required. The USP/NF standard is usually sufficient. In certain embodiments, solid oral dosage forms can further include one or more components that chemically and/or structurally predispose the composition for delivery of the gut- targeted NLRP3 antagonist and optionally the anti-TNFa agent to the stomach or the lower Gl; e.g., the ascending colon and/or transverse colon and/or distal colon and/or small bowel. Exemplary formulation techniques are described in, e.g., Filipski, K.J., et al., Current Topics in Medicinal Chemistry, 2013, 13, 776-802, which is incorporated herein by reference in its entirety.

Examples include upper-GI targeting techniques, e.g., Accordion Pill (Intec Pharma), floating capsules, and materials capable of adhering to mucosal walls.

Other examples include lower-GI targeting techniques. For targeting various regions in the intestinal tract, several enteric/pH-responsive coatings and excipients are available. These materials are typically polymers that are designed to dissolve or erode at specific pH ranges, selected based upon the Gl region of desired drug release. These materials also function to protect acid labile drugs from gastric fluid or limit exposure in cases where the active ingredient may be irritating to the upper Gl (e.g., hydroxypropyl methylcellulose phthalate series, Coateric (polyvinyl acetate phthalate), cellulose acetate phthalate, hydroxypropyl methylcellulose acetate succinate, Eudragit series (methacrylic acid-methyl methacrylate copolymers), and Marcoat). Other techniques include dosage forms that respond to local flora in the Gl tract, Pressure- controlled colon delivery capsule, and Pulsincap.

Ocular compositions can include, without limitation, one or more of any of the following: viscogens (e.g., Carboxymethylcellulose, Glycerin, Polyvinylpyrrolidone, Polyethylene glycol); Stabilizers (e.g., Pluronic (triblock copolymers), Cyclodextrins); Preservatives (e.g., Benzalkonium chloride, ETDA, SofZia (boric acid, propylene glycol, sorbitol, and zinc chloride; Alcon Laboratories, Inc.), Purite (stabilized oxychloro complex; Allergan, Inc.)).

Topical compositions can include ointments and creams. Ointments are semisolid preparations that are typically based on petrolatum or other petroleum derivatives. Creams containing the selected active agent (i.e. gut-targeted NLRP3 antagonist, and optionally the anti- TNFa agent) are typically viscous liquid or semisolid emulsions, often either oil-in-water or water- in-oil. Cream bases are typically water-washable, and contain an oil phase, an emulsifier and an aqueous phase. The oil phase, also sometimes called the“internal” phase, is generally comprised of petrolatum and a fatty alcohol such as cetyl or stearyl alcohol; the aqueous phase usually, although not necessarily, exceeds the oil phase in volume, and generally contains a humectant. The emulsifier in a cream formulation is generally a nonionic, anionic, cationic or amphoteric surfactant. As with other carriers or vehicles, an ointment base should be inert, stable, nonirritating and non-sensitizing.

In any of the foregoing embodiments, pharmaceutical compositions described herein can include one or more one or more of the following: lipids, interbilayer crosslinked multilamellar vesicles, biodegradeable poly(D,L-lactic-co-glycolic acid) [PLGA]-based or poly anhydride-based nanoparticles or microparticles, and nanoporous particle-supported lipid bilayers. Preferably the above pharmaceutical composition embodiments comprise a gut-targeted NLRP3 antagonist and an anti-TNFa agent.

Dosages

The dosages may be varied depending on the requirement of the patient, the severity of the condition being treating and the particular compound being employed. Determination of the proper dosage for a particular situation can be determined by one skilled in the medical arts. The total daily dosage may be divided and administered in portions throughout the day or by means providing continuous delivery.

In some embodiments, the gut-targeted NLRP3 antagonist is administered at a dosage of from about 0.001 mg/Kg to about 500 mg/Kg

Kits

Also provided herein are kits containing one or more (e.g., at least 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 14, 15, 16, 18, or 20) of any of the pharmaceutical compositions described herein. In some embodiments, the kits can include instructions for performing any of the methods described herein. In some embodiments, the kits can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some embodiments, the kits can provide a syringe for administering any of the pharmaceutical compositions described herein. The kits described herein are not so limited; other variations will be apparent to one of ordinary skill in the art.

EXEMPLARY SYNTHESIS OF COMPOUNDS:

All compounds of the above“Compound Table” can were synthesised in accordance with the synthesis defined in the respective cited patent application. IFM-0004911 ( , -2-(1 ,2- Dihydroxypropan-2-yl)-N'-((3,3-dimethyl-1 ,2,3,5,6,7-hexahydrodicyclopenta[b,e]pyridin-8- yl)carbamoyl)thiazole-5-sulfonimidamide), for example, corresponds to Ex. 512 of PCT/US2019/060770, and was synthesised as per the description therein, i.e. by analogous synthesis provided herein:

Synthesis of IFM-0004911

IFM-0004991 was synthesised under analogous consitions to those for the synthesis of

Compound 159 of PCT/US2019/060770 as provided below, using appropriate starting material,:

Compound 159: N'-((1.2.3.5.6.7-hexahvdrodicvdopenta[b.e1pyridin-8-yl¾carbamoyl)-5-(2- hvdroxypropan-2-yl)-1-phenyl-1 H-pyrazole-3-sulfonimidamide (scheme 2)

Step 1 : /V-(ferf-butyldimethylsilyl)-/V -((1 ,2,3,5,6,7-hexahydrodicyclopenta[b,e]pyridin-8- yl)carbamoyl) -5-(2-hydroxypropan-2-yl)-1 -phenyl-1 H-pyrazole-3-sulfonimidamide

Into a 50-mL round-bottom flask purged and maintained with an inert atmosphere of nitrogen, was placed A/-(te/f-butyldimethylsilyl)-5-(2-hydroxypropan-2-yl)-1 -phenyl-1 H-pyrazole-3- sulfonoimidamide (160 mg, 0.41 mmol) in THF (10 ml_). To the stirred solution was added NaH (60% wt. oil dispersion, 48.7 mg, 1.22 mmol) at 0^°C. The resulting solution was stirred for 10 min at RT. Then 2,2,2-trichloroethyl (1 ,2,3,5,6,7-hexahydrodicyclopenta[b,e]pyridin-8-yl)carbamate (142 mg, 0.41 mmol) was added to the reaction solution. The resulting solution was allowed to react with stirring for an additional 2 h while the temperature was maintained at 40^°C in an oil bath. The reaction was then quenched by the addition of 10 mL of H2O. The resulting solution was extracted with 5x20 mL of EtOAc and the organic layers was combined and concentrated. The residue was purified using TLC with DCM/MeOH = 10: 1. This resulted in 230 mg (95.4%) of the title compound as a light yellow solid. MS-ESI: 595 (M+1).

Step 2: /V -((1,2,3,5,6,7-hexahydrodicyclopenta[b,e]pyridin-8-yl)carbamoyl)-5-(2- hydroxypropan-2-yl) -1 -phenyl-1 H-pyrazole-3-sulfonimidamide

Into a 50-mL round-bottom flask, was placed N-(tert-butyldimethylsilyl)-N'- ((1 , 2, 3, 5,6,7- hexahydrodicyclopenta[b, e]pyridin-8-yl)carbamoyl)-5-(2-hydroxypropan-2-yl)-1 -phenyl-1 H- pyrazole-3-sulfonimidamide (230 mg, 0.39 mmol) in THF (8 mL). To the above solution was added HF-Pyridine (0.1 mL) dropwise. The resulting solution was stirred for 30 min at RT. The resulting mixture was concentrated. The crude product was purified by Prep-HPLC with the following conditions: Column, XBridge Prep C18 OBD, 19*150 mm 5 urn; mobile phase, water (10 mM NH₄HCO₃+0.1 %NH₃-H₂O) and ACN (10% to 54% gradient over 6 min); Detector, UV, 210/254 nm. This resulted in 102 mg (53.8%) of Compound 159 as an off-white solid. MS-ESI: 481 (M+1). ¹H NMR (400 MHz, DMSO-d₆) d 8.74 (s, 1 H), 7.58 (s, 2H), 7.52 (s, 5H), 6.75 (s, 1 H), 5.43 (s, 1 H), 2.79 (t, J = 7.6 Hz, 4H), 2.71 (t, J = 7.5 Hz, 4H), 2.00-1.80 (m, 4H), 1.34 (s, 6H).

Synthesis of all other examples, including comparative examples are provided in their respective “synthesis disclosed” document specified in the Compound Table.

The following protocol is suitable for testing the activity of the compounds disclosed herein. Procedure 1 : IL-1 b production in PMA-differentiated THP-1 cells stimulated with Gramicidin.

THP-1 cells were purchased from the American Type Culture Collection and sub-cultured according to instructions from the supplier. Cells were cultured in complete RPM1 1640 (containing 10% heat inactivated FBS, penicillin (100 units/ml) and streptomycin (100 pg/ml)), and maintained in log phase prior to experimental setup. Prior to the experiment, compounds were dissolved in dimethyl sulfoxide (DMSO) to generate a 30mM stock. The compound stock was first pre-diluted in DMSO to 3, 0.34, 0.042 and 0.0083 mM intermediate concentrations and subsequently spotted using Echo550 liquid handler into an empty 384-well assay plate to achieve desired final concentration (e.g. 100, 33, 11 , 3.7, 1.2, 0.41 , 0.14, 0.046, 0.015, 0.0051 , 0.0017 mM). DMSO was backfilled in the plate to achieve a final DMSO assay concentration of 0.37%. The plate was then sealed and stored at room temperature until required.

THP-1 cells were treated with PMA (Phorbol 12-myristate 13-acetate) (20 ng/ml) for 16- 18 hours. On the day of the experiment the media was removed and adherent cells were detached with trypsin for 5 minutes. Cells were then harvested, washed with complete RPMI 1640, spun down, and resuspended in RPMI 1640 (containing 2% heat inactivated FBS, penicillin (100 units/ml) and streptomycin (100 pg/ml) . The cells were plated in the 384-well assay plate containing the spotted compounds at a density of 50,000 cells/well (final assay volume 50 mI). Cells were incubated with compounds for 1 hour and then stimulated with gramicidin (5mM) (Enzo) for 2 hours. Plates were then centrifuged at 340g for 5 min. Cell free supernatant (40mI_) was collected using a 96-channel PlateMaster (Gilson) and the production of IL-1 b was evaluated by HTRF (cisbio). The plates were incubated for 18 h at 4°C and read using the preset HTRF program (donor emission at 620 nm, acceptor emission at 668 nm) of the SpectraMax i3x spectrophotometer (Molecular Devices, software SoftMax 6). A vehicle only control and a dose titration of CRID3 (100 - 0.0017 mM) were run concurrently with each experiment. Data was normalized to vehicle-treated samples (equivalent to 0% inhibition) and CRID3 at 100 mM (equivalent to 100% inhibition). Compounds exhibited a concentration-dependent inhibition of IL- 1 b production in PMA-differentiated THP-1 cells.

Procedure 2: IL-1 b production in PMA-differentiated THP-1 cells stimulated with Gramicidin.

THP-1 cells were purchased from the American Type Culture Collection and sub-cultured according to instructions from the supplier. Prior to experiments, cells were cultured in complete RPMI 1640 (containing 10% heat inactivated FBS, penicillin (100 units/ml) and streptomycin (100 pg/ml)), and maintained in log phase prior to experimental setup. Prior to the experiment THP-1 were treated with PMA (Phorbol 12-myristate 13-acetate) (20 ng/ml) for 16-18 hours. Compounds were dissolved in dimethyl sulfoxide (DMSO) to generate a 30mM stock. On the day of the experiment the media was removed and adherent cells were detached with trypsin for 5 minutes. Cells were then harvested, washed with complete RPMI 1640, spun down, resuspended in RPMI 1640 (containing 2% heat inactivated FBS, penicillin (100 units/ml) and streptomycin (100 pg/ml) . The cells were plated in a 384-well plate at a density of 50,000 cells/well (final assay volume 50 pi). Compounds were first dissolved in assay medium to obtain a 5x top concentration of 500mM. 10 step dilutions (1 :3) were then undertaken in assay medium containing 1.67% DMSO. 5x compound solutions were added to the culture medium to achieve desired final concentration (e.g. 100, 33, 1 1 , 3.7, 1.2, 0.41 , 0.14, 0.046, 0.015, 0.0051 , 0.0017 mM). Final DMSO concentration was at 0.37%. Cells were incubated with compounds for 1 hour and then stimulated with gramicidin (5mM) (Enzo) for 2 hours. Plates were then centrifuged at 340g for 5 min. Cell free supernatant (40mI_) was collected using a 96-channel PlateMaster (Gilson) and the production of I L-1 b was evaluated by HTRF (cisbio). A vehicle only control and a dose titration of CRID3 (100 - 0.0017 mM) were run concurrently with each experiment. Data was normalized to vehicle-treated samples (equivalent to 0% inhibition) and CRID3 at 100 mM (equivalent to 100% inhibition). Compounds exhibited a concentration-dependent inhibition of IL-1 b production in PMA- differentiated THP-1 cells.

STUDY 1 :

The CARD8 gene is located within the inflammatory bowel disease (IBD) 6 linkage region on chromosome 19. CARD8 interacts with NLRP3, and Apoptosis-associated Speck-like protein to form a caspase-1 activating complex termed the NLRP3 inflammasome. The NLRP3 inflammasome mediates the production and secretion of interleukin-1 b, by processing pro-l L-1 b into mature secreted I L- 1 b . In addition to its role in the inflammasome, CARD8 is also a potent inhibitor of nuclear factor N F-KB. N F-KB activation is essential for the production of pro-l L-1 b. Since over-production of I L-1 b and dyregulation of N F-KB are hallmarks of Crohn’s disease, CARD8 is herein considered to be a risk gene for inflammatory bowel disease. A significant association of CARD8 with Crohn’s disease was detected in two British studies with a risk effect for the minor allele of the non-synonymous single-nucleotide polymorphism (SNP) of a C allele at rs204321 1. This SNP introduces a premature stop codon, resulting in the expression of a severely truncated protein. This variant CARD8 protein is unable to suppress NF-KB activity, leading to constitutive production of pro-l L-1 b, which is a substrate for the NLRP3 inflammasome. It is believed that a gain-of-function mutation in an NLRP3 gene (e.g., any of the gain-of-function mutations described herein, e.g., any of the gain-of-function mutations in an NLRP3 gene described herein) combined with a loss-of-function mutation in a CARD8 gene (e.g., a C allele at rs204321 1) results in the development of diseases related to increased NLRP3 inflammasome expression and/or activity. Patients having, e.g., a gain-of-function mutation in an NLRP3 gene and/or a loss-of-function mutation in a CARD8 gene are predicted to show improved therapeutic response to treatment with an NLRP3 antagonist. A study is designed to determine: whether NLRP3 antagonists inhibit inflammasome function and inflammatory activity in cells and biopsy specimens from patients with Crohn’s disease or ulcerative colitis; and whether the specific genetic variants identify patients with Crohn’s disease or ulcerative colitis who are most likely to respond to treatment with an NLRP3 antagonist.

The secondary objectives of this study are to: determine if an NLRP3 antagonist reduces inflammasome activity in Crohn’s disease and ulcerative biopsy samples (comparing Crohn’s disease and ulcerative colitis results with control patient results); determine if an NLRP3 antagonist reduced inflammatory cytokine RNA and protein expression in Crohn’s disease and ulcerative colitis samples; determine if baseline (no ex vivo treatment) RNA levels of NLRP3, ASC, and I L-1 b are greater in biopsy samples from patients with anti-TNFa agent resistance status; and stratify the results according to presence of specific genetic mutations in genes encoding ATG16L1 , NLRP3, and CARD8 (e.g., any of the mutations in the ATG16L1 gene, NLRP3 gene, and CARD8 gene described herein).

Methods

• Evaluation of baseline expression of NLRP3 RNA and quantify inhibition of inflammasome activity by an NLRP3 antagonist in biopsies of disease tissue from patients with Crohn’s disease and ulcerative colitis.

• Determine if NLRP3 antagonist treatment reduces the inflammatory response in biopsies of disease from patients with Crohn’s disease based on decreased expression of inflammatory gene RNA measured with Nanostring.

• Sequence patient DNA to detect specific genetic mutations in the ATG16L1 gene, NLRP3 gene, and CARD8 gene (e.g., any of the exemplary mutations in these genes described herein) and then stratify the results of functional assays according to the presence of these genetic mutations.

Experimental Design

• Human subjects and tissue:

Endoscopic or surgical biopsies from areas of disease in patients with Crohn’s disease and ulcerative colitis who are either anti-TNFa treatment naive or resistant to anti-TNFa treatment; additionally biopsies from control patients (surveillance colonoscopy or inflammation-free areas from patients with colorectal cancer) are studied.

• Ex vivo T reatment Model:

Organ or LPMC culture as determined appropriate

Endpoints to be measured: Before ex vivo treatment- NLRP3 RNA level

After ex vivo treatment- inflammasome activity (either processed I L- 1 b , processed caspase-1 , or secreted IL-I b); RNA for inflammatory cytokines (Nanostring); viable T cell number and/or T cell apoptosis.

• Data Analysis Plan:

^■ Determine if NLRP3 antagonist treatment decreases processed I L- 1 b , processed caspase-1 or secreted I L- 1 b , and inflammatory cytokine RNA levels.

^■ Stratify response data according to treatment status at biopsy and the presence of genetic mutations in the NLRP3 gene, CARD8 gene, and ATG16L1 gene (e.g., any of the exemplary genetic mutations of these genes described herein).

STUDY 2: Treatment of anti-TNFa resistant patients with NLRP3 antagonists

PLoS One 2009 Nov 24;4(11):e7984, describes that mucosal biopsies were obtained at endoscopy in actively inflamed mucosa from patients with Ulcerative Colitis, refractory to corticosteroids and/or immunosuppression, before and 4-6 weeks after their first infliximab (an anti-TNFa agent) infusion and in normal mucosa from control patients. The patients in this study were classified for response to infliximab based on endoscopic and histologic findings at 4-6 weeks after first infliximab treatment as responder or non-responder. Transcriptomic RNA expression levels of these biopsies were accessed by the inventors of the invention disclosed herein from GSE 16879, the publically available Gene Expression Omnibus (https://www.ncbi.nlm.njh.QOv/Qeo/Qeo2r/7acc-GSE16879). Expression levels of RNA encoding NLRP3 and IL-1 b were determined using GE02R (a tool available on the same website), based on probe sets 207075_at and 205067_at, respectively. It was surprisingly found that in Crohn’s disease patients that are non-responsive to the infliximab (an anti-TNFa agent) have higher expression of NLRP3 and I L-1 b RNA than responsive patients (figures 1 and 2). Similar surprising results of higher expression of NLRP3 and I L-1 b RNA in UC patients that are non-responsive to infliximab (an anti-TNFa agent) compared to infliximab (an anti-TNFa agent) responsive patients (figures 3 and 4) were found.

Said higher levels of NLRP3 and I L-1 b RNA expression levels in anti-TNFa agent non responders, is hypothesised herein to lead to NLRP3 activation which in turns leads ot release of I L-1 b that induces IL-23 production, leading to said resistance to anti-TNFa agents. Therefore, treatment of Crohn’s and UC anti-TNFa non-responders or those genetically disposed to develop anti-TNFa resistance, with an NLRP3 antagonist would prevent this cascade, and thus prevent development of non-responsiveness to anti-TNFa agents. Indeed, resistance to anti-TNFa agents is common in other inflammatory or autoimmune diseases. Therefore, use of an NLRP3 antagonist for the treatment of inflammatory or autoimmune diseases will block the mechanism leading to non-responsiveness to anti-TNFa agents. Consequently, use of NLRP3 antagonists will increase the sensitivity of patients with inflammatory or autoimmune diseases to anti-TNFa agents, resulting in a reduced dose of anti-TNFa agents for the treatment of these diseases. Therefore, a combination of an NLRP3 antagonist and an anti-TNFa agent can be used in the treatment of diseases wherein TNFa is overexpressed, such as inflammatory or autoimmune diseases, to avoid such non-responsive development of patients to anti-TNFa agents. Preferably, this combination threatment can be used in the treatment of IBD, for example Crohn’s disease and UC.

Further, use of NLRP3 antagonists offers an alternative to anti-TNFa agents for the treatment of diseases wherein TNFa is overexpressed. Therefore, NLRP3 antagonists offers an alternative to anti-TNFa agents inflammatory or autoimmune diseases, such as IBD (e.g. Crohn’s diesease and UC).

Systemtic anti-TNFa agents are also known to increase the risk of infection. Gut restricted (or gut targeted) NLRP3 antagonists, however, offers a gut targeted treatment (i.e. non-systemic treatment), preventing such infections. Therefore, treatment of TNFa gut diseases, such as IBD (i.e. Crohn’s diesase and UC), with gut restricted/targeted NLRP3 antagonists has the additional advantage of reducing the risk of infection compared to anti-TNFa agents.

Proposed Experiment:

Determine the expression of NLRP3 and caspase-1 in LPMCs and epithelial cells in patients with non-active disease, in patients with active disease, in patients with active disease resistant to corticosteroids, patients with active disease resistant to TNF-blocking agents. The expression of NLRP3 and caspase-1 in LPMCs and epithelial cells will be analyzed by RNAScope technology. The expression of active NLRP3 signature genes will be analyzed by Nanostring technology. A pilot analysis to determine feasibility will be performed with 5 samples from control, 5 samples from active CD lesions, and 5 samples from active UC lesions.

STUDY 3:

It is presented that NLRP3 antagonists reverse resistance to anti-TNF induced T cell depletion/apoptosis in biopsy samples from IBD patients whose disease is clinically considered resistant or unresponsive to anti-TNF therapy.

A study is designed to determine: whether NLRP3 antagonists inhibit inflammasome function and inflammatory activity in cells and biopsy specimens from patients with Crohn’s disease or ulcerative colitis; and whether an NLRP3 antagonist will synergize with anti-TNFa therapy in patients with Crohn’s disease or ulcerative colitis.

The secondary objectives of this study are to: determine if an NLRP3 antagonist reduces inflammasome activity in Crohn’s disease and ulcerative biopsy samples (comparing Crohn’s disease and ulcerative colitis results with control patient results); determine if an NLRP3 antagonist reduced inflammatory cytokine RNA and protein expression in Crohn’s disease and ulcerative colitis samples; determine if an NLRP3 antagonist in the absence of co-treatment with anti-TNFa antibody induces T cell depletion in Crohn’s disease and ulcerative colitis biopsy samples; and determine if baseline (no ex vivo treatment) RNA levels of NLRP3, ASC, and IL-1 b are greater in biopsy samples from patients with anti-TNFa agent resistance status.

Methods

• Determine if there is synergy between an NLRP3 antagonist and anti-TNF antibody with respect to effects on T cell depletion/apoptosis in biopsies of disease from patients with

Crohn’s disease and ulcerative colitis.

Experimental Design

• Human subjects and tissue:

• Ex vivo T reatment Model:

Organ or LPMC culture as determined appropriate

• Ex vivo T reatments:

NLRP3 antagonist (2 concentrations), negative control (vehicle), positive control (caspase-1 inhibitor) each in the presence or absence of anti-TNF antibody at a concentration appropriate to distinguish differences in the T cell apoptotic between biopsies from anti-TNF resistant and anti-TNF-sensitive Crohn’s disease patients. Each treatment condition is evaluated in a minimum in duplicate samples.

• Endpoints to be measured:

Before ex vivo treatment- NLRP3 RNA level After ex vivo treatment- inflammasome activity (either processed I L- 1 b , processed caspase-1 , or secreted I L- 1 b) ; RNA for inflammatory cytokines (Nanostring); viable T cell number and/or T cell apoptosis.

• Data Analysis Plan:

^■ Determine if NLRP3 antagonist co-treatment increases T cell apoptosis/deletion in response to anti-TNF.

^■ Determine if the level of NLRP3 RNA expression is greater in TNF-resistant Crohn’s disease and ulcerative colitis samples compared to anti-TNF treatment-naive samples.

Biological Assay - Nigericin-stimulated I L-1 b secretion assay in THP-1 cells

Monocytic THP-1 cells (ATCC: TIB-202) were maintained according to providers’ instructions in RPMI media (RPMI/Hepes +10% fetal bovine serum + Sodium Pyruvate + 0.05 mM Beta-mercaptoethanol (1000x stock) + Pen-Strep). Cells were differentiated in bulk with 0.5 mM phorbol 12-myristate 13-acetate (PMA; Sigma # P8139) for 3 hours, media was exchanged, and cells were plated at 50,000 cells per well in a 384-well flat-bottom cell culture plates (Greiner, #781986), and allowed to differentiate overnight. Compound in a 1 :3.16 serial dilution series in DMSO was added 1 : 100 to the cells and incubated for 1 hour. The NLRP3 inflammasome was activated with the addition of 15 pM (final concentration) Nigericin (Enzo Life Sciences, #BML- CA421-0005), and cells were incubated for 3 hours. 10 pL supernatant was removed, and I L-1 b levels were monitored using an HTRF assay (CisBio, #62IL1 PEC) according to manufacturers’ instructions. Viability and pyroptosis was monitored with the addition of PrestoBlue cell viability reagent (Life Technologies, #A13261) directly to the cell culture plate.

The tables and respective number referred to in each example below is specific to said example.

EXAMPLE 1 : PO f30 mo/kcri Dosing of IFM-4911 to Balb/c Mice

In Vivo Colon/Plasma Distribution Studies

Male Balb/c mice were used for the colon/plasma distribution study. The mice were maintained on standard laboratory diet and water ad libitum. On the study day, the mice were allowed access to food and water. Mice were administered a single 30 mg/kg PO dose (n=3 per time point) by gastric gavage. Dosing solutions of IFM-4911 was prepared in 0.5% CMC solution. Mice were euthanized in a CO2 chamber at 0.25, 1 , 2, 4, 8, 24, and 48 h post-dose. Whole blood was collected from jugular vein into Microtainer tubes containing EDTA-K2 and stored on ice until centrifuged for the preparation of plasma. Colon were collected, rinsed with PBS, and weighed. Solid contents were removed from the colon tissue samples by flushing with 2 ml_ of saline. The colon samples were further washed by placing the sample in 5 ml_ of physiological saline and shaking for 1 minute. The colon samples were then pat dried, spread on a glass slide, and the mucosa layer was gently scraped onto another slide, and then transferred to a weighed tube. Tissue samples were placed in weighed tubes immediately frozen on dry ice upon collection until analysis.

Dose Formulation:

LC/MS/MS System

HPLC Conditions Mobile Phase for plasma: Mobile Phase for others (colon):

100% Water 95% Water (0.1 % Formic

Solution A: Solution A:

(0.1% Formic Acid) Acid): 5% Acetonitrile 95% Acetonitrile

100% Acetonitrile

Solution B: Solution B: (0.1 %Formic acid): 5%

(0.1%Formic acid)

Water

Gradient

Flow rate: 0.6 mL/min Time (min) A (%)

0.01 100

0.30 100

1.70 0.00

2.10 0.00

2.1 1 100

2.50 100

Injection volume: 2 pL

Mouse Plasma Sample Preparation and Analysis

The desired serial concentrations of working solutions were achieved by diluting stock solution of analyte with 50% acetonitrile in water solution. 5 mI_ of working solutions (1 , 2, 4, 20, 100, 200, 1000, 2000, 4000ng/ml_) were added to 10 mI_ of the blank Balb/c Mouse plasma to achieve calibration standards of 0.5-2000 ng/mL (0.5, 1 , 2, 10, 50, 100, 500, 1000, 2000 ng/mL) in a total volume of 15 mI_. Five quality control samples at 1 ng/mL, 2 ng/mL, 50 ng/mL, 800 ng/mL and 1600 ng/mL for plasma were prepared independently of those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards.

15 pL of calibratoin standards, 15 pL of QC samples, and 15 pL of example samples (10 pL of plasma, and 5 pL of blank solution of 50% acetonitrile in water) were separately quenched with acetonitrile containing IS mixture to a total volume of 200 pL of for precipitating protein. The samples were then vortexed for 30 s. After centrifugation at 4 °C, 4000 rpm for 15 min, the supernatant was diluted 3 fold with water. 2 pl_ of diluted supernatant was injected into the LC/MS/MS system for quantitative analysis.

Results:

BLOQ = Below quantifiable limit of 0.5 ng/mL These results are presented as a graph in Figure 23.

Mouse Colon Tissue Sample Preparation and Analysis

Mouse colon tissue samples were diluted with water by colon weight (g) to water volume (ml_) ratio 1 :3 for homogenization. The homogenization process involved weighing the 2 ml_ tube, collecting the tissue in the weighted tube and recording the final total weight. Water was added to the sample by tissue weight in ratio of 3:1 and the samples homogenized using a tissue homogenizer until a homogenous mixture was formed. A similar process was adapted for the homogenization of all tissue samples. The desired serial concentrations of working solutions were achieved by diluting stock solution of analyte with 50% acetonitrile in water solution. 5 mI_ of working solutions (1 , 2, 4, 20, 100, 200, 1000, 2000, 4000 ng/mL) were added to 10 pL of the blank Balb/c Mouse Colon homogenate to achieve calibration standards of 0.5-2000 ng/mL (0.5, 1 , 2, 10, 50, 100, 500, 1000, 2000 ng/mL) in a total volume of 15 pL. Five quality control samples at 1 ng/mL, 2 ng/mL, 50 ng/mL, 800ng/mL and 1600 ng/mL for colon homogenate were prepared independently of those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards.

15 pL of each calibration standard, 15 pL of each QC sample, and 15 pL of each example sample (10 pL of colon homogenate, and 5 pL of blank solution of 50% acetonitrile in water) were separately quenched with acetonitrile containing the IS mixture to a total volume of 200 pL of for precipitating protein. The samples were then vortexed for 30 s. After centrifugation at 4 °C, 4000 rpm for 15 min, the supernatant was diluted 3 fold with water. 2 pL of diluted supernatant was injected into the LC/MS/MS system for quantitative analysis. Results:

All of the colon samples were added with water by colon weight (g) to water volume (mL) ratio 1 :3 for homogenating. The actual concentration (ng/g) is the detected value (ng/mL) multiplied by 4.

Colon: Plasma Ratio for IFM-0004911 :

BLOQ = Below quantifiable limit of 0.5 ng/mL

Analogous experiments were conducted for all other examples to provide the colomplasma ratio provided herein.

EXAMPLE 2: MDCK P_ann Measurement

Study Design

1. Preparation of Madin-Darby Canine Kidney cells (MDCK Cells)

1) 50 pL and 25 mL of cell culture medium were added to each well of the transwell insert and reservoir, respectively. The HTS transwell plates were then incubated at 37 °C, 5% CO2 for 1 hour before cell seeding.

2) MDCK cells (Netherlands Cancer Institute (NKI)) were diluted to 1.56x10^® cells/mL with culture medium and 50 pL of cell suspension were dispensed into the filter well of the 96- well HTS transwell plate. Cells were cultivated for 4-8 days in a cell culture incubator at 37 °C, 5% CO2, 95% relative humidity. Cell culture medium was replaced every other day, beginning no later than 24 hours after initial plating.

The cell culture medium composition is shown below:

Item Content (%) Supplier Cat. No.

DMEM 89 Hyclone SH30022.01 FBS 10 Corning 35-081 -CV

Penicillin (10000 u/mL),

Streptomycin (10000 pg/mL) 1 Solarbio P1400

mixture (100X)

NEAA Mixture (100X) 1 Lonza 13-114E

2. Preparation of Stock Solutions

10 mM stock solutions of test compounds and of positive controls were prepared in DMSO. Metoprolol, digoxin and imatinib were used as control compounds in this assay.

3. Assessment of Cell Monolayer Integrity

1) Medium was removed from the reservoir and each transwell insert and replaced with pre-warmed fresh culture medium.

2) Transepithelial electrical resistance (TEER) across the monolayer was measured using Millicell Epithelial Volt-Ohm measuring system (Millipore, USA).

3) The plate was returned to the incubator once the measurement was done.

The TEER value was calculated according to the following equation: TEER measurement (ohms) x Area of membrane (cm²) = TEER value (ohnvcm²) TEER value should be greater than 42 ohnvcm², which indicates the well-qualified MDCK monolayer.

4. Assay Procedures

1) The MDCK cell plate was removed from the incubator and washed twice with pre warmed HBSS (10 mM HEPES, pH 7.4), and then incubated at 37 °C for 30 minutes.

2) The stock solutions of test compounds were diluted in DMSO to obtain 0.2 mM solutions and then diluted with HBSS (10 mM HEPES, pH 7.4) to obtain 1 mM working solutions. The stock solutions of controls were diluted in DMSO to obtain 0.2 mM solutions and then diluted with HBSS (10 mM HEPES, pH 7.4) to get 1 mM working solutions. The final concentration of DMSO in the incubation system was 0.5%.

3) To determine the rate of drug transport in the apical to basolateral direction, 75 mI_ of 1 mM working solution of test compound was added to the transwell insert (apical compartment) and the wells in the receiver plate (basolateral compartment) were filled with 235 mI_ of HBSS (10 mM HEPES, pH 7.4). 75 mI_ of 1 mM working solution of control compound was added to the transwell insert (apical compartment) and the wells in the receiver plate (basolateral compartment) were filled with 235 mI_ of HBSS (10 mM HEPES, pH 7.4). The assay was performed in duplicate. 4) To determine the rate of drug transport in the basolateral to apical direction, 235 pl_ of 1 mM working solution of test compound was added to the receiver plate wells (basolateral compartment) and then the transwell inserts (apical compartment) were filled with 75 mI_ of HBSS (10 mM HEPES, pH 7.4). 235 mI_ of 1 mM working solution of control compound was added to the receiver plate wells (basolateral compartment) and the transwell inserts (apical compartment) were filled with 75 mI_ of HBSS (10 mM HEPES, pH 7.4). Time 0 samples were prepared by transferring 50 mI_ of 1 mM working solution to wells of the 96- deepwell plate, followed by the addition of 200 mI_ cold methanol containing appropriate internal standards (100 nM alprazolam, 200 nM labetalol, 200nM caffeine and 200 nM diclofenac).

5) The plates were incubated at 37 °C for 2 h.

6) At the end of the incubation, 50 mI_ samples from donor sides (apical compartment for Ap BI flux, and basolateral compartment for BI Ap) and receiver sides (basolateral compartment for Ap BI flux, and apical compartment for BI Ap) were transferred to wells of a new 96-well plate, followed by the addition of 4 volumes of cold methanol containing appropriate internal standards (100 nM alprazolam, 200 nM labetalol, 200nM caffeine and 200 nM diclofenac). Samples were vortexed for 5 minutes and then centrifuged at 3,220 g for 40 min. An aliquot of 100 mI_ of the supernatant was mixed with an appropriate volume of ultra-pure water before LC-MS/MS analysis.

7) To determine the Lucifer yellow dye leakage after 2 h transport period, stock solution of Lucifer yellow was prepared in water and diluted with HBSS (10 mM HEPES, pH 7.4) to reach the final concentration of 100 mM. 100 pL of the Lucifer yellow solution was added to each transwell insert (apical compartment), followed by filling the wells in the receiver plate (basolateral compartment) with 300 pL of HBSS (10 mM HEPES, pH 7.4). The plates were Incubated at 37 °C for 30 mins. 80 pL samples were removed directly from the apical and basolateral wells (using the basolateral access holes) and transferred to wells of new 96 wells plates. The Lucifer yellow fluorescence (to monitor monolayer integrity) signal was measured in a fluorescence plate reader at 485 nM excitation and 530 nM emission.

5. Data Analysis

The apparent permeability coefficient (P_app), in units of centimeter per second, can be calculated for MDCK drug transport assays using the following equation:

Papp = (VAX[d rug]acceptor)/(AreaxTimex[drug]initial, donor)

Where VA is the volume (in mL) in the acceptor well, Area is the surface area of the membrane (0.143 cm² for transwell-96 well permeable supports), and time is the total transport time in s. The criteria that was used to calibrate the permeability values and correlated to human oral bioavailability was adapted from that done using Caco-2 cell line and is shown

below:

- Low permeability: P_{a p} < 1 x 10⁶ cm/s

- Moderate permeability: 1 x 10⁶ cm/s £ P_app < 5 x 10⁶ cm/s

- High permeability: P_app ³ 5 x 10⁶ cm/s

The efflux ratio can be determined using the following equation:

Efflux Ratio=Papp(B-A)/^papp(A-B)

Where P_{app (B-A)} indicates the apparent permeability coefficient in basolateral to apical direction, and P_{app (A-B)} indicates the apparent permeability coefficient in apical to basolateral direction.

The recovery can be determined using the following equation:

Recovery %=(VAX[drug]acceptor+VDX[drug]donor)/(V_Dx[drug]initial, donor)

Where V_A is the volume (in mL) in the acceptor well (0.235 mL for Ap BI flux, and 0.075 mL for BI Ap), V_D is the volume (in mL) in the donor well (0.075 mL for Ap BI flux, and 0.235 mL for BI Ap)

The leakage of Lucifer yellow dye, in unit of percentage (%), can be calculated using the following equation:

%LY leakage ⁼ 1 00x[LY]acceptorX0.3/([LY]donorX0.1 +[LY]acceptorX0.3)

LY leakage of <1% is acceptable to indicate the well-qualified MDCK monolayer.

EXAMPLE 3: Efficacy of IFM-0004911 in Inhibiting IL-1 B and IL-18 Production in an Acute LPS Challenge Model in C57BL/6 Mice

Summary

In this study, C57BL/6 mice were dosed orally with either the gut- restricted NLRP3 antagonist IFM-0004911 (15, 50 or 150 mg/kg) or a systemic NLRP3 antagonist control compound IFM- 0000514 (100 mg/kg), beginning 1 hour prior to intra-peritoneal (i.p.) administration of lipopolysaccharide (LPS). In vehicle-treated mice, LPS induced significant increases in the serum levels of the NLRP3-signature cytokines I L-1 b and IL-18 within 5 hours. Treatment with IFM- 000514 inhibited the production of IL-1 b and IL-18 by 81% and 93% respectively. In contrast, treatment with IFM-0004911 at 15, 50 or 150 mpk did not significantly inhibit production of either I L-1 b or IL-18. The effects of IFM-0000514 and IFM-0004911 on IL-Ib and IL-18 correlated with end-of-study plasma level of these compounds since, as expected, IFM-0004911 did not achieve significant plasma concentrations to achieve systemic efficacy following oral dosing.

Background Intraperitoneal (i.p.) injection of LPS in mice induces an increase in circulating levels of proinflammatory cytokines such as I L- 1 b , IL-18, TNFa, and IL-6. Mice deficient in caspase-1 and dual caspase-1/ IL-1 R-deficient mice have increased resistance to endotoxin-mediated shock (Li, P., et al. Mice deficient in IL-1 beta-converting enzyme are defective in production of mature IL-1 beta and resistant to endotoxic shock. Cell. 80(1995) 401-411; Glaccum, M., et al. Phenotypic and functional characterization of mice that lack the type I receptor for IL-1. J. Immunol. 159 (1997) 3364-3371). Furthermore, mice deficient in NLRP3 or ASC are resistant to LPS and fail to produce circulating I L-1 b or IL-18 while still producing normal amounts of TNFa and IL-6 in response to LPS challenge ( Sutterwala , F., et al. Critical role for NALP3/CIAS1/Cryopyrin in innate and adaptive immunity through its regulation of caspase-1. Immunity. 24 (2006) 317-327; He, Y., et al., TLR agonists stimulate Nlrp3-dependent IL-Ib production independently of the purinergic P2X7 receptor in dendritic cells and in vivo. J Immunol. 190 (2013) 334-339). Thus, acute mouse LPS challenge represents a screening model to measure the ability of compounds to selectively inhibit NLRP3 in vivo.

In the current study, C57BL/6 mice were dosed orally beginning 1 hour prior to i.p. administration of LPS. The animals were sampled 5 hours after LPS challenge (6 hours after compound dosing) to measure serum cytokine levels and plasma drug concentration. The activity of IFM-0000514 and IFM-0004911 was evaluated by comparing the circulating levels of I L-1 b and IL-18 in drug- treated versus vehicle-treated control animals. Concomitant assessment of circulating IL-6 and TNFa gave a measure of specificity since, as described above, induction of these cytokines is largely independent of NLRP3.

Objective

The objective of this study was to evaluate the effect of IFM-0000514 and IFM-0004911 relative to vehicle, when dosed orally, on the LPS-induced release of NLRP3-dependent cytokines. Plasma levels of IFM-0000514 and IFM-000491 1 were measured and correlated with efficacy to establish a pharmacokinetic/pharmacodynamic (PK/PD) relationship.

Materials

C57BL/6 mice (female 18-20g) from Jackson Labs arrived and allowed to acclimate in the holding rooms for a minimum of at least 3 days before being put on study. All mice were maintained at animal facilities at IFM-Ann Arbor under a 12 hour light/dark cycle and had food and water ad libitum.

Study Design

A Schematic Summary of In Vivo Procedures is presented in Figure 5.

Key Study Parameters

Test Articles

Vehicle Control: 0.5% CMC in H₂0

Test Compounds: IFM-0004911 , IFM-0000514

Storage: Room temperature

Stimuli

Stimuli Manufacturer Catalog No.

Lipopolysaccharides (LPS)

Sigma L2630 From Escherichia coli 0111 : B4

Readouts

Readout Manufacturer Catalog No.

Mouse I L-1 b R&D Systems DY401

Mouse IL-6 R&D Systems DY406 Mouse TNFa R&D Systems DY410

D047-3 (capture)

Mouse IL-18 MBL International

D048-6 (detection)

Methods

Oral Dosing Formulation

The vehicle was prepared by adding 5 grams of CMC to 1 L of water, to a final solution of 0.5% CMC. IFM-0000514 and IFM-0004911 were weighed out and ground with mortar and pestle. A small amount of vehicle was added and ground again to further break up any large particles. Vehicle was continually added until the correct concentration was achieved. A combination of stirring, vortexing, and sonication was used after mortar and pestle, as necessary. Lower doses of compounds were made by diluting the high dose with vehicle. Compounds were prepared once at the beginning of the study, and vortexed/sonicated prior to each dosing to ensure the material was in fine suspension.

Induction of Peritonitis

On day -1 mice were weighed and assigned to groups in a balanced manner to achieve similar average weight across the groups at the start of the study. On Day 0, Hour -1 (1 hour before LPS injection) all mice were dosed once by oral gavage with vehicle, IFM-0000514 or IFM-0004911 , per the study design table below.

Study Design Table

At Hour 0 (one hour after dosing), all mice were injected intraperitoneally with 20 mg/kg LPS in 0.1 mL of PBS. One hour after LPS injection (two hours after compound dosing), blood was collected from the retro-orbital sinus of all mice, and at least 50 m\- of plasma was isolated for subsequent PK analysis at 7^th Wave laboratories. At Hour 5 (five hours after LPS injection), blood was collected from the retro-orbital sinus of all mice into Gel Clot Activator tubes. Serum was prepared and stored at -80°C in two aliquots for each mouse. One aliquot was used for cytokine analysis by ELISA, the other was used for PK analysis at 7^th Wave laboratories. Measurement of Serum Cytokines

Mouse IL-6, TNFa, IL-18, and IL-1 b in serum were measured by ELISA according to the manufacturer’s recommendations. A single analysis was performed on each sample. Concentrations were determined relative to a standard calibration curve, and total levels reported in pg/mL. The cytokine data in pg/mL were converted to % inhibition using the formula:

% inhibition = 100 - 100*(test/average vehicle)

Table 1A Serum Cytokine Levels Tables

S.E.M 49.79 31.38 32.66 39.33 44.52

Measurement of Plasma Drug Concentrations

Quantification of IFM-0004911 and IFM-0000514 in plasma and colon samples was performed at 7^th Wave Laboratories by HPLC using standard reverse-phase conditions to ensure separation from the solvent front and adequate peak shape (Study SW18-4645). Concentrations were determined relative to a standard calibration curve, and total levels reported in ng/mL (plasma) or ng/mg (colon). Result in Table below. Method

Table 2A Total Levels of IFM-0004911 and IFM-0000514 in Plasma and Colon at the End of the Study

Results

In vehicle-treated mice, LPS treatment induced significant increases in all serum cytokines tested. Treatment with a systemic control molecule, IFM-000514, inhibited the production of I L-1 b and IL-18 by 81 % and 93% respectively. In contrast, treatment with the gut-targeted molecule, IFM- 0004911 at 15, 50 or 150 mg/Kg, did not inhibit production of either IL-1 b or IL-18 (Table 1A, above, and Figure 6).

The effects of IFM-0000514 and IFM-000491 1 on I L-1 b and IL-18 correlated with end-of-study plasma level of these compounds. While IFM-0000514 achieved comparable exposure in gut and systemic compartments, IFM-0004911 demonstrated preferential exposure in the gut with little systemic exposure observed (Table 2A, and Figure 7). Figure 7 depicts results from left to right starting with IFM-00049911 15 mpk, 50 mpk, 150 mpk, and IFM-0000514 100 mpk.

Conclusions

This study confirmed that IFM-000491 1 achieved preferential gut exposure relative to IFM- 0000514, a systemic control compound. Consistent with its gut-restricted exposure, and in contrast to IFM-0000514, IFM-000491 1 did not significantly inhibit systemic levels of the NLRP3- signature cytokines IL-1 b and IL-18 induced by LPS. Thus IFM-000491 1 is gut-targeted across a range of doses and does not have systemic activity.

EXAMPLE 4: Efficacy of IFM-0004911 in Inhibiting Acute DSS-lnduced Colitis in C57BL/6

Mice with Analysis of Biomarkers

SUMMARY

C57BL/6 mice received drinking water containing 3.5% dextran sulfate sodium (DSS) for 5 days. During this period, mice were dosed orally twice a day (b.i.d.) with either vehicle, the gut- restricted NLRP3 antagonists IFM-000491 1 (15, 50 or 150 mg/kg) starting on the first day of DSS administration. Disease severity was monitored on-study by body weight and terminally evaluated by histological analysis. End of study drug concentrations in plasma and colon tissue were also measured.

In vehicle-treated mice, DSS induced body weight loss relative to naive controls. Treatment with IFM-000491 1 inhibited DSS-induced weight loss and increase in colon weight/length ratio.

Treatment with IFM-000491 1 also inhibited the DSS-induced histological measures of disease severity, including overall inflammatory score, mucosal inflammation, submucosal inflammation, mucosal ulceration and mucosal erosion. End of study serum and colon drug concentrations confirmed that IFM-000491 1 showed an expected gut targeted exposure profile in this study. DSS-induced release of mature, active, caspase 1 into the feces. This release of activated caspase 1 was blocked by treatment with the gut-targeted NLRP3 antagonist, IFM-000491 1. This data indicates that DSS-induced activation of NLRP3 is responsible for release of active caspase 1 in feces and suggests that measuring active caspase 1 in feces may provide a biomarker for inhibition of NLRP3 by gut-restricted NLRP3 antagonists such as IFM-0004911. DSS-induced an increase in processing of I L-1 b into the mature form in colon interstitial extract. Levels of mature I L- 1 b was blocked by treatment with the gut-targeted NLRP3 antagonist, IFM- 0004911 (15, 50 or 150 mg/kg). This data indicates that IFM-0004911 blocks DSS-induced activation of NLRP3-mediated processing of I L-1 b in colon tissue. Treatment with IFM-0004911 also blocked DSS-induced release of mature, active, caspase 1 into the feces. This data indicates that DSS-induced activation of NLRP3 is responsible for release of active caspase 1 in feces and suggests that measuring active caspase 1 in feces may provide a biomarker for inhibition of NLRP3 by gut-restricted NLRP3 antagonists such as IFM-0004911.

BACKGROUND

Administration of dextran sulphate sodium (DSS) to mice in their drinking water causes epithelial cell injury and results in the induction of an acute colonic inflammation characterized by mucosal erosions/ulcers, loss of crypts, and infiltration of granulocytes. Disease can be monitored in-life by weight loss, which typically peaks 2-5 days after colonic challenge.

Preclinical evidence for involvement of NLRP3 in the pathogenesis of DSS-induced colitis can be derived from studies in which NLRP3-knockout mice treated with DSS exhibited attenuated colitis and lower mortality (4, 5). Furthermore, inhibition of NLRP3 by a pharmacologic inhibitor (MCC950), or of IL-1 b signaling with an I L- 1 b R antagonist (anakinra), also attenuated colitis in a DSS-colitis model (6).

In the current study, C57BL/6 mice received drinking water containing 3.5% dextran sulfate sodium (DSS) for 5 days. During this period, they were dosed orally with IFM-000491 1 twice a day. Disease severity was monitored on-study by body weight and terminally evaluated by biomarker analysis. Disease severity was also monitored on-study by body weight and terminally evaluated by analysis of exploratory biomarker endpoints. End of study drug concentrations in plasma and colon tissue were also measured.

Objective

The purpose of this study was to evaluate the effect of IFM-0004911 , on exploratory tissue and feces biomarker endpoints for NLRP3 activation, in a DSS-driven model of IBD.

Materials

Mice Female C57BL/6 mice were purchased from Jackson Laboratories. All mice were maintained at animal facilities at IFM Ann Arbor under a 12hr light/dark cycle and had food and water ad libitum. The animals were acclimatized for 1 week prior to study initiation. The study was approved by the Animal Care and Use Committee.

Key Study Parameters

Test Articles

Vehicle Control: H₂0

Test Compound: IFM-0004911

Storage: Room temperature Stimuli and Other reagents

Tissue Sample Reagents

Additional Reagents and Materials

Methods

Compound Formulation and Dosing

In Vivo Procedures

After acclimatization, mice were weighed on Day 0 then placed in cages with ad libitum access to drinking water containing 3.5% dextran sulfate sodium (DSS) for 5 days. The mice were observed and weighed over this period to assess the induction of colitis. During this time, IFM- 0004911 (15, 50 & 150 mg/kg) was administered by oral gavage b.i.d. Compounds were given in a volume of 0.2 ml in 0.5% CMC at ~ 7 a.m. and 3 p.m. over this 12-day period. After 5 days of dosing, 6 hours after the last dose, mice were anesthetized by isoflurane inhalation and a terminal blood draw was taken by cardiac puncture. The mice were then sacrificed by cervical dislocation and the abdomen was opened and the colon length measured from the cecum to the anus and recorded. The tissue was then removed, and the weight of this segment recorded. Feces, along with homogenate prepared from ~1 cm piece of colon proximal the anus was harvested for WES system biomarker analysis.

Table 1B Group Designation and Dose Levels

A Schematic Summary of In Vivo Procedures is presented in Figure 8.

Biomarker Sample Preparation

Interstitial extract, colon homogenate, and feces homogenate samples were thawed on ice. 12 pL of each interstitial extract was mixed with 3 pL of 2x sample buffer. Homogenate samples were centrifuged at 3000 x g for 10 min (4°C) and 70 pL of supernatant was transferred to a Multiscreen 96-well format filter placed on top of a V-bottom 96-well plate, making sure not to disturb the pellet. The homogenate samples were then centrifuged on the filter plate at 3000 x g for 10 min (4°C). Protein content was quantified by BCA assay with 1 :10 dilution of the samples (2.5 pl_ sample in 22.5 mI_ lysis buffer). 8 mI_ lysate mix was prepared for each sample, containing 28.2 pg total protein. 2 mI_ of 5x Fluorescent Master Mix was then added prior to heating the samples for 5 min at 95°C. The samples were cooled, briefly vortexed and centrifuged.

Capillary Electrophoresis

4 pL from each sample were loaded into a ProteinSimple pre-fiiled microplate. Primary antibodies were diluted 1 :5Q with ProteinSimple antibody diluent 2. Primary antibody mix was then loaded into the microplate. Ail other reagents were prepared as per the manufacturer’s instructions (detection module). Following a centrifugation of the microplate at 1000 x g for 5 min, samples were loaded onto a ProteinSimple 12-230 kDa capillary cartridge.

Results and analysis

In vehicle-treated mice, DSS induced significant body weight loss relative to naive controls (Table 2B). Treatment with IFM-0004911 inhibited DSS-induced weight loss and increase in colon weight/length ratio (Tables 2B and 3B). DSS-induced release of mature, active, caspase 1 into the feces (Figure 9, 10). This release of activated caspase 1 was blocked by treatment with the gut-targeted NLRP3 antagonist, IFM-0004911. This data indicates that DSS-induced activation of NLRP3 is responsible for release of active caspase 1 in feces and suggests that measuring active caspase 1 in feces is expected to provide a biomarker for inhibition of NLRP3 by gut-restricted NLRP3 antagonists such as IFM-0004911.

In vehicle-treated mice, DSS-induced an increase in processing of I L-1 b into the mature form in colon interstitial extract (Figure 11). Levels of mature I L-1 b was blocked by treatment with the gut-targeted NLRP3 antagonist, IFM-0004911 (15, 50 or 150 g/kg). This data indicates that IFM-0004911 blocks DSS-induced activation of NLRP3-mediated processing of I L-1 b in colon tissue.

Table 2B Group Means for Animal Weight from Mouse DSS-induced Colitis Model

The below results are the averages for each treatment group.

Table 3B Group Means for Colon Length/Weight and Percent Weight Loss from

Mouse DSS-lnduced Colitis Model

The below results are the averages for each treatment group.

RESULTS:

The results of the three experiments are depicted in figures 9 to 11.

Figure 9. Effect of IFM-0004911 on the release of active caspase-1 in feces in a mouse DSS-induced colitis model. DSS induces NLRP3 activity, resulting in release of active caspase 1 in feces. Active caspase 1 detected and quantitated with anti-caspase 1 antibodies using capillary electrophoresis. Results are shown for each treatment group with mean +/- SEM. The 150 mg/kg IFM-0004911 treatment and Naive control groups were statistically different vs vehicle DSS control group (p < 0.05). Statistical significance was calculated in GraphPad Prism 8 using an unpaired t test. Figure 10. Effect of IFM-0004911 on the release of active caspase-1 in feces in a mouse DSS-induced colitis model. DSS induces NLRP3 activity, resulting in release of active caspase 1 in feces. Active caspase 1 detected with anti-caspase 1 antibodies using capillary electrophoresis. LPS + gramicidin-stimulated mouse bone marrow-derived macrophage extract was used as a positive control for active caspase 1 (p20).

Figure 11. Effect of IFM-0004911 on levels of processed (mature) IL-1 b in colon interstitial extract from a mouse DSS-induced colitis model. DSS induces NLRP3 activity, resulting in release of mature I L-1 b in colon interstitial extract. Mature I L-1 b detected and quantitated with anti-IL-1 b antibodies using capillary electrophoresis. Results are shown for each treatment group with mean +/- SEM. Data was plotted in GraphPad Prism 8.

Discussion and Conclusions

Gut- restricted NLRP3 antagonist IFM-0004911 inhibited release of mature IL-1 b in colon interstitial extract and fecal release of active caspase-1 in an acute mouse DSS model of colitis.

EXAMPLE 5: Efficacy of IFM-0004911 in Inhibiting Acute DSS-induced Colitis in C57BL/6

Mice

SUMMARY

C57BL/6 mice received drinking water containing 3.5% dextran sulfate sodium (DSS) for 9 days followed by normal tap water for 3 days. During this 12-day period, mice were dosed orally twice a day (b.i.d.) with either vehicle, the gut-restricted NLRP3 antagonists IFM-0004911 (15, 50 or 150 mg/kg), or the systemic control compound IFM-0000514 (100 mg/kg) starting on the first day of DSS administration. Disease severity was monitored on-study by body weight and terminally evaluated by histological analysis. End of study drug concentrations in plasma and colon tissue were also measured. In vehicle-treated mice, DSS induced significant body weight loss relative to naive controls. Treatment with IFM-0004911 inhibited DSS-induced weight loss and increase in colon weight/length ratio. Treatment with IFM-0004911 also inhibited the DSS- induced histological measures of disease severity, including overall inflammatory score, mucosal inflammation, submucosal inflammation, mucosal ulceration and mucosal erosion. End of study serum and colon drug concentrations confirmed that IFM-0004911 showed an expected gut targeted exposure profile in this study.

In the current study, C57BL/6 mice received drinking water containing 3.5% dextran sulfate sodium (DSS) for 9 days followed by normal tap water for 3 days. During this 12-day period, they were dosed with orally with either vehicle, IFM-0004911 , or IFM-0000514 twice a day. Disease severity was monitored on-study by body weight and terminally evaluated by histological analysis. End of study drug concentrations in plasma and colon tissue were also measured.

Objective The purpose of this study was to evaluate the efficacy of IFM-0004911 in an acute DSS-driven model of IBD.

Materials

Mice

Female C57BL/6 mice were purchased from Jackson Laboratories. All mice were maintained at animal facilities at IFM Ann Arbor under a 12-h light/dark cycle and had food and water ad libitum. The animals were acclimatized for 1 week prior to study initiation. The study was approved by the Animal Care and Use Committee.

Key Study Parameters

Test Articles

Vehicle Control: H₂0

Test Compounds: IFM-0004911 , IFM-0000514

Storage: Room temperature

Stimuli and Other reagents

Readouts

Methods

Compound Formulation and Dosing

The vehicle was prepared by adding 5 grams of CMC to 1 L of water, to a final solution of 0.5% CMC. IFM-0000514 and IFM-0004911 were weighed out and ground with mortar and pestle. A small amount of vehicle was added and ground again to further break up any large particles. Vehicle was continually added until the correct concentration was achieved. A combination of stirring, vortexing, and sonication was used after mortar and pestle, as necessary. Lower doses of compounds were made by diluting the high dose with vehicle. Compounds were prepared once at the beginning of the study, and vortexed/sonicated prior to each dosing to ensure the material was in fine suspension. In Vivo Procedures

After acclimatization, mice were weighed on Day 0 then placed in cages with ad libitum access to drinking water containing 3.5% dextran sulfate sodium (DSS) and normal feed for 9 days. After 9 days of DSS mice were returned to normal tap water for the remaining 3 days of the study. The mice were observed and weighed over this period to assess the induction of colitis. During this time, IFM-0004911 (15, 50 & 150 mg/kg), or IFM-0000514 (100 mg/kg) was administered by oral gavage b.i.d. Compounds were given in a volume of 0.2 ml in 0.5% CMC at ~ 7 a.m. and 3 p.m. over this 12-day period. After the 12 days of dosing, mice were anesthetized by isoflurane inhalation and a terminal blood draw was taken by cardiac puncture. The mice were then sacrificed by cervical dislocation and the abdomen was opened and the colon length measured from the cecum to the anus and recorded. The tissue was then removed, and the weight of this segment recorded. Feces, along with ~1 cm piece of colon proximal the anus was harvested and sent to Bonn for WES system biomarker analysis. Cytokine measurement by ELISA were performed on plasma samples aliquots were sent to 7th Wave labs for PK analysis.

Table 1C Group Designation and Dose Levels

A Schematic Summary of In Vivo Procedures is presented in Figure 12.

Histology Procedures

For histologic analysis, tissues were fixed in OCT, cut into sections, and stained with

Hematoxylin and Eosin (H&E). Blinded histology scoring for individual mice was performed on microscopic cross-sections of the colon and disease severity was graded from 0 to 4 (0, no signs of inflammation; 1 , very low level; and 2, low level of leukocytic infiltration; 3, high level of leukocytic infiltration, high vascular density, thickening of the colon wall; 4, transmural infiltrations, loss of goblet cells, high vascular density, thickening of the colon wall). Sections were also graded for evidence of inflammation in mucosal and submucosal regions as well as for evidence of damage, such as mucosal ulceration and erosion.

Measurement of Plasma and Colon Drug Concentrations

On day 12, 6 hours after compound dosing, blood was collected and purified serum was stored at -80 °C. Colon tissue was also collected at the same time point in pre-weighed 1.5 mL

Eppendorf tubes. The colon weight was measured, and samples were stored at -80 °C. Serum and colon sample quantification of IFM-0004911 was performed by HPLC using standard reverse-phase conditions to ensure separation from the solvent front and adequate peak shape. Concentrations were determined relative to a standard calibration curve and total levels reported in either ng/mL (plasma) or ng/mg (colon).

Peak Name: Tolbutamide

Use as Internal Standard

Q1/Q3 Masses: 271.00/155.00 Da

Peak Name: IFM-4911

Internal Standard: Tolbutamide

Q1/Q3 Masses: 466.20/203.10 Da

Fit Quadratic Weighting 1/x aO -0.0609

a1 0.267

a2 -0.0000815

Correlation coefficient 0.9995

Use Area

Peak Name: Tolbutamide

Use as Internal Standard

Q1/Q3 Masses: 271.00/155.00 Da

Peak Name: IFM-4911

Internal Standard: Tolbutamide

Q1/Q3 Masses: 466.20/203.10 Da

Fit Quadratic Weighting 1/x aO -0.000614

a1 0.265

a2 0.0000784

Correlation coefficient 0.9998

Use Area Results and analysis

In vehicle-treated mice, DSS induced significant body weight loss relative to naive controls. Treatment with IFM-0004911 , a gut-targeted NLRP3 antagonist, inhibited DSS-induced weight loss and increase in colon weight/length ratio (Table 2C, and Figures 13 and 14). Treatment with IFM-0004911 also inhibited the DSS-induced histological measures of disease severity, including overall inflammatory score, mucosal inflammation, submucosal inflammation, mucosal ulceration and mucosal erosion (Table 3C and Figures 14-17).

Serum and colon drug concentrations, measured at trough (6 hours post last compound dose on day 12) confirmed that IFM-0004911 showed a gut-targeted exposure profile relative to IFM- 000514 (figure 18). Thus, efficacy of IFM-0004911 correlated with its gut exposure in this model. Significantly, IFM-0004911 was active in this colitis model at doses that do not achieve sufficient peripheral exposure to demonstrate systemic activity.

Discussion and Conclusions

IFM-0004911 inhibited body weight loss and histological disease scores in an acute mouse DSS model of colitis. Specifically, IFM-0004911 improves:

i. body weight loss and colon weight to length

ii. overall histology scores at all doses

iii. both mucosal and sub-mucosal inflammation measures

iv. measures of mucosal damage with significant effects on mucosal erosions

IFM-0004911 also demonstrated gut-restricted exposure that correlated with efficacy in this model.

Tables and Figures

Table 2C Average Animal Weight and Colon Length/Weight from Mouse DSS- induced Colitis Model

The below results are the averages for each treatment group.

Table 3C Histologic Scoring of Colon Samples from Mouse Acute DSS-lnduced

Colitis Model

RESULTS DEPICTED IN GRAPHS:

Figure 13. Effect of IFM-0004911 on body weight loss in a mouse DSS-induced colitis model. Change in body weight, expressed as percent of starting weight. Results are shown as mean +/- SEM for each treatment group. The naive control group and all three IFM-0004911 treatment groups were statistically different vs vehicle DSS control group (p < 0.05). Statistical significance was calculated in GraphPad Prism 8 using an unpaired t test.

Figure 14. Effect of IFM-0004911 on increased colon weight/length ratio in a mouse DSS- induced colitis model. Change in colon weight/length ratio, expressed mg/cm. Results are shown for each treatment group with mean +/- SEM. The naive control group and all three IFM- 0004911 treatment groups were statistically different vs vehicle DSS control group (p < 0.05). Statistical significance was calculated in GraphPad Prism 8 using an unpaired t test.

Figure 15. Effect of IFM-0004911 on the histological disease score in a mouse DSS- induced colitis model. The histological disease score was calculated as described. Results are shown for each treatment group with mean +/- SEM. The naive control group and all three IFM-0004911 treatment groups were statistically different vs vehicle DSS control group (p < 0.05). Statistical significance was calculated in GraphPad Prism 8 using an unpaired t test. Figure 16. Effect of IFM-0004911 on the mucosal and submucosal inflammation score in a mouse DSS-induced colitis model. The inflammation score was calculated as described. Results are shown for each treatment group with mean +/- SEM. For mucosal inflammation score, the naive control group and all three IFM-0004911 treatment groups were statistically different vs vehicle DSS control group (p < 0.05). For submucosal inflammation score, the naive control group and the 50 mg/kg and 150 mg/kg IFM-0004911 treatment groups were statistically different vs vehicle DSS control group (p < 0.05). Statistical significance was calculated in GraphPad Prism 8 using an unpaired t test.

Figure 17. Effect of IFM-0004911 on mucosal ulceration and erosion scores in a mouse DSS-induced colitis model. The inflammation score was calculated as described. Results are shown for each treatment group with mean +/- SEM. For mucosal erosion, the naive control group and all three IFM-0004911 treatment groups were statistically different vs vehicle DSS control group (p < 0.05). Statistical significance was calculated in GraphPad Prism 8 using an unpaired t test.

Figure 18. Plasma and colon exposure of IFM-0004911 at end of study in a mouse DSS- induced colitis model. Data was plotted in GraphPad Prism 8.

Table: Trough PK Assessment of IFM-4911 in Acute DSS Model

Time of analysis is at 12 hours after last dose (at trough)

Analogous experiments to the above were also carried out for IFM-0003674, the results of which are provided below in examples 6 and 7.

EXAMPLE 6: Efficacy of IFM-0003764 in Inhibiting IL-1 B and IL-18 Production in an Acute

LPS Challenge Model in C57BL/6 Mice

Summary

In this study, C57BL/6 mice were dosed orally with the gut-restricted NLRP3 antagonist IFM- 0003764 (16.5, 50 or 150 g/kg) or a systemic NLRP3 antagonist control compound IFM- 0000514 (100 mg/kg), beginning 1 hour prior to intra-peritoneal (i.p.) administration of lipopolysaccharide (LPS). In vehicle treated mice, LPS induced significant increases in the serum levels of the NLRP3-signature cytokines I L-1 b and IL-18 within 5 hours. Treatment with IFM- 000514 inhibited the production of I L-1 b and IL-18 by 81.2% and 96.8% respectively. In contrast, treatment with IFM-0003764 at 15, 50 or 150 mpk did not inhibit production of either IL-1 b or IL- 18. The effects of IFM-0000514 and IFM-0003764 on I L-1 b and IL-18 correlated with end-of-study plasma level of these compounds since, as expected, IFM-0003764 did not achieve significant plasma concentrations to achieve systemic efficacy following oral dosing.

In the current study, C57BL/6 mice were dosed orally beginning 1 hour prior to i.p. administration of LPS. The animals were sampled 5 hours after LPS challenge (6 hours after compound dosing) to measure serum cytokine levels and plasma drug concentration. The activity of IFM-0000514 and IFM-0003764 was evaluated by comparing the circulating levels of I L-1 b and IL-18 in drug- treated versus vehicle-treated control animals. Concomitant assessment of circulating IL-6 gave a measure of specificity since, as described above, induction of this cytokine is largely independent of NLRP3.

A Schematic Summary of In Vivo Procedures is presented in Figure 19.

Test Articles

Vehicle Control: 0.5% CMC in H₂0

Test Compounds: IFM-0003764, IFM-0000514

Storage: Room temperature

Readouts

Results:

In vehicle treated mice, LPS treatment induced significant increases in all serum cytokines tested. Treatment with a systemic control molecule, IFM-000514, inhibited the production of I L-1 b and IL-18 by 81.2% and 96.8% respectively. In contrast, treatment with the gut-targeted molecule,

IFM-0003764 at 15, 50 or 150 mg/Kg, did not inhibit production of either I L-1 b or IL-18 (Table 2D and Figure 21). The effects of IFM-0000514 and IFM-0003764 on I L-1 b and IL-18 correlated with end-of-study plasma level of these compounds. While IFM-0000514 achieved comparable exposure in gut and systemic compartments, IFM-0003764 demonstrated preferential exposure in the gut with little systemic exposure observed (Figure 20). Figure 20 depicts results from left to right starting with IFM-0003764 16.5 mpk, 50 mpk, 150 mpk, and IFM-0000514 100 mpk.

Table 2D: (Plasma) Serum Cytokine Levels Tables

Conclusion:

This study confirmed that IFM-0003764 achieved preferential gut exposure relative to IFM- 0000514, a systemic control compound. Consistent with its gut-restricted exposure, and in contrast to IFM-0000514, IFM-0003764 did not significantly inhibit systemic levels of the NLRP3- signature cytokines IL-1 b and IL-18 induced by LPS. Thus IFM-0003764 is gut-targeted across a range of doses and does not have systemic activity.

EXAMPLE 7: Efficacy of IFM-0003764 in Inhibiting Acute DSS-lnduced Colitis in C57BL/6 Mice with Analysis of Colon Biomarkers

SUMMARY

C57BL/6 mice received drinking water containing 3.5% dextran sulfate sodium (DSS) and were dosed orally twice a day (b.i.d.) with either vehicle, the gut-restricted NLRP3 antagonist IFM- 0003764 (15, 60, or 150 mg/kg) or the systemic control compound IFM-0002384 (60 mg/kg). After 5 days, 5 animals from each group were sacrificed for colon biomarker analysis.

Biomarker Sample Preparation

Interstitial extract, colon homogenate, and feces homogenate samples were thawed on ice. 12 pL of each interstitial extract was mixed with 3 pL of 2x sample buffer. Homogenate samples were centrifuged at 3000 x g for 10 min (4°C) and 70 pL of supernatant was transferred to a Multiscreen 96-well format filter placed on top of a V-bottom 96-well plate, making sure not to disturb the pellet. The homogenate samples were then centrifuged on the filter plate at 3000 x g for 10 min (4°C). Protein content was quantified by BCA assay with 1 :10 dilution of the samples (2.5 pL sample in 22.5 pL lysis buffer). 8 pL lysate mix was prepared for each sample, containing 28.2 pg total protein. 2 pL of 5x Fluorescent Master Mix was then added prior to heating the samples for 5 min at 95°C. The samples were cooled, briefly vortexed and centrifuged.

Capillary Electrophoresis

4 pL from each sample were loaded Into a ProteinSimple pre-fiiled mieroplate. Primary antibodies were diluted 1 :50 with ProteinSimple antibody diluent 2. Primary antibody mix was then loaded into the microplate. Ail other reagents were prepared as per the manufacturer's instructions (detection module). Following a centrifugation of the mieroplate at 1000 x g for 5 min, samples were loaded onto a ProteinSimple 12-230 kDa capillary cartridge.

RESULTS AND ANALYSIS

DSS induced an increase in biomarkers of NLRP3 activation in the gut, including increased activation of caspase 1 , maturation of IL-1 b and cleavage of gasdermin D, relative to naive controls over the 5 days of the study (Figure 22). Both IFM-0003764- and IFM-0002384-treated mice showed reduction of activated caspase 1 , processed I L- 1 b , and cleaved Gasdermin D in colon tissue extract, demonstrating pathway inhibition by IFM-0003764 and IFM-0002384 in the gut. End of study (5 day) serum and colon drug concentrations confirmed that IFM-0003764 showed a gut targeted exposure profile in this study.

DISCUSSION AND CONCLUSIONS

IFM-0003764 demonstrated gut-restricted exposure that correlated with efficacy in this model.

Consequently, these experiments show that significant effects in colitis efficacy models (as supported by the DSS efficacy models) is obtained with the gut-targeted NLRP3 antagonists at doses that did not achieve sufficient systemic exposure to have systemic efficacy (as supported by the LPS PK-PD models). Therefore, these gut-targeted compounds provide efficacy without systemic drug exposure, which is expected to avoid potential unwanted systemic effects (for example reduce the risk of infection).

Additionally, gut-targeted NLRP3 antagonists can be used in combination with other drugs for IBD, for example colitis, where a systemic NLRP3 antagonist may be not be tolerated in a combination setting with systemic immunosuppressive therapy. As a specific example: TNFa blockade is commonly used to treat colitis. We have demonstrated, however, that NLRP3 activity leads to production of I L1 b, and NLRP3 is hypothesized to drive the disease. Therefore, gut-targeted NLRP3 antagonists can be used as a monotherapy or in combination with anti- TNFa agents, especially in TNFa blockade resistant colitis. Indeed, earlier combination trials of systemic TNFa blockade and systemic anti-l L1 treatment was not tolerated. However, systemic TNFa blockade and gut-targeted NLRP3 antagonists is expected to be tolerated owing to the absense of or extremely low systemic exposure of the NLRP3 antagonist, and thus provide benefit for patients with TNFa blockade resistant colitis. OTHER EMBODIMENTS

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Claims:

1. A gut-targeted NLRP3 antagonist for use in the treatment or the prevention of a condition mediated by TNF-a, in a subject in need thereof, wherein the NLRP3 antagonist is administered to said subject at a therapeutically effective amount.

2. The gut-targeted NLRP3 antagonist for use according to claim 1 , wherein said subject is resistant to treatment with an anti-TNFa agent.

3. The gut-targeted NLRP3 antagonist for use according to any preceeding claim, wherein the condition is a gut disease or disorder.

4. The gut-targeted NLRP3 antagonist for use according to any preceeding claim, wherein the condition is Inflammatory Bowel Disease.

5. The gut-targeted NLRP3 antagonist for use according to any preceeding claim, wherein the condition is Crohn’s Disease, or Ulcerative Colitis.

6. A pharmaceutical composition comprising a gut-targeted NLRP3 antagonist and at least one pharmaceutically acceptable excipient, for use according to any preceding claim.

7. A combination comprising a gut-targeted NLRP3 antagonist, or a pharmaceutically acceptable salt thereof, and at least one other therapeutically active agent, for use according to any one of claims 1 to 5.

8. The pharmaceutical composition of claim 6, or combination of claim 7, comprising an anti- TNFa agent.

9. The pharmaceutical composition or the combination of claim 8, wherein the anti-TNFa agent is Infliximab, Etanercept, Certolizumab pegol, Golimumab or Adalimumab.

10. The pharmaceutical composition or the combinatoin of claim 9, wherein the anti-TNFa agent is Adalimumab.