Compositions and methods for reducing immune intolerance and treating autoimmune disorders

Abstract

Compositions and methods for reducing immune intolerance and treating autoimmune disorders are provided. [Solution] Provided herein are compounds and pharmaceutically acceptable salts thereof, lipid particles comprising such compounds or pharmaceutically acceptable salts thereof, and compositions thereof, 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, e.g., in combination with protein therapy or gene therapy. The compounds have the following structural formula (I): JPEG2026042844000040.jpg23165 wherein the 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. The undesirable immune responses can lead to neutralization of the enzyme / protein or alteration of its pharmacokinetics. In many situations, patients lack 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 for treating many congenital disorders and other diseases. The immunogenicity of carriers and / or the genetic material carried therein is a major challenge for the clinical application of gene therapy. The presence of anti-carrier antibodies is a contraindication for some approved gene therapy treatments. Furthermore, nascent anti-carrier antibodies may prevent repeated administration in subjects receiving 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, potentially with debilitating and devastating effects. Current approaches to treating autoimmune disorders rely on general immunosuppression at the humoral, cellular, and / or complement levels, which leaves 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., autoantigens 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 autoantigens. 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, 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 pharmaceutically 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 pharmaceutically acceptable salts 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 are compositions (eg, pharmaceutical compositions) comprising a plurality of lipid particles described herein.

[0011] Also provided herein are methods 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 are methods for tolerizing a subject in need thereof to an antigen and inhibiting or reducing the titer of antigen-specific antibodies in the subject, comprising administering to the subject the 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 for 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 comprising an antigen or an immunogenic fragment of an antigen).

[0014] Also provided herein are methods for 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 comprising 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 thereof, 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 antigen therapy treatment, 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 the antigen therapy, or a therapeutically effective amount of a composition described herein that includes antigen therapy.

[0017] Also provided herein are compounds or compositions (e.g., pharmaceutical compositions) of the present disclosure for the uses 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 are uses of compounds of the present disclosure or compositions described herein for the manufacture of a medicament for the uses described herein (e.g., treatment of an autoimmune disorder, treatment of a disease, disorder, or condition treatable with antigen therapy).

[0018] The foregoing will be apparent from the following more particular description of exemplary embodiments. [Brief explanation 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] 1 shows the dose-response relationship of Compound 2 in vivo on FoxP3+ / CD4+ Tregs.

[0022] [Figure 2C] 1 shows the dose-response relationship of Compound 2 in vivo 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 Compound 2 at the indicated doses in the murine EAE model described in Example 4.

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

[0028] [Figure 4B] 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] 1 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 for 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] 1 shows the therapeutic effect of early and sustained use of Compound 2 compared to DMF in the study described in Example 8.

[0034] [Figure 5D] 1 shows the therapeutic effect of Compound 2 versus natalizumab in treatment escalation cohorts from the study described in Example 8.

[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 for the study described in Example 9.

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

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

[0039] [Figure 6D] 1 shows the survival rates of the 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 compared to 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 in 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 cutaneous lupus in mice over time, showing the incidence of cutaneous lupus in the untreated control group and the Compound 2-treated group in the early-induced lupus model described in Example 13.

[0045] [Figure 10] 1 shows the percentage change in body weight 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 INVENTION

[0046] Exemplary embodiments are described below.

[0047] definition

[0048] The compounds described herein include those compounds generally described and further exemplified by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise specified. For purposes of this 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, compound names 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. Typically, 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 refer to an acceptable standard deviation, as practiced in the art. Alternatively, "about" may refer to a range of ±20%, e.g., ±10%, ±5%, or ±1% of the given value. It should be understood that the term "about" can precede any particular value specified herein, except for 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 a (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, 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, wherein 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, although 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 substitutions 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, azide, sulfhydryl, alkylthio, sulfate, sulfonate, sulfamoyl, sulfonamide, sulfonyl, cycloalkyl, heterocyclyl, aralkyl, aryl, or heteroaryl.Those skilled in the art will understand that, where appropriate, the substituent itself can be substituted.Thus, the substituent 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, in some embodiments, independently refer to halo, (C-C)alkoxy, (C-C)haloalkoxy, (C-C)alkyl, or (C-C)haloalkyl, or optionally substituted (C-C 15 ) aryl or (C5-C 15In 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, (C-C)alkoxy, (C-C)haloalkoxy, (C-C)alkyl, or (C-C)haloalkyl, (e.g., halo, (C-C)alkoxy, (C-C)haloalkoxy, (C-C)alkyl, or (C-C)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, (C-C)alkoxy, (C-C)haloalkoxy, (C-C)alkyl, or (C-C)haloalkyl. In some embodiments, an optionally substituted (e.g., substituted) alkyl is independently selected from halo (e.g., fluoro), (C-C)alkoxy, (C-C)haloalkoxy (e.g., (C-C)fluoroalkoxy), (C-C 15 ) aryl or (C5-C 15 ) heteroaryl; and

[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 that includes the value 0, such as , , , and p, where p is as described herein, means that the substitution is optional; thus, the atom or moiety designated as "optionally substituted" can be unsubstituted or substituted. In some embodiments, an optionally substituted group is unsubstituted. As used herein, when an optionally substituted group indicated by a variable followed by a subscript number that includes the value 0 is unsubstituted, the subscript number following the variable is 0. In some embodiments, an optionally substituted group is substituted. As used herein, when an optionally substituted group indicated by a variable followed by a subscript number that includes the value 0 is substituted, the subscript number following the variable is other than 0. The term "substituted" means that the group designated herein is unsubstituted. Unless otherwise indicated, for example, as in the case of the terms "substituted" or "optionally substituted," the group designated herein is unsubstituted.

[0062] As used herein, the term "compounds of the disclosure" refers to compounds of any of the structural formulas 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 substitution), 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 exist, salts are likewise included, particularly pharmaceutically 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 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 indicating 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 with respect to its corresponding optical isomer(s). When a disclosed compound is depicted by a structure indicating stereochemistry and the compound has one or more chiral centers, the stereochemistry refers to the absolute configuration of substituents around one or more chiral centers. "R" and "S" can also, or alternatively, be used to refer to the absolute configuration of 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 each other, 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 calculated using 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 calculated using the following formula:

number

[0070] Unless otherwise specified, the compounds of this disclosure include compounds that differ only in the presence of one or more isotopically enriched atoms, for example, by the replacement of hydrogen with deuterium or tritium, or carbon with 13 C or 14 Compounds produced by substituting C-enriched carbons are within the scope of the present invention. In all structures provided, any hydrogen atom may be independently substituted with 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 "pharmaceutically acceptable" means that the substance or composition 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 excessive 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 tissues without undue toxicity, irritation, allergic response, etc., within the normal scope of sound medical judgment, and is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. 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 pharmaceutically 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, and picoline; and salts derived from N-methylamine, trimethylamine, and picoline. +((C1-C4) alkyl) salts. Representative alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, barium, and the like. Further pharmaceutically 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 alkyl sulfonates, and aryl sulfonates.

[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 pharmaceutically acceptable salt thereof). A prodrug may become active upon reaction under biological conditions (and, e.g., be biologically inactive), or may have activity in its unreacted form. A prodrug may be reduced in metabolism under physiological conditions (e.g., due to the presence of a hydrolyzable group), thereby increasing the circulating half-life (e.g., in blood) of the prodrug. Thus, in some embodiments, a prodrug contains a hydrolyzable group, as in the case of an ester prodrug, e.g., an alkyl ester prodrug. Prodrugs are generally described in detail 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 (ed.).

[0077] As used herein, the term "hydrolyzable group" refers to a moiety that, when present in a prodrug, yields a carboxylic acid or its salt upon hydrolysis. Hydrolysis can occur spontaneously, for example, under acidic or basic conditions in a physiological environment (e.g., blood, metabolically active tissues such as the liver, kidneys, lungs, and 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 circulation half-life, improved uptake, improved duration of action, or improved onset of action.

[0078] Examples of hydrolyzable groups include (C-C), each optionally substituted with one or more independently selected halo (e.g., fluoro). 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 (C-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 described herein (e.g., one or more independently selected halo (e.g., fluoro)). 10 ) alkyl).

[0079] The compounds described herein may exist as "solvates" or "hydrates." A "hydrate" is a compound present in a composition containing one or more water molecules. A hydrate may contain a stoichiometric amount of water, such as a monohydrate or dihydrate, or may contain a random amount 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, or diethyl ether. Mixtures of such solvates or hydrates can also be prepared. The source of such solvates or hydrates may be derived 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 autoantigens, antigens produced by the body under normal conditions or as part of a disorder, or foreign antigens, non-self antigens. Examples of autoantigens 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 thereof (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), and alleviating symptoms caused by a disease or condition.

[0083] A "therapeutically effective amount" is an amount effective, at the dosages and for the period of time necessary, to achieve the desired therapeutic result (e.g., induction of immune tolerance, reduction of immune intolerance, treatment, cure, suppression, or amelioration of a physiological response or condition, etc.). The full therapeutic effect does not necessarily occur by administration of a single 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 the desired response in an individual.

[0084] As used herein, a "subject" includes humans, domestic animals, e.g., laboratory animals (e.g., dogs, monkeys, pigs, rats, mice, etc.), household pets (e.g., cats, dogs, rabbits, etc.), and livestock (pig, cow, sheep, goat, horse, 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 pharmaceutically 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 are each independently -(R 30 ) p is replaced by Each R 30 are independently halo, (C-C)alkoxy, (C-C)haloalkoxy, (C-C)alkyl, or (C-C)haloalkyl, or two R attached to adjacent ring atoms 30 together - (CH2) q - or -O(CH2) r O- is formed, 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 substituted with one, two, or three independently selected (C6-C 15 ) aryl or (C5-C 15 ) methyl substituted with heteroaryl. R 30 The values ​​of the remaining variables including and 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., 1, 2, or 3) 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). R 30 The values ​​of the remaining variables, including , and 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, independently selected from phenyl, naphthyl, pyridinyl, quinolinyl, isoquinolinyl, or benzo[d]imidazolyl (C-C 15 ) aryl or (C5-C 15 (C1-C3) alkyl (and in some preferred embodiments, methyl) substituted with (C1-C3) heteroaryl). 30 The values ​​of the remaining variables, including , and 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 its first to seventh aspects.

[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 aspects 1 through 10 thereof.

[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 aspects 1 through 11 thereof.

[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 its first to fourteenth aspects.

[0101] A second embodiment is a compound of the following structural formula: [ka] or a prodrug thereof, or a pharmaceutically 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 R 3 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 the 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 pharmaceutically acceptable salt thereof, wherein: X 1 , X 2 , and X 3 are > C(H), respectively, X 1 is N and X 2 and X 3 are > C(H), respectively, 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 3are >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 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 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 the ring atom of ring A that is para to R 3 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 pharmaceutically 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 pharmaceutically 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 pharmaceutically 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 pharmaceutically acceptable salt thereof. In some embodiments, the compound of structural formula I is selected from the compounds listed in Table 1, or a pharmaceutically 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 Typically, the compound of the present disclosure is formulated with one or more pharmaceutically acceptable carriers for administration to a subject.The present disclosure provides such compositions, including pharmaceutical compositions.Therefore, one embodiment is a composition (e.g., pharmaceutical composition) comprising the compound of the present disclosure and a pharmaceutically acceptable carrier.For example, the composition described herein can be used in the method described herein to provide 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) comprising one or more lipids and a compound of the present disclosure.

[0116] Also referred to herein is at least one phospholipid (e.g., a saturated C4-C phospholipid such as dimyristoylphosphatidylcholine (DMPC)). 30 C4-C of acyl chain 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 comprising 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., above 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, phosphatidyl alcohol, and phosphatidylglycerol. Phospholipids can be saturated or unsaturated, i.e., contain one or more unsaturated units, and can contain acyl chains of various lengths. In some embodiments, phospholipids are C4-C6 30 Acyl chains, 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 a variety of natural and synthetic sources. 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, the 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 a 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 a 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 about 1% to about 99%, e.g., about 1% to about 50%, 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%.

[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 preferably can be embedded in whole or in part in a lipid bilayer (e.g., liposome) by covalent or non-covalent bonding. 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 bilayer of liposomes so 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, for example, PS.

[0123] In some embodiments, the one or more lipids comprise a phospholipid or a pharmaceutically acceptable salt thereof, such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) or a pharmaceutically acceptable salt thereof. In some embodiments, the phospholipid is a saturated phospholipid, such as a C4-C 30 In some embodiments, the phospholipid is an unsaturated phospholipid, e.g., a 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, herpesviruses, adenoviruses, lentiviruses, rabies viruses, lentiviruses, VSV, poxviruses (e.g., vaccinia viruses, smallpox viruses, canarypox viruses), 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 the 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 the gene therapy and the 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 pharmaceutically acceptable carrier.

[0127] The compositions described herein, and therefore 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, intra-articular, intra-arterial, intra-synovial, 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 solutions.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 oily phase and mixed with an emulsifying agent and / or suspending agent.If desired, certain sweeteners, flavorings, or coloring agents 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 pharmaceutically 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) humectants 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) humectants 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 form may also comprise buffering agents.

[0131] Liquid dosage forms for oral administration include pharmaceutically 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 (e.g., 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 can also contain adjuvants such as wetting agents, emulsifiers and suspending agents, sweeteners, flavoring agents, coloring agents, fragrances, and preservatives.

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

[0133] Solid compositions of a similar type 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. Solid dosage forms such as 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 can 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 other substances other than inert diluent, such as tableting lubricant and other tableting aids, for example, 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 gastrointestinal 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 a compound of the present disclosure and a delayed-release component. Such compositions allow for targeted release of the compound to the lower gastrointestinal tract, e.g., the small intestine, large intestine, colon, and / or rectum. In certain embodiments, the delayed-release compositions further include an enteric or pH-dependent coating, such as cellulose acetate phthalate and other phthalates (e.g., polyvinyl acetate phthalate, methacrylates (Eudragits)). Alternatively, the delayed-release compositions can provide controlled release in the small intestine and / or colon by providing pH-sensitive methacrylate coatings, pH-sensitive polymer microspheres, or polymers that undergo hydrolytic degradation. The delayed-release compositions can be formulated with hydrophobic or gelling excipients or coatings. Colonic delivery can further be provided by coatings 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 azoaromatic bond coatings.

[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 oleaginous suspension. This suspension can be formulated according to techniques known in the art using suitable dispersing or wetting agents (e.g., Tween® 80, etc.) 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. Additionally, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland 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 pharmaceutically 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 pharmaceutically 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 pharmaceutically 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 glycol.

[0139] The compositions described herein may also be administered topically, particularly 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 pharmaceutically acceptable carriers.Alternatively, the composition can be formulated into 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 ester 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, and 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 with or without a preservative, such as benzylalkonium chloride. Alternatively, for ophthalmic use, the composition may be formulated into 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 by 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 pharmaceutically 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 solubilizing derivatives, can also be effectively used as pharmaceutically 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, e.g., 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 embodiments, the composition is in a liquid dosage form, and in further embodiments, 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 the compounds of the present disclosure.

[0148] Some embodiments provide combinations (e.g., pharmaceutical combinations) comprising a compound of the present disclosure (e.g., a composition described herein comprising a compound of the present disclosure) and one or more additional therapeutic agents (e.g., one or more compositions comprising 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 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 the additional therapeutic agent(s)). In some aspects, the kit further comprises written instructions for administering to a 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 with respect to 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 depending, for example, on the subject being treated and the particular mode of administration. Typically, a unit dosage form contains about 1 to about 1,000 mg of active ingredient(s), e.g., 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), e.g., about 0.1 mg to about 50 mg, about 0.1 mg to about 10 mg, or 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%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.08%, 0.07%, 0.06 ... %, 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. The range 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 one or more therapeutic agents provided in the pharmaceutical composition ranges from 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%, or 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 higher affinity than, for example, its natural ligand, phosphatidylserine, 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 costimulatory 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] We now show that TIM receptor agonists inhibit the activity of at least toll-like receptors (TLR) 3 and TLR 7 without substantially inhibiting the activity of TLRs 2 and 4, which primarily recognize patterns displayed by bacteria. Toll-like receptors (TLRs) form a family of pattern recognition receptors expressed on innate immune cells and constitute the immune system's first line of defense against microorganisms. To date, 10 human TLR subtypes have been identified. TLRs 1, 2, 4, 5, 6, and 10 are expressed on the cell surface, while TLRs 3, 7, 8, and 9 are localized in the endoplasmic reticulum, endosomes, and lysosomes. TLRs 1, 2, and 6 recognize and bind bacterial lipoproteins and glycolipids. TLRs 3, 7, 8, and 9 recognize viral dsRNA (TLR 3), ssRNA (TLR 7, TLR 8), and unmethylated CpG. TLRs recognize and bind to nucleic acids such as DNA (TLR9). TLR4 recognizes and binds to fibronectin and LPS. TLR5 recognizes and binds to bacterial flagellin. Without being bound by any particular theory, it is believed that the compounds of the present disclosure do not produce 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 present disclosure (e.g., a therapeutically effective amount of a compound of the present disclosure). In some aspects, the compound of the present 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 the activity of TLR3, TLR7, TLR8, and / or TLR9 by a compound of the present 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 the 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 cells are immune cells, e.g., T cells such as regulatory T cells, natural killer (NK) cells, macrophages, neutrophils, myeloid-derived suppressor cells, or dendritic cells. In some embodiments, the immune cells are FoxP3+ and / or CD4+, e.g., FoxP3+ and / or CD4+ T cells. In some embodiments, the immune cells are B cells, such as regulatory B cells. In some embodiments, the immune cells (e.g., regulatory B cells) are 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 thereof (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 present disclosure, e.g., in the form of a composition described herein.

[0161] Another embodiment is a method of immune tolerization (e.g., antigen therapy) in a subject in need thereof, 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 described herein 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 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 reducing and / or eliminating an immune response to, for example, an antigen. The immune response may be evidenced, for example, by increased immunological activity, 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 to, for example, reducing immunological activity, 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 administered antigen therapy.

[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. "Immune tolerization" contemplates a gradual improvement along this continuum toward 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 tolerance 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 can 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 and eosinophils. In some embodiments, subjects exhibiting immune intolerance or subjects with immune intolerance (e.g., subjects with an autoimmune disease, such as an autoimmune disease described herein) have a measurable cytokine response. For example, a subject with rheumatoid arthritis may have a measurable TNF-α response. In some embodiments, subjects exhibiting immune tolerance or subjects with immune tolerance (e.g., subjects with 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, resulting in, for example, 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) an increase in the number of regulatory T cells and / or an increase in the activity or level of tolerogenic T cells (e.g., FoxP3+ / CD4+ T cells, CD4+ / CD25 T cells, etc.); 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+ 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 These indicators include (a) a decrease in antigen-specific antibody titers and / or B cell numbers, including antigen-specific memory B cells; (b) a decrease in IL-6 and / or IL-17; (c) an increase in TGF-β, IL-10, IL-35, CD40, CD80, and / or CD86; (d) 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 are 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 interfere with innate immune responses, such as those triggered by innate immune cells responding to danger signals from pathogens, but rather 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 embodiments, immune tolerization is general adaptive immune tolerization. In some embodiments, immune tolerization is antigen-specific, resulting in, for example, immune intolerance to a specific antigen(s) or reduced immune tolerance to a specific 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 present 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 present 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. An immunogenic fragment of an antigen may induce an immune response in a subject that is as strong as the immune response induced by the antigen itself, but does not necessarily induce an immune response that is as strong 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, e.g., in the form of a composition described herein. Some aspects include administering to the subject a composition described herein 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.

[0171] In some embodiments 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 derived 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 Cry j1 and Cry j2 (cedar). The source of the allergen listed in parentheses 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 a therapeutically effective amount of a compound of the present disclosure to the subject, e.g., 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 described herein 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.

[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 regsThe compounds of the present disclosure are also believed to induce a population of regulatory T cells by expanding a population of regulatory T cells, e.g., regulatory T cells that are 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 regulatory T cells from 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 immunosuppression.

[0174] In some embodiments, regulatory T cells are FoxP3+, e.g., FoxP3+ / TIM3+, FoxP3+ / NRP1+. Whether regulatory T cells are positive (+) or negative (-) for any of the above-mentioned markers can be determined, 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 present 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 a therapeutically effective amount of a compound of the present disclosure to the subject, e.g., 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 described herein 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.

[0177] Compounds of the present disclosure have been shown 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 for 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 autoimmune disorders, 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. Accordingly, in some aspects of methods for treating an autoimmune disorder, the method further comprises administering (e.g., co-administering) to the subject 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 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 immunogenic fragment thereof. In some aspects, the autoantigen or immunogenic fragment thereof and the compound of the present disclosure are administered to the subject in separate formulations.

[0179] Specific examples of autoimmune disorders that can be treated 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), Barrow's disease, Behcet's disease, and benign mucosal fibroids. 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 Inflammation 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 inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis , juvenile diabetes mellitus (type 1 diabetes), juvenile myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Much-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 rheumatoid arthritis (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, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing multiple sclerosis 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-person 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, 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-Barré 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-Barré 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 thereof, 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.

[0184] Clinical management of multiple sclerosis typically follows one of two paradigms: the escalation paradigm or the induction / maintenance paradigm. In the escalation paradigm, a more potent and effective disease-modifying therapy (DMT) (and associated with a higher risk of serious adverse events) is administered after treatment failure with a less potent and effective DMT. Standard treatment in the escalation paradigm typically includes treatment with glatiramer acetate, interferon beta, and / or teriflunimidyl esterase (TMS), escalating to fingolimod and / or dimethyl fumarate upon treatment failure, further escalating to natalizumab and / or anti-B-cell therapy upon treatment failure, and 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 in the treatment of 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 highly potent DMTs include, but are not limited to, natalizumab, alemtuzumab, anti-B-cell agents, 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 the α4β1 and α4β7 integrins, which are expressed on the surface of all leukocytes except neutrophils, and inhibits α4-mediated adhesion of leukocytes to their counterreceptor(s). Natalizumab injection is indicated as monotherapy for the treatment of relapsing forms of multiple sclerosis in adults, including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease. Glatiramer acetate injection is indicated for the treatment of relapsing forms of multiple sclerosis in adults, including clinically isolated syndrome, relapsing-remitting disease, and active secondary progressive disease. Oral dimethyl fumarate is indicated for the treatment of relapsing forms of multiple sclerosis.

[0188] In some embodiments, the multiple sclerosis has not been previously treated. In alternative embodiments, the multiple sclerosis has been previously treated with standard therapy, such as, for example, natalizumab (TYSABRI®), glatiramer acetate and / or dimethyl fumarate, or a 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 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 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 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 maintenance therapy comprising a compound of the present disclosure, e.g., in the form of a pharmaceutical composition.

[0191] In some embodiments (e.g., when the autoimmune disease is multiple sclerosis), the compound of the present 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. In alternative embodiments, the autoimmune disorder has been previously treated with a standard therapy, such as natalizumab (TYSABRI®) or glatiramer acetate for multiple sclerosis.

[0193] Examples of autoantigens associated with autoimmune disorders include thyroid stimulating hormone receptors 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 1 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 typically 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, by, for example, inhibiting unwanted immune responses to the antigen therapy and / or by allowing for the 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 to the subject a compound of the present disclosure, 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 to the subject antigen therapy (e.g., a therapeutically effective amount of the antigen therapy), e.g., simultaneously or sequentially (e.g., co-administering) 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 antigen therapy, e.g., a composition comprising a plurality of lipid particles, each lipid particle comprising a compound of the present disclosure and 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 embodiments, 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, and uveitis) and infliximab (Remicade®; for Crohn's disease, pediatric Crohn's disease, ulcerative colitis, pediatric ulcerative colitis, rheumatoid arthritis, ankylosing spondylitis, psoriatic arthritis, and plaque psoriasis), and golimumab. These include prazolam (Simponi®, for rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, and polyarticular juvenile idiopathic arthritis), etanercept (Enbrel®, for rheumatoid arthritis, polyarticular juvenile idiopathic arthritis, psoriatic arthritis, ankylosing spondylitis, and plaque psoriasis), and certolizumab pegol (Cimzia®, for Crohn's disease, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, non-radiographic axial spondyloarthritis, and 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, α-L-iduronidase for Hurler syndrome, and adenosine deaminase for adult adenosine deaminase deficiency.

[0197] In some embodiments, the antigen therapy is gene therapy. Gene therapy typically works through one of three mechanisms: (1) by providing a healthy copy of a disease-causing gene to the subject (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 involves DNA (e.g., antisense oligonucleotides (ASO)) and / or RNA (e.g., siRNA), which can be delivered to a subject in vivo or ex vivo via various products. In vivo gene delivery products include plasmid DNA, viral vectors (e.g., 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 therapies include the voretigene neparvovec-rzyl (Luxturna®, for retinal dystrophy) and the onasemnogene abeparvovec-xioi (Zolgensma®, for childhood spinal muscular atrophy).

[0198] In some embodiments, gene therapy involves 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, herpesviruses, adenoviruses, lentiviruses, rabies viruses, lentiviruses, VSV, poxviruses (e.g., vaccinia virus, smallpox virus, 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, often underlying transplant rejection. Accordingly, another embodiment is a method for treating graft-versus-host disease in a subject in need thereof, 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 thereof, 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 of the methods described herein, the method further comprises administering an antigen or an immunogenic fragment thereof to the subject. In some further embodiments, the antigen or an immunogenic fragment thereof and a compound of the present disclosure are co-administered. For example, it may sometimes be desirable to induce antigen-specific immune tolerance (e.g., when a compound of the present disclosure is administered to tolerize a subject to antigen therapy). When antigen-specific immune tolerance is desired, the antigen or an immunogenic fragment thereof and a compound of the present disclosure, for example, in the form of a composition as described herein, are preferably co-administered.

[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 present disclosure and an antigen) at the same or near-same site on a subject's body via the same route of administration.

[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, antigen, or 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) occurs within 24 hours, preferably within 12 hours, of administration of the first composition, e.g., within 10, 5, 4, 3, 2 hours, 60, 30, 15, 10, or 5 minutes of administration of the first composition. Typically, when co-administration is sequential, administration of the subsequent composition(s) follows immediately after completion of administration of the first composition, taking into account any operations that the clinician administering the compositions or the subject may need to perform to prepare for administration of the subsequent composition(s).

[0206] When co-administration is oral, administration site is mouth, and whether the two or more co-administered drugs are administered in a single preparation or in separate preparations, they are orally administered to the same site.However, when co-administration is by injection of two or more compositions, more generally, administration site is approximately the same.In this situation, the anatomical site of administration is 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 embodiments, the antigen or immunogenic fragment thereof and the compound of the present disclosure are co-administered. In a further embodiment, the antigen or immunogenic fragment thereof is administered prior to the administration of the compound of the present disclosure. In an alternative further embodiment, the compound of the present disclosure is administered prior to the administration of the antigen or immunogenic fragment thereof. In yet another further embodiment, the compound of the present disclosure and the antigen or immunogenic fragment thereof are administered simultaneously.

[0208] Co-administration can be by any of the routes of administration described herein. In some embodiments, the compounds of the present disclosure and the antigen or immunogenic fragment thereof are co-administered orally. In some embodiments, 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, 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 containing the gene therapy and the compound of the present disclosure to promote 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, 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 embodiments where effective co-presentation of a compound of the present disclosure and an antigen to a subject's immune system is facilitated by incorporating the compound of the present disclosure and the antigen into lipid particles comprising the compound of the present disclosure and the antigen.

[0211] The compounds of the present disclosure can 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 can be administered before, after, or simultaneously with the other therapy(ies) (e.g., additional therapeutic agent(s)). When administered simultaneously (e.g., concurrently), the compounds of the present disclosure and the other therapy can be in separate formulations or the same formulation. Alternatively, the compounds of the present disclosure and the other therapy can be administered sequentially as separate compositions at approximately the same time or at different times. When the compounds of the present disclosure and the other therapy (e.g., therapeutic agents) are administered as separate formulations or compositions, the compounds of the present disclosure and the other therapy can be administered by the same or 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 diseases, conditions, or disorders described herein.

[0212] In some aspects, the methods described herein further comprise administering to the subject additional non-antigenic therapy(ies) (e.g., a therapeutically effective amount), e.g., in combination with a compound of the present disclosure or a composition described herein. In some aspects, the compound or composition of the disclosure described herein is administered before the additional therapy(ies). In some aspects, the compound or composition of the disclosure described herein is administered after the additional therapy(ies). In some aspects, the compound or composition of the disclosure described herein is administered simultaneously with the additional therapy(ies).

[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 of body weight per treatment. Determining the dosage for a particular agent, subject, and disease is well within the capabilities of one of ordinary skill in the art. Preferably, the dosage causes no or minimal adverse 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, ingestion, 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), 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 compounds or compositions disclosed herein can be administered prophylactically according to the methods disclosed herein, as when a compound or composition disclosed herein is co-administered with antigen therapy to a subject with no known immune intolerance to the antigen therapy. The compounds or compositions disclosed 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., an allergic reaction, transplant rejection). Thus, in some embodiments, the subject does not have known immune intolerance to the antigen, e.g., because the subject is naive to the antigen. In some embodiments, the subject does not have known immune intolerance to the antigen after administration and / or exposure to the antigen. In some embodiments, the subject is immune intolerant to the antigen, e.g., 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 immunogenic fragment thereof (e.g., antigen therapy, such as a therapeutically effective amount of antigen therapy) to the subject in the absence of a compound of the present disclosure or a composition described herein.

[0218] However, a subject's immune intolerance may increase over time following the methods described herein, e.g., after subsequent exposure(s) to the antigen. In such cases, the methods described herein can be repeated, e.g., in the same manner as repeated "booster" vaccinations, 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, ingestion, topical, transdermal, rectal, parenteral (e.g., intra-arterial, intravenous, intramuscular, subcutaneous, or intradermal injection), intravenous infusion, and inhalation (e.g., intrabronchial, intranasal, or oral inhalation, intranasal infusion), depending on the compound and the particular condition 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. Accordingly, also provided herein is a method for 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 acyl chain-containing phospholipids), 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 or other therapeutic agents of the present disclosure can be administered at doses ranging from about 0.001 mg / kg to about 100 mg / kg of body weight, or at doses 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, suitable doses 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 of body weight per treatment. In some embodiments, suitable doses (e.g., daily doses) are about 0.1 mg / kg to about 10 mg / kg of 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 of body weight. Suitable doses 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 mentioned above may be required. The specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including, for example, the activity of the specific drug used, age, body weight, overall health, sex, diet, administration time, excretion rate, drug combination, severity and course of the disease, condition or symptom, the subject's predisposition to the 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 ability of one skilled in the art. [Example]

[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, we used the co-crystal structure of dicaproyl PS bound to TIM4 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 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. The endogenous ligand bound to the protein, phosphatidylserine (PS), was removed from the binding site. All water molecules were removed except for those in the binding site that formed a bridge between PS and the protein through hydrogen-bonding interactions. Charges were calculated for all atoms in the protein at physiological pH (7.4), the same pH used in in vitro experiments to measure binding affinity. Charges for 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 were performed for all molecules, and the top 20 binding poses were identified.

[0226] To validate the docking method, the endogenous ligand PS, which had previously been removed from the protein complex, was redocked into the binding site. The RMSD of the pose closest to the correct (native) pose was 2.213 Å and was identified among the top 11 poses. The binding affinity of PS to the protein was calculated computationally 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] The docking model showed that the polar groups of compound 2 aligned similarly to the natural ligand, forming 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 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. Anti-FoxP3 and anti-CD4 antibodies for flow cytometry were obtained from eBioscience. Compounds 1–3 were formulated using 10% DMPC in PBS. PS liposomes were synthesized at a PS:lipid molar ratio of 30:70.

[0230] Splenocytes 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 The compounds were administered at 0.1 μM. Cells were incubated for 72 hours before FoxP3+ / CD4+ T cell phenotypic analysis. Flow cytometry analysis was performed to assess changes in the proportion 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 an EC 50 and EC 90 I got both.

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

[0232] 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 resulting model after fitting the data to a four-parameter dose-response model. Table 2 shows the EC values ​​obtained from the model fitting. 50 and EC 90 This is a summary of the values ​​of [Table 2]

[0233] Lower and upper bounds on the percentage of FoxP3+ / CD4+ T cells were obtained from model fitting as a function of increasing dose for each compound. 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 the EC 50 and EC 90 The potency of compound 3 measured by α- and β-glucan-1 was two and one orders of magnitude higher (more potent) than that of PS, respectively. This is consistent with the results of molecular modeling 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 after 5 days of treatment.

[0237] 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. 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 assignment. Details of the treatment groups are shown in Table 3. Five days after oral administration, 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 removed for splenocyte analysis, and single-cell suspensions were prepared for cell phenotyping by flow cytometry. Cells were stained and gated for CD4. The percentages of FoxP3+ / CD4+ T cells and FoxP3+ / NRP1+ were assessed using flow cytometry. For dose-response analysis, dose-response data were fitted to four- and five-parameter log-logistic models using the "drc" package in R. Model fitting yielded EC 50 and EC 90 I got both.

[0240] The percentage of FoxP3+ / CD4+ T cells increased dose-dependently 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 is 4.88 μg, ED90 The ED was estimated to be 82.1 μg. Figure 2B shows the fit of the dose-PD model and the associated confidence interval 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 also shown.

[0241] The FoxP3+ / CD4+ T cell population was further analyzed for neuropilin-1 (NRP1) expression. Several reports have correlated NRP1 expression on T cells with the state of immune tolerance. NRP1 expression was observed at low doses of Compound 2. This was a nearly binary response, and the ED 50 is 4.32E -2 was estimated to be μg (Fig. 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-regs) increased by 150% from the lowest dose group to the highest dose group. Without being bound by 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 levels is considered sufficient to induce tolerance.

[0243] Example 4. MOG 35-55 Therapeutic efficacy of Compound 2 compared with standard treatments, anti-α4 mAb and glatiramer acetate, in an induced mouse EAE model 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 it, 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. While there is no known cure for MS, blocking the ability of autoreactive T cells to enter the CNS has proven to be an effective treatment option for ameliorating 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 offer a potential cure for MS. Compound 2 has been proposed as a T cell immunoglobulin mucin protein family receptor (TIM) agonist, capable of inducing 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. However, all anti-TIM3 antibodies that have demonstrated some functional efficacy in vivo and in vitro interfere with TIM3 binding to PS, suggesting that the PS-TIM3 interaction, even if low affinity, is key to TIM3 function.

[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. Furthermore, PS-mediated T cell arrest was blocked by anti-PS antibodies. Collectively, 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 Compound 2, a TIM agonist, on disease progression compared with standard-of-care anti-α4 mAb and glatiramer acetate. 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 within 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 listed 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 directed 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. 35-55 Mice are immunized with the peptide by subcutaneous injection (0.1 mL emulsion / mouse) at the base of the tail under anesthesia. On the day of injection (day 0) and two 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 an overview of the study. To evaluate the therapeutic effect of Compound 2, treatment was initiated 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 one of the treatment groups listed in Figure 3A or a placebo group. The treatment groups are listed 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 10 μg oral dose of Compound 2 was as effective as the full anti-α4 integrin dosing regimen equivalent to Tysabri, a third-line drug used for severe MS, in controlling MS symptoms after disease onset in a mouse EAE model. Switching from anti-α4 integrin to a 10 μg oral dose of Compound 2 after one or two injections of anti-α4 integrin also resulted in disease control after disease onset. 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 the 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. Boc-Dap-OH (1.2, 0.506 g) was then added, and the reaction mixture was stirred at room temperature for an additional 2 h. Ice was added, and the solution was acidified to pH 3. The isolated product was extracted with EtOAc (2 × 25 mL), washed with water, dried (NaSO), 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 a maximum of 1% AcOH / EtOAc over 20 min. Fractions were pooled after TLC. The residue was dissolved in EtOAc, n-heptane was added, and volatiles were removed under reduced pressure to remove traces of acetic acid. This process was repeated three times using minimal amounts of 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 hours. It was then warmed to room temperature and concentrated to half its 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 begins 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 over 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, ice was added, and then acidified 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. Boc-Dap-OH (0.349 g) was then 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 (NaSO), 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 over 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 4 N 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 CsCO (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 NaSO), 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 over 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 overnight at room temperature. The solution was concentrated under reduced pressure, ice was added, and the mixture was acidified 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. Fmoc-Dap-OH (0.217 g) was then added, and the reaction mixture was stirred at room temperature for an additional 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) over 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 using 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 hours. 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 overnight under vacuum in a desiccator.

[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 a 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 NaSO, 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 an additional 10 minutes. Then, a solution of Me2CO3 (3.5 mL) was added. The reaction mixture was stirred at −78° C. for 1 hour. The reaction mixture was quenched with saturated ammonium chloride solution (15 mL). The solution was extracted with EtOAc (2×75 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 first with 5% EtOAc / hexane, then up to 40% EtOAc / hexane 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 NaSO, 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, ice was added, and then acidified with 2N HCl (dropwise). The solution was extracted with EtOAc (2×100 mL), washed with brine, and dried (NaSO). 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 sticky material was extracted with EtOAc (2 x 75 mL). The combined organics were washed with water (2 x 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 approximately 50% EtOAc / hexane (20 mL), and air-dried under vacuum to give 126 mg of product.

[0276] MS: 666.3124 (M+H: 666.3139); 688.2954 (M+Na: 688.2959) indicated that the major product was the desired product coupled 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. A 4N HCl / dioxane solution (0.6 mL) of (S)-2-((tert-butoxycarbonyl)amino)-3-(2-(5-(naphthalen-1-ylmethoxy)pyridin-2-yl)acetamido)propanoic acid (0.050 g) was stirred at 0° C. for 3 hours. It was then warmed to room temperature and concentrated to half its 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 Whole 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 on ice for 30 minutes. Cells were then washed with PBS and stained with a viability dye to exclude dead cells. The 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 in the presence of T cells. 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 of 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 Whole 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 minutes. Then, cells were washed with PBS and stained with a viability dye to exclude dead cells. The 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 using FlowJov10. The results are shown in Figure 4B.

[0283] Treatment with 3 ng / 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% (a 606% increase). In total PBMCs, treatment with 3 ng / ml of Compound 2 increased the percentage of CD4+ / Foxp3+ T cells from 1.9% to 5.2% (a 171% increase). 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 x 10 5 Cells / well were incubated in 96-well round-bottom 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 the transcription factors Foxp3 and RORγt. Cells were washed and fixed for data acquisition on an Attune NXT flow cytometer. Data were analyzed using FlowJov10.

[0285] The results, shown in Figure 4C, demonstrate that compound 2 maintains Foxp3 expression and inhibits RORγt expression in a Th17-polarizing 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 Th17 expression 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 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), followed by viability dye 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 were analyzed using FlowJov10. The results are shown in Figure 4D.

[0287] In Figure 4D, blood-derived T cells from 5 of 6 MS patients responded to treatment with Compound 2 ex vivo, as evidenced 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 β-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 U.S. Food and Drug Administration (FDA) for the treatment of relapsing MS. This antibody is generally recommended for patients who have previously had an inadequate response to or are intolerant to alternative MS therapies. Natalizumab was diluted to a final concentration of 15 mg / ml with 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 a 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 the best preclinical model of multiple sclerosis (MS). More than 6,000 scientific papers have been published on this model. EAE is characterized by an immune response directed against CNS tissue and can be induced by immunizing animals against CNS proteins. In the active EAE model, MOG emulsified in complete Freund's adjuvant (CFA) is used. 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 100 mg of pertussis toxin (PT) in PBS at 200 ng / mouse / dose (0.1 mL) on the day of injection (day 0) and two 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 signs and the scoring criteria used in this study.

[0292] This study was designed to evaluate the initial sustained use of Compound 2 versus DMF (sustained use cohort) in a treatment escalation paradigm, and Compound 2 versus 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 in the 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 Figure 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 the 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 the score of 2.5 was recorded.

[0296] Overall survival: Death due to disease was monitored in the initial sustained use cohort and the escalation cohort. Death due to disease (clinical score 5) was recorded as an event "1", and death due to ulceration or ex vivo analysis was recorded as a "0". Animals surviving to the end of the study were recorded as a "0" on day 27. Survival analysis was performed in GraphPad Prism 9.

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

[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 up 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 randomly assigned to the DMF group, 10 mice to the untreated control group, and 9 mice to the Compound 2 group (3:1:1 randomization). All animals in the DMF-treated group (n = 30) progressed to a disease score of 2.5 within 7 days of disease onset. In contrast, only 5 of the 9 animals randomly assigned to the Compound 2-treated 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 receive natalizumab, and 9 were randomized to receive Compound 2.

[0301] As shown in Figure 5C, animals that continued DMF therapy progressed at the same rate as untreated control animals, with all animals achieving a clinical score of 5 by the end of the study. In contrast, animals treated with Compound 2 after disease onset had better overall disease control during treatment (days 1–14) and after treatment cessation (day 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 group, 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 with 6 of 9 animals switched from DMF to natalizumab.

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

[0304] In many cases, DMTs are reserved for patients who have failed at least one or two less effective treatments. This is primarily due to the risk / benefit profile of more effective DMTs in milder cases. The main problems with this paradigm are the high failure rate and intolerance of older, less effective DMTs and injectables. Furthermore, the early use of less effective drugs means missing the opportunity to control the disease early.

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

[0306] Because the disease progresses rapidly in the mouse MOG EAE model, the full escalation paradigm currently used in clinical practice and described above is not possible. Instead, we used a shorter version of the paradigm with only two escalation steps.

[0307] It is noteworthy that many published EAE models are performed prophylactically. In prophylactic models, animals are treated after immunization before clinical symptoms are observed. In the EAE studies described in this example, treatment was administered therapeutically after disease symptoms appeared. This difference may explain the poor performance of therapies such as glatiramer acetate and DMF in the models described herein, which also highlights the superior efficacy of Compound 2.

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

[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 symptom onset. 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 conducted 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) lists 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 administered 30 μg of Compound 2 / mouse subcutaneously once daily for up to 7 days, or for 3 consecutive days when the disease score was less than 2.5, at which point animals were administered a maintenance dose of 3 μg / mouse subcutaneously once daily for 14 days. All animals in all groups were monitored for disease score and survival for 14 days after the final dose.

[0314] Disease control (induction therapy): Disease control was defined as maintenance of a clinical score of 2.5 or less 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 either the DMF or Compound 2 maintenance groups according to Figure 6A. 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 ulcer formation or ex vivo analysis was recorded as "0". Animals surviving 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. As shown in Figure 6A, animals were randomized to treatment groups when they reached a disease score of 2.5. By day 15 after immunization, all animals were assigned to treatment groups.

[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 up 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 to the untreated control group, and 10 mice 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 in the Compound 2 (30 μg SC group) progressed to a score >2.5 (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 the 18 animals randomized to the natalizumab group, 11 / 18 survived the treatment induction period. Of the 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, four were randomly assigned to the DMF maintenance group and four 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 (day 7 after the start of DMF maintenance) was 4.37 (SD = 1.2). The last measured score for the Compound 2 group, measured on day 7 after the start 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 the DMF maintenance group 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 (Figure 6F).

[0324] The induction / maintenance treatment paradigm in the management of MS is an emerging paradigm. Patients with progressive disease are administered a highly potent (and less safe) DMT to induce disease control and then switched to a safer maintenance therapy. In this study, the therapeutic effects of Compound 2 were evaluated both in the induction phase versus natalizumab and in the maintenance phase versus DMF. Furthermore, the effects of sustained use of Compound 2 were evaluated as both an induction and maintenance agent 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 Figure 6A. A high dose of Compound 2 (30 μg / mouse SC) was superior to natalizumab in controlling disease and preventing escalation during the induction period. Furthermore, the mean clinical scores of animals treated with Compound 2 decreased during the induction period in at least 4 of 10 mice randomized to Compound 2, indicating improved clinical scores compared with 0 / 18 in the natalizumab group. 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 a low number of animals were available for randomization to the DMF or Compound 2 maintenance cohorts. However, the 1:1 randomization resulted in an n=4 in each group. The mortality rate for animals assigned to DMF maintenance therapy was 75%, while the mortality rate for the Compound 2 maintenance cohort was 0%. 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 14 days. This observation is consistent with previous studies showing that Compound 2 can reduce clinical scores in animals with stable high scores over time.

[0327] The results of this study demonstrate that early and sustained use of Compound 2 as both induction and maintenance medications can replace current induction / maintenance regimens. Furthermore, Compound 2 is a highly effective molecule and 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 with 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 with subcutaneous prednisone (1 μg / mouse) daily for 10 days after disease onset, or with 3 μg of Compound 2 daily for 10 days after disease onset, or left untreated, one day after adoptive transfer. Minimal disease expression was defined as a return to a clinical score of 1 after progression (a score of 2 or higher). The time to return to the minimum score was recorded, and the probability of reversion was plotted for untreated animals, animals treated with standard of care (SOC; prednisone), and Compound 2.

[0329] The results are shown in Figure 7. By day 10, 63% of animals treated with Compound 2 had returned to minimal disease expression (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 myelopathy, resulting in paralysis and visual loss. An NMO mouse model was established using the AQP4-p201-p220 peptide (Genemed Synthesis Inc.). Briefly, C57BL / 6 mice (n = 30) were immunized with 100 micrograms of AQP4-p201-p220 peptide emulsified in CFA on day 0. Mice were injected subcutaneously at the base of their tails under anesthesia. On the day of injection (day 0) and two days later, mice were intraperitoneally injected with pertussis toxin (PTx) in PBS at 200 ng / mouse / dose (0.1 ml).

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

[0332] Fundus examinations were performed on day 6 after immunization and approximately every four days thereafter, i.e., on days 9, 13, 16, 22, and 26. Each eye was scored for optic disc inflammation and overt retinal damage as determined by the appearance of "exudates" (specks) on fundus images. Eye samples were collected 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 tissue.

[0333] In the pilot study, exudates, a symptom of inflammation, were the most common finding. NMO is an optic nerve disease, and exudates have 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 effects 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 35-55Mice were immunized with 100 mg of Compound 2. At the onset of disease (clinical score 1), mice were randomly assigned to two treatment groups and received subcutaneous injections of 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 onset of systemic disease. On day 60, mice were randomly assigned to a treatment group (n = 12) (compound 2, 3 μg subcutaneous injection daily) or a control group (n = 11), and treatment was initiated. Mice were monitored daily for the development of cutaneous lupus by checking for the appearance of skin lesions.

[0337] Interim data from the study are 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). Furthermore, mice treated with Compound 2 experienced a two-day delay in the onset of skin symptoms compared to untreated control mice.

[0338] Example 14. Dextran sulfate sodium (DSS)-induced IBD model IBD was induced in 6- to 8-week-old female C57 / B6 mice by adding 3% DSS (Sigma-Aldrich) to their drinking water for 3 days on two separate occasions: days 0, 1, and 2 of the induction phase, and days 11, 12, and 13 of the rechallenge 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 rechallenge phase. The control group (n = 5) did not receive any treatment. Mouse weights were monitored daily from day 0.

[0339] As shown in Figure 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) will be 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 a single ascending oral dose in healthy adult volunteers, and (2) to evaluate the safety and tolerability of Compound 2 after single ascending / multiple ascending oral doses in adult subjects with PPMS, RRMS, NMO, or MOGAD. Secondary objectives of the study are: (1) to evaluate the pharmacokinetics of Compound 2 after a single ascending oral dose in healthy adult volunteers, and (2) to evaluate the pharmacokinetics of Compound 2 after a single ascending / multiple ascending oral dose 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 treatment + placebo + 2 enrolled in 1:1 and 1:4 designs). Cohorts 1, 2, and 3 will enroll healthy volunteers. Cohorts 4 and 5 will enroll subjects with PPMS or RRMS. Cohort 6 may enroll healthy volunteers or subjects with PPMS or RRMS.

[0342] After Cohort 3 clears SAD, Phase I MAD will begin. 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 terminate after 14 days of follow-up or as needed for the SAD study. Subjects with RRMS or PPMS will receive a single oral dose of Compound 2 during the SAD portion of the study and daily oral doses of Compound 2 for up to 28 days during the MAD portion of the study. During this phase 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 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] The 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 Reproductive Pathology (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 their 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, socioeconomic, or educational status. 4. Body mass index (BMI) at screening: 18.0 to 35.0 kg / m 2 (including borderline values) (under 56 years old), BMI at screening 18.0 to 30.0 kg / m 2 (Borderline values ​​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 autoantibody NMO-IgG test. 7. Subject has been confirmed to have 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 recurrent 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 of 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 met from the time of first administration of study drug through follow-up visits (for the subject and their partner), including an additional 90-day period (for the subject).

[0346] Exclusion criteria included the following: 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, brainstem, 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. Subjects with 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 immunodepleting therapy within 3 months prior to blood collection for this study or PBMC sample collection and processing, or is expected to require immunosuppressive therapy 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 on a 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, or COVID-19 convalescent plasma. 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 infertility 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 ideation / 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: a maximum of 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, randomized 4:1 treatment to placebo. The total sample size per cohort will be determined by dose-limiting toxicities (DLTs). 12-24 subjects with RRMS and / or PPMS will be enrolled in cohorts 4, 5, and 6 without randomization to dose. The total sample size per cohort will be determined by DLTs. 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 RRMS to PPMS ratio 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 different concentrations of Compound 2 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 an appropriate container. To facilitate dissolution, alternating sonication and magnetic stirring was used to obtain a clear solution. The solution was then dispensed into amber PET bottles, appropriately labeled, and stored 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 for at least 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. Potency was maintained after three months of storage and several freeze-thaw cycles between -80°C and room temperature, demonstrating 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] While 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. The present invention provides, for example, the following items. (Item 1) A compound of the following structural formula: [ka] or a pharmaceutically 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 are independently chloro or fluoro; R 3 are selected independently (C6-C 15 ) aryl or (C5-C 15 ) (C1-C3) alkyl substituted with heteroaryl, 15 ) aryl and (C5-C 15 ) heteroaryl, each independently, is —(R30 ) p is replaced by Each R 30 are independently halo, (C-C)alkoxy, (C-C)haloalkoxy, (C-C)alkyl, or (C-C)haloalkyl, or two R attached to adjacent ring atoms 30 together - (CH2) q - or -O(CH2) r O- is formed, 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. (Item 2) The compound according to item 1, wherein ring A is phenyl or pyridinyl. (Item 3) 3. The compound according to item 1 or 2, wherein ring A is phenyl. (Item 4) In the formula, L is -(CH2)- n 4. The compound according to any one of items 1 to 3, wherein (Item 5) In the formula, R 1 is H. (Item 6) In the formula, R 3 However, each independently -(R 30 ) p substituted with one, two or three independently selected (C-C 15 ) aryl or (C5-C 15 6. The compound according to any one of items 1 to 5, wherein R is methyl substituted with heteroaryl. (Item 7) In the formula, R 3 However, each independently -(R 30 ) pone, two, or three independently selected from phenyl, naphthyl, pyridinyl, quinolinyl, isoquinolinyl, or benzo[d]imidazolyl, substituted with 15 ) aryl or (C5-C 15 ) methyl substituted with heteroaryl. (Item 8) In the formula, R 3 However, each independently -(R 30 ) p one or two independently selected (C-C 15 ) aryl or (C5-C 15 8. The compound according to any one of items 1 to 7, wherein R is methyl substituted with heteroaryl. (Item 9) 9. The compound according to any one of items 1 to 8, wherein n is 1. (Item 10) 10. The compound according to any one of items 1 to 9, wherein m is 0. (Item 11) 11. The compound according to any one of items 1 to 10, wherein each p is independently 0, 1, or 2. (Item 12) Item 12. The compound according to item 11, wherein each p is 0. (Item 13) 13. The compound according to any one of items 1 to 12, wherein each q is independently 3 or 4. (Item 14) 14. The compound according to any one of items 1 to 13, wherein each r is independently 1 or 2. (Item 15) A compound according to any one of items 1 and 5-14 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof, wherein: X 1, X 2 , and X 3 are >C(H), respectively, X 1 is N and X 2 and X 3 are >C(H), respectively, 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, or a pharmaceutically acceptable salt thereof. (Item 16) In the formula, X 1 , X 2 , and X 3 and each are >C(H). (Item 17) In the formula, X 1 is N and X 2 and X 3 and each are >C(H). (Item 18) In the formula, -OR 3 But L or -(CH2) n - is attached to a ring atom of ring A that is meta or para to (Item 19) The compound according to item 1 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. (Item 20) The compound according to item 1 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. (Item 21) The compound according to item 1 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. (Item 22) The compound according to item 1 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. (Item 23) The compound according to item 1 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. (Item 24) The compound according to item 1 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. (Item 25) The compound according to item 1 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. (Item 26) The compound according to item 1 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. (Item 27) The compound according to item 1 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. (Item 28) The compound according to item 1 of the following structural formula: [ka] or a pharmaceutically acceptable salt thereof. (Item 29) 29. A composition comprising the compound according to any one of items 1 to 28 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. (Item 30) 30. The composition of item 29, further comprising an antigen. (Item 31) 30. A method for treating an autoimmune disorder in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the composition of item 29. (Item 32) 32. The method of claim 31, further comprising administering to the subject an autoantigen associated with the autoimmune disorder, or an immunogenic fragment thereof. (Item 33) 31. A method for treating an autoimmune disorder in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the composition of claim 30, wherein the antigen is an autoantigen associated with the autoimmune disorder, or an immunogenic fragment thereof. (Item 34) The autoimmune disorder may be 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), Barrow's disease, Behçet's disease, benign mucous membrane pemphigoid, bullous pemphigoid, Castleman's disease (CD), or ), celiac disease, Chagas disease, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing 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 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's syndrome purpura (HSP), herpes gestationis or pemphigoid of pregnancy (PG), hidradenitis suppurativa (HS) (acne inversa), hypogammaglobulinemia, IgA nephropathy, IgG4-related sclerosing disease, immune thrombocytopenic purpura (ITP), inclusion body myositis (IBM), interstitial cystitis (IC), juvenile arthritis, juvenile diabetes mellitus (type 1 diabetes), juvenile Myositis (JM), Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear immunoglobulin A disease (LAD), lupus, chronic Lyme disease, Meniere's disease, microscopic polyangiitis (MPA), mixed connective tissue disease (MCTD), Mooren's ulcer, Much-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 rheumatoid arthritis (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, progestational dermatitis, psoriasis, psoriatic arthritis, pure red cell aplasia (PRCA), pyoderma gangrenosum, Raynaud's phenomenon, reactive arthritis, reflex sympathetic dystrophy, relapsing polychondritis, restless legs syndrome (RLS) LS), retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-person syndrome (SPS), subacute bacterial endocarditis (SBE), Susac's 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, ulcerative colitis (UC), undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vitiligo, or Vogt-Koyanagi-Harada syndrome. (Item 35) 34. The method of any one of items 31 to 33, wherein the autoimmune disorder is 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-Barré syndrome, Hashimoto's thyroiditis, steroid-responsive encephalopathy with autoimmune thyroiditis (SREAT), neurosarcoidosis, optic neuritis, or transverse myelitis. (Item 36) 34. The method of any one of items 31 to 33, wherein the autoimmune disorder is multiple sclerosis, neuromyelitis optica, myelin oligodendrocyte glycoprotein antibody-associated disease (MOGAD), rheumatoid arthritis, or myasthenia gravis. (Item 37) 34. The method of any one of items 31 to 33, wherein the autoimmune disorder is multiple sclerosis. (Item 38) 1. A method of treating a disease, disorder, or condition in a subject in need thereof by antigen therapy, said method comprising administering to said subject: (i) a therapeutically effective amount of said antigen therapy, and immunotolerizing said subject to said antigen therapy; 29. The composition of claim 29, in an amount sufficient to (ii) A therapeutically effective amount of the composition of item 30, wherein the antigen in the composition is the antigen therapy. (Item 39) 37. The method of any one of items 32 to 36, wherein the composition is co-administered with the autoantigen or antigen therapy. (Item 40) 40. The method of claim 38 or 39, wherein the antigen therapy is gene therapy or enzyme replacement therapy. (Item 41) 31. A method for immune tolerization of a subject in need thereof, comprising administering to said subject a therapeutically effective amount of the composition of item 29 or 30. (Item 42) 1. A method of immune tolerization to an antigen in a subject in need thereof, the method comprising administering to said subject: (i) an antigen, or an immunogenic fragment thereof, and a therapeutically effective amount of the combination described in item 29 Composition, or (ii) A therapeutically effective amount of the composition according to item 30, wherein the antigen in the composition is the antigen to which the subject is to be tolerized or an immunogenic fragment of the antigen to which the subject is to be tolerized. (Item 43) A method for inducing a regulatory T cell population in a subject, comprising administering to the subject a therapeutically effective amount of the composition according to item 29 or 30. (Item 44) 31. A method for inducing a regulatory T cell population in a subject in response to an antigen, comprising administering to the subject a therapeutically effective amount of the composition of claim 30, wherein the antigen in the composition is the antigen in response to which the regulatory T cell population is to be induced, or an immunogenic fragment of the antigen in response to which the regulatory T cell population is to be induced. (Item 45) 1. A method of inhibiting or reducing antigen-specific antibody titers in a subject, the method comprising administering to said subject: (i) an antigen and a therapeutically effective amount of the composition according to item 29; or (ii) A therapeutically effective amount of the composition of item 30, wherein the antigen in the composition is the antigen against which the antibody titer is to be inhibited or reduced, or an immunogenic fragment of the antigen against which the antibody titer is to be inhibited or reduced. (Item 46) 31. A method for increasing the activity or level of tolerogenic T cells in a subject, comprising administering to the subject a therapeutically effective amount of the composition of item 29 or 30. (Item 47) A method for inducing a regulatory B cell population in a subject, comprising administering to the subject a therapeutically effective amount of the composition according to item 29 or 30. (Item 48) 46. ​​The method of any one of items 42, 44, and 45, wherein the antigen, or the immunogenic fragment thereof, and the composition are co-administered. (Item 49) 49. The composition of item 30 or the method of any one of items 42, 44, 45, and 48, wherein the antigen is a protein. (Item 50) 50. The composition of item 30 or the method of any one of items 42, 44, 45, 48, and 49, wherein the antigen is an autoantigen. (Item 51) 50. The composition of item 30 or the method of any one of items 42, 44, 45, 48, and 49, wherein the antigen is a foreign antigen. (Item 52) 52. The composition or method of item 51, wherein the foreign antigen is antigen therapy. (Item 53) 53. The composition or method of item 51 or 52, wherein the foreign antigen is a therapeutic protein. (Item 54) 54. The composition or method according to any one of items 51 to 53, wherein the foreign antigen is an enzyme replacement therapy. (Item 55) 53. The composition or method of item 51 or 52, wherein the foreign antigen is a cell therapy or gene therapy. (Item 56) 56. The method of any one of items 32 to 55, further comprising administering to the subject an additional therapeutic agent. (Item 57) 57. The method according to any one of items 32 to 56, wherein the composition is administered orally.

Claims

[Claim 1] The invention described in the specification.

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