CRILA® AND EGCG COMPOSITIONS FOR THE TREATMENT OF UTERIFIOBIOMA

Abstract

In certain embodiments, the present application discloses, in part, biologically active compositions including Crila® compositions or admixtures of Crina oleracea dry extract (Crila®) and epigallocatechin gallate (EGCG), ratios of Crina oleracea dry extract to EGCG, and their uses in treating intramyocellular or subserosal fibroids and various associated diseases.

Description

[Technical field]

[0001] Related Applications This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 315,101, filed March 1, 2022, which is incorporated by reference in its entirety.

[0002] FIELD OF THEINVENTION The present invention relates to biologically active compositions and formulations, and combinations thereof, for the treatment of uterine fibroids and diseases associated with intramyocellular and submucosal fibroids. [Background technology]

[0003] 2. Background of the Invention Uterine leiomyomas (fibroids) affect 30-50% of women of reproductive age and are a significant cause of infertility. Intramural and submucosal fibroids decrease the chances of pregnancy compared to unaffected women (RR = 0.3-0.7).

[0004] Surgical removal of uterine fibroids may restore fertility; however, recurrence rates are high and benefits are often temporary. Significant postoperative consequences, such as adhesions, may adversely affect a woman's fertility and general health. Hysterectomy (i.e., surgical removal of the uterus) is a common procedure for uterine fibroids and is indicated in up to one-third of all cases. Hysterectomy to treat uterine fibroids can be highly effective, but is associated with many undesirable side effects, such as loss of fertility, open abdominal surgery, sexual dysfunction, and long recovery times. Hysterectomy procedures are also associated with significant morbidity (e.g., sepsis, bleeding, peritonitis, bowel and bladder injury), mortality, and costs. Other invasive techniques for the treatment of uterine fibroids include surgical myomectomy, such as laparoscopic myomectomy, hysteroscopy (i.e., the use of a thin fiber-optic camera to take pictures inside the uterus), uterine artery embolization, endometrial ablation, myolysis, and needle myolysis.

[0005] Hormonal therapy to induce medical menopause can be used to reduce the size of uterine fibroids, but these therapies also prevent pregnancy. Current treatments for uterine fibroids include medical treatment with NSAIDS, estrogen-progesterone combinations, and GnRH analogs. Drug therapy with GnRH analogs is limited due to side effects such as hot flashes, vaginal dryness, mood swings, and reduced bone density. Drug therapy is relatively ineffective and palliative rather than curative. U.S. Patent No. 10,155,004 discloses a method for treating uterine fibroids, among other diseases, which includes administering an estra-4,9-dien-3-one derivative. U.S. Patent No. 5,472,977 discloses a method for inhibiting uterine fibroid disease, which includes administering various substituted triphenyldropyran derivatives.

[0006] Crinum latifolium is widely used in Asian folk and traditional medicines (Vietnam, Ayurveda, China) as a tonic and for inflammation, infection, allergic disorders and tumors. See Tram, NTN et al., Crinum L. (Amaryllidaceae). Fitoterapia 2002, 73 (3), 183-208; Gasca-Silva, CA et al., Recent updates on Crinum latifolium L. (Amaryllidaceae): A review of ethnobotanical, phytochemical, and biological properties. South African Journal of Botany 2022, 146, 162-173.

[0007] Several in vitro studies have been published on Crinum latifolium. See Jenny, M. Et al., Crinum latifolium leave extracts suppress immune activation cascades in peripheral blood mononuclear cells and proliferation of prostate tumor cells. Scientia pharmaceutica 2011, 79 (2), 323-336. One study showed that aqueous extracts have immune modulating properties in human peripheral blood mononuclear cells. See Zvetkova, E. Et al., Aqueous extracts of Crinum latifolium (L.) and Camellia sinensis show immunomodulatory properties in human peripheral blood mononuclear cells. International immunopharmacology 2001, 1 (12), 2143-2150.

[0008] The plant has had a long tradition of use for the treatment of several ailments, including prostate problems in men and reproductive problems in women, but there was no data confirming the ethnobotanical use of the medicinal plant. Of the 12 subspecies of the genus Crinum, only one subspecies was found to be associated with specific pharmacological properties, a specific cultivar, Crinum latifolium L. var. crilae Tram & Khanh, which has been the focus of research for the last 20 years. This cultivar has been cultivated and propagated, and processed plant material is currently sold internationally under the trade name Crila®.

[0009] Crila® contains various phytochemicals, such as crinamidin-like compounds and various types of amaryllidaceae alkaloids. Crila® has been shown to be able to be used as a non-hormonal therapy that can reduce the size of uterine fibroids, relieve symptoms associated with uterine fibroids, and in some cases restore fertility. See Burton, TC Et al. In Examining the Estrogenicity of Crinum latifolium L. var. crilae Tram & Khanh, var n. (Amaryllidaceae) Using Cell-based and Receptor-based Assays, MENOPAUSE; The Journal of the North American Menopause Society, Lippincott Williams & Wilkins, 530 Walnut St. Philadelphia, PA 19106-3621 USA: 2014; pp 1335-1335.

[0010] In vivo, researchers have shown that chemically induced tumors in rats orally treated with aqueous extracts of Crinum latifolium (L.) and Camellia sinensis grow more slowly. See Zvetkova, E. et al., Aqueous extracts of Crinum latifolium (L.) and Camellia sinensis show immunomodulatory properties in human peripheral blood mononuclear cells International immunopharmacology 2001, 1 (12), 2143-2150. Acute toxicity studies in mice have shown that dry extracts of Crinum latifolium (L.) at levels 20, 30, 80 and 100 times the normal human dose or at doses up to 25 gm / kg per day produced no toxicity after 48 hours. LD 50was 49.7 gm / kg. See Yen, HT et al., The evaluation of acute and subchronic toxicities of An Phu Khang capsules in experimental animals. Tap chi Nghien cuu Y hoc 2021, 148 (12), 86-95. Findings of low toxicity were confirmed in rats, rabbits and dogs, and in physiological models. See Zvetkova, E. et al., supra.

[0011] Standardized Crila® has been publicly available in Vietnam since 2005 and more recently in other countries. Crila® is widely used internationally with no reported adverse events requiring medical attention. Standardized formulations of Crila®, imported under FDA "prior notice" inspection, have been sold in the United States since 2010.

[0012] Clinical Trials with Crila®: There are two well-described clinical trials of Crila® for uterine fibroid tumors and benign prostatic hyperplasia (BPH). The BPH trial was a single-arm, open-label study conducted in 2005 in 189 men over a 2-month period in three hospitals in Vietnam. Of the 157 completers, 89.2% showed beneficial responses in terms of International Prostate Symptom Score (p<.001), prostate volume reduction (p<.05), and residual urine sensation (p<.05). Another 3-month trial of 195 women with uterine fibroids (leiomyomas) was conducted successfully in 2007 in three hospitals. See Hoa, VT, To Evaluate the Effect and Possibility of Accepting of Crila in Uterus Fibroid Tumor Treatment. Vietnam Ministry of Health 2007.

[0013] Crila® reduced tumor size or stopped tumor growth in 79.5% of women, while tumor growth continued at a slower rate in the other women. Mean tumor diameter decreased from 48.5 mm to 44.4 mm (p<.01). Thirty-six percent of women reported heavy menstrual flow before taking Crila®, which decreased to 1% after treatment. There were no significant differences between baseline and study endpoints in vital signs, liver enzymes, creatinine, or white blood cells. Red blood cells were slightly elevated within normal ranges. There were no changes in urinalysis by microscopy. Twenty patients (12.73%) experienced mild, transient side effects possibly attributable to the study drug, mainly gastrointestinal (17), dizziness (4), rash (2), and insomnia (1). In this open-label study of uterine fibroids (n = 195, 3 months), there were no changes in vitals, liver and kidney function, or blood counts from baseline to final follow-up. The study had three post-baseline assessments. Reported side effects included nausea, vomiting, headache, vaginal dryness, and hot flashes. These side effects tended to decrease over time and with changes in dosing, and no medical intervention was required. Fifteen percent experienced mild nausea at the end of the first 30 days, but this number decreased to 5% at the end of the 90 days. A preliminary Crila® in uterine fibroids trial is mentioned in the report but is not fully described. The preliminary trial involved 40 uterine fibroids patients and the results were "satisfactory."

[0014] In 2005, the Vietnamese Ministry of Health approved Crila® for use in hospitals and pharmacies nationwide for men with benign prostatic hyperplasia (BPH), and in 2007, the Vietnamese Ministry of Health approved Crila® for use in hospitals and pharmacies nationwide for women with uterine leiomyomas.

[0015] Epigallocatechin gallate (EGCG): Green tea leaves contain polyphenols such as catechins or flavin-3-ols, including epicatechin (EC), epigallocatechin (EGC), epigallocatechin gallate (EGCG), and alkaloids. See Singh, BN et al., Green tea catechin, epigallocatechin-3-gallate (EGCG): mechanisms, perspectives and clinical applications. Biochemical pharmacology 2011, 82 (12), 1807-1821 and Zhang, Y. et al., A review for physiological activities of EGCG and the role in improving fertility in humans / mammals. Biomedicine & Pharmacotherapy 2020, 127, 110186. Catechins are the major component of tea-derived phenols, accounting for approximately 30-42% of the dry weight of green tea. Studies have shown that EGCG catechin is the most abundant and active compound responsible for much of green tea's health-promoting role, explaining the positive research results cited in the medical literature for the use of green tea extracts. EGCG is the most abundant polyphenol and the primary antioxidant in green tea.

[0016] A study conducted by the United States Department of Agriculture reported that green tea has a strong antineoplastic effect on a wide range of human tumor cells and that EGCG inhibits key pathways of tumor growth. See Khan, N. et al., Tea polyphenols for health promotion. Life sciences 2007, 81 (7), 519-533. EGCG is believed to inhibit each stage of tumor formation by regulating signaling pathways involved in cell proliferation, transformation, inflammation, apoptosis, oxidative stress and invasion. EGCG polyphenols inhibit key pathways of tumor growth by regulating signaling pathways involved in cell proliferation, transformation, inflammation, apoptosis, metastasis and invasion. See Zhang, D. et al., Green tea extract inhibits proliferation of uterine leiomyoma cells in vitro and in nude mice. American Journal of Obstetrics and Gynecology 2010, 202 (3), 289. e1-289. e9.

[0017] Al-Hendy et al. showed that the levels of catechol-O-methyltransferase (COMT) are increased in uterine leiomyomas compared to the adjacent myometrium and described its important role in the pathogenesis of uterine fibroids. Zhang, D.; Rajaratnam, V.; Al-Hendy, O.; Halder, S.; Al-Hendy, A., Green tea extract inhibition of human leiomyoma cell proliferation is mediated via catechol-O-methyltransferase. Gynecologic and obstetric investigation 2014, 78 (2), 109-118. In addition, it was revealed that EGCG exerts a strong COMT inhibitory effect, which also contributes to its effective anti-uterine fibroid activity. See Zhang, D.; Al-Hendy, M.; Richard-Davis, G.; Montgomery-Rice, V.; Rajaratnam, V.; Al-Hendy, A., Antiproliferative and proapoptotic effects of epigallocatechin gallate on human leiomyoma cells. Fertility and sterility 2010, 94 (5), 1887-1893. Zhang et al. demonstrated the utility of EGCG in suppressing uterine leiomyoma tumor formation in vivo in a nude mouse model. See Zhang, D. et al., American journal of obstetrics and gynecology 2010, supra. Al-Hendy et al. also reported that EGCG acts as an anti-uterine leiomyoma agent through the regulation of multiple signaling pathways. This group further demonstrated that EGCG acts at the genetic level to inhibit proliferation and induce apoptosis in human uterine leiomyoma cells. The study also showed that EGCG-treated human uterine fibroid cells had 14-fold increased expression of the BMP2 gene compared to untreated control cells.The significant increase in BMP2 secretion from uterine fibroids by EGCG treatment may overcome uterine fibroid-induced endometrial BMP resistance through increased binding to BMP receptors, thereby improving endometrial decidualization and subsequently enhancing implantation, fertility, and pregnancy outcomes. Furthermore, studies have shown that EGCG significantly reduces TGF-β3 production by human uterine fibroid cells. See Ciebiera, M. et al.; Role of transforming growth factor β in uterine fibroid biology. International Journal of Molecular Sciences 2017, 18 (11), 2435. Since TGF-β3 is the main cytokine responsible for endometrial BMP resistance, such effects of EGCG are expected to lead to improved endometrial receptivity.

[0018] The foregoing examples and limitations of the related art are intended to be illustrative and not exhaustive. Other limitations of the related art will become apparent to those of ordinary skill in the art upon reading this specification and studying the figures or drawings provided herein. [Brief description of the drawings]

[0019] [Figure 1] FIG. 1 is a representative chromatograph showing the main components of Crila®.

[0020] [Diagram 2] FIG. 2 is a representative chromatograph of a magnified and labeled portion of FIG. 1, with the major peaks labeled sequentially as peaks A, B, C, D, E, F, G, H, I, J and K along with their retention times. Summary of the Invention

[0021] Summary of the Invention As disclosed in this application, the inventors have recognized a continuing need for effective, non-hormonal, non-surgical and fertility-friendly treatment options for uterine fibroids and related diseases. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The following embodiments, aspects and variations thereof are exemplary and illustrative and are not intended to be limiting in scope.

[0023] In certain embodiments, the present application discloses novel biologically effective compositions and formulations for the treatment of uterine fibroids, and methods of their use.

[0024] In one embodiment, the present application discloses a therapeutically active composition comprising an admixture of Crinum latifolium L. dry extract (Crila®) and epigallocatechin gallate (EGCG), wherein the ratio of Crila® to EGCG in the composition ranges from 1:10 wt / wt to 100:1 wt / wt. In one variation, the ratio of Crila® to EGCG in the composition ranges from 10:1 wt / wt to 1:100 wt / wt. In another variation, the ratio of Crila® to EGCG in the composition is 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20, or 1:10 wt / wt. In another variation, the ratio of EGCG to Crila® in the composition is 1:100, 1:90, 1:80, 1:70, 1:60, 1:50, 1:40, 1:30, 1:20 or 1:10 wt / wt. In some variations, the therapeutically active composition is a pharmaceutical composition. As used herein, the ratio of Crila® to EGCG expressed as wt / wt means the weight-to-weight ratio of Crila® to EGCG, and can be expressed in milligrams, such as mg / mg, or in grams, such as g / g (or gm / gm). As used herein, the term "dry extract" refers to a mixture or extract of ingredients obtained from Crila® and as defined herein, which may be formulated as a mixture of single ingredients or specifically isolated ingredients (or compounds or alkaloids), such as a Crila® composition as disclosed herein; or the term may include all of the ingredients, including all of the alkaloids and other compounds, obtained from the extract of Crila®.

[0025] In another embodiment, therapeutically active compositions are provided that include isolated Crila® compositions 1-20 of Table 1. As referred to herein, the therapeutically active composition that includes the dried extract of Acanthus nigra can be Crila® or a Crila® composition that includes an isolated mixture as described in Table 1. Thus, for example, Crila® composition 1 (in Table 1) includes the components A , B , C , D , E , F , G , H , I , J and K Composition 2 (Table 1) contains the ingredient A and B and Crila® composition 8 (in Table 1) contains the ingredient D , E , F , G and H In some embodiments, the composition comprises the components A , B , C , D , E , F , G , H , I , J and K Crila® composition 1 comprising: A and B Crila® composition 2, comprising: A , B and C Crila® composition 3, comprising: A , B , C and D Crila® composition 4, comprising: A and C Crila® composition 5, comprising: A , C and DCrila® composition 6, comprising: A , B , G and H Crila® composition 7, containing the ingredients D , E , F , G and H Crila® composition 8, containing the ingredients A , D , E , F , G and H Crila® composition 9, containing: A , F , G and H Crila® composition 10 comprising: A , B , C , I , J and K Crila® composition 11, comprising: C , D , E and F Crila® composition 12, comprising: C , D , E , F , G , H and I Crila® composition 13 containing the ingredients D , E and F Crila® composition 14, containing the ingredients D , E , F , G and H Crila® composition 15 containing the ingredients A , D , E and F Crila® composition 16 containing the ingredients C , D , G , H and ICrila® composition 17 containing the ingredients D , E , F , J and K Crila® composition 18 containing the ingredients A , B , C , D , E , F and G Crila® composition 19 comprising: E , F , G and H The present invention relates to an isolated Crila® composition selected from the group consisting of Crila® compositions 20 comprising:

[0026] In one embodiment of the composition, the ratio of Crila® to EGCG in the composition ranges from 1:1 wt / wt to 10:1 wt / wt. In another embodiment of the composition, the ratio of Crila® to EGCG in the composition ranges from 1:1 wt / wt to 5:1 wt / wt. In another embodiment, the ratio of Crila® to EGCG in the composition ranges from 4.0:1 wt / wt to 5.5:1 wt / wt. In another embodiment of the composition, Crila® and EGCG are formulated as a dry powder. In yet another embodiment, Crila® and EGCG are formulated in a capsule formulation or capsule dosage form.

[0027] In some variations of the above compositions, the capsule is a softgel capsule. In other variations, the capsule is a hard capsule. In other variations, the capsule is an enteric coated capsule, such as capsules made from cellulose derivatives, such as HPMC AS-LF and HP-55, and acrylic / methacrylic acid derivatives, such as EUDRAGIT L100 and S100, with and without plasticizers.

[0028] In another embodiment of the present application, there is provided a pharmaceutical composition comprising the above therapeutically active composition comprising an admixture of Indian Crinum dried extract (Crila®) and epigallocatechin gallate (EGCG, purity >95%); and pharmaceutically acceptable excipients, wherein the composition is effective for the treatment of intramyocellular or subserosal fibroids (uterine leiomyomas).

[0029] In another embodiment, the present application provides a method of at least one of treating, preventing, delaying progression of, and reversing one or more symptoms of intramyocellular fibroids or subserosal fibroids (uterine leiomyomas) in a subject in need of such treatment or at risk of having one of intramyocellular fibroids or subserosal fibroids, comprising: administering to a subject a therapeutically active composition comprising an admixture of a dried extract of Crina alba (Crila®) and epigallocatechin gallate (EGCG), wherein the ratio of the dried extract of Crina alba to EGCG in the composition is in the range of 1:10 wt / wt to 100:1 wt / wt per day, to prevent, slow and / or arrest intramural or subserosal fibroids. A method is disclosed.

[0030] In one variation, the present application discloses a composition comprising an admixture of Crinum xanthoides dry extract (Crila®) and epigallocatechin gallate (EGCG), wherein the ratio of Crinum xanthoides dry extract to EGCG in the composition ranges from 1:10 wt / wt to 100:1 wt / wt, for use in treating intramyocellular or subserosal fibroids (uterine leiomyomas) in a subject in need of such treatment. In another variation, the present application discloses a composition comprising an admixture of Crila® and EGCG, wherein the ratio of Crinum xanthoides dry extract to EGCG in the composition ranges from 1:10 wt / wt to 100:1 wt / wt, for use as a medicament in treating intramyocellular or subserosal fibroids (uterine leiomyomas) in a subject in need of such treatment.

[0031] In one embodiment of the method, the ratio of Crila® to EGCG in the composition is 1:1 wt / wt to 10:1 wt / wt. In another embodiment, the ratio of Crila® to EGCG in the composition is in the range of 1:1 wt / wt to 5:1 wt / wt. In another embodiment of the method, the ratio of Crila® to EGCG in the composition is in the range of 4.0:1 wt / wt to 5.5:1 wt / wt. In yet another embodiment, Crila® and EGCG are formulated as a dry powder. In another embodiment of the method, Crila® and EGCG are formulated in a capsule formulation or capsule dosage form.

[0032] In another embodiment, a method for the treatment of cancer in a patient is provided, comprising administering to the patient a composition comprising: A , B , C , D , E , F , G , H , I , J and K Crila® composition 1 comprising: A and B Crila® composition 2, comprising: A , B and C Crila® composition 3, comprising: A , B , C and D Crila® composition 4, comprising: A and C Crila® composition 5, comprising: A , C and D Crila® composition 6, comprising: A , B , G and H Crila® composition 7, containing the ingredients D , E ,F , G and H Crila® composition 8, containing the ingredients A , D , E , F , G and H Crila® composition 9, containing: A , F , G and H Crila® composition 10 comprising: A , B , C , I , J and K Crila® composition 11, comprising: C , D , E and F Crila® composition 12, comprising: C , D , E , F , G , H and I Crila® composition 13 containing the ingredients D , E and F Crila® composition 14, containing the ingredients D , E , F , G and H Crila® composition 15 containing the ingredients A , D , E and F Crila® composition 16 containing the ingredients C , D , G , H and I Crila® composition 17 containing the ingredients D , E , F , J and K Crila® composition 18 containing the ingredients A ,B , C , D , E , F and G Crila® composition 19 comprising: E , F , G and H Methods are provided that include administering to a patient in need of such treatment a therapeutically effective amount of a composition comprising an isolated Crila® composition selected from the group consisting of Crila® composition 20. In another embodiment, the cancer is selected from the group consisting of leukemia, neuroblastoma, glioblastoma, cervical cancer, colorectal cancer, pancreatic cancer, renal cancer, and melanoma. In another embodiment of the method, the cancer is selected from the group consisting of lung cancer, breast cancer, prostate cancer, ovarian cancer, and head and neck cancer.

[0033] The disclosed compositions comprising Crila® and EGCG separately, or both Crila® and EGCG together in a composition, or the Crila® composition may contain other ingredients such as oleic acid, Kolliphor® EL (polyoxyl castor oil or Cremophor EL), Vitamin E TPGS (D-alpha-tocopherol polyethylene glycol-1000 succinate), Maisine® CC (glyceryl monolinoleate), Gelucire® 44 / 14 (lauroyl polyoxyl-32 glyceride), Miglyol® 812N (ester of caprylic fatty acid and glycerin derived from saturated coconut and palm kernel oils), Plurol® Oleique (polyglyceryl-6 dioleate), Lauro glycol. TM 90 (Propylene Glycol Monolaurate (Type II), Labrasol® (Caprylocaproyl Polyoxyl-8 Glyceride), Kolliphor® EL (Polyoxyl Castor Oil), Captisol® (SBE-Beta-Cyclodextrin), Peceol TM(glycerol / glyceryl monooleate (type 40)), sodium deoxycholate, deoxycholic acid and Labrafil® M2125CS (linoleoyl polyoxyl-6 glycerides).

[0034] In some variations, the dose of Crila® or Crila® composition administered is between 50 mg and 5,000 mg per day; and the dose of EGCG administered is between 50 mg and 1,000 mg per day. In another variation, the dose of the composition is between 100 mg per day and 7,000 mg per day. In some variations, the composition is administered daily or every other day for at least 30, 60, 90, or 120 days. In another variation, the composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months.

[0035] In another embodiment, the present application provides a pharmaceutical composition comprising a therapeutically effective amount of a composition comprising an admixture of the Indian Crinum dried extract (Crila®) of any one of the above embodiments and aspects and epigallocatechin gallate (EGCG), or a mixture thereof; and a pharmaceutically acceptable excipient, Uterine fibroids; and conditions associated with intramyocellular or subserosal fibroids (uterine leiomyomas), and methods for the treatment of uterine fibroids, A pharmaceutical composition is disclosed.

[0036] In some variations of the above compositions, the composition is a nutraceutical composition, a dietary supplement, or a pharmaceutical composition. A nutraceutical composition refers to a substance or composition that is considered a food or part of a food that provides a medical or health benefit and can be used to aid in the prevention or treatment of one or more diseases.

[0037] In another embodiment, there is provided a method of treating intramyocellular or subserosal fibroids in a patient in need of treatment, comprising: administering to the patient a therapeutically active composition comprising an admixture of a dried extract of Crina officinalis (Crila®) and epigallocatechin gallate (EGCG), wherein the ratio of the dried extract of Crina officinalis to EGCG in the composition is in the range of 1:10 wt / wt to 100:1 wt / wt. A method is provided.

[0038] In another embodiment, a method of treating, preventing, slowing progression of, and / or ameliorating one or more symptoms of intramyocellular fibroids or subserosal fibroids (uterine leiomyomas) in a subject in need of such treatment or in a subject at risk of having one of intramyocellular fibroids or subserosal fibroids is provided, comprising: administering to a subject a therapeutically active composition comprising a dried extract of Crinum xanthoides (Crila®) and epigallocatechin gallate (EGCG), wherein the ratio of the dried extract of Crinum xanthoides to EGCG in the composition is in the range of 1:10 wt / wt to 100:1 wt / wt, thereby preventing, slowing the progression of, and / or arresting intramural or subserosal fibroids. A method is provided.

[0039] In one embodiment of the above method, in administering Crinum xanthoides dry extract (Crila®) and epigallocatechin gallate (EGCG), the ratio of Crinum xanthoides dry extract to EGCG in the composition is 1:10 wt / wt to 100:1 wt / wt, and the administration is concomitant administration of Crila® and EGCG.

[0040] As used herein, simultaneous administration of Crila® and EGCG means that Crila® and EGCG are administered or given to a patient at the same time or at about the same time, e.g., EGCG is given after Crila®, or Crila® is given after EGCG, or EGCG is given after Crila® on the same day, or Crila® is given after EGCG on the same day.

[0041] In another embodiment of the method, the administration of Crila® is 920 mg, 2 capsules BID (i.e., twice a day), corresponding to 3680 mg per day, for 90 days, and the administration of EGCG is 400 mg, 1 capsule BID, corresponding to 800 mg per day, for 90 days. In another embodiment of the method, the administration of EGCG is 150 mg, 1 capsule BID, corresponding to 300 mg per day, and the administration of Crila® is 920 mg, 2 capsules BID, corresponding to 3680 mg per day, for 90 days. In yet another embodiment of the method, the administration of EGCG is a combination of 150 mg 1 capsule BID, and the administration of Crila® is a combination of 920 mg, 2 capsules BID, corresponding to 3680 mg per day, for 90 days.

[0042] In yet another embodiment of the above method, the dose of Crila® administered is 50 mg to 5,000 mg per day and the dose of EGCG administered is 50 mg to 1,000 mg per day, wherein a composition comprising Crila® and EGCG is administered daily for at least 30 days, 60 days, 90 days, or 120 days.

[0043] Also included in the above embodiments, aspects and variations are salts of amino acids such as arginate, gluconate, and galacturonate.Also provided is a pharmaceutical composition comprising a pharma- ceutically acceptable excipient and a therapeutically effective amount of the composition.

[0044] The pharmaceutical compositions of the invention may be formulated as a powder, such as a lyophilized powder for oral or parenteral administration. The powder may be reconstituted by adding a suitable diluent or other pharma- ceutically acceptable carrier before use. The liquid formulation is generally a buffered, isotonic, aqueous solution. Examples of suitable diluents include normal isotonic saline solution, 5% dextrose in water or buffered sodium or ammonium acetate solution. Such formulations are particularly suitable for parenteral administration, but may also be used for oral administration. Additives such as polyvinylpyrrolidinone, gelatin, hydroxycellulose, acacia, polyethylene glycol, mannitol, sodium chloride, or sodium citrate may be added. Alternatively, the compounds may be encapsulated, tableted, or made into emulsions or syrups for oral administration. Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition or to facilitate preparation of the composition. Liquid carriers include syrup, peanut oil, olive oil, glycerin, saline, alcohol, or water. Solid carriers include starch, lactose, calcium sulfate, dihydrate, terra alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar, silicon dioxide or gelatin. The carrier may also include a sustained release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but may be between about 20 mg and 1 g per dosage unit. Pharmaceutical preparations are produced according to conventional techniques of pharmacy, including milling, mixing, granulation, and optionally compressing for tablets; or milling, mixing, and filling for hard gelatin capsules. When a liquid carrier is used, the preparation will be in the form of a syrup, elixir, emulsion, aqueous suspension or non-aqueous suspension. Such liquid preparations may be administered orally directly or filled into gelatin capsules.

[0045] In some embodiments, the disclosed compositions are formulated into capsules, such as gel capsules. In certain applications, the capsule is a hard capsule, and the capsule shell can comprise gelatin, hydrolyzed starch, or cellulose derivatives, such as hydroxypropylmethylcellulose (hypromellose). In some variations, the capsule can comprise a plurality of particles and one or more pharma- ceutically acceptable additives. The additives can be selected from gelling polymers, fillers, effervescent systems, glidants, ion-exchange resin powders, or combinations thereof; or as disclosed herein. In some variations, the capsule can comprise one or more additives. In some variations, the capsule can comprise one or more gelling polymers, where the gelling polymers are hydrophilic gelling polymers that absorb water or solvent and / or swell to form a viscous mixture or gel. Examples of suitable hydrophilic gelling polymers include cellulose ethers (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, methylcellulose, hydroxyethyl cellulose, etc.), polyalkylene oxides (e.g., polyethylene oxide, polypropylene oxide, etc.), natural gums (e.g., glucomannan, guar gum, gum arabic, tragacanth gum, tara gum, alginate, alginic acid, fucoidan, laminarin, agar, carrageenan, xanthan gum, gellan gum, dextran, welan gum, diutan gum, pullulan, etc.), polyacrylic acid or crosslinked polyacrylic acid (e.g., carbomer), polyvinyl alcohol, polyvinylpyrrolidone, polyamines, or combinations of gelling polymers. The average molecular weight of the gelling polymer may range from about 30,000 to about 15,000,000. When a gelling polymer is used in a capsule, the amount of gelling polymer can range from about 0.1% to about 50% by weight of the capsule contents; or from about 0.1% to 10%, from about 10% to 20%, from about 20% to 30%, from about 30% to 40%, or from about 40% to 50% by weight of the capsule contents.

[0046] In another variation, the capsule may further comprise one or more fillers or diluents. Suitable fillers may include cellulose, microcrystalline cellulose, cellulose derivatives (e.g., calcium carboxymethylcellulose, ethylcellulose), starch (such as potato starch), modified starch, pregelatinized starch, glucose / dextrose, fructose, sucrose, lactose, mannitol, sorbitol, xylitol, calcium carbonate, calcium sulfate, calcium phosphate, calcium silicate, magnesium carbonate, magnesium oxide, or combinations thereof. The amount of the bulking agent may range from about 0.1% to about 50% by weight of the capsule contents; or from about 0.1% to about 10%, about 10% to 20%, about 20% to 30%, about 30% to 40%, or about 40% to 50% by weight of the capsule contents.

[0047] Suitable formulations for each of these methods of administration can be found in Remington: The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition, Lippincott, Williams & Wilkins, Philadelphia, Pa.

[0048] In addition to the exemplary embodiments, aspects and variations described above, further embodiments, aspects and variations will become apparent by reference to the drawings and figures, and by study of the descriptions below.

[0049] Detailed Description of the Invention Definition: Unless otherwise specified herein, the definitions of the terms used are standard definitions used in the art of organic synthesis and pharmaceutical sciences. Exemplary embodiments, aspects and variations are illustrated in the figures and drawings, and it is intended that the embodiments, aspects and variations disclosed herein, as well as the figures and drawings, be considered as illustrative and not limiting.

[0050] "Pharmaceutically acceptable salt" refers to a salt composition that is generally considered to have the desired pharmacological activity, is safe, non-toxic, and acceptable for veterinary and human pharmaceutical use. Such salts include acid addition salts formed with inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, and the like; or organic acids, such as acetic acid, propionic acid, hexanoic acid, malonic acid, succinic acid, malic acid, citric acid, gluconic acid, salicylic acid, and the like.

[0051] "Therapeutically effective amount" means an amount of a compound or agent that elicits any of the biological effects described herein. EXAMPLES

[0052] experiment: The following procedure can be used to prepare the compositions of the present application: The compositions include a mixture of the active ingredients of Crila® powder, as well as selected and isolated ingredients and mixtures of ingredients in Crila® powder.

[0053] Crila® Powder: Crila® contains a dried plant extract of the Amaryllidaceae cultivar Crilae var. crilae Tram & Khanh. The dried plant extract can be obtained from this cultivar in several ways.

[0054] For example, leaves from living plants are picked at the desired time of the growing days and size of the cultivar to optimize the amount and ratio of active ingredients. Plant leaves are generally harvested after about 2-3 years, depending on growing conditions and climate. In one method, the leaves are collected and washed with water to remove dirt and foreign matter. In one variation, the leaves are thoroughly sun-dried for at least 24-48 hours to reduce the moisture content. Alternatively, the leaves are placed in a withering tray and hot air (about 45°C) is directed onto the leaves for the time necessary to obtain dried leaves with the desired moisture content, which is generally less than about 5% water; or less than 5% moisture content is obtained. The moisture content may be measured using the Karl Fischer (KF) method or other standard methods known in the art. Dried leaves may be stored in a dry, dark storage location for up to one or two years.

[0055] Fermentation and Extraction Methods: Next, approximately the same weight of water is added to the dried leaves and the slurry is allowed to stand for about 3 hours. The resulting slurry is then gently pressed or repeatedly rolled with a roller for at least 3 hours to break down the leaf cells and mix the chemical elements. Rolling of the leaves can be carried out at room temperature or below about 40° C. for a desired period of time, such as 6 to 12 hours or up to about 48 hours.

[0056] The resulting aqueous slurry of leaves is then warmed to about 45-50°C and stirred for at least 6-12 hours to allow mixing and the "fermentation process" of the active substances in solution. The slurry is then further processed by an extraction process using a solvent or solvent mixture, such as the addition of water or a mixture of water and an alcohol, such as ethanol. The mixture of water and ethanol can be 10% ethanol in water, 20% ethanol in water, 30% ethanol in water, or about 40% ethanol in water. The resulting water-alcohol slurry is then stirred for at least 3 hours, about 3-12 hours, or up to about 24-30 hours to extract the active substances from the slurry containing the leaves and leaf pieces. The time used in the extraction process is based in part on the water-alcohol ratio and the amount of aqueous slurry being processed.

[0057] Alternatively, the Crinum umbellata plant (or leaf) extract can be obtained by an extraction process using an organic solvent selected from the group consisting of ethanol, methanol, diethyl ether, ethyl acetate, methyl acetate, acetone, MTBE, MEK, ethylene glycol, hexane, heptane, chloroform, dichloromethane or mixtures thereof. In another variation, the Crinum umbellata extract can be obtained by an extraction process using a combination of hot water (about 45°C to 90°C) and ethanol. In another variation, the Crinum umbellata extract can be obtained by an extraction process using a mixture of water and the organic solvent or solvent mixtures mentioned above. In one variation, the extraction solvent is selected from a mixture of solvents such as hot water at about 50°C to 60°C, ethyl acetate and water, ethyl acetate and methanol, and ethyl acetate and hexane.

[0058] When a solvent mixture is used in the extraction process, the more polar solvent in the solvent mixture can be increased over time during the extraction process. For example, when a solvent mixture such as ethyl acetate and methanol is used in the extraction process, the initial solvent mixture can be about 5% methanol in ethyl acetate, and the amount of methanol can be increased stepwise over time, such as in the second or subsequent extraction steps. The increase in the amount of methanol can be, for example, from 5% methanol in ethyl acetate, to 10% methanol, to 20% methanol, to 30% methanol, to 40% methanol, to 50% methanol, to 60% methanol, to 70% methanol, to 80% methanol, and to 90% methanol in ethyl acetate. Alternatively, the extraction can be performed using supercritical extraction with liquid CO2, as known in the art.

[0059] The slurry is then filtered or strained to remove large pulp fibers by filtering or straining through a stainless steel 28 or 38 mesh screen, a 28 or 38 mesh basket strainer, or through an in-line 28 or 38 mesh strainer. The filtrate is agitated for about 3 hours and transferred to a low pressure distillation still where the water and ethanol distillates are distilled under reduced pressure at about 45° C. The resulting brownish green slurry is transferred to a holding tank and optionally a bulk of potato starch and silicon dioxide are added to the slurry and agitated for about 3 hours.

[0060] Isolation by spray drying: The resulting slurry is spray dried using standard parameters known in the art, such as using a spray dryer equipped with a pressure nozzle. The concentration of the plant slurry can be between 10-15% or up to 25% dry matter content. The inlet air temperature, feed temperature and pump speed for spray drying were selected in the ranges of 100-150°C, 5-30°C and 20-40% (50 L / hr-100 L / hr), respectively. The spray dried material is removed from the spray dryer and then stored in a dry holding tank before being analyzed for quality control. If the product passes the desired quality standards, the Crila® powder is encapsulated and bottled.

[0061] Figure 1 shows a representative spectrograph of a dried Crila® powder sample prepared for analysis using a Thermo TSQ Vantage Triple Stage Quadrupole LC / MS / MS / MS system equipped with a Thermo Finnigan TSQ Vantage and a HESI II Source with two mechanical pumps, an Xcalibur Data System, providing optimal assay precision and accuracy. The TSQ Vantage is equipped with an ion source, second generation (G2) ion optics, and a hyperbolic quadrupole, providing high sensitivity with low chemical noise. The spectrum is displayed as a full time trace off of the LC / MS / MS / MS. The chromatograph shows the results of the scanned data. The second graph is the EIC method developed with an internal standard. Separation was performed using a 0.5 x 50 mm Targa C-18 AQ column at a flow rate of 40 μl / min.

[0062] FIG. 2 is an enlarged portion of the spectrograph of FIG. 1, showing the respective A (7.15), B (7.63), C (7.65), D (8.40), E (8.57), F (8.76), G (8.91), H (9.10), I (9.63), J (9.85) and K The peak with the relative retention time (RT, in minutes) labeled as (9.97) A ~ K ) is used to indicate the separation and designation of the various main components of Crila®. As can be observed from the chromatograph, for example peaks C The peak designated (7.65) has at least a second unresolved component (or peak) present at an RT of 7.66.

[0063] The major alkaloid constituents identified from Crila® include lycorine, clinamidin, flexinine, ambellin, epoxyambellin, triphaeridine, 11,12-dehydroanhydro bowdensine, macronine, 1-epidemethybowdensine, lycobetaine, crinine, crinamine or 8-O-demethyl-homolycorine, norgalanthamine, homolycorine, crypowellin B, lycorine dimer, flexinine and 1-O-acetyllycorine. See Nguyen Thi Ngoc Tram et al, Fitoterapia, 2002 Jun; 73(3); 183-208 and Silva, LC et al, Molecules, 2002, May 6; 27(9); 2976. LC / MS chromatograms of the Crila® composition can be used to separate the various alkaloid components and identify the alkaloids or alkaloid mixtures associated with the different fractions that can be isolated and recovered for biological evaluation.

[0064] Figure 3 is a representative graph showing quantification of an extracted peak in a mixture that is crinamidine, showing complete MS data information and confirmatory ions derived from the crinamidine molecule. The system shows transmission of MW 318.156, which is a singly charged ion MW+H of crinamidine with an accurate mass of 317.1257742 Da. Since this system is a triple quadrupole system and not an exact mass system, slight deviations from the true calculated mass are observed. Daughter ions generated after passing through the collision cell are believed to represent loss of C2H3O, or ring fragmentation, resulting in ions with molecular weights of 272.099-272.101 Da that are monitored. Generally, daughter ions are analyzed and identified by injecting the parent molecule directly into solution and optimizing energy and collision cell parameters to generate fragments of the parent molecule. After identifying potential monitoring ions, a method can be developed to analyze complex samples and identify transition ions with the least interference and best overall signal, and these ions can be utilized as quantitative / qualitative ion selections for method validation series.

[0065] The liquid chromatographic (LC) gradient programs for the various solvent systems using formic acid were as shown in the table below, using water (H2O), methanol (MeOH) with 0.1% formic acid. [Table 1]

[0066] Crila® Alkaloid Ingredients for the Treatment of Uterine Fibroids: Based on the results obtained from the spectrographs of Crila® samples analyzed by LC / MS / MS / MS using a Thermo TSQ Vantage Triple Stage Quadrupole LC / MS / MS / MS system, it is possible to design an optimal method for scale-up preparative HPLC isolation. A (7.15), B (7.63),C (7.65), D (8.40), E (8.57), F (8.76), G (8.91), H (9.10), I (9.63), J (9.85), and K (9.97) and their various mixtures ("Crila® Compositions No. 1-20") summarized in Table 1 below were obtained, and the volatile solvents were removed by freeze-drying or lyophilization to obtain alkaloid residues or mixtures of alkaloid residues (also called "Crila® dried extract") for biological evaluation. [Table 2]

[0067] As mentioned above, the ingredients C A particular composition, such as the composition identified as (7.65), i.e., the designation "Crila® composition" (also listed in the table above), may contain, for example, one or more alkaloids or other natural product compounds contained in Crila®. The Crila® compositions described above may lack a distinct active ingredient and constitute a subset of the botanical composition, drug, or complex mixture obtained from the natural product that is Crila® and contains multiple botanical ingredients.

[0068] Cytotoxicity of isolated Crila® compositions: MTS proliferation assay using SK-N-AS cells Day 1: SK-N-AS cells were plated at 5x10 cells per well in 100 μL of appropriate growth medium into Falcon 96-well tissue culture plates, one plate for each sample of Crila® or Crila® composition being tested. 3Column 1 was blank; it contained medium but no cells. Plates were incubated overnight at 37° C., 5% CO2 to allow adhesion.

[0069] Day 2: Add Crila® or Crila® Composition diluted in culture medium to the cells in 4 separate doses at concentrations ranging from 0.005 nM to 10 μM. After 48-72 hours of exposure to Crila® or Crila® Composition, cells are incubated for 48-72 hours at 4°C for 1 h using CellTiter 96. (登録商標) As per Aqueous Non-Radioactive Cell Proliferation Assay (MTS), Promega, MTS agent is added to all wells and incubated for 1-6 hours (37°C, 5% CO2) depending on cell type. Plates are processed using a Bio-Tek Synergy HT Multi-detection microtiter plate reader at a wavelength of 490 nanometers and data processed with KC4V.3 software. Data plots of absorbance versus concentration of Crila® or Crila® compositions are drawn and the concentration resulting in 50% inhibition (IC) is determined for each of the (isolated) Crila® or Crila® compositions tested. 50 ) is extrapolated.

[0070] Crila® or Crila® Composition and EGCG for the Treatment of Uterine Fibroids: Effect of Crila® or Crila® Composition (Table 1) and / or EGCG on Uterine Fibroid Growth: In one study, human leiomyoma (HuLM) cells are used for the initial evaluation of Crila® and / or EGCG treatment. Crila® is used alone or in combination with EGCG to evaluate its ability to inhibit the proliferation of HuLM cells in the experimental approach described below. Similarly, Crila® composition is evaluated for its ability to inhibit the proliferation of HuLM cells in the experimental approach described below.

[0071] Effects of single or combined drug treatment on HuLM cell proliferation: Crila® and / or EGCG were analyzed at various concentrations and time points as specified. [Table 3] [Table 4] [Table 5] As the figure above shows, when Crila® was used alone, it effectively reduced the viability of HuLM cells from concentrations above 1000 μg / ml, but only after 5 and 7 days of treatment, with IC50 values ​​of 971.5±223.9 μg / ml and 902.5±76.2 μg / ml, respectively. This inhibitory effect observed on day 5 was statistically significant (*p-value < 0.05, **p< 0.01). [Table 6] [Table 7]

[0072] As shown in the figure above, when EGCG was used alone, it effectively reduced the viability of HuLM cells from concentrations above 50 μM after 5 days of treatment and from 25 μM after 7 days of treatment. This inhibitory effect observed at these doses on days 5 and 7 was statistically significant (*p-value < 0.05, ****p< 0.0001).

[0073] Because one-day treatment with both agents produced some inconsistent data, the following studies treating cells with combined drugs focused on 2, 3, 5, and 7-day treatments. The treatment design and results with Crila® in combination with EGCG are shown below. [Table 8] [Table 9]

[0074] As shown in the figures above, when combined with 50 μM EGCG (hatched box), all tested Crila® concentrations effectively reduced the viability of HuLM cells after 3, 5, and 7 days of treatment compared to Crila® alone (white box). This inhibitory effect, which continued from day 3 to day 7 at these doses combined with EGCG, was statistically significant (*p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001, respectively). These experiments were repeated twice separately.

[0075] The results show that when combined with EGCG, Crila® at test concentrations of 100-1000 μg / ml for 3-7 days showed a higher inhibitory effect on HuLM viability, which was seen from the lowest concentration of 100 μg / ml Crila® at 3 days of treatment.

[0076] Similarly, Crila® compositions are evaluated for their ability to inhibit HuLM cell proliferation in experimental approaches as described above, at concentrations of 100-1000 μg / ml showing a higher inhibitory effect on HuLM viability when combined with EGCG for 3-7 days of treatment.

[0077] Synergistic inhibitory effect of Crila® or Crila® Composition and EGCG on HuLM cell proliferation: To further explore whether treatment with Crila® combined with EGCG has a synergistic effect on the viability of HuLM cells, based on the above test, we focused on treatment with 10 μM, 25 μM, and 50 μM EGCG for 3 days, 5 days, and 7 days in the following test. Treatment design and results of Crila® combined with EGCG are shown below. [Table 10] [Table 11]

[0078] As shown in the above figure, when combined with 25 μM and 50 μM EGCG, all tested Crila® concentrations showed synergistic inhibitory effects on HuLM cell viability after 3, 5 and 7 days of treatment. However, when combined with 10 μM EGCG, the tested Crila® concentrations did not show consistent synergistic inhibitory effects on HuLM cell proliferation, especially the third point (500 μg / ml Crila® combined with 10 μM EGCG), which had the opposite effect. These experiments were repeated twice separately.

[0079] We conclude that Crila® in combination with EGCG showed synergistic inhibitory effects on HuLM viability when treated for 3-7 days at test concentrations of 100-1000 μg / ml for Crila and 25-50 μM for EGCG. These effects were not consistently seen when Crila® was combined with 10 μM EGCG.

[0080] Similarly, the Crila® composition in combination with EGCG produces a synergistic effect on the viability of HuLM cells at 3, 5 and 7 days of treatment using 10 μM, 25 μM and 50 μM EGCG.

[0081] Crila and EGCG treatment inhibit HuLM cell proliferation by decreasing proliferation but not by increasing apoptosis: Based on the established complex concentrations, we assessed whether the inhibition of HuLM cell proliferation was caused by the suppression of proliferation markers such as PCNA or the pro-apoptotic marker BAX or both, at both mRNA and protein levels, using qRT-PCR and Western blotting analysis, respectively. [Table 12] [Table 13]

[0082] The real-time RT-PCR results in the figure above showed that when combined with 50 μM EGCG, Crila® used at concentrations of 100-500 μg / ml resulted in a significant decrease in the levels of the proliferation marker PCNA mRNA compared to single agent treatment, however this effect reached a plateau at this point as Crila® at 1000 μg / ml in combination with 50 μM EGCG did not result in a more significant decrease in PCNA expression compared to Crila® 1000 μg / ml alone.

[0083] Real-time RT-PCR analysis of BAX showed that when combined with 50 μM EGCG, 1000 μg / ml Crila® alone significantly increased levels of the apoptotic marker BAX mRNA compared to single agent treatment, but this effect was not seen with less than 1000 μg / ml Crila® in combination with 50 μM EGCG.

[0084] Similarly, real-time RT-PCR analysis of BAX suggests that when combined with 50 μM EGCG, 1000 μg / ml Crila® composition alone significantly increases levels of the apoptotic marker BAX mRNA compared to single-agent treatment.

[0085] We also tested both markers expression at the protein level. As shown in the figure below, the results of the Western blot analysis showed that when combined with 50 μM EGCG, Crila® at the concentrations of 100-250 μg / ml used in this study significantly reduced the levels of the proliferation marker PCNA protein compared to single agent treatment. This effect reached a maximum at this time point, since Crila® at 500 μg / ml and 1000 μg / ml combined with 50 μM EGCG did not result in a significant reduction of PCNA protein compared to Crila® alone. For BAX, the combination of Crila® with 50 μM EGCG caused a significant increase in BAX protein levels compared to single agent treatment. [Table 14] [Table 15] [Table 16] [Table 17]

[0086] Based on these studies, combined Crila® and EGCG treatment inhibited HuLM cell proliferation by reducing proliferation, but not by increasing apoptosis. Real-time RT-PCR and Western blot data confirmed these results.

[0087] Similarly, it is observed that the combined treatment of Crila® composition with EGCG inhibits the proliferation of HuLM cells by reducing their proliferation. Real-time RT-PCR and Western blot data can be used to confirm these results.

[0088] material and method: Reagent preparation: 250 mg of dried Crila® powder was dissolved in 50 ml of cell culture medium (DMEM / F12 with 10% FBS) with constant stirring at room temperature for 1 h. The extract was then sterile filtered through a 0.22 μm PES filter and stored at room temperature until further use. EGCG was dissolved in DMSO to a 100 mM stock solution or in dH2O to a 10 mM stock solution.

[0089] Cell proliferation assay: Cell proliferation assay was performed by dimethylthiazolyl diphenyltetrazolium bromide (MTT) assay.

[0090] Human uterine leiomyoma (HuLM) cells were cultured in DMEM / F12 containing 10% fetal bovine serum. HuLM cells were seeded at 3000 cells / well in 96-well plates and then treated with various concentrations for various time points as described in the figures below, with DMSO vehicle added to control wells.

[0091] Samples (from treated and control wells) were measured after 1-2, 3, 5 and 7 days. Medium was replaced with fresh complete medium every 48 h. Relative cell viability was determined using the MTT assay.

[0092] At each time point, cell supernatant was removed and 150 μl of 0.5 mg / ml MTT solution in PBS was added to each well, and the plate was incubated at 37°C for 4 h, then aspirated and 200 μl of dimethyl sulfoxide (DMSO) was added to each well and gently agitated on a shaker for 15 min to 1 h, protected from light, until the insoluble formazan was dissolved in DMSO. Absorbance was measured at 570 nm on a Varioskan Lux microplate reader (ThermoFisher).

[0093] Data are presented as % survival relative to vehicle-only control for each drug or drug combination at each time point. Each data point is the mean ± SEM of quadruplicate determinations. *p < 0.05, **p < 0.01 ***p < 0.001, ****p < 0.0001.

[0094] Combination Index To determine synergy, various combinations of doses and time courses of Crila® and EGCG were selected based on the time courses of single and combined treatments, and combination index (CI) scores were calculated using the Chou-Talalay method and CompuSyn software. For this analysis, the combined treatment data along with data obtained from previous single treatments were entered into CompuSyn to determine the CI value for each combination point. The CI value quantitatively defines antagonism (CI > 1.5), additivity (1 < CI < 1.5) and synergy (CI < 1), and the results are presented as classical isobolograms.

[0095] Western Blot Analysis: Proteins were harvested after 48 h of treatment, extracted by sonication on ice and dissolved in RIPA buffer (Sigma). Proteins were separated by SDS-PAGE and then transferred to PVDF membranes (Bio-Rad). Membranes were blocked with 5% skim milk in TBS-T (150 mmol / L NaCl, 10 mmol / L TRIS, PH 7.6 and 0.1% TWEEN-20) for 1 h at room temperature and incubated with primary antibodies overnight at 4°C. All primary antibodies were obtained from Genetex and Abcam. Membranes were then washed three times for 10 min with TBS-T and incubated with the appropriate HRP-conjugated secondary antibodies for 1 h at room temperature. Proteins were then measured using ChemiDoc (Bio-Rad). Bands were quantified using Fiji software.

[0096] Quantitative real-time PCR: Total RNA was harvested from cells after 48 hours of treatment and extracted using RNeasy Plus mini kit (Qiagen). cDNA was synthesized by SuperScript Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Target expression levels were detected using SsoAdvanced SYBR Green supermix (Bio-Rad). The primers used in this study are shown in the table below. Data were obtained as Ct values ​​and used to determine ΔCt values ​​(Ct of target gene - Ct of housekeeping gene GAPDH). These values ​​were used to calculate the mean ΔCt value ± SD for each treatment and used for statistical comparison. Visual representation of the data was performed using the formula 2- ΔΔ This was done by converting the ΔCt values ​​to fold change data relative to the ΔCt values ​​for control vehicle only cells using Ct. [Table 18]

[0097] Statistical analysis: Statistical analysis was performed using GraphPad Prism, and data were expressed as mean ± SD. Means of data from two or more groups were compared using two-way analysis of variance (ANOVA) followed by Tukey's. *p < 0.05, **p < 0.01, ***p < 0.001.

[0098] Supplementary Data: Cell viability results from separate studies combining Crila® with 50 μM EGCG for 2, 3, 5 and 7 days of treatment are shown below, with the Y-axis showing relative cell viability compared to control vehicle-only treatment.

[0099] Separate Test 1: [Table 19]

[0100] Separate Test 2: [Table 20]

[0101] Synergy results from separate studies of Crila® with 10 μM, 25 μM, and 50 μM EGCG for 3, 5, and 7 days are shown below, with combination index (CI) data corresponding to the isobolograms shown next to each isobologram, respectively.

[0102] Separate Test 1: [Table 21]

[0103] Separate Test 2: [Table 22]

[0104] In one embodiment, the present application discloses a study in human leiomyoma cells and describes that compositions containing Crila® and EGCG, such as ABC-105 and ABC-205, which are combination formulations of Crila® and EGCG, show an unexpected synergistic effect of significantly reducing cell viability in patients with uterine fibroids. ABC-105 constitutes a composition containing 920 mg of Crila® and 400 mg of EGCG formulated in a gel cap. ABC-205 constitutes a composition containing 920 mg of Crila® and 150 mg of EGCG formulated in a gel cap.

[0105] Similarly, a combined formulation of the Crila® composition and EGCG shows an unexpected synergistic effect in significantly reducing cell viability in patients with uterine fibroids.

[0106] A hepatic safety analysis must be performed before initiating a randomized, double-blind, placebo-controlled, multicenter prospective clinical trial of ABC-105 / ABC-205 in women with uterine fibroids to evaluate changes in the size of uterine fibroids (UFs) and related symptoms. Hepatic safety is of paramount concern, as therapeutic agents, mainly biologics, that target uterine fibroids often face problems with their hepatic safety profile. The hepatic safety of the combination treatment of ABC, Crila® and EGCG is unknown. A four-arm study may be performed in women with uterine fibroids to allow comparison between these groups. The results of this study may be used to confirm the hepatic safety of a larger multicenter ABC study.

[0107] Results: In patients with uterine fibroids of various stages, levels, and sizes, the size of the uterine fibroids is reduced by 10% to 30% after 30 days of treatment. In some studies, it has been observed that in patients with uterine fibroids of various sizes, the size of the uterine fibroids is reduced by 10% to 50% after 60 days of treatment. Furthermore, in patients with uterine fibroids of various sizes, the size of the uterine fibroids is reduced by 50% to more than 90% after 90 days of treatment.

[0108] While numerous exemplary embodiments, aspects, and variations are provided herein, those of skill in the art will recognize certain modifications, permutations, additions, and combinations, and specific subcombinations, of the embodiments, aspects, and variations, and it is intended that the claims below be construed to include all such modifications, permutations, additions, and combinations, and specific subcombinations, of the embodiments, aspects, and variations.

[0109] The entire disclosures of all documents cited throughout this application are hereby incorporated by reference.

Claims

1. A composition comprising an aqueous solution containing a dried extract of Indian Crinum (Crinum latifolium L.) var. crilae Tram & Khanh cultivar at a concentration of 100 μg / mL to 1000 μg / mL and epigallocatechin gallate (EGCG) at a concentration of 10 μM to 50 μM.

2. 2. The composition of claim 1, wherein the solution of dried extract of Crinum var. crilae Tram & Khanh cultivar is present in a concentration of 100 μg / mL to 250 μg / mL.

3. 2. The composition of claim 1, wherein the EGCG in the composition is present at a concentration of 10 μM to 25 μM.

4. 2. The composition of claim 1, wherein the solution of the dried extract of Crinum var. crilae Tram & Khanh cultivar is present at a concentration selected from 100 μg / mL, 250 μg / mL, 500 μg / mL and 1000 μg / mL, and the EGCG is present at a concentration selected from 10 μM, 25 μM and 50 μM.

5. 2. The composition of claim 1, wherein the solution of dried extract of Crinum var. crilae Tram & Khanh cultivar is present in a concentration of 250 μg / mL to 500 μg / mL.

6. 2. The composition of claim 1, wherein the solution of dried extract of Crinum var. crilae Tram & Khanh cultivar is present in a concentration of 500 μg / mL to 1000 μg / mL.

7. 2. The composition of claim 1, wherein the solution of dried extract of Crinum var. crilae Tram & Khanh cultivar is present in a concentration of 100 μg / mL to 500 μg / mL.

8. 2. The composition of claim 1, wherein the solution of dried extract of Crinum var. crilae Tram & Khanh cultivar is present in a concentration of 250 μg / mL to 1000 μg / mL.

9. 2. The composition of claim 1, wherein the EGCG in the composition is present at a concentration of 25 μM to 50 μM.

10. 1. A composition comprising a dried extract of Crinum umbellata var. crilae Tram & Khanh cultivar and dried epigallocatechin gallate (EGCG), wherein, when the composition is administered to a patient, the cultivar has a maximum serum concentration of 100 μg / mL to 1000 μg / mL and the EGCG has a maximum serum concentration of 10 μM to 50 μM.

11. i) to upregulate BAX mRNA expression in a cell or a patient; or ii) to reduce the expression of PCNA in a cell or a patient; or iii) To inhibit the proliferation of HuLM cells in tissues or patients. The composition according to any one of claims 1 to 10.

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