JP2008128819A - Evaluation and screening method of substance having preventing or treating action with respect to pollinosis, medicine for preventing or treating pollinosis, and its manufacturing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an easy and efficient evaluation and screening method of a substance having preventing or treating action with respect to pollinosis using the new knowledge related to the sideration mechanism of pollinosis, a medicine for effectively preventing or treating pollinosis and an efficient manufacturing method of the medicine. <P>SOLUTION: This evaluation and screening method of the substance having preventing or treating action with respect to pollinosis includes at least a process for evaluating whether a substance to be inspected suppresses the activation of pollen or a natural immunological mechanism due to a natural immunologically active component. The manufacturing method of the medicine for preventing or treating pollinosis and the medicine containing a substance having action for suppressing the activation of the natural immunological mechanism as an effective component to prevent or treat pollinosis are also disclosed. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a method for evaluating and screening a substance having a preventive or therapeutic action against hay fever, and a method for preventing or treating hay fever, utilizing the inventors' new knowledge regarding the onset mechanism of hay fever. The present invention relates to a drug and a method for producing the same.

  The allergic symptom of hay fever caused by pollen such as cedar pollen is considered to be an immediate allergic reaction via IgE (antibody). That is, when the antigens, Cryj1 and Cryj2 proteins in cedar pollen enter the body, the information is transmitted from antigen-presenting cells such as macrophages to T cells. Furthermore, IgE that reacts with these pollen antigens is produced from B cells under the direction of T cells. The produced IgE binds to mast cells via a receptor. In such a state, when the antigens, Cryj1 and Cryj2 proteins in the cedar pollen enter the body again, they bind to IgE on mast cells, and as a result, mast cells are activated, histamine is released, sneezing and runny nose Cause various allergic reactions. It is thought that allergic symptoms of hay fever are caused through such an acquired immune mechanism.

Moreover, antiallergic drugs, antihistamines and the like have been widely used as hay fever drugs. However, anti-allergic drugs have the problem that it takes time until their effects appear, and anti-histamine drugs are effective in relieving allergic symptoms that have already occurred, such as sneezing, runny nose, nasal congestion, and itchy eyes. Although there is a nature, there is a problem that severe symptoms are not very effective and there are further side effects.
On the other hand, in recent years, extensive research has been conducted on the development of hay fever drugs that are free from side effects. For example, a hay fever therapeutic agent containing an IgE antibody production inhibitor containing sphingolipids as an active ingredient. Etc. have been proposed (see Patent Document 1).

  However, since the onset mechanism of hay fever has not yet been fully elucidated, and there are many patients suffering from symptoms, new knowledge on the onset mechanism of hay fever and new knowledge using the above knowledge are available. At present, development of drugs for hay fever is desired.

Japanese Patent Laid-Open No. 2004-43359

  An object of the present invention is to solve the conventional problems and achieve the following objects. That is, the present invention obtains new knowledge about the onset mechanism of hay fever, and uses the knowledge to easily and efficiently evaluate and screen a substance having a preventive or therapeutic action against hay fever. And it aims at providing the chemical | medical agent for the prevention or treatment of effective hay fever, and its efficient manufacturing method.

  In order to solve the above-mentioned problems, the present inventors have made extensive studies and as a result, obtained the following findings. That is, it is a finding that innate immunity activation is involved in the onset mechanism of hay fever.

  Conventionally, allergic symptoms of hay fever are the antigens in cedar pollen, Cryj1 and Cryj2 proteins bound to IgE (antibody) in blood, resulting in activation of mast cells and release of histamine from the mast cells. It was thought to cause various allergic reactions such as sneezing and runny nose. That is, the allergic symptom of hay fever was considered to be an immediate allergic reaction via IgE (allergic reaction via acquired immune mechanism).

On the other hand, the present inventors found that silkworm larvae died when the inner membrane fraction of cedar pollen was administered using silkworm larvae, and these deaths were caused by azathioprine, an immunosuppressive agent. It has been found that it is improved (Examples, described later). Since the silkworm is an organism having only an innate immune mechanism that does not have an acquired immune mechanism, the above results indicate that the onset mechanism of hay fever without the acquired immune mechanism, that is, the onset of hay fever via the innate immune mechanism. This suggests the existence of a mechanism.
This indicates that a new evaluation system and screening system using activation of the innate immune mechanism as an index is useful for developing a drug for hay fever. Moreover, it has shown that the substance which can suppress activation of an innate immune mechanism is useful as a chemical | medical agent for hay fever.

  The activation of the innate immune mechanism is involved in the onset mechanism of hay fever, and furthermore, the development of drugs for hay fever can be easily and efficiently performed using this newly discovered onset mechanism. What can be done has never been known before, and is a new finding of the present inventors.

The present invention is based on the above findings by the present inventors, and means for solving the above problems are as follows. That is,
<1> A method for evaluating whether a test substance has a preventive or therapeutic action against hay fever,
(A) a step of administering pollen or its innate immunity activation component and the test substance to an organism having an innate immunity mechanism;
(B) detecting the activation of the innate immune mechanism by the pollen or its innate immune activation component in the organism having the innate immune mechanism; and
(C) It is a method characterized by including the process of evaluating whether the said to-be-tested substance suppresses activation of the innate immune mechanism by the said pollen or its innate immunity activation component.
<2> A method for screening a substance having a preventive or therapeutic action against hay fever,
(A) a step of administering pollen or its innate immune activation component and a test substance to an organism having an innate immune mechanism;
(B) detecting the activation of the innate immune mechanism by the pollen or its innate immune activation component in the organism having the innate immune mechanism;
(C) a step of evaluating whether or not the test substance suppresses activation of the innate immune mechanism by the pollen or its innate immune activation component; and
(D) A method comprising a step of selecting a substance evaluated to suppress activation of the innate immune mechanism by the pollen or its innate immunity activation component.
<3> The method according to any one of <1> to <2>, wherein the organism having an innate immune mechanism is an organism having only an innate immune mechanism.
<4> The method according to <3>, wherein the organism having only the innate immune mechanism is an organism belonging to insects.
<5> The method according to <4>, wherein the organism belonging to insects is a silkworm.
<6> A method for producing a drug for preventing or treating hay fever,
(A) a step of administering pollen or its innate immune activation component and a test substance to an organism having an innate immune mechanism;
(B) detecting the activation of the innate immune mechanism by the pollen or its innate immune activation component in the organism having the innate immune mechanism;
(C) a step of evaluating whether the test substance suppresses activation of an innate immune mechanism by the pollen or an innate immune activation component thereof,
(D) selecting a substance evaluated to suppress the activation of the innate immune mechanism by the pollen or its innate immunity activation component;
(E) generating the substance selected in step (d), and
(F) A production method comprising the step of mixing the substance produced in the step (e) with a pharmaceutically acceptable carrier.
<7> The production method according to <6>, wherein the organism having the innate immune mechanism is an organism having only the innate immune mechanism.
<8> The production method according to <7>, wherein the organism having only the innate immune mechanism is an organism belonging to insects.
<9> The production method according to <8>, wherein the organism belonging to insects is a silkworm.
<10> A drug for preventing or treating hay fever, comprising a substance having an action of suppressing activation of an innate immune mechanism as an active ingredient.
<11> The drug according to <10>, wherein the substance having an action of suppressing the activation of the innate immune mechanism is a substance having an action of suppressing the activation of the innate immune mechanism by pollen or an innate immune activation component thereof It is.
<12> The drug according to any one of <10> to <11>, wherein the substance having an action of suppressing activation of an innate immune mechanism is azathioprine.

  According to the present invention, various problems in the past can be solved, and the new knowledge of the present inventors regarding the onset mechanism of hay fever can be used to easily and efficiently a substance having a preventive or therapeutic action against hay fever. It is possible to provide an effective evaluation method and screening method, and a drug for effective prevention or treatment of hay fever and an efficient production method thereof.

(Evaluation method, screening method)
The evaluation method of the present invention is a method for evaluating whether a test substance has a preventive or therapeutic action against hay fever, and includes the following steps (a) to (c), and further if necessary: Including other processes.
The screening method of the present invention is a method for screening a substance having a preventive or therapeutic action against pollinosis, and includes the following steps (a) to (d), and further includes other steps as necessary. .
Whether the evaluation method and the screening method (hereinafter sometimes simply referred to as the “method”) suppress the activation of the innate immune mechanism in the evaluation or screening of a substance having a preventive or therapeutic action against hay fever. It is characterized in that it is performed as an index.

<Process (a)>
In the said method, pollen or its innate immunity activation component, and a test substance are first administered to an organism having an innate immunity mechanism (step (a)).

-Organisms with innate immune mechanisms-
The “organism having an innate immune mechanism” is not particularly limited and can be appropriately selected from any multicellular organism according to the purpose. Among them, “an organism having only an innate immune mechanism” is used. It is preferable that the activation of the innate immune mechanism in the step (b) described later can be detected easily and efficiently.

The “innate immunity mechanism” means an immune biological defense mechanism (innate immunity mechanism) that does not depend on an acquired immunity (acquired immunity) mechanism. Vertebrate animals have an acquired immune mechanism that protects the living body using molecules that specifically recognize invaders such as antibodies against the invasion of pathogens, but invertebrates and plants have such an acquired immune mechanism. I don't have it. That is, the “organism having only the innate immunity mechanism” is, in other words, invertebrates and plants having no acquired immunity mechanism.
Therefore, the “organism having only the innate immunity mechanism” can be appropriately selected from invertebrates and plants according to the purpose, and among them, organisms belonging to insects are preferable. The “insects” refers to a class of arthropods from the Greater Submaxillary, which is composed of four subclasses of caterpillars, flying beetles, sterile insects, and moth insects. The “organism belonging to insects” is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably a larva from the viewpoint of convenience of handling. There is no restriction | limiting in particular as said larva, It can select suitably according to the objective, For example, the larva of the Lepidoptera (including moth and butterfly), Coleoptera (including beetle), etc. are mentioned. The larva is preferably a large larva from the viewpoint of ease of administration of the pollen or its innate immune activation component or the test substance. The “large larva” refers to a larva having a body length of 1 cm or more. Examples of the larva include silkworm (larvae) larvae.
The organism having only the innate immunity mechanism other than the organism belonging to the insects is not particularly limited and can be appropriately selected according to the purpose. For example, arthropods other than insects such as spiders and scorpions , Mollusks such as slugs and snails, annelids such as earthworms, terrestrial animals such as starfish and sea urchins, linear animals such as leopards and roundworms, coelenterates such as hydra, sea anemones and jellyfish, rice, radish, etc. And other plants.
In addition, mutants that are vertebrates but do not have acquired immune mechanisms (for example, immunodeficient (SCID) mice that do not have T cells or B cells) and the like are also examples of organisms having only the innate immune mechanism. .

  Further, not only organisms having only the innate immunity mechanism but also organisms having both the innate immunity mechanism and the acquired immunity mechanism can be used in the method. For example, a mammal has both an innate immune mechanism and an acquired immune mechanism, but the mammal's brain does not have an acquired immune mechanism and protects against infection only by the innate immune mechanism. Therefore, it is also preferable to use a mammalian brain for the above-mentioned method because the activation of the innate immune mechanism in the step (b) described later can be detected easily and efficiently.

-Pollen or its innate immune activation component-
The “pollen or its innate immunity activation component” is not particularly limited as long as it is the whole or a part of pollen that invades the body and causes allergic symptoms of hay fever, and is appropriately selected depending on the purpose. Examples thereof include an inner pollen fraction obtained by the preparation method described in Reference Examples described later.
The type of pollen is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include pollen such as Japanese cedar, Japanese cypress, Japanese apricot, Japanese maple, ragweed, and Japanese mugwort.

-Test substance-
The “test substance” is not particularly limited, and any substance for which it is desired to evaluate whether it has a preventive or therapeutic action against hay fever can be used, for example, cell extract, cell culture supernatant, fermentation Microbial product, marine organism extract, plant extract, purified protein, crude protein, peptide, non-peptidic compound, artificially synthesized compound, naturally derived compound, existing immunosuppressant, existing pollinosis For example.

-Administration-
The method for administering the pollen or its innate immunity activation component or the test substance to the organism having the innate immunity mechanism is not particularly limited and can be appropriately selected according to the purpose, for example, oral administration , Intraperitoneal administration, injection into blood, injection into the intestine, addition to feed (food), and the like.
Also, the dose of the pollen or its innate immunity activation component or the test substance to the organism having the innate immunity mechanism is not particularly limited and can be appropriately selected according to the purpose.

<Step (b)>
Next, in the method, activation of the innate immune mechanism by the pollen or its innate immune activation component in the organism having the innate immune mechanism is detected (step (b)).
The type of activation of the innate immune mechanism to be detected is not particularly limited and can be appropriately selected according to the type of the organism having the innate immune mechanism, for example, killing of the organism having the innate immune mechanism. Melanin pigment production, inflammatory reaction, body hair handstand, release of cytokines and reactive oxygen species (ROS) from cells responsible for innate immunity.
The method for detecting the activation of the innate immune mechanism is not particularly limited, and can be appropriately selected from, for example, known detection methods according to the type of activation of the innate immune mechanism to be detected.

<Step (c)>
Next, in the method, it is evaluated whether or not the test substance suppresses activation of the innate immune mechanism by the pollen or its innate immune activation component (step (c)).
The method for evaluating whether the test substance suppresses the activation of the innate immune mechanism is not particularly limited and can be appropriately selected depending on the purpose. For example, in parallel with the step (a) Preparing a control (control) in which only the pollen or its innate immunity activation component is administered without administering the test substance, and administering the test substance in the step (a) as compared with this control In this case, when the degree of activation of the innate immunity mechanism by the pollen or its innate immunity activation component in the organism having the innate immunity mechanism is reduced or prevented, the test substance prevents hay fever. Alternatively, it can be evaluated as having a therapeutic action.
As mentioned above, the said evaluation method can be performed by the said process (a)-process (c). By the evaluation method, it is possible to easily and efficiently evaluate whether or not the test substance has a preventive or therapeutic action for hay fever.

<Step (d)>
In the screening method, a substance evaluated to suppress the activation of the innate immune mechanism by the pollen or its innate immune activation component in the step (c) is further selected (step (d)).
Using various test substances, the evaluation by the steps (a) to (c) is performed, and then in this step (d), the pollen or its innate immunity activation component is selected from various test substances. By selecting a substance that suppresses the activation of the innate immune mechanism, a substance having a preventive or therapeutic action against hay fever can be screened easily and efficiently.

  The substance having a preventive or therapeutic action against hay fever evaluated or screened by the above method can be appropriately generated by a technique such as chemical synthesis or separation / purification. The use of the substance is not particularly limited and may be appropriately selected depending on the purpose. For example, it may be used as it is for the prevention or treatment of hay fever, or for the development of a drug for hay fever. It may be used for such experiments. Moreover, you may use as an active ingredient of the chemical | medical agent manufactured by the manufacturing method of this invention mentioned later, or the chemical | medical agent of this invention mentioned later.

(Production method)
The production method of the present invention is a method for producing a drug for preventing or treating hay fever, and includes the following steps (a) to (f), and further includes other steps as necessary.

<Step (a) to Step (d)>
Steps (a) to (d) in the production method are the same as steps (a) to (d) in the evaluation method of the present invention and the screening method of the present invention, respectively. By the steps (a) to (d), a substance having a preventive or therapeutic action against hay fever can be easily and efficiently selected.

<Process (e)>
In the manufacturing method, next, a substance having a preventive or therapeutic action against hay fever selected in the steps (a) to (d) is generated (step (e)).
There is no restriction | limiting in particular as said production | generation means, For example, according to the structure of the said substance, origin, etc., it can select suitably from well-known production | generation means, such as chemical synthesis and isolation | separation purification.

<Step (f)>
In the production method, the substance produced in the step (e) is then mixed with a pharmaceutically acceptable carrier (step (f)).
-Pharmaceutically acceptable carrier-
The pharmaceutically acceptable carrier is not particularly limited and may be appropriately selected depending on, for example, the desired dosage form of the drug to be produced. Moreover, there is no restriction | limiting in particular as said dosage form, For example, the dosage form enumerated by the item of the chemical | medical agent of this invention mentioned later etc. are mentioned.

-Mixed-
The method for mixing the substance produced in the step (e) and the pharmaceutically acceptable carrier is not particularly limited and can be appropriately selected depending on the purpose. An acceptable carrier may be added, the substance may be added to the pharmaceutically acceptable carrier, or both may be simultaneously added to the container and mixed. Moreover, as said mixing method, it can select suitably from the mixing method of each component in the manufacturing method of a well-known chemical | medical agent, for example.
Moreover, there is no restriction | limiting in particular as a usage-amount ratio of the said substance and the said pharmaceutically acceptable carrier at the time of the said mixing, According to the objective, it can select suitably.

<Other processes>
There is no restriction | limiting in particular as said other process, According to the objective, it can select suitably, For example, the shaping | molding process etc. which shape | mold the mixture obtained at the said process (f) are mentioned.

As mentioned above, the manufacturing method of this invention can be performed by the said process (a)-process (f), and, thereby, the chemical | medical agent for prevention or treatment of pollinosis can be manufactured efficiently.
There is no restriction | limiting in particular as a usage form of the chemical | medical agent for the prevention or treatment of hay fever obtained by the said manufacturing method, According to the objective, it can select suitably, For example, similarly to the chemical | medical agent of this invention mentioned later. Can be used.

(Drug)
The drug of the present invention is a drug for the prevention or treatment of hay fever and contains a substance having an action of suppressing activation of the innate immune mechanism as an active ingredient, and further contains other ingredients as necessary. Do it. The drug may be a drug manufactured by the above-described manufacturing method of the present invention.

-Substances that have the effect of suppressing the activation of the innate immune mechanism-
The substance having the action of suppressing the activation of the innate immune mechanism is not particularly limited as long as it is a substance that can suppress the activation of the innate immune mechanism in an organism having the innate immune mechanism. Examples thereof include azathioprine, FK506, immunosuppressive agents such as steroidal drugs, and the like. Moreover, as the substance having an action of suppressing the activation of the innate immune mechanism, a substance having an action of suppressing the activation of the innate immune mechanism by pollen or its innate immune activation component is preferable, and among them, azathioprine is particularly preferable. . The “innate immunity mechanism”, “activation of innate immunity mechanism”, “organism having innate immunity mechanism”, and “pollen or its innate immunity activation component” are the evaluation method of the present invention described above, the present invention. As described in the item of the screening method.

The substance having the action of suppressing the activation of the innate immune mechanism may be a substance having the action of suppressing the activation of only the innate immune mechanism, or the acquired immune mechanism together with the innate immune mechanism. The substance which has the effect | action which also suppresses activation of may be sufficient. The “acquired immune mechanism” is as described in the items of the evaluation method of the present invention and the screening method of the present invention.
In addition, the substance having the action of suppressing the activation of the innate immune mechanism is evaluated or screened as having a preventive or therapeutic action against hay fever by, for example, the evaluation method of the present invention and the screening method of the present invention described above. The material can also be used.

The content of the substance (active ingredient) having an action of suppressing the activation of the innate immune mechanism in the drug is not particularly limited and can be appropriately selected according to the purpose. The active ingredient itself may be sufficient.
Moreover, the substance (active ingredient) which has the effect | action which suppresses activation of the said innate immune mechanism may be used individually by 1 type, and may use 2 or more types together. There is no particular limitation on the content ratio of each active ingredient in the drug when two or more are used in combination, and the ratio can be appropriately selected according to the purpose.

-Other ingredients-
There is no restriction | limiting in particular as said other component, In the range which does not impair the effect of this invention, it can select suitably according to the objective, For example, a pharmacologically acceptable carrier etc. are mentioned. The carrier is not particularly limited and may be appropriately selected depending on, for example, the dosage form of the drug described later. Moreover, there is no restriction | limiting in particular also as content of the said other component in the said chemical | medical agent, According to the objective, it can select suitably.

-Dosage form-
The dosage form of the drug is not particularly limited, and can be appropriately selected according to a desired administration method as described later. For example, oral solids (tablets, coated tablets, granules, powders, capsules) Agents), oral liquids (internal solutions, syrups, elixirs, etc.), injections (solutions, suspensions, solid preparations for erection, etc.), ointments, patches, gels, creams, external powders, Examples include sprays and inhaled powders.

Examples of the oral solid preparation include, for example, excipients and further additives such as binders, disintegrants, lubricants, colorants, flavoring and flavoring agents as necessary, in addition to the active ingredients. Can be manufactured.
Examples of the excipient include lactose, sucrose, sodium chloride, glucose, starch, calcium carbonate, kaolin, microcrystalline cellulose, and silicic acid. Examples of the binder include water, ethanol, propanol, simple syrup, glucose solution, starch solution, gelatin solution, carboxymethylcellulose, hydroxypropylcellulose, hydroxypropyl starch, methylcellulose, ethylcellulose, shellac, calcium phosphate, polyvinylpyrrolidone and the like. It is done. Examples of the disintegrant include dry starch, sodium alginate, agar powder, sodium hydrogen carbonate, calcium carbonate, sodium lauryl sulfate, stearic acid monoglyceride, and lactose. Examples of the lubricant include purified talc, stearate, borax, and polyethylene glycol. Examples of the colorant include titanium oxide and iron oxide. Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like.

The oral solution can be produced by a conventional method, for example, by adding additives such as a flavoring / flavoring agent, a buffering agent and a stabilizer to the active ingredient.
Examples of the flavoring / flavoring agent include sucrose, orange peel, citric acid, tartaric acid and the like. Examples of the buffer include sodium citrate. Examples of the stabilizer include tragacanth, gum arabic, and gelatin.

As the injection, for example, a pH adjuster, a buffer, a stabilizer, an isotonic agent, a local anesthetic, etc. are added to the active ingredient, and subcutaneous, intramuscular and intravenous use are performed by a conventional method. Etc. can be manufactured.
Examples of the pH adjusting agent and the buffering agent include sodium citrate, sodium acetate, sodium phosphate and the like. Examples of the stabilizer include sodium pyrosulfite, EDTA, thioglycolic acid, thiolactic acid, and the like. Examples of the isotonic agent include sodium chloride and glucose. Examples of the local anesthetic include procaine hydrochloride and lidocaine hydrochloride.

The ointment can be produced, for example, by mixing a known base, stabilizer, wetting agent, preservative and the like with the active ingredient and mixing them by a conventional method.
Examples of the base include liquid paraffin, white petrolatum, white beeswax, octyldodecyl alcohol, and paraffin. Examples of the preservative include methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and the like.

  As the patch, for example, a cream, gel or paste as the ointment can be applied to a known support by a conventional method. Examples of the support include woven fabric, nonwoven fabric, soft vinyl chloride, polyethylene, polyurethane and other films made of cotton, suf, and chemical fibers, and foam sheets.

−Use−
The said medicine can be used by administering to a patient with hay fever, for example.
The animal to which the drug is administered is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include humans, mice, rats, cows, pigs, monkeys and the like.
The method for administering the drug is not particularly limited, and can be appropriately selected according to, for example, the dosage form of the drug. Oral administration, intraperitoneal administration, injection into the blood, Examples thereof include injection, addition to feed (food), administration into the nasal cavity by applying an ointment, and administration into the nasal cavity by spraying.
The dose of the drug is not particularly limited and can be appropriately selected according to the age, weight, desired degree of effect, etc. of the patient to be administered. The amount of active ingredient per administration is preferably 1 μg to 1 g, more preferably 1 μg to 1 mg.
Further, the administration timing of the drug is not particularly limited and can be appropriately selected according to the purpose. For example, it may be administered prophylactically before the onset of allergic symptoms of hay fever, It may be administered therapeutically after the onset of allergic symptoms.
In addition, the kind of pollen that causes hay fever to be prevented or treated by the drug is not particularly limited, and can be appropriately selected according to the purpose. For example, cedar, cypress, sparrow, and camouflage. , Pollen of ragweed, mugwort and the like.

[effect]
According to the evaluation method and screening method of the present invention, a substance having a preventive or therapeutic action against hay fever can be evaluated or screened easily and efficiently using whether or not the activation of the innate immune mechanism is suppressed as an index. can do. In addition, according to the production method of the present invention, it is possible to efficiently produce a drug for the prevention or treatment of hay fever, comprising as an active ingredient the substance having a preventive or therapeutic action against hay fever as evaluated or screened. Can do. Therefore, the evaluation method, screening method, and production method are very useful for the development of a new drug for hay fever.
In the evaluation method, screening method, and production method, preferably, by using an organism having only an innate immune mechanism such as a silkworm larva, it is possible to more easily and more efficiently prevent hay fever or A substance having a therapeutic action can be evaluated or screened, and a drug for preventing or treating hay fever can be produced more efficiently. An organism having only the innate immune mechanism is low in cost and easy to handle. Therefore, it is possible to reduce experimental costs and experimental space, that is, it is possible to develop a drug for hay fever more easily and at a lower cost.
In addition, since the drug of the present invention contains a substance having an action of suppressing the activation of the innate immune mechanism as an active ingredient, according to the drug of the present invention, an organism having an innate immune mechanism, that is, any multicellular organism , The activation of the innate immune mechanism by pollen or its innate immunity activation component can be suppressed, and the onset of hay fever can be suppressed. Therefore, the drug is very useful for preventing or treating hay fever.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

(Reference example: destruction of cedar pollen inner membrane by ultrasonic treatment and preparation of cedar pollen inner membrane fraction)
Conventionally, the phenomenon that pollen undergoes a morphological change under alkaline conditions such as runny nose is known in the art, and this phenomenon is caused by cracking of the outer membrane (cell wall) of pollen and covering the inner membrane (cell membrane). It is also called “destruction” or “rupture” of pollen because the protoplasm is released. It is known that pollen that has entered the eyes and nose and has been “destroyed” or “ruptured” releases the allergen antigens Cryj1, Cryj2, etc., and causes allergic symptoms of hay fever such as sneezing and runny nose.
In this reference example, it was confirmed that the cedar pollen was “broken” and “ruptured” under alkaline conditions (pH 7 to), and an inner membrane appeared, and an inner membrane fraction was prepared.

Distilled water (pH 6), LB10 (bacterial medium, pH approximately 7), and, 25 mM phosphate buffer Various _{(KH 2 PO 4 (pH4.7)} , K 2 HPO 4 (pH8.9), NaH 2 PO 4 ( Sugi pollen was suspended in pH 4.6) and Na ₂ HPO ₄ (pH 9.2) and allowed to stand at room temperature for 2 hours. Pollen suspended in acidic liquids, including distilled water, did not change shape, but when suspended in alkaline (pH 7 ~) liquid, it changed shape and the inner membrane structure appeared. It was confirmed (FIGS. 1A-B, before sonication, left panel). Next, pollen was collected by centrifugation at 3,000 rpm for 5 minutes, suspended in distilled water, then subjected to ultrasonic treatment (Branson ultrasonic treatment device), and observed with a phase contrast microscope. About pollen which caused the shape change and the intima structure appeared, the intima structure was destroyed by the ultrasonic treatment (FIGS. 1A and B, the right panel after the ultrasonic treatment).

  Each crushed material after sonication was collected by ultracentrifugation (45,000 rpm, 30 minutes, Beckman ultracentrifuge, rotor 50.2 Ti) and suspended in 0.9% NaCl to obtain a cedar pollen inner membrane fraction. . Each obtained cedar pollen inner membrane fraction was used in the following Examples 1-4.

(Example 1: Toxicity of cedar pollen inner membrane fraction to silkworm larvae)
50 μl of each cedar pollen inner membrane fraction prepared in the above Reference Example (approximately 250 μg of protein amount, equivalent to 25 mg of cedar pollen), silkworm larvae (5th day, 2nd day (no feeding after 4th-year sleep)), 1 g body weight ), The inner membrane fraction prepared under conditions where the inner membrane appears (alkaline (pH 7-)) is toxic to silkworm larvae, and almost all silkworm larvae die after 33 hours. did. On the other hand, silkworm larvae were resistant to the inner membrane fraction prepared under conditions (acidic) in which no inner membrane appeared, and most of them survived even 61 hours after injection. The photograph on the left side of FIG. 2 shows an untreated normal silkworm, and the photograph on the right side of FIG. 2 shows a silkworm larvae that died by injecting a cedar pollen endothelium fraction into a body fluid. Also, FIG. 3 shows the types of solutions in which cedar pollen is suspended, the presence or absence of intima appearance when left in each solution for 2 hours, and silkworms at 33 hours and 61 hours after administration of each intima fraction. Indicates the survival number (survival rate in parentheses).

Silkworms are organisms that have only an innate immune mechanism and no acquired immune mechanism. Therefore, from the result of this Example 1 that the silkworm larvae died by administration of the cedar pollen inner membrane fraction, it was shown that cedar pollen acts on the innate immune mechanism and causes symptoms of hay fever.
Conventionally, after pollen invades the body, only the mechanism that causes the symptoms of hay fever is known through the production of IgE, that is, through the acquired immune mechanism. Therefore, it has never been known that cedar pollen has a mechanism that causes symptoms of hay fever through only the innate immune mechanism, not through the acquired immune mechanism, and a new finding by the present inventors. It is.

(Example 2: Toxicity to beetle larvae of cedar pollen inner membrane fraction)
100 μl of the cedar pollen inner membrane fraction prepared in the above reference example (the inner membrane fraction prepared under conditions where the inner membrane appears) (about 500 μg as the amount of protein, equivalent to 50 mg of cedar pollen) were used as two beetle larvae (After about 5 months after hatching), the body was injected into the back blood of body weight 30 g). Two days later, one of the animals became blackened (FIG. 4, upper left). In the other head, the injection site was blackened (FIG. 4, upper right). None of the other two animals injected with saline showed a blackening reaction (FIG. 4, lower panel). This blackening is due to the production of melanin, and melanin production is well known as an innate immune response of insects. Therefore, the results of the experiment also showed that cedar pollen acts on the innate immune mechanism.

(Example 3: Toxicity of cedar pollen inner membrane fraction to mice)
-Intracerebral injection-
In the brain of an MCH (ICR) mouse (female, 8 weeks old), 50 μl of the cedar pollen inner membrane fraction prepared in the above Reference Example (the inner membrane fraction prepared under conditions where the inner membrane appears) was approximately 250 μg as the protein amount. Cedar pollen equivalent to 25 mg). The control group was injected with the same amount of physiological saline. No apparent abnormality was observed in any of the mice until at least 2 hours after the injection. After 14 hours, no abnormality was observed in the mice injected with physiological saline (FIG. 5, upper left), but mice injected with the cedar pollen inner membrane fraction died (FIG. 5, upper right).

  Mammals are organisms that have an acquired immune mechanism, but the brain has no acquired immune mechanism and protects against infection only by the innate immune mechanism. As a result of the experiment, when a mouse cedar pollen inner membrane fraction was injected into the mouse brain, the mouse died several hours later. The cedar pollen inner membrane fraction stimulates the mouse's innate immune system (microglial cells in the brain) by some mechanism, and the stimulation becomes hypersensitive, and as a result, it has a significant effect on nerve activity. Conceivable. Therefore, the experiment also showed that cedar pollen acts on the innate immune mechanism.

-Intravenous injection into immunodeficient mice-
Intravenous SCID mice were injected with 100 μl of cedar pollen inner membrane fraction (inner membrane fraction prepared under conditions where the inner membrane appears) prepared in the above Reference Example (approximately 500 μg of protein, equivalent to 50 mg of cedar pollen). The control group was injected with the same amount of physiological saline and observed after 9 hours. No particular abnormality was observed in the mice injected with physiological saline (FIG. 5, lower left side), but in the mice injected with the cedar pollen inner membrane fraction, the whole body hairs were observed to stand upside down (FIG. 5, Bottom right)

  SCID mice are immunodeficient mice that have no immune system cells that work in the acquired immune mechanism, such as T cells and B cells. When this mouse was intravenously injected with a cedar pollen endothelium fraction, the whole body hair was upside down, that is, a systemic inflammatory reaction was observed. Therefore, the experiment also showed that cedar pollen acts on the innate immune mechanism.

(Example 4: Effect of immunosuppressant azathioprine on toxicity of cedar pollen inner membrane fraction)
Using a mortar, an azathioprine bulk powder (manufactured by Tanabe Seiyaku Co., Ltd.) was mixed with artificial silkworm larvae (silk mate, manufactured by Nippon Nosan Co., Ltd.) at each concentration shown in FIG. 6 to prepare a bait. The prepared bait is given to silkworm larvae, and 50 μl of the cedar pollen inner membrane fraction prepared in the above reference example (the inner membrane fraction prepared under conditions where the inner membrane appears) (approximately 250 μg as the amount of protein, equivalent to 25 mg of cedar pollen) After injection into body fluids of silkworm larvae (5th day, 2nd day (4th year after no sleep), body weight 1 g), the number of survivors after 26 hours was examined. In the azathioprine non-administered group (0 mg), the total number died, but when 0.98 mg and 1.83 mg of azathioprine were added per 1 g of food, all silkworm larvae survived (FIG. 6).

  Silkworm larvae to which azathioprine is administered die from infectious bacteria such as Staphylococcus aureus and O157, which have a smaller number of bacteria than silkworm larvae to which azathioprine is not administered. That is, azathioprine exhibits an immunosuppressive effect against silkworm larvae. As a result of the experiment, it was found that killing of pollen against silkworm larvae can be treated by administration of a human immunosuppressant azathioprine. Since silkworm larvae have no acquired immune mechanism and only an innate immune mechanism, azathioprine is thought to act on the innate immune mechanism. Therefore, the experiment also showed that cedar pollen acts on the innate immune mechanism. Moreover, it was suggested that the immunosuppressive agent which can suppress activation of innate immune mechanisms, such as azathioprine, can become a chemical | medical agent for prevention or treatment of hay fever.

  As described above, the present inventors have found for the first time in the world that activation of the innate immune mechanism is involved in the pathogenesis of hay fever. This indicates that a new evaluation system and screening system using activation of the innate immune mechanism as an index is useful for developing a drug for hay fever. Moreover, it has been shown that a substance capable of suppressing the activation of the innate immune mechanism, which has never been considered before, is useful as a drug for hay fever.

  The evaluation method of the present invention, the screening method of the present invention, and the production method of the present invention are very useful for the development of a new drug for hay fever, and the drug of the present invention prevents or treats hay fever. Very useful to.

FIG. 1A is a diagram showing destruction of the cedar pollen inner membrane by ultrasonic treatment. FIG. 1B is a diagram showing destruction of the cedar pollen inner membrane by ultrasonic treatment. FIG. 2 is a graph showing the toxicity of the cedar pollen inner membrane fraction to silkworm larvae. FIG. 3 is a diagram showing the difference in toxicity to the silkworm larvae of the cedar pollen inner membrane fraction suspended in each solution. FIG. 4 is a diagram showing the influence of a cedar pollen inner membrane fraction on beetle larvae. FIG. 5 is a graph showing the toxicity of the cedar pollen inner membrane fraction to mice. FIG. 6 is a graph showing the effect of azathioprine administration on silkworm larvae administered with the cedar pollen inner membrane fraction.

Claims (8)

A method for evaluating whether a test substance has a preventive or therapeutic action against hay fever,
(A) a step of administering pollen or its innate immunity activation component and the test substance to an organism having an innate immunity mechanism;
(B) detecting the activation of the innate immune mechanism by the pollen or its innate immune activation component in the organism having the innate immune mechanism; and
(C) A method comprising evaluating whether or not the test substance suppresses activation of an innate immune mechanism by the pollen or its innate immunity activation component. A method for screening a substance having a preventive or therapeutic action against hay fever,
(A) a step of administering pollen or its innate immune activation component and a test substance to an organism having an innate immune mechanism;
(B) detecting the activation of the innate immune mechanism by the pollen or its innate immune activation component in the organism having the innate immune mechanism;
(C) a step of evaluating whether or not the test substance suppresses activation of the innate immune mechanism by the pollen or its innate immune activation component; and
(D) A method comprising a step of selecting a substance evaluated to suppress activation of an innate immune mechanism by the pollen or its innate immunity activation component.   The method according to claim 1, wherein the organism having an innate immune mechanism is an organism having only an innate immune mechanism.   The method according to claim 3, wherein the organism having only the innate immune mechanism is an organism belonging to insects.   The method according to claim 4, wherein the organism belonging to insects is a silkworm. A method for producing a drug for the prevention or treatment of hay fever,
(A) a step of administering pollen or its innate immune activation component and a test substance to an organism having an innate immune mechanism;
(B) detecting the activation of the innate immune mechanism by the pollen or its innate immune activation component in the organism having the innate immune mechanism;
(C) a step of evaluating whether the test substance suppresses activation of an innate immune mechanism by the pollen or an innate immune activation component thereof,
(D) selecting a substance evaluated to suppress the activation of the innate immune mechanism by the pollen or its innate immunity activation component;
(E) generating the substance selected in step (d), and
(F) A production method comprising the step of mixing the substance produced in the step (e) with a pharmaceutically acceptable carrier.   A drug for preventing or treating hay fever, comprising a substance having an action of suppressing activation of an innate immune mechanism as an active ingredient. The drug according to claim 7, wherein the substance having an action of suppressing activation of the innate immune mechanism is azathioprine.