WO2002031497A1 - Means of examining ability of angiogenesis - Google Patents

Abstract

A means of examining the ability of angiogenesis of a patient which comprises simultaneously measuring the concentrations of vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) and endostatin in the blood of the patient.

Description

 Specification

Angiogenesis test method Industrial application field

 An object of the present invention is to provide an optimal technique for a non-invasive treatment means in cancer treatment. Specifically, a novel immunotherapy established by the present inventors [induction of interleukin 12 (IL-12), activation of natural killer T cells (NKT cells), and biology based on inhibition of angiogenesis] Cancer therapy with biological response modifiers (BRM)) to provide a therapeutic means focusing on the inhibition of vascular neoplasia. Conventional technology

In the selection of a substance useful for the prevention or treatment of cancer, its direct action on cancer cells has been conventionally regarded as important. The immunostimulant was found to be useful for treating cancer, but all of the compounds obtained as immunostimulants had weak anticancer effects, and were treated with immunotherapy alone or in combination with chemotherapy However, sufficient therapeutic effects on cancer have not been achieved. The inventor of the present invention, Dr. Yagida's medical science, first focused on the usefulness of a substance that induces interleukin 12 (IL-12) in vivo as an epoch-making technique in cancer treatment. Discovered that it has this function, and established a cancer treatment that could be called a new immunotherapy. Conventionally, IL-12 itself has an anticancer effect, but there is a fact that if IL-12 itself is directly administered into a living body, side effects occur and patients cannot tolerate treatment, so it can be used as an anticancer drug. I didn't come. However, Yagida's reported preparations containing AHCC achieved significant healing and prolonging effects in the treatment of cancer. In other words, Yagida is IL By administering an effective amount of AHCC capable of inducing 12 in vivo, the purpose of treating cancer was achieved (Japanese Patent Application Laid-Open No. H10-139670). Furthermore, in some cancer patients, administration of AHCC does not sufficiently induce the production of IL-112, resulting in no therapeutic effect. The effect may not be obtained. Therefore, Yagida aims to develop a new therapeutic agent for cancer that works by a mechanism different from the anticancer effect of AHCC, and is formulated with IL-X (a ratio of Shiitake 2, Suehiro mushroom 2, and Reishi mushroom 1). (Patent pending) and NK T cell activator (patent pending). In addition, Yagida focused on the usefulness of shark cartilage as an angiogenesis inhibitor, and achieved optimal formulation (Patent No. 3103513), which has been applied to cancer patients since 1990. Oral ingestion (Beta-shark MCZO C: Seishin Co., Ltd.) achieved a remarkable cancer treatment effect in combination with an IL_12 inducer. Angiogenesis in solid tumors causes cancer cells to grow rapidly and cause metastasis, leading to worsening of the symptoms that the cancer causes to the host. In recent years, various studies have revealed that tumor cells produce substances that induce angiogenesis. So far vascular endothelial growth factor (VEGF: vascular endothelial growth factor) , basic fibroblast growth factor (b FGF: basic fibroblast growth factor ) number of angiogenic factors, such as, and en Dosutachin _(en dostatin) etc. Angiogenesis inhibitors have been reported. Among them, VEGF (Vascular Endothelial Cell Proliferation Factor) is regarded as important because it has strong angiogenesis and promotes vascular permeability, that is, the production of pleural effusion and ascites that determine the prognosis of patients. Disclosure of the invention

 The problem to be solved by the present invention is to elucidate the in vivo kinetic relationship between an angiogenesis inhibitor and an angiogenesis promoting factor in the treatment with an angiogenesis inhibitor in the above-described neoimmunotherapy, It is to provide a more reliable means. Yet another object of the present invention is to elucidate the relationship between vascular endothelial cell growth factor and cancer status. In order to solve the above-mentioned problems, the present inventors have performed immunological measurement and vascular endothelial growth factor (VEGF), basic fibroblast proliferation in a case where the new immunotherapy is being treated. The present invention has been completed by measuring the blood kinetics of a factor (b FGF: basic fibroblast growth factor) and endostatin and analyzing the relationship with its therapeutic effect. .

That is, the present invention provides

 1. As a means of examining the angiogenesis ability of a patient, the patient's vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF), and A means for testing angiogenesis, characterized in that blood levels of endostatin are simultaneously measured;

2. A test means for determining the dormancy of cancer, characterized by measuring the vascular endothelial growth factor (VEGF) of the patient.

 3. A test for compatibility between the angiogenesis inhibitor and / or the anticancer drug and the patient is performed by the test means described in 1 or 2 above. A test or determination means for the effectiveness of the drug.

4. New inhibitors of angiogenesis performed by measuring 1 or 2 above Screening method.

 5Mechanism that implements the examination means described in the preceding paragraphs 1 and Z or 2 and / or the verification means described in claim 3, a mechanism that determines the test results, and a mechanism that performs therapeutic procedures based on the determination A medical business system that performs treatment as a trinity.

 Mosquitoes. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a graph showing changes in blood concentrations of endostatin, VEGF (vascular endothelial growth factor) and bFGF (basic fibroblast growth factor) from the left side of the figure.

 FIG. 2 is a graph showing the correlation between VEGF (vascular endothelial growth factor) and blood concentration of endostatin. In the figure, before treatment (12 cases), after treatment (29 cases), response cases (CR, PR: 15 cases), effective cases (CR + PR + Long NC: 22 cases), and others (Short NC: 4 cases) , NC / Short NC + Long NC: 11 cases, PD: 3 cases).

 FIG. 3 is a diagram showing a VEGF / endostatin ratio. In the figure, before treatment (12 cases), after treatment (29 cases), response cases (CR, PR: 15 cases), effective cases (CR + PR + Long NC: 22 cases), and others (Short NC: 4 cases) , NC / Short NC + Long NC: 11 cases, PD: 3 cases).

 FIG. 4 is a graph showing the correlation between the blood concentration of jVEGFI (vascular endothelial growth factor) and IL-11 induction.

 FIG. 5 is a graph showing the correlation between the blood concentration of endostatin and induction of IL-12.

FIG. 6 is a graph showing the correlation between the blood concentration of VEGF (vascular endothelial growth factor) and IF Ny. FIG. 7: Blood concentration of VEGF (vascular endothelial growth factor).

BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in detail.

The outline of the new immunotherapy practiced by Asahikuni Yagida, a medical doctor, is as follows. The pillar is immunological cancer treatment with biological response modifiers (BRM preparations), which has made IL-11 induction, activation of NKT cells, and inhibition of angiogenesis. 1) As an angiogenesis inhibitor, shark cartilage preparation (patent No. 3103513), Bettashark MC or LO (Seisin Corporation), 20 _{g /} day, 2) IL — For the 12-lead method, a) For patients with T hl, apply IL-1X (East West Pharmaceutical Research Laboratories) for 3 to 20, preferably 6.0 g / day (AH CC 6.O g Z day: currently discontinued) b) PSK (Sankyo) 1-20, preferably 3. Og Z day or every other day for Th2 patients, 3) NK T cell activation method Is stimulating NKR_P1. As the first pillar, angiogenesis inhibitors, select and improve the best shark cartilage in terms of efficacy, safety, ease of use, and economic efficiency from various basic studies In addition, Better Shear MC or LO was developed. At present, 20 g per day is orally administered on an empty stomach in 2-3 divided doses. The second pillar, IL-12 inducer, includes IL-X (formerly AHCC), which acts on mice or humans whose Th1 immune response is mainly functioning, in early cancer and For postoperative cases, for example, about 3 g days, for advanced cancer For example, it is administered orally in about 6 days. According to the results of comparison of the IL-12 production ability over time between IL-X administration and AHCC administration, untreated tumor-bearing mice (B10) peaked at day 7 and peaked at day 10 by day 10. IL-12 Not produced. On the other hand, in mice administered with AHCC (lg / KgZ day), IL-11 production capacity peaked on day 7 after tumor (LLca) transplantation, declined markedly on day 10 and decreased on day 14 It has decreased significantly in the eyes. IL-X (Shitake mushroom 2, Suehiro mushroom 2, Reishi mushroom 1) (1 g / Kg / day) maintains IL-11 production ability on day 7, 10 and 14 Have been. In addition, krestin, which responds to advanced cancer, terminal cancer, and Th2 mouse human, was administered, for example, at about 3 g / day or every other day. Based on these, various natural forms of total vitamin, urso (30 mg or 600 mg / day), and active vitamin D3 (1.0 mg Og Z days) are co-administered. . We also add PSK, OK432 or live BCG vaccines as needed. Regarding the third pillar, NKT cells, in the patients treated with the above two pillars, complete healing despite no IL-12 production

(CR: Complete Response) or partial healing (PR: Partial Response) were observed in the same proportion as those in which IL-12 was produced and treatment was effective. Therefore, NK T cells are

In order to examine whether they are activated by (ITC), the T cell receptor Va24 Vj31 1 of NKT cells and the NK cell receptor NKR-P1 (C D 3 XCD 16 1) was studied. As a result, the NKR-P1 receptor activated by saccharides having a 1-3 structure, such as Nig mouth sugar and oligosaccharides, showed a positive correlation with IFNy and IL-12. Va 2 4 V | 3 11 activated by lipid showed a negative correlation. It also showed a high correlation with NKR-P1 in CR and PR cases. In other words, in cases where NITC therapy was effective, it was suggested that cytotoxic T cells (CTLs) and another NKT cell might work as effector cells. The following describes the results of a study on the blood kinetics of angiogenesis inhibitory factors and angiogenesis promoting factors in patients treated with the above-mentioned new immunotherapy (NITC). In 29 outpatients receiving new immunotherapy (NITC), randomized immunological measurements and vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (b FGF: basic fibroblast growth factor) and angiogenesis inhibitor endostatin (enzyme linked immunosorbent assay) (ACCUCYTE Human VEGF, ACCUCYTE Human bFGF, ACCUCYTE Human Endostatin: CYTIMMUNE Sciences Inc. ). As shown in Table 1, 6 cases of complete healing (CR), 9 cases of partial healing (PR), and no change (NC: no change) 1 1 case (7 cases showed no tumor cell growth for more than 6 months) The long term NC / Long NC group, and the remaining 4 cases were the short term NC / Short NC group, which means no tumor cell growth for less than 6 months. : Progressive Disease) 3 cases, for a total of 29 cases. The sum of CR and PR was 15/29 (51.7%), which was half the response rate. Most notably, 11 cases of NC (37.9%) were notable. Of these, the long term NC is as large as 7 cases, and the average survival period is 13.43 months, indicating tumor dormancy. Recently, it has been proposed that the long term NC should be added to the response rate, but this is considered to be 75.9% (Table 1).

n Mean SD

 CR 6 17.50 9.63 31 24 22 12 10 6

 PR 9 11.22 9.65 28 25 16 9 9 5 3 3 3

Long NC 7 13,43 6.50 25 18 16 10 9 9 7

 Short NC 4 3.50 1.00 5 3 3 3

 PD 3 6.67 3.06 10 6 4

 Total 29 11.52 8.56

 (M: F = 10: 19)

CR (6) Long-term NC (7) Short NC (4)

 breast cancer 3 gastric cancer 1 lung cancer 丄

 large bowel cancer 2 lung- cancer 1 gastric cancer 1 gastric cancer 1 breast cancer 2 breast cancer 1

PR (9) large bowel cancer pancreatic cancer 1 breast cancer 2 liver cancer 1 PD (3)

 gastric cancer 2 prostatic cancer 1 pharyngeal cancer 1 uterine body cancer ovarian cancer 1 ovarian cancer 1 uterine body cancer 1

 small bowel sarcoma

liver cancer 1

(Blood kinetics of VEGF and bFGF in neoimmunotherapy (NITC)) The serum concentrations of VEGF and bFGF of pro-angiogenic factors are shown in FIG. Compared to the VEGF concentration in the pre-treatment case, that in the CR case was significantly lower. Among the NCs, the VEGF concentration tended to decrease in the long term NC. It should be noted here that the difference between long term NC and short term NC among NCs also depends on how the angiogenic factors are suppressed in b FGF concentration and VEGF concentration. Maybe. In other words, it is suggested that whether or not the tumor is in a dormancy state depends on whether or not the angiogenic factors are suppressed. Similarly, it was suggested that the difference between CR and PR may depend on how the angiogenesis promoting factor is suppressed. A similar tendency was observed for bFGF, which is an angiogenesis promoting factor. Therefore, by measuring the angiogenesis-promoting factor in the serum of the patient, it is possible to examine the angiogenesis of the patient and to test the effect of treating cancer.

(Blood kinetics of VEGF in new immunotherapy (NITC))

 FIG. 7 shows the results of measuring VEGF in NITC-treated patients. NC (77 cases) 2 9 1 pg Zml decreased (p <0.001), PR (55 cases) 2 compared to the average of 4 7 pg_ml in 19 7 cases before treatment 94.3 pg Zml was significantly reduced (p <0.01), and CR (48 cases) was significantly reduced to 251.8 pg Zml (p <0.01) . Also, as compared with the PD65 cases (5 16.8 pg / m1), the values were significantly lower in NC, PR and CR. Thus, measurement of VEGF is considered to be an important evaluation method for not only PR and CR but also NC dormancy.

The samples were separated from serum within 30 minutes after blood collection, stored frozen, and subjected to business measurement. The VEGF assay was performed by ELISA (kit name: Quant). ikinehuman VEGF, R & D Systems).

(Blood kinetics of endostatin in neo-immunotherapy (NITC)) Endostatin has a similar concept to that of angiostatin, that is, the anti-angiogenesis inhibitor produced by tumors in mice. It was separated from Ma. It is a protein with a molecular weight of 20 KDa and is a C-terminal fragment of type XVIII collagen from amino acid analysis. Endostatin production in 12 patients before treatment and 6 CRs in NITC showed significantly lower CR (p <0.01). In addition, a significant difference was observed with PD cases (p <0.05). The pro-angiogenic factors bFGF and VEGF also tend to decrease when they become CR, which is a suitable purpose when considered as an effect of shark cartilage. It is interesting, however, that endostatin production is also decreasing (Figure 1). Furthermore, when judging the effectiveness of shark cartilage or the usefulness of immunotherapy, it should be judged based on the decrease in endostatin production. More preferably, it would be more reliable to judge based on a decrease in both the angiogenesis promoting factors VEGF and endostatin. In addition, and b FGF instead of VEGF, it may be subjected to judgment Ri by measuring the and E down Dosutachin _c

(Correlation between VEGF and endostatin)

The correlation between VEGF and endostatin was examined in 12 cases before treatment, 29 cases during treatment, and 15 responders with CR and PR in NITC (Fig. 2). It can be seen that as the efficacy of NITC increases during the course of treatment, the production of VEGF decreases as compared to endostatin. When the ratio of VEGF / endostatin was examined in patients undergoing NITC (Fig. 3), the former significantly decreased after treatment and before treatment. In addition, in the effective examples (CR, PR, LongNC), the numerical value of the ratio was further reduced. This suggested that NITC treatment reduced VEGF earlier and more abundantly than endostatin. Therefore, examining the ratio of VEGF / endostatin would allow a more accurate grasp of the state of angiogenesis inhibition.

(Correlation between I L 1 1 2 and V E G F)

 The correlation between IL-12 and VEGF in patients undergoing new immunotherapy (NITC) was examined (Fig. 4). IL-12 has also been found to exhibit an anti-angiogenic effect in the presence of IFNy. A positive correlation was observed in 12 patients before treatment, but a negative correlation was observed after treatment. In other words, it is unclear from this fact whether the reduction in tumor was the result of IL-12 acting as an angiogenesis inhibitory agent or as a result of exerting an immunological antitumor effect. It is inferred that the result of 12 shows an antitumor effect.

(Correlation between IL-1 and Endostatin)

 The correlation between IL-112 and endostatin in NITC patients was examined before and after treatment. There was an inverse correlation before treatment, but a positive correlation after treatment (Fig. 5). This fact suggests that the implementation of new immunotherapy (NITC) may be effective for both IL-12 and endostatin in antitumor properties. (Correlation between I F N y and V E G F)

IFN y and VEGF in patients treated with new immunotherapy (NITC) The correlation with was examined. As can be seen from the correlation with IL-112, the correlation between IFN and VEGF was inversely correlated even in the treated patients compared to before treatment (Fig. 6). This suggests that 1-12 and 1] ^ 0 act together to suppress angiogenesis and that CTLs and NKT cells cooperatively exert antitumor effects.

(Details of substances used in new immunotherapy (NITC))

 Therapeutic agents for cancer that have the ability to activate NK T cells by selectively acting on N KR-P1 (natural killer P1) of NK T cells used in new immunotherapy (NITC) At least one selected from mushroom mycelium processed products is the active ingredient. Specific examples include SPG (sizofiran: Kaken Pharmaceutical Co., Ltd.) (a polysaccharide obtained from the oral liquid of the culture of Suehiro mushroom mycelium), SCP (oral preparation of a processed product of Suehiro mushroom mycelium) (limited Schizophyllum Commune Fries (Science name: Schizophyllum Commune Fries), Mycelium-derived product, PARA (Krestin), etc., Straw mushroom mycelium, AH CC and L EM (Noda edible bacteria) A processed product of mycelium of Shiitake mushrooms such as Kogyo Co., Ltd. is effective. Suehiro mushroom fungi SPG (sizofiran: Kaken Pharmaceutical Co., Ltd.), a polysaccharide derived from mycelium culture mouth liquid, is used as an anticancer drug for some limited cancers (Taito Co., Ltd., Kaken Pharmaceutical Co., Ltd.) . The same applies to PSK (Krestin).

Substances that have the ability to activate NKT cells by selectively acting on NKR-P1 (natural killer P1) of NK T cells can be expressed in the form of —-1,3 and / or α-1,4 darcoside bonds. There is a saccharide component having a structure, particularly preferably at least an α-1,3 darcoside linkage structure. The substance having the activity of activating cells may be a polysaccharide having this structure and / or a substance containing 2 to 10 oligosaccharides. NK T cells NKR- P l (natural killer PI) Other substances sugars Ru substance der of _alpha 1 → 3 conformation selectively acts with the ability to activate NKT cells, Nigeroori pentasaccharide (3 — Ο— α—D—Dalcovirano Silose D—A saccharide containing glucose as a constituent unit) (Example product: Nige Mouth Oligosaccharide Liquid Sugar / Seller Takeda Food Industry Co., Ltd. Glucose: ① Nigerose _α -D-Glcp. (1 → 3) -D-Glc ②-Geguchi Syl glucose _α -D-Glcp- (1 → 3)-a -D-Glcp- (1 → 4) ) -D-Glc ③2 Gero Sino-Remaltoose a -D-Glcp- (1 → 3) -a -D-Glcp- (1 → 4)-a -D-Glcp-(1 → 4) -D-Glc , Glc is glucose, p (italic) is an abbreviation for vilanose), fucoidan (exemplified substance)

Fucoidan from Okizumi Mozuku (Equation 1)

o O

 Fucoidan from Okinawan Mozuku

S = S0 ₃ H

_F- fucoidan / sulfated fucan derived from gagome kelp: a sugar consisting solely of fucose (Formula 2)

 [Equation 2]

sSO ¾ =

、 ガゴメ コンブ由来 G —フコィ ダン/硫酸化フコガラタ タ ン ： ガラ ク ト ス と フ コースから成る糖 （式 3 )

G-fucoidan / sulfated fucogalatatan from Gagome kelp: sugar consisting of galactose and fucose (Formula 3)

 [Equation 3]

ガコメコンブ由来 G-フコィダン

G-Fucoidan

S = S0 ₃ H

U-fucoidan / sulfated fucodalclon mannan derived from gagome kelp, sugar consisting of mannose and fuuse (Formula 4)

 [Equation 4]

ガゴメコンブ由来 U-フコィダン

U-fucoidan from Gagome kelp

S = S0 ₃ H

(Fucoidan is a sulfated fucose-containing polysaccharide.French kelp is crushed, chipped, aqueous components are extracted, and the extraction residue is removed by centrifugation. Ultra-filtration removes low molecular substances such as etc. and freeze-dried to formulate the product.Sold by Takara Shuzo Co., Ltd., oligosaccharides (for example, Sales Company / Shiroko Co., Ltd .: Extract from sea squirrel laver) The main components are at least one selected from α1 → 3 linked galactan sulfate oligosaccharides and α1 → 3 bond and galactan sulfate oligosaccharides composed of | 31-4 bonds). One is the active ingredient. The compound is not limited to these, and is a saccharide substance having an _α1 → 3 steric structure (a saccharide component having a 1 → 3 glucosidic bond structure), and furthermore, NK R—P 1 (natural killer) of Ν Κ Τ cells A wide range of substances that selectively act on P 1) and have the ability to activate cells. The dose of these compositions is about 1 to 200 mg / Kg body weight per day, and is taken orally in 10 to 12 months, 1 to several times a day. Of course, parenteral ingestion is also possible by reducing the dosage and preparing a parenteral therapeutic agent for cancer. Examples of a composition containing a substance derived from a mushroom mycelium used in a new immunotherapy (NITC) include a spleen mouth such as an SCP, a processed mushroom mycelium, and a spleen mushroom mycelium culture port such as an SPG. Contains at least two types selected from liquid-derived polysaccharides, processed shiitake mycelia such as AH CC and LEM, and perennial mushroom mycelium additives such as MAK (Noda Shokubai Kogyo Co., Ltd.) and SM. . It is widely known that most of these have been used as immunostimulants for anticancer effects. In the new immunotherapy (NITC), these combinations and IL-1 We found a relationship between the ability to induce production and the ability of NKT cells to selectively act on NKR-P1 (natural killer P1) to activate NKT cells. It has established an advantage that is incomparable with the anticancer effect (20% effective rate) of anticancer drugs. The optimal combination is a composition that combines three types of processed products of Suehi or Mushroom mycelium, or a polysaccharide derived from oral liquid of Sue or Mushroom mycelium, Shiitake mushroom mycelium, and Perennial mushroom mycelium. Yes, more specifically, 20 to 60, preferably 30 to 50 weight of polysaccharide derived from a succulent mycelium mycelium culture product or a phloem cultivated by mycelium schizophrenia. / ₀ , 20 to 60% or 30 to 50% by weight of Shiitake mushroom mycelium, and 5 to 40 or preferably 10 to 30% by weight of Perennial mushroom mycelium It is. This combination composition is effective as a therapeutic agent for cancer or a health supplement food preparation for oral ingestion that is taken in expectation of anticancer effects. The oral intake of the combination composition of the present invention is usually about lSOOO rag ZK g body weight per day for an adult. The dosage may be adjusted depending on the amount of IL-11 induced to produce and the degree of activation of NKT cells by selective action of NKT or NKT cells on NKR-P1 (natural killer P1). The administration period is 10 days to 12 months, and the frequency of administration is one to several times a day. Polysaccharides obtained from mycelium cultured port solution of Sue human B mushrooms, is already SPG (si _Z o ί iran) and to Kaken and Taiwan Sugar Corporation and _c and their preparation are commercialized, JP-B The method described in JP-A No. 524-26434, JP-B No. 52-444634, etc. is exemplified.

Processed mycelium of shiitake mushrooms such as L EM and reishi (manne mushrooms) mycelium such as MAK The product has already been commercialized by Noda Shokubai Kogyo Co., Ltd. One example of these production methods is to add rice bran to bagasse (sugar cane juice residue), mix well, adjust the water content, fill in a certain container, create a solid culture medium, and perform high-pressure steam sterilization. Inoculate the hypha of each bacterium, which has been cultivated beforehand, into the culture medium, and culture the hypha in a culturing room at 23 ° C for four months. After crushing the culture medium in which the mycelium spreads and performing autolysis, extract the processed shiitake mycelium and the perennial mushroom mycelium with hot water for 15 hours. After the extract is sterilized with a membrane filter, the filtrate is concentrated and dried to obtain a powder (Japanese Patent Publication No. Hei 7-14435, Japanese Patent Laid-Open Publication No. Hei 1-131980, Japanese Patent Publication No. 5 1-19013, Japanese Patent Publication No. 53-18591). The processed mycelium of Suehiro mushroom is a mycelium component and is oil-soluble. Therefore, the extract is extracted by a suitable organic solvent such as acetone extraction treatment, the bacteria are removed by a filter, and the filtrate is concentrated and dried to obtain a powder. In the present invention, this oral preparation [SCP (Tozai Pharmaceutical Laboratory Co., Ltd.)] was used. Other mushroom mycelium processed products can be prepared by the same treatment according to their solubility characteristics (water-soluble / oil-soluble). Oral preparations are prepared into tablets, powders, capsules, syrups and the like. The preparation can also be prepared by blending known additives such as excipients, disintegrants, binders, and lubricants, and using conventional means. If necessary, flavoring agents, coloring agents, fragrances, stabilizers, bactericides, preservatives and the like can be added. As described above, the present invention focuses on angiogenesis inhibitory and angiogenesis-promoting functions in new immunotherapy (NITC), and promotes angiogenesis-promoting and angiogenesis-inhibiting factors such as VEGF, bFGF, and endostatin. Blood movement It clarifies the relationship between the condition and the condition of cancer and the therapeutic effect. That is, the present invention provides a means for testing angiogenesis, which comprises simultaneously measuring the blood levels of VEGF or bFGF and endostatin in a patient. From the values of the concentrations of VEGF or bFGF and endostatin in blood obtained by the detection means of the present invention, the effect of the substance used for treatment, for example, an angiogenesis inhibitor can be assayed. Specifically, for example, the blood of a patient is collected before and after the administration of the angiogenesis inhibitor, and the concentrations of VEGF or bFGF and endstatin in the serum are measured. If these concentrations after administration are lower than before administration, it is determined that the angiogenic ability has decreased. If the concentrations of these factors do not change or increase, it is determined that the angiogenesis inhibitor used is not effective, and it can be determined that another angiogenesis inhibitor is selected. That is, the present invention 'provides a means for assaying an angiogenesis inhibitor, in which an assay for compatibility between an angiogenesis inhibitor and a patient is performed by the above-described examination means.

Further, by measuring blood angiogenesis-promoting factors and angiogenesis-inhibiting factors in animals, it is possible to screen substances capable of reducing these values. For example, using a tumor-bearing mouse as described above, administering the compound thereto, collecting blood before and after administration, preparing serum, and preparing angiogenic factors and angiogenesis in the serum. The concentration of the inhibitor is measured and compounds that can reduce these concentrations after administration compared to before administration are selected. The obtained compound is a compound that can reduce the angiogenesis ability, and can be used for diseases related to angiogenesis such as solid cancer. VEGF, bF GF, tumor necrosis factor (TNF) -α, transforming growth factor (ΤGF) -β, interleukin-11] 3, type III prostaglandin and the like. Preferably, VEGF or bFGF is measured. It is preferable to measure endostatin as an angiogenesis inhibitor. The animal used for the screening may be an animal in which angiogenesis has been induced, and for example, a mouse transplanted with cancer can be suitably used. In addition, the present invention focuses on angiogenesis inhibition and angiogenesis promotion functions in new immunotherapy (NITC), and is a factor relating to angiogenesis promotion and angiogenesis inhibition. For example, blood kinetics of VEGF, bFGF, and endostatin and cancer Since the relationship between the disease state and the therapeutic effect has been clarified, if these are carried in a commercial medium, they will be a means of differentiating the value of the product. Therefore, those that carry such information in commercial media are extremely useful. If such information is used commercially, it will be a means of differentiating the value of the product, and the commercial method using this information is extremely useful. As a result, the present invention provides a mechanism for implementing the new immunotherapy (NITC) with the above-mentioned examination means and / or the above-mentioned examination means, a mechanism for judging the test results, and a mechanism for performing a therapeutic procedure based on the judgment. It can be provided as a medical business system that performs cancer treatment as a trinity. For example, a system to provide cancer immunotherapy in cooperation with an immune function testing organization, an immune function evaluation organization, a treatment and / or prescription organization (medical institution) will be formed. For example, based on the system, when blood of a cancer patient is transferred to an immune function testing laboratory after collection, at least VEGF or bFGF and endostatin are collected at the testing laboratory. Are measured, and the obtained measurement results are sent to the affiliated immune function evaluation organization and evaluated based on the test results. Further, it is preferable to conduct a test for immune functions such as IL-112, NKT activity, IFNγ and the like. As a result, therapeutic agents and treatment methods that are compatible with the blood kinetics, immune function, and stage of cancer progression of factors related to angiogenesis of the cancer patient are selected from the above-mentioned respective prescriptions, and the information is used as the treatment and / or prescription. It is transmitted to an institution (medical institution), and performs cancer immunotherapy based on the notification. Such a new cancer immunotherapy system is extremely useful. In addition, the cancer immunotherapy using each of the above-mentioned prescriptions also has extremely high utility.

【Example】

 The present invention will be described in more detail with reference to clinical examples and examples, but the present invention is not limited to these, and various applications are possible without departing from the technical idea of the present invention. .

 [Clinical case 1]

(Preparation and ingestion of formulation)

40% by weight of processed product of Suehiro mushroom mycelium (SCP: Tozai Pharmaceutical Research Laboratories Co., Ltd.) and 40% by weight of processed product of Shiitake mushroom mycelium (LEM (registered trademark): Noda Shokubai Kogyo Co., Ltd.) Part, Reishi (Mannentake mushroom), 1 Kg of a composition (IL-X) consisting of 20% by weight of a mycelium processed product (ΜΑ Κ: Noda Shokubai Kogyo Co., Ltd.) Three types of granules were obtained by a fluidized granulation method using a spray-dry method. The granules were orally ingested 6 g per day. In addition, a shark cartilage preparation (Patent No. 3135131), Bettashark MC (Seishin K.K.), was orally ingested 20 g / day. In addition, patients with Th2 were ingested PSK (Sankyo) 3.Og / day, and combined with IL-X. For ingestion. The number of patients was 29, and the results are shown in Table 2 below.

[Table 2]

Metastasis, its nature 24 + / Th1 / endosta

 Disease name IL-12 IF IL-10 FGF-B VEGF

Other Age ^ ⁰⁰¹⁶¹ Th2 Chin

 1 1 +

 Left cancer blind left left stomach mean mean mean

 Intestinal milk ng ml ng / ml ng / ml

 Female 56 0.05 10.9 9.4 7.8 2.4 97 30.12 36.45 0.78 CR 31 Liver metastasis Chest P

 Transferred transmembrane transfer 59 0.03 20.3 8.3 34.1 57.4 226 7.80 4.22 1.15 CR 10 Relocated bone}

 Female 50 0.04 25.7 3.5 7.8 23.4 723 7.80 28.06 1.46 CR 2 Female 35 13.5 12 15.6 46.3 276 13.69 17.53 0.8 24 60 19.1 12 10.9 13.5 631 16.62 12.35 0.97 6 60 8.9 6.3 17.5 37 373 15.20 18.51 0.78 22 72 9.1 4.3 7.8 5.6 551 29.17 47.07 2.75 9 76 0.05 11.4 4.5 7.8 3.1 329 45.52 48.26 1.21 9

男 59 0.02 11.8 4.7 18.8 44.5 295 18.25 15.45 0.78 NC 25 70 0.05 9.8 2.7 7.8 10.8 169 23.67 51.01 1.28 28 F 0 0.02 21.2 9.4 43.F 69.7 426 51.37 14.20 1.69 25 F 63 0.02 14.8 1.8 7.8 4 598 27.03 31.51 3.62 PR 3 M 68 0.04 15.6 0 8.9 73.3 599 23.38 23.97 1.38 PR 5 female 36 0.25 16.4 14 7.8 3.5 103 24.16 19.65 1.82 PR 3 female 58 0.44 13.5 4.3 7.8 30 246 32.65 16.30 1.21 PR 16 male 70 0.08 15.3 18 53.5 93.6 99 28.29 24.89 1.92 PR 3

Male 59 0.02 11.8 4.7 18.8 44.5 295 18.25 15.45 0.78 NC 25

Half c c c p p p p 9V

 R R R R R R R R

Between medical treatments ix

/ D 前^α CD3V

 End star

 Disease Miya cancer name Metastasis, its sex Age ^ IL-10 FGF-B VEGF

 Other TCRVi8 CD I IL-12 IFN r

 Th2 chin

 1 1 +

 17 Liver Right surgery Pulmonary turnover Lung turnover Left lung turnover Pulmonary turnover 57 0.06 17.2 13 236 136 253 27.12 16.00 0.78 NC 18 18 Left lung, post-pulmonary metastasis breast metastasis transfer transfer node transfer bone 53 0.06 9.8 1 1 7.8 4.6 198 15.02 45.53 2.10 NC 16 Metastasis cancer transfer bone transfer ffi ^

 19 Gastric cancer 51 0.12 17.7 16 7.8 49.6 338 26.56 10.91 2.09 NC 10

20 Right breast gender gender female gender 50 0.05 14.5 5.6 14.9 36.6 305 22.23 15.14 1.71 NC 9 ₉₁ Right breast 56 0.09 13 4.6 7.8 4.4 443 18.17 12.20 0.78 NC 9

22 rectum 61 0.68 14.5 5.7 7.8 4 154 14.42 10.37 0.78 NC

23 Gastric cancer 50 0.04 9.1 7.1 7.8 6.9 180 17.24 39.40 1.29 NC 5

24 Breast cancer 72 0.12 20.8 4.8 7.8 17.4 565 33.1 1 25.38 1.37 NC 3

25 lung 67 0.03 16.2 3.9 7.8 1.5 359 44.17 28.15 4.32 NC 3

26 Tongue 69 0.04 14.6 16 23 31.6 320 42.31 21.64 1.44 NC 3 ₂₇ Ovarian 52 52 0.04 15.4 8 7.8 1.5 115 40.39 50.68 1.56 PD 4

28 Female 62 0.24 10.6 5.1 7.8 2 401 33.53 20.78 1.43 PD 10

29 Male 57 0.04 12.9 3.9 19.6 14.7 961 25.95 12.77 2.35 PD 6

Half

 Fruit

Period treatment The method for measuring each factor shown in the table is shown below.

 (Measurement of vascular endothelial cell growth factor / VEGF and basic fibroblast growth factor / bFGF and angiogenesis inhibitor endostatin / endostatin)

 The enzyme-linked immunosorbent assay (ELISA) of commercially available kits was used for the measurement (ACCUCYTE Human VEGF, ACCUCYTE Human bFGF, ACCUCYTE Human Endostatin: CYTIMMUNE Sciences Inc.). (Measurement of NK T cells)

Among the activation of NKT cells, the measurement of the activation by the action on NKR-1 is based on the determination of the cell surface antigens (CD3 and CD161) which are specifically present on the cell surface of the Ν KT Τ cells. The measurement can be performed by examining the increase in the number of cells. Specifically, lymphocytes in peripheral blood are assayed for CD3-positive cells and CD166-positive cells. That is, CD3 and CD161, which are cell surface antigens of Ν Ν cells, are measured by Two Co1 or test using flow cytometry using a monoclonal antibody. I do. “Activated NKT cells” means that the percentage of NKT cells in lymphocytes is 10% or more. The NKT cell activating ability means a function of increasing the ratio of NKT cells to 10% or more, or a function of further increasing the ratio of NKT cells before administration of a certain substance. Using the blood of a cancer patient, the percentage of cells that are positive for the cell surface antigens CD3 and CD161 in blood cells was determined by Two Co1 or flow cytometry. It was measured as usual. CD 3 and C As the monoclonal antibody against D161, CD3-PC5 manufactured by Coulter and CD161 manufactured by Becton Dickinson were used, respectively. Of the activation of NKT cells, the measurement of activation by the action on Va24Vj3111 was performed by measuring cell surface antigens (Va24 and Vβ1 The measurement can be performed by examining the increase in the number of Ν Κ Τ cells by the measurement in 1). Specifically, for lymphocytes in peripheral blood, Vα24-positive cells and 11-positive cells are assayed. In other words, Va24 and V / 311, which are cell surface antigens of NKT cells, are conjugated to monoclonal antibodies (TCR-VQ: 24PE, TCR-Vi311 FITC; Beckman Cou lter). Was measured by Two Co1 or inspection using flow cytometry. (Preparation of sample for measuring site force-in)

First, a mononuclear cell fraction is separated and prepared from the blood of a cancer patient. Heparin-added peripheral blood obtained from a cancer patient was diluted 2-fold with phosphate buffered saline (PBS) and mixed, and then F £: 011—. The mononuclear cell fraction was collected after layering on 0 & 1 & ₇ solution (specific gravity 1.077), centrifuging at 400 G for 20 minutes. After washing, RPMI-164 medium supplemented with 10% fetal bovine serum (FBS) was added to adjust the cell number to 1 × 10 6. Magnoretinin (Phytohemagglutinin; hereinafter abbreviated as PHA) (manufactured by DIFCO) (DIFCO) was added to 200 μl of the obtained cell suspension to a concentration of 20 / ig / ml. The cells were cultured for 24 hours at 37 ° C in the presence of 5% CO 2 in a well microplate, and used as a sample for measuring cytokine in the cultured cell solution. (Measurement of IL-12)

 IL-12 was measured by the ELISA method using a kit made by R & D SYSTEMS. In practice, 50 μl of the assay diluent Assay Diluent RD 1F, 200 μl of the standard solution or the sample prepared in Example 1 was placed in each well of the 96-well microplate. After each dispensing, the mixture was allowed to stand at room temperature and reacted for 2 hours. Thereafter, horseradishpelexidase (hereinafter, abbreviated as HRP) -labeled anti-IL-11 antibody was dispensed in a quantity of 200 μl each, and allowed to stand at room temperature for 2 hours. After removing the reaction solution in each well and washing three times, the chromogenic substrate solution was dispensed one by one, and allowed to stand at room temperature for 20 minutes, and then the enzyme reaction stop solution was dispensed in 50 μl portions. Using 550 nm as a control, the absorbance of each well at 450 nm was measured by Etn ax (Wako Pure Chemical Industries, Ltd.). The IL-l-l2 amount is expressed as pg Zml. The ability to induce IL-11 production is a function that enhances the amount of IL-11 produced by peripheral blood mononuclear cells upon stimulation to 7.8 pg Zml or more (7.8 is the measurement limit value), Alternatively, it means a function of enhancing the production amount of IL-112 before administering a certain substance.

(Measurement of IFNγ)

 The measurement of IF N y is based on the Io of Bio SouracEuroeS.

It was measured by enzyme immunoassay (EIA method) using the FNY EASIA kit. Actually, 50 μl of a standard solution or a 2-fold diluted sample prepared above was dispensed into each well of a 96-well microplate, and 50 μl of HRP-labeled anti-IFN-γ antibody was dispensed. The mixture was dispensed one by one and reacted at room temperature for 2 hours while shaking. After removing the reaction solution from each well and washing three times, the coloring substrate solution was dispensed in 200 μl aliquots, and reacted at room temperature for 15 minutes with shaking. The enzyme reaction stop solution was dispensed at 50 μl each. The absorbance of each well at 450 nm and 490 nm was measured by E max (Wako Pure Chemical Industries) using 630 nm as a control. The amount of IFN _y is expressed as I UZ ml. (Measurement of IL-10)

 IL-10 was measured by a solid-phase enzyme immunoassay (ELISA method) using an IL-10 EASIA kit from BioSourceEuropeS. The method was performed according to the method for measuring IF γ except that an anti-IL 110 antibody was used. The IL 110 amount is expressed as pg / ml.

(Measurement of T h 1 Z T h 2 cell ratio)

 The Th1 / Th2 cell ratio was assayed by a routine method by a helper T (Th) cell line Threcco1or analysis by flow cytometry. Th1 / Th2 is the ratio of helper T cells that have the cell surface antigen CD4 that produce IFNγ (Th1) to cells that produce IL-4 (Th2). And expressed as CD 4 XIFNT // IL—4.

First, the blood of a cancer patient was treated with phorbol 1 2 —myristate 13 —acetate (phorbol 12 2 — myristate 13 A aceetate) and ionomycin (Ionomycin) for 37 hours for 4 hours. Cells in the blood were stimulated to produce cytokines. Then, the production reaction was stopped by adding preferdin A (B referdin A), and CD4, an anti-CD4 antibody, was used as a cell surface marker, using CD5-PC5 (Beckman Couter). After staining the cells and fixing the cells, FACSL ysing Solution (Nippon Vector (Kinson). After that, cell membrane permeabilization treatment was performed by FACSP ermeabi 1 izing Solution (Nippon Vector Dickinson Company), and anti-IFN y antibody anti-IL-14 antibody (FASTI MMU NEIFN y FITC / IL-4PE, Intracellular cytokins were stained with Betaton Dickinson (Japan) and measured and analyzed using a flow cytometer (FACSC alibur, Becton Diekinson).

[Example 1]

 Immune function testing institutions, medical institutions, and immunological competence testing and treatment policy guidance agencies.

A medical institution collects blood from a patient by a known collection method corresponding to a target test measurement item in the blood component. The medical institution separates serum and blood cell components from the blood, if necessary, depending on the intended test measurement items. The medical institution will identify the blood, serum, and / or blood cell components and the patients from which they are derived, and immediately send them to the affiliated immunological testing laboratory. Immune function testing institutes conduct tests for specific immune-related functions determined by the guidance of an institution that conducts immunological ability tests and guidance on treatment policies (hereinafter referred to as “instruction bodies”) (for example, IL-11 production ability, NKT cell activity). , IFN y value, TNF a value, etc.) are performed on blood, serum, and / or blood cell components sent from medical institutions. If necessary, serum and blood cell components are separated from the blood. In addition to testing for immune function-related functions, the immunological function testing organization conducts tests for cancer markers and tests for cancer-related factors (for example, angiogenesis promoting factors and angiogenesis inhibitory factors) if necessary. Inform the instructor of the test results I do. Based on the notified test results, the instructor should at least assay the subject's IL-12 production ability, NKT cell activity, IFNy level, TNFa level, and promote angiogenesis factors (such as VEGF and bFGF), angiogenesis inhibitors (eg endostatin), and various cancer markers as needed. Based on the results of the assay, the instructing institution shall apply the assay results based on the prescription examples of IL-12 inducer, NKT cell activator, other BRM preparations, shark cartilage preparations, etc. To identify. The test results and treatment examples are immediately transmitted to the affiliated medical institutions.

 The medical institution treats the target patient with cancer by referring to the specified prescription example. In addition, the guidance organization determines the effectiveness and ineffectiveness of the treatment by examining the results of various tests performed on the patient's blood components in the same manner as above during the course of treatment, and determines whether the treatment is effective or ineffective. Provide appropriate prescription examples to medical institutions. Guidance agencies collect guidance fees by presenting the test results and prescription examples to medical institutions. Industrial applicability

In the present invention, the blood levels of VEGF (vascular fibroblast growth factor) and VEGF (basic fibroblast growth factor) and endostatin in a patient are simultaneously measured as a means of testing angiogenesis. In addition, we found the correlation and provided an optimal means for the patient's condition and treatment procedure, and to maintain cancer dormancy (Cancerdormancy) for a long time, that is, 1 ong-NC (6 months or more). NC) as well as CR and PR, In order to lead this cancer to a dormant state, attention has been focused on the angiogenesis inhibitory effect, and it has been found that VEGF inhibition is particularly important.

Claims

The scope of the claims 1. As a means of examining the angiogenesis potential of a patient, the patient's vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF), A method for testing angiogenesis, characterized by simultaneously measuring the blood levels of endostatin and endostatin.  2. A test means for determining the dormancy of cancer, characterized by measuring the vascular endothelial growth factor (VEGF) of the patient.  3. A method for testing or judging the effectiveness of an angiogenesis inhibitor and / or an anticancer drug, which is performed by the testing means according to claim 1 or 2, wherein the compatibility of the drug with the patient is tested. 4. A screening method for a novel angiogenesis inhibitor, which is performed by performing the measurement according to claim 1 or 2.  5. A mechanism that implements the inspection means described in Claims 1 and / or 2 and the verification means described in Z or Claim 3; a mechanism that determines the inspection result; A medical business system that treats cancer as a trinity. .