Methods for treating endometriosis

Abstract

The present invention describes methods of treating endometriosis by modulating nuclear receptor subfamily 4 group A member 1 (NR4A1) activity, the methods comprising administering to an individual in need thereof a NR4A1 ligand. In one embodiment, the compound comprises a methylene substituted diindolylmethane.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 981,431, filed on February 25, 2020, the entire contents of which are incorporated herein by reference.

[0003] Government Licensing Statement

[0004] This invention was made with government support under Grant P30-ES029067 awarded by the National Institutes of Health. The government has certain rights in this invention. Background Art

[0005] Endometriosis is a common but complex inflammatory disease that primarily affects women of childbearing age, with an estimated 5,500,000 women in the United States and 176,000,000 women worldwide experiencing symptoms of endometriosis. Endometriosis develops when endometrial cells implant in distal sites, which may include the pelvic region, peritoneal surfaces, ovaries, ligaments, bowel, and bladder. Endometriosis results in part from retrograde menstrual bleeding; the resulting endometriotic lesions are variable in severity and pain, as well as overall short-term or long-term adverse health effects. Once diagnosed, the stage of the disease (i.e., stages I-IV) and its location are important in determining appropriate treatment options, which may include surgical removal of endometriotic tissue and hormonal therapy, including progesterone, oral contraceptives, and GnRH antagonists. The use of hormonal therapy to treat endometriosis in women of childbearing age is problematic, and there is a need to develop less toxic, hormone-independent treatments.

[0006] There remains a need for a treatment approach that avoids or reduces the significant side effects of conventional hormonal therapy for endometriosis.

[0007] Signaling pathways activated in endometriosis are similar to those observed in cancer and include inflammation-mediated responses associated with macrophage recruitment and activation, activation of pro-growth and survival genes / pathways, and angiogenic / pro-migration genes / pathways. For example, mTOR signaling is activated in endometriosis. However, the use of mTOR inhibitors to treat this disease is limited by toxicity issues.

[0008] Orphan nuclear receptors: Nuclear receptor subfamily 4 group A member 1 (NR4A1) (such as according to SEQ ID NO.1), nuclear receptor subfamily 4 group A member 2 (NR4A2) and nuclear receptor subfamily 4 group A member 3 (NR4A3) are immediate early genes induced by a variety of stressors, and NR4A receptors play an important role in maintaining cellular homeostasis and disease. Increasing evidence suggests a role for these receptors in metabolism, cardiovascular and neurological function, as well as in inflammation and inflammatory diseases, as well as in immune function and cancer. NR4A1 regulates cancer cell proliferation, survival, cell cycle progression, migration and invasion in lung, melanoma, pancreatic, colon, cervical, ovarian and gastric cancers, and rhabdomyosarcoma cell lines, and these responses are inhibited by bisindole-derived NR4A1 ligands that act as antagonists in cancer cells.

[0009] There is also evidence that NR4A1 is overexpressed in endometriosis, and it has been reported that levels of this receptor were significantly reduced after ultrasound-guided ethanol sclerotherapy in patients with high NR4A1 serum expression. Phosphorylated NR4A1 levels are higher in ovarian endometriotic tissue compared with normal endometrium; moreover, loss of NR4A1 stimulates fibrosis, and activation of NR4A1 by the NR4A1 agonist cytosporoneβ suggests that this receptor protects against fibrosis. Ishikawa endometrial carcinoma cells are frequently used as a model of endometriotic epithelial cells, and our recent studies clearly demonstrated that NR4A1 exhibits pro-endometriotic activity that is inhibited by NR4A1 antagonists. Summary of the invention

[0010] The present disclosure provides such methods of treating endometriosis, along with other related advantages. In this regard, the present disclosure demonstrates that NR4A1 regulates multiple pro-endometriosis genes / pathways that are inhibited by NR4A1 antagonists, and in one aspect, provides a method of treating endometriosis in an individual by modulating nuclear receptor subfamily 4 group A member 1 (NR4A1) activity, the method comprising administering to the individual a therapeutically effective amount of a compound of the present disclosure, such as an NR4A1 ligand.

[0011] In one embodiment, the NR4A1 ligand has the formula:

[0012]

[0013] or a salt thereof,

[0014] in,

[0015] R1, R2, R1' and R2' are each independently selected from hydrogen, a straight chain alkyl group containing one to about ten carbon atoms, and a branched chain alkyl group containing one to about ten carbon atoms;

[0016] R3, R4, R5, R6, R3', R4', R5' and R6' are each independently selected from hydrogen, halogen, a straight chain alkyl group containing one to about ten carbon atoms, a branched chain alkyl group containing one to about ten carbon atoms, an alkoxy group containing one to about ten carbon atoms, and a nitro group;

[0017] R7 is selected from hydrogen, a straight chain alkyl group containing one to about ten carbon atoms, a branched chain alkyl group containing one to about ten carbon atoms, a cycloalkyl group containing one to about ten carbon atoms, and an aryl group;

[0018] R8, R9, R 10 , R 11 and R 12 independently selected from H, halogen, a straight chain alkyl group containing one to about ten carbon atoms, a branched chain alkyl group containing one to about ten carbon atoms, an alkoxy group containing one to about ten carbon atoms, a haloalkyl group containing one to about ten carbon atoms, a nitro group, a hydroxyl group, and a haloalkoxy group containing one to about ten carbon atoms;

[0019] Among them, R8, R9, R 10 , R 11 and R 12 At least one of is OH, and

[0020] When R 10 When it is OH, R8, R9, R 11 and R 12 At least one of the is not hydrogen.

[0021] This summary is provided to introduce some concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key features of the claimed subject matter, nor is it intended to be used as an aid in determining the scope of the claimed subject matter. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The foregoing aspects of the claimed subject matter and many of the attendant advantages will become more readily appreciated as the claimed subject matter is better understood by reference to the following detailed description when taken in conjunction with the accompanying drawings, wherein:

[0023] Figure 1A-Figure 1D The effect of NR4A1 knockdown in endometriotic cells is shown, wherein ESECT-7 and ESECT-40 cells were transfected with oligonucleotides targeting downregulation of NR4A1 [siNR4A1(1) and siNR4A1(2)], and the effect on cell proliferation ( Figure 1A ), NR4A1 expression ( Figure 1B ), growth promotion ( Figure 1C ) and pro-apoptotic ( Figure 1D ) The effects of gene products;

[0024] Figure 1E shows the effect of NR4A1 knockout on Annexin V staining according to one embodiment of the present disclosure;

[0025] Figure 2A and Figure 2B The effects of NR4A1 ligands on transactivation and growth in endometriotic cells are shown, wherein ESECT-7 ( Figure 2A ) or ESECT-40( Figure 2B ) cells were transfected with UAS-luc / GAL4-Luc or NBRE-luc / flag-NR4A1 and treated with DIM-C-pPhOH(OH), DIM-C-pPhOH-3,5-Br2(4-OH-3,5-Br2), DIM-C-pPhOH-3-Cl-5-OCH3(4-OH-3-Cl-5-OCH3), or DIM-C-pPhOH-3-Cl(4-OH-3-Cl);

[0026] Figure 2C and Figure 2D The effect of DIM-C-pPhOH and DIM-C-pPhOH-3-Cl-5-OCH3 on cell growth ( Figure 2C ) and growth-promoting gene products ( Figure 2D )

[0027] Figure 3A-3C shows how NR4A1 antagonists induce apoptosis in endometriotic cells according to one embodiment of the present disclosure, wherein ESECT-7 ( Figure 3A ) and ESECT-40( Figure 3B ) were treated with NR4A1 antagonists and Annexin V staining was determined by fluorescence, where ESECT-7 and ESECT-40 cells were treated with NR4A1 ligands for 24 h and whole cell lysates were analyzed by Western blotting for pro-apoptotic gene products ( Figure 3C );

[0028] Figure 4A-4D The effect of NR4A1 ligand on normal endometrial (NEM) cells (where NEM cells were treated with NR4A1 ligand for 24 hours) and on cell growth ( Figure 4A ), growth-promoting genes ( Figure 4B ), Annexin V staining ( Figure 4C ) and pro-apoptotic gene products ( Figure 4D )

[0029] Figure 5A and Figure 5B The effect of NR4A1 on endometriotic cell fibrosis according to one embodiment of the present disclosure is shown, wherein ESECT-7 cells are treated with a NR4A1 antagonist ( Figure 5A ) or transfected with siNR4A1(1) or siNR4A1(2) ( Figure 5B );

[0030] Figure 5C A graphical representation of the effect on gene expression of fibrosis markers in ESECT-7 cells treated with NR4A1 ligand for 24 hours according to one embodiment of the present disclosure;

[0031] Fig. 6A and Figure 6B According to one embodiment of the present disclosure, NR4A1 antagonists and receptor knockouts were transfected with siNR4A1 oligonucleotides ( Fig. 6A ) or treated with NR4A1 antagonist for 24 h ( Figure 6B ) of IHEEC cells;

[0032] Figure 6C and Fig.6D According to one embodiment of the present disclosure, NR4A1 antagonists and receptor knockouts were transfected with siNR4A1 oligonucleotides ( Figure 6C ) or treated with NR4A1 antagonist for 24 h ( Fig.6D )'s Ishikawa cells;

[0033] Figure 7A-7C The normal mouse uterus ( Fig. 7A ), eutopic endometrium ( Figure 7B ) and ectopic lesions ( Figure 7C ) in Nr4a1 expression;

[0034] Fig.7D and Fig. 7E Epithelial cells of the uterus, eutopic endometrium, and ectopic lesions are shown ( Fig.7D ) and matrix ( Fig. 7E ) The IHC signal density of Nr4a1 in

[0035] Fig. 8A Schematically illustrates a treatment method and a control method according to an embodiment of the present disclosure;

[0036] Figure 8B and Figure 8C are images of ectopic lesions isolated from mice with endometriosis treated with vehicle (Panel B) and C-DIM (Panel C) according to one embodiment of the present disclosure;

[0037] Fig.8D graphically illustrating the volume of isolated ectopic lesions treated with vehicle versus C-DIM according to one embodiment of the present disclosure;

[0038] Fig. 8E graphically showing body weight of mice treated with vehicle versus C-DIM according to one embodiment of the present disclosure;

[0039] Fig. 9A Schematically illustrating an experiment for measuring the inhibition of the growth of human ectopic lesions in SCID mice by DIM-C-pPhOH-3-Cl-5-OCH3 (C-DIM) according to one embodiment of the present disclosure, wherein the human ectopic lesions were generated by injecting human endometrial cells expressing luciferase into SCID mice, and at week 3 after endometriosis induction, the mice were randomly divided into two groups, wherein one group of mice was treated with 25 mg / kg of C-DIM (once a day) and the mice in the other group were treated with vehicle (once a day) for 30 days according to one embodiment of the present disclosure;

[0040] Fig. 9B shows luciferase activity of human ectopic lesions measured at 4, 7, 16, 23 and 30 days after drug treatment according to one embodiment of the present disclosure; and

[0041] Fig. 9C A diagram is shown according to an embodiment of the present disclosure, based on Fig. 9B Luciferase activities shown in , relative folds of luciferase activity compared to drug treatment on day 0. DETAILED DESCRIPTION

[0042] Endometriosis is an inflammatory disease that primarily affects women of childbearing age, and new hormone-independent treatment options are needed because current hormone therapies are of concern. Orphan nuclear receptor 4A1 (NR4A1, Nur77), such as according to SEQ ID NO.1, is expressed in patient-derived (stromal) endometriotic cells as well as epithelial cell lines, and the present disclosure demonstrates that knockout of NR4A1 in patient-derived ESECT-7 and ESECT-40 cells reduces cell proliferation and induces apoptosis. In addition, treatment of these cells with the bisindole-derived NR4A1 ligand 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH) and its supported 3-chloro-5-methoxy analog (DIM-C-pPhOH-3-Cl-5-OCH3) inhibits cell growth and induces apoptosis and related genes. The compounds of the present disclosure exhibit NR4A1 antagonist activity in both functional and transactivation assays, while these effects were not observed in normal (NEM) endometrial cells. The present disclosure also demonstrates that NR4A1 knockout and treatment with NR4A1 antagonists reduce fibrosis, α-smooth muscle actin (α-SMA) and related profibrotic genes in ESECT-7 and ESECT-40 cells, and similar results are observed in endometriotic cell lines derived from epithelial cells. In addition, in an endometriosis mouse model with autologous transplantation, and also in SCID mice transplanted with human endometriotic cells, treatment with 25 mg / kg / day DIM-C-pPhOH-3-Cl-5-OCH3 significantly inhibited the growth and expansion of endometriotic lesions. Therefore, the present disclosure demonstrates that bisindole-derived NR4A1 ligands are a new class of drugs for non-hormonal therapy of endometriosis.

[0043] Thus, in one aspect, the present disclosure provides methods for treating endometriosis. In one embodiment, these methods include treating endometriosis in an individual that can be treated by modulating NR4A1 activity, which includes administering to the individual a therapeutically effective amount of a compound, such as an NR4A1 ligand. In one embodiment, the compound is a methylene-substituted diindolylmethane. In certain embodiments, compounds according to the present disclosure are variously referred to as "CDIM" or "C-DIM" compounds. As further discussed herein, in certain embodiments, such compounds are methylene-substituted diindolylmethane compounds, such as the chemical formula according to embodiments of the present disclosure.

[0044] In some embodiments, modulating NR4A1 activity comprises binding a compound described elsewhere herein to an NR4A1 protein. In some embodiments, the compound has antagonistic activity, i.e., the compound has reduced efficacy or no efficacy in stimulating the associated function of a receptor (e.g., an antagonist ligand). In some embodiments, an antagonist ligand blocks the constitutive function of a receptor and the ability of its stimulatory associated ligand to bind to the NR4A1 protein and activate NR4A1-dependent genes. In some embodiments, the NR4A1 ligand may be a tissue-specific, response-specific, or gene-specific agonist. In one embodiment, the NR4A1 ligand is effective in endometriotic cells of an individual. Such endometriotic cells are contrasted with normal endometriotic (NEW) cells of an individual.

[0045] The term "antagonist" refers to a compound that can combine with the NR4A1 receptor to reduce or inhibit the activity of molecules and cells. Antagonists can be ligands that bind directly to the receptor. Alternatively, antagonists can combine indirectly with the receptor, for example by (a) forming a complex with another molecule or protein that binds directly to the receptor, or (b) otherwise causing modification of another compound so that the other compound binds directly to the NR4A1 receptor.

[0046] The term "agonist" refers to a compound that can combine with the NR4A1 receptor to produce or increase molecular and cellular activity. An agonist can be a ligand that binds directly to the receptor. Alternatively, an agonist can combine indirectly with the receptor, for example by (a) forming a complex with another molecule or protein that binds directly to the receptor, or (b) otherwise causing modification of another compound so that the other compound binds directly to the NR4A1 receptor.

[0047] The term "activation" and variations thereof refer to any measurable increase in molecular and cellular activity.

[0048] In one embodiment, the cell is contacted with the compound or pharmaceutical composition in vitro. In one embodiment, the cell is contacted with the compound or pharmaceutical composition in vivo by administering an effective amount of the compound or pharmaceutical composition to a subject.

[0049] In one embodiment, modulation of NR4A1 induces downregulation of a protein selected from the group consisting of EFGR, cMyc, survivin, Bcl-2, SMA, and combinations thereof. Figure 1C , Figure 1D , Figure 2D and Figure 3CAs shown, the compounds of the present disclosure are suitable for inducing downregulation of EFGR, cMyc, survivin, Bcl-2, such as in endometriotic cells and relative to a control when administered to a subject. In one embodiment, EFGR is according to SEQ ID NO.2. In one embodiment, cMyc is according to SEQ ID NO.3. In one embodiment, survivin is according to SEQ ID NO.4. In one embodiment, Bcl-2 is according to SEQ ID NO.5. As shown in Examples 4 and Figure 6B As shown, the compounds of the present disclosure are suitable for inducing down-regulation of SMA, such as in endometrial cells and relative to a control. In one embodiment, SMA is according to SEQ ID NO.6.

[0050] In one embodiment, modulation of NR4A1 activity induces upregulation of proteins including Bax. Figure 1C , Figure 1D , Figure 2D and Figure 3C As shown, the compounds of the present disclosure are suitable for inducing downregulation of Bax when administered to a subject, such as in endometriotic cells and relative to a control. In one embodiment, Bax is according to SEQ ID NO.7.

[0051] In one embodiment, modulation of NR4A1 activity induces a decrease in the mRNA level of a fibrosis marker selected from FN, Col1A1, CTGF, and a combination thereof. Figure 6B As shown, the compound is suitable for inducing a decrease in mRNA levels of fibrosis markers such as FN, Col1A1, CTGF when administered to a subject, such as in endometrial cells and relative to a control. In one embodiment, FN is according to SEQ ID NO. 8. In one embodiment, Col1A1 is according to SEQ ID NO. 9. In one embodiment, CTGF is according to SEQ ID NO. 10.

[0052] In one embodiment, modulation of NR4A1 activity cleaves PARP and caspase-3. As shown, compounds according to the present disclosure are suitable for inducing cleavage of PARP and caspase-3 when administered to a subject, such as in endometrial cells and relative to a control. In one embodiment, PARP is according to SEQ ID NO. 11. In one embodiment, caspase-3 is according to SEQ ID NO. 12.

[0053] Although specific embodiments of proteins are described, it will be understood that other homologs or isoforms of these proteins are also within the scope of the present disclosure.

[0054] In one embodiment, modulation of NR4A1 activity induces apoptosis in ovarian endometrioma.

[0055] In one embodiment, modulation of NR4A1 activity does not inhibit the growth of normal endometrial cells relative to a control.

[0056] In one embodiment, modulation of NR4A1 activity inhibits the progression of fibrosis, such as relative to a control.

[0057] In one embodiment, modulation of NR4A1 activity inhibits the intrinsic transcriptional activity of NR4A1 in endometriotic cells relative to a control. In one embodiment, modulation of NR4A1 activity inhibits the growth of ectopic lesions relative to a control.

[0058] In one embodiment, the NR41 ligand of the present disclosure is according to the formula:

[0059]

[0060] or a salt thereof,

[0061] in,

[0062] R1, R2, R1' and R2' are each independently selected from hydrogen, a straight chain alkyl group containing one to about ten carbon atoms, and a branched chain alkyl group containing one to about ten carbon atoms;

[0063] R3, R4, R5, R6, R3', R4', R5' and R6' are each independently selected from hydrogen, halogen, a straight chain alkyl group containing one to about ten carbon atoms, a branched chain alkyl group containing one to about ten carbon atoms, an alkoxy group containing one to about ten carbon atoms, and a nitro group;

[0064] R7 is selected from hydrogen, a straight chain alkyl group containing one to about ten carbon atoms, a branched chain alkyl group containing one to about ten carbon atoms, a cycloalkyl group containing one to about ten carbon atoms, and an aryl group;

[0065] R8, R9, R 10 , R 11 and R 12 independently selected from H, halogen, a straight chain alkyl group containing one to about ten carbon atoms, a branched chain alkyl group containing one to about ten carbon atoms, an alkoxy group containing one to about ten carbon atoms, a haloalkyl group containing one to about ten carbon atoms, a nitro group, a hydroxyl group, and a haloalkoxy group containing one to about ten carbon atoms;

[0066] Among them, R8, R9, R 10 , R 11 and R 12At least one of is OH, and

[0067] When R 10 When it is OH, R8, R9, R 11 and R 12 At least one of the is not hydrogen.

[0068] As will be understood by those skilled in the art, chemical moieties referred to as monovalent chemical moieties (e.g., alkyl, aryl, etc.) also include structurally permitted multivalent moieties. For example, while an "alkyl" moiety generally refers to a monovalent group (e.g., CH3CH2-), an "alkyl" moiety may also refer to a divalent group (e.g., -CH2CH2-, which is equivalent to an "alkylene" group) in appropriate circumstances. Similarly, where a divalent moiety is desired, those skilled in the art will understand that the term "aryl" refers to the corresponding divalent arylene group.

[0069] As used herein, the terms may be preceded and / or followed by a single dash "-" or a double dash "=" to indicate the bond order of the bond between the specified substituent and its parent moiety; a single dash indicates a single bond, and a double dash indicates a double bond. In the absence of a single dash or a double dash, it is understood that a single bond is formed between the substituent and its parent moiety; furthermore, unless otherwise indicated by a dash, the substituents are intended to be read "from left to right." For example, C1-C6 alkoxycarbonyloxy and -OC(O)C1-C6 alkyl indicate the same functionality; similarly, arylalkyl and -alkylaryl indicate the same functionality.

[0070] All atoms are understood to have their normal valence numbers for forming bonds (e.g., carbon is 4, N is 3, O is 2, and S is 2, 4, or 6 depending on the oxidation state of the atom). Sometimes, a moiety may be defined as, for example, (A) a B, wherein a is 0 or 1. In such cases, when a is 0, the moiety is B, and when a is 1, the moiety is AB.

[0071] When the number of atoms or groups of the same type in a substituent can vary (e.g., an alkyl group can be C1, C2, C3, etc.), the number of repeating atoms or groups can be represented by a range (e.g., C1-C6 alkyl), which includes every number in the range and any and all subranges. For example, C1-C3 alkyl includes C1, C2, C3, C 1-2 , C 1-3 and C 2-3 alkyl.

[0072] Unless otherwise indicated, the term "alkyl" as used herein means a straight or branched hydrocarbon containing 1 to 10 carbon atoms. Representative examples of alkyl include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, and n-decyl. When an "alkyl" group is a linking group between two other moieties, it may also be straight or branched; examples include, but are not limited to -CH2-, -CH2CH2-, -CH2CH2CHC(CH3)-, -CH2CH(CH2CH3)CH2-.

[0073] The term "cycloalkyl" as used herein means a monocyclic or bicyclic cycloalkyl ring system. A monocyclic ring system is a cyclic hydrocarbon group containing 3 to 8 carbon atoms, wherein such groups may be saturated or unsaturated, but not aromatic. In certain embodiments, the cycloalkyl group is fully saturated. Examples of monocyclic cycloalkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cycloheptyl, and cyclooctyl. A bicyclic cycloalkyl ring system is a bridged monocyclic or fused bicyclic ring. The bridged monocyclic ring contains a monocyclic cycloalkyl ring, wherein two non-adjacent carbon atoms of the monocyclic ring are connected by an alkylene bridge having an additional carbon atom between one and three (i.e., in the form of -(CH2) w -, wherein w is 1, 2 or 3).

[0074] "Alkoxy" means an alkyl group, as defined herein, attached to the parent molecular moiety through an oxygen atom. Representative examples of alkoxy include, but are not limited to, methoxy, ethoxy, propoxy, 2-propoxy, butoxy, tert-butoxy, pentoxy, and hexoxy.

[0075] The term "aryl" as used herein means phenyl (i.e., monocyclic aryl) or a bicyclic ring system containing at least one benzene ring or an aromatic bicyclic ring containing only carbon atoms in the aromatic bicyclic ring system. The bicyclic aryl can be azulenyl, naphthyl, or a phenyl fused to a monocyclic cycloalkyl, a monocyclic cycloalkenyl, or a monocyclic heterocyclic group. The bicyclic aryl is connected to the parent molecular moiety by any carbon atom contained in the phenyl portion of the bicyclic system, or any carbon atom in the naphthalene ring or the azulenyl ring. The fused monocyclic cycloalkyl or monocyclic heterocyclic moiety of the bicyclic aryl is optionally substituted by one or two oxy groups and / or the group.

[0076] "Halogen" refers to a chlorine, bromine, fluorine or iodine atom radical. The term "halogen" also contemplates the terms "halo" or "halide".

[0077] The terms "haloalkyl," "haloalkenyl," and "haloalkoxy" refer to an alkyl, alkenyl, or alkoxy group, as the case may be, substituted with one or more halogen atoms.

[0078] The term "nitro" as used herein refers to a -NO2 group.

[0079] The term "substituted" as used herein means that a hydrogen radical of a designated moiety is replaced with a radical of a designated substituent, provided that the substitution results in a stable or chemically feasible compound. The term "substitutable" when used with reference to a designated atom means that attached to the atom is a hydrogen radical, which can be replaced with a radical of a suitable substituent.

[0080] In another embodiment, the present disclosure relates to a method for treating endometriosis in an individual by regulating NR4A1, the method comprising administering a bisindole-derived compound as described in WO2021 / 022220 to a subject in need thereof. The disclosure of WO2021 / 022220 is hereby incorporated by reference herein for its disclosure. In some embodiments, the method further comprises binding to at least one of nuclear receptor 4A1 (NR4A1) and nuclear receptor 4A2 (NR4A2) by a bisindole-derived compound. In some embodiments, the bisindole-derived compound (CDIM) includes two or more substituents on its phenyl ring. In some embodiments, bisindole derived compounds include, but are not limited to, 1,1-bis(3'-indolyl)-1-(p-chlorophenyl)methane (DIM-C-pPhCl; 4-Cl), 1,1-bis(3'-indolyl)-1-(4-chloro-3-trifluoromethylphenyl)methane (3-CF3-4-Cl), 1,1-dimethyl-1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (N-Me-4-OH), 1,1-bis(3'-indolyl)-1-(4-bromo-2-hydroxy-phenyl)methane (2-OH-4-Br), 1-bis(3'-indolyl)-1-(p-bromophenyl)methane (DIM-C-pPhBr), 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (CDIM8), 3,5-disubstituted analogs of CDIM8, and CDIM9. 8-3,5-(CH3)2, CDIM8-3,5-Br2, CDIM8-3,5-Cl2, CDIM8-3-Br-5-OCH3, CDIM8-3-Cl-5-OCH3, CDIM8-3-Cl-5-Br, CDIM8-3-Cl-5-F, CDIM, 3,5-disubstituted analogs of CDIM, CDIM-3,5-Br2, CDIM-2,5-Br2, CDIM-3,5-Cl2, CDIM-3,5-(CH3)2, CDIM-3-Br-5-OCF3, CDIM-3-Br-5-OCH3, CDIM-3-Br-5-CF3, CDIM-3-Cl-5-OCH3, CDIM-3-Cl-5-OCF3, CDIM-3-Cl-5-CF3 and combinations thereof. In one embodiment, the bisindole derived compound is CDIM-3-Br-5-CF3.

[0081] 2-Hydroxy Ligand

[0082] In some embodiments, R8 is OH. In some cases, because the OH group is located on the central phenyl group, such ligands are referred to herein as "2-hydroxy" and / or "2-OH" ligands. In certain such embodiments, R9, R 10 , R11 and R 12 Each is H. In certain other embodiments, R9, R 10 , R 11 and R 12 is independently selected from halogen, CH3, OCCl3, CF3, tert-butyl, OCH3, OH, C6H5 and CN. In one embodiment, R 10 is OCH3. In one embodiment, R 11 In one embodiment, R9 and R 11 For Br.

[0083] In one embodiment, the composition of the present disclosure has one of the following structures:

[0084]

[0085]

[0086] and its salts.

[0087] 3-Hydroxy Ligand

[0088] In some embodiments, R9 is OH. In some cases, because the OH group is located on the central phenyl group, such ligands are referred to herein as "3-hydroxy" and / or "3-OH" ligands. In certain such embodiments, R8, R 10 , R 11 and R 12 Each is H. In certain other embodiments, R8, R 10 , R 11 and R 12 R8 is independently selected from halogen, CH3, OCCl3, CF3, tert-butyl, OCH3, OH, C6H5 and CN. In certain embodiments, R8 is halogen.

[0089] In one embodiment, the composition of the present disclosure has one of the following structures:

[0090]

[0091]

[0092] and its salts.

[0093] 4-Hydroxy Ligand

[0094] In some embodiments, R 10In some cases, because the OH group is located on the central phenyl group, such ligands are referred to herein as "4-hydroxy" and / or "4-OH" ligands. In certain such embodiments, R8, R9, R 11 and R 12 is independently selected from halogen, CH3, OCCl3, CF3, tert-butyl, OCH3, OH, C6H5 and CN. In certain other embodiments, R9 is halogen and R 11 Selected from H, halogen and OCH3.

[0095] In one embodiment, the composition of the present disclosure has one of the following structures:

[0096]

[0097] and its salts.

[0098] The C-DIM compounds disclosed herein can be prepared by the condensation of substituted benzaldehydes with indoles or substituted indoles. These compounds can be synthesized by incubating two portions of indoles or substituted indoles with one portion of benzaldehyde or substituted benzaldehyde in dilute acetic acid at 80°C-90°C for 24 hours-48 hours. The solids are recovered by filtration and crystallized from benzene or benzene / hexane to obtain C-DIM in 70%-90% yield. Using a single indole starting material will produce symmetrical products, while using two different indole starting materials will result in asymmetrical products.

[0099] The preparation and characterization of representative C-DIM compounds are described, for example, in US Pat. No. 7,232,843, which is incorporated herein by reference in its entirety.

[0100] A person of ordinary skill in the art will be able to determine the most effective dose and time for administering the composition, taking into account the route of delivery, metabolism of the compound and other pharmacokinetic parameters, such as volume of distribution, clearance rate, age of the subject, and the like. For example, a NR4A1 ligand such as a NR4A1 antagonist can be administered by any well-known method, such as by topical administration, oral administration, intravenous injection, intraperitoneal injection, intramuscular injection, intranasal administration, transdermal administration, rectal administration, or by any means of delivering an effective amount of the active agent to the tissue or site to be treated. A suitable dose is a dose that achieves the desired endpoint. It should be understood that different doses may be required to treat different disorders. An effective amount of an agent is, for example, an amount that causes a cessation or significant reduction in neoplastic cell count, growth, size, cell migration or cell invasion.

[0101] The composition can be administered with a pharmaceutical carrier and / or diluent. The agent can also be administered in combination with other agents, for example, in combination with other compounds suitable for treating endometriosis. Examples of pharmaceutical carriers or diluents that can be used in the present invention include any physiological buffer medium, i.e., a physiological buffer medium having a pH of about 7.0 to 7.4 and containing a suitable water-soluble organic carrier. Suitable water-soluble organic carriers include, but are not limited to, corn oil, dimethyl sulfoxide, gelatin capsules, and the like.

[0102] As used herein, the term "therapeutically effective amount" refers to an amount of a compound or composition sufficient to treat a particular disorder, condition, or disease such as to improve, alleviate, mitigate, and / or delay one or more of its symptoms. With reference to endometriosis or other unwanted cell proliferation, an effective amount includes an amount sufficient to cause a reduction in endometriotic lesions and / or reduce the growth rate of endometriotic lesions. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent occurrence and / or recurrence. An effective amount can be administered in one or more administrations.

[0103] "Pharmaceutically acceptable carriers" for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences, 18th edition (Easton, Pa.: Mack Publishing Company, 1990). For example, sterile saline and phosphate buffered saline at physiological pH can be used. Preservatives, stabilizers, dyes and even flavoring agents can be provided in the pharmaceutical composition. For example, sodium benzoate, sorbic acid and parabens can be added as preservatives. As described in the aforementioned document on page 1449. In addition, antioxidants and suspending agents can be used. As described in the aforementioned document.

[0104] Suitable excipients for non-liquid formulations are also known to those skilled in the art. A detailed discussion of pharmaceutically acceptable excipients and salts can be found in Remington's Pharmaceutical Sciences, 18th edition (Easton, Pa.: Mack Publishing Company, 1990).

[0105] In addition, auxiliary substances, such as wetting agents or emulsifiers, biological buffer substances, surfactants, etc. may be present in such solvents. The biological buffer may be any solution that is pharmacologically acceptable and provides the desired pH for the formulation, i.e., a pH within a physiologically acceptable range. Examples of buffer solutions include saline, phosphate buffered saline, Tris buffered saline, Hank buffered saline, etc.

[0106] Depending on the intended mode of administration, the pharmaceutical composition may be in the form of a solid, semisolid or liquid dosage form, such as, for example, tablets, suppositories, pills, capsules, powders, liquids, suspensions, creams, ointments, lotions, etc., preferably in a unit dosage form suitable for single administration of precise doses. These compositions will contain an effective amount of a selected drug in combination with a pharmaceutically acceptable carrier, and may also contain other agents, adjuvants, diluents, buffers, etc.

[0107] The present disclosure includes pharmaceutical compositions comprising a compound of the present disclosure, including isomers, racemic or non-racemic mixtures of isomers, or a pharmaceutically acceptable salt or solvate thereof, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic and / or prophylactic ingredients.

[0108] Typically, the compounds of the present invention will be administered in a therapeutically effective amount by any of the acceptable modes of administration. Suitable dosage ranges depend on a variety of factors, such as the severity of the disease to be treated, the age and relative health of the subject, the efficacy of the compound used, the route of administration and form, the indications for which administration is directed, and the preferences and experience of the relevant practitioners. Those of ordinary skill in the field of treating such diseases will be able to rely on personal knowledge and the disclosure of the application to determine the therapeutically effective amount of the compounds of the present invention for a given disease without undue experimentation.

[0109] Thus, the compounds of the present disclosure may be administered as pharmaceutical formulations, including those suitable for oral (including buccal and sublingual), rectal, nasal, topical, pulmonary, vaginal or parenteral (including intramuscular, intraarterial, intrathecal, subcutaneous and intravenous) administration, or in a form suitable for administration by inhalation or insufflation. The preferred mode of administration is intravenous or oral administration using a convenient daily dosage regimen, which can be adjusted according to the degree of affliction.

[0110] For solid compositions, conventional non-toxic solid carriers include, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. Liquid pharmaceutically administrable compositions can be prepared, for example, by dissolving and dispersing the active compounds described herein and optional pharmaceutical adjuvants in excipients such as, for example, water, saline, aqueous glucose solution, glycerol, ethanol, etc., to form a solution or suspension. If desired, the pharmaceutical composition to be administered may also contain a small amount of non-toxic auxiliary substances, such as wetting agents or emulsifiers, pH buffers, etc., such as sodium acetate, sorbitan monolaurate, triethanolamine sodium acetate, triethanolamine oleate, etc. The actual method of preparing such dosage forms is known or will be apparent to those skilled in the art, for example, see Remington's Pharmaceutical Sciences cited above.

[0111] Yet another embodiment is the use of permeation enhancer excipients including polymers such as: polycations (chitosan and its quaternary ammonium derivatives, poly-L-arginine, aminated gelatin); polyanions (N-carboxymethyl chitosan, polyacrylic acid); and thiolated polymers (carboxymethylcellulose-cysteine, polycarbophil-cysteine, chitosan-thiobutylamidine, chitosan-thioglycolic acid, chitosan-glutathione conjugates).

[0112] For oral administration, the composition is usually in the form of tablets, capsules, soft capsules, or can be aqueous or non-aqueous solutions, suspensions or syrups. Tablets and capsules are preferred oral administration forms. Tablets and capsules for oral use may include one or more commonly used carriers, such as lactose and corn starch. Lubricants such as magnesium stearate are also usually added. Generally, the compounds of the present disclosure can be combined with oral, non-toxic, pharmaceutically acceptable inert carriers such as lactose, starch, sucrose, glucose, methylcellulose, magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, sorbitol, etc. In addition, when necessary or necessary, suitable binders, lubricants, disintegrants and colorants can also be incorporated into the mixture. Suitable binders include starch, gelatin, natural sugars such as glucose or β-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth or sodium alginate, carboxymethyl cellulose, polyethylene glycol, wax, etc. Lubricants used in these dosage forms include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Disintegrants include, but are not limited to, starch, methyl cellulose, agar, bentonite, xanthan gum, etc.

[0113] Thus, for example, capsules can be prepared by conventional procedures so that the dosage unit is 100 mg of the compound of the present disclosure, 100 mg of cellulose, and 10 mg of magnesium stearate. A large number of unit capsules can also be prepared by filling standard two-piece hard gelatin capsules each with 100 mg of powdered active ingredient, 150 mg of lactose, 50 mg of cellulose, and 10 mg of magnesium stearate. Alternatively, tablets can be prepared by conventional procedures so that the dosage unit is 100 mg of the compound of the present disclosure, 150 mg of lactose, 50 mg of cellulose, and 10 mg of magnesium stearate. A large number of tablets can also be prepared by conventional procedures so that the dosage unit is 100 mg of the compound of the present disclosure, and the other ingredients can be 0.2 mg of colloidal silicon dioxide, 5 mg of magnesium stearate, 250 mg of microcrystalline cellulose, 10 mg of starch, and 100 mg of lactose. Appropriate coatings can be applied to increase palatability or delay absorption.

[0114] When a liquid suspension is used, the active agent can be combined with any oral, non-toxic, pharmaceutically acceptable inert carrier such as ethanol, glycerol, water, etc., as well as with an emulsifier and a suspending agent. If desired, flavoring agents, colorants, and / or sweeteners may also be added. Other optional components for incorporation into the oral formulations herein include, but are not limited to, preservatives, suspending agents, thickening agents, and the like.

[0115] Parenteral preparations can be in conventional form, either as liquid solutions or suspensions, in solid forms that can sink to dissolve or suspend in liquids before injection, or as emulsion preparations. Preferably, according to techniques known in the art, suitable carriers, dispersants or wetting agents and suspending agents are used to prepare sterile injectable suspensions. Sterile injectable preparations can also be sterile injectable solutions or suspensions in parenterally acceptable nontoxic diluents or solvents. Acceptable solvents and solvents that can be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils, fatty esters or polyols are generally used as solvents or suspension media. In addition, parenteral administration can involve the use of slow-release or slow-acting systems to maintain a constant dosage level.

[0116] Parenteral administration includes intra-articular, intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes, and includes aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions, these aqueous and non-aqueous isotonic sterile injection solutions may contain antioxidants, buffers, antibacterial agents and solutes that make the preparation isotonic with the blood of the intended recipient, these aqueous and non-aqueous sterile suspensions may include suspending agents, solubilizers, thickeners, stabilizers and preservatives. Administration via some parenteral routes may involve the introduction of the preparation of the present invention into the patient's body by a needle or catheter driven by a sterile syringe or some other mechanical devices such as a continuous infusion system. The preparation provided by the present disclosure may be administered by a needle, a syringe, a pump or any other device recognized in the art for parenteral administration.

[0117] Preferably, according to technology known in the art, use suitable carrier, dispersant or wetting agent and suspending agent to prepare sterile injectable suspension.Sterile injectable preparation can also be sterile injectable solution or suspension in parenterally acceptable nontoxic diluent or solvent.Acceptable solvent and solvent that can be used are water, Ringer's solution and isotonic sodium chloride solution.In addition, sterile fixed oil, fatty ester or polyol are usually used as solvent or suspension medium.In addition, parenteral administration can involve the use of slow release or slow-acting system to maintain constant dosage level.

[0118] Preparations according to the disclosure for parenteral administration include aseptic aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or vehicles are propylene glycol, polyethylene glycol, vegetable oils (such as olive oil and corn oil), gelatin and injectable organic esters (such as ethyl oleate). Such dosage forms may also contain adjuvants, such as preservatives, wetting agents, emulsifiers and dispersants. They may be sterilized by, for example, filtering through a bacteria-retaining filter, by incorporating a sterilizing agent into the composition, by irradiating the composition or by heating the composition. They may also be manufactured using sterile water or some other sterile injectable media before use.

[0119] The formulation may optionally contain an isotonic agent. The formulation preferably contains an isotonic agent and glycerol is the most preferred isotonic agent. When glycerol is used, its concentration is within the range known in the art, such as, for example, about 1 mg / mL to about 20 mg / mL.

[0120] The pH of the parenteral formulation can be controlled by a buffer such as phosphate, acetate, TRIS or L-arginine. The concentration of the buffer is preferably sufficient to provide pH buffering during storage to maintain the pH at the target pH ± 0.2 pH units. When measured at room temperature, the preferred pH is between about 7 and about 8.

[0121] Other additives such as pharmaceutically acceptable solubilizers, such as Tween (Polyoxyethylene (20) sorbitan monolaurate), Tween (polyoxyethylene (20) sorbitan monopalmitate), Tween (polyoxyethylene (20) sorbitan monooleate), Pluronic (polyoxyethylene polyoxypropylene block copolymer) and PEG (polyethylene glycol) are added to the preparation, and these additives can be used if the preparation will contact plastic materials. In addition, parenteral preparations may contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc.

[0122] Sterile injectable solutions are prepared by mixing one or more compounds of the compounds of the present disclosure with various other ingredients listed above (as required) in a suitable solvent and then filtering and sterilizing. Typically, dispersions are prepared by mixing various sterilized active ingredients into a sterile solvent containing a basic dispersion medium and other ingredients required from those listed above. With regard to sterile powders for the preparation of sterile injectable solutions, preferred preparation methods are vacuum drying and freeze drying techniques, which produce powders of active ingredients plus any additional desired ingredients from previously sterile filtered solutions of active ingredients plus any additional desired ingredients. Therefore, for example, parenteral compositions suitable for administration by injection are prepared by stirring 1.5% by weight of active ingredients in 10% by volume propylene glycol and water. The solution is made isotonic and sterilized with sodium chloride.

[0123] Alternatively, the pharmaceutical compositions of the present disclosure can be administered in the form of suppositories for rectal administration. These suppositories can be prepared by mixing the medicament with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol.

[0124] The pharmaceutical compositions of the present disclosure may also be administered by nasal aerosol or inhalation. Such compositions are prepared according to techniques well known in the art of pharmaceutical formulation using benzyl alcohol or other suitable preservatives, absorption promoters to improve bioavailability, propellants such as fluorinated hydrocarbons or nitrogen, and / or other conventional solubilizing or dispersing agents and may be prepared as saline solutions.

[0125] Preferred formulations for topical drug delivery are ointments and creams. Ointments are semisolid preparations, which are usually based on petrolatum or other petroleum derivatives. As known in the art, creams containing selected active agents are viscous liquids or semisolid emulsions, which are oil-in-water or water-in-oil. The cream base is washable and contains an oil phase, an emulsifier, and an aqueous phase. The oil phase is sometimes also referred to as the "internal" phase, which is usually composed of petrolatum and fatty alcohols such as cetyl alcohol or stearyl alcohol; although not necessarily, the aqueous phase usually exceeds the oil phase in volume and usually contains a wetting agent. The emulsifier in the cream formulation is usually a nonionic, anionic, cationic or amphoteric surfactant. As will be understood by those skilled in the art, the specific ointment or cream base to be used is the base that will provide the best drug delivery. Like other carriers or solvents, the ointment base should be inert, stable, non-irritating and non-sensitizing.

[0126] Preparations for oral administration include tablets, lozenges, gels, etc. Alternatively, oral administration can be performed using a transmucosal delivery system known to those skilled in the art. The disclosed compound may also use a conventional transdermal drug delivery system, i.e., wherein the medicament is generally contained in a laminated structure used as a drug delivery device to attach to the transdermal "patch" of the body surface, delivered by skin or mucosal tissue. In this structure, the pharmaceutical composition is generally contained in a layer or "medicine box" below the upper backing layer. The laminated device may include a single medicine box, or it may include multiple medicine boxes. In one embodiment, the medicine box includes a polymer matrix of a pharmaceutically acceptable contact adhesive material for attaching the system to the skin during drug delivery. Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylene, polysiloxane, polyisobutylene, polyacrylate, polyurethane, etc. Alternatively, the medicine box and skin contact adhesive containing the medicine exist as separate and different layers, and the adhesive below the medicine box may be a polymer matrix as described above in this case, or may be a liquid or gel medicine box, or may take some other forms. The backing layer in these laminates serves as the upper surface of the device, acting as the primary structural element of the laminate structure, and providing a great deal of flexibility to the device.The material selected for the backing layer should be substantially impermeable to the active agent and any other materials present.

[0127] Compounds of the present disclosure can be formulated for aerosol administration, particularly respiratory administration and including intranasal administration. The compound will generally have a small particle size of, for example, about 5 microns or less. Such a particle size can be obtained by means known in the art, for example, by micronization. The active ingredient is provided in a pressurized package together with a suitable propellant such as chlorofluorocarbon (CFC, for example dichlorodifluoromethane, trichlorofluoromethane or dichlorotetrafluoroethane), carbon dioxide or other suitable gas. Aerosols can also conveniently contain surfactants, such as lecithin. Dosage can be controlled by a metering valve. Alternatively, the active ingredient can be provided in the form of a dry powder, for example, with a powder mixture of a compound in a suitable powder matrix such as lactose, starch, starch derivatives such as hydroxypropyl methylcellulose and polyvinyl pyrrolidone (PVP). The powder carrier will form a gel in the nasal cavity. The powder composition can be present in a unit dosage form in, for example, a capsule or box of a gelatin or blister package, and the powder can be applied from these capsules or boxes by an inhaler.

[0128] The composition of pharmaceutical or therapeutic effective amount is delivered to the subject. The precise effective amount will vary with the subject, and will depend on the species, age, size and health of the subject, the nature and severity of the disease being treated, the advice of the treating physician, and the therapeutic agent or therapeutic agent combination selected for administration. Therefore, the effective amount in a given situation can be determined by routine experiments. For the purpose of the present disclosure, in at least one dose, the therapeutic amount will generally be in the range of about 0.01 mg / kg to about 250 mg / kg, more preferably about 0.1 mg / kg to about 10 mg / kg body weight. In larger mammals, the daily dose shown can be about 1 mg to 300 mg, once or multiple times a day, more preferably in the range of about 10 mg to 200 mg. According to the need to reduce and / or alleviate the signs, symptoms or causes of the obstacle in question, or any other desired changes in the biological system, multiple doses can be applied to the subject. When necessary, a preparation with an enteric coating suitable for sustained-release or controlled-release administration of the active ingredient can be prepared.

[0129] The pharmaceutical formulation is preferably in unit dosage form. In this form, the formulation is subdivided into unit doses containing appropriate quantities of the active ingredient. The unit dosage form can be a packaged formulation, the package containing discrete quantities of the formulation, such as bagged tablets, capsules, and powders in vials or ampoules. In addition, the unit dosage form can itself be a capsule, tablet, cachet, or lozenge, or it can be any of these packaged forms in an appropriate number of packages.

[0130] The subject can be any animal, such as a mammal, bird, reptile, or fish. Exemplary mammal classes include rodents, primates, canines, felines, ungulates, lagomorphs, and the like. For example, the subject can be a human, monkey, ape or other primate, mouse, rat or other rodent, dog, cat, pig, horse, cow, rabbit, and the like.

[0131] As used herein, the term "treatment" means providing an improvement, cure or preventive effect on a disorder or condition. In some embodiments, treatment includes preventing the escalation or progression of the condition, or slowing the rate of escalation or progression of the condition (compared to no treatment or other treatment). In the case of endometriosis (described in detail below), treatment includes slowing or preventing the rate of cell growth or cell division, slowing or preventing cell migration and / or slowing or preventing cell invasion. In one embodiment, the treatment of endometriosis includes reducing the volume of ectopic lesions and / or inhibiting the growth rate of ectopic lesions.

[0132] As shown in the examples and figures of the present disclosure, according to one embodiment of the present disclosure, the C-DIM / NR4A1 antagonists of the present disclosure inhibit the growth of ESECT-7 and ESECT-40 cells isolated from ovarian endometriomas by activating apoptosis, but do not inhibit the growth of NEM (normal) endometrial cells, even though NR4A1 is expressed in NEM cells. DIM-C-pPhOH and DIM-C-pPhOH-3-Cl-5-OCH3, as NR4A1 agonists, induce genes involved in the glucose uptake process in C2C12 myocytes compared to endometriotic cells. These cellular differences in CDIM / NR4A1 ligand activity (e.g., antagonist / agonist / inactive) are often observed for selective receptor modulators that exhibit tissue / cell-specific activity, suggesting that CDIM is a selective NR4A1 modulator. The selectivity of these compounds in terms of effectiveness in endometriotic cells but not normal endometrial cells may be due to several reasons, including differential expression of essential cofactors, which are currently being investigated.

[0133] The development of fibrosis can lead to reduced fertility and pain during endometriosis. Zeng and colleagues found that knockout of NR4A1 in normal endometrial tissue (NESC) and endometriotic tissue (EESC) enhanced fibrosis, while cytosporoneβ (NR4A1 agonist) treatment inhibited TGFβ-induced fibrosis in NESC and EESC cells. Zeng et al., Cell Physiol Biochem., 2018, Vol. 46, No. 3: pp. 1078-1090. According to one embodiment of the present disclosure, in contrast to this observation, knockout of NR4A1 or treatment with C-DIM (NR4A1 antagonist) inhibited the progression of fibrosis in ESECT-7 cells and IHEECs compared with controls. In an endometriosis mouse model, the present disclosure demonstrated that NR4A1 levels were higher in endometriotic tissue compared to normal endometrial tissue, and that DIM-C-pPhOH-3-Cl-5-OCH3 treatment inhibited endometriosis progression in mice with endometriosis without any changes in body weight, and similar results were observed in SCID mice bearing HEC. Why are there apparently conflicting reports on the role of NR4A1 in endometriosis? First, Zeng et al. induced endometriosis into Nr4a1 full KO mice by autologous transplantation of Nr4a1 KO uterine fragments. Therefore, as described by Zeng et al., we were unable to determine the role of NR4A1 in ectopic lesions on the progression of endometriosis due to interference in systemic Nr4a1 KO female recipients. In the SCID mouse model, Zeng et al. treated mice with 17-β estradiol to stimulate endometriosis progression. However, in the examples of the present disclosure, 17-β estradiol was not injected into the mouse recipients. High concentrations of exogenous 17-β estradiol exposure significantly altered intracellular signaling in SCID mice compared to untreated SCID mice. Without wishing to be bound by theory, it is believed that this type of experimental difference alters the role of NR4A1 in the progression of endometriosis.

[0134] In summary, the present disclosure demonstrates that NR4A1 is a novel pro-endometriosis transcription factor that stimulates the progression of endometriosis, and that the bisindole-derived NR4A1 antagonists of the present disclosure can be used as novel non-hormonal therapies for the treatment of endometriosis and that such treatments minimize or eliminate the side effects of traditional hormonal therapies.

[0135] Example

[0136] Example 1: Materials and Methods

[0137] Primary human endometriotic stromal cells from endometriosis patients: Ovarian endometriomas were isolated from patients with endometriosis in the proliferative phase according to an IRB-approved protocol. The isolated ectopic lesions were digested at 37°C for 90 minutes with type 3 collagenase (300 μg / mL) and DNaseI (40 μg / mL), and the tissues were then filtered through 150 μm, 100 μm, and 40 μm sieves to separate endometrial stromal cells. The collected endometrial stromal cells were cultured in DMEM / F12 supplemented with 10% FBS and 1× antibiotic / antimycotic solution, and then verified by flow cytometry using CD90 antibody (a mesenchymal cell marker). Hereafter, we refer to the stromal cells as ovarian endometriomas isolated from ectopic endometrium (ESECT). As a control, primary normal endometrial stromal cells were isolated from biopsies of eutopic endometrium (NEM) of normal women in the proliferative phase. All cells were incubated in a CO2 incubator at 37°C in a humidified atmosphere of 5% CO2 and 95% air.

[0138] Reagents and antibodies: Annexin V cell death apoptosis kit (V13241) was purchased from Invitrogen. The primary antibodies used were EGFR (4267, 1:1000), survivin (2808, 1:1000), c-caspase-3 (9661, 1:1000), c-PARP (9541, 1:1000) from Cell Signaling Technology (Danvers, MA), c-Myc (sc-40, 1:500), Bcl-2 (sc-509, 1:500) and Bax (sc-20067, 1:500) from Santacruz Biotechnology (Santacruz, CA), NR4A1 (ab109180, 1:3000) and α-SMA (ab32575, 1:3000) from Abcam (Cambridge, MA), and dapoxetine (ab32610, 1:3000) from Sigma Aldrich. Corporation (Milwaukee, WI) β-actin (A1978, 1: 10000), and COL1A1 (GTX112731, 1: 2000), CTGF (GTX124232, 1: 2000) and FN (GTX112794, 1: 2000) from GeneTex, Inc. (Irvine, CA). Secondary antibodies of rabbit (7074), mouse (7076) and anti-rabbit Alexa Fluor (4412) were purchased from Cell Signaling Technology (Danvers, MA). The two siNR4A1 oligonucleotides used in this study were SASI_Hs02_00333289 and SASI_Hs02_00333290, and non-targeting scrambled small interfering RNA (iGL2) from Sigma Aldrich Corporation (The Woodlands, TX) was used as a control. The bisindole-derived NR4A1 ligands 1,1-bis(3′-indolyl)-1-(p-hydroxyphenyl)methane (DIM-C-pPhOH), 1,1-bis(3′-indolyl)-1-(3-chloro-5-methoxyphenyl)methane (DIM-C-pPhOH-3-Cl-5-OCH3), 1,1-bis(3′-indolyl-1-(3,5-dibromo-4-hydroxyphenyl)methane (DIMC-C-pPhOH-3,5-Br2), and 1,1-bis(3′-indolyl)-1-(3-chloro-4-hydroxyphenyl)methane (DIM-C-pPhOH) were prepared as described.

[0139] Cell proliferation assay: Patient-derived endometriotic cells ESECT-7 and ESECT-40 were seeded into 96-well plates, and the cells were then treated with DMSO or different concentrations of DIM-C-pPhOH and DIM-C-pPhOH-3-Cl-5-OCH3 for 24 hours. ESECT-7 and ESECT-40 were treated with two siNR4A1 oligonucleotides to downregulate NR4A1. Non-target siRNA was used as a control for siRNA. Afterwards, the culture medium was removed, and MTT solution diluted in PBS was added to the cell culture. After incubation for 3 hours, the culture medium was aspirated and washed with PBS. Dimethyl sulfoxide (DMSO) was added and incubated at 37°C for 10 minutes, and the absorbance at 570nM was measured.

[0140] Western blotting: ESECT-7 and ESECT-40 cells (2×105) were seeded and allowed to attach for 24 hours, and then treated with DMSO or different concentrations of DIM-C-pPhOH and DIM-C-pPhOH-3-Cl-5-OCH3 for 24 hours. The cells were then lysed, and the whole cell lysate was resolved in a 10% SDS-PAGE gel and the proteins were transferred by Western blotting using a PVDF membrane, followed by incubation of primary and secondary antibodies, and detection using ECL reagents as described.

[0141] Annexin V staining: ESECT-7 and ESECT-40 cells were seeded on Nunc chamber coverslips and subsequently treated with various drugs. The cells were then washed with ice-cold PBS and 5 μL of Alexa Fluor 488 Annexin V and 100 μg / mL PI (according to the manufacturer's instructions) were added to the cells and incubated for 15 min, and the cells were observed using a Zeiss confocal fluorescence microscope.

[0142] Immunofluorescence: ESECT-7 and ESECT-40 cells were seeded in Nunc chamber coverslips and subsequently treated with various drugs. Cells were fixed with 4% paraformaldehyde in PBS at 37°C for 20 minutes. The cells were then blocked and incubated with α-SMA primary antibody in buffer (5% bovine serum albumin in PBS) at 4°C overnight, followed by incubation with Alexa Fluor-conjugated secondary antibodies at a dilution of 1:250 for 2 hours at room temperature. Finally, cells were observed using a Zeiss confocal fluorescence microscope.

[0143] RNA interference: ESECT-7 and ESECT-40 cells were seeded in six-well plates and grown to 60% confluency (24 hours) and then transfected with Lipofectamine 2000 according to the manufacturer's protocol. Both siNR4A1 oligonucleotides and non-targeting control small interfering RNA were used. Six hours after transfection, the medium was replaced with fresh medium, left to stand for 72 hours, cells were harvested, and protein expression was determined.

[0144] Quantitative real-time PCR: Total RNA was isolated from cultured cells according to the manufacturer's instructions (Zymo Research (Irvine, CA)). The concentration and purity of RNA samples were determined using a Nanodrop spectrophotometer. Total RNA was reverse transcribed using a CFX384 Real-Time PCR System (Bio-Rad) and an iTaq Universal SYBR Green One-Step Kit (Thermo Fisher Scientific (Grand Island, NY)) using the manufacturer's protocol. The comparative cycle threshold method was used for relative quantification of samples. The values ​​for each gene were normalized to the expression level of TATA-binding protein. The sequences of the primers used for real-time PCR included the following sequences: α-SMA, 5'-GGC CGA GAT CTC ACT GAC TAC-3' (sense, SEQ ID NO.13) and 5'-TTC ATGGAT GCC AGC AGA-3' (antisense, SEQ ID NO.14); COL1A1, 5'-CAG CCG CTT CAC CTA CAG C-3' (sense, SEQ ID NO.15) and 5'-TTT TGT ATT CAA TCA GTG TCT TGC C-3' (antisense, SEQ ID NO.16); CTGF, 5'-TTG GCC CAG ACC CAA CTA TG-3' (sense, SEQ ID NO.17) and 5'-CAG GAGGCG TTG TCA TTG GT-3' (antisense, SEQ ID NO.18); FN, 5'-GGG AGC CTC GAA GAG C-3' (sense, SEQ ID NO.19) and 5'-AAC AAG TAC AAA CCA ACG CA-3' (antisense, SEQ ID NO.20); and TATA binding protein, 5'-GAT CAG AAC AAC AGC CTG CC-3' (sense, SEQ ID NO.21) and 5'-TTC TGA ATAGGC TGT GGG GT-3' (antisense, SEQ ID NO.21).

[0145] Luciferase assay: ESECT-7 and ESECT-40 cells were seeded in DMEM / F12 supplemented with 2.5% charcoal-treated FBS on 12-well plates. After overnight attachment and growth, different amounts of DNA [i.e., UASx5-Luc (500 ng), GAL4-NR4A1 (50 ng), NBRex3-Luc (800 ng), NurREx3-Luc (800 ng), and Flag-NR4A1 (80 ng)] were co-transfected into each well using Lipofectamine 2000 reagent (Invitrogen (Carlsbad, CA)) according to the manufacturer's protocol. Six hours after transfection, cells were treated with inoculation medium (as above) containing solvent (DMSO) or the indicated concentrations of compounds for 18 hours. Cells were then lysed and cell extracts were used for chemiluminescent quantification of luciferase activity. Luciferase activity values ​​were normalized to the corresponding protein concentration values ​​determined by the Bradford assay. Both constructs, GAL4-NR4A1 and Flag-NR4A1, contain the full-length NR4A1 coding sequence, and all plasmids used in this study were described previously.

[0146] Endometriosis mouse model with autologous transplantation: One uterine horn was isolated from a female mouse (6 weeks old, C57BL / 6J) under anesthesia, and then the uterine horn was cut longitudinally. Using a 2 mm skin biopsy punch, an endometrial fragment was obtained from the isolated uterus and then sutured to the mesentery attached to the intestine of the same mouse. The abdominal incision was then closed by suturing. The mouse estrous cycle was determined using vaginal cytology before harvesting the ectopic lesions. Ectopic lesions were isolated from mice with endometriosis at the estrus of the 3rd week after endometriosis induction. As an endometriosis control, the uterus was isolated from C57BL / 6J female mice without endometriosis at the estrus of 10 weeks of age.

[0147] Endometriosis mouse model with immortalized human endometrial cell xenografts: Luciferase-stable immortalized human endometrial epithelial cells (LIHESC) and luciferase-stable immortalized human endometrial stromal cells (LIHESC) were generated from IHESCs and IHEECs, and then the expression of luciferase was verified by luciferase activity assay. IHESCs were isolated from the endometrium obtained by hysterectomy for benign conditions, and IHEECs were generated from endometriomas by transduction of luciferase expressing lentivirus. Matrigel containing a mixture of LIHESCs (1×106 cells) and LIHEECs (1×106 cells) was injected into the peritoneal cavity of severe combined immunodeficient (SCID) female mice (6 weeks old) to induce endometriosis. Hereafter, we refer to the mixture of LIHEECs and LIHESCs as human endometrial cells (HECs). Ectopic lesions developed well in about 80% of SCID mice transplanted with HEC, and immunostaining with antibodies against vimentin and cytokeratin 18 showed that the ectopic lesions developed successfully from HEC. To noninvasively determine the progression of human ectopic lesions, we injected 150 mg / kg of D-luciferin intraperitoneally into mice 5 minutes before imaging, and then measured the luciferase activity of human ectopic lesions in mice using the InVivo Image System live imaging system.

[0148] Treatment of endometriosis with C-DIM: Endometriosis was induced into mice using the autologous and xenograft methods as described above. After the establishment of ectopic lesions in mice (week 3 after induction of endometriosis), we randomly divided mice with endometriosis into two groups, and then injected mice in the experimental group with 25 mg / kg of C-DIM intraperitoneally for three weeks (once a day, daily), and injected mice in the control group with vehicle (5% DMSO and 10% 2-hydroxypropyl-β-cyclodextrin, once a day) for three weeks. In the case of the autologous transplant model, we isolated ectopic lesions of mice treated with C-DIM vs. vehicle from mice with endometriosis during the estrus period of the third week after drug treatment, and then determined their volume. In the case of the xenograft model, we determined the luciferase activity of human ectopic lesions treated with C-DIM vs. vehicle in SCID mice with endometriosis during drug treatment.

[0149] Immunohistochemistry: Immunostaining was performed on 10% neutral buffered, formalin-fixed and paraffin-embedded mouse tissue sections as previously described. For immunostaining, sections were dewaxed, rehydrated and boiled in 10 mM citrate buffer (pH 6.0) for 10 minutes. To reduce nonspecific binding of antibodies, sections were washed again in phosphate-buffered saline (PBST) containing 0.1% Tween-20 and pre-incubated with 10% goat serum in PBST for 1 hour at room temperature. We measured Nr4a1 levels in the uterus, eutopic endometrium and ectopic lesions using antibodies against Nr4a1 (NB100-5674, Novus, 1:300). Specific antigens were visualized using a DAB (3,3'-diaminobenzidine) substrate kit. Immunostaining intensity was quantified using the ImageJ program.

[0150] Statistical analysis: All experiments were repeated at least three times. Data are expressed as mean ± standard error (SE). Statistical significance was determined using one-way analysis of variance. P values ​​< 0.05 were considered statistically significant.

[0151] Example 2: Expression of NR4A1 and Effects of Receptor Knockout

[0152] Recent studies have shown that NR4A1 is expressed in endometrial cancer cells (Ishikawa and Hec1B) and plays an important role in regulating cell growth, survival, migration / invasion, and related genes, as previously observed in cancer cells derived from other solid tumors. Endometriotic cells also express NR4A1, and Figure 1A Results in this study showed that knockdown of NR4A1 by RNA interference (RNAi) reduced the growth of patient-derived ESECT-7 and ESECT-40 endometriotic cells. Figure 1B As shown, the knockdown efficiency of both oligonucleotides was >85%, and the expression of the growth-promoting genes EGFR and cMyc was reduced in parallel with the loss of NR4A1 ( Figure 1C We also observed that knocking down NR4A1 in endometriotic cells reduced the expression of prosurvival survivin and Bcl-2 gene products and induced the cleavage of Bax, caspase-3, and PARP, all markers of apoptosis ( Figure 1D ). In addition, NR4A1 knockout also induced Annexin V staining in ESECT-7 and ESECT-40 cells ( Figure 1E ), and these results are comparable to those previously observed in endometrial cancer cells.

[0153] Example 3: Bisindole-derived NR4A1 ligands: transactivation and function

[0154] Our previous studies identified 1,1-bis(3'-indolyl)-1-(p-hydroxyphenyl)methane (DIM-D-pPhOH, CDIM8) as an NR4A1 antagonist, and we also developed several supported (3,5-substituted) CDIM8 analogs to reduce in vivo metabolism (conjugation) of the hydroxyl group and enhance activity. Figure 2A The results in the study showed that CDIM8 and 3,5-dibromo (4-OH-3,5-Br2), 3-chloro-5-methoxy (4-OH-3-Cl-3-OCH3), and 3-chloro-(4-OH-3-Cl)-supported DIM-C-pPhOH analogs inhibited the intrinsic transcriptional activity of GAL4-NR4A1 and NR4A1 in ESECT-7 cells transfected with GAL4-NR4A1 chimera / upstream activating sequence (UAS)-luciferase (luc) and NR4A1 / NGFI-B response element (NBRE)-luc constructs. The UAS-luc construct contains five tandem GAL4 binding sites, and the NBRE-luc construct contains a NBRE site that binds NR4A1. The same group of compounds reduced the intrinsic transcriptional activity of NR4A1 in ESECT-40 cells ( Figure 2B ). Thus, the bisindole-derived NR4A1 antagonists effectively inhibited the intrinsic transcriptional activity of NR4A1 in human endometriotic cells and endometrial cancer cells. Comparative analysis of NR4A1 ligand derivatives showed that DIM-C-pPhOH-3-Cl-5-OCH3 effectively inhibited the intrinsic transcriptional activity of NR4A1 compared with other NR4A1 ligands. For example, 5μM-10μM DIM-C-pPhOH-3-Cl-5-OCH3 effectively inhibited the growth of ESECT-7 and ESECT-40 cells compared with 15μM-25μM DIM-C-pPhOH ( Figure 2C ) and down-regulate the expression of EGFR and cMyc in the same cell line ( Figure 2D ). In addition, compared with 25 μM DIM-C-pPhOH, 10 μM DIM-C-pPhOH-3-Cl-5-OCH3 in ESECT-7 ( Figure 3A ) and ESECT-40( Figure 3B ) cells. Compared with 25 μM DIM-C-pPhOH, 5 μM DIM-C-pPhOH-3-Cl-5-OCH3 treatment also significantly induced several apoptotic markers in ESECT-7 and ESECT-40 cells, including downregulation of survivin and Bcl-2, and induced Bax and cleaved PARP and caspase-3 ( Figure 3C ).

[0155] However, compared with human endometriotic stromal cells (ESECT), 15 μM-25 μM DIM-C-pPhOH and 5-10 μM DIM-C-pPhOH-3-Cl-5-OCH3 treatment did not inhibit the growth of normal endometrial NEM cells ( Figure 4A ) and did not reduce cMyc and EGFR expression compared with DMSO control ( Figure 4B In addition, 25 μM DIM-C-pPhOH and 10 μM DIM-C-pPhOH-3-Cl-5-OCH3 treatments also did not enhance Annexin V staining compared to DMSO controls ( Figure 4C ), the levels of cleaved forms of PARP and Bax in NEM cells ( Figure 4D NEM cells also express NR4A1, however, NR4A1 antagonists exhibited cell context-dependent effects and did not affect these cells, and this cell-dependent specificity is often observed for selective receptor modulators that rely not only on the receptor but also on the expression of specific cofactors that may vary between cell lines.

[0156] Previous studies on stroma-derived endometriotic cells have shown that knockdown of NR4A1 enhances TGFβ1-induced fibrogenic gene expression in human ectopic endometrial stromal cells (EESCs) and normal endometrial stromal cells (NESCs). To validate this observation, we inhibited the intrinsic transcriptional activity of NR4A1 in ESECT-7 endometriotic stromal cells ( Figure 5A ) and reduced NR4A1 levels by siNR4A1 ( Figure 5B ) and then determined the progression of fibrosis. Inhibition of NR4A1 reduced the expression level of SMA in ESECT-7 cells compared with controls ( Figure 5A and Figure 5B In addition to SMA, 20 μM-25 μM DIM-C-pPhOH and 5 μM-10 μM DIM-C-pPhOH-3-Cl-5-OCH3 also reduced the mRNA levels of fibrotic markers such as FN, Col1A1, and CTGF in ESECT-7 cells compared with the DMSO control ( Figure 5C ).

[0157] Example 4: Effects of NR4A1 antagonists on fibrosis in endometriotic cells versus endometrial cancer cells.

[0158] To verify the function of NR4A1 in the fibrotic progression of human endometrial cells, we used immortalized human endometrial epithelial cells (IHEECs) as normal human endometrial epithelial cells because IHEECs are not transformed in SCID mice (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC1892381 / ). As a control, we used epithelial-derived Ishikawa endometrial carcinoma cells because NR4A1 inhibitors prevented the growth and survival of Ishikawa cells. Compared with the control, knockout of NR4A1 ( Fig. 6A ) and DIM-C-pPhOH and DIM-C-pPhOH-3-Cl-5-OCH3 treatment ( Figure 6B ) reduced the expression of fibrotic markers (α-SMA, COL1A1, FN, and CTGF) in IHEECs. Knockdown of NR4A1 ( Figure 6C ) and DIM-C-pPhOH and DIM-C-pPhOH-3-Cl-5-OCH3 treatment ( Fig.6D ) also reduced the expression of fibrotic markers (α-SMA, COL1A1, FN, and CTGF) in Ishikawa cells. Thus, activation of NR4A1 stimulates fibrotic progression in endometriosis, as observed in endometrial epithelial cancer cell lines.

[0159] Example 5: Compared with normal uterus, the expression level of Nr4a1 in endometriotic tissue is increased.

[0160] To determine whether the expression level of NR4A1 is increased in endometriotic tissues compared with normal endometrium, we surgically induced endometriosis into mice using autologous transplantation. Ectopic lesions and eutopic endometrium were isolated from mice with endometriosis at estrus 3 weeks after induction of endometriosis. As a control, we also isolated uteri from female mice (10 weeks old) without endometriosis at estrus.

[0161] Immunohistochemistry (IHC) using NR4A1 antibody showed that NR4A1 levels were significantly increased in the epithelium of ectopic lesions compared with the epithelium of the uterus (2.6-fold, p<0.001) ( Fig. 7A , Figure 7C and Fig.7D In addition to ectopic lesions, NR4A1 levels were also elevated in the epithelium of the eutopic endometrium compared with the epithelium of the normal uterus (1.27-fold, p=0.002) ( Fig. 7A , Figure 7B and Fig.7D). Thus, the epithelium of ectopic lesions and eutopic endometrium of mice with endometriosis had higher levels of NR4A1 compared to the epithelium of the normal uterus. In addition to the epithelium, NR4A1 levels were slightly elevated in the stroma of ectopic lesions compared to the stroma of the uterus (1.1-fold, p=0.012) ( Fig. 7E ). However, NR4A1 levels were not elevated in the stroma of eutopic endometrium compared with the stroma of normal uterus ( Fig. 7E ). Overall, increased levels of NR4A1 in endometriotic tissue are associated with the progression of endometriosis.

[0162] Example 6: DIM-C-pPhOH-3-Cl-5-OCH3 treatment effectively inhibits the growth of ectopic lesions in mice with endometriosis.

[0163] Although NR4A1 expression levels were significantly increased in ectopic lesions compared with normal uterus, it is unclear whether the increase in NR4A1 induces endometriosis or is simply a consequence of endometriosis. To address this question, we used the NR4A1-specific antagonist DIM-C-pPhOH-3-Cl-5-OCH3 to investigate whether inhibition of NR4A1 in ectopic lesions would suppress the daily progression of endometriosis in mice with endometriosis. Based on our previous studies, we treated mice with endometriosis ( Fig. 8A Compared with vehicle, 25 mg / kg DIM-C-pPhOH-3-Cl-5-OCH3 treatment significantly reduced (2.4-fold, p=0.015) the volume of ectopic lesions ( Figure 8B , Figure 8C and Fig.8D However, DIM-C-pPhOH-3-Cl-5-OCH3 treatment did not change the body weight of mice compared with vehicle ( Fig. 8E ). Therefore, DIM-C-pPhOH-3-Cl-5-OCH3 treatment effectively inhibited the growth of ectopic lesions in mice with endometriosis with minimal side effects.

[0164] The above observations raised the question whether DIM-C-pPhOH-3-Cl-5-OCH3 also inhibits the growth of human ectopic lesions. To address this question, we induced endometriosis in SCID mice using HEC carrying the luciferase gene. Mice bearing human ectopic lesions were treated with 25 mg / kg DIM-C-pPhOH-3-Cl-5-OCH3 compared to vehicle-treated controls ( Fig. 9AIn addition to mouse ectopic lesions, DIM-C-pPhOH-3-Cl-5-OCH3 treatment also significantly reduced luciferase activity in ectopic lesions compared with vehicle images (13.4-fold, p=0.022) ( Fig. 9B and Fig. 9C Since luciferase activity recapitulates the growth of human ectopic lesions in mice, DIM-C-pPhOH-3-Cl-5-OCH3 treatment with the NR4A1 antagonist inhibited the growth of human ectopic lesions in SCID mice compared to vehicle and confirmed the in vivo efficacy of the NR4A1 antagonist as an inhibitor of endometriosis.

[0165] abbreviation:

[0166] ●C-DIM, methylene-substituted diindolylmethane;

[0167] ●COLIA1, type alpha 1 collagen;

[0168] CTGP, connective tissue growth factor;

[0169] ●DAB, diaminobenzidine;

[0170] ●DIM, 3,3'-diindolylmethane;

[0171] ·DIM-C-pPhOH, 1,1-bis(3'-indolyl)-1-(4-hydroxyphenyl)methane;

[0172] EGFR, epidermal growth factor receptor;

[0173] FN, fibronectin; HEC, human endometrial cells;

[0174] IHC, immunohistochemistry;

[0175] NBRE, nerve growth factor β response element;

[0176] NR4A, nuclear receptor 4A;

[0177] LIHESC, luciferase-stable immortalized human endometrial cells;

[0178] PBS, phosphate buffered saline;

[0179] PVDF, polyvinylidene fluoride;

[0180] SMA, smooth muscle actin.

[0181] It should be understood that, unless explicitly stated, any embodiment, feature, element, definition or general description provided for any aspect of the present disclosure may be applied to any other aspect of the present disclosure without limitation. Therefore, any embodiment discussed herein may be implemented with respect to any method, medicament or composition of the present invention, and conversely, any method, medicament or composition of the present invention may be implemented with respect to any embodiment discussed herein. In addition, the medicaments and compositions of the present invention may be used to implement the methods of the present invention.

[0182] When used in conjunction with the term "comprising" herein, the use of the word "a" or "an" may mean "one", but it is also consistent with the meaning of "one or more", "at least one", and "one or more than one".

[0183] Use of the term "or" is intended to mean "and / or" unless explicitly stated to refer to only alternatives or the alternatives are mutually exclusive, although the present disclosure supports a definition referring to only alternatives and "and / or."

[0184] Throughout this application, the term "about" is used to indicate that a value includes the inherent error variation for the device, the method being employed to determine the value, or the variation that exists between study subjects.

[0185] The terms "comprises", "having" and "includes" are open-ended linking verbs. Any form or tense of one or more of these verbs, such as "comprises / comprising", "has / having" and "includes / include" are also open-ended. For example, any method that "comprises", "has" or "includes" one or more steps is not limited to having only those one or more steps, and also covers other unlisted steps. In addition to "comprising", or as an alternative, any embodiment of this document may be cited as "consisting of". The conjunction "consisting of" excludes any element, step or component not specified in the claims. Words using the singular or plural also include the plural and singular, respectively. In addition, when used in this application, the words "herein", "above" and "below" and words of similar meaning should refer to this application as a whole, rather than any specific part of this application.

[0186] Publications cited herein and the subject matter cited are hereby specifically incorporated by reference in their entirety.

[0187] While the preferred embodiment of the invention has been shown and described, it will be appreciated that various changes can be made without departing from the spirit and scope of the invention.

Claims

1. Use of a compound of the following formula or a salt thereof in the preparation of a medicament for treating endometriosis in an individual by modulating the activity of nuclear receptor subfamily 4 group A member 1 (NR4A1): in, R1, R2, R1' and R2' are each independently selected from hydrogen, a straight chain alkyl group containing one to ten carbon atoms, and a branched chain alkyl group containing one to ten carbon atoms; R3, R4, R5, R6, R3', R4', R5' and R6' are each independently selected from hydrogen, halogen, a straight chain alkyl group containing one to ten carbon atoms, a branched chain alkyl group containing one to ten carbon atoms, an alkoxy group containing one to ten carbon atoms and a nitro group; R7 is selected from hydrogen, a straight chain alkyl group containing one to ten carbon atoms, a branched chain alkyl group containing one to ten carbon atoms, a cycloalkyl group containing one to ten carbon atoms, and an aryl group; R8, R9, R 11 and R 12 independently selected from H, halogen, a straight chain alkyl group containing one to ten carbon atoms, a branched chain alkyl group containing one to ten carbon atoms, an alkoxy group containing one to ten carbon atoms, a haloalkyl group containing one to ten carbon atoms, a nitro group, a hydroxyl group, and a haloalkoxy group containing one to ten carbon atoms; Where R 10 is OH, and Among them, R8, R9, R 11 and R 12 At least one of the is not hydrogen.

2. The use according to claim 1, wherein R8, R9, R 11 and R 12 Independently selected from halogen, CH3, OCCl3, CF3, tert-butyl, OCH3, and OH.

3. The use according to claim 1, wherein R9 is halogen and R 11 Selected from H, halogen and OCH3.

4. The use according to claim 1, wherein the compound is selected from: ； ； ; and their salts.

5. The use according to any one of claims 1 to 4, wherein modulation of NR4A1 induces downregulation of a protein selected from the group consisting of EFGR, cMyc, survivin, Bcl-2, SMA and combinations thereof.

6. Use according to any one of claims 1 to 4, wherein modulation of NR4A1 activity induces upregulation of proteins including Bax.

7. The use according to any one of claims 1 to 4, wherein modulation of NR4A1 activity induces a decrease in the mRNA level of a fibrosis marker selected from the group consisting of FN, Col1A1, CTGF and a combination thereof.

8. Use according to any one of claims 1 to 4, wherein modulation of NR4A1 activity cleaves PARP and caspase-3.

9. The use according to any one of claims 1 to 4, wherein modulation of NR4A1 activity induces apoptosis in ovarian endometrioma.

10. The use according to claim 9, wherein modulation of NR4A1 activity does not inhibit the growth of normal endometrial cells relative to a control.

11. The use according to any one of claims 1 to 4, wherein modulation of NR4A1 activity inhibits the progression of fibrosis.

12. The use according to any one of claims 1 to 4, wherein modulation of NR4A1 activity inhibits the intrinsic transcriptional activity of NR4A1 in endometriotic cells relative to a control.

13. The use according to any one of claims 1 to 4, wherein modulation of NR4A1 activity inhibits the growth of ectopic lesions relative to a control.

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