Compositions and methods for reducing immune intolerance and treating autoimmune disorders - Patents.com

Abstract

Provided herein are compounds and pharma- ceutically acceptable salts thereof, lipid particles comprising such compounds or pharma- ceutically acceptable salts thereof, and compositions as described above, which can be used to reduce immune intolerance in a subject, for example to treat an autoimmune disorder, or to improve the efficacy of antigen therapy, for example in combination with protein therapy or gene therapy.The compounds have the following structural formula (I): JPEG2024531259000041.jpg23165 where variables (e.g., rings A, L, R 1 , R 2 , R 3 , m) are as described herein.

Description

[Technical field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 233,039, filed August 13, 2021, the entire teachings of which are incorporated herein by reference. [Background technology]

[0002] Enzyme and protein replacement therapy is a successful therapeutic strategy for treating congenital disorders in which endogenous proteins are mutated, missing, or abnormal. However, clinical administration of exogenous enzymes or proteins is associated with the development of undesirable immune responses against the enzymes or proteins. Undesirable immune responses can lead to neutralization of the enzyme / protein or alteration of its pharmacokinetics. In many situations, patients have no alternative treatment options, and undesirable immune responses to treatment are a major challenge faced by recipients of enzyme and protein replacement therapy.

[0003] Similarly, gene therapy offers a promising approach to treat many congenital disorders and other diseases. The immunogenicity of the carrier and / or the genetic material carried therein is a major challenge to the clinical application of gene therapy. The presence of anti-carrier antibodies is a contraindication to some approved gene therapy treatments. Moreover, nascent anti-carrier antibodies may prevent repeated administration in subjects who receive the first dose of gene therapy.

[0004] Autoimmune disorders are a group of disorders in which the body lacks or loses tolerance to self-antigens. As a result, the body's immune system attacks healthy cells, which can have debilitating and devastating effects. Current approaches to treat autoimmune disorders rely on general immunosuppression at the humoral, cellular and / or complement levels, which renders patients immunocompromised and susceptible to opportunistic infections.

[0005] Thus, there is a need for compositions that can reduce immune intolerance to foreign antigens (e.g., enzyme replacement therapy, gene therapy) or endogenous antigens (e.g., self-antigens that cause autoimmune disorders), for example, by mitigating the immunogenicity of enzyme and protein replacement therapy and / or gene therapy, or by increasing self-tolerance to self-antigens. Summary of the Invention [Means for solving the problem]

[0006] The technology described herein relates to tolerance induction to foreign antigens (e.g., antigen-specific and / or antigen-exclusive tolerogenesis) or to self-antigens. The technology is based on engaging and regulating (e.g., activating) the T cell immunoglobulin mucin protein (TIM) family of receptors.

[0007] As used herein, compounds of the following structural formula: [ka] or a pharma- ceutically acceptable salt thereof, wherein the variables (e.g., rings A, L, R 1 , R 2 , R 3 , m) are as described herein.

[0008] Also provided herein are lipid particles comprising one or more lipids, or a pharma- ceutically acceptable salt thereof, and a compound of the present disclosure.

[0009] Also provided herein are compositions (eg, pharmaceutical compositions) comprising the compounds of the present disclosure.

[0010] Also provided herein is a composition (eg, a pharmaceutical composition) comprising a plurality of lipid particles described herein.

[0011] Also provided herein is a method of tolerizing a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition described herein.

[0012] Also provided herein is a method of tolerizing a subject in need thereof to an antigen and inhibiting or reducing antigen-specific antibody titers in a subject, comprising administering to the subject an antigen and a therapeutically effective amount of a composition described herein, or administering to the subject a composition described herein comprising the antigen or an immunogenic fragment of the antigen.

[0013] Also provided herein are methods of inducing a population of regulatory T cells in a subject (e.g., in response to an antigen) and increasing the activity or level of tolerogenic T cells in a subject, comprising administering to the subject a therapeutically effective amount of a composition described herein (e.g., a composition described herein that includes an antigen or an immunogenic fragment of an antigen).

[0014] Also provided herein is a method of inducing a population of regulatory B cells in a subject (e.g., in response to an antigen), comprising administering to the subject a therapeutically effective amount of a composition described herein (e.g., a composition described herein that includes an antigen or an immunogenic fragment of an antigen).

[0015] Also provided herein are methods for treating an autoimmune disorder in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a composition described herein (e.g., a composition described herein that includes an autoantigen associated with the autoimmune disorder).

[0016] Also provided herein are methods for treating a disease, disorder, or condition in a subject in need of treatment with antigen therapy, comprising administering to the subject antigen therapy (e.g., a therapeutically effective amount of antigen therapy) and a composition described herein in an amount sufficient to immunotolerize the subject to antigen therapy, or a therapeutically effective amount of a composition described herein that includes antigen therapy.

[0017] Also provided herein is a compound or composition (e.g., a pharmaceutical composition) of the present disclosure for a use described herein (e.g., treatment of an autoimmune disorder, treatment of a disease, disorder, or condition treatable with antigen therapy), wherein the composition is a composition described herein. Also provided herein is the use of a compound of the present disclosure or a composition described herein for the manufacture of a medicament for a use described herein (e.g., treatment of an autoimmune disorder, treatment of a disease, disorder, or condition treatable with antigen therapy).

[0018] The above will be apparent from the following more particular description of the exemplary embodiments. [Brief description of the drawings]

[0019] [Figure 1] 1 shows the change in the ratio of FoxP3+ / CD4+ T cells by the treatment described in Example 2.

[0020] [Figure 2A] FIG. 1 is a diagram of the study described in Example 3.

[0021] [Figure 2B] FIG. 1 shows the in vivo dose-response relationship of Compound 2 for FoxP3+ / CD4+ Tregs.

[0022] [Figure 2C] FIG. 1 shows the in vivo dose-response relationship of Compound 2 for FoxP3+ / NRP1+ / CD4+ Tregs.

[0023] [Figure 3A-1] FIG. 1 is a diagram of the study described in Example 4. [Figure 3A-2] FIG. 1 is a diagram of the study described in Example 4.

[0024] [Figure 3B] 1 shows the mean clinical scores of mice treated with the indicated doses of Compound 2 in the mouse EAE model described in Example 4.

[0025] [Figure 3C] 1 shows the mean clinical scores of mice treated with the full anti-α4 integrin regimen, 10 μg of Compound 2 administered orally once daily, or one or two doses of anti-α4 integrin followed by 10 μg of Compound 2 administered orally once daily in the murine EAE model described in Example 4.

[0026] [Figure 3D] 1 shows the mean clinical scores of mice treated with glatiramer acetate, anti-α4 integrin, or the indicated doses of Compound 2 in the mouse EAE model described in Example 4.

[0027] [Figure 4A] FIG. 1 is a graph of the percentage of IgM+CD71+ cells versus compound 2 concentration, showing that compound 2 enhances human regulatory B cells (CD19+IgM+CD71+).

[0028] [Figure 4B] FIG. 1 is a bar graph showing that Compound 2 acts directly on T cells to enhance Tregs (FoxP3+) in the presence and absence of other immune cells.

[0029] [Figure 4C] Graph of T cell percentage versus concentration of Compound 2 showing that Compound 2 maintains FoxP3 expression and inhibits RORγt expression in a Th17 polarizing environment in vitro.

[0030] [Figure 4D] 2 is a graph of the percentage of FoxP3+ / CD4+ T cells in multiple sclerosis patient samples before and after treatment with Compound 2, showing the changes in patient samples in response to Compound 2.

[0031] [Figure 5A] 1 shows the study design and randomization schedule of the study described in Example 8.

[0032] [Figure 5B] 1 shows the probability of disease control (score < 2.5) in Compound 2 or dimethyl fumarate (DMF) treated groups from the study described in Example 8.

[0033] [Figure 5C] The therapeutic effect of early and sustained use of Compound 2 versus DMF in the study described in Example 8 is shown.

[0034] [Figure 5D] The therapeutic efficacy of Compound 2 versus natalizumab in treatment escalation cohorts from the study described in Example 8 is shown.

[0035] [Figure 5E] 1 shows overall survival rates for various treatment groups from the study described in Example 8.

[0036] [Figure 6A] 1 shows the study design and randomization schedule of the study described in Example 9.

[0037] [Figure 6B] 1 shows the probability of disease control (score ≦2.5) in the treatment arm with induction therapy of the study described in Example 9.

[0038] [Figure 6C] 1 shows the clinical scores of treatment groups in the induction regimen of the study described in Example 9.

[0039] [Figure 6D] 1 shows survival rates of treatment groups at the end of the induction therapy period of the study described in Example 9.

[0040] [Figure 6E] The therapeutic effect of the indicated maintenance therapy following induction therapy of compound 2 from the study described in Example 9 is shown versus natalizumab.

[0041] [Figure 6F] 1 shows overall survival in the maintenance cohort of the study described in Example 9.

[0042] [Figure 7] The treatment group of the study described in Example 10 demonstrates the potential for reversion to minimal expression of the disease.

[0043] [Figure 8] 1 shows the mean clinical scores of mice treated with Compound 2 or Compound 8 in the mouse EAE model described in Example 12.

[0044] [Figure 9] 1 is a graph of the incidence of mouse cutaneous lupus over time, showing the onset of cutaneous lupus in untreated control and Compound 2-treated groups in the early-induced lupus model described in Example 13.

[0045] [Figure 10] 1 shows the percentage of body weight change over time in the untreated control group and the Compound 2-treated group in the dextran sulfate sodium (DSS)-induced IBD model described in Example 14. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Exemplary embodiments are described below.

[0047] definition

[0048] The compounds described herein include those described generally and further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of the present invention, chemical elements are defined as defined in the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75 thFurther, general principles of organic chemistry are identified in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5 th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the relevant contents of which are incorporated herein by reference.

[0049] Unless otherwise specified herein, the nomenclature used herein generally follows the examples and rules set forth in Nomenclature of Organic Chemistry, Sections A, B, C, D, E, F, and H, Pergamon Press, Oxford, 1979, the chemical structure names and chemical structure naming rules of which are incorporated herein by reference. Optionally, the names of compounds may be generated using a chemical naming program (e.g., CHEMDRAW®, version 17.0.0.206, PerkinElmer Informatics, Inc.).

[0050] When introducing elements disclosed herein, the articles "a," "an," "the," and "said" are intended to mean that there is one or more elements. Furthermore, the one or more elements may be the same or different.

[0051] "About" means within an acceptable error range of a particular value as determined by one of ordinary skill in the art. Usually, the acceptable error range of a particular value depends at least in part on how the value is measured or determined, e.g., on the limitations of the measurement system. For example, "about" may mean an acceptable standard deviation according to convention in the art. Alternatively, "about" may mean a range of ±20%, e.g., ±10%, ±5%, or ±1% of a given value. It should be understood that the term "about" may precede any particular value specified herein, except for the particular values ​​used in the examples.

[0052] "Alkyl" refers to a branched or straight-chain monovalent hydrocarbon radical having the specified number of carbon atoms. Thus, "(C1-C8)alkyl" refers to a radical having from 1 to 8 carbon atoms in a branched or straight-chain arrangement. In some embodiments, alkyl is any of the groups represented by (C1-C 30 ) alkyl, for example (C5-C 30 ) alkyl, (C1-C 25 )Alkyl, (C5-C 25 ) alkyl, (C1-C 15 ) alkyl, (C1-C 10 )alkyl, (C1-C6)alkyl, or (C1-C5)alkyl. Examples of alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, isopentyl, neopentyl, 2-methylpentyl, n-hexyl, and the like. In some embodiments, the alkyl is optionally substituted, for example, with one or more substituents described herein.

[0053] "Aryl" refers to a monocyclic or polycyclic (e.g., bicyclic, tricyclic) aromatic hydrocarbon ring system having the specified number of ring atoms. Thus, "(C6-C 15 "Aryl" refers to a ring system having 6 to 15 ring atoms. Examples of aryl include phenyl, naphthyl, and fluorenyl. In some embodiments, aryl is optionally substituted, for example, with one or more substituents described herein.

[0054] "Heteroaryl" refers to a monocyclic or polycyclic (e.g., bicyclic, tricyclic) aromatic hydrocarbon ring system having the specified number of ring atoms, in which at least one carbon atom in the ring system is replaced with a heteroatom selected from nitrogen, sulfur, and oxygen. Thus, "(C5-C 15"Heteroaryl" refers to a heteroaromatic ring system having 5 to 15 ring atoms consisting of carbon, nitrogen, sulfur, and oxygen. Heteroaryl can contain 1, 2, 3, or 4 (e.g., 1, 2, or 3) heteroatoms independently selected from nitrogen, sulfur, and oxygen. Typically, heteroaryl is a (C5-C 20 ) Heteroaryl, for example (C5-C 15 )heteroaryl, (C5-C 12 ) heteroaryl, C5 heteroaryl, or C6 heteroaryl. Monocyclic heteroaryls include, but are not limited to, furan, oxazole, thiophene, triazole, triazene, thiadiazole, oxadiazole, imidazole, isothiazole, isoxazole, pyrazole, pyridazine, pyridine, pyrazine, pyrimidine, pyrrole, tetrazole, and thiazole. Bicyclic heteroaryls include, but are not limited to, indolizine, indole, isoindole, indazole, benzimidazole, benzofuran, benzothiazole, purine, quinoline, isoquinoline, cinnoline, phthalazine, quinazoline, quinoxaline, naphthyridine, and pteridine. In some embodiments, heteroaryls are optionally substituted, for example, with one or more substituents described herein.

[0055] "Alkoxy" refers to an alkyl radical attached through an oxygen linking atom, where alkyl is as described herein. Examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, and the like.

[0056] "Halogen" and "halo" are used interchangeably herein and refer to fluorine, chlorine, bromine, or iodine, respectively. In some aspects, halo is fluoro, chloro, or bromo. In some aspects, halo is fluoro or chloro. In some aspects, halo is fluoro.

[0057] "Haloalkyl" includes mono-, poly-, and perhaloalkyl groups, where each halogen is independently selected from fluorine, chlorine, bromine, and iodine (e.g., fluorine, chlorine, and bromine), and alkyl is as described herein. In one aspect, the haloalkyl is a perhaloalkyl (e.g., perfluoroalkyl). Examples of haloalkyl include, but are not limited to, trifluoromethyl and pentafluoroethyl.

[0058] "Haloalkoxy" refers to a haloalkyl radical attached through an oxygen linking atom, where haloalkyl is as described herein. Examples of haloalkoxy include, but are not limited to, trifluoromethoxy.

[0059] The term "substituted" refers to the replacement of a hydrogen atom with a suitable substituent. Typically, a hydrogen atom bonded to a carbon atom is replaced with a suitable substituent, but a hydrogen bonded to a heteroatom, such as a nitrogen, oxygen, or sulfur atom, may also be replaced with a substituent. It will be understood that "substituted" or "substituted" includes the implicit condition that such substitution is in accordance with the allowed valence of the substituted atom. It is also preferred that the substituents and substitutions result in stable compounds that do not undergo spontaneous transformation, for example, by rearrangement, cyclization, elimination, and the like. Suitable substituents for use herein include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. For example, suitable substituents may include halogen, hydroxyl, carbonyl (such as carboxyl, alkoxycarbonyl, formyl, or acyl), thiocarbonyl (such as thioester, thioacetate, or thioformate), alkyl, alkoxy, alkylthio, acyloxy, phosphoryl, phosphate, phosphonate, amino, amido, amidine, imine, cyano, nitro, azido, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, cycloalkyl, heterocyclyl, aralkyl, aryl, or heteroaryl.Those skilled in the art will understand that the substituents themselves can be substituted where appropriate.Thus, the substituents may further include, for example, acetamide.

[0060] The permissible substituents can be one or more and the same or different for appropriate organic compounds. Thus, "optionally substituted" groups can in some embodiments, independently, be halo, (C-C)alkoxy, (C-C)haloalkoxy, (C-C)alkyl, or (C-C)haloalkyl, or optionally substituted (C-C 15 )Aryl or (C5-C 15) heteroaryl. In some embodiments, the optionally substituted aryl or heteroaryl is substituted with 0-5 (e.g., 0-3, 0, 1, 2, 3, 4, 5) substituents independently selected from halo, (C1-C6)alkoxy, (C1-C6)haloalkoxy, (C1-C6)alkyl, or (C1-C6)haloalkyl, (e.g., halo, (C1-C5)alkoxy, (C1-C5)haloalkoxy, (C1-C5)alkyl, or (C1-C5)haloalkyl). In some embodiments, an "optionally substituted" aryl or heteroaryl is substituted with 0-5 (e.g., 0-3, 0, 1, 2, 3, 4, 5) substituents independently selected from halo, (C1-C3)alkoxy, (C1-C3)haloalkoxy, (C1-C3)alkyl, or (C1-C3)haloalkyl. In some embodiments, an optionally substituted (e.g., substituted) alkyl is independently selected from halo (e.g., fluoro), (C1-C6)alkoxy, (C1-C6)haloalkoxy (e.g., (C1-C6)fluoroalkoxy), (C6-C6)haloalkoxy, ... 15 )Aryl or (C5-C 15 ) heteroaryl.

[0061] As used herein, or -(R 30 ) p (In the formula, R 30The term "optionally substituted", as indicated by a variable followed by a subscript number containing the value 0, such as , and p are as described herein, means that the substitution is optional, and thus the atom or moiety designated as "optionally substituted" may be unsubstituted or substituted. In some embodiments, an optionally substituted group is unsubstituted. When an optionally substituted group, as indicated herein by a variable followed by a subscript number containing the value 0, is unsubstituted, the subscript number following the variable is 0. In some embodiments, an optionally substituted group is substituted. When an optionally substituted group, as indicated herein by a variable followed by a subscript number containing the value 0, is substituted, the subscript number following the variable is other than 0. The term "substituted" means that the group specified herein is unsubstituted. Unless otherwise indicated, for example, as in the case of the terms "substituted" or "optionally substituted", the group specified herein is unsubstituted.

[0062] As used herein, the term "compounds of the disclosure" refers to any compound of the structural formulae set forth herein (e.g., compounds of structural formula I, exemplary compounds), as well as isomers such as stereoisomers (including diastereoisomers, enantiomers, and racemates) and tautomers thereof, isotopically labeled variants thereof (including those with deuterium substitutions), prodrugs (e.g., alkyl ester prodrugs), and inherently formed moieties thereof (e.g., polymorphs and / or solvates such as hydrates). Where moieties capable of forming salts are present, salts are likewise included, particularly pharma- ceutically acceptable salts thereof.

[0063] The compounds of the present disclosure may have asymmetric centers, chiral axes, and chiral planes (e.g., as described in E.L. Eliel and S.H. Wilen, Stereo-chemistry of Carbon Compounds, John Wiley & Sons, New York, 1994, pages 1119-1190), unless otherwise specified, and may exist as racemic mixtures, individual isomers (e.g., diastereomers, enantiomers, geometric isomers (including cis- and trans-double bond isomers), conformational isomers (including rotamers and atropisomers), tautomers) and mixtures of intermediates, along with all possible isomers and mixtures thereof.

[0064] When a disclosed compound is depicted by a structure without showing stereochemistry and the compound has one or more chiral centers, the structure should be understood to encompass one enantiomer or diastereomer of the compound separated or substantially separated from the corresponding optical isomer(s), racemic mixtures of the compound, and mixtures in which one enantiomer or diastereomer is enriched relative to its corresponding optical isomer(s). When a disclosed compound is depicted by a structure showing stereochemistry and the compound has one or more chiral centers, the stereochemistry refers to the absolute configuration of the substituents around the one or more chiral centers. "R" and "S" can also, or alternatively, be used to indicate the absolute configuration of the substituents around one or more chiral carbon atoms. D- and L- can also, or alternatively, be used to designate stereochemistry.

[0065] "Enantiomers" are a pair of stereoisomers that are non-superimposable mirror images of one another, most commonly because they contain an asymmetrically substituted carbon atom that acts as a chiral center.

[0066] "Diastereomers" are stereoisomers that are not mirror-image related, most commonly because they contain two or more asymmetrically substituted carbon atoms.

[0067] As used herein, a "racemate" or "racemic mixture" refers to a mixture containing equimolar amounts of two enantiomers of a compound. Such a mixture does not exhibit optical activity (i.e., does not rotate the plane of polarized light).

[0068] Enantiomeric excess (ee) is defined as the absolute difference between the mole fractions of each enantiomer multiplied by 100% and is represented by the following formula:

number

[0069] Diastereomeric excess (de) is defined as the absolute difference between the mole fractions of each diastereomer multiplied by 100% and is represented by the following formula:

number

[0070] Unless otherwise specified, compounds of the present disclosure include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, by replacing a hydrogen with deuterium or tritium, or a carbon with 13 C or 14 Compounds produced by substituting C-enriched carbons are within the scope of the invention. In all structures provided, any hydrogen atom may be independently substituted with a deuterium ( 2 H), tritium ( 3 H) and / or fluorine (18 F). Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents according to the present invention.

[0071] The phrase "pharmacologically acceptable" means that the substance or composition which it modifies is, within the normal scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, or the like, commensurate with a reasonable benefit / risk ratio.

[0072] As used herein, the term "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with mammalian tissue without excessive toxicity, irritation, allergic response, etc., within the normal scope of sound medical judgment, and commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge et al. describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, the relevant teachings of which are incorporated herein by reference in their entirety. Pharmaceutically acceptable salts of the compounds described herein include salts derived from suitable inorganic and organic acids and suitable inorganic and organic bases.

[0073] Examples of salts derived from suitable acids include salts of amino groups formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, and perchloric acid, or organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, or malonic acid, or by using other methods used in the art such as ion exchange. Other pharma- ceutically acceptable salts derived from suitable acids include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, cinnamate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, glutarate, glycolate, hemisulfate, heptanoate, hexanoate, hydroiodide, hydroxybenzoate, 2-hydroxyethanesulfonate, Examples of the salts include hydroxymaleate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 2-phenoxybenzoate, phenylacetate, 3-phenylpropionate, phosphate, pivalate, propionate, pyruvate, salicylate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, and valerate.

[0074] Mono-, di-, or tri-acid salts can be formed, and such salts can exist in either a hydrated, solvated or substantially anhydrous form.

[0075] Salts derived from appropriate bases include salts derived from inorganic bases such as alkali metal bases, alkaline earth metal bases, and ammonium bases; salts derived from aliphatic, alicyclic, and aromatic organic amines such as methylamine, trimethylamine, picoline, and the like; and salts derived from N +Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium, and the like. Further pharma- ceutically acceptable salts include non-toxic ammonium, quaternary ammonium, and amine cations, formed where appropriate using counterions such as halides, hydroxides, carboxyls, sulfates, phosphates, nitrates, lower alkylsulfonates, and arylsulfonates.

[0076] The compounds described herein may be provided in the form of a prodrug, e.g., an ester prodrug. As used herein, the term "prodrug" refers to a compound that can be hydrolyzed, oxidized, metabolized, and / or reacted under biological conditions (e.g., in vivo) to provide a compound of structural formula I or a salt thereof (e.g., a pharma- ceutically acceptable salt thereof). A prodrug may become active upon reaction under biological conditions (and, e.g., is biologically inactive), or may have activity in its unreacted form. A prodrug may have reduced metabolism under physiological conditions (e.g., due to the presence of a hydrolyzable group), resulting in an increased circulating half-life (e.g., in blood) of the prodrug. Thus, in some embodiments, the prodrug includes a hydrolyzable group, as in the case of an ester prodrug, e.g., an alkyl ester prodrug. Prodrugs are generally described in Burger's Medicinal Chemistry and Drug Discovery (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5 th They can be prepared using well-known methods, such as those described in (E. G., et al., ed.).

[0077] As used herein, the term "hydrolyzable group" refers to a moiety that, when present in a prodrug, hydrolyzes to yield a carboxylic acid or its salt. Hydrolysis can occur spontaneously, for example, under acidic or basic conditions in a physiological environment (e.g., blood, metabolically active tissues such as liver, kidney, lung, brain), or can be catalyzed by an enzyme(s) (e.g., esterases, peptidases, hydrolases, oxidases, dehydrogenases, lyases, or ligases). Hydrolyzable groups can confer advantageous properties to the prodrug in vivo, such as improved water solubility, improved blood circulation half-life, improved uptake, improved duration of action, or improved onset of action.

[0078] Examples of hydrolyzable groups include (C-C 10 )Alkyl, (C2-C 10 ) alkenyl, (C2-C 10 ) alkynyl, (C1-C 10 )Alkoxy(C1-C 10 ) alkyl, or (C1-C 10 )Alkoxy(C1-C 10 )Alkoxy(C1-C 10 For example, the hydrolyzable group can be (C1-C 10 ) alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, hexyl or heptyl, allyl, ethoxymethyl, methoxyethyl, methoxyethoxymethyl, or methoxyethoxyethyl. When a compound of structural formula I is provided in the form of an ester prodrug, the hydrogen of the carboxylic acid of the compound of structural formula I may be optionally substituted with a hydrolyzable group, such as a hydrolyzable group as described herein (e.g., one or more independently selected halo (e.g., fluoro) (C1-C 10 ) alkyl).

[0079] The compounds described herein may exist as "solvates" or "hydrates". A "hydrate" is a compound that exists in a composition that contains one or more water molecules. A hydrate may contain stoichiometric amounts of water, such as monohydrates or dihydrates, or may contain random amounts of water. A "solvate" is similar to a hydrate, except that water is replaced with a solvent other than water, such as methanol, ethanol, dimethylformamide, diethyl ether, etc. Mixtures of such solvates or hydrates can also be prepared. The source of such solvates or hydrates may be from the solvent of crystallization, may be inherent in the solvent of preparation or crystallization, or may be adventitious to such solvent.

[0080] "Pharmaceutically acceptable carrier" refers to a non-toxic carrier or excipient that does not destroy the pharmacological activity of the drug with which it is formulated and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the drug. Pharmaceutically acceptable carriers that may be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, saturated vegetable fatty acids such as glycine, sorbic acid, potassium sorbate, protamine sulfate, water, partial glyceride mixtures of salts or electrolytes, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat.

[0081] As used herein, "antigen" refers to any substance that can be recognized by the immune system. "Antigen" broadly encompasses proteins such as enzymes, peptides such as polypeptides, carbohydrates such as polysaccharides, haptens, nucleic acids, and grafts. Antigens can be self-antigens, antigens produced by the body under normal conditions or as part of a disorder, or foreign antigens, non-self-antigens. Examples of self-antigens include autoantigens associated with autoimmune disorders, including any of the autoantigens described herein. Examples of foreign antigens include antigen therapy (e.g., therapeutic proteins, gene therapy, cell therapy), allergens, and alloantigens.

[0082] As used herein, "treating" refers to taking steps to deliver therapy to a subject, such as a mammal in need of treatment (e.g., by administering one or more therapeutic agents to the mammal). "Treating" includes inhibiting a disease or condition (e.g., by slowing or halting its progression or causing regression of the disease or condition), as well as alleviating symptoms caused by a disease or condition.

[0083] A "therapeutically effective amount" is an amount effective to achieve a desired therapeutic result (e.g., induction of immune tolerance, relief of immune intolerance, treatment, cure, suppression, or remission of a physiological response or condition, etc.) at the dosage and for the period of time required. A complete therapeutic effect does not necessarily occur by administration of one dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount may be administered in one or more administrations. The therapeutically effective amount may vary depending on factors such as the disease state, age, sex, and weight of the mammal, the mode of administration, and the ability of the therapeutic agent or combination of therapeutic agents to elicit a desired response in an individual.

[0084] As used herein, a "subject" includes humans, domestic animals, such as laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e.g., cats, dogs, rabbits, etc.), and livestock (pigs, cows, sheep, goats, horses, etc.), as well as non-domestic animals. In some embodiments, the subject is a human.

[0085] compound A first embodiment is a compound of the following structural formula: [ka] or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, wherein: Ring A is phenyl or (C5-C6)heteroaryl; L is -(CH2) n -, -C(O)-, or -C(OH)-; R 1 is H or (C1-C5) alkyl, Each R 2 is independently chloro or fluoro; R 3 are one or more independently selected (C6-C 15 )Aryl or (C5-C 15 ) (C1-C3) alkyl substituted with heteroaryl; (C6-C 15 )Aryl and (C5-C 15 ) Heteroaryl is each independently -(R 30 ) p is replaced by Each R 30 are independently halo, (C1-C5)alkoxy, (C1-C5)haloalkoxy, (C1-C5)alkyl, or (C1-C5)haloalkyl, or two R attached to adjacent ring atoms; 30 Together - (CH2) q - or -O(CH2) r Forming O- n is 1, 2, or 3; m is 0, 1, 2, 3, or 4; each p is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each q is independently 3, 4, 5, or 6; and Each r is independently 1, 2, 3, or 4.

[0086] In a first aspect of the first embodiment, Ring A is phenyl or pyridinyl. The values ​​of the remaining variables are as described in the first embodiment.

[0087] In a second aspect of the first embodiment, ring A is phenyl. The values ​​of the remaining variables are as described in the first embodiment or first aspect thereof.

[0088] In a third aspect of the first embodiment, R 1 is H. The values ​​of the remaining variables are as described in the first embodiment, or its first or second aspect.

[0089] In a fourth aspect of the first embodiment, R 3 are each independently -(R 30 ) p 1, 2, or 3 independently selected (C6-C 15 )Aryl or (C5-C 15 ) methyl substituted with heteroaryl. 30 and the values ​​of the remaining variables, including p, are as described in the first embodiment or the first to third aspects thereof.

[0090] In a fifth aspect of the first embodiment, R 3 are each independently -(R 30 ) p one or more (e.g., one, two, or three) independently selected from phenyl, naphthyl, pyridinyl, quinolinyl, isoquinolinyl, or benzo[d]imidazolyl, substituted with 15 )Aryl or (C5-C 15 ) heteroaryl-substituted (C1-C3) alkyl (and in some preferred embodiments, methyl). R30 and the values ​​of the remaining variables, including p, are as described in the first embodiment or the first to fourth aspects thereof.

[0091] In a sixth aspect of the first embodiment, R 3 are each independently -(R 30 ) p substituted with one or two independently selected (C6-C 15 )Aryl or (C5-C 15 ) heteroaryl (and in some preferred embodiments, is independently selected from phenyl, naphthyl, pyridinyl, quinolinyl, isoquinolinyl, or benzo[d]imidazolyl (C6-C 15 )Aryl or (C5-C 15 (C1-C3)alkyl (and in some preferred embodiments, methyl) substituted with (C1-C3)heteroaryl). 30 and the values ​​of the remaining variables, including p, are as described in the first embodiment or the first to fifth aspects thereof.

[0092] In a seventh aspect of the first embodiment, n is 1. The values ​​of the remaining variables are as described in the first embodiment, or its first to sixth aspects.

[0093] In an eighth aspect of the first embodiment, m is 0. The values ​​of the remaining variables are as described in the first embodiment, or aspects 1 to 7 thereof.

[0094] In a ninth aspect of the first embodiment, each p is independently 0, 1, or 2. The values ​​of the remaining variables are as described in the first embodiment, or its first through eighth aspects.

[0095] In a tenth aspect of the first embodiment, each p is 0. The values ​​of the remaining variables are as described in the first embodiment, or its first to ninth aspects.

[0096] In an eleventh aspect of the first embodiment, each q is independently 3 or 4. The values ​​of the remaining variables are as described in the first embodiment, or its first through tenth aspects.

[0097] In a twelfth aspect of the first embodiment, each r is independently 1 or 2. The values ​​of the remaining variables are as described in the first embodiment, or its first through eleventh aspects.

[0098] In a thirteenth aspect of the first embodiment, -OR 3 is attached to a ring atom of ring A that is meta or para to L. 3 The values ​​of the variables including are as described in the first embodiment or the first to twelfth aspects thereof.

[0099] In a fourteenth aspect of the first embodiment, -OR 3 is attached to a ring atom of ring A that is para to L. 3 The values ​​of the variables including are as described in the first embodiment or the first to thirteenth aspects thereof.

[0100] In a fifteenth aspect of the first embodiment, n is 1 or 2. The values ​​of the remaining variables are as described in the first embodiment, or aspects 1 to 14 thereof.

[0101] A second embodiment is a compound of the following structural formula: [ka] or a prodrug thereof, or a pharma- ceutically acceptable salt thereof. 1 , R 2 , R 3 , n, m) are as described in the first embodiment, or any aspect thereof.

[0102] In a first aspect of the second embodiment, -OR 3 is -(CH2) n - is attached to a ring atom of ring A that is meta or para to R3 The values ​​of the variables including are as described in the first embodiment, or any aspect thereof.

[0103] In a second aspect of the second embodiment, -OR 3 is -(CH2) n - is attached to a ring atom of ring A that is para to R 3 The values ​​of the variables including are as described in the first embodiment, or any aspect thereof.

[0104] A third embodiment is a compound of the following structural formula: [ka] or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, wherein: X 1 , X 2 , and X 3 are > C(H), or X 1 is N and X 2 and X 3 are > C(H), or X 1 and X 2 are >C(H), and X 3 is N, or X 1 and X 3 are >C(H), and X 2 is N. The remaining variables (e.g., R 1 , R 2 , R 3 , n, m) are as described in the first embodiment, or any aspect thereof.

[0105] In a first aspect of the third embodiment, X 1 , X 2 , and X 3 and are >C(H), respectively. The values ​​of the remaining variables are as described in the first embodiment, or any aspect thereof, or the third embodiment.

[0106] In a second aspect of the third embodiment, X 1 is N and X 2 and X 3 and are >C(H), respectively. The values ​​of the remaining variables are as described in the first embodiment, or any aspect thereof, or the third embodiment.

[0107] In a third aspect of the third embodiment, -OR 3 is -(CH2) n - is attached to a ring atom of ring A that is meta or para to R 3 The values ​​of the variables, including: are as described in the first embodiment, or any aspect thereof, or the third embodiment, or the first or second aspect thereof.

[0108] In a fourth aspect of the third embodiment, -OR 3 is -(CH2) n - is attached to a ring atom of ring A that is para to R 3 The values ​​of the variables, including: are as described in the first embodiment, or any aspect thereof, or the third embodiment, or the first or second aspect thereof.

[0109] A fourth embodiment is a compound of structural formula I, or a pharma- ceutically acceptable salt thereof, wherein the variables (e.g., rings A, L, R 1 , R 2 , R 3 , m) are as described in the first embodiment, or any aspect thereof.

[0110] A fifth embodiment is a compound of structural formula II, or a pharma- ceutically acceptable salt thereof, wherein the variables (e.g., ring A, R 1 , R 2 , R 3 , n, m) are as described in the first or second embodiment, or any aspect thereof.

[0111] A sixth embodiment is a compound of formula III, or a pharma- ceutically acceptable salt thereof, wherein the variables (e.g., R 1, R 2 , R 3 , n, m, X 1 , X 2 , X 3 ) are as described in the first or third embodiment, or any aspect thereof.

[0112] Examples of compounds of structural formula I include the compounds listed in Table 1, or a prodrug thereof, or a pharma- ceutically acceptable salt thereof. In some embodiments, the compound of structural formula I is selected from the compounds listed in Table 1, or a pharma- ceutically acceptable salt thereof.

[0113] Methods for making the compounds of the present disclosure are described in the Examples herein and / or are within the capabilities of one of ordinary skill in the art.

[0114] Compositions and kits Usually, the compound of the present disclosure is formulated with one or more pharma- ceutically acceptable carriers for administration to a subject.The present disclosure provides such compositions, including pharmaceutical compositions.Thus, one embodiment is a composition (e.g., pharmaceutical composition) that comprises the compound of the present disclosure and a pharma- ceutically acceptable carrier.For example, the composition described herein can be used in the method described herein to deliver the compound of the present disclosure.

[0115] The compounds and compositions described herein may be in the form of a lipid particle formulation, such as a liposomal formulation. Thus, one embodiment is a lipid particle (e.g., a liposome) that comprises one or more lipids and a compound of the present disclosure.

[0116] Also included herein is at least one phospholipid (e.g., a saturated C4-C phospholipid such as dimyristoylphosphatidylcholine (DMPC)). 30 C4-C of acyl chain etc. 30Also provided are solid lipid particles (e.g., liposomes) comprising a phospholipid (containing an acyl chain) and a therapeutic agent (e.g., a compound of the present disclosure) that can be embedded in the lipid bilayer of the lipid particle. It has been found that oral administration of such solid lipid particles can be used, for example, to target the lipid particle (and thereby the therapeutic agent) to immune cells and / or lymph node(s), thereby enhancing co-localization of the lipid particle and immune cells (e.g., in the lymph node) and / or enhancing uptake of the lipid particle into the lymph node.

[0117] As used herein, "lipid particle" refers to a particle that includes at least one lipid, e.g., a phospholipid, such as a lysophospholipid. Examples of lipid particles include liposomes, micelles, and lipid nanoparticles. Lipid particles, such as liposomes, can be unilamellar or multilamellar. Lipid particles, such as liposomes, can have a fluid lipid membrane, or a gel-like or solid lipid membrane, e.g., a lipid membrane that melts at a temperature above normal human body temperature, i.e., about 37°C. In some embodiments, the lipid particle is a liposome. In some embodiments, the lipid particle is a lipid nanoparticle. In some embodiments, the lipid particle is solid. In some embodiments, the lipid particle has a melting temperature above about 37°C, e.g., above about 40°C, above about 45°C, above about 50°C, above about 55°C, or about 55°C.

[0118] Examples of phospholipids include dimyristoylphosphatidylcholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine 18:1 Δ9-Cis PC (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine 18:0 (DSPC), 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine 16:0-18:1 (POPC), phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine, phosphatidylinositol, bisphosphatidylglycerol, phosphatidic acid, phosphatidylalcohol, and phosphatidylglycerol. Phospholipids may be saturated or unsaturated, i.e., contain one or more unsaturated units, and may contain acyl chains of various lengths. In some embodiments, phospholipids are C4-C 30 Acyl chain, e.g., C8-C 26 , C 12 -C 22 , C 10 -C 25 , C 14 -C 18 , or C 16 -C 26 Contains acyl chains. Phospholipids can be obtained from various sources, natural and synthetic. For example, PS can be obtained from pig brain PS or plant-based soybean (soybean) PS. Egg PC, PS, and synthetic PC are commercially available. In some embodiments, phospholipid is not PS or its salt (e.g., its pharmaceutically acceptable salt).

[0119] Other lipids suitable for inclusion in the lipid particles described herein include N 4 -cholesteryl-spermine, or a salt thereof, e.g., N 4 -cholesteryl-spermine HCl salt. 4 -Cholesteryl-spermine HCl salt, also known as Genzyme Lipid 67 (GL67), is cholesterol derivatized with spermine to produce the HCl salt of the cationic lipid.

[0120] Typically, the molar percentage of the therapeutic agent (e.g., a compound of the present disclosure) in a lipid particle (e.g., a liposome) containing the therapeutic agent is about 1% to about 50%, e.g., about 1% to about 35%, about 1% to about 25%, about 1% to about 15%, about 3% to about 10%, about 5% to about 50%, about 5% to about 45%, about 15% to about 40%, about 25% to about 35%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 25%, about 30%, or about 35%. In some embodiments, the molar percentage of the therapeutic agent (e.g., a compound of the present disclosure) in a lipid particle (e.g., a liposome) containing the therapeutic agent is less than 35%, e.g., less than 30%, less than 15%, or about 1% to about 10%.

[0121] Typically, the molar percentage of lipids (taken individually or collectively) in the lipid particles (e.g., liposomes) described herein is from about 50% to about 99%, e.g., from about 50% to about 75%, from about 85% to about 99%, about 70%, about 75%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or 99%. The molar percentage of each lipid in the lipid particles (e.g., liposomes) described herein can be from about 1% to about 99%, e.g., from about 1% to about 50%, from about 1% to about 35%, from about 1% to about 25%, from about 1% to about 15%, from about 3% to about 10%, from about 5% to about 50%, from about 5% to about 45%, from about 15% to about 40%, from about 25% to about 35%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 25%, about 30%, or about 35%.

[0122] The compounds of the present disclosure can be encapsulated in lipid particles such as liposomes described herein, or can be attached (covalently or non-covalently) to lipid head groups, or can be embedded in whole or in part in lipid bilayers (e.g., liposomes) covalently or non-covalently.Without wishing to be bound by any particular theory, it is believed that the compounds of the present disclosure can be embedded in the lipid bilayers of liposomes such that the amino acid residues of the compounds of the present disclosure remain exposed to the outside of the liposome, thereby mimicking natural surface presentation, e.g., PS.

[0123] In some embodiments, the one or more lipids comprise a phospholipid or a pharma- ceutically acceptable salt thereof, such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or a pharma- ceutically acceptable salt thereof. In some embodiments, the phospholipid is a saturated phospholipid, such as C4-C 30 In some embodiments, the phospholipid is an unsaturated, e.g., C4-C 30 It is the unsaturated phospholipid that contains acyl chain.In some embodiments, phospholipid is selected from DMPC, DSPC, DOPC or POPC, or its pharmaceutically acceptable salt.In some embodiments, phospholipid is DMPC or DSPC, or its pharmaceutically acceptable salt.

[0124] In some embodiments, the lipid particle (e.g., liposome) further comprises an antigen, e.g., any of the antigens described herein. Thus, in some embodiments, the lipid particle further comprises a gene therapy. In some further embodiments, the gene therapy comprises DNA and / or RNA and a viral vector. In some embodiments, the viral vector is derived from an adeno-associated virus (AAV), e.g., a recombinant AAV. In some embodiments, the AAV is AAV9. Other examples of viral vectors suitable for use with the present disclosure include viral vectors derived from retroviruses, herpes viruses, adenoviruses, lentiviruses, rabies viruses, lentiviruses, VSV, poxviruses (e.g., vaccinia viruses, smallpox viruses, canarypox), reoviruses, Semliki Forest viruses, yellow fever viruses, Sindbis viruses, togaviruses, baculoviruses, bacteriophages, alphaviruses, and flavaviruses. In some embodiments, the antigen, e.g., a gene therapy agent comprising DNA and / or RNA and a viral vector, is encapsulated within the lipid particle.

[0125] Lipid particles further comprising an antigen, and formulations comprising such lipid particles, are believed to be particularly useful for applications involving delivery of gene therapy agents (e.g., gene therapy agents comprising DNA and / or RNA) to a subject. The lipid particles are expected to facilitate co-presentation of gene therapy and compounds of the present disclosure to the immune system. Such particles can be formulated for oral and / or parenteral (e.g., subcutaneous, intramuscular, intravenous, intradermal) administration, e.g., injection.

[0126] Another embodiment is a composition (e.g., a pharmaceutical composition) comprising a plurality of lipid particles (e.g., a plurality of lipid particles comprising a compound of the present disclosure). In some aspects, the composition further comprises a pharma- ceutically acceptable carrier.

[0127] The compositions described herein, and thus the compounds of the present disclosure, may be administered orally, parenterally (including subcutaneously, intramuscularly, intravenously, and intradermally), by inhalation spray, topically, rectally, nasally, bucally, intravaginally, or via an implanted reservoir. As used herein, the terms "parenteral" and "parenterally" include subcutaneous, intradermal, intravenous, intramuscular, intraocular, intravitreal, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, intrahepatic, intraperitoneal, intralesional, and intracranial injection or infusion techniques. In some embodiments, the compositions described herein can be administered intravenously and / or intraperitoneally. In some embodiments, the compositions described herein can be administered orally. In some embodiments, the compositions described herein can be administered subcutaneously. Preferably, the compositions described herein are administered orally, subcutaneously, intraperitoneally, or intravenously.

[0128] The compositions provided herein can be orally administered in any orally acceptable dosage form, including but not limited to capsules, tablets, aqueous suspensions, dispersions, and liquids.For tablets for oral use, commonly used carriers include lactose and cornstarch.Lubricants such as magnesium stearate are also commonly added.For oral administration in capsule form, useful diluents include lactose and dried cornstarch.When aqueous suspensions and / or emulsions are required for oral use, the active ingredient can be suspended or dissolved in an oil phase and mixed with emulsifying and / or suspending agents.If desired, certain sweeteners, flavors, or colorants can also be added.

[0129] In some embodiments, the oral formulations are formulated for immediate release or sustained / delayed release.

[0130] Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, the active compound is mixed with at least one inert pharma- ceutically acceptable excipient or carrier, 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 carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose, and acacia; (c) wetting agents, such as glycerol; (d) disintegrating agents, such as agar-agar, calcium carbonate, potato starch, or tapioca starch; (e) solution retarding agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium salts; (g) wetting agents, such as acetyl 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 forms may also comprise buffering agents.

[0131] Liquid dosage forms for oral administration include pharma- ceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs.In addition to the compounds of the present disclosure, liquid dosage forms may contain inert diluents commonly used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethyl alcohol (ethanol), isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (specifically cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil, and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol, and fatty acid esters of sorbitan, or mixtures thereof.In addition to inert diluents, oral compositions may also contain adjuvants, such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, aromatic agents, and preservatives.

[0132] Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, where the active ingredient is formulated with a carrier such as sugar and acacia, tragacanth, or gelatin and glycerin.

[0133] Similar types of solid compositions may also be used as fillers in soft and hard-filled gelatin capsules, using excipients such as lactose or milk sugar, and high molecular weight polyethylene glycols. The solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical formulation art. These dosage forms may optionally contain opacifying agents, and may be of a composition that releases the active ingredient(s) only, or preferentially, in a certain part of the intestinal tract, optionally in a delayed manner. Examples of embedding compositions that can be used include polymeric substances and waxes.

[0134] The compound of the present disclosure can also be in microencapsulated form with one or more of the above-mentioned excipients.In such solid dosage form, compound can be mixed with at least one inert diluent such as sucrose, lactose or starch.According to common practice, such dosage form can also contain additional material other than inert diluent, such as tableting lubricant and other tableting aids, such as magnesium stearate and microcrystalline cellulose.

[0135] Compositions for oral administration may be designed to protect the active ingredient against degradation as it passes through the digestive tract, for example by an outer coating of the formulation on a tablet or capsule.

[0136] In another embodiment, the compounds of the present disclosure can be provided as extended (or "delayed" or "sustained") release compositions. The delayed release compositions include the compounds of the present disclosure and a delayed release component. Such compositions allow for targeted release of the compounds, for example, to the lower gastrointestinal tract, for example, the small intestine, large intestine, colon, and / or rectum. In certain embodiments, the delayed release compositions further include enteric or pH-dependent coatings, such as cellulose acetate phthalate and other phthalates (e.g., polyvinyl acetate phthalate, methacrylates (Eudragits)). Alternatively, the delayed release compositions can provide controlled release to the small intestine and / or colon by providing pH-sensitive methacrylate coatings, pH-sensitive polymer microspheres, or polymers that undergo degradation by hydrolysis. The delayed release compositions can be formulated with hydrophobic or gelling excipients or coatings. Colonic delivery can further be provided by coatings that are digested by bacterial enzymes such as amylose or pectin, by pH-dependent polymers, by hydrogel plugs that swell over time (Pulsincap), by time-dependent hydrogel coatings, and / or by acrylic acid linked to an azoaromatic bond coating.

[0137] The compositions described herein can also be administered subcutaneously, intraperitoneally, or intravenously, for example, in the form of a sterile injectable preparation, for example, as a sterile injectable aqueous or oily suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents (such as, for example, Tween® 80) and suspending agents. The sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol. Acceptable vehicles and solvents that can be used include mannitol, dextrose, water, Ringer's solution, lactated Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conventionally used as a solvent or suspending medium. For this purpose, any non-irritating fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharma- ceutically acceptable oils such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, or carboxymethylcellulose or similar dispersants commonly used in the formulation of pharma- ceutically acceptable dosage forms such as emulsions and / or suspensions. Other commonly used surfactants such as Tween® or Span® and / or other similar emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma-ceutically acceptable solid, liquid or other dosage forms may also be used for formulation purposes.

[0138] The compositions described herein can also be administered in the form of suppositories for rectal administration. These can be prepared by mixing the compounds of the present disclosure with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature, thereby melting in the rectum and releasing the drug. Such materials include cocoa butter, beeswax, and polyethylene glycols.

[0139] The compositions described herein may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, such as diseases of the eye, the skin, or the lower intestinal tract. Suitable topical formulations are readily prepared for each of these areas or organs.

[0140] Topical application for the lower intestinal tract can be effected in a rectal suppository formulation (see above) or in a suitable enema formulation. Topically-transdermal patches can also be used.

[0141] For topical application, the composition can be formulated into a suitable ointment containing the active ingredient suspended or dissolved in one or more carriers. Carriers for topical administration of the compounds described herein include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compounds, emulsifying wax, and water, and penetration enhancers. Alternatively, the composition can be formulated into a suitable lotion or cream containing the active compound suspended or dissolved in one or more pharma- ceutically acceptable carriers. Alternatively, the composition can be formulated in a suitable lotion or cream containing the active compound suspended or dissolved in a carrier with a suitable emulsifier. Suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol, and water. Suitable carriers also include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water, as well as penetration enhancers.

[0142] For ophthalmic use, the composition may be formulated as a micronized suspension in isotonic, pH-adjusted, sterile saline, or preferably as a solution in isotonic, pH-adjusted, sterile saline, either in the presence or absence of a preservative, such as benzylalkonium chloride. Alternatively, for ophthalmic use, the composition may be formulated in an ointment, such as petrolatum.

[0143] The composition can also be administered by nasal aerosol or inhalation. Such compositions can be prepared according to well-known techniques in the field of pharmaceutical formulations, and can be prepared as a solution in saline, using benzyl alcohol or other suitable preservatives, absorption enhancers to enhance bioavailability, fluorocarbons, and / or other conventional solubilizers or dispersants. Without being bound to any particular theory, it is believed that the local delivery of the compositions described herein, such as can be achieved by nasal aerosol or inhalation, can reduce the risk of systemic effects of the composition, such as effects on red blood cells.

[0144] Other pharma- ceutically acceptable carriers that can be used in the compositions described herein include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-α-tocopherol polyethylene glycol 1000 succinate, surfactants such as Tween® used in pharmaceutical dosage forms, or other similar polymeric delivery matrices, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. For example, to enhance delivery of the agents described herein, cyclodextrins, e.g., α-, β-, and γ-cyclodextrin, or chemically modified derivatives thereof, e.g., hydroxyalkyl cyclodextrins, including hydroxypropyl-β-cyclodextrin, e.g., 2- and / or 3-hydroxypropyl-β-cyclodextrin, or other solubilized derivatives, can also be effectively used as pharma- ceutically acceptable carriers in the compositions described herein.

[0145] One embodiment is a composition comprising a compound of the present disclosure and a cyclodextrin or a chemically modified derivative thereof. In some aspects, the cyclodextrin or a chemically modified derivative thereof comprises a hydroxyalkyl cyclodextrin, such as hydroxypropyl-β-cyclodextrin. In some aspects, the compound of the present disclosure and the cyclodextrin (e.g., a hydroxyalkyl cyclodextrin, such as hydroxypropyl-β-cyclodextrin) are present in a ratio of about 1 to about 50 weight / weight (w / w) to about 1 to about 250 w / w, such as about 1 to about 50 w / w to about 1 to about 100 w / w, about 1 to about 80 w / w, or about 1 to about 166 w / w. In some aspects, the composition further comprises a diluent, such as water. In some aspects, the composition further comprises a sweetener and / or a flavoring agent.

[0146] In some aspects, the composition is in a liquid dosage form, and in further aspects, the composition is in a liquid dosage form for oral administration.

[0147] In some embodiments, the compositions described herein further comprise one or more additional therapeutic agents, eg, for use in combination with a compound of the present disclosure.

[0148] Some embodiments provide combinations (e.g., pharmaceutical combinations) that include a compound of the present disclosure (e.g., a composition described herein that includes a compound of the present disclosure) and one or more additional therapeutic agents (e.g., one or more compositions that include one or more additional therapeutic agents). Such combinations are particularly useful, for example, when the compound of the present disclosure and the one or more additional therapeutic agents are administered separately. In the combinations provided herein, the compound of the present disclosure and the one or more additional therapeutic agents can be administered by the same route of administration or by different routes of administration.

[0149] One embodiment is a kit comprising a compound of the present disclosure (e.g., a composition described herein comprising a compound of the present disclosure) and an antigen (e.g., any of the antigens described herein, such as an antigen therapy). In one aspect, the kit comprises a therapeutically effective amount of a compound of the present disclosure (e.g., an amount sufficient to immunotolerize the subject to the antigen to be administered; a therapeutically effective amount of the compound for treating a disease, disorder, or condition described herein). In some aspects, when the antigen is an antigen therapy, the kit comprises a therapeutically effective amount of the antigen therapy for treating the disease, disorder, or condition. In some aspects, the kit further comprises additional therapeutic agent(s) (e.g., a composition comprising additional therapeutic agent(s)). In some aspects, the kit further comprises written instructions for administering to the subject a compound of the present disclosure and / or the antigen and / or additional agent(s) for treating a disease, disorder, or condition described herein.

[0150] Suitable additional therapeutic agents include those agents described herein in connection with combination therapy.

[0151] The compositions described herein can be provided in unit dosage form. The amount of active ingredient that can be combined with a carrier to produce a unit dosage form varies, for example, depending on the subject being treated and the particular mode of administration. Usually, a unit dosage form contains about 1 to about 1,000 mg of active ingredient(s), for example, about 1 to about 500 mg, about 1 to about 250 mg, about 1 to about 150 mg, about 0.5 to about 100 mg, or about 1 to about 50 mg of active ingredient(s). In some embodiments, a unit dosage form contains about 0.01 mg to about 100 mg of active ingredient(s), for example, about 0.1 mg to about 50 mg, about 0.1 mg to about 10 mg, about 0.5 mg to about 50 mg of active ingredient(s). In some embodiments, the unit dosage form contains from about 1 mg to about 5,000 mg of the active ingredient(s), e.g., from about 10 mg to about 2,500 mg, from about 15 mg to about 1,000 mg, or from about 100 mg to about 1,000 mg of the active ingredient(s). In some embodiments, the unit dosage form contains about 15 mg, about 30 mg, about 50 mg, about 100 mg, about 125 mg, or about 150 mg of the active ingredient(s).

[0152] In some embodiments, the concentration of one or more therapeutic agents provided in the pharmaceutical composition is 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05 ... %, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or 0.0001% w / w, w / v, or v / v, and / or 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%, 4%, 3.75%, 3.50%, 3.25%, 3%, 2.75%, 2.50%, 2.25%, 2%, 1.75%, 1.50%, 125% ,1%,0.5%,0.4%,0.3%,0.2%,0.1%,0.09%,0.08%,0.07%,0.06%,0.05%,0.04%,0.03%,0.02%,0.01%,0.009%,0.008%,0.007%,0.006%,0.005%,0.004%,0.003%,0.002%,0.001%,0.0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002%, or greater than 0.0001% w / w, w / v, or v / v.

[0153] In some embodiments, the concentration of one or more therapeutic agents provided in the pharmaceutical composition is from about 0.0001% to about 50%, from about 0.001% to about 40%, from about 0.01% to about 30%, from about 0.02% to about 29%, from about 0.03% to about 28%, from about 0.04% to about 27%, from about 0.05% to about 26%, from about 0.06% to about 25%, from about 0.07% to about 24%, from about 0.08% to about 26%, from about 0.09% to about 27%, from about 0.10% to about 12%, from about 0.11% to about 12%, from about 0.12% to about 12%, from about 0.13% to about 13%, from about 0.14% to about 13%, from about 0.15% to about 13%, from about 0.16% to about 13%, from about 0.17% to about 13%, from about 0.18% to about 13%, from about 0.19% to about 14%, from about 0.20% to about 25%, from about 0.21% to about 25%, from about 0.22% to about 25%, from about 0.23% to about 25%, from about 0.24% to about 25%, from about 0.25% to about 25%, from about 0.26% to about 25%, from about 0.27% to about 25%, from about 0.28% to about 25%, from about 0.29% to about 25%, from about 0.30% to about 25%, from about 0.31% to about 25%, from about 0.32% to about 25%, from about 0.33% to about 25%, The range of the active ingredient is from about 0.08% to about 23%, from about 0.09% to about 22%, from about 0.1% to about 21%, from about 0.2% to about 20%, from about 0.3% to about 19%, from about 0.4% to about 18%, from about 0.5% to about 17%, from about 0.6% to about 16%, from about 0.7% to about 15%, from about 0.8% to about 14%, from about 0.9% to about 12%, or from about 1% to about 10% w / w, w / v, or v / v. In some embodiments, the concentration of the one or more therapeutic agents provided in the pharmaceutical composition is in the range of about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w / w, w / v, or v / v.

[0154] How to use It has now been found that various compounds of the present disclosure and compositions described herein can bind to TIM with greater affinity than its natural ligand, phosphatidylserine, for example, and reduce the immune response.

[0155] One embodiment is a method of modulating T cell immunoglobulin and mucin domain (TIM) receptor expression or activity, comprising contacting a cell (e.g., a cell expressing a TIM receptor, such as an immune cell) with a compound of the present disclosure (e.g., a therapeutically effective amount of a compound of the present disclosure). TIM receptors are type 1 cell surface glycoproteins, and the TIM receptors expressed in humans, TIM1, TIM3, and TIM4, have been identified as phosphatidylserine receptors. TIM1 is preferentially expressed on T helper 2 cells and functions as a potent co-stimulatory molecule for T cell activation. TIM3 is preferentially expressed on T helper 1 cells, type 1 T cells, and dendritic cells, and generates an inhibitory signal that triggers apoptosis of T helper 1 cells and type 1 T cells. TIM4 is expressed on antigen presenting cells and mediates phagocytosis of apoptotic cells to promote tolerance. In some aspects, the TIM receptor is a TIM3 receptor. In some aspects, the TIM receptor is a TIM4 receptor. In some aspects, the TIM receptor is a TIM1 receptor. "TIM" is also referred to in the literature as, for example, "Tim".

[0156] Agonists of TIM receptors have now also been found to inhibit the activity of at least toll-like receptors (TLR) 3 and TLR 7, without substantially inhibiting the activity of TLR 2 and 4, which primarily recognize patterns presented by bacteria. Toll-like receptors (TLRs) form a family of pattern recognition receptors expressed on innate immune cells and constitute the first line of defense of the immune system against microorganisms. To date, ten human TLR subtypes have been identified. TLR 1, 2, 4, 5, 6, and 10 are expressed on the cell surface, whereas TLR 3, 7, 8, and 9 are localized in the endoplasmic reticulum, endosomes, and lysosomes. TLR 1, 2, and 6 recognize and bind bacterial lipoproteins and glycolipids. TLR 3, 7, 8, and 9 recognize and bind nucleic acids such as viral dsRNA (TLR 3), ssRNA (TLR 7, TLR 8), and unmethylated CpG DNA (TLR 9). TLR 4 recognizes and binds fibronectin and LPS. TLR5 recognizes and binds to bacterial flagellin. Without being bound to any particular theory, it is believed that the compounds of the present disclosure do not provide general immunosuppression, but may exert their effects in a more selective and specific manner.

[0157] Another embodiment is a method of modulating (e.g., inhibiting) the activity of TLR3, TLR7, TLR8, and / or TLR9, comprising contacting a cell (e.g., a cell expressing TLR3, TLR7, TLR8, and / or TLR9; an immune cell) with a compound of the disclosure (e.g., a therapeutically effective amount of a compound of the disclosure). In some aspects, the compound of the disclosure selectively modulates (e.g., inhibits) the activity of TLR3, TLR7, TLR8, and / or TLR9, e.g., modulates (e.g., inhibits) the activity of TLR3, TLR7, TLR8, and / or TLR9 to a greater extent than it modulates the activity of TLR1, 2, 4, 5, 6, and / or 10. For example, modulation (e.g., inhibition) of TLR3, TLR7, TLR8 and / or TLR9 activity by a compound of the disclosure can be more than 2-fold, e.g., more than 5-fold, more than 10-fold, more than 25-fold, or more than 100-fold, compared to modulation (e.g., inhibition) by the compound of activity of TLR1, 2, 4, 5, 6, and / or 10. In some embodiments, the compound does not measurably modulate (e.g., inhibit) the activity of TLR1, 2, 4, 5, 6, and / or 10.

[0158] In some embodiments of the methods described herein, the cell is an immune cell, e.g., a T cell, such as a regulatory T cell, a natural killer (NK) cell, a macrophage, a neutrophil, a myeloid-derived suppressor cell, or a dendritic cell. In some embodiments, the immune cell is a FoxP3+ and / or CD4+, e.g., a FoxP3+ and / or CD4+ T cell. In some embodiments, the immune cell is a B cell, such as a regulatory B cell. In some embodiments, the immune cell (e.g., a regulatory B cell) is CD19+, CD71+, IgM+, CD24+, CD38+, and / or CD27+.

[0159] In some embodiments of the methods described herein, the methods are performed in vitro. In other embodiments of the methods described herein, the methods are performed in vivo. Thus, in some embodiments, the cell (e.g., an immune cell) is in a subject (e.g., a subject having a disease, disorder, or condition described herein).

[0160] Another embodiment is a method of tolerizing a subject in need of tolerization (e.g., a subject having an autoimmune disorder, such as an autoimmune disorder described herein), comprising administering to the subject a therapeutically effective amount of a compound of the disclosure, e.g., in the form of a composition described herein.

[0161] Another embodiment is a method of immune tolerization (e.g., antigen therapy) of a subject in need of immune tolerization to an antigen, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, e.g., in the form of a composition described herein. Some aspects include administering to the subject an antigen, or an immunogenic fragment thereof, and a therapeutically effective amount of a compound of the present disclosure, e.g., in the form of a composition described herein. Some aspects include administering to the subject a composition comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof, e.g., a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof. In some aspects, the antigen, or an immunogenic fragment thereof, and the compound of the present disclosure are administered to the subject in separate formulations.

[0162] As used herein, "immune tolerization" refers to, for example, reducing and / or eliminating an immune response to an antigen. The immune response may be evidenced, for example, by immunological hyperactivity, inflammatory cytokine release, and / or activation of immune cells, such as macrophages, neutrophils, eosinophils, T cells, and B cells. As used herein, "immune tolerization" refers, for example, to reducing immunological hyperactivity, inhibiting inflammatory cytokine release, and / or inhibiting activation and / or neutralizing immune cells, such as macrophages, neutrophils, eosinophils, T cells, and B cells. In a clinical setting, immune tolerization may be evidenced, for example, by a reduction in the severity of an autoimmune disease and / or an improvement in the activity of an antigen therapy administered.

[0163] Thus, the process of immune tolerization can be viewed along a continuum from immunological hyperactivity to immunological hypoactivity to, for example, immunological unresponsiveness to an antigen. By "immune tolerization" we contemplate the gradual progression along this continuum towards immunological unresponsiveness, as well as the induction of immunological hypoactivity or immunological unresponsiveness. In other words, immune tolerization includes reducing the level of immune intolerance and inducing immune tolerance. In certain preferred embodiments described herein, the method induces immune tolerance.

[0164] In some embodiments, a subject exhibiting immune intolerance or a subject with immune intolerance has a measurable immune response, e.g., to an antigen, e.g., measurable antibody production in response to the antigen. In some embodiments, a subject exhibiting immune tolerance or a subject with immune intolerance does not have a measurable immune response, e.g., to an antigen, e.g., measurable antibody production in response to the antigen. ELISAs and / or activity assays, including those described herein, are known in the art and can be used to measure antibody production indicative of immune intolerance.

[0165] In some autoimmune diseases, antibodies are not always present. Immune intolerance in such cases may be evidenced by clinical symptoms of the autoimmune disease, and / or the presence of autoreactive T cells or B cells, and / or an increase in other inflammatory immune cells, such as neutrophils, eosinophils, etc. In some embodiments, subjects who exhibit immune intolerance or subjects who are immune intolerant (e.g., subjects who have an autoimmune disease, such as an autoimmune disease described herein) have a measurable cytokine response. For example, subjects suffering from rheumatoid arthritis may have a measurable TNF-α response. In some embodiments, subjects who exhibit immune tolerance or subjects who are immune tolerant (e.g., subjects who have an autoimmune disease, such as an autoimmune disease described herein) do not have a measurable cytokine response.

[0166] Tolerization can be achieved in a general or antigen-specific manner, e.g., resulting in general or antigen-specific tolerance (e.g., general or specific, acquired or adaptive tolerance), respectively. Indicators of general tolerization include, for example: (a) immunological hyperactivity and / or lack and / or reduction in anti-inflammatory cytokine release, (b) neutralization of immune cells such as macrophages, neutrophils, eosinophils, T cells, and B cells, (c) increased numbers of regulatory T cells and / or increased activity or levels of tolerogenic T cells (e.g., FoxP3+ / CD4+ T cells, CD4+ / CD25 T cells, and / or increased levels of inflammatory cytokines), (d) increased immune response and / or ... hi / Foxp3+ / CTLA4+ / Tim3+ / NRP1+ / ICOS-T cells;CD4+ / CD25 hi / Foxp3+ / CTLA4+ / Tim3+ T cells; and / or CD4+ / CD25 hi Indicators of antigen-specific immune tolerization include, for example: (a) an increase in the number of antigen-specific regulatory T cells (e.g., CD4+ / FoxP3+ T cells; CD4+ / CD25+ / FoxP3+ / CTLA4+ / NRP1+ / ICOS- T cells); and / or (d) an increase in the number of regulatory B cells (e.g., CD19+ / CD71+ / IgM+ / CD24+ / CD38+ / CD27+ B cells; and / or CD19+ / CD71+ / IgM+ B cells). hi / Foxp3+ / CTLA4+ / Tim3+ / NRP1+ / ICOS- T cells;CD4+ / CD25 hi / Foxp3+ / CTLA4+ / Tim3+ T cells; and / or CD4+ / CD25 hi / Foxp3+ / CTLA4+ / NRP1+ / ICOS- T cells), (b) a decrease in antigen-specific antibody titers and / or B cell numbers, including antigen-specific memory B cells, (c) a decrease in IL-6 and / or IL-17, (d) an increase in TGF-β, IL-10, IL-35, CD40, CD80, and / or CD86, (e) a decrease in responsiveness after rechallenge with antigen, and / or (f) an increase in the number of antigen-specific regulatory B cells (e.g., CD19+ / CD71+ / IgM+ / CD24+ / CD38+ / CD27+ B cells, and / or CD19+ / CD71+ / IgM+ B cells). Techniques for assessing these indicators are known in the art and described herein. For example, some of the aforementioned indicators can be assessed using culture conditions.

[0167] In autoimmune diseases, treatment with the compounds of the present disclosure results in the proliferation of natural regulatory T cells. Such treatment does not prevent innate immune responses such as those triggered by innate immune cells that respond to danger signals from pathogens, but results in general adaptive immune tolerance. Thus, immune tolerization can be achieved herein without general innate immune suppression, so that, for example, a subject can still mount an innate immune response to an antigen (e.g., a pathogen). In some aspects, immune tolerization is general adaptive immune tolerization. In some aspects, immune tolerization is antigen-specific, for example, resulting in immune intolerance to a particular antigen(s) or reduced immune tolerance to a particular antigen(s).

[0168] It is understood that antigen-specific immune tolerization can be achieved by administering to a subject a particular antigen and a therapeutically effective amount of a compound of the disclosure or a composition described herein, in accordance with the methods described herein, and additionally, or alternatively, by administering to a subject an immunogenic fragment of the particular antigen and a therapeutically effective amount of a compound of the disclosure or a composition described herein.

[0169] As used herein, an "immunogenic fragment" of an antigen refers to a fragment of the antigen that induces an immune response against the antigen. The immunogenic fragment of an antigen may induce an immune response in a subject to the same extent as the immune response induced by the antigen itself, but does not necessarily induce an immune response to the same extent as the antigen itself, as long as the fragment has an immune tolerizing effect when administered according to the methods described herein.

[0170] Another embodiment is a method for inhibiting or reducing antigen-specific antibody titers in a subject, comprising administering to the subject an antigen, or an immunogenic fragment thereof, and a therapeutically effective amount of a compound of the present disclosure, for example in the form of a composition described herein. Some aspects include administering to the subject a composition described herein that comprises a compound of the present disclosure and an antigen, or an immunogenic fragment thereof, for example a composition that comprises a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof. In some aspects, the antigen, or an immunogenic fragment thereof, and the compound of the present disclosure are administered to the subject in separate formulations.

[0171] In some aspects of the methods described herein, the antigen is an allergen, such as a food allergen or a latex allergen. Examples of food allergens include peanut allergens, such as Ara hI or Ara hII, walnut allergens, such as Jug rI, Brazil nut allergens, such as albumin, shrimp allergens, such as Pen aI, egg allergens, such as ovomucoid, milk allergens, such as bovine β-lactoglobin, wheat gluten antigens, such as gliadin, and fish allergens, such as parvalbumin. An example of a latex allergen is Hey b7. Other allergens include antigen E, or protein antigens from grasses, such as Amb aI (ragweed pollen), Lol p1 (grass), house dust mite allergens, such as Der pI and Der PII (house dust mite), Fel dI (house cat), Bet v1 (birch), and protein antigens from tree pollens, such as Cry j1 and Cry j2 (cedar). The source of the allergen listed in brackets following each allergen indicates the source with which the indicated allergen is normally associated.

[0172] Another embodiment is a method for inducing a population of regulatory T cells in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, for example in the form of a composition described herein. Some aspects further comprise administering to the subject an antigen, or an immunogenic fragment thereof, in response to which a population of regulatory T cells is induced. Some aspects comprise administering to the subject a composition as described herein, comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof, for example a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof. In some aspects, the antigen, or an immunogenic fragment thereof, and the compound of the present disclosure are administered to the subject in separate formulations.

[0173] Without wishing to be bound by any particular theory, it is believed that the compounds of the present disclosure primarily stimulate natural regulatory T cells (nT regs The compounds of the present disclosure are also believed to induce a population of regulatory T cells by expanding a population of regulatory T cells that are, for example, FoxP3+ / NRP1+. regs, e.g., FoxP3+ T cells, FoxP3+ / TIM3+ T cells). Thus, in some embodiments, the method of inducing a regulatory T cell population is, for example, a method of expanding a population of natural regulatory T cells (e.g., regulatory T cells that are FoxP3+ / NRP1+) without substantially inducing inducible regulatory T cells. As described herein, for example, neuropilin-1 (Nrp1) expression can be used to distinguish natural and inducible regulatory T cells. Thus, in some embodiments, the method of inducing a regulatory T cell population is, for example, a method of inducing a population of regulatory T cells that express Nrp1 (e.g., FoxP3+ / NRP1+ T cells) by expanding a population of natural regulatory T cells. Without wishing to be bound by any particular theory, for example, the ability to expand a population of natural regulatory T cells (e.g., regulatory T cells that are FoxP3+ / NRP1+) without substantially inducing inducible regulatory T cells is believed to be useful in treating autoimmune diseases without affecting general immune suppression.

[0174] In some embodiments, the regulatory T cells are FoxP3+, for example, FoxP3+ / TIM3+, FoxP3+ / NRP1+.Regulatory T cells can be determined to be positive (+) or negative (-) for any of the above markers, for example, by flow cytometry analysis.

[0175] Another embodiment is a method of increasing the activity or level of tolerogenic T cells in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the disclosure, e.g., in the form of a composition described herein.

[0176] Another embodiment is a method for inducing a population of regulatory B cells in a subject, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, for example in the form of a composition described herein. Some aspects further comprise administering to the subject an antigen, or an immunogenic fragment thereof, in response to which a population of regulatory B cells is induced. Some aspects comprise administering to the subject a composition as described herein comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof, for example a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an antigen, or an immunogenic fragment thereof. In some aspects, the antigen, or an immunogenic fragment thereof, and the compound of the present disclosure are administered to the subject in separate formulations.

[0177] Compounds of the present disclosure have been found to increase the expression of certain regulatory markers on B cells, such as CD19, CD71, and IgM, thereby inducing a population of CD19+ / CD71+ / IgM+ B cells. In some embodiments, regulatory B cells are CD19+, CD71+, IgM+, CD24+, CD38+, and / or CD27+, e.g., CD19+ / CD71+ / IgM+. Whether regulatory B cells are positive (+) or negative (-) for any of the aforementioned markers can be determined, for example, by flow cytometry analysis.

[0178] Another embodiment is a method of treating an autoimmune disorder in a subject (e.g., a subject in need of treatment), comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, e.g., in the form of a composition described herein. It is understood that in an autoimmune disorder, it may be desirable to induce general adaptive immune tolerization (e.g., immune tolerance), e.g., by inducing a population of regulatory T cells, or specific immune tolerization (e.g., immune tolerance), e.g., by tolerizing the subject to an autoantigen or immunogenic fragment thereof associated with the autoimmune disorder. Thus, in some aspects of the method of treating an autoimmune disorder, the method further comprises administering to the subject (e.g., co-administering) an autoantigen or immunogenic fragment thereof associated with the autoimmune disorder. Some aspects include administering to the subject a composition described herein comprising a compound of the present disclosure and an autoantigen, or an immunogenic fragment thereof, e.g., a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an autoantigen, or an immunogenic fragment thereof. In some aspects, the autoantigen, or an immunogenic fragment thereof, and the compound of the present disclosure are administered to the subject in separate formulations.

[0179] Specific examples of autoimmune disorders treatable according to the methods described herein include achalasia, Addison's disease, adult Still's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome, autoimmune angioedema, autoimmune dysautonomia, autoimmune encephalomyelitis, autoimmune hepatitis, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune oophoritis, autoimmune orchitis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune urticaria, axonal neuropathy (AMAN), Baro's disease, Behcet's disease, and benign mucosal keratoderma. Pemphigoid, Bullous Pemphigoid, Castleman's Disease (CD), Celiac Disease, Chagas Disease, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Recurrent Multifocal Osteomyelitis (CRMO), Churg-Strauss Syndrome (CSS) or Eosinophilic Granulomatosis (EGPA), Cicatricial Pemphigoid, Cogan's Syndrome, Cold Agglutinin Disease, Congenital Heart Block, Coxsackie Myocarditis, CREST Syndrome, Crohn's Disease, Dermatitis Herpetiformis, Dermatomyositis, Devic's Disease (Neuromyelitis Optica), Discoid Lupus, Dressler's Syndrome, Endometriosis, Eosinophilic Esophagitis (EoE), Eosinophilic Fasciitis, Erythema Nodosum, Essential Mixed Combined cryoglobulinemia, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), giant cell myocarditis, glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis, Graves' disease, Guillain-Barré syndrome, Hashimoto's thyroiditis, hemolytic anemia, Henoch purpura (HSP), herpes gestationis or pemphigoid of pregnancy (PG), hidradenitis suppurativa (HS) (acne contralateral), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis , Juvenile Onset Diabetes Mellitus (Type 1 Diabetes), Juvenile Myositis (JM), Kawasaki Disease, Lambert-Eaton Syndrome, Leukocytoclastic Vasculitis, Lichen Planus, Lichen Sclerosus, Lignin Conjunctivitis, Linear Immunoglobulin A Disease (LAD), Lupus, Chronic Lyme Disease, Meniere's Disease, Microscopic Polyangiitis (MPA), Mixed Connective Tissue Disease (MCTD), Mooren's Ulcer, Mucha-Habermann Disease, Multifocal Motor Neuropathy (MMN) or MMNCB, Multiple Sclerosis, Myasthenia Gravis, Myositis, Narcolepsy, Neonatal Lupus, Neuromyelitis Optica, Neutropenia, Ocular Cicatricial Pemphigoid, Optic Neuritis, Relapsing Rheumatism (PR), PANDAS,Paraneoplastic cerebellar degeneration (PCD), paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, pars planitis (peripheral uveitis), Parsonage-Turner syndrome, pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia (PA), POEMS syndrome, polyarteritis nodosa, polyglandular syndrome types I, II, and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, primary biliary cirrhosis, primary sclerosing cholangitis, progesterone dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, recurrent multiple myelopathy, These include: chronic chondritis, restless legs syndrome (RLS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt syndrome, scleritis, scleroderma, Sjögren's syndrome, sperm and testicular autoimmunity, stiff-body syndrome (SPS), subacute bacterial endocarditis (SBE), Susac syndrome, sympathetic ophthalmia (SO), Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), thyroid eye disease (TED), Tolosa-Hunt syndrome (THS), transverse myelitis, type 1 diabetes mellitus, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, and Vogt-Koyanagi-Harada syndrome.

[0180] In some embodiments, the autoimmune disorder is a neurological autoimmune disorder. Examples of neurological autoimmune disorders include multiple sclerosis, neuromyelitis optica, myasthenia gravis, anti-myelin oligodendrocyte glycoprotein antibody disease (MOG), MOG antibody-associated disorder (MOGAD, e.g., MOG-associated pediatric demyelinating disease), autoimmune encephalitis, acute disseminated encephalomyelitis (ADEM), chronic meningitis, central nervous system vasculitis, Guillain-Barre syndrome, Hashimoto's thyroiditis, steroid-responsive encephalopathy with autoimmune thyroiditis (SREAT), neurosarcoidosis, optic neuritis, and transverse myelitis.

[0181] In some embodiments, the autoimmune disorder is rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease (IBD), multiple sclerosis, type 1 diabetes, Guillain-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, psoriasis, Graves' disease, Hashimoto's thyroiditis, myasthenia gravis, or vasculitis. In some embodiments, the autoimmune disorder is systemic lupus erythematosus. In some embodiments, the autoimmune disorder is IBD.

[0182] In some embodiments, the autoimmune disorder is multiple sclerosis, neuromyelitis optica, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), rheumatoid arthritis, or myasthenia gravis. In some embodiments, the autoimmune disorder is multiple sclerosis. In some embodiments, the autoimmune disorder is neuromyelitis optica. In some embodiments, the autoimmune disorder is MOGAD. In some embodiments, the autoimmune disorder is rheumatoid arthritis. In some embodiments, the autoimmune disorder is myasthenia gravis.

[0183] Another embodiment is a method of treating multiple sclerosis in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a compound of the disclosure, e.g., in the form of a composition described herein.

[0184] Clinical management of multiple sclerosis usually follows one of two paradigms: the escalation paradigm or the induction / maintenance paradigm. In the escalation paradigm, a more effective and efficacious disease-modifying treatment (DMT) is administered after treatment with a less effective and efficacious DMT has failed. Standard treatment in the escalation paradigm usually includes treatment with glatiramer acetate, interferon beta, and / or teriflunimide, escalating to fingolimod and / or dimethyl fumarate upon treatment failure, further escalating to natalizumab and / or anti-B cell upon treatment failure, and still further escalating to alemtuzumab and / or mitoxantrone upon treatment failure.

[0185] The induction / maintenance treatment paradigm for the clinical management of multiple sclerosis involves an induction phase followed by a maintenance phase in which patients are treated with a high-potency DMT to induce disease control, followed by a switch to a safer, lower-potency DMT as maintenance therapy.

[0186] Disease-modifying therapies (DMTs) used to treat multiple sclerosis include interferon beta-1a (e.g., AVONEX®, REBIF®), interferon beta-1b (e.g., BETASERON®, EXTAVIA®), glatiramer acetate (e.g., COPAXONE®, GLATOPA®), ofatumumab (e.g., KESIMPTA®), pegylated interferon beta-1a (e.g., PLEGRIDY®), teriflunomide (e.g., AUBAGIO®), monomethyl fumarate (e.g., BAFIERTAM™), dimethyl fumarate (e.g., TECFIDERA®), fingolimod (e.g., GILENYA®), cladribine (e.g., MAVENCLAD®), siponimod (e.g., MAYZENT®), ponesimob (e.g., PONVORY®), diroximel fumarate (e.g., VUMERITY®), ozanimob (e.g., ZEPOSIA®), alemtuzumab (e.g., LEMTRADA®), mitoxantrone (e.g., NOVANTRONE®), ocrelizumab (e.g., OCREVUS®), and natalizumab (e.g., TYSABRI®). Examples of high efficacy DMTs include, but are not limited to, natalizumab, alemtuzumab, anti-B cells, and mitoxantrone. Examples of lower potency DMTs include, but are not limited to, glatiramer acetate, interferon beta, teriflunimide, DMF, and fingolimov.

[0187] Natalizumab is a recombinant humanized IgG4κ monoclonal antibody produced in mouse myeloma cells. Natalizumab binds to the α4 subunit of α4β1 and α4β7 integrins expressed on the surface of all leukocytes except neutrophils, and inhibits α4-mediated adhesion of leukocytes to their counter-receptor(s). Natalizumab injection is indicated as monotherapy for the treatment of relapsing forms of multiple sclerosis, including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults. Glatiramer acetate injection is indicated for the treatment of relapsing forms of multiple sclerosis, including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease, in adults. Dimethyl fumarate for oral use is indicated for the treatment of relapsing forms of multiple sclerosis.

[0188] In some embodiments, the multiple sclerosis has not been previously treated, hi alternative embodiments, the multiple sclerosis has been previously treated with standard therapies, such as, for example, natalizumab (TYSABRI®), glatiramer acetate and / or dimethyl fumarate, or DMT.

[0189] In some embodiments, the multiple sclerosis is primary progressive multiple sclerosis (PPMS). In some embodiments, the multiple sclerosis is relapsing remitting multiple sclerosis (RRMS). In some embodiments, the multiple sclerosis is clinically isolated syndrome (CIS). In some embodiments, the multiple sclerosis is secondary progressive multiple sclerosis (SPMS).

[0190] In some embodiments (e.g., when the autoimmune disease is multiple sclerosis), the method includes administering to the subject a therapeutically effective amount of an induction therapy comprising a compound of the present disclosure, e.g., in the form of a pharmaceutical composition. In some embodiments (e.g., when the autoimmune disease is multiple sclerosis), the method includes administering to the subject a therapeutically effective amount of a maintenance therapy comprising a compound of the present disclosure, e.g., in the form of a pharmaceutical composition. In some embodiments (e.g., when the autoimmune disease is multiple sclerosis), the method includes administering to the subject a therapeutically effective amount of an induction therapy comprising a compound of the present disclosure, e.g., in the form of a pharmaceutical composition, and (e.g., followed by) a therapeutically effective amount of a maintenance therapy comprising a compound of the present disclosure, e.g., in the form of a pharmaceutical composition.

[0191] In some embodiments (e.g., where the autoimmune disease is multiple sclerosis), the compound of the disclosure is administered in combination with a DMT, such as natalizumab and / or glatiramer acetate and / or dimethyl fumarate, and in some further embodiments, the method further comprises administering to the subject a DMT, such as natalizumab and / or glatiramer acetate and / or dimethyl fumarate.

[0192] In some embodiments, the autoimmune disorder has not been previously treated, hi alternative embodiments, the autoimmune disorder has been previously treated with a standard of care therapy, such as, for example, natalizumab (TYSABRI®) or glatiramer acetate for multiple sclerosis.

[0193] Examples of autoantigens associated with autoimmune disorders include the thyroid stimulating hormone receptor of the thyroid gland (Graves' disease), thyroid antigens such as thyroid peroxidase (Hashimoto's thyroiditis), beta cell antigens such as glutamic acid decarboxylase and insulin (type I diabetes), cytochrome P450 antigens (Addison's disease), myelin proteins such as myelin basic protein (multiple sclerosis), uveal antigens (uveitis), H +These include gastric parietal cell antigens such as ATPase and intrinsic factor (pernicious anemia), transglutaminase (gluten enteropathy), cardiomyocyte proteins such as myosin (myocarditis, rheumatic heart disease), platelet antigens such as GP IIb / IIIa (idiopathic thrombocytopenic purpura), erythrocyte membrane proteins (autoimmune hemolytic anemia), neutrophil membrane proteins (autoimmune neutropenia), basement membrane antigens such as type IV collagen α3 chain (Goodpasture's disease), intrahepatic bile duct / mitochondrial antigens such as 2-oxoacid dehydrogenase complex (primary biliary cirrhosis), hepatocyte antigens such as cytochrome P450 and 206 (autoimmune hepatitis), acetylcholine receptors (myasthenia gravis), and desmoglein (pemphigus and other bullous diseases). The disorder listed in parentheses next to each autoantigen indicates the autoimmune disorder with which the indicated autoantigen is usually associated.

[0194] The compounds of the present disclosure and compositions described herein are expected to be useful adjunctive therapies for antigen therapy, e.g., gene therapy, for example, by inhibiting undesired immune responses to the antigen therapy and / or by allowing administration and / or repeated administration of the antigen therapy. Another embodiment is a method of treating a disease, disorder, or condition in a subject in need of treatment with antigen therapy, comprising administering a compound of the present disclosure to the subject, e.g., in the form of a composition described herein. In some aspects, the compound of the present disclosure is administered in an amount sufficient to immunotolerize the subject to the antigen therapy. In some aspects, the method further comprises administering (e.g., co-administering) an antigen therapy (e.g., a therapeutically effective amount of the antigen therapy) to the subject, e.g., simultaneously or sequentially with the compound of the present disclosure. Some aspects include administering to the subject a composition described herein comprising a compound of the present disclosure and an antigen therapy, e.g., a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and an antigen therapy. In some aspects, the antigen therapy and the compound of the present disclosure are administered to the subject in separate formulations.

[0195] In some aspects, the antigen therapy is an antibody therapy (e.g., monoclonal antibody therapy), including chimeric antibody therapy, humanized antibody therapy, and fully human antibody therapy. Specific examples of antibody therapy include anti-tumor necrosis factor (anti-TNF) therapy, such as adalimumab (Humira®; for rheumatoid arthritis, juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, Crohn's disease, ulcerative colitis, plaque psoriasis, hidradenitis suppurativa, uveitis) and infliximab (Remicade®; for Crohn's disease, pediatric Crohn's disease, ulcerative colitis, pediatric ulcerative colitis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis), golimumab (Humira®; for rheumatoid arthritis, juvenile idiopathic arthritis, juvenile idiopathic arthritis, ankylosing spondylitis, psoriatic arthritis, plaque psoriasis), golimumab (Humira®; for rheumatoid ... mab (Simponi®, for rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, polyarticular juvenile idiopathic arthritis), etanercept (Enbrel®, for rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, plaque psoriasis), and certolizumab pegol (Cimzia®, for Crohn's disease, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, non-radiographic axial spondyloarthritis, plaque psoriasis).

[0196] In some embodiments, the antigen therapy is a protein replacement therapy, e.g., enzyme replacement therapy. Examples of protein replacement therapy include replacement therapy for clotting disorders, such as Factor VIII and Factor IX for hemophilia A and B, enzyme replacement therapy for lysosomal storage diseases, such as alglucosidase alfa (Myozyme® and Lumizyme®) for Pompe disease, alpha-L-iduronidase for Hurler syndrome, and adenosine deaminase for adult adenosine deaminase deficiency.

[0197] In some embodiments, the antigen therapy is a gene therapy. Gene therapy typically works by one of three mechanisms: (1) by providing the subject with a healthy copy of the disease-causing gene (e.g., as voretigene neparvovec-rzyl (Luxturna®) does), (2) by inactivating the disease-causing gene (e.g., ASO and siRNA), or (3) by introducing a gene into the body to help treat the disease. Gene therapy includes DNA (e.g., antisense oligonucleotides (ASO)) and / or RNA (e.g., siRNA), which can be delivered to the subject in vivo or ex vivo via a variety of products. In vivo gene delivery products include plasmid DNA, viral vectors (AAV, such as AAV9), and non-viral vectors, such as bacterial vectors or lipid nanoparticles. Other examples of non-viral vectors suitable for in vivo gene delivery include exosomes, polymer particles, inorganic particles, and lipid-polymer hybrid particles. Ex vivo gene delivery products include subject-derived cellular gene therapy products. Gene therapy also includes gene editing technologies, such as CRISPR. Gene editing technologies such as CRISPR can be conveniently delivered to a subject via any of the in vivo gene delivery products described herein.Specific examples of gene therapy include the voretigene neparvovec-rzyl (Luxturna®, for retinal dystrophy) and the onasemnogene abeparvovec-xioi (Zolgensma®, for pediatric spinal muscular atrophy).

[0198] In some embodiments, gene therapy includes DNA and / or RNA and viral vectors. In some embodiments, the viral vector is derived from an adeno-associated virus (AAV), such as a recombinant AAV. In some embodiments, the AAV is AAV9. Other examples of viral vectors suitable for use with the present disclosure include viral vectors derived from retroviruses, herpes viruses, adenoviruses, lentiviruses, rabies viruses, lentiviruses, VSV, poxviruses (e.g., vaccinia viruses, smallpox viruses, canarypox), reoviruses, Semliki Forest viruses, yellow fever viruses, Sindbis viruses, togaviruses, baculoviruses, bacteriophages, alphaviruses, and flavaviruses.

[0199] In some embodiments, the antigen therapy is a cell therapy. One example of a cell therapy is axicabtagene ciloleucel (Yescarta®, for relapsed or refractory large B-cell lymphoma). Another example of a cell therapy is CAR-T cells.

[0200] Alloantigens are antigens that are present in some but not all individuals of a species and are recognized as foreign by those who do not possess them, and are often the basis of transplant rejection.Accordingly, another embodiment is a method for treating graft-versus-host disease in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure or a composition described herein.

[0201] Examples of alloantigens include, but are not limited to, major histocompatibility complex (MHC) class I and class II antigens, minor histocompatibility antigens, endothelial glycoproteins such as blood group antigens, and carbohydrate determinants.

[0202] Another embodiment is a method of promoting wound healing in a subject in need of treatment, comprising administering to the subject a therapeutically effective amount of a compound of the present disclosure, e.g., in the form of a composition described herein.

[0203] In some embodiments of any method described herein, the method further comprises administering an antigen or its immunogenic fragment to the subject. In some further embodiments, the antigen or its immunogenic fragment is co-administered with the compound of the present disclosure. For example, it may be sometimes desirable to induce antigen-specific immune tolerance (e.g., when the compound of the present disclosure is administered to tolerize the subject to antigen therapy). When antigen-specific immune tolerance is desired, the antigen or its immunogenic fragment is preferably co-administered with the compound of the present disclosure, for example in the form of a composition as described herein.

[0204] As used herein, "co-administer," "co-administration," and the like refer to the simultaneous or near simultaneous but sequential administration of two or more agents (e.g., a compound of the disclosure and an antigen) via the same route of administration at the same or near the same site on a subject's body.

[0205] When co-administration is simultaneous (e.g., concurrent), the first agent (e.g., a compound of the present disclosure) and the second agent (e.g., an additional therapeutic agent, an antigen, or an immunogenic fragment thereof) can be present in separate formulations or in the same formulation. Alternatively, the first and second agents can be administered sequentially as separate compositions. When co-administration is sequential, administration of the subsequent composition(s) is performed within 24 hours, preferably within 12 hours, for example, within 10 hours, 5 hours, 4 hours, 3 hours, 2 hours, 60 minutes, 30 minutes, 15 minutes, 10 minutes, or 5 minutes, of administration of the first composition. Usually, when co-administration is sequential, administration of the next composition(s) follows immediately after completion of administration of the first composition, taking into account the actions that the clinician or subject administering the composition may need to take to prepare for administration of the next composition(s).

[0206] When co-administration is oral, administration site is mouth, and whether co-administered two or more drugs are administered in a single formulation or in separate formulations, they are orally administered at the same site.However, when co-administration is by injection of two or more compositions, administration site is more generally approximately the same.In such a situation, the anatomical sites of administration are generally less than 2 inches apart from each other, for example, less than about 0.5 inches, less than about 1 inch, or less than about 1.5 inches apart from each other.

[0207] In some aspects, the antigen or immunogenic fragment thereof and the compound of the present disclosure are co-administered. In a further aspect, the administration of the antigen or immunogenic fragment thereof precedes the administration of the compound of the present disclosure. In an alternative further aspect, the administration of the compound of the present disclosure precedes the administration of the antigen or immunogenic fragment thereof. In yet another further aspect, the administration of the compound of the present disclosure and the antigen or immunogenic fragment thereof are simultaneous.

[0208] Co-administration can be by any route of administration described herein. In some aspects, the compounds of the present disclosure and the antigen or immunogenic fragment thereof are co-administered orally. In some aspects, the compounds of the present disclosure and the antigen or immunogenic fragment thereof are co-administered subcutaneously.

[0209] Without wishing to be bound by any particular theory, it is believed that it may be desirable for the subject's immune system to encounter the antigen and the compound of the present disclosure together, or for the antigen and the compound of the present disclosure to be "co-presented" to the subject's immune system. When the compound of the present disclosure is co-administered with the antigen, and the antigen is a protein, such as in protein replacement therapy, co-administration, for example by injecting the antigen and the compound of the present disclosure in separate formulations, is expected to provide effective co-presentation of the compound of the present disclosure and the antigen to the subject's immune system. In such applications, the compound of the present disclosure may, but need not, be incorporated into lipid particles. In a preferred embodiment of such applications, the co-administration is subcutaneous administration, for example by injection. In applications involving the delivery of gene therapy (e.g., gene therapy including DNA and / or RNA), it may be desirable to formulate the gene therapy and the compound of the present disclosure into lipid particles that include the gene therapy and the compound of the present disclosure to facilitate effective co-presentation of the gene therapy and the compound of the present disclosure to the subject's immune system. In preferred embodiments, such particles are formulated for oral and / or parenteral (eg, subcutaneous, intramuscular, intravenous, intradermal) administration, eg, by injection.

[0210] Also, without wishing to be bound by any particular theory, it is believed that certain compounds of the present disclosure identified herein are embedded in liposomes.For example, the use of such compounds according to the methods disclosed herein can be advantageous in aspects in which the effective co-presentation of the compounds of the present disclosure and antigens to the immune system of a subject is promoted by incorporating the compounds of the present disclosure and antigens into lipid particles that contain the compounds of the present disclosure and antigens.

[0211] The compounds of the present disclosure may also be administered in combination with one or more non-antigenic therapies to treat a disease, disorder, or condition. When administered "in combination" with such a non-antigenic therapy, the compounds of the present disclosure may be administered before, after, or simultaneously with the other therapy(s) (e.g., additional therapeutic agent(s)). When administered simultaneously (e.g., concurrently), the compounds of the present disclosure and the other therapy may be in separate formulations or in the same formulation. Alternatively, the compounds of the present disclosure and the other therapy may be administered as separate compositions, at about the same time or sequentially at different times. When the compounds of the present disclosure and the other therapy (e.g., therapeutic agent) are administered as separate formulations or compositions, the compounds of the present disclosure and the other therapy may be administered by the same route of administration or by different routes of administration. A skilled clinician can determine the appropriate timing for administering each therapy used in combination (e.g., sufficient timing to allow for overlap of the pharmaceutical effects of the therapies). Typically, the combination therapy provides the beneficial effects of the drug combination in treating the disease, condition, or disorder described herein.

[0212] In some aspects, the methods described herein further include administering to the subject an additional non-antigenic therapy(s) (e.g., a therapeutically effective amount), e.g., in combination with a compound disclosed herein or a composition described herein. In some aspects, the compound or composition disclosed herein is administered prior to the additional therapy(s). In some aspects, the compound or composition disclosed herein is administered after the additional therapy(s). In some aspects, the compound or composition disclosed herein is administered simultaneously with the additional therapy(s).

[0213] The therapeutically effective amount of the agent to be administered can be determined by a clinician of ordinary skill in the art using the guidance provided herein and other methods known in the art. For example, a suitable dose may be about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.01 mg / kg to about 1 mg / kg body weight per treatment. Determining the dosage for a particular agent, subject, and disease is well within the capabilities of a person of ordinary skill in the art. Preferably, the dosage will not cause adverse side effects or will cause minimal side effects.

[0214] The compounds of the present disclosure, compositions described herein, antigens, or other therapeutic agents can be administered via a variety of routes of administration, including, for example, oral, feeding, topical, transdermal, rectal, parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous injection, and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal instillation), depending on the compound, antigen, and / or therapeutic agent, respectively, and the particular disease to be treated. Administration can be local or systemic. The preferred mode of administration can vary depending on the particular compound or agent.

[0215] In some embodiments, administration (e.g., of a compound of the present disclosure or a composition and / or antigen described herein) is oral. In some embodiments, administration (e.g., of a compound of the present disclosure or a composition and / or antigen described herein) is intravenous. In some embodiments, administration (e.g., of a compound of the present disclosure or a composition and / or antigen described herein) is subcutaneous.

[0216] The disclosed compounds or compositions described herein can be administered prophylactically according to the methods disclosed herein, as well as when the disclosed compounds or compositions described herein are co-administered with antigen therapy to a subject with no known immune intolerance to antigen therapy. The disclosed compounds or compositions described herein can also or alternatively be administered therapeutically according to the methods disclosed herein, as when a subject has demonstrated immune intolerance to an antigen (e.g., allergic reaction, graft rejection). Thus, in some embodiments, the subject does not have a known immune intolerance to the antigen, for example, because the subject is naive to the antigen. In some embodiments, the subject does not have a known immune intolerance to the antigen after the antigen is administered and / or after exposure to the antigen. In some embodiments, the subject is immune intolerant to the antigen, for example, develops immune intolerance after administration and / or exposure to the antigen, or is inherently immune intolerant to the antigen.

[0217] The methods described herein aim to reduce immune intolerance to an antigen over the long term of a subject's life, e.g., for the period of time necessary to treat a disease, disorder, or condition with an antigen therapy described herein. Thus, in some aspects of the methods described herein, the method further comprises administering an antigen or an immunogenic fragment thereof (e.g., an antigen therapy, such as a therapeutically effective amount of an antigen therapy) to the subject in the absence of a compound of the present disclosure or a composition described herein.

[0218] However, the subject's immune intolerance may increase over time following the methods described herein, e.g., following subsequent exposure(s) to the antigen. In such cases, the methods described herein can be repeated, e.g., in the same manner that "booster" vaccinations are repeated, to re-tolerize the subject to the antigen.

[0219] The compounds of the present disclosure, or other therapeutic agents described herein, can be administered via a variety of routes of administration, including, for example, oral, feeding, topical, transdermal, rectal, parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous injection, intradermal injection), intravenous infusion, and inhalation (e.g., intrabronchial, intranasal or oral inhalation, intranasal instillation) routes of administration, depending on the compound and the particular disease to be treated. Administration can be local or systemic. In some embodiments, administration (e.g., of a compound of the present disclosure) is oral. In some embodiments, administration (e.g., of a compound of the present disclosure) is intravenous. Preferred modes of administration can vary depending on the particular compound or agent. Typically, a compound of the present disclosure or other therapeutic agent is administered about 1 to about 6 times per day (e.g., 1, 2, 3, 4, 5, or 6 times), also or alternatively, as an infusion (e.g., continuous infusion). In some aspects, administration (e.g., of a compound of the present disclosure) is QD or BID (e.g., QD). In some embodiments, administration (eg, of a compound of the present disclosure) is daily.

[0220] Orally administered liposomes as described herein can reach lymph nodes and co-localize with immune cells, including B cells and T cells, in the lymph nodes. Thus, also provided herein is a method of delivering a therapeutic agent (e.g., a compound of the present disclosure) to a lymph node of a subject, comprising orally administering to the subject a therapeutically effective amount of a composition containing a plurality of lipid particles (e.g., solid lipid particles), wherein each lipid particle comprises at least one phospholipid (e.g., C4-C in dimyristoylphosphatidylcholine (DMPC)). 30 Acyl chains, e.g., saturated C4-C 30 The lipid particle includes a lipid bilayer that contains a phospholipid containing an acyl chain, and a therapeutic agent (e.g., a compound of the present disclosure) that can be embedded in the lipid bilayer of the lipid particle.

[0221] The compounds of the present disclosure or other therapeutic agents can be administered at a dose ranging from about 0.001 mg / kg body weight to about 100 mg / kg body weight, or at a dose ranging from about 1 mg / dose to about 5,000 mg / dose every 4 to 120 hours, or according to the requirements of the particular agent. For example, a suitable dose can be about 0.001 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 100 mg / kg, about 0.01 mg / kg to about 10 mg / kg, or about 0.01 mg / kg to about 1 mg / kg body weight per treatment. In some embodiments, a suitable dose (e.g., daily dose) is about 0.1 mg / kg to about 10 mg / kg body weight per treatment, e.g., about 0.1 mg / kg to about 5 mg / kg, about 0.1 mg / kg to 2.5 mg / kg, or about 0.2 mg / kg to about 2.4 mg / kg body weight. A suitable dose may be about 0.001 mg / dose to about 100 mg / dose, about 0.01 mg / dose to about 100 mg / dose, about 0.1 mg / dose to about 50 mg / dose, about 0.1 mg / dose to about 10 mg / dose, about 0.5 mg / dose to about 50 mg / dose, about 1 mg / dose to about 10,000 mg / dose, about 1 mg / dose to about 7,500 mg / dose, about 1 mg / dose to about 5,000 mg / dose, about 10 mg / dose to about 2,500 mg / dose, or about 100 mg / dose to about 1,000 mg / dose. In some embodiments, a suitable dose (e.g., daily dose) is about 10 mg / dose to about 1,000 mg / dose, e.g., about 15 mg / dose to about 1,000 mg / dose, about 10 mg / dose to about 500 mg / dose, about 10 mg / dose to about 250 mg / dose, about 15 mg / dose to about 150 mg / dose, about 15 mg / dose, about 30 mg / dose, about 50 mg / dose, about 100 mg / dose, about 125 mg / dose, or about 150 mg / dose.

[0222] Doses lower or higher than those listed above may be necessary.The specific dosage and treatment regimen for any particular patient depends on a variety of factors, including, for example, the activity of the specific drug used, age, body weight, general health, sex, diet, administration time, excretion rate, drug combination, severity and course of disease, condition or symptom, predisposition of the subject to disease, condition or symptom, and the judgment of the treating physician.Determining the dosage for a particular drug, subject, and disease, disorder, or condition is within the capabilities of those skilled in the art. EXAMPLES

[0223] Example 1. Design of TIM agonists The co-crystal structure of dicaproylphosphatidylserine (PS) bound to TIM4 was published in 2007, and the "apo" structure of TIM4 in the presence of high concentrations of tartaric acid as a precipitant was also published in the same publication. Herein, we describe compounds designed to mimic PS, the endogenous ligand of the TIM-4 receptor, which induces an agonist effect.

[0224] To guide compound design, the co-crystal structure of dicaproyl PS bound to TIM4 was used to define the "active" 3D conformation of the metal ion-dependent ligand binding site (MILIBS) and the key interactions between the ligand and the MILIBS site. Docking was used to calculate the binding affinity of the compounds to TIM4.

[0225] In preparation for docking, the X-ray crystal structure of the protein (PDB ID: 3BIB) was downloaded from the PDB database. Phosphatidylserine (PS), an endogenous ligand bound to the protein, was removed from the binding site. All water molecules were removed except for the water molecule that is in the binding site and forms a bridge between PS and the protein through hydrogen bond interactions. Charges were calculated for all atoms in the protein at physiological pH (7.4), which is the same pH used in the in vitro experiments to measure binding affinity. Charges of atoms of all docked molecules were also calculated at physiological pH. The docking method was validated by redocking PS into its binding site. Both rigid and flexible docking was performed for all molecules, and the top 20 binding poses were identified.

[0226] To validate the docking method, the endogenous ligand PS, which had been previously removed from the protein complex, was redocked into the binding site. The pose closest to the correct (native) pose had an RMSD of 2.213 Å and was identified among the top 11 poses. The binding affinity of PS to the protein was computed through docking to be 6.0 kcal / mol.

[0227] To calculate the binding affinity of the proposed compounds to TIM4, the proposed structures were docked. The range of binding affinity values ​​for the proposed structures and the top 20 binding poses obtained for the proposed structures using rigid (and flexible) docking are shown in Table 1. [Table 1-1] [Table 1-2]

[0228] Docking models showed that the polar groups of compound 2 aligned similarly to the natural ligand and formed interactions with the protein's binding site similar to those of the natural ligand, PS.

[0229] Example 2. In vitro dose-response analysis of compounds 1-3 in mouse T cells All animal studies were performed in accordance with Tufts University / Tufts Medical Center & Human Nutrition Research Center on Aging under IACUC number B2020-91. Animals were housed four per cage and had free access to food and water. Anti-FoxP3 and anti-CD4 for flow cytometry were obtained from eBioscience. Compounds 1-3 were formulated with 10% DMPC in PBS. PS liposomes were synthesized with a PS:lipid molar ratio of 30:70.

[0230] Spleen cells from naive C57BL / 6 mice were stained with CFSE and 2 × 10 5 Cells / well were seeded and each compound was diluted to 1e using a log2 dilution scheme. -4 μM~3.0e -9 Compounds were administered at 0.1 μM. Cells were incubated for 72 h before FoxP3+ / CD4+ T cell phenotypic analysis. Flow cytometer analysis was performed to evaluate the change in the percentage of FoxP3+ / CD4+ T cells as a function of compound and dose. Concentration responses were fitted to a four-parameter log-logistic model using the “drc” package in “R”. Model fitting yielded EC 50 and E.C. 90 I got both.

[0231] At the end of the incubation period, the cells were observed microscopically and all cells appeared healthy and suitable for flow cytometry analysis.

[0232] A visual inspection of the model fit suggested that a four-parameter dose-response model was sufficient to capture the data for the compounds tested. Figure 1 shows the model that resulted from fitting the data to a four-parameter dose-response model. Table 2 shows the EC values ​​obtained from the model fitting. 50 and E.C. 90 This is a summary of the values ​​of . [Table 2]

[0233] Lower and upper limits of the percentage of FoxP3+ / CD4+ T cells as a function of increasing dose of each compound were obtained from model fitting. The mean FoxP3+ / CD4+ T cell values ​​increased from a mean (SEM) of 1.32% (0.12), 1.47% (0.49), 2.20% (0.18), and 1.92% (0.10) for low doses of Compound 3, Compound 1, Compound 2, and PS liposomes, respectively, to a mean (SEM) of 3.48% (153), 5.18% (4.36), 4.54 (0.14), and 4.87 (0.53) for high doses of Compound 3, Compound 1, Compound 2, and PS liposomes, respectively.

[0234] Compounds 1-3 produced a dose-dependent increase in FoxP3+ / CD4+ T cells, but only compound 2 showed a clear plateau. Treatment with PS liposomes also produced a clear plateau, but not EC 50 and E.C. 90 The potency of compound 3 measured by was two and one orders of magnitude higher (more potent) than that of PS, respectively. This is consistent with the molecular modeling results described in Example 1, which showed that compound 2 had the lowest docking score among compounds 1 to 3, while compounds 1 and 3 had docking scores close to that of PS.

[0235] Compound 3 is attractive due to its increased potency and aqueous solubility compared to PS.

[0236] Example 3. Dose Pharmacodynamics of Compound 2 Following Single Oral Ascending Dose in Mice This study evaluated the pharmacodynamic (PD) effects of a single subcutaneous dose of Compound 2 in mice following 5 days of treatment.

[0237] All animal studies were performed in accordance with Tufts University / Tufts Medical Center & Human Nutrition Research Center on Aging under IACUC number B2020-91. Animals were housed four per cage and had free access to food and water. Compound 2 was formulated using 10% DMSO in PBS buffer.

[0238] This study was designed to evaluate the PD effect of a single dose of Compound 2 in mice. Animals were administered a single oral gavage dose according to group allocation. Details of the treatment groups are shown in Table 3. Five days after oral dosing, animals were sacrificed and spleens were removed for T cell phenotyping. Figure 2A is a schematic diagram of the study design. [Table 3]

[0239] For ex vivo analysis, spleens were harvested for splenocyte analysis and single cell suspensions were prepared for cell phenotyping by flow cytometry. Cells were stained and gated for CD4. The percentage of FoxP3+ / CD4+ T cells and the percentage of FoxP3+ / NRP1+ were assessed using flow cytometry. For dose-response analysis, dose-response was fitted to four- and five-parameter log-logistic models using the “drc” package in “R”. Model fitting yielded EC 50 and E.C. 90 I got both.

[0240] The percentage of FoxP3+ / CD4+ T cells increased in a dose-dependent manner, from a mean of 10.7% (SD=2.2) in the lowest dose group to 25.4% (SD=0.31) in the highest dose group. Data were fitted to a four-parameter dose-response model. ED 50 4.88μg, ED90 The ED was estimated to be 82.1 μg. Figure 2B shows the fit of the dose-PD model and the associated confidence intervals around the mean of the model-predicted dose-PD. Overall, the model was able to capture the data well. Figure 2B shows the ED 50 and ED 90 Both values ​​are shown.

[0241] The FoxP3+ / CD4+ T cell population was further analyzed for neuropilin-1 (NRP1) expression. Several reports have shown that NRP1 expression on T cells correlates with a state of immune tolerance. NRP1 expression was observed at low doses of Compound 2. This was a nearly binary response, with ED 50 is 4.32E -2 was estimated to be μg (Figure 2C).

[0242] The data from this study support the hypothesis that Compound 2 can induce a tolerogenic immune response in a dose-dependent manner. The number of FoxP3+ / CD4+ T cells (T-reg) increased by 150% from the lowest dose group to the highest dose group. Without being bound to any particular theory, an increase of more than 100% (e.g., a doubling of FoxP3+ / CD4+ T cells) is believed to increase tolerance to the target antigen. Therefore, Compound 2 was administered at its ED 50 Repeated administration at these doses appears to be sufficient to induce tolerance.

[0243] Example 4. MOG 35-55 The therapeutic effect of Compound 2 in an induced mouse EAE model compared with standard-of-care anti-α4 mAb and glatiramer acetate Multiple sclerosis is a chronic, often disabling disease of the human central nervous system (CNS). When tolerance to myelin is lost, the immune system attacks the myelin, resulting in the clinical manifestations of the disease. This is mediated by pathogenic autoreactive T cells that recognize autoantigenic peptides complexed with major histocompatibility complex (MHC) molecules. There is no known cure for MS, but blocking the ability of autoreactive T cells to invade the CNS has proven to be an effective treatment option to ameliorate MS symptoms. Anti-α4 mAbs such as Tysabri are approved for the management of MS, as is glatiramer acetate. Other treatment options rely on systemic immunosuppressants such as steroids.

[0244] Addressing the presence of autoreactive T cells and inducing their transition to more tolerogenic T cells may provide a curative therapy for MS. Compound 2 has been proposed as a T cell immunoglobulin mucin protein family receptor (TIM) agonist that can induce tolerogenic T cells. TIMs play an important role in adaptive and innate immune responses and are associated with the control of autoimmunity and cancer. Several ligands, including PS, are known to bind to TIMs.

[0245] The affinity of PS for different members of the TIM family varies substantially, with TIM3 having a lower affinity for PS than TIM4, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 109, 109, 109, 109, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,

[0246] The tolerogenic potential of PS has been exploited by tumors. For example, PS in the human ovarian tumor microenvironment can induce T cell signaling arrest. Moreover, PS-mediated T cell arrest was blocked by anti-PS antibodies. Taken together, published data on TIM and its role in immune tolerance suggest that TIM is a potential target for the treatment of autoimmune disorders.

[0247] This study evaluates the effect of the TIM agonist Compound 2 compared to standard of care anti-α4 mAb and glatiramer acetate on disease progression. Additionally, the study evaluates the dose response of Compound 2 in experimental autoimmune (allergic) encephalomyelitis (EAE) and the effect of switching from anti-α4 mAb treatment to Compound 2 treatment.

[0248] All animal studies were conducted in accordance with Tufts University / Tufts Medical Center & Human Nutrition Research Center on Aging under IACUC number B2020-91. Animals were housed in designated facilities at the Tufts University-Tufts Medical Center & Human Nutrition Research Center on Aging. Animals were housed four per cage and had free access to food and water.

[0249] Materials and reagents are shown in Table 4. [Table 4]

[0250] Experimental autoimmune (allergic) encephalomyelitis (EAE) is considered the best preclinical model of multiple sclerosis (MS). EAE is characterized by an immune response against CNS tissue and can be induced by immunizing animals against CNS proteins. In an active EAE model, MOG emulsified in complete Freund's adjuvant (CFA) is administered to mice. 35-55 Mice are immunized with the peptides by subcutaneous injection (0.1 mL of emulsion / mouse) at the base of the tail under anesthesia. On the day of injection (day 0) and 2 days later, mice are injected intraperitoneally with pertussis toxin (PT) in PBS at 600 ng / mouse / dose (0.1 mL).

[0251] Symptoms typically develop in mice 9-14 days after immunization (day 0). Daily observation and scoring of mice begins on day 9 and continues until the end of the study. Table 5 details expected clinical signs and the scoring criteria used in this study. [Table 5-1] [Table 5-2]

[0252] Figure 3A shows the outline of the study. To evaluate the therapeutic effect of Compound 2, treatment was started after the first clinical symptoms were observed. Once a mouse had a clinical score of 1, it was randomly assigned by a random sampling algorithm to the treatment groups listed in Figure 3A or to a group receiving placebo. The treatment groups are shown in Table 6. Mice in all groups were monitored and scored daily for clinical symptoms. [Table 6]

[0253] At the end of the study, spleens were removed from a representative sample of mice from each group for splenocyte analysis. Single cell suspensions were prepared for cell phenotyping by flow cytometry. Cells were stained for CD4 + Gates were set for T cells. FoxP3 + / CD4 + The percentage of T cells was assessed by flow cytometry.

[0254] Figure 3B shows that compound 2 dose-dependently reduced MS clinical scores after disease onset. Figure 3C shows that a daily oral dose of 10 μg of compound 2 was as effective as the full dosing regimen of anti-α4 integrin, equivalent to Tysabri, a third-line drug used for severe MS, in controlling MS symptoms after disease onset in the mouse EAE model. Disease control after disease onset was also obtained by switching from anti-α4 integrin to a 10 μg oral dose of compound 2 after one or two injections of anti-α4 integrin. Figure 3D shows that all compound 2 doses and regimens were more effective than glatiramer acetate, an FDA-approved first-line treatment for mild MS, in controlling MS symptoms after disease onset. Example 5. Synthesis of Compounds 1 to 3

[0255] Compound 1 HCl, (S)-2-amino-3-(2-(4-(benzyloxy)phenyl)acetamido)propanoic acid hydrochloride, was synthesized according to the following synthetic route: [ka]

[0256] (S)-3-(2-(4-(benzyloxy)phenyl)acetamido)-2-((tert-butoxycarbonyl)amino)propanoic acid 1.3. To a solution of benzylphenylacetic acid derivative 1.1 (0.60 g) in DMF (6 mL) was added DIEA (0.95 mL) and HATU (0.99 g). The reaction mixture was stirred at room temperature for 15 min. Then Boc-Dap-OH (1.2, 0.506 g) was added and the reaction mixture was stirred at room temperature for another 2 h. Ice was added and the solution was acidified to pH 3. The separated product was extracted with EtOAc (2×25 mL), washed with water, dried (Na2SO4) and the solvent was removed under reduced pressure. The product was applied to a 10 g precolumn attached to a 25 g Gold column and eluted with 50% (1% AcOH / EtOAc) / hexane for 5 min followed by up to 1% AcOH / EtOAc for 20 min. Fractions were pooled after TLC. Traces of acetic acid were removed by dissolving the residue in EtOAc, adding n-heptane, and removing the volatiles under reduced pressure. This process was repeated three times with minimal EtOAc and n-heptane. The product was further purified by crystallization from EtOAc / hexane.

[0257] (S)-2-Amino-3-(2-(4-(benzyloxy)phenyl)acetamido)propanoic acid, 1, HCl. A solution of compound 1.3 (0.218 g) in 4N HCl / dioxane (7 mL) was stirred at 0° C. for 2 h. It was then warmed to room temperature and concentrated to half the volume under reduced pressure. Ether was then added to precipitate the product. The product was filtered, washed with excess ether, and traces of solvent were removed under high vacuum overnight to give compound 1 HCl.

[0258] Compound 2 can be prepared by using a process that starts with O-alkylation of methyl(4-hydroxyphenyl) acetate (3.1) with 1-(bromomethyl)naphthalene (2.1, e.g., in the presence of an inorganic base such as cesium carbonate in a polar aprotic solvent such as DMF) to give 2.2. Ester hydrolysis of 2.2 (e.g., using potassium hydroxide in water) gives 2.3. Amide coupling of 2.3 with an α-N-protected (S)-2,3-diaminopropionic acid such as Boc-Dap-OH (1.2) in the presence of a cross-coupling reagent such as 1,1'-carbonyldiimidazole (CDI) in a polar aprotic solvent such as DMF gives 2.4. N-deprotection of 2.4 (e.g., using HCl in 1,4-dioxane) gives compound 2.

[0259] Compound 2 HCl, (S)-2-amino-3-(2-(4-(naphthalen-1-ylmethoxy)phenyl)acetamido)propanoic acid, HCl, was synthesized according to the following synthetic route: [ka]

[0260] Methyl 2-(4-(naphthalen-1-ylmethoxy)phenyl)acetate 2.2. To a solution of compound 3.1 (0.810 g) in DMF (6 mL) was added Cs2CO3 (2.38 g) and 1-bromomethylnaphthalene (1.13 g). The reaction mixture was stirred at room temperature overnight. The reaction mixture was quenched with ice and water. The solution was extracted with EtOAc (2×35 mL), washed with water (2×20 mL) and brine (20 mL), dried (Na2SO4), and the solvent was removed under reduced pressure. The crude product was applied to a 25 g precolumn attached to a 25 g Gold column and eluted with 5% EtOAc / Hexanes for 10 min, followed by 5-30% EtOAc / Hexanes over 20 min.

[0261] 2-(4-(Naphthalen-1-ylmethoxy)phenyl)acetic acid 2.3. To a solution of compound 2.2 (1.32 g) in MeOH (60 mL) was added KOH (1.69 g) in water (15 mL). The reaction mixture was stirred at room temperature overnight. The solution was concentrated under reduced pressure and ice was added before acidifying to pH 3. The precipitated solid was filtered, washed with water, dried under suction, and then dried under high vacuum overnight.

[0262] (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(4-(naphthalen-1-ylmethoxy)phenyl)acetamido)-propanoic acid 2.4. To a solution of naphthylphenylacetic acid derivative 2.3 (0.500 g) in DCM (15 mL) was added DIEA (0.90 mL) and EDAC (0.328 g). The reaction mixture was stirred at room temperature for 15 min. Then Boc-Dap-OH (0.349 g) was added and the reaction mixture was stirred at room temperature for 3 days. The reaction mixture was washed with water (2×20 mL) and brine (15 mL), dried (Na2SO4) and the solvent was removed under reduced pressure. The product was applied to a 10 g precolumn attached to a 25 g Gold column and eluted with 30% (1% AcOH / EtOAc) / Hexane for 5 min followed by up to 1% AcOH / EtOAc for 20 min. Fractions were pooled after TLC. The residue was dissolved in EtOAc, n-heptane was added, and the volatiles were removed under reduced pressure to remove traces of acetic acid. This process was repeated three times with minimal EtOAc and n-heptane.

[0263] (S)-2-Amino-3-(2-(4-(naphthalen-1-ylmethoxy)phenyl)acetamido)propanoic acid, 3, HCl. A solution of compound 2.4 (0.289 g) in 4N HCl / dioxane (7 mL) was stirred at 0° C. for 2 h. It was then warmed to room temperature and concentrated to half the volume under reduced pressure. Ether was then added to precipitate the product. The product was filtered, washed with excess ether, and traces of solvent were removed under high vacuum overnight.

[0264] Compound 3, (S)-2-amino-3-(2-(4-(benzhydryloxy)phenyl)acetamido)propanoic acid, was synthesized according to the following synthetic route: [ka]

[0265] Methyl 2-(4-(benzhydryloxy)phenyl)acetate 3.2. To a solution of compound 3.1 (1.00 g) in dimethylformamide (DMF, 9 mL) was added a solution of Cs2CO3 (2.94 g) and benzhydryl bromide (1.56 g) in DMF (5 mL). The reaction mixture was stirred at room temperature (RT) overnight. The reaction mixture was quenched with ice and water. The solution was extracted with ethyl acetate (EtOAc, 2×35 mL), washed with water (20 mL) and brine (20 mL), dried (over Na2SO4), and the solvent was removed under reduced pressure. The isolated product was applied to a 25 g precolumn attached to a 25 g Gold column and eluted with 10% EtOAc / Hexanes for 5 min, followed by 10–30% EtOAc / Hexanes for 30 min.

[0266] 2-(4-(Benzhydryloxy)phenyl)acetic acid 3.4. To a solution of compound 3.3 (0.800 g) in methanol (MeOH, 25 mL) was added a solution of KOH (0.945 g) in water (10 mL). The reaction mixture was stirred at room temperature overnight. The solution was concentrated under reduced pressure and ice was added before acidifying to pH 3. The precipitated solid was filtered, washed with water, dried under suction, and then dried under high vacuum overnight.

[0267] (S)-2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-3-(2-(4-(benzhydryloxy)phenyl)acetamido)-propanoic acid 3.6. To a solution of the benzhydrylacetic acid derivative 3.4 (0.212 g) in DMF (3 mL) was added diisopropylethylamine (DIEA, 0.35 mL) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 0.279 g). The reaction mixture was stirred at room temperature for 25 min. Then Fmoc-Dap-OH (0.217 g) was added and the reaction mixture was stirred at room temperature for another 2 h. Ice was added and the solution was acidified to pH 3. The precipitated solid was filtered, washed with water and dried in a vacuum desiccator overnight. The product was applied to a 5 g precolumn attached to a 25 g Gold column and eluted with 0.25% AcOH / DCM for 5 min, followed by 0-5% MeOH / (0.25% AcOH-DCM) for 30 min. Fractions were pooled after thin layer chromatography (TLC). Traces of acetic acid were removed by dissolving the residue in EtOAc and adding n-heptane followed by removal of volatiles under reduced pressure. This process was repeated twice with minimal DCM and n-heptane.

[0268] (S)-2-Amino-3-(2-(4-(benzhydryloxy)phenyl)acetamido)propanoic acid 3. To a solution of compound 3.6 (0.220 g) in DMF (3.5 mL) was added piperidine (0.80 mL) and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was poured onto ice. The separated solid and the aqueous mixture were extracted with ether (3×20 mL). The separated aqueous layer was acidified to pH 3 and the separated solid was filtered and washed with excess water. It was dried under vacuum desiccator overnight.

[0269] Example 6. Synthesis of Compound 8 Compound 8 was synthesized using the following reaction. [ka]

[0270] 5-((Tert-Butyldimethylsilyl)oxy)-2-methylpyridine. To a solution of 6-methylpyridin-3-ol (2.50 g) in DMF (40 mL) was added imidazole (3.12 g). The reaction mixture was stirred under nitrogen atmosphere. To this solution was slowly added a solution of tert-butyldimethylsilyl chloride (TBDMS-Cl, 4.66 g) in DMF (10 mL). The reaction mixture was stirred at room temperature for 3 h. Thin layer chromatography (TLC) showed the reaction was complete. The reaction mixture was quenched with water. The solution was extracted with ethyl acetate (EtOAc, 2×100 mL), washed with water (2×30 mL) and brine (20 mL), dried over Na2SO4, and the solvent was removed under reduced pressure. The crude compound was applied to a precolumn attached to a 40 g gold column and eluted with 0-40% EtOAc / Hexane over 30 min. Fractions were pooled after TLC. The desired product was eluted with approximately 15-25% EtOAc / hexanes to give 5-((tert-butyldimethylsilyl)oxy)-2-methylpyridine (4.76 g). [ka]

[0271] Methyl 2-(5-((tert-butyldimethylsilyl)oxy)pyridin-2-yl)acetate. To a solution of 5-((tert-butyldimethylsilyl)oxy)-2-methylpyridine (2.31 g) in tetrahydrofuran (THF; 50 mL) at -78°C was slowly added lithium diisopropylamide (LDA) in THF (41.2 mL, 1 M). The reaction mixture was stirred at -78°C for another 10 min. Then a solution of Me2CO3 (3.5 mL) was added. The reaction mixture was stirred at -78°C for 1 h. The reaction mixture was quenched with saturated ammonium chloride solution (15 mL). The solution was extracted with EtOAc (2 x 75 mL), washed with water (2 x 30 mL) and brine (20 mL), dried over Na2SO4 and the solvent was removed under reduced pressure. The crude compound was applied to a precolumn attached to a 40 g gold column and eluted first with 5% EtOAc / Hexanes followed by up to 40% EtOAc / Hexanes over 60 min. Fractions were pooled after TLC and concentrated to give methyl 2-(5-((tert-butyldimethylsilyl)oxy)pyridin-2-yl)acetate (0.720 g). [ka]

[0272] Methyl 2-(5-hydroxypyridin-2-yl)acetate. To a solution of methyl 2-(5-((tert-butyldimethylsilyl)oxy)pyridin-2-yl)acetate (0.698 g) in dimethylformamide (DMF; 8 mL) was added CsF (0.75 g). The reaction mixture was stirred at room temperature overnight. The reaction mixture was diluted with EtOAc (25 mL) and the solution was washed with water (2×10 mL) and brine (10 mL), dried over Na2SO4, and the solvent was removed under reduced pressure to give methyl 2-(5-hydroxypyridin-2-yl)acetate (0.415 g). [ka]

[0273] Methyl 2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetate. To a solution of methyl 2-(5-hydroxypyridin-2-yl)acetate (0.405 g) in DMF (9 mL) was added Cs2CO3 (1.58 g) and 1-bromomethylnaphthalene (0.562 g). The reaction mixture was stirred at room temperature for 3 h. TLC showed the reaction was complete. The reaction mixture was quenched with ice and water. The solution was extracted with EtOAc (2×25 mL), washed with water (2×10 mL) and brine (15 mL), dried over Na2SO4, and the solvent was removed under reduced pressure. The crude compound was applied to a precolumn attached to a 12 g gold column and eluted with 10-40% EtOAc / Hexane over 40 min. The product was eluted with 30%-40% EtOAc / Hexane. Fractions were pooled after TLC and concentrated to give methyl 2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetate (510 mg). [ka]

[0274] 2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetic acid. To a solution of methyl 2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetate (0.495 g) in methanol (40 mL) was added KOH (0.50 g) in water (6 mL). The reaction mixture was stirred at room temperature overnight. TLC showed the reaction was complete. The solution was concentrated under reduced pressure and ice was added followed by acidification with 2N HCl (dropwise). The solution was extracted with EtOAc (2×100 mL), washed with brine and dried (Na2SO4). The solvent was removed under reduced pressure to give 2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetic acid (0.470 g). HPLC: 97.8%, MS: 294.1122 (M+1);316.0953 (M+23). [ka]

[0275] (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetamido)propanoic acid. To a solution of 2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetic acid (0.420 g) in DMF (10 mL) was added diisopropylethylamine (DIEA, 0.28 mL) and 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU, 0.561 g). The reaction mixture was stirred at room temperature for 2.5 hours. Then (S)-3-amino-2-(tert-butoxycarbonyl)aminopropionic acid (Boc-Dap-OH, 0.292 g) and DIEA (0.27 mL) were added and the reaction mixture was stirred at room temperature for 4 hours. TLC and high performance liquid chromatography (HPLC) showed the reaction was complete. The reaction was quenched with ice and the precipitated gummy material was extracted with EtOAc (2×75 mL). The combined organics were washed with water (2×50 mL) and brine (30 mL) and the solvent was removed under reduced pressure. The residue was recrystallized from EtOAc, filtered, washed with EtOAc (5 mL), followed by ca. 50% EtOAc / hexanes (20 mL) and air-dried under suction to give 126 mg of product.

[0276] MS: 666.3124 (M+H: 666.3139); 688.2954 (M+Na: 688.2959) indicated the major product was from the coupling of the desired product with Boc-Dap-OH. The compound was washed with EtOAc (30 mL) and the solvent was removed under vacuum. The solvent was removed from the mother liquor under reduced pressure and the crude product was applied to a 25 g precolumn attached to a 25 g Gold column and eluted with 2-10% (0.25% AcOH / DCM) / MeOH over 40 min. After TLC confirmation, fractions were pooled to give 59 mg of (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetamido)propanoic acid. MS: 480.2119 (M+H: 480.2134);502.1951 (M+Na: 502.1954). [ka]

[0277] (S)-2-amino-3-(2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetamido)propanoic acid. (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetamido)propanoic acid (0.050 g) in 4N HCl / dioxane solution (0.6 mL) was stirred at 0° C. for 3 h. It was then warmed to room temperature and concentrated to half the volume under reduced pressure (maximum 40° C.). Ether was then added to the colloidal solution. The separated product was filtered, washed with excess ether, and traces of solvent were removed under high vacuum overnight to give (S)-2-amino-3-(2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetamido)propanoic acid (40 mg).

[0278] HPLC: 96.3%, MS: 380.1605 (M+H: 380.1611);402.1418 (M+Na: 402.1430), 1 H NMR (700 MHz, dmso) δ 13.93 (s, 1H), 8.62 (s, 1H), 8.56 (s, 1H), 8.47 - 8.38 (m, 3H), 8.13 (d, J = 8.1 Hz, 1H), 7.98 (dd, J = 18.0, 8.1 Hz, 2H), 7.91 (s, 1H), 7.72 (d, J = 7.0 Hz, 1H), 7.63 - 7.52 (m, 4H), 5.71 (s, 2H), 4.02 (s, 1H), 3.82 (s, 2H), 3.65 (dd, J = 12.6, 7.0 Hz, 1H), 3.53 - 3.49 (m, 1H).

[0279] Example 7. Pharmacology of Compound 2 in Human PBMCs A series of experiments were performed to understand the pharmacology of compound 2 in human peripheral blood mononuclear cells (PBMCs).

[0280] 2×10 5 Total PBMCs or T cell-depleted PBMCs were cultured in 96-well plates for 5 days in the presence of increasing doses of Compound 2 ranging from 0 to 10 ng / ml. On day 5, cells were washed and stained for CD19, CD71, and IgM for 30 minutes on ice, after which cells were washed with PBS and stained with a viability dye to exclude dead cells. Results are shown in Figure 4A.

[0281] Compound 2 inhibits regulatory B cells (CD19 + CD71 + IgM + ). More specifically, Compound 2 enhanced the expression of regulatory markers (CD71, IgM) on human B cells (T cell-depleted PBMCs) in the absence of T cells, and the expression of these regulatory markers increased when T cells were present. This suggests that the effect of Compound 2 on regulatory B cells may be further enhanced by T cell / B cell interactions. For example, treatment with 10 ng / ml Compound 2 increased the percentage of B-regs in T cell-depleted PBMCs and whole PBMC cultures from 6.19% to 14.0% and 17.5%, respectively. Other regulatory B cell markers of interest include CD24, CD38, and CD27.

[0282] 2×10 5 Total PBMCs or sorted T cells (CD3 + ) were cultured in 96-well plates for 5 days in the presence of increasing doses of compound 2 ranging from 0 to 3 ng / ml. On day 5, cells were washed and stained for CD3 and CD4 on ice for 30 min, after which cells were washed with PBS and stained with a viability dye to exclude dead cells. Stained cells were fixed / permeabilized and further stained for Foxp3. Finally, cells were washed and fixed for data acquisition on an Attune NXT flow cytometer. Data were analyzed by FlowJov10. The results are shown in Figure 4B.

[0283] Treatment with 3ng / ml of Compound 2 increased the percentage of CD4+ / Foxp3+ T cells in cultures of isolated T cells (sorted) from 0.32% to 2.27% (an increase of 606%). In total PBMCs, treatment with 3ng / ml of Compound 2 increased the percentage of CD4+ / Foxp3+ T cells from 1.9% to 5.2% (an increase of 171%). These results indicate that Compound 2 acts directly on T cells to enhance Foxp3 expression in the presence and absence of other immune cells.

[0284] Total human PBMCs isolated from healthy human volunteers were cultured in vitro in a Th17 polarizing environment as follows. Day 0: 2×10 5 Cells / well were incubated in 96-well round-bottom well plates with (i) anti-CD3 / CD28 microbeads for T cell activation (1 bead / 10 cells), (ii) 10 ng / ml IL-6, 2 ng / ml TGFβ and 10 ng / ml IL-23, and (iii) 10 μg / ml anti-IFNγ and 10 μg / ml anti-IL-4 (final volume 200 μl). Day 4: Each well was split into two wells and 100 μl of medium containing the same cytokine / antibody cocktail used on day 0 was added to each well in the presence of increasing doses of Compound 2 (0, 0.1, 0.3, 1, and 3 ng / ml). Day 9: Cells were washed and stained with surface stains (CD3, CD4) followed by viability dye to exclude dead cells. Nuclear staining was performed after cell fixation and permeabilization for transcription factors Foxp3 and RORγt. Cells were washed and fixed for data acquisition on an Attune NXT flow cytometer. Data was analyzed by FlowJov10.

[0285] The results shown in Figure 4C show that compound 2 maintains Foxp3 expression and inhibits RORγt expression in a Th17 polarized environment (representing a proinflammatory environment) in vitro. In a strongly proinflammatory environment, treatment with compound 2 inhibited Th17 as evidenced by a statistically significant (t-test) decrease in the percentage of Th17 from an average of 5.8% to an average of 1.1%, corresponding to an 80% decrease in RORγt cells.

[0286] Total human PBMCs were isolated from six subjects with a confirmed diagnosis of MS. On day 0, 2 × 10 5 Cells / well were incubated in 96-well round-bottom well plates alone or with up to 2.5 μM compound 2 for 5 days. On day 5, cells were harvested, washed, and stained with surface stains (CD3, CD4) and then with viability dyes to exclude dead cells. Nuclear staining was performed after cell fixation and permeabilization for the transcription factor Foxp3. Cells were washed and fixed for data acquisition on an Attune NXT flow cytometer. Data was analyzed by FlowJov10. Results are shown in Figure 4D.

[0287] In FIG. 4D, blood-derived T cells from 5 of 6 MS patients responded to ex vivo treatment with Compound 2, as shown by an increase in the percentage of Foxp3+ / CD4+ T cells.

[0288] Example 8. MOG 35-55 Evaluation of the therapeutic efficacy of Compound 2 in an escalating treatment paradigm in an induced mouse EAE model All animal studies were conducted in accordance with Tufts University / Tufts Medical Center & Human Nutrition Research Center on Aging under IACUC number B2020-91. Animals were housed four per cage and had free access to food and water.

[0289] Table 7 shows the materials and reagents used in this experiment. [Table 7]

[0290] Compound 2 (0.03 mg / ml) was formulated with hydroxypropyl beta cyclodextrin (HβCD) 1:80 (Compound 2:HβCD) and stored at 4°C until use. Natalizumab, an anti-α4β1 integrin antibody, is a monoclonal antibody approved by the US Food and Drug Administration (FDA) for the treatment of relapsing MS. This antibody is generally recommended for patients who have previously responded inadequately to or cannot tolerate alternative MS therapies. Natalizumab was diluted to a final concentration of 15 mg / ml in PBS prior to administration. DMF is a disease-modifying therapy (DMT) recommended for the treatment of active relapsing multiple sclerosis. DMF (1 g) was dissolved in 50 ml of 4% solution of METHOCEL® to obtain a 20 mg / ml DMF solution. The solution was stored at 4°C until use.

[0291] Experimental autoimmune (allergic) encephalomyelitis (EAE) is considered to be the best preclinical model of multiple sclerosis (MS). More than 6,000 articles have been published in scientific journals on this model. EAE is characterized by an immune response against CNS tissue and can be induced by immunizing animals against CNS proteins. In an active EAE model, MOG emulsified in complete Freund's adjuvant (CFA) is administered to mice. 35-55 The peptide was injected subcutaneously at the base of the tail under anesthesia (100 μg MOG per mouse). 35-33 Mice are immunized by immunization with 0.1 mL of pertussis toxin (PT) in PBS at 200 ng / mouse / dose (0.1 mL) on the day of injection (day 0) and 2 days later (day 2). Mice typically develop symptoms 9-14 days after immunization (day 0). Daily observation and scoring of mice begins on day 7 and continues until the end of the study. Table 5 (see Example 4 above) details expected clinical symptoms and the scoring criteria used in this study.

[0292] This study was designed to evaluate the initial sustained use of Compound 2 vs. DMF (sustained use cohort) in a treatment escalation paradigm, and Compound 2 vs. natalizumab after DMF failure (escalation cohort). All animals in the escalation cohort received 14 doses of DMF or Compound 2, or 3 doses of natalizumab, according to group assignment. Figure 5A shows the design and randomization schedule for animals in the study.

[0293] Probability of disease control: Disease control was defined as a clinical score of less than 2.5. After 3:1:1 randomization, animals in the different treatment groups were monitored. A score of 2.5 was recorded as an event and analyzed by survival analysis in GraphPad Prism9. Animals that did not reach a clinical score of 2.5 by day 14, the end of the treatment period, were assigned a score of 0 for survival analysis.

[0294] Efficacy of Compound 2 vs. DMF in early sustained use: As shown in Figure 5A, animals in the DMF group that reached a score of 2.5 were randomized 1:1:1. Animals randomized to continue DMF treatment received the remainder of the 14 DMF doses. Animals from the initial randomization treated with Compound 2 after disease onset continued treatment for a total of 14 doses. After the 14th dose, treatment was discontinued and clinical scores and overall survival were monitored.

[0295] Efficacy of Compound 2 vs. Natalizumab in Treatment Escalation Cohorts: As shown in FIG. 5A, animals in the DMF group that reached a score of 2.5 were randomized 1:1:1. Animals randomized to natalizumab received 100 μg / mouse intravenously every 3 days for a total of 3 doses, starting on the same day that a score of 2.5 was recorded. Animals randomized to Compound 2 received 3 μg / mouse subcutaneously once daily for a total of 14 doses, starting on the same day that a score of 2.5 was recorded.

[0296] Overall Survival: Mortality due to disease was monitored in the initial sustained use cohort and the escalation cohort. Mortality due to disease (clinical score 5) was recorded as event "1" and death due to ulceration or ex vivo analysis was recorded as "0". Animals surviving to the end of the study were recorded as "0" on day 27. Survival analysis was performed in GraphPad Prism9.

[0297] Results: The disease incidence in this study was 97% (87 / 90 mice). Disease onset began on day 9 after immunization. Animals were randomized into treatment groups when they reached a disease score of "1," as shown in FIG. 5A. All animals were assigned to treatment groups by day 15 after immunization.

[0298] IACUC guidelines require that animals that develop ulcers at the immunization site be sacrificed. If a sacrificed animal was assigned to a treatment group, data from that animal were used to calculate the average clinical score until the day the animal was euthanized. Euthanasia due to ulcers at the immunization site was not assigned a clinical score of "5."

[0299] At the onset of disease (clinical score 1), a total of 30 mice were randomized to the DMF group, 10 mice were randomized to the untreated control group, and 9 mice were randomized to the Compound 2 group (3:1:1 randomization). All animals in the DMF treatment group (n=30) progressed to a score of 2.5 within 7 days of disease onset. In contrast, only 5 of the 9 animals randomized to the Compound 2 treatment group progressed to a disease score of 2.5 over the entire treatment period (14 days) (Figure 5B).

[0300] After reaching a score of 2.5, animals in the DMF treatment group were randomized 1:1:1 according to the study design. Animals in the compound 2 group continued treatment with compound 2, as shown in Figure 5A. Of the 30 animals that progressed on DMF therapy, 9 were randomized to continue DMF therapy, 9 were randomized to natalizumab, and 9 were randomized to compound 2.

[0301] As shown in Figure 5C, animals that continued on DMF therapy progressed at the same rate as untreated control animals, with all animals reaching a clinical score of 5 by the end of the study. In contrast, animals subjected to Compound 2 treatment after disease onset had better overall disease control during treatment (days 1-14) and after treatment cessation (days 14-end of study). As shown in Figure 5D, animals switched from DMF to Compound 2 had a mean clinical score of 2.6 (SD=1.1) and a median score of 2.5 by day 14 after the switch. Animals switched from DMF to natalizumab had a mean clinical score of 3.6 (SD=1.6) and a median score of 3.75 after the switch.

[0302] Animals treated with compound 2 had better overall survival regardless of cohort (initial sustained use or escalation cohort). In the initial sustained use, all animals treated with compound 2 survived to day 27. In contrast, all animals in the DMF-treated group died by day 26 (Figure 5E). In the escalation cohort, 8 of 9 animals switched from DMF to compound 2 survived to day 27 compared to 6 of 9 animals switched from DMF to natalizumab.

[0303] Clinical management of MS patients currently follows one of two paradigms: the escalation paradigm or the induction / maintenance paradigm. In the escalation paradigm, more potent and effective (and higher risk of serious adverse events) medications are administered after less potent and effective medications have failed. Potent disease-modifying therapies (DMTs) include natalizumab, alemtuzumab, anti-B cells, and mitoxantrone. Less effective treatments include glatiramer acetate, interferon beta, teriflunimide, DMF, and fingolimov. Typically, in the escalation treatment paradigm, treatment begins with glatiramer acetate, interferon beta, and / or teriflunimidyl, escalates to fingolimod and / or dimethyl fumarate upon treatment failure, escalates further to natalizumab and / or anti-B-cell upon treatment failure, and escalates still further to alemtuzumab and / or mitoxantrone upon treatment failure.

[0304] DMTs are often reserved for subjects who have failed at least one or two less effective therapies. This is mainly due to the risk / benefit of high-potency DMTs in mild cases. The main problems with this paradigm are the high failure rate and intolerance of older, less effective DMTs and injectables. Moreover, early use of less effective drugs means missing the opportunity to control the disease early.

[0305] There is a clinical need for highly efficacious molecules with favorable safety / tolerability profiles for early and sustained use. Compound 2 has been shown to be a highly efficacious molecule. Furthermore, the mechanism of action of Compound 2 is hypothesized to be restoration of immune tolerance, which is expected to provide a better overall safety profile compared to immunomodulators / suppressants.

[0306] Due to the rapid progression of disease in the mouse MOG EAE model, a full escalation paradigm as currently used in the clinic and described above is not possible, so instead we used a shorter version of the paradigm with only two escalation steps.

[0307] It is worth noting that many published EAE models are performed prophylactically. In prophylactic models, animals are treated after immunization before clinical symptoms are observed. In the EAE study described in this example, treatment is performed therapeutically after disease symptoms appear. This difference may explain the poor performance of therapies such as glatiramer acetate and DMF in the model described herein, which at the same time highlights the excellent efficacy of compound 2.

[0308] This study demonstrated that early and sustained use of compound 2 was superior to DMF, preventing disease escalation and improving overall survival compared to DMF. Furthermore, switching from DMF to compound 2 after disease escalation resulted in better disease control and improved overall survival compared to DMF followed by switching to natalizumab.

[0309] Example 9. MOG 35-55 Evaluation of the therapeutic efficacy of Compound 2 in an induction / maintenance treatment paradigm in an induced mouse EAE model The data in Example 8 suggest that treatment with Compound 2 is superior to treatment with DMF in controlling disease and preventing symptom escalation when initiated at the onset of symptoms. Furthermore, Compound 2 was superior to natalizumab even after failure of disease control with DMF.

[0310] An emerging clinical treatment paradigm is the induction / maintenance treatment paradigm, in which a potent disease-modifying therapy (DMT) is used to induce disease control (induction phase) and then switched to a safer maintenance therapy (maintenance phase). This study evaluated the use of compound 2 versus natalizumab in the induction phase, and also evaluated the use of compound 2 versus DMF in the maintenance phase of treatment.

[0311] All animal studies were performed in accordance with the Tufts University / Tufts Medical Center & Human Nutrition Research Center on Aging. Animals were housed five per cage and had free access to food and water.

[0312] Table 7 (see Example 8) shows the materials and reagents used in this study. Compound 2, natalizumab, and DMF were prepared as described in Example 8. Table 5 (see Example 4) details the expected clinical symptoms and scoring criteria used in this study.

[0313] This study was designed to evaluate the therapeutic effects of compound 2 versus natalizumab in a treatment induction / maintenance paradigm (induction cohort), and compound 2 versus DMF after natalizumab (maintenance cohort). In the induction cohort, animals were randomized 2:1:1 natalizumab:compound 2:control once they reached a score of 2.5, as shown in Figure 6A. Animals in the natalizumab cohort received three doses of 100 μg natalizumab / mouse every 3 days before randomization to the maintenance cohort. Animals randomized to the maintenance cohort after natalizumab received a maintenance dose of DMF or compound 2 for 14 days according to group assignment (1:1 randomization). Animals in the Compound 2 induction cohort were dosed subcutaneously with 30 μg Compound 2 / mouse once daily for up to 7 days or for 3 consecutive days with a disease score below 2.5, at which point animals were dosed subcutaneously with a maintenance dose of 3 μg / mouse once daily for 14 days. All animals in all groups were monitored for disease scores and survival for 14 days after the final dose.

[0314] Disease control (induction therapy): Disease control was defined as maintenance of a clinical score ≤2.5 after randomization. After 2:1:1 randomization, animals in the different treatment groups were monitored. Clinical scores were recorded daily for all animals and plotted over time. Scores >2.5 were recorded as events and analyzed by survival analysis in GraphPad Prism9. Animals that did not reach a clinical score of 2.5 by the end of the induction period (day 7) were assigned a score of 0 for survival analysis.

[0315] Survival (induction therapy): Death due to disease during the treatment induction period (clinical score of 5) was recorded as event "1" and animals that survived to the end of the induction period on day 7 after randomization were censored at day 7 for the purposes of survival analysis.

[0316] Disease control (maintenance therapy): At the end of the natalizumab induction period, animals were randomized 1:1 to DMF or Compound 2 maintenance groups according to Figure 6 A. Animals were dosed for 14 days and clinical scores were monitored daily.

[0317] Survival (maintenance therapy): Death due to disease (clinical score 5) was recorded as event "1", death due to ulceration or ex vivo analysis was recorded as "0". Animals that survived to the end of the study were recorded as "0" on the last day of the study. Survival analysis was performed in GraphPad Prism9.

[0318] Results: The disease incidence in this study was 97% (87 / 90 mice). Disease onset began on day 9 after immunization. Animals were randomized into treatment groups when they reached a disease score of "2.5," as shown in Figure 6A. All animals were assigned to treatment groups by day 15 after immunization.

[0319] IACUC guidelines require that animals that develop ulcers at the immunization site be sacrificed. If an animal was assigned to a treatment group, data from that animal were used to calculate the average clinical score until the day the animal was euthanized. Euthanasia due to ulcers at the immunization site was not assigned a clinical score of "5."

[0320] Animals were randomized at a clinical score of 2.5. A total of 18 mice were randomized to the natalizumab group, 9 mice were randomized to the untreated control group, and 10 mice were randomized to the Compound 2 30 μg / mouse group (2:1:1 randomization). Of the 18 animals in the natalizumab group, 12 animals progressed to a clinical score >2.5. In contrast, 0 / 10 animals progressed to a score >2.5 in the Compound 2 (30 μg SC group) (Figure 6B). The mean clinical scores by day 7 were 4.44 (SD=0.85), 3.61 (SD=1.17), and 2.25 (SD=0.26) for the untreated control, natalizumab, and Compound 2 30 μg SC groups, respectively (Figure 6C). Of 18 animals randomized to the natalizumab group, 11 / 18 survived the treatment induction period. Of 9 animals randomized to the Compound 2 30 μg SC group, 9 / 9 survived the induction period.

[0321] Of the 11 animals in the natalizumab group that survived the induction period, 4 were randomly assigned to the DMF maintenance group and 4 to the Compound 2 group. The mean clinical scores for animals randomly assigned to DMF and Compound 2 maintenance therapy were 2.5 (SD=0.4) and 2.8 (SD=0.3), respectively. The last measured score for the DMF maintenance group (7 days after initiation of DMF maintenance) was 4.37 (SD=1.2). The last measured score for the Compound 2 group, measured 7 days after initiation of Compound 2 maintenance, was 2.5 (SD=0). Animals in the Compound 2 maintenance group were monitored until the end of the 14-day dosing period. The mean clinical score at the end of day 14 was 2.3 (SD=0.3) (Figure 6E).

[0322] All nine animals randomized to the Compound 2 induction group were switched to Compound 2 maintenance therapy. The mean score on the first day of maintenance therapy was 2.3 (SD=0.4). Clinical scores remained stable throughout the maintenance treatment period, with a clinical score of 2.2 (SD=0.3) recorded on the last day of Compound 2 administration.

[0323] Of the animals randomized to DMF maintenance after natalizumab induction, 3 of 4 progressed to a clinical score of 5 (death from disease). All animals randomized to maintenance with compound 2 after natalizumab induction survived to the end of the study (FIG. 6F).

[0324] The induction / maintenance treatment paradigm in the management of MS is an emerging paradigm. Patients with progressive disease are administered highly potent (less safe) DMTs to induce disease control and then switched to safer maintenance therapy. In this study, the therapeutic effect of Compound 2 was evaluated both in the induction phase versus natalizumab, and in the maintenance phase versus DMF. Furthermore, the effect of sustained use of Compound 2 was evaluated as both induction and maintenance drug by varying the dose between the induction and maintenance phases.

[0325] To mimic malignant disease justifying the induction / maintenance paradigm, animals were allowed to reach a clinical score of 2.5 before randomization according to FIG. 6A. High doses of Compound 2 (30 μg / mouse SC) were superior to natalizumab in disease control and prevention of escalation during the induction period. Furthermore, the mean clinical scores of animals treated with Compound 2 declined during the induction period in at least 4 of 10 mice randomized to Compound 2, indicating improved clinical scores compared to 0 / 18 in the natalizumab group, and these animals were switched to a maintenance dose of Compound 2 before the end of the treatment induction period.

[0326] Surprisingly, 38.8% of mice randomized to natalizumab induction therapy died before completion of induction therapy. This unexpected result meant that fewer animals were available for randomization to DMF or compound 2 maintenance cohorts. However, the 1:1 randomization resulted in n=4 in each group. Animals assigned to DMF maintenance therapy had a 75% mortality rate, compared with 0% mortality in the compound 2 maintenance cohort. Furthermore, the clinical scores of animals randomized to the compound 2 maintenance cohort improved from 2.8 at the start of maintenance to 2.2 by the end of the 14 days. This observation is consistent with previous studies showing that compound 2 can reduce the clinical scores of animals with stable high scores for extended periods of time.

[0327] The results of this study indicate that early and sustained use of Compound 2 as both induction and maintenance agents can replace current induction / maintenance regimens. Furthermore, Compound 2 is a highly effective molecule that can replace less effective maintenance therapy after highly effective DMTs such as natalizumab. Overall, treatment with Compound 2 resulted in better disease control, improved clinical scores, and better survival rates compared to regimens lacking Compound 2, regardless of regimen or sequence.

[0328] Example 10. Use of Compound 2 in Myasthenia Gravis (MG) In this passive MG model in B6 mice, donor mice (n = 20, 6-8 weeks old) were immunized weekly for 4 weeks with an emulsion of complete Freund's adjuvant (CFA) and nicotinic acetylcholine receptor (AChR). Seven days after the last immunization, splenocytes were harvested and cultured at 10 6 RAG2 cells / mouse - / -The recipient mice (n=10 / group) were treated 1 day after adoptive transfer with prednisone (1 μg / mouse) administered subcutaneously daily for 10 days after disease onset, with 3 μg of Compound 2 administered subcutaneously daily for 10 days after disease onset, or left untreated. Minimal expression of disease was defined as a return to a clinical score of 1 after progression (score ≥2). The time to return to the minimum score was recorded and the probability of reversion was plotted for untreated animals, standard of care (SOC; prednisone) and Compound 2 treated animals.

[0329] The results are shown in Figure 7. By day 10, 63% of animals treated with Compound 2 had reverted to minimal expression of disease (score 1), compared to 46% of SOC (prednisone)-treated animals and 28% of untreated animals.

[0330] Example 11. Use of Compound 2 in Neuromyelitis Optica (NMO) NMO is a CNS autoimmune inflammatory demyelinating disease that causes optic nerve spinal cord injury, resulting in paralysis and vision loss. The NMO mouse model was established using AQP4-p201-p220 peptide (Genemed Synthesis Inc.). Briefly, C57BL / 6 mice (n=30) were immunized on day 0 with 100 micrograms of AQP4-p201-p220 peptide emulsified in CFA. Mice were injected subcutaneously at the base of the tail under anesthesia. On the day of injection (day 0) and 2 days later, mice were injected intraperitoneally with pertussis toxin (PTx) in PBS at 200 ng / mouse / dose (0.1 ml).

[0331] Prior to immunizing the mice on day 0, both eyes of each mouse were imaged by ophthalmoscopy. Ophthalmoscopy was used to examine (a) optic disc inflammation, and (b) retinal damage (exudate as evidence of inflammation). Mice were monitored daily for the development of disease one week after immunization (day 7) according to the scoring system developed by Ramadan et al.,Brain 2016 for clinical symptoms. Mice that showed a score of 1.0 or optimal symptoms were immediately randomized into either group A or group B treatment regimen for up to 14 days. Group A (n=10) received 3 μg of compound 2 subcutaneously daily, while group B (n=8) served as control and was treated with placebo.

[0332] Fundus examinations were performed on day 6 after immunization and approximately every 4 days thereafter, i.e., on days 9, 13, 16, 22, and 26. Each eye was scored for optic disc inflammation and obvious retinal damage as determined by the appearance of "exudate" (spots) on fundus images. Eye samples were taken during disease development and at least 9-12 days after randomization after treatment, and on day 14 (end of study) to evaluate both optic nerve and retinal tissues.

[0333] In the pilot study, exudate, a symptom of inflammation, was the most common finding. Although NMO is an optic nerve disease, exudate has been reported clinically in the retina of NMO patients. The MS-like clinical symptoms in NMO are generally mild, and in this study, the scores were very mild. Table 8 summarizes the study results. [Table 8]

[0334] Example 12. MOG 35-55 Therapeutic efficacy of Compound 2 and Compound 8 in an induced mouse EAE model 100 μg of MOG emulsified with CFA and pertussis toxin as described in Example 4 35-55At the onset of disease (clinical score of 1), mice were randomized into two treatment groups and injected subcutaneously with either Compound 2 or Compound 8 at 3 μg / mouse / day.

[0335] FIG. 8 shows that after 10 days of treatment, no statistical difference was observed between the Compound 2 and Compound 8 groups.

[0336] Example 13. Pristane-induced lupus model Four-week-old female Balb / c mice were intraperitoneally injected with 0.5 ml of pristane (Sigma Aldrich). Eight weeks after pristane injection, all mice showed positive anti-dsDNA antibody titer tests (FUJIFILM Wako Pure Chemical Corporation), indicating the development of systemic disease. On day 60, mice were randomly assigned to treatment group (n=12) (compound 2, daily subcutaneous injection of 3 μg) or control group (n=11), and treatment was initiated. Mice were monitored daily for symptoms of cutaneous lupus by checking for the appearance of skin lesions.

[0337] Interim data from the study is shown in Figure 9. Although the study is ongoing, after two weeks of treatment, mice treated with Compound 2 had a lower incidence of cutaneous lupus compared to untreated controls (8% vs. 73%, respectively). Additionally, mice treated with Compound 2 showed a two-day delay in the onset of skin symptoms compared to untreated control mice.

[0338] Example 14. Dextran Sodium Sulfate (DSS)-Induced IBD Model IBD was induced in 6-8 week old female C57 / B6 mice by adding 3% DSS (Sigma Aldrich) to the drinking water for 3 days on two different occasions (days 0, 1, and 2 of the induction phase, and days 11, 12, and 13 of the re-exposure phase). One group (n=5) was treated with Compound 2 (30 μg subcutaneously, for 7 days) during the disease induction phase, followed by a low dose of Compound 2 (3 μg subcutaneously, for 7 days) during the disease re-exposure phase. The control group (n=5) did not receive any treatment. The body weight of the mice was monitored daily from day 0.

[0339] As shown in FIG. 10, in both disease cycles, animals treated with Compound 2 lost less weight and recovered more quickly than untreated animals, suggesting that Compound 2 may be an effective treatment for IBD.

[0340] Example 15. Phase I Clinical Trial A placebo-controlled study in healthy adult volunteers and adult subjects with a confirmed diagnosis of primary progressive multiple sclerosis (PPMS), relapsing-remitting multiple sclerosis (RRMS), neuromyelitis optica (NMO), and myelin oligodendrocyte glycoprotein antibody disease (MOGAD) is conducted to evaluate the safety, tolerability, and pharmacokinetics of Compound 2 after single and multiple doses, as well as the pharmacodynamics and early response in these subjects after 12 weeks of oral administration of Compound 2. The primary objectives of this study are: (1) to evaluate the safety and tolerability of Compound 2 after single ascending oral doses of Compound 2 in healthy adult volunteers, and (2) to evaluate the safety and tolerability of Compound 2 after single ascending / multiple ascending oral doses of Compound 2 in adult subjects with PPMS, RRMS, NMO, or MOGAD. Secondary objectives of the study are: (1) to evaluate the pharmacokinetics of Compound 2 following single ascending oral doses in healthy adult volunteers, and (2) to evaluate the pharmacokinetics of Compound 2 following single ascending / multiple ascending oral doses in adult subjects with PPMS, RRMS, NMO, or MOGAD.

[0341] This is a single-blind, single ascending dose / multiple ascending dose (SAD / MAD) Phase I study in healthy volunteers and patients with PPMS, RRMS, NMO, or MOGAD. Phase I SAD consists of six cohorts of eight subjects each (6+2 treatment+placebo, enrolled in 1:1 and 1:4 designs). Cohorts 1, 2, and 3 will enroll healthy volunteers. Cohorts 4 and 5 will enroll subjects suffering from PPMS or RRMS. Cohort 6 may enroll healthy volunteers or subjects suffering from PPMS or RRMS.

[0342] Phase I MAD will begin after cohort 3 clears SAD. The MAD portion of the study will consist of three cohorts of 10 subjects in each cohort suffering from RRMS or PPMS. Subjects will be dosed daily for up to 28 days. Subjects will be allowed to maintain concurrent MS medication during the MAD portion of the study.

[0343] Healthy volunteers will receive a single dose of Compound 2 according to cohort assignment. Participation of healthy volunteers in the study will end after 14 days of follow-up or as needed for the SAD study. Subjects suffering from RRMS or PPMS will receive a single oral dose of Compound 2 during the SAD portion of the study and will receive daily oral doses of Compound 2 for up to 28 days during the MAD portion of the study. At this stage of the study, subjects with RRMS and PPMS will be allowed to continue their MS medication. Subjects in the MAD portion of the study will be able to choose whether or not to continue in the Phase Ib study. Subjects who do not wish to continue will be removed from the study after the follow-up period (e.g., 30 days, 6 months). The expected doses for the Phase I SAD / MAD study are shown in Table 9. [Table 9]

[0344] Safety and tolerability of Compound 2 will be assessed based on recorded adverse events (AEs), physical examination, vital sign measurements, electrocardiograms, and clinical laboratory evaluations. Adverse events will be coded using the Medical Dictionary for Clinical Trials (MedDRA). Adverse events and laboratory values ​​will be graded using the NCI CTCAE v5.0. Blood / plasma samples will be collected at designated time points to assess the pharmacokinetics of Compound 2 using a validated liquid chromatography-mass spectrometry / mass spectrometry (LC-MS / MS) assay.

[0345] Inclusion criteria included: 1. The subject, or his / her legally authorized representative, must be willing and able to provide appropriate written informed consent. 2. Healthy volunteers with no known acute or chronic pathologies (respiratory, gastrointestinal, renal, hepatic, hematologic, lymphatic, neurological, cardiovascular, psychiatric, musculoskeletal, genitourinary, immune, dermatological, endocrine, etc.) at the time of enrollment. 3. All men or non-pregnant women aged 18-70 years will be included in the study, regardless of ethnicity, socio-economic or educational status. 4. Body mass index (BMI) at screening: 18.0 to 35.0 kg / m 2 (including borderline values) (age <56 years), BMI at screening 18.0-30.0 kg / m 2 (Borderline included) (56 years and older). 5. Subjects with a definitive diagnosis of PPMS or RRMS according to the 2017 revised McDonald criteria. 6. Subjects with a confirmed diagnosis of NMO based on neurological examination, MRI scan, and positive NMO-IgG autoantibody test. 7. Subjects with a confirmed diagnosis of MOGAD by meeting three of the following criteria: (a) laboratory findings: seropositive MOG-IgG by cell-based assay; (b) clinical findings of any of the following symptoms: (i) ADEM; (ii) optic neuritis, including chronic relapsing optic neuropathy (CRION); (iii) transverse myelitis (short or long segment), (iv) brain or brainstem syndrome with demyelination, or (v) Any combination of the above. 8. Kurtzke Expanded Disability Status Scale (EDSS) score 0-9.5. 9. Subjects must have undergone a 3T MRI brain and / or spinal cord within 6 months to 1 year prior to enrollment. 10. Subject is willing and able to comply with the requirements of the study protocol, including scheduled clinic visits, maintenance of an investigational drug diary, clinical examinations, and other study procedures such as EKG, 3T MRI brain and / or spinal cord, and ophthalmologic examination. 11. Laboratory evaluations performed within the past 6 months include White Blood Cells (WBC), Hemoglobin (Hgb), Platelets (PLT), Alanine Transaminase (ALT), Aspartate Transaminase (AST), Alkaline Phosphatase (ALP), Total Bilirubin (T.Bili), Lipase, BUN, Creatinine, Prothrombin Time (PT), and Partial Thromboplastin Time (PTT) within acceptable normal reference ranges (>Grade 1 abnormalities will exclude from study). 12. Female subjects of reproductive age and subjects with female partners of childbearing potential must agree to use an acceptable method of contraception. This criterion must be followed from the time of first administration of study drug through follow-up visits (for the subject and her partner), including an additional period of 90 days (for the subject herself).

[0346] Exclusion criteria included: 1. Subjects diagnosed with CIS, which refers to a first episode of neurological symptoms lasting at least 24 hours caused by inflammation or demyelination of the central nervous system, usually occurring in young adults and affecting the optic nerves, brain stem, and / or spinal cord. 2. Blood loss of more than 250 mL or blood donation within 56 days or plasma donation within 7 days since study screening. 3. Subject has a history of HIV or HIV-related disease, Hepatitis B or C, or other infectious diseases. 4. Grade 3 lymphopenia (<500-200 / mm) in the past 6 months 3 or <0.5~0.2*10e9 / L). 5. Subject has received treatment with immunodepleting drugs within 3 months prior to blood collection for this study or PBMC sample collection and processing, or is anticipated to require immunosuppressive treatment within the next 6 months. 6. History of cancer treatment with chemotherapy and / or radiation therapy within the past 5 years prior to study enrollment. 7. Subject has COVID-19 positive status during study enrollment (confirmed by clinical signs and symptoms and a positive COVID test result by SARS-CoV-2 NAAT) or has received a recent COVID-19 vaccination including a booster dose within the past 30 days or has received antiviral therapy for the prevention of COVID-19, e.g., paxlovid, remdesivir, molnupiravir, interferon, anti-SARS-CoV-2 monoclonal antibodies, IVIG-SARS-CoV-2, COVID-19 convalescent plasma, etc. 8. Recent administration of a live attenuated vaccine, such as influenza, MMR, shingles, chickenpox, yellow fever, or rotavirus vaccine, or an inactivated vaccine, such as Hepatitis A or rabies vaccine, within the past 30 days. 9. Subject is participating in another clinical trial involving an investigational drug, biologic, or device within 60 days or 5 half-lives (whichever is longer) prior to the first vaccine dose. 10. Women who are pregnant or breastfeeding, currently undergoing fertility treatment, or planning pregnancy at the time of study enrollment. 11. Any psychiatric condition, including recent (e.g., within 1 year of study enrollment) or active suicidal thoughts / behavior, or abnormal laboratory findings, that may increase the risk of study participation or that, in the investigator's judgment, would render the subject unsuitable for the study.

[0347] A minimum of 54 subjects will be enrolled in the study: up to 24 healthy volunteers and 30 subjects with RRMS or PPMS (4:1 ratio). In the SAD study, approximately 12-24 healthy volunteers will be enrolled in each of cohorts 1, 2, and 3 with 4:1 randomized treatment to placebo. The total sample size per cohort will be determined by dose-limiting toxicity (DLT). 12-24 subjects with RRMA and / or PPMA will be enrolled in cohorts 4, 5, and 6 without randomization to dose. The total sample size per cohort will be determined by DLT. In the MAD study, approximately 30 subjects with RRMS or PPMS will be enrolled in MAD cohorts 1, 2, and 3. Subjects will be randomized to a 4:1 ratio of RRMS to PPMS in each cohort.

[0348] Example 16. Pharmaceuticals USP <795> Compounding is performed according to guidelines. Briefly, compounding is performed by transferring 24,000 mg of hydroxypropyl beta cyclodextrin (HPBCD) and 300 mg of compound 2 into a suitable container. Approximately 100 mL of water is added to the container, and stirring and sonication are repeated until a clear solution is obtained. The final preparation is transferred to a PET container for clinical use.

[0349] Oral dose solutions of Compound 2 at different concentrations were formulated using a Compound 2:HPBCD ratio of 1:80, ranging from 0.02 mg / ml to 3 mg / ml. Briefly, formulation was performed by weighing HPBCD and dissolving it in sterile HPLC grade water. Compound 2 was then weighed and added to the HPBCD solution in a suitable container. To facilitate dissolution, alternating sonication and magnetic stirring were used to obtain a clear solution. The solution was then dispensed into amber PET bottles, appropriately labeled, and kept refrigerated until administration.

[0350] A pilot stability study was conducted on a 0.7 mg / ml oral solution of Compound 2 at three temperatures: -20° C., 4° C., and room temperature (RT). Potency was maintained over a minimum of 3 months under all storage conditions.

[0351] Samples were also subjected to three freeze-thaw cycles (-20°C to room temperature). The clarity of the solutions was assessed. No precipitation was observed and all solutions maintained their clarity after the freeze-thaw cycles. The potency of these solutions was also measured using an LC / MS assay. The potency was maintained even after three months of storage and several freeze-thaw cycles between -80°C and room temperature, indicating the stability of this formulation.

[0352] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0353] Although exemplary embodiments have been particularly shown and described, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the embodiments encompassed by the appended claims.

Claims

1. A compound of the following structural formula: 【Chemistry 15】 or a pharmaceutically acceptable salt thereof, wherein: Ring A is phenyl or (C 5 -C 6 )heteroaryl; L is —(CH 2 ) n —, —C(O)—, or —C(OH) 2 —; R 1 is H or (C 1 -C 5 ) alkyl; each R 2 is independently chloro or fluoro; R 3 is (C 1 -C 3 )alkyl substituted with one or more independently selected (C 6 -C 15 )aryl or (C 5 -C 15 )heteroaryl, wherein said (C 6 -C 15 )aryl and (C 5 -C 15 )heteroaryl are each independently substituted with -(R 30 ) p ; each R 30 is independently halo, (C 1 -C 5 )alkoxy, (C 1 -C 5 )haloalkoxy, (C 1 -C 5 )alkyl, or (C 1 -C 5 )haloalkyl, or two R 30 attached to adjacent ring atoms together form —(CH 2 ) q — or —O(CH 2 ) r O—; n is 1, 2, or 3; m is 0, 1, 2, 3, or 4; each p is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; each q is independently 3, 4, 5, or 6; and The compound, or a pharmaceutically acceptable salt thereof, wherein each r is independently 1, 2, 3, or 4.

2. A compound according to claim 1 of the following structural formula: 【Chemistry 16】 or a pharmaceutically acceptable salt thereof, wherein: X 1 , X 2 , and X 3 are each >C(H); X 1 is N, X 2 and X 3 are each >C(H); X 1 and X 2 are each >C(H) and X 3 is N; or The compound, or a pharmaceutically acceptable salt thereof, wherein X 1 and X 3 are each >C(H) and X 2 is N.

3. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein X 1 , X 2 , and X 3 are each >C(H).

4. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein X 1 is N, and X 2 and X 3 are each >C(H).

5. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein --OR 3 is bonded to a ring atom of ring A that is meta or para to --(CH 2 ) n --.

6. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein R 1 is H.

7. The compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein R 3 is methyl substituted by one or two independently selected (C 6 -C 15 )aryl or (C 5 -C 15 )heteroaryl, each independently substituted by -(R 30 ) p .

8. The compound of claim 2 or a pharmaceutically acceptable salt thereof, wherein R 3 is methyl substituted by one or two independently selected (C 6 -C 15 )aryl or (C 5 -C 15 )heteroaryl independently selected from phenyl, naphthyl, pyridinyl, quinolinyl, isoquinolinyl or benzo[d]imidazolyl, each independently substituted by -(R 30 ) p .

9. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein n is 1.

10. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein m is 0.

11. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein each p is independently 0, 1, or 2.

12. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein each q is independently 3 or 4, and each r is independently 1 or 2.

13. A compound of any of the following structural formulas: 【Chemical 27】 【Chemical Formula 28】 or a pharmaceutically acceptable salt thereof.

14. The compound of claim 13 having the following structural formula: 【Chemistry 19】 or a pharmaceutically acceptable salt thereof.

15. The compound of claim 13 having the following structural formula: 【Chemistry 24】 or a pharmaceutically acceptable salt thereof.

16. A composition comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

17. The composition of claim 16, formulated for oral administration.

18. A combination comprising the compound of claim 1 or a pharmaceutically acceptable salt thereof and a further therapeutic agent.

19. A composition comprising the compound of claim 2 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

20. The composition of claim 19, formulated for oral administration.

21. A combination comprising the compound of claim 2 or a pharmaceutically acceptable salt thereof and a further therapeutic agent.

22. A compound of the following structural formula: 【Chemistry 24】 or a pharmaceutically acceptable salt thereof.

23. A composition comprising the compound of claim 22 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

24. The composition of claim 23, formulated for oral administration.

25. A combination comprising the compound of claim 22 or a pharmaceutically acceptable salt thereof and a further therapeutic agent.

26. A compound of the following structural formula: 【Chemistry 24】 。 27. A compound of the following structural formula: 【Chemistry 24】 A pharmaceutically acceptable salt of 28. The pharmaceutically acceptable salt of claim 27, wherein the pharmaceutically acceptable salt is a hydrochloride salt.

29. The composition of claim 23, formulated as a liquid dosage form for oral administration.

30. The composition of claim 29, wherein the liquid dosage form is a solution.

31. A composition comprising a compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 15, 22, and 26, or a pharmaceutically acceptable salt thereof according to claim 27 or 28; a composition according to any one of claims 16, 17, 19, 20, 23, 24, 29, and 30; or a combination according to any one of claims 18, 21, and 25, for treating an autoimmune disorder in a subject in need thereof.

32. The composition or combination of claim 31, wherein the autoimmune disorder is multiple sclerosis, neuromyelitis optica, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), rheumatoid arthritis, or myasthenia gravis.

33. The composition or combination of claim 31, wherein the autoimmune disorder is multiple sclerosis.

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