JP2015051970A - Composition for producing adjuvant for cancer patients receiving chemotherapy - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a composition for producing adjuvant for cancer patients receiving chemotherapy.SOLUTION: A composition for producing an adjuvant for cancer patients receiving chemotherapy of the invention mainly contains soybean fermentation extract, green tea extract, spirulina, turmeric extract, Antrodia Cinnamomea mycelium, and grape seed extract. The composition provides development of colon polyps and inhibition of canceration, increased survival ratio of cancer patients, particularly increased efficacy of chemotherapy comprising increased disease-free survival rate, and/or a reduction effect of side effects caused by chemotherapy. And the composition of the invention also provides anti-inflammatory activity which is effective in production reduction of inflammatory mediators, such as prostaglandin E2 (PGE2), nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6).

Description

  This application claims the benefit of Taiwan Patent Application No. 102132310, filed on September 6, 2014, the entire disclosure of which is incorporated herein by reference.

  The present invention relates to a composition for producing an adjuvant for chemotherapy, and in particular, the present invention is effective in inhibiting the growth and canceration of colorectal polyps and increasing the effectiveness of chemotherapy and compliance with chemotherapy. The present invention relates to a composition for producing an adjuvant for cancer patients undergoing chemotherapy, which reduces side effects caused by chemotherapy.

  Soybean fermented extract, green tea, spirulina, turmeric, venix mushroom, and grape seed are all natural treasures for health, and each of these ingredients has health benefits in many biochemical tests. It is shown. The main pharmacological activity of curcumin contained in turmeric is to inhibit tumor growth, and the polysaccharides abundantly contained in Benicus mushrooms can enhance immune function and kill tumor cells. A variety of single-component products are commercially available, but there are no related studies on the combined effects of the six components on reducing the side effects of chemotherapy in cancer treatment.

  Taking colorectal cancer as an example, colorectal cancer includes colon cancer and rectal cancer, and is one of the most common malignant tumors in the world. Although it is not completely clear what causes colorectal cancer, colorectal cancer is generally thought to be highly related to diet. For example, both increased consumption of processed foods, animal fats, protein-rich foods, and insufficient intake of fiber-rich foods lead to an increase in the incidence of colorectal cancer, a modern world disease. Fortunately, although colorectal cancer occurs frequently, lesions are relatively easy to find and colorectal cancer takes a relatively long time to progress. Because most early colorectal cancers progress about 5 to 10 years after polyps, colorectal cancers are generally easily controlled if colon polyps are detected at an early stage on routine examination. However, symptoms in the early stages of colorectal cancer are not noticeable and have low specificity, making it easier for patients and physicians to overlook those symptoms and miss opportunities for early detection and treatment.

  Colorectal cancer is usually the most common three types of treatment: surgery, radiation therapy, and chemotherapy. Above all, along with understanding of tumors, currently chemotherapy of colorectal cancer mainly uses combination chemotherapy based on fluorouracil (5-FU) to reduce cancer recurrence and metastasis and improve survival rate.

  FOLFOX4 is a chemotherapy regimen currently used for the treatment of stage II and III and metastatic colorectal cancer and consists of drugs including fluorouracil (5-FU), leucovorin and oxaliplatin. However, these drugs have toxic side effects including fever, nausea, vomiting, diarrhea, bone marrow suppression, numbness in the extremities of the limbs, and poor circulation. Patients often suffer from poor quality of life and life-threatening complications due to the symptoms of side effects caused by drugs.

  Therefore, there is a need to find adjuvants for cancer patients who receive chemotherapy to reduce discomfort and improve quality of life.

  Furthermore, it is known that chronic inflammation and immune system activation are related to tumor progression. There is some evidence to suggest involvement of chronic immune activation, including an association between the use of non-steroidal anti-inflammatory drugs (NSAIDs) and risk mitigation of cancer progression. Mediators of inflammatory responses play an important role in tumorigenesis and are targets for therapeutic intervention for cancer. There is a need to regulate the inflammatory response when receiving chemotherapy.

  In view of the foregoing, an object of the present invention is to provide a composition for producing an adjuvant for cancer patients undergoing chemotherapy.

  Another object of the present invention is to provide a composition for producing an anti-inflammatory agent.

  In particular, the composition of the present invention includes a fermented soybean extract, a green tea extract, a spirulina, a turmeric extract, a venomus mycelia mycelium, a grape seed extract.

  According to an embodiment of the present invention, the composition comprises a fermented soybean extract, a green tea extract, a spirulina, a turmeric extract, a venix mushroom mycelium, and a grape seed extract in a weight ratio of 12-30: 4: 1-2. : 1-2: 1-2: 1-2.

  According to one embodiment of the invention, the cancer patient is a colorectal cancer patient.

  According to one embodiment of the invention, the cancer patient receives chemotherapy that administers a chemotherapeutic agent selected from the group consisting of 5-fluorouracil, leucovorin, oxaliplatin and any combination thereof.

  According to one embodiment of the invention, the composition of the invention is administered orally.

  In some embodiments, the composition of the invention inhibits colonic polyp growth and canceration, and reduces tumor size or prolongs survival (especially progression-free survival), compliance with chemotherapy It is effective for improving the effectiveness of chemotherapy including improvement and reduction of side effects caused by chemotherapy.

  In particular, the composition of the present invention is effective in reducing the production of prostaglandin E2 (PGE2), nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin-6 (IL-6). is there.

  These and other features of the present invention will become more apparent from the following preferred embodiments and description with reference to the drawings. Nevertheless, changes and modifications may be made without departing from the spirit and scope of the novel concepts disclosed herein.

  The foregoing summary, as well as the following detailed description of the present invention, will be better understood when taken in conjunction with the accompanying drawings. For the purpose of illustrating the invention, there are shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

1 is a graph showing an abnormal crypt nest (ACF) in a rat colon. (A) MB-6 (B1 = 17.3 mg / rat / day, B2 = 34.6 mg / rat / day, B3 = 69.2 mg / rat / day) treated rats were treated with C1 (1,2-dimethylhydrazine (DMH ) Control) Showed a significant reduction in total ACF / colon (40.6-51.0%, * P <0.05) compared to rats. (B) The number of crypts per ACF was classified into 1-3 crypts, 4-6 crypts, 7 crypts or more. MB-6 significantly suppressed the formation of chemically induced ACF, and the highest inhibition rate (81.7%, * P <0.05 versus C1 group) was observed in the group with 7 or more crypts. It is a graph which shows the survival condition of the colon tumor CT-26-VD carrying | support BALB / c mouse | mouth treated with the chemotherapy of MB-6 and leucovorin / 5-fluorouracil. Median survival was 27.5 days (tumor only), 30.5 days (NC), 34.5 days (PSK), 35 days (MB-6-L), 39 days (MB-6-M) ), 39 days (MB-6-H). NC is negative control, PSK is polysaccharide K, MB-6-L is 0.625 g / kg MB-6, MB-6-M is 1.25 g / kg, MB-6-H is 2.5 g / kg MB-6 is shown. It is a graph which shows that progression free survival (PFS) in the number of all cases (intention to treat, ITT) was extended significantly.

  Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

  As used herein, the singular forms also include a plurality of referents unless the context clearly singular. That is, for example, reference to “a component” includes a case where there are a plurality of such members and their equivalents known to those skilled in the art.

  In the present invention, the term “therapeutically effective amount” as used in connection with administration of a biologically active agent or composition refers to the biological effect intended in the treated subject as compared to an untreated control group. Refers to the dose of biologically active agent or composition sufficient to produce Specifically, an effective amount of the composition of the present invention increases the effectiveness of chemotherapy, including prolonging survival, particularly progression-free survival, inhibiting colonic polyp growth and / or canceration, Refers to an amount that increases compliance with chemotherapy and / or reduces side effects caused by chemotherapy. The amount can be adjusted based on the route of administration and the conditions of treatment including age, weight, symptoms, therapeutic effect, dosage regimen, treatment time. For example, in certain embodiments, the oral dose of the composition used in the present invention is 10 to 1,000 mg / kg per day, specifically 10 to 800 mg / kg per day, more specifically 1 10 to 500 mg / kg per day, more specifically 10 to 250 mg / kg per day, more specifically 10 to 150 mg / kg per day.

  As used herein, the term “individual” or “subject” refers to non-human animals, including humans and companion animals (dogs, cats, etc.), livestock (cattle, sheep, pigs, horses, etc.), or laboratory animals ( Rat, mouse, guinea pig, etc.).

  In accordance with the present invention, the composition of the present invention can be used as an adjuvant for chemotherapy. In one embodiment, a therapeutically effective amount of a composition of the present invention can be prepared with a pharmaceutically acceptable carrier into a pharmaceutical composition in a form suitable for delivery and absorption purposes. Depending on the mode of administration, the pharmaceutical composition of the present invention is preferably about 10% to about 100%, or about 20% to about 100%, or about 40% to about 100%, or about 60% to about 100%. Containing the active ingredient by weight, of which the weight percentage is calculated based on the weight of the entire composition.

  The term “carrier” or “pharmaceutically acceptable carrier” as used herein refers to diluents or excipients for pharmaceuticals, including those known to those skilled in the pharmaceutical arts.

  The present invention provides a composition mainly comprising a soybean fermented extract, green tea, spirulina, turmeric extract, venix mushroom mycelium, grape seed extract, said composition for cancer patients undergoing chemotherapy It can be used for the production of an adjuvant.

  Specifically, the fermented soybean extract used in the present invention refers to an extract produced by fermentation of an aqueous soy extract and at least one lactic acid bacterium and optionally at least one yeast. In one embodiment, the at least one lactic acid bacterium is a Lactobacillus species and the at least one yeast is a Saccharomyces species. In a particular embodiment, the fermentation is carried out using a Lactobacillus heterogeneous culture, for example a culture of 5 or 10, 15, 20, 25, 30 strains of Lactobacillus and preferably at least one yeast is said Lactobacillus. Added to different cultures. The Lactobacillus strains that can be used for the fermentation are Lactobacillus acidophilus CCRC (Taiwan Food Industry Development Laboratory Bioresource Conservation and Research Center, Bioresource Collection and Research Center at Food Research and Development 64 and 14065, 10695). 14079, Lactobacillus delbrueckii bulgaricus CCRC 10696 and / or 14007, 14009, 14010, 14069, 14071, 14098, 16054, Lactobacillus lactis lactis CCRC 10791 and / or 12267, 12306, 12312, 12315, 12323, 14016, 14015, 14117, Lactobacillus kefir CCRC 14011 and / or Lactobacillus kefiranofaciens CCRC 16059. Yeast strains that can be used for the fermentation include Saccharomyces cerevisiae CCRC 20577 and / or 20578, 20581, 21494, 21550, 21597, 21805, 21138, 22234, 22337, 22731, 27728, and / or Candida kefyr CCRC 21269 and ( Or) including, but not limited to, 21742, 22057. Specifically, the fermentation is followed by steps such as sterilization, filtration, concentration, lyophilization or any combination thereof. Preferably, sterilization, for example, by heating is performed after the fermentation, and filtration and concentration are selectively performed. More preferably, the soybean fermented extract is dried by freeze drying, for example, to obtain a powdered fermented soybean extract. In a specific embodiment, the soybean fermented extract of the present invention comprises (a) fermenting an aqueous soybean extract with at least one lactic acid bacterium and at least one yeast to form a fermentation liquor, (b) the fermentation Manufactured by a process comprising the steps of: sterilizing a liquid; (c) collecting the filtrate by filtering the sterilized fermentation broth; and (d) concentrating the filtrate to obtain a concentrated soybean fermented extract. . In a specific embodiment, soy is mixed with distilled water (eg, in a ratio of about 1:10) and the mixture is heated at about 100 ° C. for about 30 minutes and then filtered. This yields an aqueous soy extract, which is further fermented in a suitable medium with at least one lactic acid bacterium and at least one yeast and cultured at 36-43 ° C. for 45-50 hours. The fermented extract is sterilized, filtered, and concentrated (eg, removing about 95% water content in the filtrate) to obtain a concentrated form of soy fermented extract. In a specific embodiment, the specific gravity of the soy fermentation extract is 1.136 g / ml, 71.49% moisture, 5.15% ash, 0.16% crude fat, 5.45% Of crude protein, 0.15% crude fiber, and carbohydrates. Also, multiple vitamins and minerals such as 0.004 mg / 100 g vitamin B1, 0.12 mg / 100 g vitamin B2, 2.17 mg / 100 g iron, 113.55 mg / 100 g calcium, 379.19 mg / 100 g phosphorus Can be included. The soybean fermented extract used in the present invention is prepared according to US Pat. Nos. 6,855,350 and 6,733,801, which are incorporated herein by reference in their entirety.

  Furthermore, the green tea extract, the turmeric extract, and the grape seed extract are made by a standard procedure that includes extraction with water and ethanol. Spirulina is obtained by drying after harvesting into a powder or tablet. The above-mentioned venix mushroom mycelium can be obtained by drying, concentrating and / or polishing to a powder after general liquid fermentation. All of these components are commercially available from the manufacturer and can be purchased.

  In accordance with some embodiments of the present invention, the composition of the present invention comprises a fermented soy extract, green tea extract, spirulina, turmeric extract, venix mushroom mycelium, grape seed extract, and their weight ratio Is 12 to 30: 4: 1 to 2: 1 to 2: 1 to 2: 1 to 2, preferably 12 to 20: 4: 1 to 2: 1 to 2: 1 to 2: 1 to 2. . According to a specific embodiment of the present invention, the weight ratio of soybean fermented extract, green tea extract, spirulina, turmeric extract, venix mushroom mycelium, grape seed extract is 12: 4: 2: 1: 1. : 1. According to another specific embodiment of the present invention, the weight ratio of soy fermentation extract, green tea extract, spirulina, turmeric extract, venix mushroom mycelium, grape seed extract is 12: 4: 1: 1. : 2: 1. Under the above weight ratio, the weight ratio of each component of the composition can be appropriately adjusted based on different situations.

  The composition of this invention can be manufactured as a foodstuff or a pharmaceutical. Food-type products can be prepared by any conventional technique using conventional excipients or fillers, and additives can be mixed as needed. In addition, pharmaceuticals can generally be prepared by pharmaceutical techniques or methods using a therapeutically effective amount of the composition of the present invention and a pharmaceutically acceptable carrier. Administration of the composition of the present invention includes oral administration or injection, and may further comprise a pharmaceutically acceptable carrier, diluent and / or excipient. The carriers described above can be solvents, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, absorption delaying agents, and the like. The diluent can be sterile water for injection (BWFI), phosphate buffer, Ringer's solution, glucose solution, or the like. The excipient may be calcium carbonate, sodium carbonate, calcium phosphate, sodium phosphate, lactose, starch, gelatin, alginic acid, stearic acid, magnesium stearate and the like.

  In some specific embodiments, the compositions of the invention have the effect of inhibiting colonic polyp growth and / or canceration. Specifically, as shown in the embodiments, the composition of the present invention can inhibit abnormal crypt foci (ACF) induced by 1,2-dimethylhydrazine (DMH) and proliferate colon polyps. And / or has an effect of inhibiting canceration.

  In some specific embodiments, the compositions of the invention have the effect of improving the effectiveness of chemotherapy, including prolonging survival of cancer patients, particularly progression-free survival. As shown in the embodiments, subjects receiving chemotherapy with the composition of the present invention have a longer life than subjects receiving chemotherapy without the composition of the present invention, in particular, progression-free survival. It was done.

  In some specific embodiments, the compositions of the invention have the effect of improving compliance with chemotherapy. As shown in the embodiments, subjects receiving chemotherapy with the composition of the present invention showed significantly higher chemotherapy compliance than subjects receiving chemotherapy without the composition of the present invention. .

  In some specific embodiments, the compositions of the invention have the effect of reducing the side effects caused by chemotherapy. As shown in the embodiment, when the composition of the present invention is given during the cancer chemotherapy process, the composition can reduce the side effects of chemotherapy, especially kidney damage and neutropenia It turns out that.

  In addition, based on the present invention, the compositions described herein can be used as anti-inflammatory agents. Specifically, the composition of the present invention includes prostaglandin E2 (PGE2), nitric oxide (NO), tumor necrosis factor α (TNF-α), interleukin-6 (IL-6), macrophages and the like. Effectively reduces inflammatory mediator production in inflammatory cells.

  The present invention includes prostate cancer, breast cancer, uterine cancer, blood cancer, ovarian cancer, endometrial cancer, cervical cancer, colorectal cancer, testicular cancer, lymphoma, rhabdomyosarcoma, nerve Blastoma, pancreatic cancer, lung cancer, brain cancer, skin cancer, stomach cancer, oral cancer, liver cancer, laryngeal cancer, gallbladder cancer, thyroid cancer, liver cancer, kidney cancer, nasopharyngeal cancer Applicable to patients with cancer, including According to one embodiment of the invention, the patient is a colorectal cancer patient. According to an embodiment of the present invention, the chemotherapy is treatment using a drug comprising 5-fluorouracil, leucovorin, oxaliplatin.

  The present invention will be further clarified by the following embodiments. The following embodiments are provided for purposes of illustration and not limitation. Those skilled in the art will be able to make many changes in the disclosed specific embodiments based on the disclosure of the present invention and obtain the same or similar results without departing from the spirit and scope of the present invention. It is necessary to understand what can be done.

Inhibition of abnormal crypt nest (ACF) formation induced by 1,2-dimethylhydrazine (DMH)

1.1 Materials and methods

  Animal experiments were approved by the National Taiwan University Animal Experiment Committee and the National Committee of National Taiwan University, and all animals were processed according to standard guidelines for the care and use of laboratory animals. Approximately 6 weeks old F344 male rats were purchased from the Laboratory Animal Center of the National Taiwan University School of Medicine (College of Medicine, National Taiwan University).

  Male F344 rats were randomly grouped by 8 by weight. Rats began using Harlan AIN-76 rodent refined diet (Harlan Laboratories, Indiana, USA) containing 17.7% protein, 64.9% carbohydrate, 5.2% fat by weight from one week after acquisition. (Police) (Harlan AIN-76A purified feed data sheet, Harlan Laboratories. Source: http://www.harlan.com/download.axd/1131a6e1617348b9a99c8d2af933f796.pdf=170). Rats were kept in a stainless steel cage with free access to food and distilled water. The animal room was maintained at 23 ± 1 ° C., 50 ± 10% humidity, with a 12 hour day / night cycle. Feed intake and weight were recorded daily.

  The composition (MB-6) of the present invention includes, for example, soybean fermentation extract (prepared according to the methods of US Pat. Nos. 6,855,350 and 6,733,801), green tea extract (Camellia sinensis O.), Spirulina algi (Arthrospira platensis) , Turmeric extract (Curcuma longa L.), Benix nokitake mycelium, grape seed extract (Vitis vinifera). Table 1 shows the components of MB-6 in a specific embodiment in a weight ratio of 12: 4: 2: 1: 1: 1.

  The animals received a control group C1 that received 1,2-dimethylhydrazine (DMH) injection, a control group C2 that received saline injection, and a low dose (17.3 mg / rat / day) of the six-component formulation of the present invention. Experimental group B1 received, experimental group B2 received medium dose (34.6 mg / rat / day) of the six component formulation of the present invention, high dose (69.2 mg / rat / day) of the six component formulation MB-6 of the present invention Group) was received in the experimental group B3. DMH was dissolved in physiological saline (0.9% NaCl solution) to make a 2% solution, adjusted to pH 6.7, aliquoted, and stored at -20 ° C until needed. DMH was administered by intraperitoneal (IP) injection at a dose of 20 mg / kg weekly for 4 weeks (corresponding to 1 ml injection of 2% solution per kg body weight). The control group was injected with saline (1 ml saline per kg body weight). Tables 2 and 3 show.

  At 15 weeks, the animals were fasted overnight and euthanized by carbon dioxide aspiration the next day. The colon was then removed, length and weight were measured, and the number of abnormal crypt foci (ACF) and the number of crypts that make up each ACF were counted. Briefly, the colon was cut along the longitudinal axis, widened with the mucosal lining open upward, and cut into three parts, front, middle and posterior. Each part was placed between two sheets of filter paper in a Petri dish containing 10% buffered formalin and fixed for over 24 hours. For ACF quantification, fixed colon tissue was stained with 0.2% methylene blue (PBS as solvent), and ACF numbers were evaluated at 40-100 times magnification using a light microscope. In order to determine the number of crypts, the number of crypts per ACF was further classified into 1-3 crypts, 4-6 crypts, 7 crypts or more.

  Experimental data were analyzed by one-way analysis of variance (One-way ANOVA) using SAS software, and differences between groups were compared with Student's T-test. Results with p <0.05 indicate a significant difference.

1.2 Results

  A total of 50 F344 rats were assigned to C1 (DMH control group), C2 (standard control group), B1 (MB-6-L), B2 (MB-6-M), and B3 (MB-6-H) groups. Randomly assigned. There was no significant difference between the weights of the five groups over time. The number of ACFs per colon is shown in FIG. 1A. MB-6 treated rats showed a significant decrease in total ACF counts per colon (40.6-51.0%, P <0.05) compared to C1 (DMH control group) rats. The number of crypts per ACF was further classified into 1 to 3 crypts (small), 4 to 6 crypts (medium), and 7 or more crypts (large). MB-6 significantly suppressed the formation of chemically induced ACF, and the highest inhibition rate (81.7%, FIG. 1B) was observed in the group with 7 or more crypts. Refer also to Table 4 and Table 5.

  In summary, in animal experiments, the six-component formulation of the present invention can effectively reduce the number of abnormal crypt foci induced by 1,2-dimethylhydrazine, especially the occurrence of large abnormal crypt foci. Indicated.

Inhibition of colorectal cancer formation induced by 1,2-dimethylhydrazine (DMH)

2.1 Materials and methods

  Purchased 6 week old F344 male rats were randomly grouped one week after acquisition. There were 30 animals in each group. The animals received a control group C1 that received 1,2-dimethylhydrazine (DMH) injection, a control group C2 that received saline injection, and a medium dose (34.6 mg / rat / day) of the six-component formulation of the present invention. Grouped into experimental group B2 received. DMH was dissolved in physiological saline (0.9% NaCl solution) to make a 2% solution, adjusted to pH 6.7, aliquoted, and stored at -20 ° C until needed. DMH was administered by intraperitoneal (IP) injection at a dose of 30 mg / kg weekly for 8 weeks (equivalent to 1.5 ml injection of 2% solution per kg body weight). The control group was injected with saline (1.5 ml saline per kg body weight). Tables 6 and 7 show.

  At the end of the 32 week experimental period, the experimental animals were fasted overnight, euthanized the next day by ether anesthesia, and dissected. The animal's colon was removed and length and weight were measured and stored for subsequent tumor analysis.

  The colon, cecum, small intestine, and stomach were removed and completely incised along the longitudinal axis of these organs. After washing with phosphate buffered saline, the organs were placed flat on the filter paper with the mucosa facing up. The tumor on the sample was examined and the location, number and size of the tumor was recorded. The length (L), width (W), and depth (D) of each tumor is measured by vernier, and the volume (V) of each tumor is calculated by the formula: V = (L * W * D * π) / 6 It was done. A photograph of the sample was taken and stored.

  Experimental data were analyzed by one-way analysis of variance (One-way ANOVA) using SAS software, and differences between groups were compared with Student's T-test. Results with p <0.05 indicate a significant difference.

2.2 Results

  As shown in Table 8, with regard to the number of tumors, the 6-component preparation of the present invention effectively inhibited tumor formation induced by 1,2-dimethylhydrazine, and the inhibition rate was 38.4%, a statistically significant difference. (P <0.05).

Furthermore, as shown in Table 9, the tumor on the basis of the volume, 0 to 100 mM ³ (small), 100 to 500 mm ³ (medium size) were divided into three groups of 500 mm ³ greater (large). The six-component preparation of the present invention can inhibit tumors induced by 1,2-dimethylhydrazine, and in particular, small (0-100 mm ³ ) tumors were significantly inhibited with an inhibition rate of 49.7% (p <0. 0). 001).

Volume reduction and survival of colorectal cancer in mice

3.1 Materials and methods

  Male BALB / c mice were purchased at the National Laboratory Animal Center of Taiwan and maintained at 21 ± 2 ° C. with a 12 hour day / night cycle. AIN-76 refined feed and distilled water were constantly fed.

Mice were randomly divided into 7 groups of 12 mice and 3 mice were placed in a cage. The mouse colon cancer cell line CT-26-VD was cultured at 37 ° C. in complete Dulbecco's modified Eagle medium (DMEM, GIBCO, Cat # 11995) containing 10% fetal calf serum in an incubator containing 5% CO _2. It was. The establishment of a mouse colon cancer model has been described previously (Witek M et al., Int J Radiate Oncol Biol Phys 2014; 88: 1188-95). Briefly, mice were weighed and anesthetized with an appropriate dose of pentobarbital sodium based on body weight (Somnotol, Canada, 10 μL / g, 6.5 mg / ml). 100 μL of CT-26-VD cell suspension (2 × 10 ⁵ cells / mL, 2 × 10 ⁴ cells / mouse) was injected into the spleen of mice (day 0) and the animals were protected from hypothermia until recovery It was done. Treated mice had three different doses of MB-6 (0.625 g / kg (MB-6-L), 1.25 g / kg (MB-6-M), 2.5 g / kg (MB-6 -H)) was administered orally. The mice were then treated with LV / 5-FU (leucovorin / 5-fluorouracil) on days 3 and 6 after tumor cell injection. Sterile milliQ water and PSK (polysaccharide K, 0.325 g / kg) were administered to the negative control (NC) and positive group mice, respectively, instead of the test substance MB-6. A summary of each group is shown in Table 10.

ＮＣ、陰性対照；ＰＳＫ、多糖Ｋ；ＭＢ−６−Ｌ、０．６２５ｇ／ｋｇ ＭＢ−６；ＭＢ−６−Ｍ、１．２５ｇ／ｋｇ；ＭＢ−６−Ｈ、２．５ｇ／ｋｇ ＭＢ−６；ＬＶ／５−ＦＵ、ロイコボリン／５−フルオロウラシル。

NC, negative control; PSK, polysaccharide K; MB-6-L, 0.625 g / kg MB-6; MB-6-M, 1.25 g / kg; MB-6-H, 2.5 g / kg MB- 6; LV / 5-FU, leucovorin / 5-fluorouracil.

  The body weight of each mouse was monitored once a week. On day 15, mice were injected with luciferin (150 mg / kg IP) and anesthetized by inhalation of 3% isoflurane, then placed in a dark box equipped with a non-invasive IVIS imaging system 100 series (Xenogen, CA). It was. Bioluminescence was quantified using system software to determine tumor growth. The lifetime of each mouse was recorded up to 55 days. At the end of the study, all animals were euthanized by carbon dioxide inhalation.

3.2 Results

  Compared to the LV / 5-FU chemotherapy group, MB-6 did not affect feed intake and body weight of CT-26-VD bearing BALB / c mice. Bioluminescence quantification showed that 1.25 or 2.5 g / kg MB-6 administration significantly increased the efficacy of treatment with LV / 5-FU chemotherapy (data not shown). Median survival was 27.5 days (tumor only), 30.5 days (NC), 34.5 days (PSK), 35 days (MB-6-L), 39 days (MB-6-M) 39 days (MB-6-H) (FIG. 2). Administration of 0.625-2.5 g / kg MB-6 significantly prolonged the life of tumor-bearing mice that received chemotherapy, of which MB-6-M was 30% longer than the NC group Was extended.

Human clinical trials

4.1 Materials and methods

4.1.1 Eligible patients

  This study is a comparative group, a multicenter, parallel-group comparison, double-blind, randomized, placebo-controlled clinical study, MB-6 in mCRC patients in combination with FOLFOX4 (5-FU + LV + oxaliplatin) Efficacy and safety were evaluated in comparison with placebo. The clinical study was approved by the study review board of the six hospitals participating in the study, and all patients provided written informed consent.

  At 20 years of age or older, histologically confirmed clinical findings of CRC and / or metastatic cancer with one or more measurable lesions by either computed tomography (CT) or magnetic resonance imaging (MRI) Has an Eastern Cooperative Oncology Group (ECOG) performance status of 2 or less, suitable bone marrow reserve (hemoglobin 9 g / dL or more, absolute neutrophil count 1.5 x 109 / L or more, platelet 100 x 109 / L L or more), and liver and kidney function (total bilirubin is 1.25 × or less, creatinine is 1.25 or less, alanine aminotransferase (ALT) or aspartate aminotransferase (AST) is 2.5 × less than the upper limit of normal value) Prepared patients were eligible.

  Patients who are pregnant or breastfeeding, have not used appropriate contraceptives, have evidence of CNS metastatic cancer, have active infections that require systemic antimicrobial therapy, currently chronic diarrhea And other serious comorbidities (eg, angina pectoris, myocardial infarction, congestive heart failure, epilepsy, or other serious medical condition at the discretion of the investigator), a history of the second primary malignancy One patient (excluding basal cell carcinoma or cervical intraepithelial carcinoma of the skin that has been appropriately treated) or other concurrent anticancer treatment or treatment with another new drug within the last 4 weeks Patients who received were excluded.

4.1.2 Treatment protocol

Patients were treated with MB-6 and combined FOLFOX4 (de Gramont A et al., J Clin Oncol 2000; 18: 2938-47; Goldberg RM et al., J Clin Oncol 2004; 22: 23-30). MB-6 was administered in 6 capsules of 320 mg each 3 times a day with meals. The placebo group took the same number of capsules with only inactive ingredients. A FOLFOX4 chemotherapy regimen was administered with a 2-hour infusion of LV (200 mg / m ² ) and oxaliplatin (85 mg / m ² ), followed by a 22-hour infusion of 400 mg / m ² of 5-FU bolus and 600 mg on day 1. / ^{m 2,} and 2 hours of LV200mg / ^{m 2} infusion, were subsequently performed as infusion and day 2 600 mg / ^{m 2} 22 hours of 400mg ^/ m 2 5-FU bolus. The FOLFOX4 regimen was repeated every 2 weeks for a total of 16 weeks.

4.1.3 Follow-up evaluation

  Vital signs and body weights were measured every 2 weeks prior to randomization. Serum carcinoembryonic antigen (CEA) value and hematological and biochemical parameters (red blood cell (RBC) count, hematocrit value, white blood cell (WBC), platelet count, absolute neutrophil count, white blood cell percentage, total bilirubin, creatinine , ALT and AST) measurements were taken every 4 weeks. Tumor assessment was performed every 8 weeks. Drug compliance was recorded at each test visit.

4.1.4 Major endpoints

  The primary efficacy endpoint of this study is the best overall effect on MB-6 (complete response according to RECIST 1.1 criteria (Eisenhauer EA et al., Eur J Cancer 2009; 45: 228-47)). (CR) + partial response (PR)) and was compared with the results of patients receiving placebo.

4.1.5 Secondary endpoint

  Progression free survival (PFS) was evaluated as the period from the date of randomization to the date on which disease progression was observed. Death from any cause of death was considered an exacerbation event. Patients with no disease progression or survival follow-up were censored on the last objective tumor assessment day. Exacerbations were censored on the start of study drug if no post-baseline tumor assessment or survival follow-up was performed.

  Adverse events (AEs) that occurred at or after the start of study drug were determined by the MedDRA coding dictionary (Program CTE. Common terminology for advance events (CTCAE). Version 3.0. National Cancer Institute, another classification by Organ University 2006). Classified according to basic terms. The duration of AE, severity, relevance to study drug, response measures, and results were recorded. Whether the event was classified as a serious adverse event (SAE) was also recorded.

4.2 Results

4.2.1 Patient demographics

  A total of 72 patients with metastatic colorectal cancer were recruited. Subjects were randomized in a double-blind manner and received either MB-6 or placebo in a 1: 1 ratio using a random replacement block size. Thirty-four patients were randomized to the MB-6 group and 38 were randomized to the placebo group. A total of 60 patients (29 in the MB-6 group and 31 in the placebo group) completed the 16-week trial (83.3%). A total of 72 patients were included in the ITT (intent-to-treat) number. There was no difference in patient demographics and baseline characteristics between the two groups (p> .05).

4.2.2 Main evaluation items

  The best overall effect was 41.2% in the MB-6 group and 39.5% in the placebo group, and the difference was not significant (P = 1.000). The tumor reduction effect (CR + PR) in the final measurement according to the RECIST 1.1 criteria was 61.8% in the MB-6 group and 63.2% in the placebo group.

4.2.3 Secondary evaluation items

  None of the patients in the MB-6 group showed an exacerbation of disease during the 16-week study period, while 6 patients in the placebo group showed an exacerbation. Patients in the MB-6 group showed a significantly lower disease progression rate than those in the placebo group (0.0% vs 15.8%, P = 0.026). There were no deaths in the MB-6 group, but two died in the placebo group during the 16-week study period. Furthermore, progression-free survival in patients treated with the six-component formulation of the present invention was significantly increased after 8 weeks of discontinuation of medication (p = 0.0056). Table 11 shows.

  Furthermore, there was no significant weight loss from baseline in the MB-6 group, while the placebo group showed significant weight loss at the 4th, 6th and 8th weeks of the trial (P <. 05). However, the difference in weight change between the two groups at the end of the trial was not significant (P = .114). In addition, there was a significant decrease in serum CEA levels at week 8 with FOLFOX4 chemotherapy, and the decrease was similar in the presence of MB-6.

  The compliance of MB-6 was 93.8 ± 11.6%, and that of placebo was 94.1 ± 11.4% (P = .919). Interestingly, the MB-6 group showed significantly higher compliance with FOLOX4 chemotherapy compared to the placebo group (94.4 ± 7.8% vs. 89.0 ± 13.2%, respectively, P = 0.037). ). Table 12 shows.

  As shown in Table 12, the composition of the present invention (MB-6) increased the subject's tolerance to chemotherapy (p = 0.0370).

4.2.3 Reduced safety and side effects

  According to CTCAE 3.0, the severity of AE (side effects) was classified as 1 = mild, 2 = moderate, 3 = severe, 4 = life threatening, 5 = death. In general, the placebo group had a significantly higher incidence of Grade 4 or higher AEs than the MB-6 group (28.9% vs. 2.9%, respectively, P = .004). A total of 19 patients (6 in the MB-6 group, 13 in the placebo group) reported 28 serious adverse events (SAE). In particular, the placebo group had a significantly higher incidence of increased serum creatinine than the MB-6 group (29% vs. 5.9%, P = .014), and the MB-6 group had side effects on the kidneys caused by chemotherapy Indicates a decrease. Table 13 shows.

  Furthermore, the six-component preparation (MB-6) of the present invention can improve neutropenia caused by chemotherapy, particularly severe ones, at a high level (level 4 or higher, p = 0.0302). ). Table 14 shows.

Study of extinguishing

  Macrophages play a central role in the inflammatory response. After activation by LPS, it results in activation of downstream signaling pathways including the NF-κB pathway and the MAP kinase pathway. These signaling pathways induce the expression of various inflammatory mediators including NO, PG and inflammatory cytokines. Thus, RAW 264.7 mouse macrophage-like cells provide an excellent model for screening anti-inflammatory effects.

5.1 Materials and methods

5.1.1 Cell culture and sample processing

RAW 264.7 cells were cultured in DMEM medium supplemented with 10% FBS at 37 ° C. in humidified 5% CO ₂ air. RAW 264.7 cells were seeded in 96-well plates at a density of 0.1-1 × 10 5 cells / well. Cells are then incubated with test samples (MB-6) at a concentration of 0.0625 to 1 mg / ml or with a positive control (dexamethasone or Celebrex) and then activated with 100 ng / ml LPS for 18 hours. It was done. At the end of the culture period, cell viability was determined by a colorimetric MTT assay. Cell culture supernatants were stored and stored frozen at −20 ° C. for analysis of nitrite, PGE2, TNF-α, IL-6 levels.

5.1.2 Determination of nitrite

  The level of nitrite in the medium was determined using the Griess reagent system (Promega, Madison, Wis., USA) and assumed to reflect NO levels. Briefly, 50 μl of cell culture medium was mixed with 50 μL of sulfanilamide solution and incubated for 10 minutes at room temperature, then 50 μL of NED solution was added and incubated for an additional 10 minutes at room temperature. Absorbance was measured at 520 nm using a microplate reader (BioTek, Winooski, Vermont, USA). Fresh media was used as a blank for all experiments. The level of nitrite in the sample was read from a standard sodium nitrite curve.

5.1.3 PGE2, TNF-α, IL-6 assay

  The levels of PGE2, TNF-α, IL-6 in the medium were quantified using an EIA kit (R & D Systems, Minneapolis, Minn., USA).

5.2 Results

5.2.1 Effects of MB-6 on LPS-induced NO and PGE2 production and cell survival

  To evaluate the effect of MB-6 on LPS-induced NO and PGE2 production in RAW 264.7 cells, media was collected and nitrite and PGE2 levels were measured. MB-6 significantly inhibited LPS-induced NO production in a dose-dependent manner. Furthermore, treatment with MB-6 also reduced LPS-induced PGE2 production. Celebrex, a COX-2 inhibitor, was used as a positive control for PGE2 production. Furthermore, the cytotoxic effect of MB-6 was assessed using the MTT assay in RAW 264.7 cells, and MB-6 did not affect cell viability at the concentration used (0.0625-1 mg / ml). It was.

5.2.2 Effect of MB-6 on LPS-induced TNF-α and IL-6 release

  The effects of MB-6 on LPS-induced cytokines such as TNF-α and IL-6 release were further examined by enzyme linked immunosorbent assay (ELISA). It was found that pre-treatment of cells with MB-6 significantly reduced IL-6 and TNF-α production. The results are shown in Table 15.

Summary

  The results of this study confirmed that MB-6 increases the effectiveness of chemotherapy in patients with colon cancer. Preclinical in vivo studies have shown that MB-6 can extend the life span of colon cancer bearing BALB / c mice treated with LV / 5-FU. Results from clinical studies show that patients who received MB-6 had significantly higher PFS compared to the placebo group over the 16-week study period, and similar serum in the MB-6 and placebo groups at 8 weeks. A decrease in CEA value was observed, indicating that the addition of MB-6 does not impair the effectiveness of FOLFOX4. Furthermore, significantly higher FOLFOX4 compliance was observed in the MB-6 group, indicating higher tolerance. In the MB-6 group, the occurrence of grade 4 or higher AE was significantly low.

  Patients in the MB-6 group had significantly higher median PFS and slower disease progression, suggesting that MB-6 improved the overall survival quality of mCRC patients treated with FOLFOX4. These data are consistent with preclinical studies showing that MB-6 extended the life span of tumor-bearing mice receiving chemotherapy by more than 30%.

  Although there was no significant difference in the best overall effect (CR + PR) between the MB-6 group and the placebo group, the data showed that a previous study (Tourignand C et al.) Showed that the tumor reduction effect of FOLFOX4 was 58.5%. , J Clin Oncol 2006; 24: 394-400). This suggests that MB-6 did not impair the efficacy of FOLFOX4 in mCRC patients. Also, at the end of the study, there was no significant difference in CEA levels and immune function between the two groups, confirming that MB-6 does not adversely affect the chemotherapy regimen. That is, MB-6 did not interfere with the cytotoxic effect of FOLFOX4 in these patients with metastatic colorectal cancer (mCRC). Furthermore, the data in this study also show that MB-6 can provide anti-inflammatory activity, supporting the results of slower progression of cancer treated with MB-6.

  In summary, preclinical studies have shown that MB-6 can extend the life span of colon cancer bearing Balb / c mice treated with LV / 5-FU. Results of clinical studies show that patients receiving MB-6 have significantly higher PFS and a significantly lower grade 4 or higher AE incidence compared to the placebo group during the 16-week study period It was done.

  A person having ordinary knowledge in the technical field to which the present invention belongs will be able to expand the present invention to the maximum extent based on the description disclosed herein without the need for further illustration. Accordingly, it is to be understood that the description, examples and claims of the invention are exemplary rather than limiting of the scope of the invention.

None

Claims (10)

Use of a composition for the manufacture of an adjuvant in the treatment of cancer in patients with chemotherapy, wherein said composition is a soy fermentation extract, a green tea extract, a spirulina, a turmeric extract, a venix octopus mycelium, a grape Use of a composition for the manufacture of an adjuvant in the treatment of cancer in patients with chemotherapy, characterized in that it comprises a seed extract. Use of the composition according to claim 1, characterized in that the fermented soy extract is made by fermentation of an aqueous soy extract and at least one lactic acid bacterium and optionally at least one yeast. The fermented soybean extract (a) fermenting the aqueous soy extract with at least one lactic acid bacterium and at least one yeast to form a fermented liquid; and (b) sterilizing the fermented liquid. And (c) a step of obtaining a filtrate by filtering the sterilized fermentation broth, and (d) obtaining a concentrated soybean fermentation extract by concentrating the filtrate. Use of the composition according to claim 1, characterized in that The soybean fermented extract, the green tea extract, the spirulina, the turmeric extract, the venix mushroom mycelium, and the grape seed extract are 12-30: 4: 1 to 2: 1 in the composition. Use of the composition according to claim 1, characterized in that it is present in a weight ratio of 2: 1 to 2: 1 to 2. Use of the composition according to claim 1, characterized in that the cancer is colorectal cancer. 2. The chemotherapy according to claim 1, wherein the chemotherapy is performed by administering a chemotherapeutic agent selected from the group consisting of 5-fluorouracil, leucovorin, oxaliplatin and any combination thereof. Use of the composition. The composition is in an amount effective to inhibit the growth or canceration of colorectal polyps, improve the effectiveness of chemotherapy, improve compliance with chemotherapy, or reduce side effects caused by chemotherapy. Use of the composition according to claim 1. Use of a composition according to claim 1, characterized in that the composition has the effect of increasing survival, in particular increasing progression-free survival. Use of the composition according to any one of claims 1 to 4 in the manufacture of an anti-inflammatory agent. In an amount effective to reduce the production of prostaglandin E2 (PGE2), nitric oxide (NO), tumor necrosis factor α (TNF-α) or interleukin-6 (IL-6). Use of the composition according to claim 9, characterized in that it is present.

Images