Treating fibroids with drugs such as vitamin D and epigallocatechin gallate (EGCG)

Abstract

The present invention relates to the use of vitamin D and an agent selected from epigallocatechin gallate (EGCG), catechins, epigallocatechin gallate and epicatechin gallate (ECG) for preventing, controlling and / or treating fibroids in a subject.

Description

Field of the Invention

[0001] The present invention relates to the use of vitamin D and one or more agents selected from epigallocatechin gallate (EGCG), catechins, epigallocatechin gallate and epicatechin gallate (ECG) for preventing, controlling and / or treating fibroids in a subject. Background Art

[0002] Uterine fibroids, also known as uterine leiomyoma or myoma, are the most common tumors of the female reproductive tract. They are monoclonal benign tumors of uterine smooth muscle, and therefore originate from the myometrium of women of childbearing age (mainly between 35 and 50 years old). Uterine fibroids are well-known estrogen-dependent tumors in the uterus of premenopausal women. The hallmark of uterine leiomyoma is excessive deposition of extracellular matrix (ECM) [Malik M, et al. Matrix Biol 2012, 7-8: 389–397]. The ECM components that make up most of the volume of uterine fibroids are fibronectin, collagen, and proteoglycans [Ingber D and Folkman J. J Cell Biol 1989, 109: 317–330]. Like other tissues in the body, the ECM action in uterine fibroids stimulates cell-cell adhesion, cell migration, cell proliferation, and cell differentiation and promotes further deposition of ECM [Ingber D and Folkman J. J Cell Biol 1989, 109: 317–330]. The ECM in uterine tissue, both in normal myometrium and in fibroids, contains a large amount of collagen. The excess ECM in uterine fibroids is due to the overproduction of collagen and the reduction of collagen breakdown, and collagen deposition accounts for a greater proportion of the ECM components in uterine fibroids than in healthy uterine tissue [Leppert PC, et al. Fertil Steril 2004, 82: 1182–1187]. The increased deposition of collagen and fibronectin is partly due to the altered physical properties of leiomyoma tissue relative to healthy myometrium. The abnormal orientation and organization of collagen in the stromal matrix of uterine fibroids contributes to its rigid properties [Tomasek JJ, et al. Nat Rev Mol Cell Biol 2002, 3: 349–363]. The smooth muscle cells of the myometrium can respond to injury by synthesizing extracellular fibrous matrix.

[0003] Uterine fibroids usually develop without symptoms. They may also be associated with different symptoms, such as: excessive menstrual bleeding (menorrhagia), dysmenorrhea and intermenstrual bleeding, chronic pelvic pain, compression of surrounding organs, pain during sexual intercourse, anemia and stress symptoms, such as a feeling of swelling, increased urination frequency and bowel disturbances. In addition, they may impair reproductive function and may lead to subfertility, early pregnancy loss, late pregnancy complications, infertility, recurrent miscarriage, and premature birth.

[0004] The impact of uterine fibroids on quality of life and total cost of treatment has attracted worldwide attention.

[0005] Other fibroid diseases include fibroadenomas and prostatic hyperplasia.

[0006] Fibroadenomas are benign human breast tumors characterized by proliferation of epithelial and fibroblastic stromal elements of the terminal ductal units.

[0007] Prostatic hyperplasia, also known as benign prostatic hyperplasia (BPH), prostatic enlargement, or prostatic leiomyoma, is a noncancerous growth in the size of the prostate gland with increased deposition of extracellular matrix, or fibrous collagen.

[0008] Therefore, there remains a need to provide non-invasive and effective therapeutic approaches for the treatment of fibroids.

[0009] EGCG

[0010] Epigallocatechin gallate (EGCG) is an ester of epigallocatechin and gallic acid extracted from green tea. Its role in tumors has been thoroughly studied and has shown beneficial effects. EGCG inhibits proliferation, induces apoptosis in human uterine fibroid cells, and reduces the volume of uterine fibroid tumors in mice [Zhang, D. et al., Am. J. Obstet. Gynecol. 2010, 202, 289.e1–289.e9]. A clinical study showed that women with uterine fibroids had a 32.6% reduction in total fibroid volume after receiving EGCG treatment [E. Roshdy et al., Int. J. Women's Health, 2013: 5 477-486]. In these studies, EGCG is a regulator of proliferation, transformation, and inflammation of uterine fibroid tumors. EGCG exerts its effects by possibly regulating different factors involved in proliferation, cell cycle regulation, and apoptosis, such as proliferating cell nuclear antigen (PCNA), cyclin-dependent kinase 1 (CDK1), and B-cell lymphoma 2 (BCL2).

[0011] Vitamin D

[0012] Vitamin D is a group of fat-soluble secosteroids that exert different biological activities. Their role in increasing intestinal absorption of calcium, magnesium and phosphate is well known. In fact, vitamin D changes bone cell activity and is important for new bone formation in children and adults. Vitamin D is mainly produced in the skin from sunlight, and about 10% is also absorbed from dietary intake. The liver and kidneys convert vitamin D into the active hormone, called calcitriol.

[0013] 1,25-Dihydroxyvitamin D has been shown to inhibit uterine fibroid cell proliferation in vitro and reduce uterine fibroid size in animal models. Vitamin D (vitamin D3) reduces uterine fibroid cell proliferation in vitro and reduces uterine fibroid tumor growth in in vitro animal models. Based on in vitro experiments, uterine fibroid cells are highly sensitive to the growth inhibitory effects of the active form of vitamin D, which modulates cell proliferation and induces apoptosis by downregulating proliferating cell nuclear antigen (PCNA) and cyclin-dependent kinase 1 (CDK1).

[0014] Vitamin D deficiency is associated with an increased risk of uterine fibroids, particularly an inverse relationship between vitamin D serum levels and fibroid severity. SUMMARY OF THE INVENTION

[0016] The authors surprisingly found that the combined use of vitamin D and at least one selected from epigallocatechin gallate (EGCG), catechins, epigallocatechin gallate and epicatechin gallate (ECG) has a strong inhibitory effect on fibronectin and collagen release in fibroblasts in vitro. The combination of vitamin D and the at least one agent exerts a synergistic effect, reducing the proliferation of fibrotic components and then reducing the excessive deposition of extracellular matrix commonly found in fibroids. The increased efficacy of vitamin D in combination with the at least one agent has a major effect on the reduced proliferation of fibrotic components compared to the efficacy of vitamin D alone or the at least one agent alone.

[0017] In other words, the authors surprisingly found that the combination or co-administration of vitamin D and at least one agent selected from EGCG, catechins, epigallocatechin gallate and ECG for the treatment of fibroblasts exerts a more favorable effect than either of the individual components. To date, it has been known that the administration of vitamin D or EGCG can be used to treat uterine fibroids, but it was not known or suggested that these compounds could be used together to treat such fibroid conditions more advantageously. The evidence of a reduction in fibroid volume demonstrates an absolutely novel and achievable beneficial effect of using vitamin D and at least one agent selected from EGCG, catechins, epigallocatechin gallate and ECG.

[0018] Therefore, an object of the present invention is vitamin D and one or more agents selected from epigallocatechin gallate (EGCG), catechins, epigallocatechin gallate and epicatechin gallate (ECG) for the prevention, control and / or treatment of fibroids. Preferably, the one or more agents are selected from the group consisting of epigallocatechin-3-gallate, (-)-epigallocatechin-3-gallate, (-)-epicatechin-3-O-gallate (GCG), (-)-epicatechin-3-gallate, (+)-catechin (C), (-)-epicatechin (EC), (+)-epicatechin (GC), (-)-epicatechin (EGC), (-)-epicatechin-3-gallate, (-)-catechin-3-gallate and (+)-catechin-3-gallate. Preferably, the one or more agents are epigallocatechin gallate (EGCG). Preferably, the vitamin D is selected from the group consisting of vitamin D3 (cholecalciferol), vitamin D2 (ergocalciferol), 25(OH) vitamin D (calcifediol), 1,25 dihydroxyvitamin D (calcitriol) and combinations thereof. Preferably, vitamin D refers to vitamin D3.

[0019] Preferably, the fibroid is in a tissue containing fibroblasts. Preferably, the fibroid is a uterine fibroid, a fibroadenoma or a prostatic hyperplasia. Preferably, the fibroid is a uterine fibroid. Preferably, the uterine fibroid is intramural, subserosal and / or submucosal.

[0020] Preferably, the amount of vitamin D per day is about 1 μg to 100 μg. Preferably, the amount of vitamin D per day is about 25 μg to 50 μg, preferably the amount per day is about 50 μg. Preferably, vitamin D is administered twice a day, each administration of about 25 μg. Preferably, 1 IU vitamin D corresponds to 0.025 μg vitamin D, so that the amount of vitamin D is about 40 IU to 4000 IU.

[0021] Preferably, the amount of the one or more agents is about 5 mg to 1000 mg per day. Preferably, the amount of the one or more agents is about 300 mg per day. Preferably, the one or more agents are administered twice a day, each administration being about 120 mg to 180 mg, preferably about 150 mg. It should be understood that when more agents are present, the amount of the one or more agents may refer to each of the agents or to the sum of all agents present.

[0022] Preferably, the weight ratio of vitamin D to the one or more agents is about 1:50 to 1:1000000. Preferably, the weight ratio of vitamin D to the one or more agents is about 1:100 to 1:1000, preferably the weight ratio of vitamin D to the one or more agents is about 1:300 to 1:600. Preferably, the weight ratio of vitamin D to the one or more agents is about 1:6000, 1:50, 1:2000, 1:2500, 1:3000, 1:5000, 1:7000, 1:10000 or 1:1000000. It should be understood that when more agents are present, the weight ratio of one or more agents may refer to each of the agents or to the sum of all agents present.

[0023] In a preferred aspect, vitamin D and one or more agents as defined above are used together with at least one other agent, which is optionally selected from the group consisting of vitamin B, polyphenols and DL-α-tocopherol, preferably the at least one other agent is vitamin B6. Then, the present invention also provides vitamin D, one or more agents as defined above, together with at least one other agent, which is optionally selected from the group consisting of vitamin B, polyphenols and DL-α-tocopherol, preferably the at least one other agent is vitamin B6, for the use as defined above.

[0024] Preferably, the weight ratio of vitamin D, the one or more medicaments and the at least one other medicament is about 1:5000:100 to 1:10000:1000. Preferably, the weight ratio of vitamin D, the one or more medicaments and the at least one other medicament is about 1:5000:1000 to 1:10000:100. Preferably, the weight ratio of vitamin D, the one or more medicaments and the at least one other medicament is about 1:6000:200, 1:5000:1000, 1:10000:100 or 1:10000:1000.

[0025] Preferably, the at least one other agent is present in an amount of about 1 to 100 mg per day, preferably about 1 to 10 mg per day, preferably about 1 to 5 mg per day, preferably about 1, 5 or 10 mg per day.

[0026] Preferably, the other agent is vitamin B6. Preferably, the amount of vitamin B6 per day is about 1 to 100 mg, preferably about 1 to 10 mg per day, preferably about 1 to 5 mg per day, preferably about 1, 5 or 10 mg per day. Preferably, vitamin B6 is administered twice a day, each administration is about 5 mg.

[0027] In a preferred embodiment, the present invention provides vitamin D and one or more agents for use as defined above, wherein the one or more agents are epigallocatechin gallate (EGCG) and / or wherein the fibroid is a uterine fibroid. Then, in a preferred embodiment, the present invention provides vitamin D and epigallocatechin gallate (EGCG) for use in treating uterine fibroid.

[0028] Preferably, the one or more agents are in the form of tea leaves or extracts of tea leaves. Preferably, the tea leaves are green tea leaves. Preferably, the tea leaves contain about 30% to 95% by weight, preferably about 30%, 45%, 50% or 95% by weight of the one or more agents. It should be understood that when more agents are present, the weight % of one or more agents in the leaf may refer to each agent or to the sum of all agents present.

[0029] In a preferred embodiment of the above use, the vitamin D and the one or more agents reduce the release of fibronectin and / or collagen.

[0030] Another object of the present invention is vitamin D and one or more agents selected from epigallocatechin gallate (EGCG), catechins, epigallocatechin gallate and epicatechin gallate (ECG), for use in a method of reducing the release of fibronectin and / or collagen.

[0031] Preferably, the reduction in fibronectin and / or collagen release is determined by comparison to a suitable control group with no treatment at all, treatment with vitamin D alone, or treatment with one or more agents alone.

[0032] In a preferred embodiment of the above use, the vitamin D and the one or more agents reduce the volume of fibroids.

[0033] Another object of the present invention is vitamin D and one or more agents selected from epigallocatechin gallate (EGCG), catechins, epigallocatechin gallate and epicatechin gallate (ECG), for use in a method of reducing the volume of fibroids.

[0034] Preferably, the reduction in fibroid volume is measured by comparison to a suitable control group with no treatment at all, treatment with vitamin D alone, or treatment with one or more agents alone.

[0035] Preferably, the fibroid is a uterine fibroid, and the prevention, control and / or treatment of uterine fibroids comprises the prevention, control and / or treatment of at least one symptom of uterine fibroids. Preferably, the symptoms of uterine fibroids are selected from the group consisting of: excessive menstrual bleeding (menorrhagia), dysmenorrhea, intermenstrual bleeding, pelvic pain (acute or chronic), dyspareunia, anemia, impaired reproductive function, subfertility, infertility, early pregnancy loss, recurrent miscarriage, premature birth and pregnancy complications, fatigue and stress symptoms such as swelling, increased urination, and bowel disorders.

[0036] Preferably, vitamin D and the one or more pharmaceutical agents are administered simultaneously and / or orally and / or twice a day.

[0037] Preferably, vitamin D and the one or more agents are used in combination with other therapeutic interventions. Preferably, the other therapeutic interventions are selected from the group consisting of surgery, gonadotropin-releasing hormone agonists (GnRHa), selective progesterone receptor modulators (sPRMs), anti-hormonal agents, hormones, anti-inflammatory analgesics and tranexamic acid.

[0038] Preferably, the surgery is selected from the group consisting of: conservative surgical therapy (myomectomy), hysterectomy, myolysis, uterine artery embolization (UAE) and magnetic resonance imaging guided focused ultrasound surgery (MRgFUS). Preferably, the hormone is estrogen, progesterone or progesterone. Preferably, the anti-inflammatory analgesic is a non-steroidal anti-inflammatory drug (NSAID). Preferably, the selective progesterone receptor modulator is ulipristal acetate (UPA).

[0039] Another object of the present invention is a composition comprising:

[0040] - Vitamin D,

[0041] - one or more agents selected from the group consisting of epigallocatechin gallate (EGCG), catechins, epigallocatechin gallate and epicatechin gallate (ECG),

[0042] - an excipient or diluent, and

[0043] -optionally at least one other pharmaceutical agent.

[0044] It should be understood that any of the amounts, weight ratios, weight %'s, definitions of one or more agents, definitions of other agents, and definitions of fibroids described above also apply to the composition.

[0045] Preferably, the composition is used for preventing, controlling and / or treating fibroids, preferably the fibroids are in tissues containing fibroblasts, preferably the fibroids are uterine fibroids, fibroadenomas or prostate hyperplasia.

[0046] Preferably, in the composition, the one or more agents are selected from the group consisting of epigallocatechin-3-gallate, (–)-epigallocatechin-3-gallate, (–)-gallocatechin-3-O-gallate (GCG), (–)-gallocatechin-3-gallate, (+)-catechin (C), (–)-epicatechin (EC), (+)-gallocatechin (GC), (–)-epigallocatechin, (–)-epicatechin-3-gallate, (–)-catechin-3-gallate and (+)-catechin-3-gallate. Preferably, the one or more agents in the composition are the agent epigallocatechin gallate (EGCG). Preferably, the vitamin D is selected from the group as defined above.

[0047] Preferably, in the composition, the one or more agents are in the form of tea leaves or extracts of tea leaves as defined above. Preferably, the amount of vitamin D in the composition is about 1 μg to 100 μg, preferably about 25 μg or 50 μg. Preferably, the amount of the one or more agents in the composition is about 5 mg to 1000 mg, preferably about 300 mg or 120 mg to 180 mg, preferably about 150 mg. Preferably, in the composition, the weight ratio of vitamin D to the one or more agents is about 1:50 to 1:1000000, preferably about 1:6000, 1:50, 1:2000, 1:2500, 1:3000, 1:5000, 1:7000, 1:10000 or 1:1000000.

[0048] Preferably, the excipient and / or diluent is selected from the group consisting of calcium phosphate, dicalcium phosphate, microcrystalline cellulose, magnesium stearate, silicon dioxide, sucrose, gum arabic, corn starch, medium chain triglycerides, tricalcium phosphate, crosslinked sodium carboxymethyl cellulose, hydroxypropyl methylcellulose, polyethylene glycol, titanium dioxide, polyvinyl pyrrolidone, talc, erythritol, xylitol, steviol glycosides and sucralose.

[0049] Preferably, the at least one optional other agent in the composition is selected from the group consisting of vitamin B, polyphenols and DL-α-tocopherol. Preferably, the weight ratio of vitamin D, the one or more agents and the at least one other optional agent in the composition is about 1:5000:100 to 1:10000:1000, preferably about 1:6000:200, 1:5000:1000, 1:10000:100 or 1:10000:1000. Preferably, the amount of at least one other optional agent in the composition is about 1 to 100 mg per day, preferably about 1 to 10 mg per day, preferably about 1 to 5 mg per day, preferably about 1, 5 or 10 mg per day. Preferably, the at least one optional other agent in the composition is vitamin B6, preferably in an amount of about 1 to 100 mg per day, preferably about 1 to 10 mg per day, preferably about 1 to 5 mg per day, preferably about 1, 5 or 10 mg per day.

[0050] Preferably, the composition is administered orally. Preferably, the composition is administered once or twice a day.

[0051] Preferably, the composition is in the form of a tablet, hard capsule, soft gel capsule, powder, syrup, cachet, troche, lozenge or pastille.

[0052] Preferably, the composition is for use in combination with other therapeutic interventions. Preferably, the other therapeutic interventions are selected from: surgery, gonadotropin-releasing hormone agonists (GnRHa), selective progesterone receptor modulators (sPRMs), anti-hormonal agents, hormones, anti-inflammatory analgesics and tranexamic acid, all of which may preferably be as defined above.

[0053] Preferably, the composition is a pharmaceutical composition or a supplement.The present invention then also provides a pharmaceutical composition or a supplement comprising the components defined above for the composition.

[0054] Where the composition is a pharmaceutical composition, the excipient and / or diluent is preferably pharmaceutically acceptable. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 and 2Represents the release of fibronectin and collagen in cultured fibroblasts, respectively. Control = fibroblasts after 18 days of culture (no TGF-β1 stimulation and no treatment); 24h = fibroblasts after 18 days of culture, followed by 24 hours of TGF-β1 stimulation, and empty fresh medium added in the following 24h; D3 = fibroblasts after 18 days of culture, followed by 24 hours of TGF-β1 stimulation, and treatment with vitamin D3 in the following 24h; EGCG = fibroblasts after 18 days of culture, followed by 24 hours of TGF-β1 stimulation, and treatment with vitamin EGCG in the following 24h; D3+EGCG = fibroblasts after 18 days of culture, followed by 24 hours of TGF-β1 stimulation, and treatment with a combination of vitamin D3 and EGCG in the following 24h.

[0056] Figure 1 The effects of vitamin D and EGCG alone or in combination on the production of fibronectin in fibroblasts in vitro after 24 hours of incubation are shown: Vitamin D alone slightly increased the production of fibronectin. EGCG alone reduced the production of fibronectin. The combination of vitamin D and EGCG significantly reduced the production of fibronectin.

[0057] Figure 2 The effects of vitamin D and EGCG alone or in combination on collagen production in fibroblasts in vitro after 24 hours of incubation are shown: Vitamin D alone reduced collagen production. EGCG alone reduced collagen production. The combination of vitamin D and EGCG significantly reduced collagen production. The combination of vitamin D plus EGCG increased the reduction in collagen.

[0058] Figure 3 Shown is the release of fibronectin in cultured fibroblasts after 24 hours in the presence of vitamin D3 (A) or EGCG (B) at different concentrations. Control = fibroblasts cultured for 18 days, followed by 24 hours of TGF-β1 stimulation and empty fresh medium added in the following 24h.

[0059] Figure 4 Shown is the release of collagen in cultured fibroblasts after 24 hours in the presence of vitamin D3 (A) or EGCG (B) at different concentrations. Control = fibroblasts cultured for 18 days, followed by 24 hours of TGF-β1 stimulation and empty fresh medium added in the following 24h.

[0060] Figure 5 and 6Represents the release of fibronectin and collagen in cultured fibroblasts after 24 hours in the presence of a combination of vitamin D3 and EGCG. Control = fibroblasts cultured for 18 days, followed by 24 hours of TGF-β1 stimulation and empty fresh medium added in the following 24 hours.

[0061] In the x-axis label, the first number (before the slash) refers to the concentration of vitamin D3 in ug / L, and the second number (after the slash) refers to the concentration of EGCG in mg / L. For example, 5 / 3 means that 5 ug / L of vitamin D3 and 3 mg / L of EGCG are administered.

[0062] Figure 7 The volume of myomas (i.e. uterine fibroids) at baseline (T0) and after a 4-month study period (T1) is shown for the treated and control patient groups. The volume of myomas is in cm 3 Mean ± SEM. Treatment group = Vitamin D + EGCG for 4 months; Control group = No treatment for 4 months. p-value ≤ 0.05 was considered statistically significant. *Significant T1 vs T0. Statistical test = Unpaired t-test.

[0063] Figure 8 Shown are mean symptom severity (SS) scores for women with fibroids. Treatment group (vitamin D + EGCG for 4 months); control group (no treatment for 4 months); T0 = baseline; T1 = after the 4-month study period. p-values ​​≤ 0.05 were considered statistically significant. *Significant T1 vs T0. Statistical test = unpaired t-test.

[0064] Fig. 9 Figure 2 Changes in quality of life (QoL) and symptom severity (SS) in women with fibroids are shown. Data are presented as box plots. A: Change in quality of life (QoL). B: Change in symptom severity (SS). The change between the changes from T0 to T1 in each group (i.e., the difference between the score at T1 - the score at T0) was determined by evaluating the patients' responses in the questionnaire. Treatment group (vitamin D + EGCG for 4 months); control group (no treatment for 4 months). Wilcoxon–Mann–Whitney test was used for analysis. p-value ≤ 0.05 was considered statistically significant. DETAILED DESCRIPTION OF THE INVENTION

[0066] The definitions of various terms and expressions used to describe the present invention in this application are provided below. It should be understood that the definitions of terms and expressions used in more than one aspect or embodiment of the present invention are equally applicable to any aspect or embodiment of the present invention described herein in which these terms and expressions appear. This also applies wherever (i.e., which part) these terms are defined or discussed in this application.

[0067] In addition, although the present application mentions discrete embodiments, it should be understood that any embodiment and the features therein can be freely combined with any other embodiment and the features therein, even without explicit statements to their effect. Therefore, such a combination of one embodiment with another embodiment, or one or more features in any one embodiment with one or more features in any other embodiment, belongs to the disclosure of the present application understood by those skilled in the art.

[0068] In this application, the use of the singular (e.g., "one" or "a kind") may include the plural, unless otherwise expressly stated. As used herein, "one or more" is a synonym for "at least one" and is intended to refer to one agent or any combination of more than one agent. As used below, the singular "agent" or "a kind of agent" is intended to refer to one or more agents as defined above. When an ingredient is administered with an additional agent that is different from one or more agents, the additional agent is defined as an "other agent." In addition, the use of the term "includes" and other grammatical forms such as "includes" and "including" is not restrictive.

[0069] As used herein, the term "comprising" has a broad standard meaning of "including", "covering" or "containing". It includes the one or more elements explicitly listed, and also allows, but does not require, the presence of other or another uncited one or more elements. In addition to this broad meaning used herein, the term "comprising" also encompasses the restrictive meaning of "consisting of...", according to which only the one or more elements explicitly listed are present, and no other one or more elements are present. In addition, the term "comprising" also includes the meaning of "essentially consisting of...", which means that in addition to those explicitly listed, there may be additional one or more elements, provided that the additional presence of one or more elements does not change the technical effect achieved by the one or more elements explicitly listed.

[0070] As used herein, the terms "about", "approximately" and their synonyms, when referring to a particular value, such as one or more endpoints of a range, encompass and disclose certain variations around the specifically recited value in addition to the specifically recited value itself. Such variations may be caused, for example, by normal measurement variability, such as weighing or dispensing various substances by methods known to the skilled person. The terms "about", "approximately" and their synonyms should be understood to encompass and disclose a range of variability above and below the specific value specified, with the percentage values ​​being relative to the specifically recited value itself, as shown below. The term “about” may encompass and disclose ±5.0%, ±4.9%, ±4.8%, ±4.7%, ±4.6%, ±4.5%, ±4.4%, ±4.3%, ±4.2%, ±4.1%, ±4.0%, ±3.9%, ±3.8%, ±3.7%. ±3.6%, ±3.5%, ±3.4%, ±3.3%, ±3.2%, ±3.1%, ±3.0%, ±2.9%, ±2.8%, ±2.7%, ±2. The terms "about", "approximately" and their synonyms, with respect to a specific recited value, may include and disclose that exact specific value itself, whether or not the inclusion of that exact specific value is explicitly mentioned; even in the absence of an explicit indication that these terms include a specific exact recited value, that exact specific value is still included within the range of variation created by the terms and is therefore disclosed.

[0071] As described above, the present invention relates to a method of using vitamin D and an agent such as EGCG as defined above to control / prevent / treat a subject's fibroids. In a related aspect, the present invention relates to a method of controlling / preventing / treating a fibroid in a subject in need or potential need thereof, comprising the step of administering vitamin D and an agent such as EGCG as defined above to the subject. In another related aspect, the present invention relates to the use of vitamin D and an agent such as EGCG as defined above in the preparation of a medicament for treating / preventing / controlling a subject's fibroids.

[0072] As used herein, the term "subject" refers to a mammal, preferably a human. The subject may be a subject in need, potential need, or suspected need of prevention / treatment / control of fibroids.

[0073] The subject may be in need of treatment for fibroids. In this case, this need will usually be determined in advance by obtaining a diagnosis of fibroids. In this case, where one or more diagnoses of one or more pathological conditions already exist, the present invention as described herein can be implemented to treat the existing pathology (e.g., uterine fibroids) while preventing the development of at least one symptom thereof (e.g., excessive menstrual bleeding, dysmenorrhea, chronic pelvic pain, and pressure symptoms, such as a feeling of swelling and intestinal disturbances). However, it is also contemplated that the subject matter of the present invention as described herein can be applied to subjects suspected of having such a need. These patients may be at risk of developing such symptoms. For example, overweight or obese subjects, subjects with a positive diagnosis of endometriosis or hypertension, and subjects with a family history of uterine fibroids or more generally women who are more at risk of developing uterine fibroids.

[0074] Thus, as used herein, the term "treat" or its grammatically related variants such as "therapy", "treating", etc., means to improve (even temporarily) encompassing but not requiring complete elimination of a pathological condition or at least one symptom thereof. In the broadest sense, treatment of fibroids by the medical uses and treatment methods herein means to achieve at least partial restoration of dysfunctional fibrotic tissue to its previous functional state. Specifically, treatment in the present sense includes restoring proper organ structure and function in an organ in which fibrotic tissue has developed. For example, in the case where the organ is the uterus, treatment would include reducing the proliferation of extracellular matrix (ECM) proteins and fibroblasts in the fibrotic tissue to restore functional uterine tissue.

[0075] As used herein, the term "fibroid" refers specifically to a condition in which noncancerous muscle tissue cells and fibrous connective tissue develop in a tissue. Such fibroids can develop from abnormal muscle cells in a tissue. Fibroids can be diagnosed during routine examinations or by any one or more of the following: X-rays, ultrasound or sonography, magnetic resonance imaging, hysterosalpingography, hysteroscopy, biopsy, blood tests. As used herein, "fibroids" are preferably uterine fibroids.

[0076] The majority of the volume of a fibroid is usually composed of fibronectin, collagen and / or proteoglycans. Therefore, the control, treatment and / or prevention of a fibroid can be measured by the release of fibronectin, collagen and / or proteoglycans. Specifically, maintaining approximately constant, reducing, inhibiting and / or preventing the release of fibronectin, collagen and / or proteoglycans is an indication of fibroid control, treatment and / or prevention.

[0077] Such maintaining approximately constant, reducing, inhibiting and / or preventing can be measured against appropriate controls by techniques known in the art, including any known methods for detecting and / or quantifying fibronectin, collagen and / or proteoglycans, such as Western blots and enzyme immunoassays.

[0078] Appropriate controls include, but are not limited to, healthy subjects, subjects with fibroids who have not received treatment, subjects with fibroids who have received treatment other than the treatment methods of the present invention, subjects with fibroids who have received vitamin D treatment alone, and subjects with fibroids who have received treatment with an agent such as EGCG alone.

[0079] As used herein, the terms "uterine fibroid", "uterine leiomyoma" and "fibroid" are used interchangeably and refer to a fibroid tumor as defined above located in the uterus. Such fibroid tumors can develop from abnormal muscle cells in the uterus, which rapidly multiply due to the influence of uterine estrogen, increasing the number of ECM proteins (particularly fibronectin, collagen and / or proteoglycans) and fibroblasts, leading to altered pathological functions. Uterine fibroids can be diagnosed during routine examinations or by any one or more of the following: X-rays, transvaginal or transabdominal ultrasound or ultrasonography, magnetic resonance imaging, hysterosalpingography, hysteroscopy, endometrial biopsy, blood tests. Typically, treatments for uterine fibroids may include: surgery, gonadotropin-releasing hormone agonists (GnRHa), selective progesterone receptor modulators (sPRMs), antihormonal agents, hormones, anti-inflammatory analgesics and tranexamic acid, as defined above. Any such treatments for uterine fibroids can be used in combination with vitamin D and the agent according to the present invention.

[0080] As used herein, the terms "prostatic hyperplasia," "benign prostatic hyperplasia (BPH)," "enlarged prostate," and "prostatic leiomyoma" refer to fibroids as defined above that are located in the prostate. For example, "prostatic hyperplasia" refers to an increase in the number of cells in the prostate and / or a noncancerous growth in the size of the prostate gland. Prostatic hyperplasia may result in increased deposition of the extracellular matrix (ECM), i.e., increased release / levels of fibronectin, collagen, and / or proteoglycans. In other words, prostatic hyperplasia is a prostatic fibroid.

[0081] As used herein, the term "fibroadenoma" refers to a fibroid tumor as defined above located in the breast. Preferably, it is a benign human breast tumor characterized by proliferation of the epithelial and fibroblastic matrix components of the terminal ductal units, i.e. increased release / levels of fibronectin, collagen and / or proteoglycans. In other words, a fibroadenoma is a breast fibroma.

[0082] As used herein, the term "prevent" or its grammatically related variants such as "prevention" refers to the application of the medical uses and treatment methods described herein to avoid possible, suspected or expected occurrences of fibroids. This suspicion of fibroids (even in the absence of a corresponding diagnosis) may come from the subject's medical history, for example, if the subject has previously suffered from fibroids. In this case, there may be a reasonable suspicion that a previously treated fibroid may recur. This suspicion of the possible occurrence of fibroids (even in the absence of a corresponding diagnosis) also applies to subjects who are at risk of developing such a condition, for example, overweight or obese subjects, subjects with a positive diagnosis of endometriosis or hypertension, and subjects with a family history of fibroids or more generally women who are at greater risk of developing uterine fibroids.

[0083] Vitamin D and one or more agents or compositions as defined above may also be used to control the size and / or growth of fibroids over a long period of time (e.g., more than 1 month, about 4 months, more than 6 months, more than 1 year, or more years). Such situations may apply, for example, when the fibroids are asymptomatic or small in size so that no intervention is indicated, and the patient is advised to control the size and growth of the fibroids through regular specialist visits and echo analysis.

[0084] As used herein, the term "vitamin D" encompasses and discloses all substances commonly referred to as vitamin D, including those having the chemical formula C 27 H 44 O=Vitamin D3 (cholecalciferol), C 28 H 44 O=Vitamin D2 (ergocalciferol), C 27 H 44 O2=25(OH) vitamin D (calcifediol), C 27 H 44 O3 = a substance of 1,25 dihydroxyvitamin D (calcitriol). Preferably, vitamin D refers to vitamin D3.

[0085] Sources of vitamin D include dietary intake, such as fish oil, fatty fish, fortified foods, and vitamin supplements.

[0086] As used herein, the term "vitamin B" encompasses and discloses all substances commonly referred to as vitamin B, including but not limited to: thiamine (vitamin B1), riboflavin, niacin, pantothenic acid, biotin, vitamin B6, folic acid, vitamin B12 (cyanocobalamin). Preferably, vitamin B refers to vitamin B6. Vitamin B6 in turn encompasses and discloses all substances commonly referred to as vitamin B6, such as pyridoxine (PN), 5'-pyridoxine phosphate (P5P), pyridoxal (PL), 5'-pyridoxal phosphate (PLP), pyridoxamine (PM), 5'-pyridoxamine phosphate (PMP), 4-pyridoxic acid (PA), pyritinol.

[0087] Sources of vitamin B6 include foods of plant or animal origin, such as cereals, whole wheat flour, lentils, milk, avocados, dried fruits, peppers, spinach, broccoli, green beans, carrots, potatoes, bananas, sunflower seeds, celery seeds, offal, tuna, crustaceans, salmon, chestnuts, eggs, soy beans, mushrooms, chocolate, aromatic herbs, paprika, peppers, green leafy vegetables, brewer's yeast, fish, and peas.

[0088] As used herein, the term "EGCG" encompasses and discloses all substances commonly referred to as EGCG, including 22 H 18 O 11 Substances such as epigallocatechin gallate (EGCG), epigallocatechin-3-gallate, (–)-epigallocatechin-3-gallate, (–)-gallocatechin-3-O-gallate (GCG), and (–)-gallocatechin-3-gallate.

[0089] As used herein, the term "epicatechin gallate" (ECG) encompasses and discloses all substances commonly known as epicatechin gallate, including those having the chemical formula C 22 H 18 O 10 Substances such as (–)-epicatechin-3-gallate, (–)-catechin-3-gallate (CG) and (+)-catechin-3-gallate (CG).

[0090] As used herein, the term "catechins" encompasses and discloses all substances commonly known as catechins, including those having the chemical formula C 15 H 14 O6 substances, such as catechin, (+)-catechin (C), epicatechin, (–)-epicatechin (EC).

[0091] As used herein, the term "gallocatechin" encompasses and discloses all substances commonly known as gallocatechin, including those having the chemical formula C 15 H 14 O7 substances, such as: gallocatechin, (+)-gallocatechin (GC), epigallocatechin, (–)-epigallocatechin (EGC).

[0092] EGCG, ECG, catechins and epigallocatechin are included in a variety of foods, any one of which or any combination can be used as the source of EGCG, ECG, catechins and / or epigallocatechin in the present invention. Specifically, in the present invention, the source of EGCG, ECG, catechins and / or epigallocatechin can be: green tea, white tea, oolong tea, black tea, carob powder, blackberry, apple, raspberry, pecan, hazelnut, pistachio, blueberry, plum, peach, avocado, grape, strawberry, cocoa, chocolate, wine, fruit, vegetable and / or onion. Carob powder can be obtained from the fruit of Ceratonia plant.

[0093] Preferably, the source of EGCG, ECG, catechins and / or epigallocatechin is tea, particularly green tea, white tea, oolong tea and black tea. Any tea tree can be used as the source of EGCG, ECG, catechins and / or epigallocatechin in the present invention. Preferably, wild tea tree (Camellia sinensis) is used. Any part or the whole plant of the tea tree can be used as the source of EGCG, ECG, catechins and / or epigallocatechin in the present invention. Preferably, the leaves of the tea tree are used. Preferably, the leaves of the wild tea tree are used as the source of EGCG, ECG, catechins and / or epigallocatechin in the present invention.

[0094] Tea leaves may contain different amounts of EGCG, ECG, catechins and / or gallocatechin, in particular from 30% to 95% by weight, such as 30%, 45%, 50% and 95% by weight. Such amounts may be determined by titration and depend on the purity and processing of the raw materials.

[0095] Preferably, EGCG, ECG, catechins and / or epigallocatechin gallate are used in the present invention as a dry tea extract, preferably a green tea dry extract.

[0096] In one embodiment, the present invention is directed to a combination of Vitamin D and EGCG.

[0097] In a preferred embodiment, the present invention relates to a combination of vitamin D, EGCG and vitamin B6.

[0098] In one embodiment, the present invention relates to the respective concentrations of:

[0099] Composition of 1 tablet

[0100] Green tea extract: 333.35 mg (Epigallocatechin gallate (EGCG) 150 mg, titer (Tit.) 45%)

[0101] Vitamin B6 5mg

[0102] Vitamin D 25 μg

[0103] In one embodiment, the present invention relates to a daily intake of:

[0104] Green tea extract: 666.70 mg (Epigallocatechin gallate (EGCG) 300 mg, titer (Tit.) 45%)

[0105] Vitamin B6 10mg

[0106] Vitamin D 50 μg corresponds to 2.000 IU

[0107] The substances of the present invention, in particular vitamin D, vitamin B, EGCG, ECG, catechin and epigallocatechin gallate, can exist and be used according to the present invention in any salt, solvate, stereoisomer, zwitterion, tautomer and / or isotope form.

[0108] The salt of the substance of the present invention is preferably pharmaceutically acceptable. Suitable pharmaceutically acceptable salts include conventional non-toxic salts obtained by the salt-forming action of the reagent of the present invention with an inorganic acid (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid) or an organic acid (e.g., acetic acid, propionic acid, succinic acid, benzoic acid, sulfanilic acid, 2-acetoxybenzoic acid, cinnamic acid, mandelic acid, salicylic acid, glycolic acid, lactic acid, oxalic acid, malic acid, maleic acid, malonic acid, fumaric acid, tartaric acid, citric acid, p-toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid or naphthalenesulfonic acid). For a review of suitable pharmaceutical salts see [Berge SM et al., J. Pharm. Sci. 1977, 66, 1-19; Gould PL Int. J. Pharm 1986, 33, 201-217; Bighley et al., Encyclopedia of Pharmaceutical Technology, Marcel Dekker & Co., New York 1996, Vol. 13, pp. 453-497; and Remington, The Science and Practice of Pharmacy, Lippincott Williams and Wilkins, 2000].

[0109] In addition, pharmaceutically acceptable base addition salts can be formed with suitable inorganic salts or organic bases such as triethylamine, ethanolamine, triethanolamine, dicyclohexylamine, ammonium hydroxide, pyridine. As used herein, the term "inorganic base" has the common meaning as understood by those of ordinary skill in the art, and broadly refers to an inorganic compound that can act as a proton acceptor. As used herein, the term "organic base" has the common meaning as understood by those of ordinary skill in the art, and broadly refers to an organic compound that can act as a proton acceptor.

[0110] Other suitable pharmaceutically acceptable salts include pharmaceutically acceptable alkali metal or alkaline earth metal salts, such as sodium, potassium, calcium or magnesium salts.

[0111] The present invention relates to the use of all possible stoichiometric and non-stoichiometric forms of the salts of the substances according to the invention.

[0112] Furthermore, the substances of the present invention may exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, EtOH, and the like.

[0113] Certain substances of the present invention, particularly EGCG, ECG, catechins and epigallocatechin, may exist in stereoisomeric forms (e.g., they may contain one or more asymmetric carbon atoms). All stereoisomers of the substances of the present invention, particularly EGCG, ECG, catechins and epigallocatechin, may be used for treating fibroids according to the present invention. The use of individual stereoisomers (enantiomers and diastereomers) and mixtures thereof is included within the scope of the present invention. Preparative HPLC with a column having a chiral stationary phase may be used to separate racemic mixtures to obtain their individual enantiomers, or individual enantiomers may be separated using methods known to those skilled in the art.

[0114] The substances of the present invention may exist in zwitterionic form, all of which may be used to treat fibroids according to the present invention. Likewise, it should be understood that the substances of the present invention may exist in tautomeric forms and their use is also included within the scope of the present invention.

[0115] The present invention also includes the use of all suitable isotopic variations of the substances of the present invention. An "isotopic variation" of a substance of the present invention is defined as a substance in which at least one atom is replaced by an atom having the same atomic number but an atomic mass different from the atomic mass usually found in nature. Examples of isotopes that can be incorporated into the substances of the present invention include isotopes such as 2 H. 3 H. 13 C. 14 C. 15 N. 17 O. 18 O. 31 P. 32 P. 35 S. 18 F and 36 Cl. Certain isotopic variants of the invention, for example, radioisotopes such as 3 H or 14 C is incorporated into them, which can be used for drug and / or substrate tissue distribution studies. In addition, the use of isotopes (such as deuterium 2H) substitution may provide certain therapeutic advantages due to greater metabolic stability. Isotopic variations of the substances of the invention can generally be prepared by conventional methods, for example by the illustrative methods or by the preparation methods described in the examples below using appropriate isotopic variations of suitable reagents.

[0116] As used herein, the term "combination" generally refers to coupling an agent as defined above with at least vitamin D for co-administration to a subject within a given treatment regimen. A given treatment regimen may include multiple, repeated co-administration of vitamin D and an agent as defined above over a predetermined time course, such as over a period of more than 1 month, more than 2 months, more than 3 months, more than 4 months, more than 5 months, or more than 6 months or longer, preferably over a period of at least 4 months or about 4 months. The co-administration of an agent as defined above and at least vitamin D may be repeated multiple times per day, such as once, twice, 3 times, 4 times, 5 times or more, preferably about twice per day. That is, the co-administration of vitamin D and an agent as defined above is repeated over a predetermined time length, and the sum of the instances of co-administration over the predetermined time length constitutes a preventive or therapeutic regimen.

[0117] The coupling of vitamin D and the medicament, for example as defined above by the "combination" of these two substances, does not need to be physical, nor does it need to imply the simultaneity of administration. Therefore, the administration of vitamin D and the medicament or a combination thereof as defined above covers and discloses a variety of possibilities regarding the route and time of administration of each substance. Contained and disclosed are, for example, the coupling of vitamin D and the medicament as defined above, for simultaneous administration by the same route, simultaneous administration by different routes, chronologically staggered (i.e., non-simultaneous) administration by the same route, or chronologically staggered (i.e., non-simultaneous) administration by different routes. Any route of administration described herein can be combined in any way, although it is generally preferred that the route is oral. It is particularly preferred to administer vitamin D and the medicament as defined above simultaneously by the oral route, for example, when vitamin D and the medicament as defined above are present in a composition, for example, in one of the inventive compositions described herein, for example, in the form of tablets, hard capsules, soft gel capsules or powders, for example in the form of sachets, for ingestion.

[0118] The simultaneous administration of vitamin D and the agent as defined above by the same route may, for example, take the form of vitamin D and the agent as defined above being contained in the same physical composition, which is administered to a subject (e.g., ingested) such that vitamin D and the agent as defined above enter the body simultaneously by the same route (e.g., by an oral route). However, the coupling of vitamin D and the agent as defined above for staggered (i.e., non-simultaneous) administration in a given co-administration is also possible and is also encompassed in the present invention and disclosed by the term "combination". The order of administration of the above-mentioned vitamin D and the drug is not particularly important; staggered (i.e., non-simultaneous) administration of vitamin D and the agent as defined above in chronological order may encompass a prior administration of the agent as defined above and a subsequent administration of vitamin D, or a prior administration of vitamin D and a subsequent administration of the agent as defined above. For example, staggered (i.e., non-simultaneous) administration of vitamin D and an agent as defined above by the same route may take the form of an initial oral administration of vitamin D followed by oral administration of an agent as defined above; vitamin D and an agent as defined above coupled in this manner fall within the scope of the invention and are within the meaning of and disclosed by "combination" as used herein. Conversely, staggered (i.e., non-simultaneous) administration of vitamin D and an agent as defined above by different routes may take the form of an initial oral administration of vitamin D followed by administration of an agent as defined above, for example, by suppository; vitamin D and an agent as defined above coupled in this manner also fall within the scope of the invention and are within the meaning of and disclosed by "combination" as used herein.

[0119] As used herein, the term "non-simultaneous" refers to the administration of vitamin D and an agent as defined above in a chronologically staggered manner, i.e., at different times. Administration refers to the co-administration of vitamin D and an agent as defined above, respectively, in a broader treatment or prevention regimen. For example, one embodiment requires the administration of an agent as defined above before administering vitamin D to the subject. Another embodiment requires the administration of an agent as defined above to the subject after administering vitamin D to the subject. It should be understood that such non-simultaneous administration represents any corresponding instance of the co-administration of vitamin D and an agent as defined above in the broader context of the entire treatment regimen. As described herein, regardless of the order of administration, such as whether the agent defined above is administered first and vitamin D is administered secondly, or vice versa, the respective combined administrations may be by the same or different routes.

[0120] In another embodiment, vitamin D and the medicament as defined above are administered to the subject simultaneously. As described above, the simultaneous administration of vitamin D and the medicament as defined above can be achieved by the same or different routes. Most typically, it will be most advantageous and convenient to achieve the simultaneous administration of vitamin D and the medicament as defined above by the same route, preferably by oral administration. This is typically achieved by combining vitamin D and the medicament as defined above to be administered in the same composition, for example in the form of tablets, including but not limited to effervescent tablets, powders (particularly in sachets), hard capsules, soft gel capsules, syrups, cachets, lozenges or pastilles.

[0121] If the co-administration is staggered in time sequence (i.e., non-simultaneous), there is no particular restriction on the duration between the administration of vitamin D and the medicament as defined above, but it is usually most effective and convenient when any interval between the administration of vitamin D and the medicament as defined above is short (close to simultaneous, such as minutes). In some cases, such intervals may be extended to about one hour or several hours between each administration. Then, in some cases, if the co-administration of vitamin D and the medicament as defined above is staggered in time sequence, the interval between administrations may be as long as 12 hours in some cases. However, even in the case where vitamin D and the medicament as defined above are administered in a staggered manner in time sequence, whether by the same or different routes, the term "combination" means that the co-administration of vitamin D and the medicament as defined above within any given regimen will be completed, that is, before the next co-administration of vitamin D and the medicament as defined above (which itself can be staggered again simultaneously or in time sequence by the same or different routes to correspond to the time sequence staggering and route different from the previous co-administration), both substances will be administered, and they will occur in the same regimen.

[0122] As used herein, the term "composition" encompasses and discloses any physical entity that contains (or consists of or consists essentially of) the respective listed substances, for example, contains or consists of or consists essentially of vitamin D and the medicament as defined above. The physical form of the composition is not limited. For example, the term "composition" encompasses and discloses a powder, wherein the listed substances are each present in powder form. As another example, the term "composition" also encompasses and discloses a liquid solution, wherein the listed substances are each present in a soluble form. As another example, the term "composition" also encompasses and discloses an emulsion, wherein the listed substances are present. As another example, the term "composition" also encompasses and discloses a suspension, wherein the listed substances are present. As another example, the term "composition" also encompasses and discloses a mixture, wherein the medicament as defined above is in one form, for example, a solid, such as a powder, and vitamin D is in another form, such as a liquid. In particular, the term "composition" can be a "pharmaceutical composition" as defined below, and can be formulated for a desired route of administration. As used and disclosed herein, the term "composition" may also be a composition suitable for oral delivery, for example in the form of a tablet, including but not limited to an effervescent tablet or a multilayer tablet, a powder (e.g., in the form of a sachet), a hard capsule, a soft gel capsule, a syrup, a cachet, a lozenge or a pastille, a soft pastille (e.g., a soft candy pastille), an ointment, or a liquid preparation. In certain particularly preferred embodiments of the present invention, a "composition" comprising vitamin D and a medicament as defined above may be in the form of a soft gel capsule. In certain particularly preferred embodiments of the present invention, a "composition" comprising vitamin D and a medicament as defined above may be in the form of a powder. The term "composition" may also be a composition suitable for delivery by a non-oral route, for example, in the form of a suppository, a tablet, a hard capsule, a soft gel capsule, a cream, a gel, a patch, or a liquid. Other dosage forms of the composition and the routes of administration mentioned are listed below.

[0123] In the medical uses and methods of treatment described herein, it is contemplated that vitamin D and the agents as defined above may be administered in combination with one or more additional substances.

[0124] For example, vitamin D and the medicament as defined above can be administered in combination with a source of vitamin B6. According to the medical uses and methods of treatment listed herein, the administration of a source of vitamin B6 in combination with vitamin D and the medicament as defined above may be particularly beneficial in the case of fibroids. The intake of vitamin B6 helps to regulate hormone activity, which is considered a risk factor for uterine fibroids. This source of vitamin B6 refers to a group of chemically similar compounds: for example, pyridoxine (PN), 5′-pyridoxine phosphate (P5P), pyridoxal (PL), 5′-pyridoxal phosphate (PLP), pyridoxamine (PM), 5′-pyridoxamine phosphate (PMP), 4-pyridoxic acid (PA), pyritinol.

[0125] For example, one embodiment of the medical uses and methods of treatment described herein may require the combined administration of about 150 mg EGCG and about 25 μg vitamin D. Another embodiment of the medical uses and methods of treatment described herein may require the combined administration of about 150 mg EGCG, about 25 μg vitamin D, and 5 mg vitamin B6.

[0126] As another example, vitamin D and the agents as defined above may also be administered in combination with one or more polyphenols in the medical uses and methods of treatment described herein. Inclusion of one or more polyphenols for administration in combination with vitamin D and the agents as defined above, according to the medical uses and methods of treatment listed herein, may be particularly beneficial in the case of fibroids. For example, polyphenols may be extracted from cocoa, olive oil, grapes and pomegranates. Polyphenols are known to have a positive effect on fibroids; olive oil polyphenols, such as hydroxytyrosol, are known for their powerful antioxidant and anti-inflammatory effects.

[0127] Therefore, in one embodiment, the composition of the present invention is a pharmaceutical composition. The composition of the present invention, including the pharmaceutical composition, may also include at least one pharmaceutically acceptable ingredient. Although the composition of the present invention, including the pharmaceutical composition, can be administered to a subject by itself, it should be understood that the addition of one or more pharmaceutically acceptable ingredients in addition to vitamin D and the medicament as defined above may be beneficial to make the composition more suitable for direct administration to a subject by a given predetermined route. Therefore, a "pharmaceutical composition" can be formulated to include pharmaceutically acceptable ingredients in addition to vitamin D and the medicament as defined above, which contributes to the composition being more suitable or particularly suitable for direct administration to a subject by a given route without further examination. This suitability may involve many different routes of administration, including oral, parenteral, transmucosal, vaginal or perivaginal, topical, transdermal or intravesical, as further described below. When present, as a non-limiting illustration, these ingredients and their favorable effects on the suitability of the composition of the present invention, including pharmaceutical compositions and supplements, for different routes of administration are listed below.

[0128] a) Formulations suitable for oral administration

[0129] Compositions suitable for oral administration can be prepared, packaged or sold in the form of discrete solid dosage units, including sachets (e.g., powder in a sachet), tablets, hard or soft capsules, cachets, lozenges or pastilles, each containing a predetermined amount of vitamin D and an agent specified herein. Other formulations suitable for oral administration include powdered or granular formulations, aqueous or oily suspensions, aqueous or oily solutions or emulsions. As used herein, "oily" liquids include carbon-containing liquid molecules that exhibit less polarity than water.

[0130] Particularly preferably, the dispersed solid dosage unit of the composition of the present invention is in powder form, especially in pouch form. As used herein, the term "pouch" refers to a sealed bag containing the composition of the present invention. The pouch can be made of paper, wax paper, plasticized paper or a combination of paper and foil. The material made of the pouch is preferably impermeable to ambient moisture and other potential atmospheric pollutants, so that by sealing the pouch, the composition of the present invention in powder form contained therein remains in a free-flowing form before use.

[0131] Tablets comprising any of the above concentrations of vitamin D and an agent as defined above can be prepared, for example, by compressing or molding the vitamin D and an agent as defined above with one or more additional ingredients. Compressed tablets can be prepared by compressing vitamin D and an agent as defined above in a free-flowing form such as a powder or granular formulation in a suitable apparatus, optionally mixed with one or more of a binder, a lubricant, an excipient, a surfactant, and a dispersant. Molded tablets can be prepared, for example, by molding vitamin D and an agent as defined above, a carrier, and a liquid at least sufficient to wet the mixture in a suitable apparatus.

[0132] Acceptable excipients for making tablets include, but are not limited to, inert diluents, granulating agents and disintegrants, binders and lubricants. Known dispersants include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate, and sodium phosphate. Known granulating agents and disintegrants include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, gum arabic, pregelatinized corn starch, polyvinyl pyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silicon dioxide, and talc.

[0133] The tablets may be uncoated or they may be coated using known methods to achieve delayed disintegration in the gastrointestinal tract of a subject, thereby providing sustained release and absorption of the vitamin D and the medicament as defined above. As an example, materials such as monostearate or distearate glyceryl may be used to coat the tablets. The tablets may also contain sweeteners, flavoring agents, coloring agents, preservatives, or some combination of these to provide an attractive and palatable formulation.

[0134] Hard capsules comprising vitamin D and an agent as defined above can be prepared using a physiologically degradable composition such as gelatin or a cellulose derivative. Such hard capsules comprise vitamin D and an agent as defined above, and may further comprise additional ingredients, including, for example, an inert solid diluent, such as calcium carbonate, calcium phosphate or kaolin.

[0135] Soft gelatin capsules comprising vitamin D and an agent as defined above can be prepared using a physiologically degradable composition (e.g., gelatin) in combination with a plasticizer (i.e., glycerol) as the basic component of a soft gelatin shell. Soft gelatin capsules may contain a liquid or semisolid solution, suspension, or microemulsion preconcentrate. The soft gelatin capsule filler comprises vitamin D and an agent as defined above, which may be mixed with water or an oil medium, such as peanut oil, liquid paraffin, olive oil, soybean oil, sunflower oil, lecithin, such as soybean lecithin or sunflower lecithin, medium chain triglycerides, polyglycerol oleate, beeswax, monoglycerides and diglycerides of fatty acids, or any combination thereof.

[0136] Liquid preparations particularly suitable for oral administration may be prepared, packaged, and sold in liquid form or as a dry product for reconstitution with water or another suitable vehicle before ingestion.

[0137] Liquid suspensions can be prepared using conventional methods to realize the suspension of one or more active ingredients in aqueous or oily vehicles. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethanol, vegetable oils (such as peanut oil, olive oil, sesame oil or coconut oil), fractionated vegetable oils and mineral oils (such as liquid paraffin). Liquid suspensions may also include one or more additional ingredients, including but not limited to suspending agents, dispersants or wetting agents, emulsifiers, mitigating agents, preservatives, buffers, salts, flavoring agents, coloring agents and sweeteners. Oily suspensions may also include thickeners. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinyl pyrrolidone, tragacanth gum, gum arabic and cellulose derivatives, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcellulose. Known dispersants or wetting agents include but are not limited to naturally occurring phosphatides, such as lecithin, condensation products of alkylene oxide with fatty acids, with long-chain fatty alcohols, with partial esters derived from fatty acids and hexitol, or with partial esters derived from fatty acids and hexitol anhydrides (for example, respectively polyoxyethylene stearate, heptadecaethyleneoxyhexadecyl alcohol, polyoxyethylene sorbitol monooleate and polyoxyethylene sorbitan monooleate). Known emulsifiers include but are not limited to lecithin and gum arabic. Known preservatives include but are not limited to methyl paraben, ethyl or n-propyl paraben, ascorbic acid and sorbic acid. Known sweeteners include, for example, glycerol, propylene glycol, sorbitol, sucrose and saccharin. Known thickeners for oily suspensions include, for example, beeswax, hard paraffin and cetyl alcohol.

[0138] Powdered and granular preparations suitable for the compositions of the present invention, such as pharmaceutical compositions, can be prepared using known methods. Such preparations can be administered directly to a subject, for example, to form a pouch or tablet, fill a capsule, or to prepare an aqueous or oily suspension or solution by adding an aqueous or oily carrier thereto. Each of these preparations may also contain one or more of a dispersant or wetting agent, a suspending agent, and a preservative. Additional excipients, such as fillers and sweeteners, flavorings, or coloring agents, may also be included in these preparations.

[0139] Compositions can also be prepared, packaged or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oil phase can be a vegetable oil such as olive oil or peanut oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions can also include one or more emulsifiers, such as naturally occurring gums (e.g., gum arabic or tragacanth), naturally occurring phospholipids (e.g., soybean or lecithin phospholipids), esters or partial esters (e.g., sorbitan monooleate) derived from a combination of fatty acids and hexitol anhydrides, and condensation products of such partial esters and ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions can also include additional ingredients, including, for example, sweeteners or flavorings.

[0140] Methods for impregnating or coating materials with chemical compositions are known in the art and include, but are not limited to, methods for depositing or binding the chemical composition onto a surface, methods for incorporating the chemical composition into the structure of the material during synthesis of the material (e.g., with physiologically degradable materials), and methods for absorbing aqueous or oily solutions or suspensions into an absorbent material, with or without subsequent drying.

[0141] b) Preparations suitable for parenteral administration

[0142] For parenteral administration, the composition comprising vitamin D and an agent as defined above can be formulated for injection or infusion, such as intravenous, intramuscular or subcutaneous injection or infusion, or administered as a bolus dose and / or continuous infusion. Suspensions, solutions or emulsions in oily or aqueous vehicles may be used, optionally containing other agents such as suspending agents, stabilizing agents and / or dispersing agents, such as those described above.

[0143] By preparing with an acceptable carrier such as sterile water or sterile isotonic saline, the composition comprising vitamin D and the medicament as defined above can be made particularly suitable for parenteral administration. Such preparations can be prepared, packaged or sold in a form suitable for push injection or continuous administration. Injectable preparations can be prepared, packaged or sold in unit dosage form, such as in ampoules, crushable or other, or in a multi-dose container containing a preservative. Compositions particularly suitable for parenteral administration include but are not limited to suspensions, solutions, emulsions in oily or aqueous carriers, pastes and implantable sustained release or biodegradable preparations. Such compositions may also include one or more additional ingredients, including but not limited to suspending agents, stabilizers or dispersants. In one embodiment of a composition particularly suitable for parenteral administration, the active ingredient is provided in a dry (e.g., powder or granule) form, used to reconstitute with a suitable carrier (e.g., sterile pyrogen-free water), and then the reconstructed composition is administered parenterally.

[0144] Compositions suitable for the present invention can be prepared, packaged or sold in the form of sterile injectable aqueous or oily suspensions or solutions. The suspension or solution can be prepared according to techniques known in the art, and in addition to one or more active ingredients, may also include additional ingredients, such as dispersants, wetting agents or suspending agents as described herein. Such sterile injectable compositions can be prepared using nontoxic, parenterally acceptable diluents or solvents such as water or 1,3-butanediol. Other acceptable diluents and solvents include, but are not limited to, Ringer's solution, isotonic sodium chloride solution and fixed oils (e.g., synthetic monoglycerides or diglycerides). Other preparations that can be generally administered parenterally include those comprising active ingredients in microcrystalline forms, liposome preparations, or as components of biodegradable polymer systems. Compositions for sustained release or implantation may include acceptable polymers or hydrophobic materials, such as emulsions, ion exchange resins, slightly soluble polymers or slightly soluble salts.

[0145] c) Preparations suitable for transmucosal administration

[0146] Compositions comprising vitamin D and a medicament as defined above suitable for transmucosal administration can be formulated. The preparation may include any substance or dosage unit suitable for administration to mucosal tissue. For example, the selected active agent can be administered to the buccal mucosa in the form of an adhesive tablet or patch, administered sublingually by placing a solid dosage form under the tongue, administered to the tongue by placing a solid dosage form on the tongue, administered nasally (intra) as liquid drops or nasal sprays, nasal non-aerosol liquid preparations or nasal dry powders, placed in or near the rectum ("rectal" preparations), or administered to the urethra as suppositories, ointments, etc.

[0147] d) Preparations suitable for vaginal or perivaginal administration

[0148] Compositions comprising vitamin D and medicaments as defined above suitable for vaginal or perivaginal administration can be prepared. For this reason, suitable dosage forms may include vaginal suppositories, creams, ointments, liquid preparations, pessaries, tampons, gels, pastes, foams or sprays. Suppositories, creams, ointments, liquid preparations, pessaries, tampons, gels, pastes, foams or sprays for vaginal or perivaginal delivery include selected one or more active agents of therapeutically effective amount and one or more conventional non-toxic vehicles suitable for vaginal or perivaginal administration. The vaginal or perivaginal forms of the present invention can be manufactured using conventional processes, as disclosed in the aforementioned Remington's "Science and Practice of Pharmacy" (The Science and Practice of Pharmacy). Vaginal or perivaginal dosage units can be made to disintegrate rapidly or disintegrate within a few hours. The time of complete disintegration can be in the range of about 10 minutes to about 6 hours, for example, less than about 3 hours.

[0149] e) Formulations suitable for topical preparations

[0150] Compositions comprising vitamin D and an agent as defined above may be formulated for topical administration. To this end, suitable dosage forms may include any dosage form suitable for application to the body surface, and may include, for example, ointments, creams, gels, lotions, solutions, pastes, etc., and / or may be prepared to contain liposomes, micelles and / or microspheres. In certain embodiments, the topical formulations herein are ointments, creams and gels.

[0151] f) Preparations suitable for transdermal administration

[0152] Compositions comprising vitamin D and medicaments as defined above suitable for transdermal administration can be prepared. As known to those skilled in the art, transdermal administration involves delivering compounds by entering the patient's systemic circulation via skin channels through compounds. This may be affected by, for example, transdermal patches or iontophoresis devices. Other components except vitamin D and medicaments as defined above can also be incorporated into transdermal patches. For example, compositions and / or transdermal patches can be prepared with one or more preservatives or antibacterial agents, including but not limited to methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chloride, etc. The dosage form of the composition of the present invention for topical administration of vitamin D and medicaments as defined above may include creams, sprays, lotions, gels, ointments, eye drops, nose drops, ear drops, etc. In such dosage forms, the ingredients of the composition of the present invention can be mixed with, for example, 1% or 2% (weight / weight) benzyl alcohol, emulsifying wax, glycerol, isopropyl palmitate, lactic acid, pure water and sorbitol solution as preservatives to form a white, smooth, uniform, opaque cream or lotion. In addition, the composition can include polyethylene glycol 400. They can be mixed with, for example, 2% (weight / weight) benzyl alcohol, white petrolatum, emulsifying wax and tenox II (butylated hydroxyanisole, propyl gallate, citric acid, propylene glycol) as preservatives to form an ointment. Bandage materials such as woven pads or rolls of gauze can be impregnated with solutions, lotions, creams, ointments or other such forms of compositions, and can also be used for topical administration. The composition can also be administered topically using a transdermal system, for example, one of an acrylic acid-based polymer adhesive and a resin crosslinker is impregnated with the composition and laminated to a permeation-proof substrate.

[0153] Examples of suitable skin contact adhesive materials include, but are not limited to, polyethylene, polysiloxane, polyisobutylene, polyacrylate, polyurethane, etc. Alternatively, the drug-containing reservoir and the skin contact adhesive are separate distinct layers, with the adhesive beneath the reservoir, in which case the reservoir may be a polymer matrix as described above, or a liquid or hydrogel reservoir, or take some other form.

[0154] g) Preparations suitable for intravesical administration

[0155] The term intravesical administration is used herein in its conventional sense to mean the delivery of the drug directly into the bladder. Suitable intravesical administration methods can be found, for example, in U.S. Pat. Nos. 6,207,180 and 6,039,967.

[0156] Vitamin D and the agents as defined above may also be administered in accordance with the medical uses and methods of treatment described herein in the form of a supplement comprising Vitamin D and the agents as defined above.

[0157] It will be appreciated that the uses described herein need not satisfy any regulatory requirements, such as the definition of a drug or supplement. Preferably, the uses described herein are non-therapeutic.

[0158] As used herein, the term "supplement" or more generally "composition" refers to any one or more of the above defined ingredients of the composition preferably intended to supplement a subject whose diet lacks such ingredients or is otherwise deficient in such ingredients, and / or to induce a beneficial health effect, for example by preventing, controlling and / or treating any of the above defined conditions, disorders or symptoms. Preferably, the supplement is formulated for oral administration, such as a pill, capsule, tablet or liquid as described above.

[0159] The authors have found that providing a certain minimum amount of vitamin D and a medicament as defined above in the composition of the present invention can be beneficial to ensure that the desired treatment or prevention effect can be achieved in a reasonable number of dosings. Typically, successful treatment or prevention of fibroids can be achieved by daily dosing totaling up to about 300 mg of total EGCG and about 50 μg of total vitamin D. However, from the perspective of the impact of any such repeated dosing on the patient's quality of life, it may be advantageous to ensure that the minimum amount of vitamin D and a medicament as defined above in the composition is accordingly reduced to reach a given target amount of such vitamin D and a medicament as defined above (e.g., a target amount of vitamin D as described above and a medicament as defined above) The required daily dosing times may be advantageous.

[0160] Thus, the medical uses and methods of treatment described herein may comprise administering to a subject up to: about 300 mg of the agent as defined above and 50 μg of vitamin D, about 150 mg of the agent as defined above and 25 μg of vitamin D, about 100 mg of the agent as defined above and 20 μg of vitamin D, about 75 mg of the agent as defined above and 17.5 μg of vitamin D, about 50 mg of the agent as defined above and 10 μg of vitamin D, 41.25 mg of the agent as defined above and 6.9 μg of vitamin D, about 5 mg of the agent as defined above and 1 μg of vitamin D in one combined administration (e.g. in tablet or in powder form, e.g. provided in a sachet).

[0161] One embodiment of the medical uses and methods of treatment described herein may also include administering to the subject about 300 mg of the agent as defined above and 50 μg of vitamin D, or 150 to 300 mg of the agent as defined above and 25-50 μg of vitamin D in any one administration (e.g., in any one combined administration). One embodiment of the medical uses and methods of treatment described herein may also include administering to the subject about 5 to 1000 mg of the agent as defined above and 1-100 μg of vitamin D in any one administration (e.g., in any one combined administration). Another embodiment of the medical uses and methods of treatment described herein may also include administering to the subject about 41.25 mg of the agent as defined above and 6.9 μg of vitamin D in any one administration (e.g., in any one combined administration).

[0162] In one embodiment, if vitamin D and the medicament as defined above are provided in powder form (e.g., in a pouch) to be administered to a subject, it may contain or consist of about 150 mg of the medicament as defined above and 25 μg of vitamin D, and it may be administered to a subject. In a related embodiment, vitamin D and the medicament as defined above are provided in powder form (e.g., in a pouch) to be administered to a subject, and the powder composition may contain about 300 mg of the medicament as defined above and 50 μg of vitamin D, and it may be administered to a subject. Alternatively, in one embodiment, if vitamin D and the medicament as defined above are provided in the form of a soft gel capsule to be administered to a subject, it may contain or consist of about 150 mg of the medicament as defined above and 25 μg of vitamin D, and it may be administered to a subject. In a related embodiment, the combination is provided in the form of a soft gel capsule to be administered to a subject, and the combination in the soft gel capsule may contain 41.25 mg of the medicament as defined above and 6.9 μg of vitamin D, and it may be administered to a subject.

[0163] Especially the subsequent embodiment comprising the administration of about 150 mg of the agent as defined above and 25 μg of vitamin D is particularly advantageous since the daily target amount is reached with a single dose.

[0164] The following amounts and combinations represent preferred embodiments of the present invention:

[0165] about 5-1000 mg of an agent as defined above and 1-100 μg of vitamin D;

[0166] about 10-500 mg of an agent as defined above and 5-70 μg of vitamin D;

[0167] about 50-150 mg of an agent as defined above and 10-60 μg of vitamin D;

[0168] about 100-300 mg of an agent as defined above and 25-50 μg of vitamin D;

[0169] About 120-180 mg of an agent as defined above, in particular, 150 mg EGCG ± 20%;

[0170] About 25 μg of vitamin D;

[0171] about 150 mg of an agent as defined above and 25 μg of vitamin D;

[0172] about 41.25 mg of an agent as defined above and 6.9 μg of vitamin D;

[0173] about 82.5 mg of an agent as defined above and about 13.8 μg of vitamin D;

[0174] about 150 mg of an agent as defined above, 5 mg of vitamin B6 and 25 μg of vitamin D;

[0175] Vitamin D and the agent as defined above in a weight ratio of about 1:6000;

[0176] Vitamin D and an agent as defined above in a weight ratio of about 1:50–1:1000000;

[0177] about 150 mg of an agent as defined above and 60 μg of vitamin D;

[0178] Vitamin D and an agent as defined above in a weight ratio of about 1:2500;

[0179] about 300 mg of an agent as defined above and 100 μg of vitamin D;

[0180] Vitamin D and the agent as defined above in a weight ratio of about 1:3000;

[0181] Vitamin D and an agent as defined above in a weight ratio of about 1:50;

[0182] Vitamin D and the agent as defined above in a weight ratio of about 1:1000000;

[0183] Vitamin D and the agent as defined above in a weight ratio of about 1:2000 to 1:7000;

[0184] Vitamin D and an agent as defined above in a weight ratio of about 1:100–1:100000;

[0185] Vitamin D, vitamin B6 and the agent as defined above in a weight ratio of about 1:200:6000.

[0186] Several examples are provided below to illustrate various embodiments of the present invention, as well as the technical effects and advantages achieved by it. It should be understood that the following examples are only for illustrative purposes and are not intended to limit the claimed invention. In fact, while achieving the described technical advantages, those skilled in the art will be able to easily implement other embodiments within the spirit and scope of the claimed invention. Example

[0187] The present inventors have discovered that administration of vitamin D and an agent such as EGCG can prevent / control / treat fibroids, particularly uterine fibroids, caused by familiarity, age, obesity, hormonal activity, etc. This represents a new and unexpected method of preventing / controlling / treating fibroids.

[0188] Example 1: Composition 1

[0189] A first exemplary composition of the present invention is in the form of a round tablet (10 mm in diameter), weighs about 500 mg, has an expected shelf life of 24 months, and has the following composition:

[0190] Table 1. Composition 1

[0191]

[0192] Example 2: Composition 2

[0193] A second exemplary composition of the present invention is in the form of a round tablet (10 mm in diameter) with an expected shelf life of 24 months and has the following composition:

[0194] Table 2. Composition 2

[0195]

[0196] It should be noted that in this example, the tolerable range of EGCG titration was 120-180 mg / tablet (150±20%).

[0197] The amount of an ingredient listed in the "mg / tablet" column in Tables 1 and 2 refers to the amount of the ingredient used to prepare one tablet. The amount of an ingredient listed in the "Amount per tablet" column in Tables 1 and 2 refers to the amount of the ingredient present in one tablet. Excessive use of vitamins B6 and D3 has been associated with the following factors:

[0198] - Starting titer of raw material (title);

[0199] - the starting chemical form of the molecular substrate;

[0200] -Material losses during the production process.

[0201] It is recommended to administer one or two tablets of Composition 1 or 2 per day.

[0202] Example 3: Anti-fibrotic properties of epigallocatechin gallate and vitamin D3 alone or in combination In vitro evaluation

[0203] Materials and methods

[0204] The anti-fibrotic properties of epigallocatechin gallate (EGCG) and vitamin D3 (alone or in combination) have been initially tested in 2D cultured human normal fibroblasts. The human normal fibroblasts used in this experiment were primary cell cultures. To exclude any effects on fibroblast viability, proliferation and apoptosis were also studied.

[0205] Fibroblast culture: DMEM, 10% FBS, 1% L-Glu, 1% penicillin / streptomycin for 18 days;

[0206] Fibroblasts were grown in the above fibroblast culture for 18 days until 50% confluence was reached. The fibrogenic process was then promoted by the addition of 10 ng / ml TGF-β1 (Peprotech, catalog #100-21-10UG). 24 hours after the addition of TGF-β1, the fibroblasts were washed thoroughly with PBS and fresh fibroblast medium and the following formulation were added. The fibroblasts were then grown for another 24 hours.

[0207] The recipe is as follows:

[0208] - Vitamin D3 (ergocalciferol, CAS number 67-97-0): 0.005 μg / ml in DMSO. Vitamin D3 was dissolved in DMSO and then further diluted in PBS. Essentially, the cells received the same "dose" of DMSO, which allowed for a clear comparison of the assays. In this case, DMSO 1:10000 was used;

[0209] -EGCG (green tea dry extract 95% polyphenols; CAS-No. 84650-60-2): 0.03 mg / ml in H2O;

[0210] - Combination treatment: EGCG 0.03mg / ml + vitamin D3 0.005μg / ml;

[0211] - Control: vehicle only (DMSO);

[0212] Both fibronectin and collagen release were measured in Western blots to assess the anti-fibrotic effect. Results are reported as arbitrary OD-units. When treatment was stopped, 24 hours after the addition of treatment, cells were frozen at -80 °C overnight. Protein extraction was performed the next day followed by Western blots.

[0213] Statistical analysis was performed. The results were significant for the single treatments.

[0214] result

[0215] - Phase I of the study

[0216] Addition of vitamin D3 alone, EGCG alone, or a combination of vitamin D3 and EGCG did not significantly alter either the fibroblast proliferation rate or their viability (data not shown). Cell morphology did not change significantly between cells treated with TGF-β1 and control cells.

[0217] In control fibroblasts, a slight increase in the release of both fibronectin and collagen was noted after 24 h of culture in regular medium ( Figure 1 and 2 ). This slight increase can be regarded as a nonspecific response to the culture environment.

[0218] In vitamin D3-treated cells, vitamin D3 slightly increased fibronectin deposition while significantly reducing collagen release ( Figure 1 and 2 ).

[0219] In EGCG-treated cells, EGCG significantly reduced the release of fibronectin and collagen ( Figure 1 and 2 ).

[0220] The combination of vitamin D3 + EGCG significantly (compared with single treatment) reduced the release of fibronectin and collagen in cultured fibroblasts ( Figure 1 and 2 ).

[0221] The components that make up the majority of a fibroid's volume are fibronectin and collagen, so a decrease in the production and deposition of these two proteins may reduce the size of a fibroid.

[0222] - Phase II of the study

[0223] As described above, fibronectin and collagen released from fibroblasts were analyzed 24 hours after the addition of EGCG or vitamin D3 to previously stimulated fibroblasts. To establish a dose-response relationship, different concentrations of the two compounds (1-100 μg / L vitamin D3 in DMSO; 0.5-10 mg / L EGCG in H2O) were tested. These in vitro concentrations can be considered to represent in vivo doses of 1-100 μg vitamin D per day and 5-1000 mg EGCG per day.

[0224] Data reported in Figure 3 AB and Figure 4 AB and expressed as the fold decrease in OD-units relative to the control value, which is obtained after 24 hours of stimulation of fibroblasts with TGF-β1 (e.g., a 50% decrease between two measurements would be called a "half-fold change"). The fold change is a measure that describes how much a quantity has changed between an original measurement and a subsequent measurement. It is defined as the ratio between two quantities; for quantities A and B, the fold change of B relative to A is B / A.

[0225] Figure 3 A shows that the concentration of vitamin D3 (especially from 5 μg / L to 100 μg / L) has an effect on the reduction of fibronectin. Figure 3 B shows that the concentration of EGCG (especially from 5 mg / L to 10 mg / L) has an effect on the reduction of fibronectin.

[0226] Figure 4 A shows that the concentration of vitamin D3 (especially from 5 μg / L to 100 μg / L) has an effect on the reduction of collagen. Figure 4 B shows that the concentration of EGCG (especially from 3 mg / L to 10 mg / L) has an effect on the reduction of collagen.

[0227] By comparison Figure 5 and Figure 3 and Figure 6 and Figure 4 , additive and / or synergistic effects were also observed.

[0228] Example 4: Clinical study

[0229] Patients and methods

[0230] A clinical study was conducted at the Hospital of Orvieto between March and October 2019. All participants gave informed consent after the purpose of the study was explained. The ethical principles of the Declaration of Helsinki and national laws were followed. Inclusion criteria: 18 years and older, premenopausal stage, at least one ≥2 cm vaginal and abdominal ultrasound examination 3(intramural, subserosal and / or submucosal) fibroids, with moderate to severe fibroid-related symptoms, and no treatment other than routine observation. Exclusion criteria: pregnant or intending to become pregnant in the next 4 months, currently breastfeeding, with severe anemia or medical morbidity, surgical eligibility, elevated liver enzymes, hormonal (estrogen, progestin, oral contraceptive) therapy (in the past 3 months), corticosteroids, supplements with possible hormonal effects, use of selective progesterone receptor modulators (sPRMs) or gonadotropin-releasing hormone (GnRH) analogs in the past 6 months. Patients were divided into two groups: one group (15 patients) was treated daily by oral administration of 25μg vitamin D + 150mg EGCG + 5mg vitamin B6 twice a day for 4 months. The second group (15 patients) did not receive treatment (control) for 4 months. The primary outcome was achieved by using Voluson TM E8 ultrasound (GE Healthcare) transvaginal ultrasound (TVU) and / or transabdominal ultrasound were performed to analyze changes in fibroid volume. Secondary outcomes were changes in the number of fibroids, pain caused by bleeding during menstruation, pressure in the pelvic area, fatigue, and quality of life (QoL) and symptom severity (SS). QoL and SS were measured as described in Spies et al., UFS-QOL, a New Disease-Specific Symptom and Health-Related Quality of Life Questionnaire for Leiomyomata., The American College of Obstetricians and Gynecologists., 2002, Vol. 99, No. 2. The higher the score, the better the health-related QoL. The lower the score, the lower the SS. The subjective experience of bleeding was expressed as severe, moderate, and normal through the bleeding assessment of the self-administered questionnaire. A complete medical history was collected from all women; a careful physical examination, ultrasound instrument evaluation, and questionnaire compilation were performed at baseline (T0) and 4 months later (T1). TM Total fibroid volume was calculated using E8 ultrasound (GE Healthcare).

[0231] Statistical analysis

[0232] When two groups were compared, statistical analysis was performed by using an unpaired t-test (2018 GraphPad Software, La Jolla, CA, USA), and results are expressed as mean ± standard error of the mean (SEM). Comparison of repeated measures was assessed by one-way ANOVA for within-group analysis: values ​​are expressed as mean ± SEM.

[0233] The Wilcoxon–Mann–Whitney test was used to analyze QoL and SS: values ​​are expressed as median, 25th percentile, and 75th percentile. Statistical significance was accepted at the p-value level ≤ 0.05.

[0234] result

[0235] Thirty women with fibroids aged between 28 and 46 years were recruited. The clinical characteristics of the patients in each group at baseline (T0) are shown in Table 3.

[0236] Table 3. Clinical characteristics of patients in each group at baseline.

[0237]

[0238] Baseline characteristics of the two groups (vitamin D + EGCG-treated group vs control group). The groups were well matched and no significant differences were found. p-values ​​≤ 0.05 were considered statistically significant. All data results were non-significant. Abbreviations: BMI, body mass index; SS, symptom severity; QoL, quality of life; SEM, standard error of the mean.

[0239] At T0, all parameters were comparable in both groups. There was no dropout record in either group. No adverse reactions attributable to treatment were observed during the entire study period. The total number of fibroids in the treatment group and the control group were 23 and 21, respectively. In the treatment group, the incidence of intramural, subserosal, and submucosal fibroids were 43.75%, 12.5%, and 43.75%, respectively. In the control group, 47.4% were intramural, 10.5% were subserosal, and 42.1% were submucosal. A significant reduction in fibroid volume was observed in the treatment group (from 10.84±1.16cm at baseline 3 8.04±0.85cm after 4 months of treatment 3 , p<0.0001)( Figure 7 ), corresponding to a 34.7% reduction in fibroid volume. The reduction in volume was independent of the type of fibroid. In the control group, fibroid volume decreased from 10.17 ± 1.43 cm at T0 to 3 Significant fluctuation to 10.94±1.50cm after 4 months of observation 3 (p<0.001), corresponding to a 6.9% increase in fibroid volume ( Figure 7). At T1, there was a significant trend between the volume of fibroids in the treatment group and the control group (p=0.0930). From T0 to T1, the number of fibroids in the treatment and control groups did not change, showing unchanged mean ± SEM (1.53±0.19 in the treatment group and 1.40±0.19 in the control group, with no significant difference between the groups) (Table 3). Table 4 illustrates the incidence of major fibroid-related symptoms in the treatment and control groups, expressed as percentage (%) at T0 and T1. The change in QoL from T0 to T1 in the treatment group showed an increasing trend (median 31), while the control group decreased slightly (median -1), and the change from T0 to T1 between the two groups was statistically significantly different (p<0.0001) ( Fig. 9 A). The change in SS from T0 to T1 showed a decrease in the treatment group (median -7, corresponding to a 47% decrease in SS), while there was no change in the control group (median 0). The change from T0 to T1 between the two groups was statistically significant (p<0.0001) ( Fig. 9 B) Then, significant improvements were found in all QoL aspects and SS, including daily activities, sexual function, bleeding, and pelvic pain.

[0240] Table 4. Number, type, and incidence of associated symptoms of fibroids at visit 1 and visit 2.

[0241]

[0242]

[0243] The incidence is expressed as the percentage (%) of subjects in each group with fibroid type or related symptoms at two time points (baseline T0 or T1 after 4 months); n, number of subjects.

[0244] In conclusion, oral supplementation with vitamin D combined with EGCG has a considerable impact on female reproductive health. In fact, this combination can avoid surgical treatment, allowing women to maintain fertility and prevent gynecological complications. This combination is suitable for improving QoL and reducing SS. In addition, these findings suggest that vitamin D and EGCG can be used to treat fibroids to prevent possible increases in size and thus control fibroids. In addition, vitamin D and EGCG can be administered with other medical treatments or as a pre-treatment before surgery; this may help patients reduce side effects and reduce possible high fibroid volumes that can complicate surgery and increase risks.

Claims

1. Use of vitamin D and a pharmaceutical agent selected from the group consisting of epigallocatechin gallate (EGCG) and (–)-gallocatechin-3-O-gallate in the preparation of a pharmaceutical composition for preventing, controlling and / or treating uterine fibroids or fibroadenomas.

2. The use of vitamin D and a medicament as claimed in claim 1, wherein the vitamin D is vitamin D3 or vitamin D2.

3. The use of vitamin D and a medicament as claimed in claim 1, wherein the vitamin D is vitamin D3.

4. The use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the daily amount of vitamin D is 1 µg to 100 µg, and / or the daily amount of the medicament is 5 mg to 1000 mg and / or the weight ratio of the vitamin D to the medicament is 1:50 to 1:1000000.

5. Use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the daily amount of vitamin D is 50 µg, and / or the daily amount of the medicament is 300 mg and / or the weight ratio of the vitamin D to the medicament is 1:6000.

6. Use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the weight ratio of the vitamin D to the medicament is 1:

50.

7. Use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the weight ratio of the vitamin D to the medicament is 1:2000.

8. Use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the weight ratio of the vitamin D to the medicament is 1:2500.

9. The use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the weight ratio of the vitamin D to the medicament is 1:3000.

10. The use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the weight ratio of the vitamin D to the medicament is 1:5000.

11. The use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the weight ratio of the vitamin D to the medicament is 1:7000.

12. The use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the weight ratio of the vitamin D to the medicament is 1:10000.

13. The use of vitamin D and a medicament as claimed in any one of claims 1 to 3, wherein the weight ratio of the vitamin D to the medicament is 1:1000000.

14. The use of vitamin D and a medicament as claimed in any one of claims 1 to 3, wherein the pharmaceutical composition further comprises at least one other medicament selected from the group consisting of vitamin B, polyphenols and DL-α-tocopherol.

15. The use of vitamin D and a medicament as claimed in claim 14, wherein the weight ratio of the vitamin D, the medicament and the at least one other medicament is 1:5000:100 to 1:10000:1000.

16. The use of vitamin D and a medicament as claimed in claim 14, wherein the weight ratio of the vitamin D, the medicament and the at least one other medicament is 1:6000:

200.

17. The use of vitamin D and a medicament as claimed in claim 14, wherein the weight ratio of the vitamin D, the medicament and the at least one other medicament is 1:5000:1000.

18. The use of vitamin D and a medicament as claimed in claim 14, wherein the weight ratio of the vitamin D, the medicament and the at least one other medicament is 1:10000:

100.

19. The use of vitamin D and a medicament as claimed in claim 14, wherein the weight ratio of the vitamin D, the medicament and the at least one other medicament is 1:10000:1000.

20. The use of vitamin D and a medicament as claimed in claim 14, wherein the at least one other medicament is vitamin B6.

21. The use of vitamin D and a medicament as claimed in claim 20, wherein the daily amount of vitamin B6 is 1 to 100 mg.

22. The use of vitamin D and a medicament as claimed in claim 14, wherein the amount of at least one other medicament per day is 1 to 10 mg.

23. The use of vitamin D and a medicament as claimed in claim 14, wherein the amount of at least one other medicament per day is 1 to 5 mg.

24. The use of vitamin D and a medicament as claimed in any one of claims 1 to 3, wherein the epigallocatechin gallate (EGCG) is in the form of tea leaves or tea leaf extract.

25. The use of vitamin D and a medicament as claimed in claim 24, wherein the tea leaves are green tea leaves.

26. The use of vitamin D and a medicament as claimed in claim 24, wherein the amount of the medicament is 30% to 95% by weight.

27. The use of vitamin D and a medicament as claimed in claim 24, wherein the amount of the medicament is 30% by weight.

28. The use of vitamin D and a medicament as claimed in claim 24, wherein the amount of the medicament is 45% by weight.

29. The use of vitamin D and a medicament as claimed in claim 24, wherein the amount of the medicament is 50% by weight.

30. The use of vitamin D and a medicament as claimed in claim 24, wherein the amount of the medicament is 95% by weight.

31. Use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the vitamin D and the medicament reduce the release of fibronectin and / or collagen.

32. The use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the prevention, control and / or treatment of uterine fibroids includes the prevention, control and / or treatment of at least one symptom of uterine fibroids, wherein the symptom is selected from the group consisting of menorrhagia, dysmenorrhea, intermenstrual bleeding, acute or chronic pelvic pain, dyspareunia, anemia, impaired reproductive function, subfertility, infertility, early pregnancy loss, recurrent miscarriage, premature birth and pregnancy complications, fatigue and stress symptoms.

33. The use of vitamin D and a medicament as claimed in claim 32, wherein the stress symptoms include swelling, increased urination frequency, and intestinal disturbances.

34. The use of vitamin D and a medicament as described in any one of claims 1 to 3, wherein the vitamin D and the EGCG are administered simultaneously and / or orally and / or twice a day.

35. A composition for use in preparing a medicament or supplement for preventing, controlling and / or treating uterine fibroids or fibroadenomas, the composition comprising: - Vitamin D, - an agent selected from the group consisting of epigallocatechin gallate (EGCG) or (–)-gallocatechin-3-O-gallate, - Excipients, and / or - At least one other agent.

36. Use of the composition as claimed in claim 35, wherein the vitamin D is vitamin D3 or vitamin D2.

37. Use of the composition of claim 35, wherein the vitamin D is vitamin D3.

38. The use of a composition as claimed in any one of claims 35 to 37, wherein the medicament is in the form of tea leaves or tea leaf extract.

39. The use of a composition according to any one of claims 35 to 37, wherein the excipient is selected from the group consisting of dicalcium phosphate, microcrystalline cellulose, magnesium stearate, silicon dioxide, sucrose, gum arabic, corn starch, medium chain triglycerides, tricalcium phosphate, crosslinked sodium carboxymethylcellulose, hydroxypropyl methylcellulose, polyethylene glycol, titanium dioxide, polyvinyl pyrrolidone, talc, erythritol, xylitol, steviol glycosides and sucralose, and / or the at least one other agent is selected from the group consisting of vitamin B, polyphenols and DL-α-tocopherol, and / or the composition is for oral administration, and / or the composition is in the form of tablets, hard capsules, soft gel capsules, powders, syrups, cachets or lozenges.

40. Use of a composition as claimed in claim 39, wherein the lozenge comprises a lozenge or a pastille.

41. The use of a composition as described in any one of claims 35 to 37, wherein the drug or supplement is combined with other therapeutic interventions.

42. Use of the composition of claim 41, wherein the other therapeutic intervention is selected from the group consisting of surgery, gonadotropin-releasing hormone agonists, selective progesterone receptor modulators, antihormonal agents, hormones, anti-inflammatory analgesics and tranexamic acid.

43. The use of the composition according to any one of claims 35 to 37, characterized in that The composition includes EGCG in the form of green tea leaf extract, microcrystalline cellulose, calcium phosphate or dicalcium phosphate, vitamin D3, vitamin B6, magnesium stearate and silicon dioxide.

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