JP6773665B2 - Vascular wall strengthening agent, method of strengthening blood vessel wall and vascular network inhibitor - Google Patents

Description

The present invention is a vascular wall strengthening agent, a vascular network, for reconstructing normal blood flow in a tissue by suppressing the formation of an abnormal vascular network that extends in a disorderly manner and reinforcing immature and fragile blood vessels. Regarding formation inhibitors and vascular normalizing agents.

Angiogenesis is the process by which new blood vessels are formed from existing blood vessels to build a vascular network. The formation of mature blood vessels is mainly brought about by the adhesion of vascular parietal cells such as pericytes or vascular smooth muscle cells to vascular endothelial cells that further cover the lumen of blood vessels to stabilize the vascular structure. Generally, in a positive oxygen state, vascular parietal cells line vascular endothelial cells to stabilize the vascular structure. However, when blood vessels are needed in tissues in response to changes in various environmental factors inside and outside the bloodstream, such as hypoxia and strong inflammatory stimuli, new blood vessels rapidly extend from existing blood vessels, resulting in immaturity with reduced strength. Blood vessel formation is induced.

Normal angiogenesis is physiologically the development of body tissue in the early stages of pregnancy (placental formation and fetal development), the wound healing process (post-surgery and injury), and the formation of collateral circulation around the ischemic site (postoperative or injured). It is known to play an important role in myocardial infarction, arteriosclerosis obliterans, etc.). On the other hand, abnormal angiogenesis causes various diseases such as cardiovascular disease, skin disease and malignant tumor. For example, in tumor tissue, a disordered vascular network is formed with the rapid growth of the tissue, and the blood vessels themselves are fragile and bleeding frequently, creating a highly stressful environment, resulting in a chronic hypoxic environment or low oxygen. Nutritional status is triggered. Cancer cells that survive in such harsh environments have been reported to become more malignant and resistant to treatment.

In cancer tissues having immature tumor blood vessels in this way, blood vessels are newly formed, but hypoxia persists due to circulatory failure. In hypoxic conditions, the infiltration and metastasis ability of cancer cells is enhanced, and immature tumor blood vessels provide a route for cancer cells to enter the blood vessels, and cancer metastasis is promoted.

It has been pointed out that circulatory insufficiency of tumor tissue not only results in hypoxia and malnutrition, but also affects the delivery of anticancer drugs to tumor tissue. Therefore, systems and compositions for delivering anticancer agents to tumor tissues have been proposed. For example, vascular endothelial growth factor (VEGF), which has the effect of promoting angiogenesis, is induced by hypoxia. It has been pointed out that this VEGF-targeted anti-VEGF antibody (bevacizumab) improves blood flow by suppressing the formation of a disordered vascular network, which will improve the delivery of anticancer drugs to cancer tissues. (Non-Patent Document 1). However, the effect of the anti-VEGF antibody (bevacizumab) is temporary, and over time, mature tumor blood vessels also regress and the tumor tissue becomes hypoxic. In addition, when the dose is increased, side effects due to damage to normal vascular endothelial cells are enhanced. Therefore, it is difficult to adjust the appropriate administration period and dose for the combined use of the anti-VEGF antibody (bevacizumab) and the anticancer drug (Non-Patent Document 2).

In addition, a method of targeting APJ (7-transmembrane G protein-coupled receptor) is disclosed as a drug delivery system. However, this method does not deliver the drug to the mature blood vessels of normal tissue, but delivers the drug to the blood vessels of tumor tissue, including mature blood vessels, and increases hypoxia and hyponutrition areas, and thus in cancer. It may further increase treatment resistance (Patent Document 1).

In addition to cancer, in myocardial infarction, cerebral infarction and diabetic blood flow disorder, histiocytes are placed under chronic hypoxia and malnutrition conditions. Such tissues are known to undergo rapid angiogenesis to restore the tissue, forming immature and fragile blood vessels. Therefore, it is considered to be a cause of cerebral hemorrhage, thrombus formation and recurrence of infarction, and a disease such as diabetic retinopathy and neovascular glaucoma. Chronic inflammation can also induce ectopic and disordered angiogenesis due to the delivery of immune cells to tissues, causing exudative age-related macular degeneration, rheumatoid arthritis and psoriasis. It also becomes. In addition, such disordered angiogenesis is thought to exacerbate inflammation itself.

Recent studies have pointed out that SASP (senescence-associated secretory phenotype) associated with cellular senescence may be the main cause of chronic inflammation associated with aging. This is the idea that aging cells that accumulate in body tissues with aging contribute to increased inflammation. As a side effect of this cell aging, it is considered that promotion of carcinogenesis and chronic inflammatory disease may be caused. Age-related DNA damage, reactive oxygen species accumulation and telomere shortening activate the p53, RAS and p16 ^INK4a signaling pathways and induce cellular senescence. As a result, inflammatory cytokines such as IL-1β, IL-6 and IL-8 and vascular endothelial growth factor (VEGF) are secreted. This is SASP, which is thought to lead to further cellular senescence of peripheral cells, widespread chronic inflammation, and eventually death of individuals through senescence syndrome.

Thus, SASP causes chronic inflammation, but in the lesion area, intracellular energy consumption associated with cell proliferation and hypermetabolism causes a hypoxic environment. This hypoxic environment is thought to contribute to the exacerbation of the condition. Hypoxia in tissues weakens the adhesion of vascular endothelial cells to connective tissue, extracellular matrix and basement membrane, allowing vascular endothelial cells to move more easily. This causes branching and elongation of new blood vessels, causing (vascular remodeling) angiopathy. As a result of cell damage, various intracellular substances are released, and these cell damage-associated molecular patterns (DAMPs) provoke an inflammatory response via their receptors, causing cellular senescence of blood vessels. Aged blood vessels are formed from immature blood vessels and have poor blood circulation combined with increased interstitial pressure due to leakage of blood components, which leads to a vicious cycle in which chronic disease progresses further due to persistent hypoxia and malnutrition of tissues. Is thought to be causing. It is considered that a vascular normalizing agent having an effect of enhancing the strength of blood vessels and improving the vascular network can also exert an effect on such a chronic disease associated with aging of blood vessels due to aging.

In order to prevent and treat such cancer, diabetes, inflammatory diseases (including age-related inflammation) and cardiovascular diseases, it is desired to develop a method for normalizing abnormal blood vessels. In addition, immature angioplasty such as inflammatory bowel disease (IBD) such as ulcerative colitis and Crohn's disease, atherosclerosis and endometriosis is considered to be an important contributor. There are many diseases. Therefore, the development of methods for arranging abnormal blood vessels into a mature and normal vascular network is considered to make a great contribution to the prevention, treatment and amelioration of these diseases.

International Publication No. 2012/102363

Jain RK: Nat Med, 7: 987-989,2001 Experimental Medicine: 33 (5) 129-133, 2015

The formation of an abnormal vascular network that extends in a disorderly manner can be a factor in various diseases as described above. In addition, in tissues with an abnormal vascular network, blood vessels are immature and fragile, and circulatory insufficiency creates a special microenvironment of hypoxia and malnutrition, which may affect the delivery of therapeutic agents. It has become a big problem. Therefore, there is a need for the development of a method for suppressing the formation of an abnormal blood vessel network, strengthening the blood vessel wall, and normalizing the abnormal blood vessel.

An object of the present invention is to provide a drug and a method capable of normalizing abnormal blood vessels.

The present inventors have found that administration of Goboushi extract containing arctigenin to mice transplanted with human pancreatic cancer cells, which is a model of abnormal angioplasty, improves the blood flow condition in the tumor tissue. It was also found that administration of Goboushi extract containing arctigenin significantly reduced the hypoxic region in the tumor, increased the number of cells in the proliferative phase, and increased the pericyte-coated blood vessels. Furthermore, the present inventors have found that administration of Goboushi extract containing arctigenin forms a normal vascular network without the blood vessels inside the tumor being randomly densely packed, meandering, or entangled with adjacent blood vessels. It was. From these results, the present inventors have found that arctigenin has an action of strengthening the blood vessel wall, suppressing abnormal vascular network formation, and normalizing abnormal blood vessels, and completed the present invention.

That is, the present invention provides a blood vessel wall strengthening agent containing arctigenin as an active ingredient and increasing blood vessels coated with blood vessel wall cells.

The present invention also provides the above-mentioned blood vessel wall strengthening agent, wherein the arctigenin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.

The present invention also provides an angiogenesis inhibitor containing arctigenin as an active ingredient.

The present invention also provides the above-mentioned angiogenesis inhibitor in which burdock is derived from burdock, burdock, burdock sprout, young burdock or forsythia.

The present invention also provides a method for strengthening a blood vessel wall, which comprises the step of ingesting arctigenin and / or arctiin to a subject who needs to strengthen the blood vessel wall.

The present invention also provides a method for strengthening the vascular wall, wherein arktigenin and / or arktiin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.

The present invention also provides a method for suppressing vascular network formation, which comprises a step of ingesting arctigenin and / or arctiine to a subject who requires suppression of vascular network formation.

The present invention also provides a method for suppressing the formation of the vascular network, wherein arktigenin and / or arktiin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.

The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention improve the blood flow state by normalizing abnormal blood vessels, and prevent, improve and prevent various diseases caused by abnormal blood vessels. Can contribute to treatment.

The figure which shows the blood flow state in a tumor after administration of the Goboushi extract containing high arctigenin. The figure which shows the ratio (%) of the positive region of pimonidazole in a tumor after administration of the goboushi extract containing high arctigenin. The figure which shows the ratio (%) of BrdU positive cells in a tumor after administration of the Goboushi extract containing high arctigenin. The figure which shows the ratio (%) of the pericyte-coated blood vessels in a tumor after administration of the goboushi extract containing high arctigenin. The figure which shows the tumor vascular structure after administration of the goboushi extract containing high arctigenin. The figure which shows the concentration of CPT-11 in the blood and in a tumor after administration of the Goboushi extract containing high arctigenin. The figure which shows the appearance frequency of the ulcer tissue in a tumor after the combined administration of the goboushi extract containing high arctigenin and bevacizumab. The figure which shows the survival rate after the combined administration of the goboushi extract containing high arctigenin and bevacizumab. The figure which shows the survival rate after the combined administration of the goboushi extract containing high arctigenin and gemcitabine to the pancreatic cancer orthotopic transplanted mouse. The figure which shows the change of the tumor size by the existing chemotherapeutic agent for a pancreatic cancer model, the goboushi extract high content of arctigenin, and the combination thereof.

The present invention provides a blood vessel wall strengthening agent containing arctigenin as an active ingredient. The blood vessel wall strengthening agent of the present invention strengthens the blood vessel wall by increasing the number of blood vessels covered with blood vessel wall cells. The vascular wall strengthening agent of the present invention can strengthen the vascular wall and improve the vascular strength by increasing the frequency of covering newly formed blood vessels or existing immature blood vessels with vascular parietal cells. In addition, the blood vessel wall strengthening agent of the present invention can suppress the formation of an abnormal blood vessel network by strengthening the blood vessel wall, and can be used as a blood vessel network formation inhibitor. The vascular network inhibitor of the present invention can induce a marked reduction in the germination (sprout) of the first random microvessels that produce an abnormal vascular network.

In addition, the blood vessel wall strengthening agent and the blood vessel network formation inhibitor of the present invention can normalize blood vessels by strengthening the blood vessel wall and suppressing the formation of an abnormal blood vessel network. Therefore, the blood vessel wall strengthening agent and the blood vessel network formation inhibitor of the present invention can be used as a blood vessel normalizing agent.

As used herein, "strengthening the blood vessel wall" means increasing the number of blood vessels covered with blood vessel wall cells. In the present specification, "vascular parietal cells" include pericytes (pericytes), smooth muscle cells and the like. As used herein, "suppressing vascular network formation" means reducing the vascular network, especially the abnormal vascular network, and reducing the germination (sprout) of the first random microvessels that produce the abnormal vascular network. Including that.

As used herein, "normalizing blood vessels" means normalizing or maturating abnormal blood vessels or vascular networks to form normal blood vessels or vascular networks. As used herein, "abnormal blood vessels" or "immature blood vessels" are intertwined with immature and fragile blood vessels, malformed blood vessels, excessively abnormally tortuous blood vessels, and adjacent blood vessels that are not covered by blood vessel wall cells. Refers to blood vessels that are present, blood vessels that have excessive branching, and blood vessels whose ends are not connected to other blood vessels and do not circulate. Further, in the present specification, the "abnormal vascular network" or "disordered vascular network" refers to the above-mentioned vascular network including abnormal blood vessels, a dense and complicated vascular network, and adjacent blood vessels intertwined. A vascular network and a vascular network in which blood vessels are not connected to each other and do not circulate. As used herein, "normal blood vessels" or "mature blood vessels" include blood vessels that are continuously connected in the middle, blood vessels that are covered with vascular parietal cells, blood vessels that are not excessively meandering or branched, and terminals. A blood vessel that is connected to a blood vessel and circulates sufficiently. As used herein, the term "normal vascular network" refers to the above-mentioned vascular network consisting of normal blood vessels, a vascular network having a regular and hierarchical structure (hierarchy), a vascular network that is not dense and spreads uniformly, and terminals. Refers to a vascular network that is connected to each other and circulates sufficiently.

The vascular wall strengthening agents, vascular network inhibitor and vascular normalizing agents of the present invention are complex branched and disordered vascular networks caused by abnormal tortuosity or excessive vascular germination caused by ischemic or inflammatory conditions. It suppresses the formation or reinforces the immature and fragile blood vessels formed in this state into blood vessels that maintain normal strength. The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention finally have a vascular network having a normal circulatory function by suppressing the formation of an abnormal vascular network and strengthening the vascular wall. Can be formed.

The blood vessel wall strengthening agent, the vascular network formation inhibitor, and the blood vessel normalizing agent of the present invention can improve the blood flow state of the tissue and reduce the hypoxic region by normalizing the blood vessels. In addition, the vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention suppress the formation of a disordered vascular network and have normal strength covered with vascular wall cells from immature blood vessels. By maturing into blood vessels, the increase in vascular permeability (including suppression of bleeding tendency) can be suppressed, and by adjusting the vascular network, the normal blood flow of tissues can be reconstructed.

In addition, the vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention can improve the repair and development of damaged tissue by improving the blood flow condition, and can be applied to the tissue to be treated. The deliverability of the therapeutic agent can be improved. Furthermore, when the cause of the abnormal vascular network is a tumor or infected tissue, improving the blood flow condition also improves the delivery of diseased cells and immune cells that eliminate pathogens to the diseased tissue. be able to. In cancer, it is known that the hypoxic environment triggers a change in the cellular state of cancer cells called epithelial-mesenchymal transformation, which changes into cells with high treatment resistance. By improving the above, it is possible to suppress the malignant transformation of such cancer tissues. In addition, improvement of the oxygen state of the tumor can be expected to improve the sensitivity of radiation therapy.

The arctigenin used in the blood vessel wall strengthening agent, the blood vessel network formation inhibitor and the blood vessel normalizing agent of the present invention may be derived from a plant containing arctigenin. Plants containing forsythia are not particularly limited, but for example, forsythia, forsythia, forsythia leaves, forsythia rhizome, forsythia, young forsythia, forsythia, forsythia jasmine, American oniazami, santorisou (gibanaazami), cardon, gorotsukiazami, forsythia. Asiatic jasmine, Forsythia, Forsythia, Forsythia, Forsythia, Sesame, Momijihirugao, Shinchikuhimehagi, Chosenteikakazura, Asiatic jasmine, Muninteikakazura, Himetikakazura, Tokyochikokuto, Keteikakazura Includes Forsythia, Forsythia jasmine, Forsythia jasmine, Asiatic jasmine and Kaya. Of these, burdock, burdock, burdock sprout, young burdock and forsythia are preferable because of their high arctigenin content. Arctigenin may be contained as an extract from these plants.

In the present invention, when arctigenin is derived from Goboushi, a Goboushi extract obtained by using the method for producing Goboushi extract described later can be used. Therefore, the productivity at the time of production can be improved, and the blood vessel wall strengthening agent, the blood vessel network formation inhibitor, and the blood vessel normalizing agent can be prepared inexpensively and easily. Further, even when a plant other than Goboushi is used, an extract containing arctigenin can be easily obtained by using the production method described later.

Goboushi contains about 7% of arctiine, which is classified as a lignan glycoside, and about 0.6% of arctigenin, which is an aglycone of arctiin. Since the method for producing a goboushi extract described later can significantly increase the content of arctigenin with respect to arctiin, the extract obtained by this production method contains a high content of arctigenin. Therefore, if the extract obtained by this method for producing a goboushi extract is used, it can be used as a blood vessel wall strengthening agent, a vascular network inhibitor and a vascular normalizing agent, which have superior effects as compared with the conventional goboushi extract. can do.

In addition, the blood vessel wall strengthening agent, the blood vessel network formation inhibitor, and the blood vessel normalizing agent of the present invention may further contain arctiine. Arctiin may be derived from a plant containing arctiin, for example, forsythia viridis, forsythia viridis or forsythia viridis. The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention are contained in this Goboushi extract by containing an extract from a plant containing arctigenin, for example, a Goboushi extract obtained from Goboushi. Alctiin may be further contained.

The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention may contain arctigenin and arctiine so that the weight ratio of arktigenin / arctiin is 0.7 or more. The weight ratio of arctigenin / arctiin is not particularly limited, but may be 1.3 or less. The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention contain a plant extract containing alktigenin and alktiin in a weight ratio of arktigenin / arktiin = 0.7 to 1.3, for example, Goboushi extract. You may. In addition, the blood vessel wall strengthening agent, the blood vessel network formation inhibitor, and the blood vessel normalizing agent of the present invention may contain a Goboushi extract containing 3% or more of arctigenin. Such a goboushi extract can be obtained by a method for producing a goboushi extract, which will be described later. When the blood vessel wall strengthening agent, the vascular network formation inhibitor and the blood vessel normalizing agent of the present invention contain the conventional Goboushi extract by containing the Goboushi extract obtained by the method for producing the Goboushi extract described later. It is possible to provide a higher blood vessel normalizing effect.

As the blood vessel wall strengthening agent, the blood vessel network formation inhibitor and the blood vessel normalizing agent of the present invention, the extract powder obtained by the method for producing a Goboushi extract described later can be used as it is.

The blood vessel wall strengthening agent, blood vessel network formation inhibitor and blood vessel normalizing agent of the present invention may further contain any component. For example, the vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention include pharmaceutically acceptable bases, carriers, excipients, binders, disintegrants, lubricants and colorants. Can be provided in a form including.

Examples of carriers and excipients used in vascular wall strengthening agents, vasonetwork inhibitors and vascular normalizing agents include lactose, glucose, sucrose, mannitol, dextrin, potato starch, corn starch, calcium carbonate, calcium phosphate, sulfuric acid. Includes calcium and crystalline cellulose and the like.

Examples of binders include starch, gelatin, syrup, tragant rubber, polyvinyl alcohol, polyvinyl ether, polyvinylpyrrolidone, hydroxypropyl cellulose, methyl cellulose, ethyl cellulose, carboxymethyl cellulose and the like.

Examples of disintegrants include starch, agar, gelatin powder, crystalline cellulose, calcium carbonate, sodium hydrogen carbonate, sodium alginate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose and the like.

Examples of lubricants also include magnesium stearate, hydrogenated vegetable oils, talc and macrogol. Further, as the colorant, any colorant that is allowed to be added to the pharmaceutical product can be used.

In addition, vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent are, if necessary, sucrose, gelatin, purified cellac, gelatin, glycerin, sorbitol, ethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, polyvinylpyrrolidone, It may be coated with one or more layers of phthalate cellulose acetate, hydroxypropylmethylcellulose phthalate, methylmethacrylate, methacrylic acid polymer and the like.

In addition, a pH regulator, a buffer, a stabilizer, a solubilizer, and the like may be added to the blood vessel wall strengthening agent, the blood vessel network formation inhibitor, and the blood vessel normalizing agent, if necessary.

In addition, the blood vessel wall strengthening agent, the blood vessel network formation inhibitor and the blood vessel normalizing agent can be provided as a preparation in any form. For example, vascular wall strengthening agents, angiogenesis inhibitors and vascular normalizing agents are capsules containing, as oral administration preparations, tablets such as sugar-coated tablets, buccal tablets, coated tablets and chewable tablets, troches, pills, powders and soft capsules. It can be a liquid agent such as an agent, a granule, a suspension agent, an emulsion, a syrup agent containing a dry syrup, and an elixir agent.

In addition, vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent are intravenously injected, subcutaneously injected, intraperitoneally injected, intramuscularly injected, transdermally administered, nasally administered, and transpulmonary for parenteral administration. It can be a preparation for administration such as administration, enteral administration, oral administration and transmucosal administration. For example, it can be an injection, a transdermal tape, an aerosol and a suppository. Further, when an extract from a plant or the like is used, since the extract has a peculiar harshness, it can be used as a preparation for masking the extract or a film coating agent for coating with a coating agent. ..

The blood vessel wall strengthening agent, blood vessel network formation inhibitor and blood vessel normalizing agent of the present invention can be administered to, for example, mammals. Mammals include, for example, humans, mice, rats, rabbits, cats, dogs, cows, horses and monkeys.

The vascular wall strengthening agent, vascular network inhibitor and vascular normalizing agent of the present invention can contain arctigenin at a daily dose of 10 to 2000 mg per adult. In addition, arctigenin may be administered 1 to 7 days a week without particular limitation. For example, arctigenin may be administered daily or 5 or 6 times weekly.

The blood vessel wall strengthening agent, the blood vessel network formation inhibitor and the blood vessel normalizing agent of the present invention may be administered in combination with any drug. The arbitrary drug is, for example, a drug for preventing, ameliorating or treating any disease, for example, an anticancer drug. The blood vessel wall strengthening agent, the vascular network formation inhibitor, and the vascular normalizing agent of the present invention normalize the blood vessels of the treatment target site, thereby enhancing the delivery of the drug administered in combination to the treatment target site. be able to.

In an environment where many immature blood vessels are formed, such as a tumor or chronic inflammation, it is considered that circulatory insufficiency is likely to occur, the delivery property of the drug is deteriorated, and the drug is difficult to be effective. Therefore, if the immature blood vessels are normalized and the blood flow state is improved by the blood vessel wall strengthening agent, the vascular network formation inhibitor and the blood vessel normalizing agent of the present invention, it is considered that the combined drug becomes effective. In Test Examples 5 to 8 described later, when arctigenin and an existing anticancer drug were administered in combination, the inhibition of ulcer formation was enhanced, the tumor growth was suppressed, and the cancer stem cells were more than when the anticancer drug was administered alone. It has been shown to reduce intratumoral ratio and prolong survival. As described above, the blood vessel wall strengthening agent, the vascular network formation inhibitor and the blood vessel normalizing agent of the present invention can enhance the deliverability of the drug by normalizing the blood vessels and enhance the effect of the drug.

The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention take forms such as pharmaceuticals, foods for the sick, health foods, functional foods, foods for specified health use, dietary supplements and supplements. be able to. The vascular wall strengthening agent, vascular network formation inhibitor and vascular normalizing agent of the present invention also indicate that they have an effect of strengthening the blood vessel wall, indicate that they have an effect of suppressing vascular network formation, and normalize blood vessels. It may be in the form of a food with a functional claim, which is labeled to have the effect of causing the blood vessel.

The present invention also provides a food composition for strengthening a blood vessel wall, which contains arctigenin as an active ingredient and increases blood vessels coated with blood vessel wall cells. The present invention also provides a food composition for suppressing angiogenesis, which contains arctigenin as an active ingredient. The present invention also provides a food composition for vascular normalization containing arctigenin as an active ingredient. The food composition for strengthening blood vessel wall, the food composition for suppressing blood vessel network formation, and the food composition for normalizing blood vessel of the present invention are configured in the same manner as the above-mentioned blood vessel wall strengthening agent, blood vessel network formation inhibitor, and blood vessel normalizing agent. can do.

In the present specification, the "food composition" includes not only general foods and drinks but also foods for the sick, health foods, functional foods, foods for specified health uses, dietary supplements and supplements. Common foods and drinks include, for example, various beverages, various foods, processed foods, liquid foods (soups and the like), seasonings, energy drinks and confectionery. As used herein, the term "processed food" refers to natural foodstuffs (animals, plants, etc.) that have been processed and / or cooked, such as processed meat products, processed vegetable products, processed fruit products, and frozen foods. Includes retort foods, canned foods, bottled foods and instant foods. The food composition of the present invention may be a food with a label to strengthen the blood vessel wall, a label to suppress vascular network formation, or a label to normalize the blood vessel. Further, the food composition of the present invention may be provided in a form enclosed in a bag, a container or the like. The bags and containers used in the present invention can be any bags and containers commonly used for food.

The present invention also provides a method of strengthening a blood vessel wall, which comprises the step of ingesting arctigenin and / or arctiin to a subject in need of strengthening the blood vessel wall. The present invention also provides a method of suppressing angiogenesis, which comprises the step of ingesting arctigenin and / or arctiin to a subject in need of inhibition of angiogenesis. The present invention also provides a method of normalizing a blood vessel, which comprises the step of ingesting arctigenin and / or arctiin to a subject in need of normalization of the blood vessel.

Subjects to which the methods of the invention apply include mammals such as humans, mice, rats, rabbits, cats, dogs, cows, horses and monkeys. Subjects that require strengthening of the vascular wall, subjects that require suppression of angioplasty, and subjects that require normalization of blood vessels are those who develop abnormal angioplasty and those who have abnormal angioplasty. It is a subject suffering from a disease that is considered to be. Target diseases include, for example, cancer, cardiovascular disease, various diseases with chronic inflammation, such as diabetic complications (nephropathy, retinopathy, neuropathy), age-related luteal degeneration and neovascular glaucoma. Inflammatory bowel disease (IBD) such as endocytic vascular disease, ulcerative colitis and Crohn's disease, atherosclerosis, endometriosis, arteriosclerosis, psoriasis, cerebral and myocardial infarction, chronic kidney disease, non-alcoholic Includes steatosis, Alzheimer's disease, Parkinson's disease, obesity and lifestyle diseases, and skin photoaging.

Arctiin is a precursor of arctigenin and is known to be metabolized in vivo to become arctigenin. In the method of the present invention, only one of arctigenin or arctiin may be ingested, or both arctigenin and arctiin may be ingested.

In the method of the present invention, arktigenin and / or alktiin may be derived from a plant containing arktigenin and / or arktiin, and may be derived from, for example, burdock, burdock, burdock sprout, young burdock and forsythia. In addition, arctigenin and / or arctiin may be ingested as a goboushi extract obtained by using the method for producing a goboushi extract described later. Further, any component may be further ingested together with arctigenin and / or arctiin. Optional ingredients include, for example, pharmaceutically acceptable bases, carriers, excipients, binders, disintegrants, lubricants and colorants.

Arctigenin and / or arctiin can also be taken orally or parenterally (injections, coatings, patches, suppositories, eye drops, nasal drops, inhalants, mouthwashes, sublingual absorbents and implants, etc.) Good. In addition, arctigenin and / or arctiin may be ingested so that the daily intake is 10 to 2000 mg per adult. In addition, arctigenin and / or arctiin may be ingested 1 to 7 days a week without particular limitation. For example, arctigenin and / or arctiin may be taken daily or 5 or 6 times a week.

The method of the present invention includes an embodiment in which arctigenin and / or arctiin is ingested not only medicinally but also non-medically. For example, the method of the present invention comprises ingesting arctigenin and / or arctiin in the form of sick foods, health foods, functional foods, foods for specified health uses, dietary supplements and supplements.

[Manufacturing method of Goboushi extract]
The Goboushi extract suitable for the present invention is produced through a crude drug cutting step, an extraction step (enzyme conversion step and an extraction step with an organic solvent), a solid-liquid separation step, a concentration step and a drying step.

(Crude drug cutting process)
In the crude drug cutting process, the raw material Goboushi is cut into a size suitable for extraction. Crude drugs used as raw materials have various sizes, shapes, and hardnesses such as various parts of plants, minerals, and animals, and it is necessary to cut them according to their characteristics. Goboushi can be cut using any means known to those skilled in the art. For example, a commercially available cutting machine can be used.

In the method for producing a goboushi extract suitable for the present invention, the activity of β-glucosidase, which is an enzyme contained in goboushi, can be measured in advance, and a suitable goboushi can be selected. As a method for measuring the activity of β-glucosidase, for example, p-nitrophenyl-β-D-glucopyranoside (C12H15NO8: molecular weight 301.25) (manufactured by SIGMA-ALDRICH) is used as a substrate and produced by reacting a crushed goboushi product. Enzyme activity can be measured by measuring the change in the absorbance of p-nitrophenol at 400 nm. As a unit for expressing enzyme activity, the amount of enzyme that produces 1 micromol of p-nitrophenol per minute can be expressed as 1 unit (U).

In order to obtain a Goboushi extract suitable for the present invention, a Goboushi having an activity of β-glucosidase inherent in Goboushi of, for example, 0.4 U / g or more, preferably 1 U / g or more can be used. If it is less than 0.4 U / g, hydrolysis will be insufficient, the weight ratio of arctigenin will decrease, and the desired Goboushi extract will not be obtained efficiently.

Further, in the method for producing a goboushi extract suitable for the present invention, goboushi cut to an arbitrary particle size can be used. It is considered that the smaller the particle size of the cut goboushi, the more the enzyme conversion is promoted and the yield of the extract increases. On the other hand, if the particle size is too small, the enzyme conversion may be too fast and process control may be difficult, or accurate solid-liquid separation may be hindered in a later process.

In order to obtain a Goboushi extract suitable for the present invention, the Goboushi is cut into a particle size of 9.5 mm or less, for example, through a 9.5 mm sieve, as shown in the following examples. .. Further, in order to obtain a goboushi extract suitable for the present invention, the particle size of goboushi is more preferably 0.85 so that the entire amount of the goboushi passes through a 9.5 mm sieve, for example, 60 to 100% is distributed on a 0.85 mm sieve. It is desirable to cut so that 65-80% is distributed on a mm sieve.

(Extraction process)
The extraction step is the most important step in terms of quality in the crude drug extract powder manufacturing step. This extraction process determines the quality of the crude drug extract powder. In the method for producing a goboushi extract suitable for the present invention, in order to extract the goboushi extract, the extraction is performed in two stages, an enzyme conversion step and an extraction step using an organic solvent.

(Enzyme conversion process)
The enzyme conversion step is an important step in the method for producing a Goboushi extract suitable for the present invention. The enzyme conversion step is a step of enzymatically converting arctiin contained in goboushi into arctigenin by β-glucosidase, which is an enzyme inherent in goboushi.

Specifically, the cut goboushi prepared in the above step is kept at an appropriate temperature to allow β-glucosidase to act on the cut product to promote the reaction from arctiine to arctigenin. For example, the goboushi can be kept at an arbitrary temperature by adding an arbitrary solution such as water to the cut goboushi and stirring at a temperature of about 30 ° C.

In order to obtain a goboushi extract suitable for the present invention, the cut goboushi is kept at a temperature around 30 ° C., for example, between 20 and 50 ° C. If the temperature is lower than 20 ° C., hydrolysis will be insufficient, the weight ratio of arctigenin will decrease, and the desired Goboushi extract cannot be obtained efficiently. On the other hand, when the temperature is higher than 50 ° C., the enzyme is inactivated, the weight ratio of arctigenin is lowered, and the desired Goboushi extract cannot be obtained efficiently.

Further, the holding time is not particularly limited as long as it is held at the above temperature, and can be held for, for example, about 30 minutes. By holding between 20 and 50 ° C., an appropriate amount of arctiin is enzymatically converted to arctigenin regardless of the holding time, and a Goboushi extract suitable for the present invention can be obtained.

(Extraction process with organic solvent)
The extraction step with an organic solvent is a step of extracting arctigenin and arctiin from Goboushi using any suitable organic solvent. That is, it is a step of extracting a goboushi extract by adding an appropriate solvent in a state where the content of arctigenin is high by the above enzyme conversion step. For example, a suitable solvent is added to the Goboushi extract, and the mixture is heated and stirred for an appropriate time to extract the Goboushi extract. In addition to heating and stirring, the Goboushi extract can be extracted by using any extraction method known to those skilled in the art, such as heating reflux, drip extraction, immersion extraction, or pressure extraction.

Since arctigenin is poorly soluble in water, the yield of arctigenin can be improved by adding an organic solvent. Any organic solvent can be used as the organic solvent. For example, alcohols such as methanol, ethanol and propanol, as well as acetone can be used. In consideration of safety, it is preferable to use ethanol as an organic solvent in the method for producing a goboushi extract suitable for the present invention.

When the Goboushi extract is extracted by heating and stirring, the heating and stirring can be performed at any temperature, but in order to obtain a Goboushi extract suitable for the present invention, a temperature of 80 ° C. or higher, for example, 80 to 90 Keep at a temperature between ° C. The time for heating and stirring is not particularly limited as long as the heating and stirring are performed at the above temperature, and the alktigenin and alktiin can be extracted from the goboushi in the solvent by heating and stirring for about 30 minutes, for example, 30 to 60 minutes. ..

The yields of arctigenin and arctiin improve as the heating and stirring time increases. However, if the heating and stirring time is long, a large amount of unnecessary fats and oils are dissolved out, and the load of the concentration step becomes large. Therefore, the heating and stirring time may be appropriately determined depending on the situation.

In addition, the yield of alktigenin and arctiin increases as the amount of ethanol increases, because the solubility of arktigenin and arctiin increases. However, when the amount of ethanol is large, a large amount of unnecessary fats and oils are dissolved, and the load of the concentration process becomes large. Therefore, the input amount may be appropriately determined according to the situation. The Goboushi extract can be sterilized and sterilized at the same time by heating and stirring in this step.

(Solid-liquid separation process)
The solid-liquid separation step is a step of separating the extracted goboushi from the extract. Solid-liquid separation can be carried out using any method known to those skilled in the art. Solid-liquid separation methods include, for example, filtration methods, sedimentation methods and centrifugation methods. Industrially, the centrifugation method is desirable.

(Concentration process)
The concentration step is a step of removing the solvent from the Goboushi extract prior to drying. Removal of the solvent from the Goboushi extract can be carried out using any method known to those skilled in the art. However, it is preferable that the extract from Goboushi obtained by the above step is not exposed to a higher temperature for a long time. For example, by using the vacuum concentration method, the Goboushi extract can be concentrated without being exposed to high temperature for a long time.

Concentration of the Goboushi extract can be concentrated to a concentration at which a desired concentration of Goboushi extract can be obtained. For example, it is desirable to concentrate to the extent that drying can be performed properly in the following drying steps. In addition, when the Goboushi extract is dried into a powder formulation in the following steps, it is desirable to concentrate it to a concentration at which appropriate formulation characteristics can be obtained.

Since alktigenin and alktiin are poorly soluble in water, a large amount of alktigenin and alktiin adhere to the manufacturing apparatus in the following drying step, and the final yield is significantly reduced. Therefore, dextrin can be added to the Goboushi extract obtained in this concentration step in order to prevent the arctigenin and alktiin from adhering to the production apparatus. The amount of dextrin added is preferably about 15 to 30% with respect to the solid content of the concentrate, for example.

(Drying process)
This is a step of finishing the Goboushi extract obtained by the above step into a powder. Drying can be carried out using any method known to those skilled in the art. For example, freeze-drying and spray-drying are known as drying methods, but it is common to use the former at the laboratory level and the latter at the mass production level.

By the above manufacturing process, a Goboushi extract containing a high content of arctigenin can be obtained. This method for producing Goboushi extract must include a step of performing enzymatic conversion at a temperature of 20 ° C. to 50 ° C., but it is not necessary to include all the other steps.

Further, by the above manufacturing process, a Goboushi extract having a high concentration of arctigenin can be obtained inexpensively and easily. Therefore, by using the Goboushi extract obtained by this method, the blood vessel wall strengthening agent, the blood vessel network formation inhibitor and the blood vessel normalizing agent of the present invention can be produced inexpensively and easily.

In addition, since the arctigenin concentration of the Goboushi extract obtained by the above manufacturing process is high, it is one of the blood vessel wall strengthening agent, the vascular network formation inhibitor and the blood vessel normalizing agent as compared with the case of using the conventional Goboushi extract. The total amount of sunlight can be reduced. Therefore, the burden on the patient can be reduced.

[Example 1]
A goboushi extract containing arctigenin was prepared by the following method.

Goboushi (enzyme activity 7.82 U / g) was cut and passed through a 9.5 mm sieve and further passed through a 0.85 mm sieve, and it was confirmed that 75% remained. 80 kg of this goboushi shredded was added to 560 L of water kept at 30 to 32 ° C. and stirred for 30 minutes, then 253 L of ethanol was added to raise the temperature to 85 ° C., and the mixture was heated under reflux for another 40 minutes. This solution was centrifuged to obtain the obtained extract. The extracts obtained by repeating this operation twice were combined, concentrated under reduced pressure, 25% of dextrin was added to the solid content of the extract, and the mixture was spray-dried. The contents of arctigenin and arctiin were 6.4% and 7.2%, respectively, and a goboushi extract powder (containing 25% dextrin) having arctigenin / arctiin (weight ratio) = 0.89 was obtained.

[Test Example 1]
As a model of abnormal blood vessels, mice transplanted with tumor cells were used. Intratumoral blood flow was evaluated for functional analysis of intratumoral microenvironmental changes due to repeated administration of arctigenin-containing goboushi extract.

1 × 10 ⁶ Cells / 200 μl of human pancreatic cancer cells Suit2 were subcutaneously transplanted into BALB / cAJc1-nu / nu mice (Claire Japan). On the 21st day of transplantation, the transplanted mice were divided into four groups: (a) untreated group, (b) goboushi extract-administered group, (c) bevacizumab-administered group, and (d) gemcitabine-administered group, and treatment was continued for 4 weeks. did. As the goboushi extract, a mixture of the goboushi extract of Example 1 in a diet of 0.5% (w / w) was given to mice. Calculating the daily food intake of mice as 3-5 g, arctigenin was administered at 15-25 mg / animal / day. Bevacizumab was intraperitoneally administered at 5 mg / kg once a week using 100 mg / 4 ml (Chugai Pharmaceutical) for intravenous infusion of Avastin. Gemcitabine was intraperitoneally administered at 100 mg / kg twice a week using a gemcitabine preparation for infusion (Eli Lilly and Company).

(Tumor size)
The mean tumor size in each group 4 weeks after the start of treatment was (a) 228 mm ^{2 in} the untreated group, (b) 189 mm ^{2 in} the goboushi extract-treated group, (c) 56 mm ^{2 in} the bevacizumab-treated group, and (d). The gemcitabine-treated group was 68 mm ² .

(Incorporation of gadolinium DTPA in tumor tissue)
Four weeks after the start of treatment, the uptake of gadolinium DTPA in each group of mouse tumor tissues was evaluated. Up to 120 seconds after rapid intravenous injection of gadolinium DTPA, a dynamic contrast-enhanced T1-weighted image of the tumor (time resolution 1.8-5.3 seconds) was taken using a 9.4 Tesla MRI machine (BIOSPEC 94 / 20USR). From this image, the signal intensity of gadolinium DTPA in the tumor tissue was measured. FIG. 1 is a graph showing the average value of the signal intensity every 30 seconds when the signal intensity at 0 seconds after intravenous injection of gadolinium DTPA is set to 100. Increased signal intensity of gadolinium DTPA in tumor tissue means uptake of gadolinium DTPA into tumor tissue. The degree of uptake of gadolinium DTPA into tumor tissue is an indicator of blood flow status in tumor tissue.

(Functional analysis of intratumoral microenvironmental changes)
As shown in FIG. 1, 90 to 120 seconds after intravenous injection of gadolinium DTPA, the signal intensity in the central part of the tumor tissue after treatment in the arctigenin-containing goboushi extract-administered group increased to 118.1 ± 13.7%. On the other hand, in the untreated group, it was 102.6 ± 3.4% in 90 to 120 seconds and did not increase. Therefore, the blood flow tended to be better in the goboushi extract-administered group than in the untreated group (P <0.05, t-test). Moreover, in the gemcitabine-treated group in which the antitumor effect was observed, the signal intensity in the central part of the tumor tissue did not increase as in the untreated group (not shown). On the other hand, in the bevacizumab treatment group having an angiogenesis inhibitory effect, the signal intensity tended to increase (not shown).

From the above results, it was suggested that the administration of the arctigenin-containing goboushi extract may reduce the poor blood flow region of the tumor tissue and leave the rich blood flow region.

[Test Example 2]
Hypoxic regions (Pimonidazole) and cell proliferation (BrdU) were evaluated by immunostaining for pathological analysis of intratumoral microenvironmental changes due to repeated administration of arctigenin-containing goboushi extract. In addition, the proportion of blood vessels covered with pericytes among the blood vessels in the tumor was evaluated by the double immunostaining method of CD31 and αSMA.

Miapaca-2 (ATCC CRL 1420) 5 × 10 ⁶ Cells / 200 μl of human pancreatic cancer cells were subcutaneously transplanted into BALB / cAJc1-nu / nu mice (Claire Japan). On the 16th day of transplantation, the transplanted mice were divided into four groups: (a) untreated group, (b) goboushi extract-administered group, (c) bevacizumab-administered group, and (d) gemcitabine-administered group, and treatment was continued for 4 weeks. did. In the goboushi extract-administered group, mice were fed a diet containing 2.0% (w / w) of goboushi extract. Calculating the daily intake of mouse food as 3-5 g, arctigenin was administered at 60-100 mg / animal / day. Bevacizumab was intraperitoneally administered at 5 mg / kg once a week using 100 mg / 4 ml (Chugai Pharmaceutical) for intravenous infusion of Avastin. Gemcitabine was intraperitoneally administered at 100 mg / kg twice a week using a gemcitabine preparation for infusion (Eli Lilly and Company).

(Tumor size)
Mean tumor size of each group after treatment after 4 weeks, (a) untreated group 1851mm ^2, (b) Goboushiekisu administered group 1035 mm ^2, (c) bevacizumab group 911 mm ^2, and (d) The gemcitabine-treated group was 949 mm ² .

(Pathological analysis of intratumoral microenvironmental changes 4 weeks after the start of treatment)
Four weeks after the start of treatment, 100 μL of BrdU (Sigma-Aldrich) 20 mg / mL and 100 μL of pimonidazole 16 mg / mL were intraperitoneally administered, and tumors were collected 1 hour later. In each group of tumor sections collected, the hypoxic region (pymonidazole) and cell proliferation (BrdU) were evaluated by immunostaining for pathological analysis of intratumoral microenvironmental changes. In addition, the proportion of blood vessels covered with pericytes among the blood vessels in the tumor was evaluated by the double immunostaining method of CD31 and αSMA.

Pimonidazole is a 2-nitroimidazole compound that is reduced under strong hypoxia with an oxygen partial pressure of 10 mmHg or less, has the property of binding to intracellular proteins and forming adducts, and is currently the most widely used low oxygen compound. It is an oxygen probe.

BrdU is a thymidine analog that is incorporated into newly synthesized DNA during the S phase of the cell cycle. BrdU-labeled DNA can be detected with anti-BrdU antibodies, making anti-BrdU antibodies a tool for detecting proliferating cells that replicate DNA.

By lining the outside of the blood vessel with pericytes, blood can flow without leaking. However, in the blood vessels inside the tumor, many weak blood vessels composed only of endothelial cells are formed, so that blood leaks and delivery of the anticancer drug to the inside of the tumor is hindered. Angiogenesis inhibitors typified by bevacizumab are thought to enhance the effect of the anticancer drug used in combination by reducing the proportion of blood vessels not covered with pericytes.

(Changes in the intratumoral microenvironment after treatment)
The percentage of positive regions of pimonidazole in the tumor after each treatment is shown in FIG. The percentage of positive regions of pimonidazole in the tumor was 40% in the untreated group, 18% in the bevacizumab-treated group, 19% in the goboushi extract-treated group, and 30% in the gemcitabine-treated group. From this result, it was clarified that the hypoxic region was significantly reduced by the treatment with bevacizumab and the goboushi extract. This is thought to be due to the normalization of blood vessels.

The proportion (%) of BrdU-positive cells in the tumor after each treatment is shown in FIG. The proportion of BrdU-positive cells in the tumor (%) was 7% in the untreated group, 15% in the bevacizumab-treated group, 11% in the goboushi extract-treated group, and 6% in the gemcitabine-treated group. From this result, it was clarified that the proliferating cells were increased by the treatment with bevacizumab and the treatment with Goboushi extract. This is thought to be due to the normalization of blood vessels.

The percentage of pericyte-coated blood vessels in the tumor after each treatment is shown in FIG. The percentage of pericytes was determined by dividing the CD31 and αSMA double positive area by the CD31 positive area. The proportion of pericyte-coated blood vessels was 23.3% in the untreated group and 35.2% in the goboushi extract-administered group. Therefore, it is suggested that the administration of Goboushi extract normalized the blood vessels and increased the pericyte-coated blood vessels. This result indicates that arctigenin can be used as a vascular wall strengthening agent to increase blood vessels covered with vascular parietal cells such as pericytes.

Therefore, it is suggested that the administration of arctigenin normalized the blood vessels in the tumor tissue. In addition, it is suggested that the normalization of blood vessels creates an environment in which drugs can be easily delivered. Therefore, it is suggested that the combined administration of the anticancer drug with arctigenin enhances the delivery of the anticancer drug to the tumor tissue, and as a result, the efficacy of the anticancer drug is enhanced.

[Test Example 3]
Using the vascular template method, changes in the intratumoral vascular structure due to repeated administration of arctigenin-containing goboushi extract were investigated.

Human pancreatic cancer cells Miapaca-2 (ATCC CRL 1420) 5 × 10 ⁶ Cells / 200 μl were transplanted subcutaneously into BALB / cAJc1-nu / nu mice (Claire Japan), and the tumor size became about 200 mm ^3. The mice were selected and used as treatment target mice. The prepared transplanted mice were divided into an untreated group and a goboushi extract-administered group, and treatment was continued for 4 weeks. In the goboushi extract administration group, 750 mg / kg of an extract containing 10% arctigenin was orally administered 5 times a week.

(Creation of blood vessel template)
Four weeks after the start of treatment, the mice were deeply anesthetized, and the chest was opened, and physiological saline was perfused from the heart to remove blood. After blood removal, 10 mL of Mercox (Ladd research industry) containing 40% methyl methacrylate (Nissin EM Co., Ltd.) containing an accelerator (Ladd research industry) was perfused from the heart. Then, the mouse was immersed in warm water at 60 ° C. for 60 minutes to cure the resin. The tumor was dissected, the tissue was corroded with 20% NaOH, a vascular template sample was taken out, and structural changes in the blood vessels in the tumor were examined using a scanning electron microscope.

(Changes in tumor vascular structure after treatment)
The blood vessels inside the tumor after each treatment are shown in Figure 5. In the untreated group of tumors, the blood vessels were dense and formed a complicated and intricate structure, and the terminal part of the blood vessel also meandered and entangled with the adjacent blood vessel. On the other hand, in tumors treated with arctigenin-containing goboushi extract, the dense structure of blood vessels as seen in the untreated group was not observed, and even at the terminal part of the blood vessel, there was little meandering of the blood vessel, and it was connected to the adjacent blood vessel. No intertwined structure was found. Therefore, it is suggested that the administration of the goboushi extract containing arctigenin normalized the blood vessels and formed a normal vascular network. This result indicates that arctigenin can be used as a vasculature inhibitor that suppresses the formation of vascular networks, especially abnormal vascular networks.

[Test Example 4]
Blood and intratumoral CPT-11 concentrations were evaluated to analyze the pharmacokinetic changes of anticancer agents in tumors due to repeated administration of arctigenin-containing goboushi extract.

Human pancreatic cancer cells Miapaca-2 (ATCC CRL 1420) 5 × 10 ⁶ Cells / 200 μl were transplanted subcutaneously into BALB / cAJc1-nu / nu mice (Claire Japan), and the tumor size became about 200 mm ^3. The mice were selected and used as treatment target mice. The prepared transplanted mice were divided into an untreated group and a goboushi extract-administered group, and treatment was continued for 4 weeks. In the goboushi extract administration group, 750 mg / kg of an extract containing 10% arctigenin was orally administered 5 times a week.

Four weeks after the start of treatment, 50 mg / kg of Campto drip infusion (Yakult) was intraperitoneally administered to the untreated group and the Goboushi extract-administered group, and tumors and blood were collected under deep anesthesia 30 minutes, 1 hour, and 3 hours, respectively. did. The collected blood was centrifuged at 1200 × g for 20 minutes at 4 ° C., and plasma was collected. 200 μL of plasma was collected, an equal amount of acetonitrile was added, and centrifugation was performed at 12,000 rpm at 4 ° C. for 10 minutes. After recovery, the tumor was measured at about 100 mg, homogenized with 100 mg / 200 μL MilliQ, 300 μL of acetonitrile was added, and the tumor was centrifuged at 12,000 rpm at 4 ° C for 10 minutes. Each supernatant was collected, injected in 10 μL each, and subjected to HPLC analysis. For the standard, 10 mg of CPT-11 was dissolved in 5 mL of methanol, 10 μL was injected, and HPLC analysis was performed.

(Changes in pharmacokinetics of anticancer drugs in blood and tumor after treatment)
The concentrations of CPT-11 in the blood and in the tumor at each time after intravenous infusion of Campto to each treatment group are shown in FIG. There was no clear change in the blood concentration of CPT-11 in each time zone of the untreated group and the goboushi extract-administered group in both groups. On the other hand, the intratumoral CPT-11 concentration did not change in both groups after 30 minutes and 1 hour, but the intratumoral CPT-11 concentration 3 hours after administration was 4 μg / g tumor in the untreated group. , 8.7 μg / g tumor in the arctigenin-treated group. It was revealed that arctigenin treatment can increase the concentration of intratumoral CPT-11 without affecting the blood CPT-11 concentration.
[Test Example 5]
During the process of tumor growth, nutrients and oxygen must be supplied through blood vessels throughout the grown tumor tissue. However, in many tumors, it is believed that due to immature angioplasty, these are not sufficiently supplied, causing partial tissue necrosis and ulceration of the tissue. Therefore, in order to verify the blood vessel normalizing effect of the above-mentioned Goboushi extract from the tissue state, ulcer formation in the tumor tissue after each treatment was observed.

Human colon cancer cells LS174T (ATCC CL-188) 5 × 10 ⁵ Cells / 200 μl were subcutaneously transplanted into BALB / cAJc1-nu / nu mice (Claire Japan). On the 14th day of transplantation, the transplanted mice were divided into four groups: (a) untreated group, (b) goboushi extract administration group, (c) bevacizumab administration group, and (d) goboushi extract and bevacizumab combination administration group. , Continued treatment for 5 weeks. In the goboushi extract administration group, 250 mg / kg of an extract containing 10% arctigenin was orally administered 5 times a week. In the bevacizumab-administered group, 5 mg / kg was intraperitoneally administered once a week using 100 mg / 4 ml (Chugai Pharmaceutical) for intravenous infusion of Avastin.

The proportion (%) of mice with tumor ulcers in each treatment group is shown in FIG. The 50% ulcer formation rate was 8 days in the untreated group, 20 days in the goboushi extract-administered group, 10 days in the bevacizumab-administered group, and 24 days in the combination-administered group. Therefore, in the goboushi extract-administered group and the combined-administered group, the effect of suppressing ulcer formation in the tumor was observed as compared with the untreated group and the bevacizumab-administered group. In the bevacizumab-administered group, an inhibitory effect on ulcer formation was observed as compared with the untreated group, but it was transient. This effect is considered to be due to the angiogenesis-inhibiting effect of bevacizumab. On the other hand, when goboushi extract was co-administered in addition to bevacizumab, the inhibition of ulcer formation was enhanced. It is considered that this is because the blood flow improving effect of bevacizumab alone on the tumor and the blood flow improving effect of the blood vessel normalizing effect of the goboushi extract treatment were enhanced in combination. As described above, the combined administration with arctigenin can remarkably reduce the region of hypoxia and malnutrition by normalizing blood vessels, and is expected to suppress the malignant transformation of cancer.

(Life extension effect by combined use)
After continuing the above treatment for 5 weeks, treatment was discontinued and survival follow-up was performed. The survival rate in each group is shown in Fig. 8. As shown in FIG. 8, the 50% survival time was 103 days in the untreated group, 114 days in the goboushi extract-administered group, 107 days in the bevacizumab-administered group, and 137 days in the combination-administered group. Therefore, the combination-administered group showed a survival-prolonging effect of 33% and 20-28%, respectively, as compared with the untreated group and the single-administered group.

[Test Example 6]
The antitumor effect of the combined administration of goboushi extract and gemcitabine was evaluated.

Human pancreatic cancer cells Miapaca-2 (ATCC CRL 1420) 5 × 10 ⁶ Cells / 200 μl were transplanted under the armpit of BALB / cAJcl nu / nu mouse (Claire Japan), and the tumor size became about 100 mm ^3. Was selected and used as a target mouse for treatment.

Four groups of mice to be treated were (a) untreated group, (b) gemcitabine-administered group, (c) goboushi extract-administered group, and (d) goboushi extract and gemcitabine in combination (combination-administered group). The treatment was continued for 4 weeks. As the goboushi extract, 250 mg / kg of the goboushi extract of Example 1 (25 mg / kg or more as arctigenin) was orally administered every day (5 times a week). Gemcitabine was intraperitoneally administered at 150 mg / kg twice a week using a gemcitabine preparation for infusion (Eli Lilly and Company).

(Tumor weight)
After the treatment test, tumor weight was measured for each treatment group. As a result, the tumor weight after the treatment test was 2.05 g in the untreated group, 1.24 g in the gemcitabine-administered group, 0.98 g in the goboushi extract-administered group, and 0.82 g in the combination-administered group. Therefore, in all of the gemcitabine-administered group, the goboushi extract-administered group, and the combined-administered group, the tumor weight was suppressed by about 40 to 60% as compared with the untreated group.

From this result, it was shown that the combined use of goboushi extract and gemcitabine suppresses tumor growth. It is considered that this is because gemcitabine is more likely to act on the tumor by normalizing the blood vessels by the goboushi extract. Therefore, it was shown that the combination drug of goboushi extract and gemcitabine has significantly higher anticancer activity than expected from the effect when each of them is administered alone.

[Test Example 7]
The 5-year survival rate after diagnosis of pancreatic cancer is less than 5% as of 2013, and the average survival time is only 4-6 months. This is an extremely low value compared to other cancers, and effective countermeasures are required. In Example 6 described above, the combined treatment of gemcitabine and goboushi extract containing a high amount of arctigenin for the pancreatic tumor transplantation model showed a tendency to suppress tumor growth as compared with monotherapy. Therefore, as a model closer to the actual lesion, a pancreatic cancer orthotopic transplantation model into mouse pancreas was prepared, and the effect of combined treatment with gemcitabine and goboushi extract on survival rate was evaluated.

Human pancreatic cancer cells Miapaca-2 cells were suspended in a high-concentration Matrigel solution (BD bioscience) at 5 × 10 ⁶ cells / 50 μl. At 8 weeks of age, female BALB / cAJcl-nu / nu (Claire Japan) was laparotomized under anesthesia, and the above cell suspension was injected into and solidified into pancreatic tissue, and then sutured and closed.

The transplanted mice were divided into 7 mice in 4 groups each of an untreated group, a goboushi extract-administered group, a gemcitabine-administered group, and a group to which these were administered in combination, and treatment was continued until all the mice died. In the goboushi extract administration group, 250 mg / kg of an extract containing 10% arctigenin was orally administered 5 times a week. In the gemcitabine-administered group, 150 mg / kg was intraperitoneally administered twice a week using a gemcitabine preparation for infusion (Eli Lilly and Company).

(Analysis of survival rate)
Survival rate was calculated by calculating and drawing the survival rate curve and median survival rate MST using the Kaplan-Mayer method (GraphPad-PRISM 6.2, MDF). Significant differences were assessed by the Logrank (Mantel-COX) and Gehan-Breslow-Wilcoxon test.

The change in survival rate (%) of each treatment group is shown in the upper graph of FIG. Median survival (MST) was 65 days (32.3% increase) in the untreated group (Non treat), 86 days (32.3% increase) in the gemcitabine group, and 88 days (35.4% increase) in the goboushi extract group (GBS-01). ), Combination treatment (Combination) was 117 days (80% increase). In addition, a significant difference was found between the treated group and the untreated group regardless of whether the Gehan-Breslow-Wilcoxon test or the Log-RANK (Mantel-Cox) test was used.

As a result, in the pancreatic cancer orthotopic transplantation model, as shown in FIG. 9, the goboushi extract-administered group and the gemcitabine-administered group showed almost the same life-prolonging effect as those of the untreated group. On the other hand, the combined administration group in which these drugs were used in combination showed a remarkable life-prolonging effect as compared with the untreated group and the single administration group. Therefore, it was shown that the combined administration of an anticancer drug and arctigenin can greatly improve the survival rate even in pancreatic cancer in which it is difficult to prolong the survival period.

[Test Example 8]
Using a pancreatic cancer transplantation model, treatment was performed with various existing anticancer agents, argtigenin-rich goboushi extract, and combinations thereof, and changes in tumor size were evaluated.

Miapaca-2 (ATCC CRL 1420) 5 × 10 ⁶ Cells / 200 μl of human pancreatic cancer cells was transplanted under the armpit of BALB / cAJc1-nu / nu mice (Claire Japan), and after breeding for about 2 weeks, about 100 mm ³ Alternatively, a mouse having a size of about 600 mm ³ was used as a treatment target mouse.

The prepared transplanted mice were divided into an untreated group, an existing anticancer drug administration group which is a component having various anticancer effects, a goboushi extract administration group, and a combination administration group thereof, and for 5 to 6 tumors in each group. , The treatment was continued for about 4 weeks. Existing anticancer agents include carboplatin (CBDCA, Bristol-Myers, 60 mg / kg intraperitoneally), doxorubicin (Doxorubicin, Kyowa Hakko, 10 mg / kg peritoneally twice weekly), oxaliplatin (Oxaliplatin, Yakult, 8 mg). / kg intraperitoneally), everolimus (Everolimus, Novartis, 5 mg / kg orally 5 mg / kg), bortezomib (Bortezomib, Janssenpharma, 1 mg / kg peritoneally twice weekly), irinotecan (Yakult) , 25 mg / kg peritoneally twice a week) and hydroxychloroquine (Hydroxychloroquine, sanophy, 100 mg / kg peritoneally 5 times a week) were used. As for goboushi extract, 750 mg / kg of an extract containing 10% arctigenin was orally administered 5 times a week.

(Measurement of tumor size)
Every 3-4 days after the start of treatment, the mice were retained and the major axis (L), minor axis (W) and height (H) of the tumor were measured with a precision digital caliper, and the tumor size = 4/3 * pi * The size was calculated using the formula L / 2 * H / 2 * W / 2 (pi = 3.14). If the height could not be measured, the tumor size = 4/3 * pi * L / 2 * (W / 2) 2 was simply used.

The variation in tumor size of each treatment group is shown in FIG. As a result of the combination treatment of the existing anticancer drug and Goboushi extract, carboplatin and oxaliplatin were 2.3 to 2.1 times (P <0.05) compared with Goboushi extract alone and 1.8 to 1.9 times (P <0.05) compared with the anticancer drug alone. A decrease in tumor size was observed. In addition, irinotecan showed a 2.1-fold (P <0.05) reduction in tumor size compared to the Goboushi extract alone and a 1.5-fold reduction in tumor size compared to the anticancer drug alone. With doxorubicin, a decrease in tumor size of 1.4 times compared with Goboushi extract alone and 1.5 times (P <0.05) compared with anticancer agent alone was observed. Everolimus showed a 2.1-fold (P> 0.05) reduction in tumor size compared to Goboushi extract alone and a 1.4-fold reduction in tumor size compared to anticancer drug alone. Bortezomib showed a 1.8-fold (P <0.05) reduction in tumor size compared to goboushi extract alone and a 1.4-fold reduction in tumor size compared to anticancer drug alone. Hydrochychloroquine, a therapeutic agent for malaria and autoimmune diseases, showed a 1.9-fold reduction in tumor size compared to the Goboushi extract alone and a 2.5-fold reduction (P <0.05) compared to the anticancer drug alone.

From the above results, it was shown that the goboushi extract containing a high amount of arctigenin is a drug that can be expected to have a strong antitumor effect when combined with existing anticancer agents having various medicinal mechanisms.

The vascular wall strengthening agent, vascular network inhibitor and vascular normalizing agent of the present invention can be used for prevention, treatment and amelioration of various diseases caused by abnormal blood vessel formation.

Claims (4)

A vascular wall-strengthening agent that increases blood vessels covered by vascular parietal cells and contains arctigenin as an active ingredient (unless applied to cancer and retinal angiopathy ). The blood vessel wall strengthening agent according to claim 1, wherein the arktigenin is derived from burdock, burdock, burdock sprout, young burdock or forsythia. An inhibitor of vascular network formation containing arctigenin as an active ingredient (except when applied to cancer and retinal angiopathy ). The angiogenesis inhibitor according to claim 3, wherein the arktigenin is derived from burdock, burdock, burdock sprout, young burdock or forsythia.

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