WO2012145254A2

WO2012145254A2 - Methods of using inhibitors of rorϒt to treat disease

Info

Publication number: WO2012145254A2
Application number: PCT/US2012/033707
Authority: WO
Inventors: Xiaohu Wang; Chen Dong
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 2011-04-16
Filing date: 2012-04-15
Publication date: 2012-10-26
Also published as: WO2012145254A9

Abstract

Ursolic acid ("UA") and analogs of UA are taught as RORϒt inhibitors useful in the treatment of a diseases or conditions ameliorated by the expression or production of IL-17, IL-17F, IL-21, and IL-22, and the differentiation and/or development of TH17 cells.

Description

M ETHODS OF USING INHI BITORS OF RORyt TO TREAT DISEASE FIELD OF THE INVENTION

The present disclosure is directed to compounds that inhibit RORyt and their application as a pharmaceutical for the treatment of disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of US provisional patent application number 61/476,265 filed April 16, 201 1 which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under ROI-AR050772 awarded by the National Institute of Health. The government has certain rights in the invention.

THE NAMES OF THE PARTIES TO A JOINT RESEARCH AGREEMENT

None.

REFERENCE TO SEQUENCE LISTING

None.

BACKGROUND OF INVENTION

TH 17 cells have recently emerged as a major player in inflammatory and autoimmune diseases due to the production of IL- 17 and IL- 17F, two related cytokines with potent proinflammatory activities. Kx>rn, T., et al., IL-17 and Thl 7 Cells, Annu Rev Immunol. 27:485- 5 17 (2009). Currently, there is no effective therapy for controlling excessive TH 17 cell responses and related diseases or disorders.

BRIEF SUMMARY OF TH E INVENTION

Methods for treating disease and disorders related to TH 1 7 cell responses in a patient in need of such treatment are provided herein. The described methods comprise the step of administrating to a patient in need thereof a therapeutically effective amount of an inhibitor of RORyt (also referred to herein as a "RORyt inhibitor"). Also, provided are methods of inhibiting the differentiation of TH 17 cells, the expression of IL- 17, IL- 17F, IL-21 and EL22 and/or the production of IL-17, IL-17F, IL-21 and IL22. Each of the methods comprises the step of administrating to a patient in need thereof a therapeutically effective amount of a RORyt inhibitor. The RORyt inhibitors taught herein include, but are not limited to, ursolic acid ("UA") and analogs of UA such as those presented below in the examples. The present disclosure also contemplates RORyt inhibitors for the use in the manufacture of a medicament for the treatment of a diseases or condition ameliorated by the inhibition of RORyt and IL- 17 expression.

As described herein, ursolic acid (UA) and its analogs, including but not limited to the analogs provided in Examples 1 to 19 below, can selectively and effectively blocked the function of RORyt and associated immune responses in both differentiated and developing Th l 7 cells. As a result, these small molecules are useful to modulate the development and differentiation of Th l 7 cells, the expression of IL-17, IL- 17F, IL21 and IL22 and/or the production of IL-17, IL- 1 F, IL21 and IL22 and treat disease and/or disorders associated with the same.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

Figures 1 A, IB, 1 C and ID show that UA dose-dependently inhibits Th I 7 differentiation. Figures 2A, 2B and 2C show that UA selectively blocks the function of RORyt but not

RORa.

Figures 3 A and 3B show that UA suppresses IL- 17 production in mature Th l 7 cells. Figures 4A, 4B, 4C and 4D show that UA treatment ameliorated EAE disease in mice. Figures 5A and 5B show the effect of UA on other T lineage differentiation. Figure 5A shows the effect of UA on human Th l , Th2, and iTreg cell differentiation. Figure 5B shows the effect of UA on mouse Th l , Th2, and iTreg cell differentiation.

Figure 6 shows that UA selectively blocks RORyt - dependent reporter activity. EL4 cells were transfected with CNS2-I117 reporter together with RORa, RORyt or control plasmids. UA or DMSO was added after transfection and the cells were harvested next day for dual- luciferase activity assays after PMA/Inomycinrestimulation. The data were normalized to an internal control renilla luciferase. The reporter gene assay was repeated 2 times with consistent results.

DETAILED DESCRIPTION OF THE INVENTION

Th l 7 cells have recently emerged as a major player in inflammatory and autoimmune diseases via the production of pro-inflammatory cytokines IL- 1 7, IL- 17F, IL-21 and IL-22. Th l 7 ceils have been recently discovered as the third effector CD4⁺ T helper subset. Park, H., et al., A Distinct Lineage of CD4 T Cells Regulates Tissue Inflammation by Producing Interleukin 17, Nat Immunol. 6: 1 133- 1 141 (2005); Harrington, L.E., et al., Interleukin 17-Producing CD4⁺ Effector T Cells Develop via a Lineage Distinct From the T Helper Type J and 2 Lineages, Nat Immunol. 6: 1 123-1 132 (2005). Th l 7 cells produce IL-17, IL-17F and IL-22. Dong, C, T_H17 Cells in Development: An Updated View of Their Molecular Identity and Genetic Programming, Nat Rev Immunol. 8:337-348 (2008); Korn, T., et al., IL-17 and Th 17 Cells, Annu Rev Immunol. 27:485-517 (2009).

Although Th l 7 cells play important roles in host defense against bacterial and fungal infections, they have been also linked to many immune-related diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases, periodontal diseases and asthma airway inflammatory diseases. Korn, T., et al., IL-17 and Thl 7 Cells, Annu Rev Immunol. 27:485-517 (2009); Tesmer, L.A., et al., Thl 7 Cells in Human Disease, Immunol Rev. 223:87- 1 13 (2008). An anti-IL- 17 antibody was recently shown to have good efficacy in treatment of multiple human diseases. Hueber, W., et al., Effects of AIN457, A Fully Human Antibody to Interleukin- 17 A, on Psoriasis, Rheumatoid Arthritis, and Uveitis, Sci Transl Med. 2:52ra72 (2010).

In Thl 7 cells, the transcription of IL- 17 and IL-17F is mediated by Th l 7-specific transcriptional regulators RORyt and RORa, though the latter plays a less significant role in mice. Ivanov, 1.1., et al., The Orphan Nuclear Receptor RORyt Directs the Differentiation Program of Proinflammatory IL-17⁺ T Helper Cells, Cell 126: 1 121 - 1 133 (2006); Yang, X.O., et al., T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORa and RORy, Immunity 28:29-39 (2008). Mice deficient in RORy and those deficient in both RORyt and RORy are defectively in production of IL- 17 and IL- 1 7F, and are resistant to experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. Ivanov, 1.1., et al., The Orphan Nuclear Receptor RORyt Directs the Differentiation Program of Proinflammatory IL-17⁺ T Helper Cells, Cell 126: 1 121 -1 133 (2006); Yang, X.O., et al., T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORa and RORy, Immunity 28:29-39 (2008).

Moreover, the transcription of IL- 1 7 and IL- I 7F is directly controlled by the master transcription factor RORyt together with a related transcription factor RORa. Ivanov, 1.1., et al., The Orphan Nuclear Receptor RORyt Directs the Differentiation Program of Proinflammatory IL-17⁺ T Helper Cells, Cell 126: 1 121 -1 133 (2006); Yang, X.O., et al., T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORa and RORy, Immunity 28:29-39 (2008). In addition, the differentiation of Thl 7 cells and the associated cytokine production is directly controlled by RORyt. For example, it has been reported that the expression of IL- 17 and development of Th l 7 cells are directly controlled by retinoid-related orphan receptor RORyt. Ivanov, 1.1., et al., The Orphan Nuclear Receptor RORyt Directs the Differentiation Program of Proinflammatory IL-17⁺ T Helper Cells, Cell 126: 1 121-1 133 (2006). Noteworthy, to date, only one inhibitor of RORyt (10901317) has been reported. Kumar, N. et al., The Benzenesulfoamide T090l317[N-(2.2.2-Tri uoroethyl)-N-[4-[2.2 -trifli ro-l-hydroxy-T

phenyl] -benzene.su/fonamide] Is a Novel Retinoic Acid Receptor-Related Orphan Receptor-oJy Inverse Agonist.. ol. Pharm. 77:228-236 (2010). But the effect of this inhibitor on Th l 7 cell development and IL- 17 expression has gone unnoticed and unconfirmed until now. Moreover, until the present disclosure, there has been no effective therapy for controlling of excessive TH 17 responses and related autoimmune diseases.

Now, through the inhibition of RORyt via UA and its analogs, we have shown that

RORyt inhibitors can be useful to treat autoimmune disease, inflammation, cancer, and immunity disorders related to excelliilar bacteria, fugus, and viruses, and other diseases and disorders associated with the over expression of: IL- 1 7; IL- 17F; IL-21 ; and I.L-21 , the production of one or more of the pro-inflammatory cytokines: IL- 17; IL- 17F; IL-21 ; and IL-22, and/or generally excessive Th l 7 cell response. See e.g., Korn, T., et al., IL-17 and Thl 7 Cells, Annu Rev Immunol. 27:485-517 (2009). More specifically, the inhibition of RORyt via UA and its analogs, we have shown that RORyt inhibitors can be useful to treat multiple sclerosis, asthma, inflammatory bowel disease, arthritis, melanoma, rheumatoid arthritis, systemic lupus erythematosus, allograft rejection, psoriasis, ankylosing spondilitis, scleroderma, Type I diabetes, psoriatic arthritis, osteoarthritis, and atopic dermatitis.

IL-21 regulates the differentiation of CD4+ T cells into TH 17 cells in an autocrine manner. Expression of IL-21 is induced in T cells by IL-6 via STAT3 and is necessary in the generation of TH 17 cells via STAT3-dependent upregulation of RORyt. IL-21 acts in an autocrine fashion in the differentiation of TH17 cells as IFNy does for TH 1 cells and IL-4 for TH2 cells. Furthermore, the differentiation of TH 17cells can be modulated via the IL-21 signaling pathway. Moreover, any interruption to the pathway upstream of STAT3 will prevent the activation of STAT3, the differentiation of TH 17 cells and ultimate expression of IL-21 and other cytokines expressed by the TH 17 cells. US Pub. App. No. 2010/0247547 Paragraphs 53, 54, 55, 56, 57 and 58, and Figure 12, incorporated herein by reference. As such, RORyt inhibitors taught herein suppress specific cytokine signaling pathways such as IL-2 1 and modulate the differentiation of THI7 cells. Moreover, the RORyt inhibitors may be useful in combination therapies, for example, with STAT3 small molecule inhibitors and IL-21 inhibitors to treat the diseases described herein.

The phrase "therapeutically effective" is intended to qualify the amount of active ingredients used in the treatment of a disease or disorder. This amount will achieve the goal of reducing or eliminating the said disease or disorder. The term "patient" means all mammals including humans. Examples of patients include humans, cows, dogs, cats, goats, sheep, pigs, and rabbits. Preferably, the patient is a human.

While it may be possible for the molecules which inhibit RORyt activity to be administered as the raw chemical, it is also possible to present them as a pharmaceutical formulation. Accordingly, the pharmaceutical formulation may include the molecule or a pharmaceutically acceptable a salt, ester, prodrug or solvate thereof, where appropriate, together with one or more pharmaceutically acceptable carriers thereof and optionally one or more other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington's Pharmaceutical Sciences.

The formulations of use molecules include those suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous, intraarticular, and intramedullary), intraperitoneal, transmucosal, transdermal, rectal and topical (including dermal, buccal, sublingual and intraocular) administration although the most suitable route may depend upon for example the condition and disorder of the recipient. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods may include the step of bringing into association the molecule or a pharmaceutically acceptable salt, ester, prodrug or solvate thereof ("active ingredient") with the carrier which constitutes one or more accessory ingredients. In certain instances, as noted above, it may be appropriate to administer at a RORyt inhibitor (or a pharmaceutically acceptable salt, ester, or prodrug thereof) in combination with another therapeutic agent such as a STAT3 inhibitor or IL-21 inhibitor. Or, by way of example only, the therapeutic effectiveness of the inhibitors provided herein may be enhanced by administration of an adjuvant (i.e., by itself the adjuvant may only have minimal therapeutic benefit, but in combination with another therapeutic agent such as a STAT3 inhibitor, the overall therapeutic benefit to the patient is enhanced). Or, by way of example only, the benefit of experienced by a patient may be increased by administering one molecule as described herein with another therapeutic agent (which also includes a therapeutic regimen) that also has therapeutic benefit.

Multiple therapeutic agents may be administered in any order or even simultaneously. If simultaneously, the multiple therapeutic agents may be provided in a single, unified form, or in multiple forms (by way of example only, either as a single pill or as two separate pills). One of the therapeutic agents may be given in multiple doses, or both may be given as multiple doses.

In the currently study described below, we screened a small chemical library, and identified ursolic acid (UA), a natural carboxylic acid ubiquitously present in plants, as a strong and selective inhibitor for RORyt function. UA inhibited IL- 17 production not only in developing Th l 7 cells but also in mature Th l 7 cells. Mice receiving UA were resistant to EAE, indicating that UA can be used for developing treatment of Thl 7-mediated diseases.

As such, we provide ursolic acid ("UA") a small molecule present in herbal medicine, and its analogs, can selectively and effectively inhibit the function of RORyt, resulting in greatly decreased IL- 17 expression in both developing and differentiated Thl 7 cells. In addition, treatment with UA ameliorated experimental autoimmune encephalomyelitis. UA and its analogs are viable drug products for developing treatments against Th l 7-mediatedinflammatory diseases and cancer.

As described herein, ursolic acid (UA) and its analogs shown in Examples below can selectively and effectively blocked the function of RORyt and IL- 1 7 expression in both differentiated and developing Thl 7 cells and can be used in diseases associated with IL- 17 expression and differentiation of Th l 7 cells.

UA is a relatively non-toxic natural small molecule having a long history in herbal medicine practice. UA can be useful for the treatment of liver diseases, skin cancer and non- lymphatic leukemia. In addition, treatment with UA has been shown to ameliorate a mouse model of human multiple sclerosis. Liu, J. Pharmacology of Oleanolic Acid and Ursolic Acid, J Ethnopharmacol. 49:57-68 (1995).

As noted above, to date, only one inhibitor of RORyt ( 10901317) has been reported. But the effect of this inhibitor on Th l 7 cell development and IL- 1 7 expression has gone unnoticed and unconfirmed until now. Moreover, until the present disclosure, there has been no effective therapy for controlling of excessive TH 17 responses and related autoimmune diseases. Now, through the inhibition of RORyt, we have shown that RORyt inhibitors can treat multiple sclerosis, autoimmune disease, asthma, inflammatory bowel disease, inflammation, cancer, multiple sclerosis, arthritis, rheumatoid arthritis, asthma, systemic lupus erythematosus, allograft rejection, psoriasis, ankylosing spondilitis, scleroderma, Type 1 diabetes, psoriatic arthritis, osteoarthritis, and atopic dermatitis, and immunity disorders related to excellular bacteria, fugus, and viruses, and any disease associated with the over expression of IL- 17, IL- 17F, IL-21 and IL- 21 , the production of one or more of the pro-inflammatory cytokines IL- 17, IL-17F, IL-21 and IL-22, and/or generally excessive Th 17 cell response.

We have demonstrated that ursolic acid (UA) selectively inhibits RORyt-mediated IL-17 expression in both developing and differentiated Th l 7 cells without affecting other T lineage cell development. More importantly, UA, as a natural product present in many medical herbs and human diet, has already been demonstrated to be relatively non-toxic (LD50 > 600 mg/kg body weight in rodents for intraperitoneal injection. Gautam, R. & Jachak, S. et al., Recent Developments in Anti-Inflammatory Natural Products, Med. Res. Rev. 29:767-820 (2009). Indeed, it has been already recommended for skin cancer therapy in Japan. Muto, Y., et al., Present Status of Research on Cancer Chemoprevention in Japan, Japanese J. Clin. Oncology 20:219-224 ( 1990).

The selective effect of UA on Th l 7 cells, together with the low toxicity and long-term medical practice, provides UA a great advantage over other RORyt inhibitors in clinical applications for developing treatment of Thl7 related-autoimmune diseases.

Ursolic acid (UA) is a relatively non-toxic natural pentacyclictriterpenoid carboxylic acid present in numerous plants, medical herbs and even human diet. Gautam. R. & Jachak, S. et al., Recent Developments in Anti-Inflammatory Natural Products, Med. Res. Rev. 29:767- 820 (2009). UA has been shown to have different pharmacological activities, including anti- tumor and anti-inflammation effects. Liu, J. Pharmacology of Oleanolic Acid and Ursolic Acid, J Ethnopharmacol. 49:57-68 ( 1995); Ikeda, Y., et al., Ursolic Acid: An Ami- and Pro- Inflammatory Trilerpenoid, Mol Nutr Food Res. 52:26-42 (2008). While useful in a wide variety and number of different types of treatments and disease, for the first time, however, UA and certain of its analogs are shown to be specific and selective inhibitors of RORyt and Th l 7 cells differentiation.

As taught below, in our in vitro differentiation system, we identified UA as selective and specific inhibitor for Th l 7 cells of both mouse and human sources since UA only blocked 1L- I 7 expression in Th l 7 cells but did not affect effector cytokine gene expression in other T lineage cells (FIGS. 1 & 2).

To search for the target of UA in Thl 7 cells, we first found that UA has a similar structure to hydroxycholesterols, the natural ligands for retinoic acid-related orphan receptors (Fig. 2), indicating UA may inhibit IL-17 expression and Thl7 cell development through RORyt. Jin, L., et al., Structural Basis for Hydroxycholesterols as Natural Ligands of Orphan Nuclear Receptor RORy, Mol Endocrinol. 24:923-929 (2010). To test this possibility, we investigated the effect of UA on the function of RORyt or RORa in several in vitro systems where lacking any Th l 7-specific genes or transcription factors, including ROR-dependent IL- 17 expression in T cells cultured under neutral condition, ROR-dependent IL- 17 reporter activity in EL4 cells or RORE reporter activity in 293 T cells.

RORa is nuclear receptor protein related to RORyt whereas plays a redundant but minor role in Th l 7 cells. We found 2μΜ UA effectively blocked the function of RORyt, but not RORa, under all these conditions (Fig 2 & 6). These data strongly suggest UA serves as a RORyt- specific antagonist. To further confirm this, we performed a co-activator binding assay and found that UA selectively inhibited the binding of RORyt, but not RORa, to its co- activtorpeptide with an IC50 value of 0 68 ± 0 1 μ -similar to the IC50 value of UA on Th l 7 cells (0.56 ± 0 Ι μΜ) (Fig, 2).

These data together firmly establish UA and its known analogs as a RORyt and Th l 7- specific inhibitors. In addition, we found that UA can also inhibit expression of IL-17 in mature human and mouse Th l 7 cells which is of more clinic relevance (Fig 3). More importantly, in our in vivo studies, we found that UA treatment resulted in not only a pronounced delay in disease onset but also a significantly reduced disease symptom in MOG-induced experimental autoimmune encephalomyelitis ("EAE") in mice (Fig 4) -a Th l 7 mediated mouse disease model that closely mimics human multiple sclerosis ("MS").

Taken together, these data strongly suggest UA and its analogs disclosed immediately below in the examples are valuable drug candidates for treatment of Th 17-related autoimmune diseases.

EXAMPLE 1

(4aR,5R,6aR,6aS,6bR,8aR, 1 OS, 12aR, 14bS)-5, 10-dihydroxy-2,2,6a,6b,9,9, 12a-heptame

1 ,3,4,5,6,6a,7,8,8a, 10, 1 1 , 12,13, 14b-tetradecahydropicene-4a-carboxylic acid

Echinocystic acid

EXAMPLE 2

3-Epiursolic acid

EXAMPLE 3

3-Epioleanolic acid

EXAMPLE 4

Olean- 18-en-28-oic acid,3-hydroxy-,(3b)-

Maloic acid

EXAMPLE 5

(2R,4aS,6aR,6aS,6bR,8aR, 1 OS, 12aR, 14bS)- 10-hydroxy-2,4a,6a,6b,9,9, 12a-heptamethyl- 1 ,3,4,5,6,6a, 7, 8, 8a, 10, 1 1 , 12, 13, 14b-tetradecahydropicene-2-carboxylic acid

Epikatonic acid

EXAMPLE 6

(4aS,6aR,6aR,6bR,8aR, 12aR, 14aS)-2,2,6a,6b,9,9, 12a-heptamethy 1- 10-oxo- 4,5, 6,6a, 7, 8, 8a, 11,12,13,14,14a-dodecahydro-3H-picene-4a-carboxylic acid

Moronic acid

3-Oxoolean-18-en-28-oic acid

EXAMPLE 7

(3 R,4S,4aR,6aR,6bS,8aR, 11 R, 12S, 12aR, 14aR, 14bR)-3-hydroxy-4,6a,6b,8a, 11,12,14b- heptamethyl-2,3,4a,5,6,7,8,9, 10, 11 , 12, 12a, 14, 14a-tetradecahydro-l H-picene-4-carboxylic acid

Boswellic acid

EXAMPLE 8

Urs- 12-en-28-oic acid,3,30-dihydroxy-, (3b)-

Rubifolic acid

EXAMPLE 9

Heterobetulinic acid

EXAMPLE 10

(4aS,6aS,6bR,8R, 1 OS, 12aR)-8, 10-dihydroxy-2,2,6a,6b,9,9, 12a-heptamethyl- 1 ,3,4,5,6,6a,7,8,8a, 10, 1 1 , 12, 13, 14b-tetradecahydropicene-4a-carboxylic acid

EXAMPLE 1 1

(2beta,3beta)-2,3-Dihydroxy-urs-12-en-28-oic acid

2P-Hydroxyursolic acid

EXAMPLE 12 Hederagenin

EXAMPLE 13 Urs- 12-ene-27,28-dioic acid, 3-beta-hydroxy-

EXAMPLE 14

(4aS,6aR,6aS,6bR,8aR, I OR, 1 1 R, 12aR, 14bS)- 10, 1 1 -dihydroxy-2,2,6a,6b,9,9, 12a-heptamethyl- 1 ,3,4,5,6,63,7,8,8a, 10, 1 1 , 12, 13, 14b-tetradecahydropicene-4a-carboxylic acid

Maslinic acid

EXAMPLE 1 5

(4aS,6aR,6aS,6bR,8aR, 1 OS, 12aR, 14bS)- 10-hydroxy-2,2,6a,6b,9,9, 12a-heptamethyl- 1 ,3,4,5,6,6a,7,8,8a, 10, 1 1 , 12, 13, 14b-tetradecahydropicene-4a-carboxylic acid

Oleanolic acid

EXAMPLE 16

01ean- 12-en-28-oic acid, 2,3-dihydroxy-, (2β,3β)-

2p-Hydroxyoleanolic acid

EXAMPLE 17

Gypsogenic acid

EXAMPLE 18

S,6aR,6aS,6bR,8aR, 1 OS, 12aR, 14bS)- 1 , 10-dihydroxy- 1 ,2,6a,6b,9,9, 12a-heptamethy 1-,3,4,5, 6,6a, 7, 8, 8a, 10,11,12,13,14b-tetradecahydropicene-4a-carboxylic acid

Pomolic acid

EXAMPLE 19

(4aS,6aR,6aS,6bR,8aR, 12aR, 14bS)-2,2,6a,6b,9,9, 12a-heptamethyl- 10-oxo- 3,4,5,6,6a,7,8,8a, 11,12,13,14b-dodecahydro- 1 H-picene-4a-carboxylic acid

Oleanonoc acid

Biological Activity Assays

T-cell Analysis. Human and mouse T cell differentiation and retroviral transduction were performed and analyzed by intracellular staining or by quantitative real-time RT-PCR assays as described. Yang, X.O., et al., T Helper 17 Lineage Differentiation Is Programmed by Orphan

Nuclear Receptors RORa and RORy, Immunity 28:29-39 (2008); Manel, N., et al., The

Differentiation of Human 7 Cells Requires Transforming Growth Factor-β and Induction of the Nuclear Receptor RORyt, Nat Immunol. 9:641 -649 (2008); Volpe, E., et al., A Critical

Function for Transforming Growth Factor-β, Interleukin 23 and Proinflammatory Cytokines in Driving and Modulating Human T_H-17 Responses, Nat Immunol. 9:650-657 (2008). UA (dissolved in DMSO) or DMSO was added into the culture media for inhibition assays.

Luciferase Reporter Assays. The CNS2-I117a and RORE reporter constructs were used for dual-luciferase reporter assays in EL4 and 293T cells, respectively, as reported. Yang, X.O., et al., T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORa and RORy, Immunity 28:29-39 (2008); Yang, X.O., et al., Molecular Antagonism and Plasticity of Regulatory and Inflammatoty T Cell Programs, Immunity 29:44-56 (2008). The reporter activity was normalized against renilla luciferase activity.

Co-activator Binding Assays. The effect of UA on the interaction of coactivator peptides with RORy were determined by Tb (terbium)-mediated time resolved fluorescence energy transfer assays using the LanthaScreen TR-FRET from Invitrogen. The experiments were conducted with 50 nM human RORy LBD-GST (amino acid 250-518) or RORa LBD- GST(amino acid 271 -523), 50nM Tb-anti-GST and 1 .5 μΜ Fluorescein co-activator peptide (GPQTPQAQQ SLLQQLLTE) containing LxxLL motif derived from SRC- 1 following the manufacturer's instructions. The binding signals were determined as the ratios of Emission 520 and Emission 495 and the results from three repeats were normalized relative to the binding in the absence of UA. All the reagents including LBD-GST were from Invitrogen. Binding curve was generated with Sigma-plot and followed the equation: y=min+(max- min)/[ l+( l/EC50)hillscope].

Experimental Autoimmune Encephalomyelitis (EAE). EAE was induced by immunizing mice (5 mice/group) twice with 300 g MOG35-55 peptide (amino acids 35-55; MEVGWYRSPFSROVHLYRNGK) emulsified in CFA followed by pertussis toxin injection, and analyzed as described. Yang, X.O., et al., T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORa and RORy, Immunity 28:29-39 (2008). The disease scores were assigned on a scale of 0-5 as follows: 0, none; 1 , limp tail or waddling gait with tail tonicity; 2, wobbly gait; 3, hindlimb paralysis; 4, hindlimb and forelimb paralysis; 5, death. When indicated, DMSO or UA was given to mice at a dose of -150 mg/Kg body weight by intraperitoneal (i.p.) injection every other day after first MOG immunization.

Calculations and Statistic Analysis. All our in vitro data were repeated for at least 2-5 times with consistent results. When indicated, the statistic significance was determined by student's t-test. (* represents P < 0.05; ** represents P< 0.03; *** represents P<0.01).

Results Ursolic Acid Inhibits Thl7 Differentiation. Human Thl7 differentiation system was used as the starting point to screen for Thl 7 inhibitors. We set up a high-throughput 96-well plate Thl 7 cultures in the presence of various compounds. Consistent with previous reports,TGF-beta, together with IL-Ι β, 1L-6 and IL-23 induced -6-12 % IL-17⁺ cells from human cord blood CD4⁺ naive T cells after 7 days' culture (data not shown). Manel, N., et al., The Differentiation of Human TH-17 Cells Requires Transforming Growth Factor-β and Induction of the Nuclear Receptor RORyt, Nat Immunol. 9:641-649 (2008); Volpe, E., et al., A Critical Function for Transforming Growth Factor-β, Interleukin 23 and Proinflammatory Cytokines in Driving and Modulating Human Tn-l 7 Responses, Nat Immunol. 9:650-657 (2008). After screening more than 2,000 known bioactive compounds, we identified UA as a Thl7 inhibitor. Figures 1A and I B show the effect of UA on human Thl 7 differentiation through intracellular staining (FIG. 1 A) and real time-RT PCR (normalized to GAPDH) (FIG. IB).

To confirm our above result, we performed in vitro T cell differentiation using naive CD4⁺ cells in the presence of different amounts of UA, and found that UA dose-dependently inhibited both human and mouse Th l 7 cell development and 2 μΜ UA inhibited >80% IL-17 expression in Thl 7 cells (FIGS. 1A, 1 C). This result was confirmed by real-time RT-PCR assays (FIG. I B, ID). Figures 1 C and ID show the effect of UA on mouse Thl7 cell differentiation through intracellular staining (FIG. 1C) and real-time-RT PCR (normalized to β- actin) (FIG. I D). The in vitro differentiation was repeated > 4 times and real-time RT PCR was repeated 2 times with consistent results. Interestingly, UA did not alter the mRNA level of RORa, RORyt, RUNX1 and IRF4 in Th l 7 cells (FIG. 1 & data not shown), which are known to be important transcription regulators in Thl 7 cytokine expression. Zhou, L., et al., Transcriptional Regulatory Networks in Thl 7 Cell Differentiation, Curr Opin Immunol. 21 : 146- 152 (2009). In addition, UA did not cause noticeable changes of IFN-γ, IL-4, or Foxp3 gene expression in human and mouse Thl , Th2 or iTreg cells, respectively (FIG. 5).

UA Selectively Inhibits the Function of RORy. UA has a similar structure to cholesterol and hydroxycholesterols -the putative ligands for ROR factors, suggesting that UA may target RORyt and RORa to inhibit Thl 7 cells. Jin, L., et al., Structural Basis for Hydroxycholesterols as Natural Ligands of Orphan Nuclear Receptor RORy, Mol Endocrinol. 24:923-929 (2010). To assess this, RORa or RORyt was retrovirally overexpressed in T cells differentiated under neutral condition. As previously reported, both RORa and RORyt induced significant amounts of IL-17 and IL-17F in non-polarized cells (FIG. 2A). Yang, X.O., et al., T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORa and ^ORy.Immunity 28:29-39 (2008). 2 μΜ UA strongly inhibited RORyt- but not RORa-mediated IL- 17 and IL-17F expression to almost background level.Yang, X.O., et al., T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORa and RORy, Immunity 28:29-39 (2008).

We have previously identified CNS2 as a cis-regulatory element that enhances the 1117 promoter activity in a ROR-dependent manner. Yang, X.O., et al., T Helper 17 Lineage Differentiation Is Programmed by Orphan Nuclear Receptors RORa and RORy, Immunity 28:29-39 (2008). RORyt-induced CNS2-I117p reporter activity was abolished by 2 μΜ UA whereas the RORa-dependent reporter activity was not affected (FIG. 6). In addition, we also examined the effect of UA in non-lymphoid cells by measuring the RORE reporter activity in 293 T cells. Yang, X.O., et al., Molecular Antagonism and Plasticity of Regulatory and Inflammatory T Cell Programs, Immunity 29:44-56 (2008). The results again demonstrate that UA selectively suppressed the function of RORyt but not RORa (FIG. 2B).

Inhibitory Kinetics of UA on RORyt and Thl7 Cells. The transcriptional activity of

RORyt is regulated by its co-activators, such as SRC (steroid receptor co-activator) through binding by the LxxLL motif. Xie, H., et al., RORyt Recruits Steroid Receptor Coactivators to Ensure Thymocyte Survival, J Immunol. 175:3800-3809 (2005). To determine potency of UA, we examined the effect of UA on the interaction of RORyt ligand binding domain (LBD) with an LxxLL motif-containing peptide derived from SRC 1. Mouse naive CD4⁺ T cells were differentiated under neutral condition, and infected with RORa, RORyt or control pMIG viruses on Day 1. 2 μΜ UA or DMSO was added 6 hrs after viral infection. The cells were re-stimulated on Day 4 for intracellular staining (Gated on hCD2⁺ cells) (FIG. 2A). 293 T cells were transfected with the RORE reporter together with RORa, RORyt or control plasmids. UA or DMSO was added after transfection and the cells were harvested next day for dual-luciferase activity assays. The data were normalized to an internal control renilla luciferase (FIG. 2B). The dose-dependent inhibitory results of UA on RORyt/RORa binding to its co-activator peptide or on Thl 7 differentiation were fitted to a sigmoidal dose response curve to determine the corresponding IC50 values (FIG. 2C). X-axis: log concentration (nM) of UA. Y-axis: relative binding of RORyt/RORa to its co-activator peptide, or relative percentage (%) of IL-17⁺ cells. All the assays were repeated at least 2 times with consistent results.

The results showed that UA dose-dependently inhibited the binding of RORyt-LBD to the "LxxLL" co-activator peptide (FIG. 2C). Consistent with the results of retroviral overexpression and reporter gene assays, UA did not inhibit the binding of RORa-LBD to the coactivator peptide (FIG. 2C), suggesting UA as a RORyt-specific antagonist. By fitting the inhibitory binding data to a sigmoidal dose response curve, the IC50 (half maximal inhibitory concentration) of UA to RORyt was determined to be 0.68 ± 0.1 μΜ. Using the same method, the IC50 of UA on Th l 7 cells was determined to be 0.56 ± 0.1 μΜ (Fig. 2C). The similar IC50 values further suggest RORyt as the direct target of UA in Thl 7 cells.

UA Inhibits IL-17 Expression in Mature Thl7 Cells. Our data thus far established UA as a RORyt-specific inhibitor in Thl 7 cell differentiation. To assess whether UA can inhibit the production of IL-17 in mature Thl 7 cells - which is more important in clinical settings, we first generated mature human and mouse Th l7 cells from naive CD4⁺ T cells. After pre-incubation with UA, the mature Thl 7 cells were then restimulated with plate-bound anti-CD3 overnight for cytokine secretion measurements. Specifically, after pre-incubation with UA, the mature human Thl 7 cells (FIG. 3 A) or mouse Thl7 cells (FIG. 3B) generated in vitro were then re-stimulated with anti-CD3 overnight for secreted cytokine analysis in the presence of indicated amounts of UA. Both human and mouse experiments were repeated 2-3 times with consistent results. The results demonstrated that UA indeed inhibited the secretion of IL-17 from differentiated Thl7 cells of both human and mouse sources (FIG. 3).

UA Ameliorated MOG-induced EAE in Mice. To investigate the therapeutic potential of UA in Th l 7-mediated autoimmune diseases, we examined the effect of UA on MOG-induced EAE in mice, and found that UA treatment not only delayed the onset of disease in mice (FIG. 4A), but also significantly ameliorated disease symptom (FIG. 4B) than the control group.

For EAE induction, the mice (n=4 or 5) were given either DMSO or UA by i.p. injection every other day after 1^st MOG-immunization and monitored daily for clinic symptom development after 2^nd MOG-immunization. Results are combination of two independent EAE experiments and the disease onset date of DMSO treated mice was set to Day 1 for statistic analysis. FIG. 4A shows the percentage of mice that developed EAE disease. Figure 4B shows the clinic scores of diseased mice. Figure 4C shows the total number of CD4⁺, IL-17⁺, IFN-y⁺ cells in the central nerve system of EAE mice. Figure 4D shows the effect of UA on MOG- specific IL-17 production in the spleens of EAE mice.

Consistently, the UA treated mice contained significantly less IL-17⁺ cells in their central nerve system as well as less IFN-y⁺ cells (FIG. 4C). Furthermore, in both MOG-immunized mice (data not shown) and EAE mice (FIG. 4D), UA treatment also caused a reduction in IL- 17production in the spleen. These data suggest that UA may be used in treatment of Th l 7- mediated inflammatory diseases.

UA contains many pharmacological activities, including strong hepatoprotective, antitumor and anti-inflammation effects partly through targeting NF-κΒ and STAT3. Liu, J. Pharmacology of Oleanolic Acid and Ursolic Acid, J Ethnopharmacol. 49:57-68 (1995); Ikeda, Y., et al., Ursolic Acid: An And- and Pro-Inflammatory Triterpenoid, Mol Nutr Food Res. 52:26- 42(2008); Huang, H.C., et al., Ursolic Acid Inhibits IL-Ιβ or TNF-a-Induced C6 Glioma Invasion Through Suppressing the Association ZIP/p62 with PKC-ζ and Downregulating the MMP-9 Expression, Mol Carcinog. 48:517-531 (2009); Shishodia, S., et al., Ursolic Acid Inhibits Nuclear Factor-κΒ Activation Induced by Carcinogenic Agents through Suppression of ΙκΒα Kinase and p65 Phosphorylation: Correlation with Down-Regulation of Cyclooxygenase 2, Matrix Metalloproteinase 9, and Cyclin Dl, Cancer Res. 63:4375-4383 (2003); Pathak, A. ., et al., Ursolic Acid Inhibits STAT3 Activation Pathway Leading to Suppression of Proliferation and Chemosensitization of Human Multiple Myeloma Cells, Mol Cancer Res. 5:943-955 (2007).

In Thl 7 cells, we found that UA did not affect the expression of STAT3 downstream targets, such as IL-21 and RORyt, indicating STAT3 is not the target in Thl7 cells. To confirm this, we performed a western blot to check the activation of STAT3 in Th l 7 cells and found that 2 μΜ UA did not have any effect on IL-6 induced STAT3 phosphorylation (data not shown). Moreover, UA has a relatively high IC₅o value for STAT3 and NF-tcB and inhibits these two molecules only when used at 25 μΜ or above, which is at least 30 fold higher than the IC50 value of UA for Thl7 cells (0.56 ± 0.1 uM) and RORyt (0.68 ± 0.1 μΜ), further excluding them from the targets of UA in Thl7 cells. Shishodia, S., et al., Ursolic Acid Inhibits Nuclear Factor-κΒ Activation Induced by Carcinogenic Agents through Suppression of ΙκΒα Kinase and p65 Phosphorylation Correlation with Down-Regulation of Cyclooxygenase 2, Matrix Metal proteinase 9, and Cyclin DI, Cancer Res. 63:4375-4383 (2003); Pathak, A. ., et al., Ursolic Acid Inhibits STAT3 Activation Pathway Leading to Suppression of Proliferation and Chemosensitization of Human Multiple Myeloma Cells, Mol Cancer Res. 5:943-955 (2007); Lauthier, F., et al., Ursolic Acid Triggers Calcium-Dependent Apoptosis in Human Daudi Cells, Anticancer Drugs 1 1 :737-745 (2000). In addition, we also exclude the possibility that UA may inhibit Thl7 cells through inducing apoptosis (data not shown), as reported in other cells.Ikeda, Y., et al., Ursolic Acid: An Anti- and Pro-Inflammatory Triterpenoid, Mol Nutr Food Res. 52:26- 42 (2008); Lauthier, F., et al., Ursolic Acid Triggers Calcium-Dependent Apoptosis in Human Daudi Cells, Anticancer Drugs 1 1 :737-745 (2000).

As a natural small molecule ubiquitously present in plants and even human diets, UA is relatively non-toxic and is well tolerated orally and topically in both human and rodents. The acute toxicity (LD50) of UA in rodents was determined to be >637 mg/kg for intraperitoneal injection and 8330 mg/kg for oral administration. Lee, A.W., et al., Ursolic Acid Induces Allograft Inflammatory Factor- 1 Expression via a Nitric Oxide-Related Mechanism and Increases Neovascularization, J Agric Food Chem. 58: 12941- 12949 (2010). In addition, UA has been identified as a major effective component in many medical herbs which have a long history in clinic practice in ancient China and Asian countries. Liu, J. Pharmacology of Oleanolic Acid and Ursolic Acid, J Ethnopharmacol. 49:57-68 (1995). Due to its important pharmacological activities, UA has been used for treatment of liver diseases and skin cancer. Liu, J. Pharmacology of Oleanolic Acid and Ursolic Acid, J Ethnopharmacol. 49:57-68 (1995). These clinical practices and its relatively low toxicity provide UA a great advantage over other ROR inhibitors reported in this month in developing therapeutics against Thl 7-mediated autoimmune diseases.Solt, L.A., et al., Suppress ion of TH17 Differentiation and Autoimmunity by a Synthetic ROR Ligand, Nature 472:491 -494 (201 1); Huh, J. R., et al., Digoxin and its Derivatives Suppress T_H17 Cell Differentiation by Antagonizing RORyt Activity, Nature 472:486-490 (201 1). Considering the broad functionality of the Thl 7 cell, particularly in inflammatory diseases and cancer, the therapeutic effects of UA and its analogs as described herein via inhibition of RORyt and ultimately Thl 7 cell function can serve a wide variety of therapeutic applications.

Claims

CLAIMS We claim:

1. A method of inhibiting RORyt function comprising the step of administering to a patient in need thereof a therapeutically effective amount of UA or an UA analog of Example 1 to 19.

2. A method of treating multiple sclerosis comprising the step of administering to a patient in need thereof a therapeutically effective amount of UA or an UA analog of Example 1 to Example 19,

3. A method of inhibiting IL-17 expression comprising the step of administering to a patient in need thereof a therapeutically effective amount of UA or an UA analog of Examples 1 to 19.

4. A method of inhibiting Thl7 cell differentiation comprising the step of administering to a patient in need thereof a therapeutically effective amount of UA or an UA analog of Examples 1 to 19.