JP4785110B2 - How to evaluate respiratory sensitization - Google Patents

Description

  The present invention relates to a respiratory sensitization evaluation method using a small animal to evaluate the possibility that a chemical substance or natural product reaches the trachea or lung and causes respiratory allergy, that is, respiratory sensitization induction. This method is used in fields such as searching for causative substances of respiratory allergy and qualitative or quantitative evaluation of allergenicity using known causative substances.

  In recent years, allergic diseases caused by natural products and chemical substances are increasing. However, no method has been reported so far for simply and accurately evaluating the possibility of the onset (trigger) of sensitization allergy in the respiratory tract when a substance enters the body and reaches the trachea and lungs.

  As a method for evaluating allergenicity of natural products and chemical substances, particularly low molecular weight chemical substances, a technique using guinea pigs has been widely used. For example, an inhalation chamber or a gas generator is used for guinea pigs. Inhalation exposure that has been performed and evaluated using a respiratory volume measuring device has been performed. However, since guinea pigs are medium-sized among rodents used as experimental animals, the costs of rearing equipment, feed, etc. are high, and genetic analysis is not progressing compared to mice. In addition, large-scale devices are required for inhalation exposure. Furthermore, a test system using smaller animals is required from the viewpoint of animal welfare.

  On the other hand, as a simpler evaluation method for respiratory sensitization, a method using a mouse that is smaller and has a genetic analysis advanced compared to guinea pigs has been studied. As the most commonly performed method using a mouse, for example, a substance to be evaluated is applied to the skin at the stage of causing sensitization (allergy), or the substance is administered subcutaneously or intraperitoneally, At the induction stage, the substance is inhaled nasally in a liquid state, inhaled in the form of a mist using a mist generating device, etc., or inoculated into the oral cavity and naturally inhaled. (For example, Toxicology 194 (2003) 147-161, Toxicology (2003), 184 (1), 51-68, Food and Chemical Toxicology (1999), 37 (8), 889-896, Inhalation Toxicology (1998), 10 (See (2), 131-154).

  In any of the methods described in the above documents, methods such as transdermal exposure and subcutaneous injection are used at the sensitization stage. However, these have a problem that they become a test system that does not accurately reflect physical properties related to the permeability (absorbability) of the administered substance into the body. For example, substances that are originally absorbed by the trachea and lungs may not be absorbed into the body by the skin barrier when exposed to the skin, and conversely, substances that are not originally absorbed by the trachea and lungs may be taken into the body by subcutaneous injection. . As a typical example, pollen and ovalbumin (OVA), which are protein antigens, are extremely difficult to be sensitized by dermal exposure, but are easily established by subcutaneous injection or nasal exposure described later.

  In addition, it is unlikely that the immune system in the skin and the immune system in the bronchi and lung show the same immune response to various substances. In general, skin allergies are Th1 type IV allergies, respiratory allergies are Th2 type I allergies and are classified differently from each other, and pollen that is a typical factor (allergen) of respiratory allergies Polymers are known not to cause sensitization even when exposed to the skin, and using techniques such as dermal exposure and subcutaneous injection is particularly problematic from the standpoint of judging the actual risk. is there.

  Moreover, since all of the above methods use only substances known to have respiratory sensitization, whether or not to cause sensitization to the respiratory tract when a certain chemical substance is inhaled into the trachea is determined. There is no guarantee that you can examine or accurately assess the type of allergy you develop.

  On the other hand, it is conceivable to use methods such as nasal exposure or inhalation exposure using a gas or mist generator at the sensitization stage. However, in nasal exposure, it is unavoidable that a part flows into the esophagus, and when a mist generator is used, dermal exposure occurs at the same time, both of which are lacking in quantitativeness. Furthermore, in any of the methods, since the respiration rate is not constant due to characteristics of chemical substances, such as irritation, the exposure amount cannot be accurately grasped, and it is difficult to evaluate quantitatively. In addition, there is a problem that it is difficult to evaluate a substance that is difficult to be made gaseous because it is difficult to dissolve in water or a solvent. In addition, as a quantitative evaluation method for sensitization, for example, Local Lymph Node Assay is known, which is a method for quantitatively evaluating sensitization in the skin by the difference in compound concentration, Although effective for quantitative evaluation of dermal exposure, nasal exposure and inhalation exposure were not expected to achieve the same level of quantification as dermal exposure.

Toxicology 194 (2003) 147-161 Toxicology (2003), 184 (1), 51-68 Food and Chemical Toxicology (1999), 37 (8), 889-896 Inhalation Toxicology (1998), 10 (2), 131-154

An object of the present invention relates to a sensitization evaluation method for simply and quantitatively evaluating respiratory sensitization when a chemical substance or natural product directly reaches the trachea or lung using a small animal.
The other object of this invention is related with the sensitization evaluation method which can evaluate the sensitization when a sample contacts a trachea or a lung directly.
Still another object of the present invention relates to a sensitization evaluation method capable of evaluating the sensitization when a sample comes into direct contact with the trachea or lung in the sensitization stage and / or the induction stage.

  As a result of intensive investigations to achieve the above-mentioned object, the present inventors have accurately administered the sensitization property of the sample to the trachea and lungs by directly administering the sample into the trachea in an amount that does not interfere with the respiration of small animals. And it discovered that it could evaluate quantitatively and completed this invention.

That is, the present invention relates to respiratory sensitization performed by administering a sample in two steps: a sensitization stage by the first sample administration and an induction stage by the sample administration after a lapse of a predetermined period from the first sample administration. This is a sex evaluation method, in which a biological reaction in the trachea or lung is evaluated by directly administering a sample of 100 μl or less per time into the trachea of a small animal having a body weight of 70 g or less. samples, allergic type samples for evaluation, and, at least 1 Tsudea selected from the group consisting of a pulmonary inhalation test sample is, sensitization to a sample of respiratory and / or allergic to develop by the sample on respiratory sensitization evaluation method which is characterized that you evaluate the type. For example, a mouse is used as the small animal.

In the respiratory sensitization evaluation method of the present invention, for example, a sample may be administered directly into the trachea at the sensitization stage, and any of the induction stage, the sensitization stage, and the induction stage may be used. Also good. Further, the respiratory sensitization evaluation method of the present invention is opened using an opening holding means capable of holding the open state of a small animal, a sample administration means is inserted from the mouth of the small animal, and the air is passed over the pharynx. You may carry out by the method of administering a sample directly in a pipe | tube.
In the present specification, in addition to the above invention, and characterized in that into the trachea of weight 70g following small animals, to assess the biological response in the trachea or lungs by administering the following samples 1 Kaito or 100μl directly A method for evaluating respiratory sensitization is also described.

  According to the method of the present invention, since a predetermined amount of sample is directly administered to the bronchial entrance into the trachea of a small experimental animal, the sample does not come into contact with the skin or the like, and the sensitization to the trachea or lung is quantitatively determined. And it can be evaluated easily. In addition, when the sample is directly administered into the trachea at the stage of sensitization and / or induction, a more accurate sensitization evaluation can be obtained.

  The respiratory sensitization evaluation method of the present invention is characterized in that a biological reaction in the trachea or lung is evaluated by directly administering a sample of 100 μl or less into the trachea of a small animal having a weight of 70 g or less.

  The small animal is not particularly limited as long as it is an animal having a weight of 70 g or less (for example, about 15 to 70 g), and examples thereof include small experimental animals such as mice, hamsters, and gerbils.

  The sample to be administered may be a liquid or a solid such as a powder. For example, an allergenic evaluation sample such as ovalbumin, an allergy type evaluation sample, a foreign substance lung such as copy toner or dust, etc. Various substances for the purpose of evaluating respiratory sensitization such as inhalation test samples can be used.

  As a preferred embodiment of the method for evaluating respiratory sensitization of the present invention, an opening holding means capable of holding an open state of a small animal is used to open, and a sample administration means is inserted from the mouth of the small animal so as to cross the pharynx. And a method of administering a sample directly into the trachea. According to this method, the sample can be reliably administered directly into the trachea.

  The device used for sample administration is not particularly limited as long as it is a device that can be directly and reliably administered into the trachea of a small animal. Preferably, a device provided with the opening holding means is used. As such a device, for example, a known device such as the device described in Japanese Patent Application Laid-Open No. 2001-276231 can be used, and preferably, a small animal endotracheal sample administration assist device shown in FIG. 1 is used. Used.

  FIG. 1 is a front view showing an example of the respiratory sensitization evaluation method of the present invention. In FIG. 1, a small animal 8 is grasped with a hand 10 through a holder 9, and a state in which the insertion portion 1 constituting a small animal 8 endotracheal sample administration assist device is inserted into the oral cavity of the small animal 8 is held. There is a method in which a sample is directly introduced into the trachea by inserting into the oral cavity of the small animal 7 through the trunk portion 5 from the side of the magnification observation means 4 constituting the intra-tracheal sample administration assisting device using the input means 7. It is shown.

  As shown in FIG. 1, the small animal endotracheal sample administration assist device includes an opening body 1 having a truncated cone shape, a body portion 5, an enlarged observation means 4, a tube pipe 2 a and a pump 2 b. A saliva removing means 2 configured, and a light source 3 coupled to a battery housing part / grip 6 are provided. As shown in FIG. 2, the magnification observation means 4 fixes the joint portion 4 a included in the magnification observation means 4 to a joint portion 5 a provided at one end of the trunk portion 5, so that the magnification observation means 4 It is installed so as to be able to maintain a partially opened state without completely covering one end of the body part 5. The saliva removing means 2 is inserted from the open part of the body 5 on the side where the magnification observation means 4 is provided, and is fixed along the light source 3 disposed near the center in the axial direction of the body 5. As the sample administration means 7, for example, a syringe, a dropper or the like can be used.

  When using the intra-tracheal sample administration aid shown in FIG. 1, a predetermined amount of sample can be directly administered to the bronchial entrance from the narrow oral cavity of a small experimental animal. In the endotracheal sample administration aid for small animals, the mouthpiece 1 has a truncated cone shape, so that it is difficult to damage the oral cavity and the burden on the experimental animal can be reduced, and saliva in the oral cavity is removed by the saliva removing means 2. Therefore, it is preferable from the viewpoints that sample introduction becomes easy and that a narrow oral cavity is illuminated by a light source, so that administration to the bronchial entrance can be ensured.

  In the present invention, the sample dose per time is 100 μl or less. If the dose exceeds 100 μl, the sample volume is too large for the small animal lung volume (usually 1 ml or less), which hinders breathing. The sample dose per time is preferably about 80 μl, more preferably about 50 μl, and particularly preferably about 10 μl.

  Evaluation of sensitization is performed by administering a sample in two steps, a sensitization stage and an induction stage. That is, sensitization is evaluated through a sensitization stage by the first sample administration and an induction stage after administration for a predetermined period. In the present invention, the sample may be administered directly into the trachea in at least one of the sensitization stage and the induction stage, and either the sensitization stage or the induction stage may be administered by other methods. Good. As another method for administering the sample, for example, a conventional method for causing sensitization such as transdermal exposure, subcutaneous injection, nasal exposure, and suction exposure of mist can be employed. As an aspect of sample administration for evaluating sensitization, for example, a method in which a sample is directly administered into the trachea as an induction stage (intratracheal administration), and then performed by a method other than intratracheal administration as the induction stage, Examples include a method other than intratracheal administration as the sensitization stage, and a method in which the sample is directly administered into the trachea as an induction stage, and a method in which the sample is directly administered into the trachea at both the sensitization stage and the induction stage. . Among them, a method in which a sample is directly administered into the trachea at both the sensitization stage and the induction stage is preferable.

  The sample administration frequency and administration period in each stage of sensitization and induction are not particularly limited as long as the establishment of sensitization in the lung is confirmed, but from the first administration of the sample (first sample administration at the sensitization stage) It is preferable to shorten the period until the final administration (the last sample administration at the stage of induction), for example, it is preferable to perform the period within one month.

  According to the respiratory sensitization evaluation method of the present invention, since the sample directly reaches the trachea and lungs, the sensitization of the sample to the respiratory tract can be more accurately evaluated. The present invention can also be used as a method for evaluating the presence or absence of allergenicity to the respiratory tract by administering as a sample a substance that is unknown whether or not allergic to the respiratory tract.

  For confirmation of the establishment of sensitization, known methods such as analysis of lung lavage fluid, infiltration of immunocompetent cells into lung tissue, and comparison of cytokine production patterns can be used.

  Hereinafter, the present invention will be described in more detail based on examples, but the present invention is not limited to these examples. In addition, mouse BALB / c, female, 6 weeks old was used as a small experimental animal, and ovalbumin (OVA) was used as a sample. Confirmation of the establishment of sensitization was performed by observing the presence or absence of infiltration of immunocompetent cells (lymphocytes and eosinophils) around the bronchi and the layer formation of goblet cells of bronchial epithelial cells.

Reference example 1
10 μg / 100 μl of OVA was administered intraperitoneally twice weekly for 2 weeks (sensitization) using a syringe, and 50 μg / 50 μl of OVA was nasally exposed for 3 consecutive days in the fourth week for induction.
When tissue sections around the bronchi were prepared and observed, as shown in FIGS. 3 and 4, infiltration of lymphocytes and eosinophils 14 around the blood vessels 13, bronchi 11, and goblet cells 12 of bronchial epithelial cells. This layer was observed and sensitization was confirmed.

Example 1
Intratracheal administration (sensitization) of 10 μg / 3 μl of OVA 3 times a week for 2 weeks in the manner shown in FIG. 1, followed by nasal exposure to 50 μg / 50 μl of OVA for 3 consecutive days in the fourth week went.
When tissue sections around the bronchi were prepared and observed, as shown in FIGS. 5 and 6, infiltration of lymphocytes and eosinophils 14 around the blood vessels 13, bronchi 11, and goblet cells 12 of bronchial epithelial cells. This layer was observed, confirming the establishment of sensitization.

Example 2
Intratracheal administration (sensitization) of 10 μg / 3 μl of OVA 3 times a week for 2 weeks in the manner shown in FIG. 1, and 50 μg / 3 μl of OVA in the same manner in the fourth week for 3 consecutive days And then evoked.
When tissue sections around the bronchi were prepared and observed, as shown in FIGS. 7 and 8, the infiltration of lymphocytes and eosinophils 14 around the bronchi 11 and the layer of goblet cells 12 of bronchial epithelial cells were found. Observed and confirmed the formation of sensitization.

Example 3
Intratracheal administration (sensitization) of 10 μg / 3 μl of OVA three times a week for 2 weeks in the manner shown in FIG. 1, and 50 μl of PBS (phosphate buffered saline) for 3 consecutive days in the fourth week Induction was performed by nasal exposure.
When tissue sections around the bronchi were prepared and observed, as shown in FIGS. 9 and 10, there was no infiltration of lymphocytes and eosinophils around the blood vessels 13 and bronchi 11, and there was no goblet cell of bronchial epithelial cells. No layer was observed, and no sensitization was observed.

Example 4
Intratracheal administration (sensitization) of 3 μl of PBS three times a week for 2 weeks in the manner shown in FIG. 1, and 50 μg / 3 μl of OVA were administered intratracheally in the same manner in the fourth week for 3 consecutive days. Raised.
When tissue sections around the bronchi were prepared and observed, as shown in FIGS. 11 and 12, there was no infiltration of lymphocytes and eosinophils around the blood vessels 13 and bronchi 11, and there was no goblet cell of bronchial epithelial cells. No layer was observed, and no sensitization was observed.

Example 5
Intratracheal administration (sensitization) of 3 μl of PBS 3 times a week for 2 weeks in the manner shown in FIG. 1, and 3 μl of PBS is administered intratracheally in the same manner for 3 days in the 4th week. went.
When tissue sections around the bronchi were prepared and observed, as shown in FIGS. 13 and 14, there was no infiltration of lymphocytes and eosinophils around the bronchi 11, and a layer of goblet cells of bronchial epithelial cells was also observed. No sensitization was found.

It is a schematic diagram which shows an example of the respiratory sensitization evaluation method of this invention. It is the elements on larger scale of an example of the respiratory sensitization evaluation method of this invention of FIG. 2 is a photograph of a tissue section around a bronchus in Reference Example 1. FIG. 4 is a schematic diagram of FIG. 3. 2 is a photograph of a tissue section around a bronchus in Example 1. It is a schematic diagram of FIG. 4 is a photograph of a tissue section around a bronchus in Example 2. It is a schematic diagram of FIG. 4 is a photograph of a tissue section around a bronchus in Example 3. FIG. 10 is a schematic diagram of FIG. 9. 6 is a photograph of a tissue section around a bronchus in Example 4. It is a schematic diagram of FIG. 6 is a photograph of a tissue section around a bronchus in Example 5. FIG. 14 is a schematic diagram of FIG. 13.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Insertion part 2 Saliva removal means 2a Tube pipe 2b Pump 3 Light source 4 Magnification observation means 4a Joint part 5 Torso part 5a Joint part 6 Battery accommodating part and grip 7 Sample administration means 8 Small animal 9 Holder 10 Hand 11 Bronchus 12 Bronchial epithelial cell Goblet cells 13 Blood vessels 14 Lymphocytes and eosinophils

Claims (6)

A method for evaluating respiratory sensitization performed by administering a sample in two steps, a stage of sensitization by the first sample administration and a stage of induction by sample administration after a lapse of a predetermined period from the first sample administration. ,
The biological reaction in the trachea or lung is evaluated by directly administering a sample of 100 μl or less per time into the trachea of a small animal weighing 70 g or less,
Sample, allergic attractable evaluation samples, allergic type samples for evaluation, and, Ri least 1 Tsudea selected from the group consisting of a pulmonary inhalation test sample,
Sensitization to the sample respiratory and / or respiratory sensitization evaluation method characterized that you evaluate the type of allergy to develop the sample.   The respiratory sensitization evaluation method according to claim 1, wherein the sample is directly administered into the trachea at the stage of sensitization.   The method for evaluating respiratory sensitization according to claim 1, wherein the sample is administered directly into the trachea at the stage of induction.   The respiratory sensitization evaluation method according to claim 1, wherein the sample is administered directly into the trachea in both the sensitization stage and the induction stage.   The respiratory sensitization evaluation method according to claim 1, wherein the small animal is a mouse.   2. The respiratory tract according to claim 1, wherein the respirator is opened using an opening holding means capable of holding the open state of the small animal, the sample administration means is inserted from the mouth of the small animal, and the sample is directly administered into the trachea beyond the pharynx. Sensitization evaluation method.