Oral dosing of itaconate prodrugs for skin delivery

Abstract

Disclosed are methods for treating an autoimmune and / or an inflammatory disease, disorder, or condition, including a skin disease, disorder, or condition comprising orally administering a prodrug of itaconate, 4-methyl itaconate, or 1-methyl itaconate to a subject in need of treatment thereof.

Description

ORAL DOSING OF ITACONATE PRODRUGS FOR SKIN DELIVERYCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 739,746 filed December 30, 2024, which is incorporated herein by reference in its entirety.BACKGROUND

[0002] Itaconic acid (IA, itaconate) is an endogenous immunomodulatory metabolite produced by diverting aconitate from the tricarboxylic acid (TCA) cycle during the activation of inflammatory macrophages. IA is an unsaturated dicarboxylic acid synthesized by immune-responsive gene 1 (IRG1). O'Neill and Artyomov, 2019. IA acts on multiple inflammatory pathways and is shown to be an essential functional component of activated macrophages. Ferreira et al., 2019; Lampropoulou et al., 2016.

[0003] Despite being a widely used commercial biomaterial derived from Aspergillus for decades, its identification as a mammalian immunometabolite was not established until 2011. Ferreira et al., 2019; Lawrence et al., 2012. It acts as an immunomodulator through several mechanisms: inhibition of succinate dehydrogenase (SDH), Qin et al., 2019, activation of Nrf2 via alkylation of KEAP1, Song et al., 2020, regulation of ATF3 / IκBζ inflammatory axis, Bambouskova et al., 2018, inhibition of glycolysis, Qin et al., 2019, regulation of type I IFNs, Swain et al., 2020, and inhibition of NLRP3 inflammasome. Hooftman et al., 2020.

[0004] Itaconate contains an α,β-unsaturated alkene and shares structural similarity with other metabolites, such as phosphoenolpyruvate, succinate, malonate, and fumarate. There is a growing body of evidence suggesting that itaconate, as well as its derivatives, have promise in various disease models. Song et al., 2020; Runtsch et al., 2022. Despite promising efficacy in preclinical models, clinical potential of IA is limited due to its highly polar structure, making it impermeable to biological membranes.

[0005] Several cell-permeable itaconate analogs, including dimethyl itaconate (DMI), 4-octyl itaconate (4-01), and 4-ethyl itaconate (4-EI), have been developed to mimic the actions of endogenous itaconate. DMI and 4-OI, interestingly have been shown to conserve the antiinflammatory effect of IA, Yang et al., 2021; Li et al., 2023, but fall short in their ability to convert to intracellular IA once delivered. Swain et al., 2020; ElAzzouny et al., 2017. Additionally, it has143955.601_P17708-02been reported that DMI is intracellularly converted into a mixture of 1 -methyl itaconate (1-MI) and 4-methyl itaconate (4-MI) and presumably exerts its effect through these active metabolites, rather than itaconate itself. ElAzzouny et al., 2017.SUMMARY

[0006] In some aspects, the presently disclosed subject matter provides a method for treating an autoimmune and / or inflammatory disease, disorder, or condition, the method comprising orally administering a compound of formula (la), formula (lb), or formula (Ic) to a subject in need of treatment thereof:00 OR RiaO OR2a r,b~ R1O2C

[0007] (la); (lb); or (Ic); wherein:

[0008] Ria is selected from: H, -CH2-O-C(=O)-C(CH3)3, -CH2-O-C(=O)-O-CH(CH3)2. -CH2-CH2- OO-(CH2)i5-CH3, and

[0009] Rib, R2a, and R2c are each independently selected from: -CH2-O-C(=O)-C(CH3)3, -CH2-O-C(=O)-O-CH(CH3)2, -CH2-CH2-O-(CH2)i5-CH3, and; and pharmaceutically acceptable salts thereof.

[0010] In some aspects, Riais H or -CH2-O-C(=O)-O-CH(CH3)2 and R2a, R2b. and R2c at each occurrence are -CH2-O-C(=O)-O-CH(CH3)2. In certain aspects, the compound of formula (la), formula (lb), or formula (Ic) is selected from:

[0011] (P2)

[0012] (P9); and (Pl 3)243955.601_P17708-02

[0013] In particular aspects, the compound of formula (la), formula (lb), or formula (Ic) is selected from:

[0014] (P2)

[0015] and (Pl 3)

[0016] In more particular aspects, the compound of formula (la), formula (lb), or formula (Ic) is selected from:

[0019] In some aspects. Ria, R2a. R2b, and R2c at each occurrence are -CH2-O-C(=O)-C(CH3)3. In certain aspects, the compound of formula (la), formula (lb), or formula (Ic) is selected from:

[0020] (Pl)

[0021] (P8); and (Pl 2)

[0022] In some aspects, Ria. R2a, R2t, and R2c at each occurrence are:. In certain aspects, the compound of formula (la), formula (lb), or formula (Ic) is selected from:343955.601_P17708-02

[0024] (PIO); and (Pl 4) O

[0025] In some aspects. Ria, R2a. R2b, and R2c at each occurrence are -CH2-CH2-O-(CH2)i5-CH3. In certain aspects, the compound of formula (la), formula (lb), or formula (1c) are selected from:O O HO^^^°^^^O(CH2)15CH^ H3C(H2C)15O'^^OX\<^0^

[0026] (P7)11o; (PH)0;o \OA^YOX^X / O(CH2)15CH3

[0027] and (P15)11O

[0028] In some aspects, the autoimmune and / or the inflammatory disease, disorder, or condition comprises a skin disease, disorder, or condition. In particular aspects, the skin disease, disorder, or condition alopecia areata.

[0029] In some aspects, the autoimmune and / or the inflammatory disease, disorder, or condition is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), sepsis, a viral infection, ischemia / reperfusion injury, pulmonary fibrosis, gout, abdominal aortic aneurysm (AAA), and cryopyrin-associated periodic syndrome (CAPS).

[0030] In some aspects, the autoimmune and / or the inflammatory disease, disorder, or condition is selected from type 1 diabetes, celiac disease, inflammatory bowel disease (IBD), including Crohn's Disease and ulcerative colitis, Sjogren's syndrome, Graves' disease, Hashimoto's thyroiditis, Addison's disease, vitiligo, myasthenia gravis, scleroderma, spondyloarthritis, vasculitis, sarcoidosis, chronic obstructive pulmonary disease (COPD), asthma, periodontitis, sarcoidosis, chronic inflammatory demyelinating polyneuropathy (CIDP), Behcet's disease, and achalasia.

[0031] In other aspects, the presently disclosed subject matter provides a pharmaceutical composition formulated for oral delivery of a compound of formula (la), formula (lb), or formula (Ic) to a subject in need of treatment thereof:O 0 O

[0032] 443955.601_P17708-02

[0033] wherein:

[0034] Ria is selected from: H, -CH2-O-C(=O)-C(CH3)3, -CH2-O-C(=O)-O-CH(CH3)2, -CH2-CH2- O O OO-(CH2)i5-CH3, and;

[0035] Rib, R2a, and R2care each independently selected from: -CH2-O-C(=O)-C(CH3)3, -CH2-O- O O A OC(=O)-O-CH(CH3)2, -CH2-CH2-O-(CH2)15-CH3, and ■

[0036] and pharmaceutically acceptable salts thereof.

[0037] Certain aspects of the presently disclosed subject matter having been stated hereinabove, which are addressed in whole or in part by the presently disclosed subject matter, other aspects will become evident as the description proceeds when taken in connection with the accompanying Examples and Figures as best described herein below.BRIEF DESCRIPTION OF THE FIGURES

[0038] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawings will be provided by the Office upon request and payment of the necessary fee.

[0039] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0040] FIG. 1A, FIG. IB, FIG. 1C, FIG. ID, FIG. IE, and FIG. IF show the effect of representative presently disclosed compounds P2, P9, and P13 on gene expression in Poly (I:C) (50 μg / mL) + IFNy (5 ng / mL) induced NHEK cells. The expression of chemokine and cytokine genes in NHEK cells are measured after 8 h incubation of P2, P9, and P13 (1-100 μM). The measured chemokine and cytokines include CXCL 9 (FIG. 1A), CXCL 10 (FIG. 1B), CXCL 11 (FIG. 1C), IFNβ (FIG. 1D), IL-1β (FIG. 1E), and IL-6 (FIG. 1F). Data expressed as mean ± SEM, n=3. Statistical analysis was performed using one-way ANOVA by Dunnett’s multiple comparisons test with comparing with poly (EC) alone; ***p < 0.0005, **p < 0.005; *p < 0.05.543955.601_P17708-02

[0041] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E and FIG. 2F show delivery of IA or 4-MI in plasma and skin of mice dosed with P2 and P13, respectively. (FIG. 2A and FIG. 2C), release of IA in plasma and skin, respectively, from mice dosed with P2 (FIG. 2B and FIG. 2D), release of 4-MI in plasma and skin, respectively, from mice dosed with P13, and (FIG. 2E and FIG. 2F), pharmacokinetics (PK) parameters of IA released from P2, and 4-MI released from P13, respectively. Data are expressed as means ± SEM (n = 3 per time point).

[0042] FIG. 3A and FIG. 3B demonstrate the impact of itaconate, 4-methylitaconate and its prodrugs on Toll-Like Receptor (TLR3) and interleukin 6 (IL-6) in normal human epidermal keratinocytes. Poly (I:C) and IFN γ induced expression of proinflammatory cytokines in normal human epidermal keratinocytes (NHEKs) following treatment with 500 μM of IA and 4-MI or 100 μM of P2 and P13. (FIG. 3A) Inhibition of TLR3 expression and (FIG. 3B) inhibition of IL-6 in NHEKs (n = 1 or 2). IA and 4-MI did not show any effect on TLR3 and IL-6, while P2 and P13 showed over 90% of inhibition.

[0043] FIG. 4 demonstrates that administration of P13 (10 mg / kg and 30 mg / kg equivalent, 7 days) had no significant effect on body weight. Vehicle (10% DMSO, 10% tween 80 and 80% PBS) or P13 (30 mg / kg or 100 mg / kg eq. dose per day) were intraperitoneally administered for 7 days. No significant weight loss was observed during the experiments.DETAILED DESCRIPTION

[0044] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0045] Abbreviations

[0046] itaconic acid (IA)

[0047] tricarboxylic acid (TCA)

[0048] immune-responsive gene 1 (IRG1)

[0049] succinate dehydrogenase (SDH)643955.601_P17708-02

[0050] dimethyl itaconate (DMI)

[0051] 4-octyl itaconate (4-OI)

[0052] 4-ethyl itaconate (4-EI)

[0053] 1 -methyl itaconate (1 -MI)

[0054] 4-methyl itaconate (4-MI)

[0055] pivaloyloxymethyl (POM)

[0056] isopropyloxycarbonyloxymethyl (POC)

[0057] (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (ODOL)

[0058] 3-(hexadecyloxy)propyl (HDP)

[0059] sodium iodide (Nal)

[0060] acetonitrile (ACN)

[0061] dichloromethane (DCM)

[0062] high-performance liquid chromatography (HPLC)

[0063] nuclear magnetic resonance (NMR)

[0064] ethyl acetate (EtOAc)

[0065] high resolution mass spectrometry (HRMS)

[0066] normal human epidermal keratinocyte (NHEK)

[0067] parallel artificial membrane permeability assay (PAMPA)

[0068] Methods for Treating an Autoimmune and / or Inflammatory Disease, Disorder, or Condition

[0069] In some embodiments, the presently disclosed subject matter provides a method for treating an autoimmune and / or inflammatory disease, disorder, or condition comprising orally administering a prodrug of itaconate, 4-methyl itaconate, or 1 -methyl itaconate, the structures of which are provided immediately herein below:O O

[0070] (itaconate)H°'^V °H; (4-methyl itaconate)0; (1 -methyl itaconate)743955.601_P17708-02

[0071] In some embodiments, the prodrug is selected from pivaloyloxymethyl (POM), isopropyloxycarbonyloxymethyl (POC), (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (ODOL), and 3-(hexadecyloxy)propyl (HDP):; (ODOL) ■ and (HDP) - CH2-CH2-O-(CH2)15-CH3. In such embodiments, e.g„ with POM, POC, and HDP, the prodrug moieties includes a terminal alkyl group. Representative alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, w-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, sec-pentyl, isopentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl. In particular embodiments, the alkyl group is t-butyl, isopropyl, and methyl as shown immediately hereinabove.

[0073] More particularly, in some embodiments, the presently disclosed subject matter provides a method for treating an autoimmune and / or an inflammatory disease, disorder, or condition, the method comprising orally administering a compound of formula (la), formula (lb), or formula (Ic) to a subject in need of treatment thereof:(lb); or

[0075] wherein:

[0076] Ria is selected from: H, -CH2-O-C(=O)-C(CH3)3, -CH2-O-C(=O)-O-CH(CH3)2, -CH2-CH2-O-(CH2)i5-CH3, and

[0077] Rib, R2a, and R2care each independently selected from: -CH2-O-C(=O)-C(CH3)3, -CH2-O-C(=O)-O-CH(CH3)2, -CH2-CH2-O-(CH2)i5-CH3, and; and pharmaceutically acceptable salts thereof.843955.601_P17708-02

[0078] In some embodiments, Riais H or -CH2-O-C(=O)-O-CH(CH3)2 and R2a, R2b, and R2c at each occurrence are -CH2-O-C(=O)-O-CH(CH3)2. In certain embodiments, the compound of formula (la), formula (lb), or formula (Ic) is selected from:

[0081] In particular embodiments, the compound of formula (la), formula (lb), or formula (Ic) is selected from:

[0084] In more particular embodiments, the compound of formula (la), formula (lb), or formula (Ic) is selected from:

[0087] In some embodiments, R1a, R2a, R2b, and R2cat each occurrence are -CH₂-O-C(=O)-C(CH₃)₃. In certain embodiments, the compound of formula (la), formula (lb), or formula (Ic) is selected from:943955.601_P17708-02

[0089]

[0090] In some embodiments, R1a, R2a, R2b, and R2cat each occurrence are:embodiments, the compound of formula (la), formula (lb), or formula (Ic) is selected from:

[0093] In some embodiments, Ria. Ria, Rib. and Ricat each occurrence are -CHi-CHi-O-(CHi)i5- CH3. In certain embodiments, the compound of formula (la), formula (lb), or formula (1c) are selected from:H3C(H2C)15OZ^^O / Y^Y

[0094] (P7)11O; (PH)1100 \OAY^. O^^^O(CH2)15CH3and (P15)11O

[0095] In some embodiments, the autoimmune and / or inflammatory disease, disorder, or condition comprises a skin disorder, condition, or disease. In certain embodiments, the skin disease, disorder, or condition is associated with hair loss. In particular embodiments, the skin disease, disorder, or condition associated with hair loss comprises alopecia areata.

[0096] Alopecia areata is a prevalent inflammatory cause of hair loss and is thought to be an autoimmune disease in which T-lymphocytes attack the hair follicles, causing the hair to stop growing and enter into the telogen phase. At the end of the telogen phase, the hair falls out. Three subtypes of alopecia areata exist, which are named according to their severity: (i) alopecia areata,1043955.601_P17708-02which involves mild patchy hair loss on the scalp; (ii) alopecia totalis, which involves loss of all scalp hair; and (iii) alopecia universalis which involves loss of scalp and all body hair. Other types of alopecia include, but are not limited to, androgenic alopecia, anagen effluvium, self-induced hair loss, telogen effluvium, and scarring alopecia.

[0097] Androgenetic alopecia includes male pattern baldness and female pattern baldness and accounts for 95% of all hair loss. Anagen effluvium is the sudden hair loss that occurs as a result of exposure to chemicals or radiation, such as the hair loss that results during certain types of chemotherapy or radiation treatment, or as a result of exposure to toxic chemicals, such as thallium and arsenic. In most cases hair growth will return to normal once treatment is finished or exposure to the toxin is eliminated.

[0098] Self-induced hair loss may be inflicted consciously or unconsciously. The two main types of self-induced hair loss are trichotillomania and traction alopecia. Trichotillomania is self-induced hair loss which results from the continuous pulling or plucking of the hair. Traction alopecia is usually caused by continuous and excessive pulling on the hair due to various types of hairstyling, which gradually results in hair loss that may become permanent.

[0099] Telogen effluvium is sudden or severe stress related hair loss, which appears as thinning throughout the whole scalp. A sudden or stressful event can cause the hair follicles to prematurely stop growing and enter into a resting phase. Other causes of telogen effluvium include thyroid gland malfunction (hypothyroidism or hyperthyroidism, which occurs when the thyroid gland produces too little or too much, respectively, of the thyroid hormone, thyroxin); diabetes; anemia; and the autoimmune disease systemic lupus erythematosis.

[0100] Scarring alopecia occurs as a result of inflammation of the hair follicles due to infection. Scarring alopecia may be caused by discoid lupus erythematosus, a diffuse connective tissue disease; lichenplanus, which is an inflammatory disease that strikes primarily the skin and mucous membranes; Pseudopelade of Brocq. a rare scarring alopecia which has no potential for regrowth; aplasia cutis congenita, a rare disorder that often results as a small blistered atrophied area usually in the midline of the scalp and present from birth; or congenital trichia.

[0101] Further subtypes of alopecia areata, include alopecia areata monolocularis, alopecia areata multilocularis, ophiasis, alopecia areata universalis, alopecia areata totalis, and alopecia areata barbae.1143955.601_P17708-02

[0102] Other types of hair loss include syphilitic alopecia, a secondary manifestation of syphilis; scleroderma; and tinea capitis (ringworm).

[0103] In some embodiments, the presently disclosed subject matter provides a method for treating an inflammatory or autoimmune skin disease and / or other pruritic conditions including, but not limited to, atopic dermatitis, vitiligo, non-segmental vitiligo, prurigo nodularis, lichen planus, contact dermatitis, dyshidrotic eczema, eczema, nummular dermatitis, seborrheic dermatitis, stasis dermatitis, primary localized cutaneous amyloidosis, bullous pemphigoid, skin manifestations of graft versus host disease, pemphigoid, discoid lupus, granuloma annulare, lichen simplex chronicus, pruritus, vulvar / scrotal / perianal pruritus, lichen sclerosus, post-herpetic neuralgia itch, lichen planopilaris, psoriasis, chronic hand eczema, hidradenitis suppurativa, hypereosinophilic syndrome, systemic lupus erythematosus, and foliculitis decalvans.

[0104] In some embodiments, the autoimmune and / or the inflammatory disease, disorder, or condition is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE). multiple sclerosis (MS), sepsis, a viral infection, ischemia / reperfusion injury, pulmonary fibrosis, gout, abdominal aortic aneurysm (AAA), and cryopyrin- associated periodic syndrome (CAPS).

[0105] In some embodiments, the autoimmune and / or the inflammatory disease, disorder, or condition is selected from type 1 diabetes, celiac disease, inflammatory bowel disease (IBD), including Crohn's Disease and ulcerative colitis, Sjogren's syndrome, Graves' disease, Hashimoto's thyroiditis, Addison's disease, vitiligo, myasthenia gravis, scleroderma, spondyloarthritis, vasculitis, sarcoidosis, chronic obstructive pulmonary disease (COPD), asthma, periodontitis, sarcoidosis, chronic inflammatory demyelinating polyneuropathy (CIDP), Behcet's disease, and achalasia.

[0106] As used herein, the term “treating” can include reversing, alleviating, inhibiting the progression of. preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. Preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. Accordingly, the presently disclosed compounds can be administered prophylactically to prevent or reduce the incidence or recurrence of the disease, disorder, or condition.

[0107] In general, a “therapeutically effective amount” of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response. As will be appreciated by those of1243955.601_P17708-02ordinary skill in the art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, and the like. In some embodiments, the term “therapeutically effective amount” refers to an amount sufficient to reduce or ameliorate the severity, duration, progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurrence, development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0108] The “subject” treated by the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0109] In certain embodiments, a compound of formula (la), formula (lb), or formula (Ic), or a pharmaceutically acceptable salt thereof, can be used in combination with one or more compound useful to treat inflammatory skin diseases. In some embodiments, the one or more compound is a steroid, a corticosteroid, an antibiotic, and / or an antihistamine.

[0110] The term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly a compound of formula (la), formula (lb), or1343955.601_P17708-02formula (Ic) disclosed herein and at least one other therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g„ single disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state.

[0111] Further, the compounds disclosed herein can be administered alone or in combination with adjuvants that enhance stability of the compounds, alone or in combination with one or more therapeutic agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0112] The timing of administration of a compound disclosed herein and at least one additional therapeutic agent can be varied so long as the beneficial effects of the combination of these agents are achieved. Accordingly, the phrase “in combination with” refers to the administration of a compound described herein and at least one additional therapeutic agent either simultaneously, sequentially, or a combination thereof. Therefore, a subject administered a combination of a compound described herein and at least one additional therapeutic agent can receive a compound and at least one additional therapeutic agent at the same time (i.e., simultaneously) or at different times (i.e., sequentially, in either order, on the same day or on different days), so long as the effect of the combination of both agents is achieved in the subject.

[0113] When administered sequentially, the agents can be administered within 1, 5, 10, 30. 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the compound described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions,1443955.601_P17708-02each comprising either a compound or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents.

[0114] When administered in combination, the effective concentration of each of the agents to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents may, but need not be, additive or synergistic. The agents may be administered multiple times.

[0115] In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.

[0116] Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by:

[0117] Qa / QA + Qb / QB = Synergy Index (SI)

[0118] wherein:

[0119] QA is the concentration of a component A, acting alone, which produced an end point in relation to component A;

[0120] Qais the concentration of component A, in a mixture, which produced an end point:

[0121] QB is the concentration of a component B, acting alone, which produced an end point in relation to component B; and

[0122] Qb is the concentration of component B, in a mixture, which produced an end point.

[0123] Generally, when the sum of Qa / QA and Qb / QB is greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effective1543955.601_P17708-02amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.

[0124] Pharmaceutical Compositions

[0125] In other embodiments, the presently disclosed subject matter provides a pharmaceutical composition formulated for oral delivery of a compound of formula (la), formula (lb), or formula (Ic) to a subject in need of treatment thereof:

[0127] wherein:

[0128] Riais selected from: H, -CH2-O-C(=O)-C(CH3)3, -CH2-O-C(=O)-O-CH(CH3)2, -CH2-CH2-O-(CH2)15-CH3, and

[0129] Rib. R2a. and R2care each independently selected from: -CH2-O-C(=O)-C(CH3)3, -CH2-O-C(=O)-O-CH(CH3)2, -CH2-CH2-O-(CH2)15-CH3, and; and pharmaceutically acceptable salts thereof.

[0130] The presently disclosed methods include oral administration of a compound of formula (la), formula (lb), or formula (Ic). Such oral administration can include a solid dosage form (i.e., as capsules, tablets, pills and the like). In such embodiments, the pharmaceutical compositions includes the active agent, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically-acceptable carriers. Such solid dosage forms can further comprise one or more additional components selected from fillers or extenders, such as starches, microcrystalline cellulose, lactose, dicalcium phosphate, sucrose, glucose, mannitol, and / or silicic acid; binders, such as carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; humectants, such as glycerol; disintegrating agents, such as crosscarmellose sodium, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and / or sodium carbonate; solution retarding agents, such as paraffin; absorption accelerators, such as quaternary ammonium1643955.601_P17708-02compounds; wetting agents, such as cetyl alcohol and / or glycerol monostearate; absorbents, such as kaolin and / or bentonite clay; lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and / or mixtures thereof; coloring agents; and buffering agents.

[0131] Release agents, wetting agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the pharmaceutical compositions. Examples of pharmaceutically-acceptable antioxidants include: water-soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfate, sodium sulfite and the like; oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxy toluene, lecithin, propyl gallate, alpha-tocopherol, and the like; and metal-chelating agents, such as citric acid, ethylenediamine tetraacetic acid, sorbitol, tartaric acid, phosphoric acid, and the like. Coating agents for tablets, capsules, pills and like, include those used for enteric coatings, such as cellulose acetate phthalate, polyvinyl acetate phthalate, hydroxypropyl methylcellulose phthalate, methacrylic acid, methacrylic acid ester copolymers, cellulose acetate trimellitate, carboxymethyl ethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, and the like.

[0132] The presently disclosed pharmaceutical compositions can be formulated to provide slow or controlled release of the active agent using, by way of example, hydroxypropyl methylcellulose in varying proportions; or other polymer matrices, liposomes and / or microspheres. In addition, the presently disclosed pharmaceutical compositions contain opacifying agents and may be formulated so that they release the active ingredient only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. The active agent can also be in micro-encapsulated form, if appropriate, with one or more of the above-described excipients.

[0133] Suitable liquid dosage forms for oral administration include, by way of illustration, pharmaceutically-acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. Liquid dosage forms typically comprise the active agent and an inert diluent, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (esp., cottonseed, groundnut, com, germ, olive, castor and sesame oils), oleic acid, glycerol, tetrahydrofuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Alternatively, certain liquid formulations can be converted, for1743955.601_P17708-02example, by spray drying, to a powder, which is used to prepare solid dosage forms by conventional procedures.

[0134] Suspensions suitable for oral administration, in addition to the active ingredient, or a pharmaceutically acceptable salt thereof, may contain suspending agents, such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof.

[0135] Further, one of ordinary skill in the art will recognize that the presently disclosed compounds, and pharmaceutical compositions thereof, include pharmaceutically acceptable salts. Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art, and include salts of active compounds that can be prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the compounds described herein. The parent form of the compound can differ from the various salt forms in certain physical properties, such as solubility, and the like.

[0136] When compounds of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent or by ion exchange, whereby one basic counterion (base) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, magnesium, and the like.

[0137] When compounds of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent or by ion exchange, whereby one acidic counterion (acid) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids, organic acids, and amino acids. See, for example, Berge et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19). Compounds containing both basic and acidic functionalities allow such compounds to be converted into either base or acid addition salts.

[0138] Accordingly, pharmaceutically acceptable salts suitable for use with the presently disclosed subject matter include, by way of example but not limitation, acetate, arginate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, monohydrogencarbonate, citrate, edetate, edisylate, estolate, esylate, fumarate,1843955.601_P17708-02galactonate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydriodic, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, isobutyrate, lactate, lactobionate. malate, maleate, malonate, mandelate, mesylate, methanesulfonate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate, phthalate, diphosphate, monohydrogen phosphate, dihydrogen phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, suberate. succinate, sulfate, monohydrogensulfate, tannate, tartrate, including (+)-tartrates, (-)-tartrates, and mixtures thereof including racemic mixtures, teoclate, p-toluenesulfonate and trifluoroacetate. Other pharmaceutically acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).

[0139] Unless otherwise noted, the chemical definitions and nomenclature provided herein are intended to comply with IUPAC. Compendium of Chemical Terminology, 2nd ed. (the " Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997).

[0140] The term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / - 15%, + / -10%, + / -5%, + / -4%, + / -3%, + / -2%, and + / -1%. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0141] The phrase “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0142] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.e., “one or more,” unless the context clearly dictates otherwise. The1943955.601_P17708-02present disclosure also contemplates other embodiments “comprising,” “consisting of’ and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not. Likewise, the term “include” and its grammatical variants are intended to be nonlimiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.EXAMPLES

[0143] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations can be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration and are not to be construed as limiting in any manner to make compounds of the disclosure by other methods.EXAMPLE 1

[0144] Orally Available Prodrugs Of Itaconate And Derivatives

[0145] Overview

[0146] This Example provides orally available itaconate derivatives for a representative systemic autoimmune and / or inflammatory disease, disorder, or condition, e.g., a skin disorder, including, but not limited to, alopecia areata. Four sets of prodrugs were synthesized by pairing pivaloyloxymethyl (POM), isopropyloxycarbonyloxymethyl (POC), (5-methyl-2-oxo-1.3-dioxol-4-yl) methyl (ODOL), and 3-(hexadecyloxy)propyl (HDP) pro-moieties with itaconic acid (IA), 1 -methyl itaconate (1-MI), and 4-methyl itaconate (4-MI).

[0147] Among these, POC-based prodrugs (P2, P9. P13) showed favorable stability, permeability, and pharmacokinetics. Notably, P2 and P13 significantly inhibited Poly(I: C) / IFNy-induced inflammatory cytokines in human epidermal keratinocytes. Oral studies demonstrated favorable pharmacokinetics releasing micromolar concentrations of IA or 4-MI from P2 and P13, respectively. These finding highlight the potential of prodrug strategies to enhance itaconate’ s cellular permeability and oral bioavailability, paving the way for clinical translation.

[0148] Background2043955.601_P17708-02

[0149] The immunomodulatory properties of itaconate and its derivatives make them promising pharmacologic candidates for treating inflammatory conditions, such as psoriasis, rheumatoid arthritis, systemic lupus erythematosus, and multiple sclerosis. O'Neill and Artyomov, 2019; Hooftman and O'Neill, 2019; Lin, et al., 2021. Prodrugs of itaconate and methyl itaconate and their use to induce hair growth has been previously reported. See, for example, U. S. Patent Application Publication No. US20230028516 for Prodrugs of Itaconate and Methyl Itaconate, to Slusher et al., published January 26, 2023, and U. S. Patent Application Publication No. US20230025922 for Use of Itaconate and its Derivatives / Analogues to Induce Hair Growth, to Garza et al., published January 26, 2023, each of which is incorporated by reference in its entirety.

[0150] More particularly, a topical, cell-permeable prodrug of 4-MI, termed SCD-153, as a novel treatment for alopecia areata has been recently reported. Tsai et al., 2023.SCD-153

[0151] Alopecia areata is a chronic autoimmune disorder characterized by the targeted destruction of hair follicles by CD8+ T cells, leading to hair loss. Interestingly, topical administration of SCD-153 to C57BL / 6 mice resulted in a significant increase in hair growth, exhibiting statistically superior effects compared to the vehicle (dimethyl sulfoxide), less cell-permeable itaconate analogues (4-MI and DMI), as well as the clinically used JAK inhibitor tofacitinib. SCD-153 demonstrated considerable skin concentrations, indicating that the prodrug strategy has promise in topically delivering IA to previously impermeable tissues. Tsai et al., 2023.

[0152] In contrast to previous reports, this Example provides orally available, cell-permeable prodrugs of IA or its monoesters, 1-MI and 4-MI, to target tissues, such as skin systemically. FDA-approved promoieties, such as pivaloyloxymethyl (POM), isopropyloxycarbonyloxymethyl (POC), (5-methyl-2-oxo-1,3-dioxol-4-yl) methyl (ODOL) and 3-(hexadecyloxy)propyl (HDP), were employed to mask either the 1 -carboxylate or 4-carboxylate on itaconate, thereby enhancing its permeability and pharmacokinetic properties.

[0153] Chemistry

[0154] The synthesis of IA prodrugs focused on three structural types: (a) itaconate diesters with identical ester groups, (b) itaconate monoesters with ester groups at the 4-position of the2143955.601_P17708-02carboxylate, and (c) itaconate diesters derived from 1 -methyl or 4-methyl itaconate, featuring various promoieties at the second carboxylic function. The prodrug groups utilized were pivaloyloxymethyl (POM), isopropyloxycarbonyloxymethyl (POC), (5-methyl-2-oxo-1,3-dioxol-4-yl)methyl (ODOL), and 3-(hexadecyloxy)propyl (HDP) to minimize challenges related to translation and toxicity. Hecker et al., 2008; Babu et al., 2008; Garaga et al., 2015.

[0155] For example, these groups were applied in the prodrug development of acyclic nucleoside phosphonates, including Adefovir, Tenofovir, and Cidofovir, which led to substantial improvement in pharmacokinetics and oral absorption. Hostetler, 2009; Pradere et al., 2014. We also have reported their application in successfully synthesizing prodrugs of the multiply-charged GCPII inhibitor 2-(phosphonomethyl)pentanedioic acid (2-PMPA) and demonstrated enhancement in oral bioavailability (20-69 fold) in mice and dogs. Dash et al., 2019; Majer et al., 2016.

[0156] Synthesis of itaconate diesters bis-POM (Pl), bis-POC (P2), and bis-ODOL (P3) were performed by alkylation of IA with carbonyloxymethyl chlorides or 5-methyl-2-oxo-1,3-dioxol-4-yl)methyl chloride, respectively, under basic conditions in the presence of sodium iodide (Scheme 1).

[0157] Scheme 1. Synthesis of bis-POM, bis-POC and bis-ODOL dialkyl esters Pl-3a.oitaconic acid (1)

[0158]

[0159] “Reagents and conditions: (i) K2CO3, Nal, MeCN, 40-55 °C, 16 h, 14-57 %.

[0160] Selective monoesterification of free IA to POM, POC, or ODOL was challenging; various reaction conditions were attempted, but only a nonseparable mixture of 1- and 4-monoesters were obtained. Thus, to synthesize pure 4-monoesters with a free 1 -carboxylate, 4-fe -butyl itaconate (5) was prepared according to a published protocol, Chollet et al., 2002, and alkylation was2243955.601_P17708-02performed under the same conditions as described above. The protected diesters 6-8 were subsequently treated with trifluoroacetic acid to give the desired monoesters P4, P5, and P6 (Scheme 2). Preparation of 3-(hexadecyloxy)propyl monoester (P7) was achieved by reaction of itaconic anhydride (9) with hexadecyloxypropanol (10) at 70 °C in chloroform proceeding selectively to the position 4 (Scheme 2).

[0161] Scheme 2. Synthesis of POM, POC, ODOL, and HDP itaconic monoesters P4-6 and P7a.5 2-4 6-89 10 P77’P5: R“8, P6: R=

[0162] P7: R=

[0163] “Reagents and conditions: (i) K2CO3, Nal, MeCN, 40-55 °C. 16 h, 71-85%; (ii) trifluoroacetic acid in DCM, rt, 2 h, 84-97%; (iii) CHCh, 70 °C, 16 h, 83%.

[0164] Mixed diesters P8-P15 were prepared by alkylation of 1-MI (11) or 4-MI (12) with a chloromethyl derivative (analogously as described for compounds Pl-3) or by Steglich esterification with 3-(hexadecyloxy)propanol (compounds Pll and P15, Scheme 3).

[0165] Scheme 3. Synthesis of itaconate diesters designed to release 1-MI or 4-MIa.2343955.601_P17708-02o o R1-CI (2-4) (i) R2-CI (2-4) (i) o or R1-OH (10), (ii) or R2-OH (10), (ii) 4-MI (12) P8-15 1-MI (11)P12: R1=, R2= CH3P8: R1= CH3, R2=P13: R1=, R2= CH3P9: R1= CH3, R2=P14: R1=, R2=CH3P10: R1= CH3, R2=P15: R1= P11: R1=CH3, R2=

[0166]

[0167] “Reagents and conditions: (i) K2CO3, Nal, MeCN, 40-45 °C, 16 h, 84-94%; (ii) DCC, DMAP. 0 °C to rt, 16 h, 28-68%.

[0168] Results and Discussion

[0169] In vitro chemical stability, permeability, and metabolic stability assessments

[0170] To develop orally available prodrugs for IA, 1-MI, and 4-MI, it was crucial to achieve high permeability, good chemical stability, and the ability to release active compounds upon oral absorption. Consequently, the prodrugs were assessed for their chemical stability under gastrointestinal pH conditions (1.2, 4.5, and 7.4) to ensure stability in the gut for oral delivery (Table 1).Table 1. Chemical stability of itaconate prodrugs.Chemical Stability Molecular StructureCmpd (% remaining at 1h) (Blue=active moiety; ClogPIDRed=promoiety) pH 1.2 pH 4.5 pH 7.40 O jP1 2.74 106 ± 9 92 ± 3 104 ± 17A 0 0 Y n ' Y "J0 0O:P4, Y xx, O.,0.H° | -- -¥' 1.27 100 ± 2 74±3 22 ± 4:i O Q2443955.601_P17708-02Table 1. Chemical stability of itaconate prodrugs.Chemical Stability Molecular StructureCmpd (% remaining at 1h)(Blue=active moiety; ClogPIDRed=promoiety) pH 1.2 pH 4.5 pH 7.4P8 YA x-O- \.s A\ 1.79 101 ± 2 104 ± 5 86 ± 8° f n '! lo / H 'yO--- c- bo oP12 -- Y o o A Y --'x « <c.. ■' 1.61 99 ± 2 109 ± 4 94 ± 11 II Q** '"x-.P2: Q Q1.83 104 ± 2 109 ± 6 47 ± 40P50.82 104 ± 0 79 ± 11 44 ± 10P9 \C. AKz-'xr xo. X x.ox y. Oxr1.34 98 ± 2 94 ± 2 63 ± 3” 0 0p oP13.. A "... A 0 -x <, A. J,-x J o - 1.16 108 ± 1 103 + 4 102 + 2P3-0.94 102 ± 1 91 + 2 91 + 14P6 o cAA,-x Y -0.66 109 ± 4 88 ± 1 0 ± 0 HO Y r. 'iPIO-0.13 100 ± 6 97 ± 4 69 ± 5■'■o-YV0' --'-''0" o2543955.601_P17708-02Table 1. Chemical stability of itaconate prodrugs.Chemical Stability Molecular StructureCmpd (% remaining at 1h)(Blue=active moiety; ClogPIDRed=promoiety) pH 1.2 pH 4.5 pH 7.4\ X’ / / GP14 Xo -0.14 98 ± 3 87 ± 6 99 ± 4P7HO fY0- - 8.38 91 ± 2 90 ± 10 65 ± 40Pll / 109 ±6 8.79 98 ± 4 97 ± 414 / > oX \P155 x-x,0. 103 ± 108 ±0 Tf o 8.91 75 ± 812 166qIA HO OH -0.33q ifd4-MI 0.14HO'T1°''1-MI 0.202643955.601_P17708-02

[0171] Those demonstrating good pH stability were further evaluated for their ability to permeate via the parallel artificial membrane permeation assay (PAMPA, GIT) assay. Faller, 2008. Finally, stable and highly permeable prodrugs were assessed for stability in mouse and human plasma, as well as in skin homogenates to assess release of actives at the desired site, as detailed in Table 2 below.

[0172] All prodrugs were either highly stable (greater than 80% remaining at 1 hour) or moderately stable (50-80% remaining at 1 hour) across the three pH conditions, with the exception of POM, POC. and ODOL monoesters of IA (P4, P5, and P6 respectively), which all exhibited complete instability at pH 7.4. The calculated cLogP values of the prodrugs expectedly showed significant improvement in lipophilicity, approximately 10-fold for POC and POM, and 50-fold for HDP prodrugs, compared to their respective active moieties (cLogP: -0.33. 0.14, and 0.20 for IA, 4-MI, and 1-MI, respectively) (SwissADME, Lausanne, Switzerland). Daina et al., 2017.

[0173] Given the polar nature of the ODOL promoieties, its addition did not enhance itaconate calculated lipophilicity. In terms of permeability via PAMPA-GIT assay to mimic permeability via the gastrointestinal tract, bis-POM and bis-POC-protected IA prodrugs (P1, P2), as well as POM / POC prodrugs of 1-MI (P8, P9) and 4-MI (P12, P13), demonstrated excellent in vitro permeability with a permeation index (Pe) greater than 10xl0‘6cm / s. These results are consistent with the increase in their cLogP, aligning with previous studies showing that permeability increases with lipophilicity when cLogP values are 3.5 or lower. Wils et al., 1994. Conversely, POM or POC monoesters of IA (P4, P5) displayed poor permeability, with a Pevalues less than 1×10-6cm / s, likely due to the retain charged site on one terminus. Regarding the ODOL prodrugs, the trend was similar, with the bis-ODOL ester of IA (P3) and ODOL-esters of 1-MI (P10) and 4-MI (P14) demonstrating high permeability, while the monoester of IA (P6) demonstrating limited permeability. Overall, all ODOL prodrugs (P3, P6, P10, and P14) exhibited a significant enhancement in the permeability (Peapproximately 15 × 10-6cm / s) of both IA and 4-MI. These results align with previously reported ODOL-based prodrugs, such as azilsartan medoxomil, faropenem medoxomil and 2-PMPA-tetraODOL, which demonstrated significantly enhanced oral bioavailability. Dash et al., 2019; Kawaguchi et al., 2013; Schurek et al., 2007.

[0174] In the case of itaconate and analogs, however, the ODOL promoiety did not outperform POM- and POC-based prodrugs in enhancing permeability attributed to the highly polar surface2743955.601_P17708-02area of the ODOL promoiety. Consequently, ODOL prodrugs were not further advanced into pharmacokinetic studies.

[0175] Interestingly, all HDP prodrugs displayed poor permeability (Pe< I xIO6cm / s) except for the IA monoester HDP prodrug (P7) which had a slightly better, but comparably lower permeability, (Pe=3.21xl0’6cm / s) to POM and POC analogs. This poor permeability was attributed to their exceptionally high lipophilicity, with cLogP values exceeding 5 for P7, Pll, and P15 (cLogP: 8.38, 8.79, and 8.91, respectively). Such high cLogP values also are associated with poor solubility, reflecting an unfavorable hydrophilic-lipophilic balance and resulting in suboptimal properties. Tsaioun et al., 2016. Overall, these findings suggest that HDP prodrugs were not effective candidates for improving the oral bioavailability of IA analogs.

[0176] To evaluate the potential for oral delivery of active compounds to systemic circulation in inflammatory conditions and to ensure skin targeting in the in vivo PK study, we focused on mouse and human plasma and skin homogenates. These models were selected because they closely mimic the enzymatic and metabolic environments of the in vivo matrices under investigation. As most of these compounds are ester-based prodrugs, however, we anticipate that other highly metabolic organs, such as the liver, may also play a significant role in their metabolic pathways, although this was not tested in this Example.

[0177] All POC, POM and ODOL prodrugs except the monoesters of IA (P4 and P5) were found to hydrolyze in mouse and human plasma readily. The monoesters of IA, however, were chemically unstable and showed poor permeability (Table 2) and therefore were not selected for further consideration. HDP prodrugs, interestingly, showed stability in both mouse and human plasma. When incubated in mouse skin homogenate, however, the HDP-based prodrugs were unstable (less than (less than 50% remaining at 1 h). In human skin homogenate, POM and POC-based prodrugs demonstrated instability similar to mouse skin homogenate. Interestingly, ODOL-based prodrugs, except the bis-ODOL prodrug (P3), however, had moderate stability (greater than (greater than 50% remaining after Ih) while HDP-based prodrugs were completely stable in human skin homogenates (greater than (greater than 90% remaining at 1 h).

[0178] Collectively, the POM (Pl, P8 and P12) and POC (P2, P9 and P13) based prodrugs showed the most promise, with enhanced PAMPA permeability and effective release of active moieties in plasma and skin. While the POM-based prodrugs showed comparable stability and permeability results, the well-known additional toxicity attributed to POM promoieties due to2843955.601_P17708-02carnitine depletion, Heidel and Dowd, 2019, led us to advance the three POC-based prodrugs P2 (IA), P9 (1-MI) and P13 (4-MI) for in vitro gene expression efficacy studies, and in vivo mouse pharmacokinetic evaluation as described herein below.

[0179] While the POM-based prodrugs showed comparable stability and permeability results, the well-known additional toxicity attributed to POM promoieties due to carnitine depletion, Heidel and Dowd, 2019, led us to advance the three POC-based prodrugs P2 (IA), P9 (1-MI) and P13 (4-Ml) for in vitro gene expression efficacy studies and mouse pharmacokinetic evaluation.2943955.601_P17708-02Table 2. Structure, metabolic stability, and permeability of itaconate prodrugs.Permeability Metabolic StabilityMolecular StructureCmpd (Pe, ×10-6(% remaining at 1h)(Blue=active moiety;ID cm / sec) Mouse Mouse Human Human Red=promoiety)pH 7.4 Plasma Skin Plasma Skin PlJL A.0., G. 35.8 0 ± 0 0 ± 0 0 ± 0 0 ± 00 01hI T '0 G0;P40.08 3 ± 1 14 ± 0.5 83 + 2 40 + 0.7HcyVoP8. X 35.2 0 + 0 0 ± 0 0 + 0 9.4 + 0.9 o o0 0P1233.2 0 ± 0 5.3 ± 0.4 0 + 0 15 + 2.9:\ o!!0 / Qc> / P2 \ o\ 35.9 0 ± 0 2.5 + 2.4 0 + 0 1.1 + 0.40P5 iHO’ 0 0 ± 0 4.9 ± 1.3 53 ± 1 29 ± 0.90P9. JJ.0..,. G,,0.'O' X 29.3 0 ± 0 0 ± 0 0 ± 0 0 ± 0 1 1 ' IT{ } 0P130 ”” GvW ' 35.3 0 ± 0 0 ± 0 0 ± 0 1.6 ± 0.46P317.0 0 ± 0 0 + 0 0 + 0 30 ± 0.. " °z4.3043955.601_P17708-02Table 2. Structure, metabolic stability, and permeability of itaconate prodrugs.Permeability Metabolic StabilityMolecular StructureCmpd (Pe.xlO’6(% remaining at Ih)(Blue=active moiety;ID cm / sec) Mouse Mouse Human Human Red=promoiety)pH 7.4 Plasma Skin Plasma SkinP6 00 20 ± 8 74 + 30 0 + 0 93 + 4 HO6O O - PI16.3 0 ± 0 0 ± 0 0 ± 0 56 ± 0.9i 0 / P14 oOV OV A 15.4 0 ± 0 16 ± 0.4 0 ± 0 76 ± 4(0 A- _ \ / y o0P7 / 6.. 3.2 92 ± 8 100 + 9.5 68 + 9 87 ± 7 H Q \ / XO---- / \\ _Pit y.- •\ o^ 0 89 ± 2 61 + 10 90 + 4 101 ± 8:x:0P15. Ox z'x 44 ±O' 0 67 + 11 32 + 2 94 ± 5 r Y ■' ■!100pIA. OH 0 - - HO jf y4-MI 0 - -p1-MI „ OH 0 - - o ff nd3143955.601_P17708-02

[0180] Immunomodulatory effects ofP2, P9 and Pl 3 in stimulated human epidermal keratinocytes

[0181] Without wishing to be bound to any one particular theory, it was thought that itaconate-based cell permeable prodrugs may offer benefits for alopecia areata (as we have previously shown) by counteracting immune stimulants, such as PolyLC and IFNy, as these stimulants have shown to contribute to the disease by activating the NLRP3 inflammasome activity, Shin et al., 2017; Shin et al., 2018, and JAK-STAT signaling, Xing et al.. 2014; Pratt et al.. 2017; Glickman et al., 2021; McPhee et al., 2012, IA and its derivatives have shown to suppress proinflammatory cytokines, such as IL-6, a JAK-STAT activator, and IL-ip, which disrupts hair cycling and is elevated in alopecia areata scalp lesions. Hoffmann 1999; Hoffmann et al„ 1997; Hoffmann et al., 1996; Hoffmann et al., 1994.

[0182] Thus, to validate the therapeutic potential of P2, P9 and P13, we evaluated their dosedependent cytotoxicity and ability to block PolyLC and IFNy-induced cytokines and chemokines release in vitro in normal human epidermal keratinocytes (NHEKs) as previously described (FIG.1). Tsai et al., 2023.

[0183] First, the in vitro cytotoxicity of the prodrugs was assessed to ensure they did not adversely affect cell viability. After 8 hours of treatment, NHEKs treated with P2, P9, and P13 all demonstrated good cell viability, with over 80% of cells remaining viable at the highest concentration of 100 (data not shown). The immunomodulatory effects were next evaluated by adding P2, P9 and P13 to NHEKs stimulated with poly I: C or IFNy to induce IL-6, IL-ip, IFNp, and IFN-inducible chemokines (CXCL9, CXCL10, CXCL11), which are recognized as characteristic markers of alopecia areata. Fetter et al., 2023. For IFNy-inducible chemokines (CXCL9, CXCL10. and CXCL11), P2 and P13 showed significant effects, with over 50% inhibition at 30-100 pM. P9 exhibited limited inhibitory effects, however, with less than 50% inhibition at 100 pM. This observation suggests that although the IA- and 4-MI-releasing prodrugs were effective, the 1-MI-releasing prodrug was less so, indicating that the free carboxylate at the 1-position on IA is essential for activity, and masking it resulted in a decrease in effectiveness.

[0184] Similarly, IL-ip although showed low induction (2-3 fold) when stimulated with Poly(LC) + IFNy, P2 and P13 demonstrated over 50% inhibition of this signal at concentrations >10 pM, and P9 showed effects at 100 pM. P2 and P13 also showed significant, dose-dependent inhibition of IL-6 and P2 in addition showed dose dependent inhibition of IFNp. while P9 did not, further suggesting that IA or 4-MI are more active versus 1-MI in this assay.3243955.601JT7708-02

[0185] Overall, among POC-based prodrugs, P2 and P13 showed significantly enhanced immunomodulatory effects in NHEKs, supporting our hypothesis that cell permeable prodrugs may offer benefits for alopecia areata. This was further evidenced in our screening data in NHEKs, where neither 4-MI nor 1 A affected the mRNA expression of TLR3 (Toll-like receptor 3) and IL-6 involved in inflammatory response while the prodrugs P2 and P13 caused greater than 90% inhibition (FIG. 3).

[0186] Pharmacokinetic Studies in Mice

[0187] Given their promising immunomodulatory effects in NHEKs, we next conducted pharmacokinetic evaluation of P2 and P13 at a dose of 100 mg / kg equivalent in mice. The dose was selected based on previously reported dose of 25-150 mg / kg, Chen et al., 2022; Xie et al., 2023; Aso et al., 2023, and to achieve effective concentrations (30-100 pM) in an in vitro NHEK model.

[0188] The concentration-time profiles of released IA from P2 and released 4-MI from P13 in plasma and skin are presented in FIG. 2. Following oral administration, intact P2 was not detectable in the plasma, suggesting rapid hydrolysis of P2 into IA. The Cmax of released IA in plasma following P2 administration (100 mg / kg IA equivalent) was 83.8 ± 18.8 p, observed at 15 minutes post-dose (Tmax), as shown in FIG. 2E. The Cmax of IA in skin was 65.9 ± 10.2 nmol / g. Notably, P2 showed significant distribution to skin tissue, indicated by higher skin versus plasma AUCo-t values (173 ± 30.0 nmol-h / g and 108 ± 2.68 pM-h, respectively), resulting in a skin / plasma ratio of 1.61 ± 0.29. The half-life of itaconate released from P2 was calculated to be 1.42 ± 0.24 hours in plasma and 3.32 ± 1.23 hours in skin, demonstrating longer retention and slower clearance from the skin compared to plasma.

[0189] Similarly, following oral administration of P13 (100 mg / kg eq. of 4-MI), intact P13 was undetectable in plasma, indicating that P13 was rapidly hydrolyzed into 4-MI. The released 4-MI showed high exposure in plasma, with a Cmax of 349 ± 39.3 pM and an AUCo-t of 415 ± 16.8 pM-h.4-MI released from P13 also exhibited high exposure in skin tissue, with an AUCo-t value of 234 ± 1.30 nmol-h / g. The skin / plasma ratio of 4-MI after P13 administration was calculated to be 0.59 ± 0.01. The half-life of 4-MI released from P13 was calculated to be 0.87 + 0.09 hours in plasma and 0.97 + 0.20 hours in skin. Overall, its exposure in the skin exceeded 100 pM, a concentration that resulted in over 50% inhibition of cytokines in NHEKs, as shown in FIG. 1.3343955.601_P17708-02

[0190] Collectively, P2 and P13 demonstrated efficient delivery of active moieties to systemic circulation and skin. While comparative PK data for IA and 4-MI versus their prodrugs are lacking in this study, our results clearly support the prodrug strategy. IA and 4-MI showed poor permeability in the PAMPA assay (Table 2), failed to inhibit cytokine production in NHEKs (FIG.3), and 4-MI was ineffective in vivo in the alopecia areata model, unlike P13 when compared at equimolar doses. Tsai et al., 2023.These findings highlight the limitations of the parent compounds and underscore the potential of prodrugs like P2 and P13 to overcome these challenges, aligning with prior literature on the importance of enhancing cellular permeability for itaconate derivatives. ElAzzouny et al., 2017; Hooftman and O’Neill, 2019; Mills et al., 2018; McGettrick et al., 2024.

[0191] Another limitation of this study is the absence of direct comparisons between our prodrugs and traditional treatments for alopecia areata, such as JAK inhibitors. Although future studies could address this gap, in our previous work, topical application of 5% P13 using an altemate-day, two-dose regimen significantly promoted hair growth compared to 5% tofacitinib under the same conditions. Tsai et al., 2023. Moreover, while JAK inhibitors like tofacitinib are effective, they carry risks of serious adverse effects, including venous thromboembolism and other on-target toxicities. Hoisnard et al., 2022; Mori et al., 2021.

[0192] In contrast, itaconate prodrugs act through a distinct mechanism, reducing the likelihood of such on-target side effects. In support, we assessed P13’s preliminary toxicity profile (given its robust efficacy in alopecia areata) at two doses via systemic administration. No hematological changes or weight loss were observed (FIG. 4 and Table 3), supporting the safety of P13. These findings suggest that itaconate prodrugs offer a promising, potentially safer alternative for treating alopecia areata.Table 3. Hematological parameters of mice treated with P13 were within normal range. Parameters Unit Reference Vehicle 30 mg / kg 100 mg / kg (n = 5) (n = 6) (n = 6) WBC 103 / |1L 1.06 - 56.08 7.58 ± 0.419 6.18 ± 0.439 5.53 ± 1.31 RBC 106Z(iL 3.57 - 15.2 10.69 + 0.119 10.27 ± 0.296 9.89 ± 1.334 Hemoglobin g / dL 6.1 - 21.7 15.54 ± 0.14 14.78 ± 0.411 14.36 ± 1.922 Hematocrit % 16.7 - 69.8 47.04 ± 0.543 45.88 ±1.108 43.0 ± 5.546 MCV fL 39 - 90.8 43.8 ± 0.18 44.7 ± 0.39 43.8 ± 0.65 MCH Pg 12.6 - 31 14.6 ± 0.06 14.4 ± 0.05 14.6 ± 0.07 MCHC g / dL 27 - 37.6 33.0 ± 0.17 32.2 ± 0.29 33.1 ± 0.38 Platelet 103 / j.iL 59 - 2633 219.2 ± 17.87 241 ± 14.16 245.4 ± 29.68 Neutrophil 103 / pL 0.03 - 32.03 0.53 ± 0.07 2.11 ± 0.51 0.82 ± 0.21 Lymphocyte 103 / μL 0.12 - 23.46 6.63 ± 0.34 3.58 ± 0.33 4.44 ± 1.08Monocyte 103 / piL 0- 5.08 0.22 ± 0.04 0.41 ± 0.13 0.18 ± 0.043443955.601_P17708-02Table 3. Hematological parameters of mice treated with P13 were within normal range. Parameters Unit Reference Vehicle 30 mg / kg 100 mg / kg (n = 5) (n = 6) (n = 6) Eosinophil 103 / ptL 0- 2.03 0.18 ± 0.01 0.07 ± 0.01 0.08 ± 0.03Basophil 103 / pL 0- 2.33 0.02 ± 0.005 0.01 ± 0.002 0.01 ± 0.005 Data are presented as mean ± standard error of the mean (SEM). WBC; white blood cells, RBC; red blood cells, MCV; mean cell volume, MCH; mean cell hemoglobin, MCHC; mean cell hemoglobin concentration.

[0193] Summary

[0194] In summary, we synthesized four sets of IA, 4-MI, and 1-MI prodrugs using the FDA-approved promoieties POM, POC, ODOL, and HDP by incorporating itaconate diester, itaconate monoester, and itaconate diesters derived from monomethyl itaconate. These prodrugs were evaluated in a panel of in vitro and pharmacokinetic assays. The POC-based prodrugs of IA (P2) and 4-MI (P13) demonstrated the best properties including chemical stability in gastric conditions, high permeability, release of the active moiety in skin homogenates, and positive immunomodulatory effects in NHEK assays. P2 and P13 also exhibited good pharmacokinetic properties following oral administration. The ability of these prodrugs to effectively deliver the active IA and 4-MI to skin tissue following oral administration, suggests that they hold potential as oral treatments for alopecia areata and other inflammatory skin diseases.

[0195] Experimental section

[0196] Chemicals and Materials

[0197] A commercially available reagent or HPLC-grade solvents and materials were used for the synthesis of the compounds described. All chemicals were reagent grade purchased from Sigma-Aldrich, Combi-Blocks, or Tokyo Chemical Industry (TCI) Co. Ltd. TLC was performed on Silica gel 60 F254-coated aluminum sheets (Merck) and spots were visualized with UV light and by the solution of Ce(SO4)2·4 H2O (1%) and H3P(Mo3O10)4(2%) in sulfuric acid (10%). Column chromatography was performed on silica gel 60 (0.063-0.200 mm, Fluorochem). NMR spectra were measured on Bruker AVANCE 400 instrument.1H NMR was recorded at 401 MHz, and signals of TMS (δ 0.0, CDCl3) and CDCl3(δ 7.26) were used for standardization.13C NMR spectra were recorded at 101 MHz, and the signal of CDCl3(δ 77.16) was used for standardization. The chemical shifts are given in 8 scale; the coupling constants J are given in Hz. The low-resolution ESI mass spectra were recorded using a ZQ micromass mass spectrometer (Waters). High-3543955.601JT7708-02resolution ESI mass spectra were recorded using an LTQ Orbitrap XL spectrometer (Thermo Fisher Scientific). The purity of all compounds subjected to biological testing was over 95%.

[0198] General synthetic procedure for prodrugs Pl-3

[0199] IA (200 mg, 1.53 mmol, 1 equiv.), appropriate chloride 2-4 (2.5-3 equiv.), sodium iodide (46.1 mg, 0.307 mmol, 0.2 equiv.), and potassium carbonate (3-4 equiv.) were dissolved in anhydrous MeCN (5-10 mL) and the mixture was stirred for 16 h at 45-55 °C under inert atmosphere. EtOAc (60 mL) was added, and the mixture was washed with brine (20 mL) and sat. Sodium thiosulfate (10 mL). The organic phase was dried over anhydrous Na2SO4, volatiles were evaporated, and the residue was purified by flash column chromatography on silica (various mobile phases) to afford compounds Pl-3.

[0200] Bis((pivaloyloxy)methyl) 2-methylenesuccinate (Pl). Chloromethyl pivalate (2) (579 mg, 554 pL, 3.84 mmol, 2.5 equiv.); potassium carbonate (637 mg, 4.61 mmol, 3 equiv.); MeCN (5 mL); 45 °C; mobile phase: cyclohexane / EtOAc, 5:1. Compound Pl was isolated as a colorless oil (199 mg) in 36% yield. 'H NMR (401 MHz. CDCl₃): 8H 1.20 (s, 18H), 3.38 (d, J = 1.0 Hz, 2H), 5.74 (s, 2H), 5.82 (s, 3H), 6.41 (s, 1H).13C NMR (101 MHz, CDCl₃): 8c 26.83, 26.84, 37.12, 38.75, 38.78, 79.70, 79.82, 130.52, 132.33, 164.45, 169.12, 177.07. ESI MS: 381.2 ([M + Na]+).HRMS (ESI): calcd. For C17H26O8Na 381.15199, found: 381.15158.

[0201] Bis(((isopropoxycarbonyl)oxy)methyl) 2-methylenesuccinate (P2). Chloromethyl isopropyl carbonate (3) (586 mg, 514 pL, 3.84 mmol, 2.5 equiv.); potassium carbonate (637 mg, 4.61 mmol, 3 equiv.); MeCN (5 mL); 45 °C; mobile phase: cyclohexane / EtOAc, 5:1. Compound P2 was isolated as a colorless oil (79.2 mg) in 14% yield. 'H NMR (401 MHz, CDCl₃): 8H 1.30 (s. 6H). 1.31 (s, 6H), 3.41 (s. 2H). 4.82 - 4.98 (m. 2H), 5.75 (s, 2H), 5.82 (s, 2H), 5.85 (s, 1H), 6.46 (s, 1H).13C NMR (101 MHz, CDCl₃): 8c 21.63, 36.95, 73.13, 82.00, 82.22, 131.05, 131.99, 153.26, 153.29, 164.28, 168.95. ESI MS: 385.1 ([M + Na]+). HRMS (ESI): calcd. For C15H22O10Na 385.11052, found: 385.11069.

[0202] Bis((5-methyl-2-oxo-l,3-dioxol-4-yl)methyl) 2-methylenesuccinate (P3). (4- Chloromethyl)-5-methyl-l,3-dioxol-2-one (4) (685 mg, 504 pL, 4.61 mmol, 3 equiv.); potassium carbonate (850 mg, 6.15 mmol, 4 equiv.); MeCN (10 mL); 55 °C; mobile phase: cyclohexane / EtOAc, 1:1. Compound P3 was isolated as a colorless oil (311 mg) in 57% yield. *H NMR (401 MHz, CDCl₃): 8H 2.17 (s. 3H), 2.19 (s. 3H), 3.38 (d, 7= 1.1 Hz, 2H), 4.86 (s, 2H), 4.92 (s, 2H), 5.81 (s, 1H), 6.40 (s, 1H).13C NMR (101 MHz, CDCl₃): 8c 9.48, 9.52, 37.57, 54.39, 54.50,3643955.601JT7708-02130.62, 132.59, 133.32, 133.34, 140.45, 140.49, 152.22, 165.41, 170.05. ESI MS: 279.0 ([M + Na]+). HRMS (ESI): calcd. For C11H12O7Na 279.04752, found: 279.04800.

[0203] General synthetic procedure for compounds 6-8

[0204] 3-(n?r / -Butoxycarbonyl)but-3-enoic acid (5) (400 mg, 2.15 mmol, 1 equiv.), appropriate chloride 2-4 (1.2- 1.3 equiv.), sodium iodide (64.4 mg, 0.430 mmol, 0.2 equiv.) and potassium carbonate (445 mg, 3.22 mmol, 1.5 equiv.) were dissolved in anhydrous MeCN (5 mL) and the mixture was stirred for 16 h at 45-55 °C. EtOAc (60 mL) was added, and the mixture was washed with brine (20 mL) and sat. Sodium thiosulfate (10 mL). The organic phase was dried over anhydrous Na2SO4, volatiles were evaporated, and the residue was purified by flash column chromatography on silica (various mobile phases) to afford desired compounds 6-8 as colorless oils.

[0205] 1 -( / er / -Butyl) 4-((pivaloyloxy)methyl) 2-methylenesuccinate (6). Chloromethyl pivalate (2) (421 mg, 402 pL, 2.79 mmol, 1.3 equiv.); 45 °C; mobile phase: cyclohexane / EtOAc, 6:1. Compound 6 was isolated as a colorless oil (458 mg) in 71% yield. 'H NMR (401 MHz, CDCl₃): 6H 1.21 (s, 9H), 1.48 (s, 9H), 3.33 (d, J= 1.2 Hz, 2H), 5.63 (d, J = 1.2 Hz, 1H), 5.76 (s, 2H), 6.25 (d, 7 = 1.0 Hz, 1H).13C NMR (101 MHz, CDCl₃): 5c 27.00, 28.10, 37.72, 38.90, 79.88, 81.44, 127.92, 134.86, 165.13, 169.79, 177.23. ESI MS: 323.2 ([M + Na]+). HRMS (ESI): calcd. For C15H24O6Na 323.14651, found: 323.14622.

[0206] Butyl) l-(((isopropoxycarbonyl)oxy)methyl) 2-methylenesuccinate (7).Chloromethyl isopropyl carbonate (3) (393 mg, 345 pL, 2.58 mmol, 1.2 equiv.); 50 °C; mobile phase: cyclohexane / EtOAc, 5:1. Compound 7 was isolated as a colorless oil (552 mg) in 85% yield. 'H NMR (401 MHz, CDCl₃): 5H 6.28 - 6.23 (m, 1H), 5.74 (s, 2H), 5.62 (td, J= 1.2 Hz, 1H), 4.95 - 4.84 (m, 1H), 3.36 - 3.31 (m, 2H), 1.46 (s, 9H), 1.29 (d, 7= 6.2 Hz, 6H).13C NMR (101 MHz, CDCl₃): 5c 169.6, 165.0, 153.4, 134.6, 128.2, 82.0, 81.5, 73.1, 37.7, 28.0, 21.7. ESI MS:325.1 ([M + Na]+). HRMS (ESI): calcd. for C14H22O7Na 325.12577; found: 325.12580.

[0207] 4-( / e / 7-Butyl) l-(((isopropoxycarbonyl)oxy)methyl) 2-methylenesuccinate (8). (4-Chloromethyl)-5-methyl-l,3-dioxol-2-one (4) (383 mg, 282 pL, 2.58 mmol, 1.2 equiv.); 55 °C; mobile phase: cyclohexane / EtOAc, 4:1. Compound 8 was isolated as a colorless oil (493 mg) in 77% yield. 'H NMR (401 MHz, CDCl₃): 5H1.46 (s, 9H), 2.16 (s, 3H), 3.31 (d, J = 0.8 Hz, 2H), 4.84 (s, 2H), 5.63 (td, 7= 1.1 Hz, 1H), 6.23 - 6.25 (m. 1H).13C NMR (101 MHz, CDCl₃): 5c 9.49,3743955.601_P17708-0228.05, 37.83, 54.15, 81.48, 128.05, 133.53, 134.91, 140.30, 152.15, 165.15, 170.54. ESI MS:377.0 ([M + Na]+). HRMS (ESI): calcd. for C15H14O10Na 377.04792; found: 377.04773.

[0208] General synthetic procedure for prodrugs P4-6

[0209] Compounds 6-8 (1 equiv.) were dissolved in anhydrous DCM (0.5-1 mL), trifluoroacetic acid (4-6 mL) was added, and the mixture was stirred for 2 h at room temperature. Volatiles were evaporated, and the residue was dissolved in DCM (3x15 mL) and evaporated three times. The residue was purified by flash column chromatography on silica (mobile phase: cyclohexane / EtOAc, 1:1) to afford desired prodrugs P4-6.

[0210] 2-Methylene-4-oxo-4-((pivaloyloxy)methoxy)butanoic acid (P4). Compound 6 (140 mg, 0.466 mmol); DCM (0.5 mL), trifluoro acetic acid (4 mL). Prodrug P4 was isolated as a colorless oil (110 mg) in 97% yield. 'H NMR (401 MHz, CDC13): 8H 1.21 (s. 9H), 3.37 (s, 2H), 5.77 (s, 2H), 5.86 (s, 1H), 6.49 (s, 1H), 11.12 (s, 1H).13C NMR (101 MHz, CDCI3): 8c 26.81, 36.97, 38.76, 79.66, 131.36, 132.54, 169.22, 171.11, 177.13. ESI MS: 267.1 ([M + Na]+). HRMS (ESI): calcd. for C11H16O6Na 267.08391; found: 267.08375.

[0211] 4-(((Isopropoxycarbonyl)oxy)methoxy)-2-methylene-4-oxobutanoic acid (P5).Compound 7 (500 mg, 1.65 mmol); DCM (1 mL), trifluoroacetic acid (6 mL). Prodrug P5 was isolated as a colorless solid (375 mg) in 92% yield. 'H NMR (401 MHz, CDCI3): 8H 1.30 (s, 3H), 1.32 (s, 3H), 3.40 (s, 2H), 4.84 - 4.99 (m, 1H), 5.76 (s, 2H), 5.88 (d, J = 1.1 Hz, 1H), 6.50 (s, 1H).13C NMR (101 MHz. CDCI3): 8c 21.75, 36.94, 73.31, 82.14, 131.72, 132.49, 153.44, 169.22, 171.37. ESI MS: 245.1 ([M - H]+). HRMS (ESI): calcd. for C10H13O7245.06668; found: 245.06667.

[0212] 4-((5-methyl-2-oxo-l,3-dioxol-4-yl)methoxy)-2-methylene-4-oxobutanoic acid (P6).Compound 8 (470 mg, 1.58 mmol); DCM (1 mL), trifluoroacetic acid (6 mL). Prodrug P6 was isolated as a colorless solid (321 mg) in 84% yield.NMR (401 MHz, CDCI3): 8H 2.19 (s, 3H), 3.40 (d. J = 1.1 Hz, 2H), 4.89 (s. 2H). 5.89 (d, J = 1.0 Hz. 1H). 6.52 (s, 1H), 11.46 (bs. 1H).13C NMR (101 MHz, CDCI3): 8c 9.47, 37.17, 54.34, 131.73, 132.72, 133.43, 140.43, 152.21, 170.20, 171.42. ESI MS: 265.0 ([M + Na]+). HRMS (ESI): calcd. for C10H10O7Na 265.03187; found: 265.03191.

[0213] Synthetic procedure for prodrug P7

[0214] 4-(3-(hexadecyloxy)propoxy)-2-methylene-4-oxobutanoic acid (P7). Itaconic anhydride (336 mg, 3.00 mmol, 1 equiv.) and 3-(hexadecyloxy)propanol (992 mg, 3.30 mmol, 1.1 equiv.)3843955.601JT7708-02were dissolved in CHCh (3 mL) and the mixture was stirred for 16 h at 70 °C. Volatiles were evaporated, and the residue was purified by flash column chromatography on silica (mobile phase: DCM / MeOH, 35: 1) to afford 1.03 g (83%) of prodrug P7 as a colorless solid. 'H NMR (401 MHz, CDCl₃): 8H 0.88 (t, 3H, J= 6.8, 3H), 1.20-1.35 (m, 26 H), 1.60 - 1.51 (m, 2H), 1.89 (p, 2H), 3.34 (d, 7=1.0, 2H), 3.39 (t, 7 = 6.7, 2H), 3.46 (t, 7 = 6.2, 2H), 4.20 (t, 7=6.4, 2H), 5.83 (d, 7= 1.1, 1H), 6.46 (d. 7= 0.9, 1H).13C NMR (101 MHz, CDCl₃): 6c 14.28. 22.85, 26.31, 29.12, 29.52, 29.67, 29.77, 29.80, 29.82, 29.84, 29.86, 32.08, 37.47, 62.54, 67.16, 71.34, 130.79, 133.38, 170.62, 170.93. ESI MS: 411.3 ([M - H]+). HRMS (ESI): calcd. for C24H43O5411.31160; found: 411.31088.

[0215] General synthetic procedure for prodrugs P8-10 and Pll-14

[0216] 1-MI (11) or 4-MI (12) (200 mg, 1.39 mmol. 1 equiv.), appropriate chloride 2-4 (1.2-1.3 equiv.), sodium iodide (41.6 mg, 0.278 mmol, 0.2 equiv.) and potassium carbonate (288 mg, 2.08 mmol, 1.5 equiv.) were dissolved in anhydrous MeCN (5 mL) and the mixture was stirred for 16 h at 40-45 °C. EtOAc (60 mL) was added, and the mixture was washed with brine (20 mL) and sat. sodium thiosulfate (10 mL). The organic phase was dried over anhydrous Na2SO4, volatiles were evaporated, and the residue was purified by flash column chromatography on silica (cyclohexane / EtOAc, 5:1) to afford desired prodrugs P8-10 and Pll-14.

[0217] 1 -Methyl 4-((pivaloyloxy)methyl) 2-methylenesucdnate (P8). 1-MI (11); chloromethyl pivalate (272 mg, 260 pL, 1.80 mmol, 1.3 equiv.); 40 °C. Compound P8 was isolated as a colorless oil (301 mg) in 84% yield. 'H NMR (401 MHz, CDCl₃): 6u 1.22 (s, 9H), 3.38 (s, 2H), 3.77 (s, 3H), 5.74 (dd, 7= 2.1, 1.1 Hz, 1H), 5.76 (s, 2H), 6.35 (s, 1H).13C NMR (101 MHz, CDCl₃): 8c 26.95, 37.52. 38.86, 52.29, 79.75, 129.07, 133.15, 166.43, 169.51, 177.17. ESI MS: 281.1 ([M + Na]+). HRMS (ESI): calcd. for C12H18O6Na 281.09956; found: 281.09993.

[0218] 4-(((Isopropoxycarbonyl)oxy)methyl) 1-methyl 2-methylenesuccinate (P9). 1-MI (11); chloromethyl isopropyl carbonate (254 mg, 223 pL. 1.67 mmol, 1.2 equiv.); 40 °C. Compound P9 was isolated as a colorless oil (336 mg) in 93% yield. 'H NMR (401 MHz, CDCl₃): 811 1.31 (s, 3H), 1.32 (s, 3H), 3.40 (s, 2H), 3.76 (s, 3H), 4.86 - 4.97 (m, 1H), 5.78 - 5.71 (m, 3H), 6.36 (s, 1H).13C NMR (101 MHz, CDCl₃): 8c 21.65, 37.33, 52.20, 73.12, 81.96, 129.18, 132.88, 153.31, 166.33, 169.27. ESI MS: 283.1 ([M + Na]+). HRMS (ESI): calcd. for C11H16O7Na 283.07882; found: 283.07925.3943955.601JT7708-02

[0219] 1 -Methyl 4-((5-methyl-2-oxo-l,3-dioxol-4-yl)methyl) 2-methylenesuccinate (PIO). 1-MI (11); (4-chloromethyl)-5-methyl-l,3-dioxol-2-one (268 mg, 197 pL, 1.80 mmol, 1.3 equiv.); 40 °C. Compound PIO was isolated as a colorless oil (298 mg) in 84% yield. ’H NMR (401 MHz, CDCl₃): 8H 2.16 (s, 3H), 3.36 (s, 2H), 3.76 (s, 3H), 4.85 (s, 2H), 5.74 (d, J= 1.1 Hz, 1H), 6.35 (s, 1H).13C NMR (101 MHz, CDCl₃): 8c 9.49, 37.60, 52.35, 54.25, 129.25, 133.20, 133.43, 140.34, 152.19. 166.50, 170.33. ESI MS: 279.0 ([M + Na]+). HRMS (ESI): calcd. for C11H12O7Na 279.04752; found: 279.04800.

[0220] 4-Methyl l-((pivaloyloxy)methyl) 2-methylenesuccinate (P12). 4-MI (12); chloromethyl pivalate (272 mg, 260 pL, 1.80 mmol, 1.3 equiv.); 40 °C. Compound P12 was isolated as a colorless oil (330 mg) in 92% yield. 'H NMR (401 MHz, CDCl₃): 8H 1.20 (s, 9H), 3.34 (s, 2H), 3.68 (s, 3H), 5.79 (d. J= 1.1 Hz, 1H). 5.82 (s, 2H). 6.39 (s, 1H).13C NMR (101 MHz, CDCl₃): 8c 26.95, 37.38, 38.90, 52.21, 79.91, 130.30, 133.06, 164.82, 170.90, 177.19. ESI MS:281.1 ([M + Na]+). HRMS (ESI): calcd. for C12H18O6Na 281.09956; found: 281.09921.

[0221] 1 -(((Isopropoxy carbonyl)oxy)methyl) 4-methyl 2-methylenesuccinate (P13). 4-MI (12); chloromethyl isopropyl carbonate (254 mg, 223 pL, 1.67 mmol, 1.2 equiv.); 45 °C. Compound P13 was isolated as a colorless oil (340 mg) in a 94% yield. 'H NMR (401 MHz, CDCl₃): 8H 1.33 (s, 3H), 1.34 (s, 3H), 3.38 (s, 2H), 3.72 (s, 3H), 4.86 - 5.02 (m, 1H), 5.82 - 5.88 (m, 3H), 6.46 (s, 1H).13C NMR (101 MHz, CDCl₃): 8c 21.64, 37.20, 52.12, 73.10, 82.20, 130.50, 132.76, 153.29, 164.52, 170.77. ESIMS: 283.1 ([M + Na]+). HRMS (ESI): calcd. for C11H16O7Na 283.07882; found: 283.07855.

[0222] 4-Methyl l-((5-methyl-2-oxo-l,3-dioxol-4-yl)methyl) 2-methylenesuccinate (P14). 4-MI (12); (4-chloromethyl)-5-methyl-l,3-dioxol-2-one (268 mg, 197 pL, 1.80 mmol, 1.3 equiv.); 40 °C. Compound P14 was isolated as a light- yellow oil (320 mg) in 90% yield. 'H NMR (401 MHz, CDCl₃): 8H 2.17 (s, 3H), 3.33 (s, 2H), 3.67 (s, 3H), 4.91 (s. 2H), 5.78 (d, J= 1.1 Hz, 1H), 6.35 (s. 1H).13C NMR (101 MHz. CDCl₃): 8c 9.36, 37.36, 52.13. 54.28. 129.98, 132.99. 133.36, 140.30, 152.11, 165.51, 170.91. ESIMS: 279.1 ([M + Na]+). HRMS (ESI): calcd. for C11H12O7Na 279.04752; found: 279.04757.

[0223] General synthetic procedure for prodrugs Pll and P15

[0224] 1-MI (11) or 4-MI (12) (400 mg, 2.78 mmol, 1 equiv.) were dissolved in anhydrous DCM (8 mL), 3-(hexadecyloxy)propanol (959 mg, 3.19 mmol, 1.15 equiv.) and DMAP (509 mg, 4.16 mmol, 1.5 equiv.) was added and the solution was cooled to 0 °C and stirred under inert4043955.601_P17708-02atmosphere. A solution of DCC (859 mg, 4.16 mmol, 1.5 equiv.) in anhydrous DCM (5 mL) was added dropwise for 10 minutes, and the resulting mixture was stirred for 1 h at 0°C and then overnight (15 h) at room temperature. The mixture was filtered, DCM (30 mL) was added, and the organic phase was washed with 10% KHSO4 (3x10 mL), sat. NaHCO₃ (10 mL) and brine (10 mL). The organic phase was dried over anhydrous Na₂SO₄, volatiles were evaporated, and the residue was purified by flash column chromatography on silica (mobile phase: cyclohexane / EtOAc, 80: 15) to afford prodrugs Pll and P15.

[0225] 4-(3-(Hexadecyloxy)propyl) 1-methyl 2-methylenesuccinate (Pll). 1-MI (11).Compound Pll was isolated as a colorless amorphous compound (332 mg) in 28% yield. 'H NMR (401 MHz, CDCl₃): δH 0.87 (t, J= 7.0 Hz, 3H), 1.23 - 1.33 (m, 26 H), 1.50 - 1.58 (m, 2H), 1.84 - 1.92 (p. 2H), 3.33 (d, J=1.2 Hz, 2H), 3.38 (t, J= 6.7 Hz, 2H), 3.45 (t, J= 6.3 Hz. 2H), 3.76 (s, 3H), 4.19 (t, J=6.5 Hz, 2H), 5.69 - 5.71 (m, 1H), 6.32 (d, J= 1.1 Hz, 1H).13C NMR (101 MHz, CDCl₃): δC 14.27, 22.84, 26.30, 29.14, 29.51, 29.66, 29.76, 29.78, 29.80, 29.82, 29.84, 29.86, 32.07, 37.89. 52.26, 62.43, 67.16, 71.32. 128.59, 128.73. 133.88, 166.79. 170.79. ESI MS: 449.3 ([M + Na]+). HRMS (ESI): calcd. for C25H46O5Na 449.32375; found: 449.32382.

[0226] l-(3-(Hexadecyloxy)propyl) 4-methyl 2-methylenesuccinate (P15). 4-MI (12).Compound P15 was isolated as a colorless amorphous compound (805 mg) in 68% yield. 'H NMR (401 MHz, CDCl₃): δH0.88 (t, J= 7.0 Hz, 3H), 1.21 - 1.34 (m, 26 H), 1.50 - 1.60 (m, 2H), 1.89 - 1.97 (p. 2H), 3.34 (d, J=1.2 Hz, 2H), 3.39 (t, J= 6.7 Hz, 2H), 3.48 (t, J= 6.3 Hz, 2H), 3.70 (s, 3H), 4.26 (t, J = 6.5 Hz, 2H), 5.69 - 5.71 (m, 1H), 6.31 - 6.35 (m, 1H).13C NMR (101 MHz, CDCl₃): δC 14.26, 22.84, 26.32, 29.22, 29.51, 29.66, 29.76, 29.78, 29.81, 29.82, 29.85, 29.87, 32.08, 37.69. 52.17, 62.58, 67.23, 71.36, 128.53. 134.04, 166.20. 171.27. ESI MS: 427.3 ([M + H]+). HRMS (ESI): calcd. for C25H47O5427.34180; found: 427.34158.

[0227] Chemical and Metabolic Stability

[0228] For measuring chemical stability, prodrugs were spiked (10 pM) in pH buffers (pH 1.2, 4.5, and 7.4) in triplicate. These mixtures were incubated at 37 °C for 1 h. Prodrug disappearance was monitored using the developed LC-MS / MS methods described below.

[0229] For measuring metabolic stability, prodrugs (10 pM) were spiked in mouse and human plasma and incubated in an orbital shaker at 37 °C. Aliquots (100 pL) were sampled at predetermined times (0 and 60 min) and quenched with cold acetonitrile (300 pL) containing internal standard (losartan 0.5 pM). The samples were vortexed for 30 s and centrifuged at 14,000g4143955.601_P17708-02for 10 min. The supernatant (50 pL) was diluted with water (50 pL) and transferred to a 250 pL polypropylene vial sealed with a Teflon cap. These samples were run using liquid chromatography and high-resolution mass spectrometry (LCMS). and the disappearance of prodrugs was noted over the stipulated time. Briefly, prodrugs were analyzed on a Thermo Scientific Dionex Ultimate 3000 UPLC system coupled to Dionex Ultimate 3000 pump and autosampler using EclipsePlus C18 UPLC column from Agilent [Santa Clara, CA, USA] (100 x 2.1 mm id. 1.8 pm). The autosampler was maintained at 4 °C and the column compartment at 35 °C for the duration of the LC-MS runs. Chromatographic separation was achieved using acetonitrile / water containing 0.1% formic acid as a mobile phase while pumping a flow of 0.3 mL / min for 9 min using gradient elution. The eluent was analyzed using a Thermo Scientific Q Exactive Focus mass spectrometer, equipped with a heated electrospray ionization (HESI) probe set in the positive ionization mode. Samples were introduced into the ionization source through a heated nebulized probe (350 °C). Disappearance of prodrugs was measured from the ratio of peak areas of analyte to IS.

[0230] PAMPA- GIT

[0231] To assess intestinal permeability, we utilized STIR WELL™ PAMPA sandwiches (pION INC). A 5 pL of GIT-0 lipid solution was applied to coat each well of the top (acceptor) compartment of STIR WELL™ PAMPA sandwich. Before assembling the sandwich, the bottom (donor) plate was loaded with 200 pL of test compounds and reference compounds (10 pM) dissolved in the pION buffer at pH 7.4, containing 0.2% DMSO. The acceptor plate was filled with 200 pL of pH 7.4 sink buffer. The sandwich was incubated at room temperature for 4 hours. Following the incubation, 50 pL of samples were collected from both donor and acceptor compartments and analyzed by LC-MS / MS. Pevalue is calculated using the following equation.

[0232] Pe = − (2.303 VD / A(t−τss)) · log [1 − (1 / (1−R)) · CD(t) / CD(0)]

[0233] where Peis the effective permeability coefficient (cm / s), A is the filter area (0.3 cm2) multiplied by a nominal porosity of 70% according to the manufacturer, VD and V are the volumes in the donor and acceptor phase, t is the incubation time, rssis the steady state time (s), Co(t) is the concentration (mol cm’3) of the compound in the donor phase at time t, CD(0) is the concentration (mol cm’3) of the compound in the donor phase at time 0 and R is the membrane retention factor:

[0234] R =CD(0) VD CD(0)

[0235] NHEK cytotoxicity4243955.601JT7708-02

[0236] Normal human epidermal keratinocytes (Lonza, Basel, Switzerland) were revived, allowed to expand, and seeded in 96-well plates. P2, P9 and P13 were diluted to concentrations of 100, 30, 10, and 1 pM and added to the assay plates containing NHEKs. The plates were checked for compound effects after 8 hours of incubation to determine % viability of NHEKs. Cell viability was measured with CellTiter-Glo assay (Promega, Madison, WI, USA).

[0237] poly I: C / IFNy -induced gene expression profiles in NHEKs

[0238] NHEKs were seeded in plates and incubated overnight at 37°C and 5% CO2, followed by stimulation of poly I: C (50 pg / mL) and IFNy (5 ng / mL) with or without the presence of prodrugs at the concentrations of 1, 10, 30, and 100 pM. After incubation, RNA was isolated and analyzed with TaqMan RT-PCR (Thermo Fisher Scientific, Waltham, MA, USA) to quantify fold changes in the expression of genes of IL-6, IFNp, IL-ip, CXCL-9, CXCL-10, and CXCL-11.

[0239] Pharmacokinetic Studies

[0240] All animal studies were conducted in accordance with protocols reviewed and approved by the Institutional Animal Care and Use Committee of Johns Hopkins University (JHU). C57BL6 mice weighing between 25-30 g and 8.5 weeks of age were maintained on a 12 h light-dark cycle, with access to food and water, ad libitum.

[0241] For PK studies, P2 or P13 were formulated in 10% DMSO, 80% PEG, 10% HBS v / v / v and administered PO at a dose of 100 mg / kg equivalent to either IA or MMI. All of the formulations were freshly prepared prior to the dosing. The mice were sacrificed at specified time points (0.25, 0.5, 1, 2, 4, 6, and 24 h) post drug administration. Blood samples were collected in heparinized microtubes by cardiac puncture and spun at 2000x g for 15 min to collect plasma and then 100 pL plasma was stabilized by the addition of 50 pL 2% formic acid in water and immediately frozen and stored at -80 “C.

[0242] Skin was dissected, flash frozen in liquid nitrogen, then weighed. Four microliters of 0.1% FA in 30:70 water: ACN / mg tissue was added as stabilizer. Samples were stored at -80 C.

[0243] For quantifying intact P2 or P13, IA, and 4-MI in plasma, naive mouse plasma was combined 2:1 with 2% formic acid stabilizer, aliquoted, and standard and QC stock solutions of each analyte were spiked to obtain standards (0.01-1000 nmol / mL), and QCs (0.05-500 nmol / mL). These then were protein precipitated using methanol (5 x plasma volume) containing internal standards (0.5 pM Losartan: Millipore Sigma, Burlington MA; 10 pM itaconic acid-C

[0013] 5, Cambridge isotope libraries, Tewksbury, MA; 10 pM 5-methoxy-2-methylene-5-oxopentanoic4343955.601_P17708-02acid (MMOPA), Enamine; Monmouth Jet., NJ.) Samples were prepared in a similar fashion (without spiking standard stock solutions) to match the ratio of matrix to solvent achieved in standards and QCs. The solutions were centrifuged at 16,000 g for 5 minutes at 4 °C, and the supernatant was analyzed using LC-MS / MS to quantify P2 or P13, IA and 4-MI using the bioanalytical methods described below.

[0244] For quantifying intact P2 or P13, IA, and 4-MI in the skin, naive skin was homogenized, and protein precipitated simultaneously using methanol (5 x tissue weight) containing internal standards in a Geno grinder at 1500 RPM for 3 min. This matrix was aliquoted, and standard and QC stock solutions of each analyte were spiked to obtain standards (0.01-1000 nmol / g), and QCs (0.05-500 nmol / g). Samples were prepared in a similar fashion (without spiking standard stock solutions) to match the ratio of matrix to solvent achieved in standards and QCs. These were then centrifuged at 16,000 g for 5 minutes at 4 °C, and the supernatant was analyzed using LC-MS / MS to quantify the analytes using the bioanalytical methods described below.

[0245] Bioanalysis

[0246] Chromatographic analysis was performed using an Ultimate 3000 ultrahigh-performance system consisting of coupled with a QExactive Focus orbitrap mass spectrometer (Thermo Fisher Scientific Inc., Waltham, MA). Analyte separation was achieved at 35 °C using an Agilent EclipsePlus column (100 mm x 2.1 mm i.d.) packed with 1.8 p.m C18 stationary phase. The mobile phase consisted of 0.1% formic acid in methanol and 0.1% formic acid in water with gradient elution. The [M + H]+ ion transition for P2 was m / z 363.1286 — 105.0592 and 113.0291 with losartan as internal standard. The [M+H] + transition for losartan were m / z 423.1695 — > 207.0915 and 377.1518. The [M + H]+ ion transition for P13 was m / z 261.0969 — 69.0339. 99.0474 and 127.0386 with losartan as internal standard as above. The [M - H]- ion transition for IA was m / z 129.0193 — > 85.0294 with IA-[13C]5 as internal standard. The [M - H]- ion transition for IA-[13C]5 was m / z 134.0361 —>■ 89.0429. The [M + H]+ ion transition for 4-MI was m / z 145.0495 69.0339 and 99.0474 with MMOPA as internal, standard The [M+H] + transition for MMOPA were m / z 159.0652 113.0656 and 127.0388.

[0247] Cytokine expression profiles of normal human epidermal keratinocytes

[0248] Neonatal human epidermal keratinocytes (NHEKs) isolated from neonatal foreskin were seeded at 100,000 cells per well in KGM supplemented with growth factors (KGM-GOLD Bullet kit, #192060). IA, 4-MI and prodrugs, reconstituted in DMSO, were used to pre-treat NHEKs with4443955.601_P17708-02either 0.1 % DMSO (vehicle) or the prodrug. After two days, the cells were treated with 50 pg / pL Poly(I: C) for 24 hours. Total RNA was extracted from the cultured NHEKs using the RNeasy Mini Kit (Qiagen, #74106). Reverse transcription of RNA to cDNA was performed using a reversetranscription kit with random hexamer primers (Applied Biosystems, #4368814). Quantitative real-time PCR (qRT-PCR) was conducted with gene-specific TaqMan probes and universal master mix (Applied Biosystems, #4366072). Multiplexed reactions included probes for target genes and the reference gene RPLPO. Relative mRNA fold changes were calculated using the AACt method.

[0249] Toxicity analysis of Pl 3 in mice on chronic administration

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[0300] Although the foregoing subject matter has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be understood by those skilled in the art that certain changes and modifications can be practiced within the scope of the appended claims.5043955.601_P17708-02

Claims

THAT WHICH IS CLAIMED:

1. A method for treating an autoimmune and / or an inflammatory disease, disorder, or condition, the method comprising orally administering a compound of formula (la), formula (lb), or formula (Ic) to a subject in need of treatment thereof:wherein:Ria is selected from: H, -CH2-O-C(=O)-C(CH3)3, -CH2-O-C(=O)-O-CH(CH3)2,-CH2-CH2-O-(CH2)15-CH3, and Rib, R2a, and R2care each independently selected from: -CH2-O-C(=O)-C(CH3)3, -CH2-O-C(=O)-O-CH(CH3)2, -CH2-CH2-O-(CH2)15-CH3, and and pharmaceutically acceptable salts thereof.

2. The method of claim 1, wherein Riais H or -CH2-O-C(=O)-O-CH(CH3)2and R2a, R2b, and R2cat each occurrence are -CH2-O-C(=O)-O-CH(CH3)2.

3. The method of claim 2, wherein the compound of formula (la), formula (lb), or formula (Ic) is selected from:5143955.601_P17708-024. The method of claim 3, wherein the compound of formula (la), formula (lb), or formula (Ic) is selected from:

5. The method of claim 4, wherein the compound of formula (la), formula (lb), or formula (Ic) is selected from:

6. The method of claim 1, wherein Ria, R2a, R2b, and R2cat each occurrence are -CH2-O-C(=O)-C(CH3)3.

7. The method of claim 6, wherein the compound of formula (la), formula (lb), or formula (Ic) is selected from:

8. The method of claim 1, wherein Ria, R2a, R2b, and R2cat each occurrence are:5243955.601JT7708-029 The method of claim 8, wherein the compound of formula (la), formula (Tb), or formula (Ic) is selected from:(P6) (PIO)10. The method of claim 1, wherein Ria, Ria. Rib, and Ric at each occurrence are -CH2- CH2-O-(CH2)i5-CH3.

11. The method of claim 10, wherein the compound of formula (la), formula (lb), or formula (1c) are selected from:O O(P7)11O; (PH)0; and 0 >)A / ^O^ / ^O(CH2)15CH3(P15) I' 012. A pharmaceutical composition formulated for oral delivery of a compound of formula (la), formula (lb), or formula (Ic) to a subject in need of treatment thereof:O O Owherein:5343955.601_P17708-02Riais selected from: H, -CH2-O-C(=O)-C(CH3)3, -CH2-O-C(=O)-O-CH(CH3)2,OA o o.-CH2-CH2-O-(CH2)15-CH3, and;Rib. R2a, and R2care each independently selected from: -CH2-O-C(=O)-C(CH3)3, -CH2-O- OA o o-,C(=O)-O-CH(CH3)2, -CH2-CH2-O-(CH2)15-CH3, and 1 ■and pharmaceutically acceptable salts thereof.

13. The method of claim 1, wherein the autoimmune and / or the inflammatory disease, disorder, or condition comprises a skin disease, disorder, or condition.

14. The method of claim 13, wherein the skin disease, disorder, or condition comprises alopecia areata.

15. The method of claim 1, wherein the autoimmune and / or the inflammatory disease, disorder, or condition is selected from rheumatoid arthritis, systemic lupus erythematosus (SLE), multiple sclerosis (MS), sepsis, a viral infection, ischemia / reperfusion injury, pulmonary fibrosis, gout, abdominal aortic aneurysm (AAA), and cryopyrin-associated periodic syndrome (CAPS).

16. The method of claim 1, wherein the autoimmune and / or the inflammatory disease, disorder, or condition is selected from type 1 diabetes, celiac disease, inflammatory bowel disease (IBD), including Crohn's Disease and ulcerative colitis, Sjogren's syndrome, Graves' disease, Hashimoto's thyroiditis, Addison's disease, vitiligo, myasthenia gravis, scleroderma, spondyloarthritis, vasculitis, sarcoidosis, chronic obstructive pulmonary disease (COPD), asthma, periodontitis, sarcoidosis, chronic inflammatory demyelinating polyneuropathy (CIDP), Behcet's disease, and achalasia.5443955.601JT7708-02

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