WO2006043783A1

WO2006043783A1 - Pharmaceutical compositions, comprising red ginseng acid polysaccharide and lentinus edodes, of preventing and treating cancer

Info

Publication number: WO2006043783A1
Application number: PCT/KR2005/003492
Authority: WO
Inventors: Jong Dae Park; Jae Joon Wee; Yi Seong Kwak; Yong Bum Song; Jong Su Kyung; Jai Won Yang
Original assignee: KT&G Corp; Korea Ginseng Corp
Current assignee: KT&G Corp; Korea Ginseng Corp
Priority date: 2004-10-22
Filing date: 2005-10-19
Publication date: 2006-04-27
Anticipated expiration: 2007-04-22
Also published as: KR100590999B1; KR20060035289A

Abstract

This invention relates to a pharmaceutical composition which is useful for the treatment of tumors, comprising RGAP and Lentinus edodes extract as active ingredients, wherein its selective augmentation on the level of immunoglobulin IgM may lead to rapid recovery of immunity in the body, thus promoting longevity and maximizing antitumor activity.

Description

PHARMACEUTICAL COMPOSITIONS, COMPRISING RED

GINSENG ACIDIC POLYSACCHARIDE AND LENTINUS

EDODES, OF PREVENTING AND TREATING CANCER

Technical Field

[I] This invention relates to a pharmaceutical composition which is useful for the treatment of tumors, in a manner that its selective augmentation on the level of im¬ munoglobulin IgM may lead to rapid recovery of immunity in the body, thus promoting longevity and maximizing antitumor activity.

[2]

Background Art

[3] Antitumor agents are largely divided into chemotherapeutics and biological drugs.

[4] Biological drugs, such as cytokine, immunotherapy, gene therapy and antian- giogenetic agent, attempt to stimulate the immune system to reject and destroy tumors. [5] Immunotherapy is a form of medical treatment based upon a concept of modulating the immune system to achieve a therapeutic goal by potentiating the activity of the host defense mechanism. [6] More recently, a large body of work has been devoted to cytostatic agents with higher selectivity to tumors instead of cytotoxic therapy that is limited by poor delivery and drug resistance. [7] In particular, inhibition of tumors using promising natural drugs with fewer side effects has also become a major area of research in oncology. [8] Among natural products, red ginseng is a herbal preparation processed by Korea's specific technology. It has an antitumor, anti-diabetic and immunomodulatory properties. [9] Red ginseng contains red ginseng acidic polysaccharide (hereinafter referred to as

"RGAP") as an active ingredient, which was successfully isolated by the inventor et al., as disclosed in the Korean Patent Unexamined Publication No. 2002-94725. [10] The antitumor activity of RGAP is assumed to be due to its immunomodulatory property.

[I I] That is, the major effects include the augmented proliferation of immune cells including macrophage and natural killer (NK) cells.

[12] RGAP is involved in the activation of immune cells to produce cytokines (e.g., nitric oxide, tumor necrosis factor- alpha), that play a significant role in the regulation of the extent of the immune response.

[13] Nitric oxide (NO) performs a variety of tasks in the body. At high concentration it acts against bacteria and cancer cells. Some of the activities attributed to NO include mitogenic effects on fibroblasts and activation of T-cells.

[14] TNF-α induces hemorrhagic necrosis of tumors in both animals and humans with its role of a mediator of septic shock.

[15] As RGAP has an immunomodulatory property as described above, one can expect the increasing level of immunoglobulin by RGAP that plays a major role in the body's immune system.

[16] In addition, there is an unknown antitumor immune mechanism that the levels of

IgM antibodies increase in a dose-dependent manner during the administration of RGAP, which is discovered by the inventor et al.

[17] Unlike IgG antibodies, IgM antibodies are the largest type of antibody. They are found in blood and lymph fluid and are the first type of antibody produced in response to an infection. They also cause other immune system cells to produce compounds that can destroy invading cells. In this respect, they can be mobilized at an early de¬ velopment of tumors.

[18]

[19] A study designed to investigate antitumor effects of RGAP indicated that after thirty days of administration of RGAP 100 and 300 mg/kg to mice following in¬ traperitoneal inoculation Sarcoma- 180 cells, the survival rates of mice at the two doses were 57.1% and 85.7%, respectively, while solid tumor growths in B16 melanoma bearing mice were inhibited by 39% and 53%, respectively (Y.S. Kim, K.M. Park, HJ. Shin, K. Y. Nam and J.D. Park, Antitumor activity of RGAP through stimulation of macrophage and NK cells, Journal of Pharmaceutical Society of Korea, 46-2: 113-119, 2002).

[20] In another study using different doses of RGAP (30, 100 and 300 mg/kg) in an animal model, which was released by the Korean Patent Unexamined Publication, the tumor growth inhibition rates of NK cells in three different doses were 9.9-22.0%, 16.0-26.1% and 14.9-30.0%, while those of macrophage were 14.3% (100 mg/kg) and 36.3% (300 mg/kg), respectively. Thus, it is noted that antitumor effects of RGAP is associated with increasing doses of the medication.

[21] However, the above Patent Unexamined Publication No. 2002-94725 suggests that the production cost of RGAP is very high because of its low yield (6.7%) from the high-priced red ginseng.

[22] Therefore, the inventor et al. had conducted intensive studies to discover the al¬ ternative material to RGAP and as a result, found that Lentinus edodes is equal to or better than RGAP in terms of efficacy and toxicity profiles with possible large-scale production and less production costs.

[23] Lentinus edodes has a polysaccharide compound called LC-33, separated from its fruit body, which exhibits a potent antitumor activity on Sarcoma- 180 and cellular immune response (Chihara, G., Hamuro, T. and Maeda, Y., Fractionation and pu¬ rification of the polysaccharide with marked antitumor activity especially lentinan from Lentinus edodes, Cancer Res., 30: 2776-2781, 1970. Maeda, Y. and Chihara, G., Lentinan a new immuno- accelerator of cell medicated response. Nature, 229: 634, 1971).

[24] The antitumor activity of Lentinus edodes is assumed to be due to the presence of both protein-bound polysaccharide (PBP) and lentinan.

[25] PBP is believed to induce the generation of interferon that may in turn potentiate the activity of the host defense mechanism through indirect inhibition of tumor cells (Tsunoda, A, A mushroom agents and their mechanisms, lentinan, a T-cell oriented im- munopotentiator, NY and Basel, vol. 19, p.436, 1985).

[26] The antitumor activity of PBP is assumed to be due to inductions of macrophage proliferation activity (Takehara, A., Antiviral activity of virus-like particles from lentinus edodes, Shiitake, Arch Virol, 59: 269-280, 1979).

[27] Lentinan is a purified polysaccharide with a high molecular weight (beta- 1,3 glucan) separated from its fruit body, that strengthens the immune system show to anticancer activity (Suga, T., Dhiio, T. Maeda, Y, Y. and Chihara, G, Antitumor activity of lentinan in murine syngeneic and autotochthonous hosts and its suppressive effect 3-methylcholanthrene-induced carcinogenesis, Cancer Res., 44: 5132-5137, 1984).

[28]

[29] In the process of identifying an alternative medication to RGAP, the inventor et al. discovered that both RGAP and Lentinus edodes extract contribute to specifically selective augmentation of the level of immunoglobulin IgM in a dose-dependent manner, leading to the manufacture of a pharmaceutical composition containing both RGAP and Lentinus edodes extract as the alternative material to RGAP with a minimum therapeutic dosing level of RGAP. Thus, the inventor et al. has consummated this invention by manufacturing a pharmaceutical composition which is useful for the treatment of tumors, in a manner that its selective augmentation of the level of immunoglobulin IgM may lead to rapid recovery of immunity in the body, thus promoting longevity and maximizing antitumor activity.

[30]

Disclosure of Invention Technical Problem

[31] An object of this invention is to provide a pharmaceutical composition containing both RGAP and Lentinus edodes extract as an alternative material to RGAP. [32] Another object of this invention is to provide a pharmaceutical composition which is useful for the treatment of tumors, in a manner that its selective augmentation of the level of immunoglobulin IgM may lead to rapid recovery of immunity in the body, thus promoting longevity and maximizing antitumor activity.

[33]

Technical Solution

[34] An object of this invention is to provide a combined composition comprising both

RGAP and Lentinus edodes as active ingredients.

[35] RGAP and Lentinus edodes used for this invention may be administered in a minimum therapeutic dosing level. Hence, the pharmaceutical composition of this invention achieves antitumor effect that is equal to or better than a single admin¬ istration of RGAP or Lentinus edodes.

[36] The composition of this invention is useful for the treatment of tumors, in a manner that its selective augmentation of the level of immunoglobulin IgM may lead to rapid recovery of immunity in the body, thus promoting longevity and maximizing antitumor activity.

[37] Furthermore, the level of IgM may be significantly increased by the pharmaceutical composition containing both RGAP and Lentinus edodes extract with the weight ratio of 1:1, leading to better survival rate and antitumor effects.

[38]

[39] This invention is described in more detail as set forth hereunder.

[40] RGAP of this invention is manufactured by the conventional method using red ginseng, preferably by the method described in the Korean Patent Unexamined Publication No. 2002-94725.

[41] Lentinus edodes extract of this invention is manufactured by the conventional method using water or alcohol, preferably by the method of using hot water (higher than 9O⁰C).

[42] Further, Lentinus edodes extract of this invention may contain one or more components selected from the group consisting of polysaccharide LC-33, beta- 1,3 glucan lentinan or PBP (protein-bound polysaccharide).

[43] According to this invention, RGAP and Lentinus edodes extract is mixed in an ap¬ propriate weight ratio, preferably in the weight ratio of 1:1.

[44] Unlike the conventional mechanism that suppresses tumor cells growth through stimulation of immunoresponder cells, the composition of this invention is useful for the treatment of tumors, in a manner that its selective augmentation of the level of im¬ munoglobulin IgM may lead to rapid recovery of immunity in the body and contribute to better survival rate in an earlier stage of cancer, and then exhibits synergistic antitumor effects by maximizing the longevity in animal tumor models as well as solid tumor growth inhibition effects. [45] [46] As RGAP and Lentinus edodes play a role to exert antitumor effects in a synergistic manner, the pharmaceutical composition of this invention is characterized by a smaller use of RGAP that may demonstrate equal to or better than a single large use of RGAP.

That is, even a significance-undetectable minimum dose of RGAP can be effective, when administered in a mixed form with Lentinus edodes. [47] In consequence the pharmaceutical composition of this invention is economically feasible, even if a small amount is produced from red ginseng and less side effects may be expected. [48] The composition of this invention containing RGAP and Lentinus edodes extract, which is mixed in the weight ratio of 1:1, has proven its better therapeutic outcome in terms of selectively augmented level of IgM, better survival rate and solid tumor inhibition, in comparison with a single administration of RGAP and the composition employing weight ratios exceeding 2:1. [49] The composition of this invention may contain one or more of pharmaceutically acceptable carriers and be formulated in a variety of dosage forms, such as oral preparations (e.g., tablet, hard/soft capsule, pill, powder, oral solution, syrup, suspension) or non-oral preparations (e.g., injection). [50] [51] The daily dosage of the composition of this invention in human may vary depending on types of cancer, severity, age and body conditions of individual patients, but in general, the daily dosage of the composition is preferably in the range of 5-150 mg/kg, more preferably in the range of 10-100 mg/kg.

Advantageous Effects

[52] As RGAP and Lentinus edodes play a role to exert antitumor effects in a synergistic manner, the pharmaceutical composition of this invention is characterized by a smaller use of RGAP that may demonstrate equal to or better than a single large use of RGAP. That is, even a significance-undetectable minimum dose of RGAP can be effective, when administered in a mixed form with Lentinus edodes.

[53] In consequence the pharmaceutical composition of this invention is economically feasible, even if a small amount is produced from red ginseng and less side effects may be expected.

[54] The composition of this invention is advantageous in that (1) its selective aug¬ mentation of the level of immunoglobulin IgM may lead to rapid recovery of immunity in the body, thus promoting longevity and maximizing antitumor activity, and (2) its synergistic antitumor effects may inhibit the development of chemically induced tumors at an earlier stage. [55]

Best Mode for Carrying Out the Invention

[56] Hereinafter, the present invention will be described further in more detail with reference to the following Examples that are given herein as explanatory purpose, but do not limit the scope of this invention within these Examples. [57]

[58] Example 1: Immunoglobulins test

[59] The inventor et al. determined the level of immunoglobulins, IgG and IgM in tumor-bearing mice treated with the pharmaceutical composition of this invention. [60]

[61] (a) Preparation of specimens

[62] RGAP was prepared in the same manner as described in the Korean Patent Ap¬ plication No. 2001-33115. [63] More specifically, 1 kg of red ginseng was added to 1OL of 85% ethanol and was extracted three times at 7O⁰C.

[64] Then, after being centrifuged, the residue was harvested and dried under ventilation.

[65] The dried material was extracted in 1OL of water four times at 8O⁰C to obtain a water-soluble extract. [66] The extract was dialyzed against distilled water at a refrigerator, using a dialysis membrane with a cut-off value of more than 12kD. [67] The inner dialysate solution having a cut-off value of more than 12kD, so obtained, was centrifuged at 8,000 rpm for 20 minutes, to obtain a supernatant. [68] Then, 85% ethanol corresponding to a 5-fold amount of the supernatant was added to the supernatant for precipitation. [69] The precipitated material was further centrifuged at 8,000 rpm for 30 minutes and lyophilized to obtain RGAP fraction. [70] Lentinus edodes extract was prepared in a manner that Lentinus edodes was extracted in hot water (9O⁰C), followed by filtration and concentration processes. [71] [72] Specimen 1 : This specimen was prepared by mixing both RGAP and Lentinus edodes extract in the weight ratio of 1:1. [73] [74] Specimen 2: This specimen was prepared by mixing both RGAP and Lentinus edodes extract in the weight ratio of 1:2. [75] [76] Specimen 3: This specimen was prepared by mixing both RGAP and Lentinus edodes extract in the weight ratio of 1:3.

[77] [78] (b) Test methods [79] 10 cells/ml of Sarcoma- 180 in a saline were inoculated intraperitoneally to ICR mice (20-3Og, 4-5 weeks of age, each group of 10 animals) at a dose of 100 μl. Twenty-four hours after carcinoma cell inoculation, either saline, RGAP or Lentinus edodes extract alone or a composition of both RGAP and Lentinus edodes extract was administered once daily for 7 days as per doses shown in the following Table 1. Then, blood samples were collected from the heart of animals to measure the level of im¬ munoglobulins, IgM and IgG.

[80] Saline was given to control, which did not transplant Sarcoma- 180 for the measurement of the level of two immunoglobulins (Table 1).

[81]

[82] (c) Test results

[83] Table 1

[84] [85] As shown in Table 1, the level of both IgM and IgG in all groups receiving each specimen was significantly increased. The level of IgM in two groups receiving RGAP or Lentinus edodes extract as monotherapy was increased by 24.8+6.9 mg/dl, 28.5+7.5 mg/dl and 19.5+8.1, 22.6+7.5, 24.4+8.0 mg/dl, respectively, compared with that of control (9.6+4.2 mg/dl). The level of IgG did not show any significant changes, while that of IgM was increased in a dose-dependent manner by the doses of RGAP or Lentinus edodes extract.

[86] The level of IgM in three groups receiving each specimen (1, 2, 3) was 29.4+9.8, 26.4+9.1, 25.9 mg/dl, respectively; these figures of three groups were higher than that of a single treatment group. By percentage over control, the levels of IgM in three groups were increased by 306.3%, 275.0% and 269.8%, respectively. Among the three groups, specimen- 1 showed the highest concentration increase rate. The increase rate of the level in the specimen- 1 group receiving 50 mg/kg of RGAP was higher than 296.9% of the specimen-2 groups receiving 100 mg/kg of RGAP.

[87]

[88] The pharmaceutical composition containing RGAP and Lentinus edodes extract showed an increase in the level of both IgG and IgM, while the single treatment group showed the increase only in the level of IGM.

[89] In this context, the pharmaceutical composition of this invention is useful for an earlier treatment of rapidly growing tumors through its role to selectively induce the augmented level of immunoglobulin IgM, leading to rapid recovery of immunity in the body and maximizing antitumor activity.

[90] Furthermore, as RGAP and Lentinus edodes play a role to exert antitumor effects in a synergistic manner, the antitumor effects of the pharmaceutical composition of this invention was better than that of RGAP as monotherapy, in that a significance-un- detectable minimum dose of RGAP is administered in a mixed form with Lentinus edodes. The level of IgM was significantly increased in the pharmaceutical composition containing both 50mg/kg of RGAP and 50mg/kg of Lentinus edodes extract in the weight ratio of 1:1, suggesting that the said composition ratio may contribute to the maximization of antitumor activity.

[91]

[92] Example 2: Longevity promotion test

[93] The inventor et al. determined the longevity of cancer-bearing animals by the phar¬ maceutical composition of this invention.

[94]

[95] (a) Preparation of specimens

[96] Three specimens (1, 2, 3) prepared from Example 1 were used for this test.

[97]

[98] (b) Test methods

[99] 10 cells/ml of Sarcoma- 180 in a saline were inoculated intraperitoneally to ICR mice (20-3Og, 4-5 weeks of age, each group of 10 animals) at a dose of 100 μl. Twenty-four hours after carcinoma cell inoculation, either saline, RGAP or Lentinus edodes extract alone or a composition of both RGAP and Lentinus edodes extract was administered once daily for 7 days as per doses shown in the following Table 2. The survival rates of mice were measured (Table 2). Thirty days after administration, the survival rate was calculated by the following formula:

[100]

[101] Survival rate = Number of surviving Sarcoma- 180-bearing mice/number of initial

Sarcoma- 180-bearing mice x 100 [102]

[103] (c) Test results

[104] Table 2

[105] [106] As shown in Table 2, the survival rate of mice by RGAP (50, 100 mg/kg) or Lentinus edodes extract (50, 100, 150 mg/kg) alone were 30%, 70%, 30%, 40%, and 50%, respectively. By comparison, the survival rates of three specimens were 70%, 60%, and 60%, respectively.

[107] The survival rate of the specimen- 1 group receiving RGAP 50 mg/kg was equal to that of the group receiving RGAP 100 mg/kg alone. [108] As RGAP and Lentinus edodes play a role to exert antitumor effects in a synergistic manner, the survival rate of the pharmaceutical composition of this invention is was higher than that of RGAP as monotherapy, in that a significance-undetectable minimum dose of RGAP was effective, when is administered in a mixed form with Lentinus edodes.

[109] [HO] Example 3: Solid tumor inhibition test [111] The inventor et al. determined the synergistic effect of the pharmaceutical composition of this invention using a solid tumor model.

[112] After each specimen was administered to B 16 melanoma cell-bearing mice, the tumor size was measured.

[113] [114] (a) Preparation of specimens [115] Three specimens (1, 2, 3) prepared from Example 1 were used for this test. [116] [117] (b) Test methods [118] 10⁵ cells/ml of B 16 melanoma cell in a saline were inoculated subcutaneously into the backs of C57BL/6 mice at a dose of 100 μl. Twenty-four hours after B 16 melanoma cell inoculation, either saline, RGAP or Lentinus edodes extract alone or a composition of both RGAP and Lentinus edodes extract was administered once daily for 7 days as per doses shown in the following Table 3. After twenty days of admin¬ istration, the tumor weight was measured, and the volume was calculated (Table 3).

[119]

[120] (c) Test results

[121] Table 3

[122] [123] As shown in Table 3, significant solid tumor growth inhibition by RGAP or Lentinus edodes extract alone or three specimens (1, 2, 3) was observed compared with control group.

[124] The growth inhibition rates by RGAP (50, 100 mg/kg) and Lentinus edodes extract (50, 100, 150 mg/kg) alone were 59.4%, 73.1%, 22.6%, 30.2%, and 59.9%, re¬ spectively. By comparison, the growth inhibition rates of three specimens were 92.5%, 83.0%, and 67.0%, respectively.

[125] As RGAP and Lentinus edodes play a role to exert antitumor effects in a synergistic manner, the solid tumor growth inhibition rate of the pharmaceutical composition of this invention was generally higher than RGAP as monotherapy, in that a significance- undetectable minimum dose of RGAP was effective when administered in a mixed form with Lentinus edodes.

[126] In particular, the solid tumor growth inhibition rate by two specimens containing RGAP and Lentinus edodes extract with the weight ratio of 1 : 1 and 1 :2 was sig¬ nificantly higher than that of a monotherapy group containing 2-fold RGAP or Lentinus edodes extract.

[127] This test demonstrated the synergistic effect of the pharmaceutical composition of this invention [128]

Claims

[I] A pharmaceutical antitumor composition comprising both RGAP (red ginseng acidic polysaccharide) and Lentinus edodes extract with the weight ratio of 1:1, wherein the antitumor effects of the composition may be exerted through its selective role to increase the level of IgM antibodies when administered.

[2] The pharmaceutical antitumor composition comprising both RGAP and Lentinus edodes extract with the weight ratio of 1:2, wherein the antitumor effects of the composition may be exerted through its selective role to increase the level of

IgM antibodies when administered. [3] The pharmaceutical antitumor composition comprising both RGAP and Lentinus edodes extract with the weight ratio of 1:3, wherein the antitumor effects of the composition may be exerted through its selective role to increase the level of

IgM antibodies when administered. [4] An antitumor agent containing both RGAP and Lentinus edodes extract as active ingredients and pharmaceutically acceptable carriers. [5] The antitumor agent according to claim 4, wherein Lentinus edodes extract is selected from the group consisting of a polysaccharide compound LC-33, a polymer beta-1, 3 glucan Lentinan or PBP (protein-bound polysaccharide). [6] The antitumor agent according to claim 4, wherein both RGAP and Lentinus edodes extract are contained in the weight ratio of 1:1 to 1:3. [7] The antitumor agent according to claim 4, wherein a tumor represents a solid tumor. [8] An immunopotentiator containing both RGAP and Lentinus edodes extract as active ingredients and pharmaceutically acceptable carriers. [9] The immunopotentiator according to claim 8, wherein RGAP and Lentinus edodes extract are contained in the weight ratio of 1:1 to 1:3. [10] The immunopotentiator according to claims 8 or 9, wherein the immunopo- tentiating effects are associated with selectively increasing levels of IgM.

[I I] A pharmaceutical antitumor agent formulated in an oral or non-oral dosage form comprising the composition as in any of claims 1 - 3.

[12] A pharmaceutical immunopotentiator formulated in an oral or non-oral dosage form comprising the composition as in any of claims 1 - 3.