WO2005077396A1

WO2005077396A1 - Drug for osteoporosis having inhibition effect on cancer comprising ginkgo biloba extracts as the active ingredient

Info

Publication number: WO2005077396A1
Application number: PCT/KR2005/000397
Authority: WO
Inventors: Kyu-Hyuck Chung; Seung-Min Oh; Yeon-Pan Kim
Original assignee: Meditech Korea Pharm Co., Ltd.
Priority date: 2004-02-16
Filing date: 2005-02-14
Publication date: 2005-08-25
Also published as: KR20050081597A

Abstract

Provided is a therapeutic agent for osteoporosis, comprising Ginkgo biloba leaf extract as an active ingredient. The Ginkgo biloba leaf extract in accordance with the present invention contains a large amount of phytoestrogen such as quercetin and kaempferol and thus can be usefully utilized as a therapeutic agent for treatment of osteoporosis without having adverse effects of causing breast cancer due to capability to function as a selective estrogen receptor modulator.

Description

Description DRUG FOR OSTEOPOROSIS HAVING INHIBITION EFFECT ON CANCER COMPRISING GINKGO BILOBA EXTRACTS AS THE ACTIVE INGREDIENT Technical Field

[1] The present invention relates to a therapeutic agent for osteoporosis, comprising a phytoestrogen contained in Ginkgo biloba leaf extract as an active ingredient. Specifically, the present invention relates to a therapeutic agent for osteoporosis, comprising, as an active ingredient, Ginkgo biloba leaf extract (hereinafter, referred to as "MDG5031") containing compound derivatives represented by the following general formula (I), which have excellent promotion effects upon osteoblast proliferation:

[2]

R = H : kaempferol -3-0- rutinoside R = OH : quercetin-3-O-rutinoside (rutiπ) R = OCH₃ : isorhamπetiπ-3-O-rutiπoside

(I) Background Art

[3] Bones are the physical support structure of the body and serve to reserve necessary bone mass and maintain structural integrity. In addition, bones play an important role 2+ in maintaining blood calcium levels as they serve as the body's calcium (Ca ) depository. [4] In order to perform such functions, bones always modulate decomposition and remodeling actions. Therefore, bones in normal persons actively undergo both bone absorption and bone formation, thereby resulting in a dynamic state to reach maximum in respect to metabolism.

[5] However, osteoporosis is a disease characterized by the decrease of calcium in normal bone tissues leading to thinning of the compact bone structure and subsequent expansion of marrow cavities. Bones become brittle with the progress of the disease, and may be easily fractured by weak impacts from the external surroundings. Bone mass is affected by various factors such as genetic factors, nutritive condition, changes of hormone level, exercise and life style, and osteoporosis is known to be caused by aging, lack of exercise, low body weight, smoking, low calcium diet, menopause and ovariectomy. In addition, it is known that bone mass is higher in the black due to a low bone resorption level, as compared to the white, even though there is a difference between individuals, and generally reaches the highest level around the age of 14 to 18 and then decreases by about 1% per year thereafter. Particularly, in women, decrease of bone mass begins to continue from the age of 30, and around menopause, sharply progresses due to changes in hormone secretion. That is, when reaching menopause, the concentration of estrogen rapidly decreases and vast amounts of B -lymphocytes are produced, and subsequent pre-B cell accumulation in bone marrow results in increased levels of IL-6 which in turn increases activity of osteoclasts, thus decreasing bone mass.

[6] As such, in the elderly, especially in postmenopausal women, osteoporosis is unavoidable although the severity of symptoms may vary, and therefore, a great deal of attention has been increasingly directed to osteoporosis and therapeutic agents thereof due to the aging of the general population in advanced countries. In addition, therapies associated with bone diseases are potentially worth about 130 billion dollars in terms of global market scale, and it is forecasted that further increases in demand will occur. For these reasons, many research groups and pharmaceutical companies have exerted great efforts for development of therapeutic agents for bone diseases.

[7] Therapeutic agents for osteoporosis now being used include estrogen preparations, androgenic anabolic steroid preparations, calcium preparations, phosphate preparations, fluoride preparations, ipriflavone, vitamin D3, etc. In addition, novel drugs for osteoporosis have been developed, which include aminobisphosphonate by Merck Co. (U.S.A.) in 1995 and Raloxifene which acts as a selective estrogen receptor modulator (SERM) by Eli Lilly Co. (U.S.A.) in 1997.

[8] Meanwhile, conventional therapeutic agents for osteoporosis are mostly estrogen substances which are known to cause adverse side effects such as cancer, cholelithiasis and thrombosis upon prolonged use. Long-term administration is inevitable in the treatment of osteoporosis since it is not feasible to treat such a disease with short-term administration. Therefore, drug developers seek to develop novel effective agents which have no adverse side effects even when administered for a prolonged period of time and exhibit efficacy sufficient to replace estrogen. Currently, a great deal of interest has been focused on a phytoestrogen as one of estrogen substitutes.

[9] The phytoestrogen was first reported in 1946 by Bennetts et al. They revealed that the cause of clover disease, which was named for the high increase (over 30%) of infertility of the sheep fed with red clover (Trifolium subterraneum var. Dwalganup), was an estrogen-like isoflavonoid contained in the plant, hence, the compound obtained from the plant has been named "phytoestrogen".

[10] As compounds known as phytoestrogens, mention may be made of isoflavone compounds such as daidzein and genistein, coumestan compounds such as coumestrol, lignan compounds such as enterolactone, and phenol compounds such as enterodiol.

[11] In general, phytoestrogens function similarly to animal estrogens. That is, phytoestrogens inhibit growth of breast cancer cells by binding to the estrogen receptor and have been used as an estrogen substitute in the treatment of cardiovascular diseases and other symptoms occurring in postmenopausal women.

[12] Continued estrogen deficiency leads to increased incidence of cardiovascular diseases and osteoporosis in postmenopausal women. With the increase in the postmenopausal population, postmenopausal osteoporosis poses a variety of medical and socio-economic problems. Hormone replacement therapies have been practiced to prevent and treat cardiovascular diseases and osteoporosis in postmenopausal women, and it is well known that benefits obtained by prolonged hormone replacement therapies are much greater than the risk associated with such therapies. However, resumption of menses that may occur upon implementing hormone replacement therapies and fear associated with possibly an increase in the risk of breast cancer upon long-term administration cause many women to be reluctant to receive this treatment or give it up early.

[13] For such reasons, there has been continued research to seek a promising estrogen that maintains the effects of estrogen on skeletal and cardiovascular systems without affecting uterine and breast tissues. Clues for feasibility as such an ideal estrogen could be acquired from anti-estrogens that have been used to treat breast cancer. Anti- estrogens exhibit tissue-dependent effects. It was found that they block the action of estrogen on breast cancer cells, but have functions as a complete or partial estrogen agonist in other tissues. Estrogen agonists and antagonists exhibit their effects by binding to estrogen receptors, and thereby these substances are called selective estrogen receptor modulators (SERMs).

[14] The SERMs display tissue specificity that has different functions depending upon genes or cell types. The SERMs are known to play an active role by binding to the estrogen receptors and then affecting transcriptional processes, rather than by simply binding to the estrogen receptors in competition with estrogen thereby to inhibit the action of estrogen. Various actions of SERMs are determined by various kinds of SERMs, estrogen receptors (ERα and ERβ), hormone-responsive gene regulatory region (ERE, estrogen responsive element), various transcription factors and regulatory proteins. Estrogen receptor (ER) functions as a transcription factor which is activated by a ligand. When SERMs bind to the estrogen receptors, inducing conformational changes and subsequent activation of the receptors, the estrogen receptor-SERM conjugate binds to the gene of interest to initiate transcription. It is known that the mode of transcription varies depending upon types of cells and gene promoters.

[15] When estrogen is deficient in postmenopausal women, a bone turnover rate increases and bone absorption exceeds bone formation, thereby decreasing bone mass. Upon administration of SERMs to postmenopausal women, bone density in the spine increases for one year and then is maintained at the increased level. Similar effects are also observed in hip joints. In contrast with postmenopausal women, when SERMs are administered to premenopausal women, decreases of bone density in the radii, spine and hip joints are observed. Therefore, SERMs have estrogen-like action on bones when blood estrogen level is low, while they appear to function as antagonists when blood estrogen level is high.

[16] Meanwhile, it is known that estrogen, and both tamoxifen and raloxifene as SERMs, have effects of lowering cholesterol level. Administration of raloxifene to postmenopausal women results in significant decrease of total cholesterol and LDL cholesterol concentrations, but has no effects on concentrations of HDL cholesterol and triglyceride. In breast cancer patients who have received prolonged administration of tamoxifen, an about 50% reduction in cardiovascular diseases was observed.

[17] Tamoxifen and raloxifene were developed as anti-estrogens for treating breast cancer. In the uterus, tamoxifen acts on the estrogen receptor as a partial agonist, while raloxifene acts as the antagonist.

[18] In addition, recent research and study has reported that phytoestrogens like genistein affect increase of bone formation due to increased expression of estrogen receptors (ERα and ERβ) (Heim M. et al., 2003; Bonnelye et al., 2003). Disclosure of Invention Technical Problem

[19] Therefore, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a therapeutic agent for osteoporosis, comprising Ginkgo biloba leaf extract as an active ingredient. The Ginkgo biloba leaf extract in accordance with the present invention contains a large amount of phytoestrogens such as quercetin and kaempferol and thus can be usefully utilized as the therapeutic agent for osteoporosis and can provide a safe therapeutic agent for osteoporosis since the safety to humans was already established. Technical Solution

[20] In accordance with the present invention, the above and other objects can be accomplished by the provision of a therapeutic agent for osteoporosis, comprising a phytoestrogen contained in Ginkgo biloba leaf extract as an active ingredient. Specifically, the present invention provides a therapeutic agent for osteoporosis, comprising, as an active ingredient, the Ginkgo biloba leaf extract (hereinafter, referred to as "MDG5031") containing compound derivatives represented by general formula (I) which have excellent promotion effects of osteoblast proliferation and excellent anti- carcinogenic effects. Brief Description of the Drawings

[21] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:

[22] Fig. 1 shows the results of a pS2 gene expression assay, which determines pS2 that is expressed upon binding of estrogenic substance to an estrogen receptor. As can be seen, expression (density) of pS2 mRNA induced by the Ginkgo biloba leaf extract (MDG5031) was two-fold or more than that by Quercetin, known to have therapeutic effects for osteoporosis, and was close to that of 17β-estradiol (E ), thus suggesting that the Ginkgo biloba leaf extract acts as a potent estrogen analog by binding to the estrogen receptor and thereby it can be therapeutically effective for treating osteoporosis in the body;

[23] Fig. 2 graphically shows anti-proliferative effects of MDG5031 on MDA-MB-231 cells, by a WST-1 assay;

[24] Fig. 3 shows anti-breast cancer effects of MDG5031, by DNA fragmentation;

[25] Fig. 4 shows anti-breast cancer effects of MDG5031, by a TUNEL assay; and

[26] Fig. 5 shows anti-breast cancer effects of MDG5031 , by determination of caspase-3 activity. Best Mode for Carrying Out the Invention

[27] EXAMPLES

[28] Now, the present invention will be described in more detail with reference to the following Examples. These examples are provided only for illustrating the present invention and should not be construed as limiting the scope and spirit of the present invention.

[29] Preparation Example 1 : Preparation of Ginkgo biloba leaf extract

[30] Green Ginkgo biloba leaves, harvested in the central region of Korea, from August to September, were washed with tap water three times and additionally with purified water twice, separated and dried in air.

[31] 100 kg of dried materials (Ginkgo biloba leaves) was placed in a mixed solvent (1:1 v/v) of 100 L ethanol and 100 L purified water and the mixture was subjected to extraction by warming to 60 to 70°C.

[32] Extraction was repeated twice and the resulting materials were combined. This was followed by filtration through a reduced pressure funnel filter on which repeatedly washed diatomaceous earth was laid, and ethanol washing (foreign materials and lipid materials being removed from the extracts). The thus-obtained filtrate was concentrated under reduced pressure until the solid content reached level of 28 to 30%.

[33] The resulting concentrate was dried under reduced pressure using a high vacuum drier (vacuum degree of 6 to 10 Torr) and was ground with a mill.

[34] Yield was 7 kg and the composition of the extract is as follow: 24.7% of total flavone glycosides (by HPLC), 15.26% of Quercetin, 8.06% of Kaempferol, 1.42% of Isorhamnetin and 7.64% of a total terpene lactone.

[35] Experimental Example 1 : Osteoporosis therapeutic effects by estrogenic action of MDG5031

[36] In order to confirm whether MDG5031 in accordance with the present invention exhibits estrogen-like effects and thereby can act as a therapeutic agent for osteoporosis, the following experiments were carried out. In order to confirm estrogenic effects of MDG5031, an MCF-7 cell line (Human breast cancer cell), available from KCLB (Korean Cell Line Bank), located at the Cancer Research Institute of the Seoul National University College of Medicine, Seoul, Korea, was used and osteoporosis therapeutic effects by estrogenic action were observed through experiments of cell proliferation effects by E-Screen assay and experiments of estrogen-responsive gene (pS2) expression.

[37] Meanwhile, MDG5031 is a composite material containing a large quantity of phytoestrogens such as quercetin and kaempferol, and thus, unlike single material, molar concentration thereof cannot be calculated. For such a reason, experiments were carried out in terms of weight concentration and in order to standardize concentrations between respective materials, other single materials used in experiments were also tested in terms of weight concentration as in MDG5031.

[38] 1-1) Experiments on estrogenic action (osteoporosis therapeutic effects) by E- Screen assay

[39] The E-Screen assay is a method developed to assess the estrogenic effects of test substances. In accordance with this assay, estrogenic effects are evaluated by observing whether cell proliferation effects are exhibited by substances administered to MCF-7 cells. Such effects were confirmed by treating MCF-7 cells with MDG5031 and determining the degree of cell proliferation of each cell exhibited after 6 days. As a result, as can be seen from Table 1, administration of MDG5031 exhibited dose- dependent cell proliferation effects. In particular, the value of relative proliferative effect (RPE) which observes and compares cell proliferation effects was highest at a concentration of 250 D/D. In addition, in this test system, when the RPE value is more than 8, it can be said that the substance to be tested has estrogenic action as a partial agonist. Based on such criteria, MDG5031 has a value higher than the above- mentioned range and thus it was confirmed that MDG5031 may act as an estrogenic substance in the body. Additionally, upon comparing with quercetin, a main ingredient of Ginkgo biloba leaf extract, already known to be effective for treating osteoporosis, it could be seen that MDG5031 exerted significantly higher cell proliferation effects than quercetin, thereby having stronger action. From the above-mentioned results, it was confirmed that MDG5031 acts as an estrogenic substance in the body and subsequently exhibits estrogen-supplying effects, and thereby can effectively treat osteoporosis.

[40] The relative proliferative effect (RPE) is expressed by the following equation: [41] RPE = [(S-1)/(E-1)] x lOO [42] wherein S represents a proliferation rate of a sample and E represents a proliferation rate of a positive control (E ). That is, RPE is the relative value expressed by taking a 10 , proliferation rate of 10^" M of E to be 100.

[43] Table 1 MCF-7 cell proliferation by E-screen assay

[44] 1-2) Experiment on estrogenic action (osteoporosis therapeutic effects) by measurement of estrogen-responsive gene (pS2) expression

[45] In order to confirm the estrogenic effects of MDG5031 at the gene level, the pS2 gene expression assay, an assay to quantify expression of pS2, a gene that is expressed when estrogenic substances bind to estrogen receptors, was used to confirm the effects of MDG 5031. Activity of this gene is determined by directly observing whether the treated agent acts as estrogen as changes in gene expression occurring when estrogen is introduced into cells from the outside and binds to the estrogen receptor. The pS2 gene expression assay was carried out by Reverse Transcription Polymerase Chain Reaction (RT-PCR). MDG5031 and a main constituent thereof, quercetin were administered to MCF-7 cells and changes in quantities of expressed pS2 mRNA were observed and compared with a blank control and positive control. 17β-estradiol (E ) was used as the positive control and respective substances were tested at a concentration of 150 D/D. As can be seen from Fig. 1, expression (density) of pS2 mRNA induced by MDG5031 was two-fold or more than that induced by the main constituent thereof, quercetin, and was near to that exhibited by E , thus demonstrating that MDG5031 has potent estrogenic effects by binding to the estrogen receptor. This results from direct binding of MDG5031 to estrogen receptors thus affecting expression of pS2 mRNA. Therefore, it can be seen that MDG5031 acts as estrogen via the same estrogen-regulated pathway as does E (17β-estradiol) and thereby exerts estrogenic effects in the body, thus being capable of serving as a therapeutic agent for osteoporosis.

[46] Experimental Example 2: Osteoporosis therapeutic effects by action of Selective Estrogen Receptor Modulators (SERMs)

[47] Estrogen receptors ERα and ERβ have different distributions in body tissues depending upon body organs, and selective estrogen receptor modulators (SERMs) selectively bind to each estrogen receptor (ERα and ERβ), thereby exerting different effects depending upon kinds of organs in the body. In addition, such drugs bind to estrogen receptors and thus act as estrogen agonists or antagonists depending upon estrogen concentration in the body.

[48] When estrogen is deficient in postmenopausal women, bone turnover rate increases and bone absorption surpasses bone formation, thereby resulting in decrease of bone mass leading to development of osteoporosis. Upon administration of SERMs such as currently developed raloxifene to postmenopausal women, bone density in the spine increases. Similar effects are also observed in hip joints. Therefore, in order to confirm whether, as the selective estrogen receptor modulator, MDG5031 in accordance with the present invention is capable of acting as a therapeutic agent for osteoporosis, the following experiments were carried out.

[49] Meanwhile, MDG5031 is a composite material containing a large quantity of phytoestrogens such as quercetin and kaempferol, and thus, unlike single materials, molar concentration thereof cannot be calculated. For such a reason, experiments were carried out in terms of weight concentration and in order to standardize concentrations between respective materials, other single materials used in experiments were also tested in terms of weight concentration as in MDG5031.

[50] 2-1) Measurement of selective estrogen receptor binding properties by competitive binding assay

[51] In order to examine whether MDG5031 acts as a selective estrogen receptor (ER) modulator, affinity of MDG5031 for estrogen receptors ERα and ERβ was in- vestigated. The experiment was performed by administering 2,4,6,7- [ H]E and MDG5031 to recombinant human ERα and ERβ or, followed by incubation for 4 hours. As can be seen from Table 2, the results showed that binding capacity of MDG5031 to ER increased in a concentration-dependent fashion, based on observation of a relative ratio (%) to a non-agent treated control, and MDG5031 was observed to exhibit more than 1.5-fold higher binding capacity, as compared to the main ingredient, quercetin. In addition, it could be seen that MDG5031 exhibits binding affinity for both ERα and ERβ and thus acts as an estrogen receptor modulator. Additionally, affinity of MDG5031 for the estrogen receptors was greater for ERβ than for ERα. Therefore, it was confirmed that, as the estrogen receptor modulator, MDG5031 is capable of acting as a therapeutic agent for osteoporosis by the same action mechanism as does the existing raloxifene.

[52] Table 2 The binding affinity of MDG5031 for ERα and ERβ by competitive binding assay

[53] Experimental Example 3: Osteoporosis therapeutic effects by osteoblast proliferation and differentiation

[54] In order to confirm whether MDG5031 in accordance with the present invention has effects on cell proliferation of osteoblasts, Saos-2 cells, a human osteoblast-like cell line, available from KCLB (Korean Cell Line Bank), located at the Cancer Research Institute of the Seoul National University College of Medicine, Seoul, Korea, was used. Proliferation effects of osteoblasts were observed using, as an agent for comparison, genistein that is a kind of phytoestrogen and is known to have therapeutic action upon osteoporosis. Measurement of osteoblast proliferation was performed by a WST-1 assay that determines the degree to which tetrazolium salt substrate is cleaved to soluble formazan dye by enzymatic activation of a mitochondrial "succinate- tetrazolium reductase" system, which exists in the mitochondrial respiratory chain and is active only in metabolically intact cells, and a method that determines changes in activity of alkaline phosphatase (ALP) which increases during cellular differentiation processes. In addition, phytoestrogens genistein and quercetin were employed as comparative agents as they have been intensively studied as therapeutic agents for os- teoporosis.

[55] In this connection, MDG5031 is a composite material containing a large quantity of phytoestrogens such as quercetin and kaempferol, and thus, unlike single materials, molar concentration thereof cannot be calculated. For such a reason, experiments were carried out in terms of weight concentration and in order to standardize concentrations between respective materials, other single materials used in experiments were also tested in terms of weight concentration as in MDG5031.

[56] 3-1) Cell proliferation experiment depending upon concentrations of the agents: WST-1 Assay

[57] For comparison of cell proliferation rate (%), using Saos-2 cell line which has similar properties to osteoblasts, the degree to which tetrazolium salt is cleaved by a mitochondrial "succinate-tetrazolium reductase" system in metabolically active cells was determined. Cell proliferation rate exerted by the agents was calculated as percentage of the OD of the agents administered group to the OD of a control group to which the agents were not administered. As can be seen from Table 3, the osteoblast proliferation effects by administration of MDG5031 were slightly higher than quercetin and were similar to genistein. In particular, administration of MDG5031 at a concentration of 150 D/D showed the highest proliferation effects of 108.9% and thereby it was determined that MDG5031 had therapeutic effects upon osteoporosis as evidenced by promotion of osteoblast proliferation.

[58] Table 3 Proliferation effects of MDG5031 on Saos-2 cells using WST-1 assay

[59] 3-2) Analysis of Alkaline Phosphatase (ALP) Activity [60] Osteoblasts exhibit specific alkaline phosphatase (ALP) activity upon differentiation thereof. In order to examine effects of MDG5031 on osteoblasts, using hydrolysis of p-nitrophenylphosphate to p-nitrophenol and phosphate by ALP, the ALP activity was measured as the ratio of the OD at 405 nm of the groups to which respective substances were added relative to the OD of a control group at 405 nm.

[61] As shown in Table 4, administration of MDG5031 at a concentration of 100 D/D increased the ALP activity of Saos-2 cells up to 147.2% and exhibited higher ALP activity as compared to administration of quercetin or genistein. As a result, it was determined that MDG5031 had therapeutic effects upon osteoporosis as evidenced by promotion of osteoblast differentiation and possessed stronger effects than quercetin or genistein.

[62] Table 4 Differentiation effects of MDG5031 on Saos-2 cells using ALP activity assay

[63] Experimental Example 4: Anti-breast cancer effects of MDG5031 [64] Hormone preparations that are used for treating osteoporosis in postmenopausal women generally have the risk of causing breast cancer. MDG5031 has been found to act as a selective estrogen receptor modulator, and thus directly binds to an estrogen receptor (ER), which in turn exhibits estrogenic action, thereby having therapeutic effects for osteoporosis. Therefore, MDG5031 can inhibit breast cancer via such affinity for ERβ by competition with E (17β-estradiol) for binding to ERβ.

[65] Thus, in order to determine whether MDG5031 in accordance with the present invention has inhibitory effects upon development of breast cancer that may be caused as an adverse side effect of administration of estrogenic substances, experiments were performed in the presence of E so as to test anti-proliferative effects of breast cancer cells and inhibitory effects of expression of estrogen receptor genes and observe cell killing effects.

[66] 4-1) Experiment on anti-proliferative effects of breast cancer cells [67] In order to examine anti-carcinogenic effects of MDG5031, E (17β-estradiol) exhibiting strong estrogenic potency and MDG5031 were co-administered to MCF-7 cells (human breast cancer cells) at a concentrations ranging from 10 D/D to 500 D/D and inhibitory effects of MDG5031 on cell proliferation by E were measured by an E- Screen assay. As can be seen from the results in Table 5, the groups to which MDG5031 was administered at 100 D/D, 250 D/D and 500 D/D, respectively, exhibited significantly reduced cell proliferation rate (RPE %) in a concentration-dependent fashion, as compared to the group to which E (17β-estradiol) (10^" M) alone was administered. The group to which MDG5031 at a higher dose of 500 D/D was administered was shown to have strong anti-carcinogenic effects similar to the inhibitory effects exhibited by the anticancer drug tamoxifen at 0.37 D/D that is a concentration at which tamoxifen best exerts anti-carcinogenic effects. [68] Table 5 Inhibitory effects of MCF-7 cell proliferation by E-Screen assay

[69] The relative proliferative effect (RPE) is expressed by the following equation: [70] RPE = [(S-1)/(E-1)] x 100 [71] wherein S represents a proliferation rate of a sample and E represents a proliferation rate of a positive control (E ). That is, RPE is the relative value expressed by taking a proliferation rate of 10^" M of E to be 100.

[72] 4-2) Experiment on anti-breast cancer effects by suppression of expression of estrogenic genes

[73] In order to confirm the anti-carcinogenic effects of MDG5031 at the gene level, experiment was carried out by measurement of luciferase activity that is capable of testing estrogen receptor-mediated responses. This experiment is a method that determines the degree to which luminescence is generated when an estrogenic substance bound to Estrogen Receptor Element (ERE) induces gene expression which in turn results in activation of luciferase. As can be seen from Table 6, MDG5031 significantly inhibited expression of estrogenic genes that are expressed by administration of E (17β-estradiol). In comparison to the ancicancer agent, tamoxifen, and the main ingredient of Ginkgo biloba leaf extract, quercetin, it has been found that MDG5031 has inhibitory action of expression weaker than tamoxifen, but remarkably greater than quercetin. Therefore, it was demonstrated that MDG5031 has anti-carcinogenic action and thereby can be employed as a therapeutic agent for osteoporosis without the risk of adverse side effects.

[74] Table 6 Inhibitory effects of estrogenic gene expression by determination of luciferase activity

[75] 4-3) Experiment of anti-breast cancer effects by cell death mechanism [76] In order to examine whether MDG5031 has anti-carcinogenic effects by a cell death mechanism, cell killing experiments were performed by DNA damage of MCF-7 cells (human breast cancer cell line). Among 100 MCF-7 cells, cells that exhibited DNA damage by cell death were counted so as to confirm the degree of cell death. As a result, it was observed that, as evidenced by the number of dead MCF-7 cells thus counted, the MDG5031 -administered group exhibited about 3-fold higher cell killing effects as compared to the control group or the E -administered group. Therefore, it was confirmed that MDG5031 had anti-carcinogenic effects.

[77] Table 7 Anti-breast cancer effects via cell death mechanism

[78] From experimental results as discussed above, MDG5031 in accordance with the present invention acts as the estrogen receptor modulator and then exhibits pronounced therapeutic effects upon osteoporosis, as compared to the main ingredient of Ginkgo biloba leaf extract, quercetin, and had anti-breast cancer action, thus exhibiting strong inhibition of carcinogenesis that may be caused by administration of estrogen preparations for treating osteoporosis.

[79] The Ginkgo biloba leaf extract of the present invention may be mixed with pharmaceutically acceptable carriers to prepare oral formulations such as tablets and capsules using conventional methods well known in the art. In order to obtain osteoporosis therapeutic effects, the Ginkgo biloba leaf extract is preferably administered in an amount of 80 mg to 500 mg, three times a day.

[80] Experimental Example 5: Anti-breast cancer effects of MDG5031 by apoptosis [81] In order to confirm whether MDG5031 has anti-cancer effects on breast cancer cells and to elucidate whether such effects of MDG5031 are displayed by apoptosis in ER- negative cell line, as an action mechanism, this experiment was performed using ER- negative MDA-MB-231 cells, which are free of estrogen receptors, by measurement of cytotoxicity, DNA fragmentation, TUNEL assay and caspase-3 detection assay.

[82] 5-1) Antiproliferative effects of estrogen receptor-negative breast cancer cell (MDA-MB-231)

[83] In order to examine antiproliferative effects of MDG5031 on breast cancer cell line that does not involve estrogen receptors, WST-1 assay was carried out using the estrogen receptor-negative cell line, MDA-MB-231. As a result, when MDA-MB-231 cells were treated with MDG5031 for 72 hours at different concentrations, 5%, 13%, 57% and 62% reductions in cell viability were observed at concentrations of 100 D/D, 250 D/D, 500 D/D and 1000 D/D of MDG5031, respectively (see Fig. 2). In contrast, genistein, used as the control, exhibited 5%, 8%, 25% and 42% reductions in cell viability at concentrations of 5x10 M, 10^" M, 5x10^" M and 10 M, respectively. Based on the results given above, it is demonstrated that the Ginkgo biloba extract in accordance with the present invention has pronounced cytotoxicity on cancer cells, as compared to genistein which has been intensively studied for anticancer effects, and thereby is applicable to develop and prepare chemopreventive agents.

[84] 5-2) Experiment on anti-breast cancer effects via cell death mechanism

[85] 5-2-1) DNA fragmentation analysis

[86] In order to confirm whether cytotoxicity of MDG5031 against MDA-MB-231 cells is mediated by apoptosis, DNA fragmentation that is characteristic of apoptosis was confirmed. First, when cells were treated with 1000 D/D of MDG5031, and as controls, 10^" M genistein (G), 10^" M quercetin (Q) which is the main flavonoid ingredient of MDG5031, and lO^M kaempferol (K), for 72 hours, only the MDG5031 -treated group exhibited ladder-like DNA fragmentation (see Fig. 3a). Next, in order to observe concentration-dependent apoptosis effects of MDG5031, when cells were exposed to 100, 250, 500 and 1000 D/D of MDG5031, DNA fragmentation was observed at concentrations of 500 and 1000 D/D of MDG5031 (see Fig. 3b).

[87] 5-2-2) TUNEL assay

[88] Tunel assay was carried out to investigate generation of apoptotic cells that is effects of cancer cell death expressed by MDG5031. Cancer cells were placed on a slide glass and fluorescent substance contained in a kit was added thereto. Then, antibody and substrate were added to and combined with the attached fluorescent substance, followed by observation under a microscope. Cells appearing as black color were regarded as cells that have undergone apoptosis. As shown in Fig. 4, apoptotic cells were observed 72 hours after administration of 10^" M genistein. In the case of MDG5031, more potent cell killing effects (apoptosis) than genistein were observed at 1000 D/D. [89] 5-2-3) Measurement of Caspase-3 activity

[90] The caspase family of cysteine proteases, a protein that plays a key role in induction of apoptosis, is present in cytoplasm in the form of an inactive enzyme and is activated by stimuli inducing apoptosis. Therefore, in order to examine whether cellular apoptosis is activated by MDG5031, observation was made on changes in activity of caspase-3, known as an effector caspase in a cellular apoptosis signaling process of MDA-MB-231 cells. At concentrations of 100 D/D and 250 D/D of MDG5031 anάw genistein, there was no difference in activity of caspase-3 between normal cells and the agent-treated cells, but MDG5031 at concentrations of 500 D/D and 1000 D/D exhibited more than about 2-fold increase in activity of caspase-3 as compared to normal cells (see Fig. 5).

[91] In light of the results given above, it was confirmed that MDG5031 in accordance with the present invention exhibits anticancer effects via a cell death mechanism that does not involve estrogen receptors. Therefore, MDG5031 acts as the estrogen receptor modulator, and thus has more potent osteoporosis therapeutic effects than the main ingredient of Ginkgo biloba leaf extract, quercetin, and at the same time, has strong effect to inhibit breast cancer that may be caused by administration of estrogen preparations for treating osteoporosis. Industrial Applicability

[92] Therefore, the present invention provides a therapeutic agent for osteoporosis, comprising Ginkgo biloba leaf extract as an active ingredient. The Ginkgo biloba leaf extract in accordance with the present invention contain a large amount of phytoestrogens such as quercetin and kaempferol and thus can be utilized as a therapeutic agent for osteoporosis having anti-carcinogenic effects due to capability to function as an estrogen receptor modulator by combined action of these phytoestrogens.

[93] Although the preferred embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that various modifications, additions and substitutions are possible, without departing from the scope and spirit of the invention as disclosed in the accompanying claims.

Claims

[1] A therapeutic agent for osteoporosis having anti-carcinogenic effects, comprising Ginkgo biloba leaf extract as an active ingredient.

[2] The therapeutic agent according to claim 1, wherein the extract contains quercetin and kaempferol, represented by general formula (I):

R = H : kaempferol-3-O-rutinoside R = OH : quercetin-3-O-rutinoside (rutiπ) R = OCH₃ : isorhamnetin-3-O-rutinoside

(I)