Production method of alimentary canal bleeding model animal, alimentary canal bleeding model animal and hemostatic agent screening method

Abstract

To provide a production method of an alimentary canal bleeding model animal which can reproduce alimentary canal bleeding caused by damage of an alimentary canal mucosa.SOLUTION: A production method of an alimentary canal bleeding model animal comprises an administration step for administering an anti-thrombotic drug to a non-human animal which has damage on an alimentary canal mucosa.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to a method for producing a gastrointestinal bleeding model animal, a gastrointestinal bleeding model animal, and a method for screening a hemostatic agent. [Background technology]

[0002] With the advent of an aging society, the number of people suffering from gastrointestinal cancer is increasing. Endoscopic submucosal dissection (ESD) for early gastrointestinal cancer is widely used because it is minimally invasive and has a high degree of curability. However, ESD has a postoperative complication of "delayed bleeding" that occurs within 28 days after surgery. Risk factors for delayed bleeding after ESD are taking antithrombotic drugs, maintenance dialysis, and lesion factors (number, size, site) (see Non-Patent Document 1). In particular, with the increase in the elderly taking antithrombotic drugs, the problem of delayed bleeding has become prominent. Delayed bleeding may sometimes require blood transfusion, transcatheter embolization surgery, or emergency surgery. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Waku Hatta, 37 others, “Prediction model of bleeding after endoscopic submucosal dissection for early gastric cancer:BEST-J score”, Gut, 2021, 70, 476-484 Summary of the Invention [Problem to be solved by the invention]

[0004] There is a need to develop a drug or device to suppress delayed bleeding. However, to clarify the usefulness of such a drug or device, animal experiments using model animals are necessary. There have been no reports of the creation of a model animal exhibiting delayed bleeding.

[0005] The present invention has been made in view of the above circumstances, and aims to provide a method for producing a model animal for gastrointestinal bleeding capable of reproducing gastrointestinal bleeding caused by injury to the gastrointestinal mucosa, and a model animal for gastrointestinal bleeding. Another aim of the present invention is to provide a method for screening a hemostatic agent effective against gastrointestinal bleeding caused by injury to the gastrointestinal mucosa. [Means for solving the problem]

[0006] (Production method of gastrointestinal bleeding model animal) The method for producing a gastrointestinal bleeding model animal described in the present specification includes: The method includes a step of administering an antithrombotic agent to a non-human animal having an injury to the gastrointestinal mucosa.

[0007] In the administration step, After the administration of the antithrombotic drug causes the activated clotting time of the non-human animal to exceed a reference value, the antithrombotic drug may be administered continuously.

[0008] The non-human animal is The pigs may be Crown miniature pigs aged 5 to 7 months.

[0009] The reference value is It may be 200 seconds or more.

[0010] The gastrointestinal mucosa is It may be at least one of the mucosa of the lesser curvature, anterior wall and posterior wall of each of the upper body, middle body, lower body and gastric angle of the stomach.

[0011] The injury may be It may be a mucosal defect created by an endoscopic mucosal resection method.

[0012] (Animal model of gastrointestinal bleeding) The gastrointestinal bleeding model animal described herein is A non-human animal in which 24 hours or more have passed since injury to the gastrointestinal mucosa, and which has overt gastrointestinal bleeding or blood clots or blood pools in the stomach.

[0013] (Method for screening hemostatic agents) The method for screening a hemostatic agent described herein comprises: Administering or applying a test substance to the gastrointestinal bleeding model animal produced by the method for producing the gastrointestinal bleeding model animal described in the present specification or to the gastrointestinal bleeding model animal described in the present specification; evaluating the hemostatic effect of the test substance in the gastrointestinal bleeding model animal; Includes. Effect of the Invention

[0014] According to the present invention, it is possible to reproduce gastrointestinal bleeding caused by damage to the gastrointestinal mucosa. Also, according to the present invention, a hemostatic agent effective for gastrointestinal bleeding caused by damage to the gastrointestinal mucosa can be obtained. [Brief description of the drawings]

[0015] [Figure 1] FIG. 1 shows activated clotting time (ACT) and intragastric images in the heparin administration group. [Diagram 2] FIG. 13 shows ACT and intragastric images in a non-heparin administration group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] The embodiments of the present invention will be described with reference to the drawings. Note that the present invention is not limited to the following embodiments and drawings. Note that in the following embodiments, the expressions "have", "include" and "contain" also include the meaning of "consisting of" or "consisting of".

[0017] [Embodiment 1: Method for producing a gastrointestinal bleeding model animal] The method for producing a gastrointestinal bleeding model animal according to the present embodiment includes administering an antithrombotic agent to a non-human animal having an injury to the gastrointestinal mucosa. The gastrointestinal tract is particularly the esophagus, stomach, small intestine (duodenum, jejunum, ileum), and large intestine (cecum, colon, rectum). Preferably, the gastrointestinal mucosa is at least one mucosa of the lesser curvature, anterior wall, and posterior wall of each of the upper gastric body, the middle gastric body, the lower gastric body, and the gastric angle, more preferably a plurality of these mucosa, and even more preferably all of these mucosa. The injury is not particularly limited, but a mucosal defect created by endoscopic mucosal resection (EMRL) is preferred. When the injury is caused by EMRL, the gastrointestinal bleeding model animal is suitable as a model of delayed bleeding after ESD.

[0018] The injury also includes ulcers. The injury may be drug-induced injury. Examples of drugs that cause injury to the gastrointestinal mucosa include nonsteroidal anti-inflammatory drugs, antithrombotic drugs, adrenal cortical hormones, bisphosphonates, antibacterial drugs, anticancer drugs, potassium preparations, and the like. When injury is caused to the gastrointestinal mucosa by a drug, a dose of the drug that causes injury to the gastrointestinal mucosa, determined by a preliminary experiment, may be administered to a non-human animal. The injury may be caused to the gastrointestinal mucosa by, for example, microorganisms such as Helicobacter pylori, gastric acid, stress, and the like.

[0019] The non-human animal is not particularly limited as long as it is an animal used in experiments other than humans, and examples thereof include rats and pigs. Examples of pigs include Yorkshire and minipigs. Minipigs are preferred because they mature quickly and weigh about 20 kg even when mature. Among minipigs, Crown-type minipigs aged 5 to 7 months, preferably 6 months, are suitable.

[0020] The antithrombotic agent may be an antiplatelet agent or an anticoagulant.Specific examples of the antithrombotic agent include aspirin, cyclodextrin hydrochloride, clopidogrel, prasugrel, dipyridamole, cilostazol, beraprost sodium, limaprost, eicosapentaenoic acid, sarpogrelate hydrochloride, trapidil, ozagrel sodium, warfarin, heparin, dalteparin, argatroban hydrate, fondaparinux, edoxaban, rivaroxaban, apixaban, dabigatran, and salts thereof.The antithrombotic agent is preferably heparin, and more preferably unfractionated heparin.

[0021] The mode of administration of the antithrombotic drug in the administration step is not particularly limited, and any administration route can be adopted. Preferably, the antithrombotic drug is administered via a catheter or the like inserted into a blood vessel, for example, a vein, of a non-human animal. The timing of administration of the antithrombotic drug in the administration step is not particularly limited, but is preferably within 10 minutes after the formation of a lesion in the gastrointestinal mucosa, for example, within 1 minute, within 3 minutes, within 5 minutes, within 7 minutes, or within 9 minutes after the formation of the lesion.

[0022] In the administration step, the antithrombotic drug may be administered continuously after the ACT of the non-human animal exceeds a reference value by administration of the antithrombotic drug. The ACT can be measured by a known method from blood collected from the non-human animal to which the antithrombotic drug has been administered. For example, the ACT is measured by measuring the change in viscosity accompanying blood coagulation by measuring the change in physical resistance. More specifically, in the measurement of the ACT, the formation of a clot is monitored for a sample in which whole blood is mixed with an activator. When the formation of a clot begins, the flow of blood is impeded and the movement of the blood slows down. Thus, for example, the speed at which the sample moves between the optical sensors is measured, and when the speed falls below a preset speed, it can be determined that a clot has been formed.

[0023] In the administration step, it is preferable to administer the antithrombotic drug while monitoring the ACT by measuring the ACT periodically, for example, every 1 to 30 minutes, every 5 to 20 minutes, or every 8 to 15 minutes. The antithrombotic drug may be administered multiple times until the ACT exceeds the reference value. The dosage of the antithrombotic drug per administration until the ACT exceeds the reference value is, for example, 30 to 70 U / kg, 40 to 60 U / kg, or 45 to 55 U / kg in the case of unfractionated heparin.

[0024] The reference value is set appropriately depending on the type, weight, etc. of the non-human animal used, but is a value higher than the ACT in a state in which an antithrombotic drug is not administered. For example, the reference value is 200 seconds or more, preferably 200 to 250 seconds, more preferably 210 to 240 seconds, and further preferably 210 to 230 seconds.

[0025] In the case of continuous administration of an antithrombotic drug, for example, a pressurized drug injector is used to administer a fixed amount of the antithrombotic drug to a non-human animal over a fixed period of time. The time period for continuous administration of the antithrombotic drug is, for example, 10 to 40 hours, 15 to 35 hours, 20 to 30 hours, or 22 to 26 hours. The dosage of the antithrombotic drug in the continuous administration of the antithrombotic drug is, for example, 30 to 70 U / kg / hour, 40 to 60 U / kg / hour, or 45 to 55 U / kg / hour in the case of unfractionated heparin.

[0026] The gastrointestinal bleeding model animal obtained by the production method according to the present embodiment is a non-human animal that has been in the gastrointestinal mucosa for more than 24 hours and has overt gastrointestinal bleeding or blood clots or blood pools in the stomach. This allows the reproduction of gastrointestinal bleeding caused by gastrointestinal mucosa injury. Therefore, it is useful as an animal experiment for developing a formulation and device for suppressing gastrointestinal bleeding caused by gastrointestinal mucosa injury.

[0027] [Embodiment 2: Screening method for hemostatic agents] The gastrointestinal bleeding model animal according to the above-mentioned embodiment 1 exhibits gastrointestinal bleeding caused by injury to the gastrointestinal mucosa, and can be used to screen hemostatic agents using the degree of gastrointestinal bleeding as an index. The screening method for hemostatic agents according to this embodiment includes the steps of administering or applying a test substance to the gastrointestinal bleeding model animal according to the above-mentioned embodiment 1, and evaluating the hemostatic effect of the test substance in the gastrointestinal bleeding model animal.

[0028] The test substance is not particularly limited, and may be a compound, an antibody, a peptide, a nucleic acid drug, or the like. It is preferable to select a method for administering or applying the test substance to the non-human model animal that is appropriate for the test substance. For example, when the test substance is a compound, it may be administered orally or intraperitoneally. When the test substance is an antibody, a peptide, a nucleic acid drug, or the like, it may be injected intravenously or subcutaneously. The test substance may be administered or applied once or multiple times. The concentration of the test substance may be appropriately determined depending on the type of the test substance. In addition, the test substance may be administered or applied at multiple concentrations obtained by diluting the concentration of the test substance in several steps. The timing of administration or application may be, for example, before administering the above-mentioned antithrombotic drug to the non-human animal. In this case, the test substance may be administered or applied to a non-human animal having an injury to the digestive tract mucosa, and then the antithrombotic drug may be administered to the non-human animal.

[0029] In the evaluation of the hemostatic effect, the hemostatic effect may be evaluated by any method capable of evaluating the degree of gastrointestinal bleeding. Gastrointestinal bleeding may be evaluated, for example, by the presence or absence of intragastric blood clots, the presence or absence and number of blood clots at the bottom of the ulcer, the presence or absence of hematemesis or blood in the stool, the presence or absence of tarry stools at the time of autopsy, the presence or absence of overt gastrointestinal bleeding, etc. To confirm the presence or absence of overt gastrointestinal bleeding, an endoscope may preferably be used to examine, for example, the presence or absence of blood clots or blood accumulation in the stomach.

[0030] In the evaluation of the hemostatic effect, a marker capable of evaluating the degree of gastrointestinal bleeding may be measured. Examples of the marker include fecal transferrin, hemoglobin, etc. In the evaluation of the hemostatic effect, a known hemostatic agent may be administered as a positive control to the gastrointestinal bleeding model animal for comparison in the same manner as the test substance.

[0031] According to the screening method for a hemostatic agent of this embodiment, the hemostatic effect is evaluated in a gastrointestinal bleeding model animal that can reproduce gastrointestinal bleeding caused by injury to the gastrointestinal mucosa, so that a hemostatic agent that is effective against gastrointestinal bleeding caused by injury to the gastrointestinal mucosa can be obtained.

[0032] The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples. EXAMPLES

[0033] Medical Crown minipigs (obtained from the Nonprofit Organization Medical Minipig Research Institute) aged 6 months and weighing 19 to 21 kg were used. Catheters were inserted into the external jugular veins on both sides of the minipigs under general anesthesia as a route for blood sampling and heparin administration.

[0034] A scope was inserted orally and mucosal defects were created using EMRL with a ligating device at 12 locations each in the lesser curvature, anterior wall, and posterior wall of the upper, middle, and lower body parts of the stomach and the gastric angle.

[0035] For four minipigs (heparin administration group; Examples 1 to 4), ACT was measured every 10 minutes using Hemochron (trademark) Jr. Signature+ (Heiwa Bussan Co., Ltd.), an elapsed time timer for coagulation measurement, and 50 U / kg of unfractionated heparin was repeatedly administered until ACT reached 200 seconds or more. After creating a mucosal defect site with EMRL, an endoscopic photograph of the ulcer bottom was taken, and the endoscope was removed, and the first heparin was promptly administered. After confirming that ACT was 200 seconds or more, continuous heparin administration (50 U / kg / hour) was started using a pressurized drug injector (Surefuser (trademark) A SFS-1002D, Nipro Co., Ltd.). Physiological saline (Otsuka saline injection, Otsuka Pharmaceutical Factory Co., Ltd.) was used as the solvent for unfractionated heparin. For three minipigs (non-heparin administration group; Comparative Examples 1 to 3), nothing was administered and ACT was measured in the same manner as in Examples 1 to 4.

[0036] The day after the procedure, the scope was inserted again under general anesthesia, and the presence or absence of intragastric blood clots was confirmed by endoscopic observation, and the number of blood clots at the bottom of the ulcer was counted. In addition, the presence or absence of hematemesis was confirmed the day after the procedure, and the presence or absence of tarry stool at the time of dissection was confirmed. Hemoglobin levels (Hb) were measured immediately after catheter insertion (pre-procedure Hb) and between anesthesia and endoscopic observation the day after the procedure (next-day Hb) and compared. Delayed bleeding was considered to have occurred (+) if at least one of the following conditions was met: obvious overt gastrointestinal bleeding (hematemesis, blood in the melena) and intragastric blood clots or blood accumulation on endoscopic examination 24 hours after the procedure.

[0037] (result) The change in ACT over time in Examples 1 to 4 and images of the inside of the stomach observed with an endoscope are shown in Figure 1. The change in ACT over time in Comparative Examples 1 to 3 and images of the inside of the stomach observed with an endoscope are shown in Figure 2. Of Examples 1 to 4, delayed bleeding occurred in Examples 1, 2 and 4, which had an ACT of 220 seconds or more. On the other hand, in Comparative Examples 1 to 3, no extension of ACT was observed and no delayed bleeding was observed after 24 hours.

[0038] The test results are shown in Table 1. [Table 1]

[0039] The present invention allows various embodiments and modifications without departing from the broad spirit and scope of the present invention. The above-described embodiments are for the purpose of explaining the present invention and do not limit the scope of the present invention. That is, the scope of the present invention is indicated by the claims, not the embodiments. Various modifications made within the scope of the claims and the scope of the invention equivalent thereto are considered to be within the scope of the present invention. [Industrial Applicability]

[0040] The present invention is useful for obtaining an animal model for gastrointestinal bleeding and for developing a hemostatic agent.

Claims

1. The method includes administering an antithrombotic agent to a non-human animal having an injury to a gastrointestinal mucosa. A method for producing an animal model for gastrointestinal bleeding.

2. In the administration step, After the administration of the antithrombotic drug causes the activated clotting time of the non-human animal to exceed a reference value, the antithrombotic drug is further administered continuously. The method for producing a gastrointestinal bleeding model animal according to claim 1.

3. The non-human animal is Crown miniature pigs aged 5 to 7 months. The method for producing a gastrointestinal bleeding model animal according to claim 1 or 2.

4. The reference value is 200 seconds or more, The method for producing a gastrointestinal bleeding model animal according to claim 2.

5. The gastrointestinal mucosa is At least one mucosa of the lesser curvature, anterior wall and posterior wall of each of the upper body, middle body, lower body and gastric angle of the stomach; The method for producing a gastrointestinal bleeding model animal according to claim 1 or 2.

6. The injury may be A mucosal defect created by endoscopic mucosal resection. The method for producing a gastrointestinal bleeding model animal according to claim 1 , 2 or 4 .

7. A non-human animal in which 24 hours or more have passed since injury was formed in the gastrointestinal mucosa, and which has overt gastrointestinal bleeding or blood clots or blood pools in the stomach. Animal model of gastrointestinal bleeding.

8. A step of administering or applying a test substance to a gastrointestinal bleeding model animal produced by the method for producing a gastrointestinal bleeding model animal according to any one of claims 1, 2 and 4 or the gastrointestinal bleeding model animal according to claim 7; evaluating the hemostatic effect of the test substance in the gastrointestinal bleeding model animal; A method for screening a hemostatic agent, comprising:

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