JP6281874B2 - Method for producing chronic cystitis model non-human animal - Google Patents

Description

  The present invention relates to a method for producing a chronic cystitis model non-human animal.

  Interstitial cystitis (IC), a type of chronic cystitis, is an organic disease of the bladder that exhibits symptoms such as frequent urination, increased urinary urgency, urgency, inflammation, and bladder pain. Then, the quality of life is significantly reduced. Although the underlying cause of interstitial cystitis has not been clarified, three pathophysiological mechanisms have been suggested so far: epithelial dysfunction, mast cell activation and neuropathitis. In recent years, interstitial cystitis has been reported to have a high prevalence, and as the degree of recognition increases, the need for therapeutic agents in clinical settings is also increasing.

  However, at present, effective treatments for interstitial cystitis have not been established, and all the therapeutic agents currently used in Japan are for off-label use other than the approved indications. Therapeutic drugs suitable for interstitial cystitis or symptoms associated with interstitial cystitis have hardly been put to practical use worldwide. Thus, one of the main causes of the progress of development of therapeutic agents for interstitial cystitis is the absence of an appropriate model animal for interstitial cystitis.

  Until now, as a model animal of interstitial cystitis, a model animal administered with cyclophosphamide (Non-patent Documents 1 and 2), a model animal administered with acetic acid or LPS and protamine sulfate (Non-patent Document 3) 4) and model animals of knockout (Non-patent Document 5) have been reported. However, although these model animals are complicated to produce and exhibit symptoms of cystitis, it is difficult to say that they are model animals that appropriately represent the pathology of interstitial cystitis, which is chronic cystitis. For example, the most typical cystitis model animal, cyclophosphamide-induced cystitis model animal, has a very high degree of cystitis, and single dose administration can only evaluate acute cystitis (non-patented There are problems such as literature 2). In addition, since cystitis is cured immediately, it is necessary to administer a low dose of cyclophosphamide multiple times in a short time interval (Non-patent Document 3). This is not chronic cystitis, It is thought to reflect acute cystitis. Accordingly, there is a strong demand for providing a model animal that appropriately represents the pathology of chronic cystitis including interstitial cystitis.

Neurosci Lett. 2007 Jun 29; 421 (3): 250-2.Epub 2007 Jun 2. Pharmacological validation of a model of cystitis pain in the mouse. Wantuch C, Piesla M, Leventhal L. NeurourolUrodyn. 2011 Nov; 30 (8): 1659-65.doi: 10.1002 / nau.21180.Epub 2011 Jun 29.Functional characterization of a chronic cyclophosphamide-induced overactivebladder model in mice.Boudes M, Uvin P, Kerselaers S, Vennekens R, VoetsT, De Ridder D. J Urol. 2004 Oct; 172 (4 Pt 1): 1529-32. Intravesical botulinum toxin aadministration produces analgesia against acetic acid induced bladder painresponses in rats. Chuang YC, Yoshimura N, Huang CC, Chiang PH, Chancellor MB. J Urol. 1996 Mar; 155 (3): 1133-8. Bladder injury model induced in ratsby exposure to protamine sulfate followed by bacterial endotoxin. Stein PC, Pham H, Ito T, Parsons CL. Sci Rep. 2012; 2: 317.Epub 2012 Mar 19.BALB / c-Fcgr2bPdcd1 mouseexpressing anti-urothelial antibody is a novel model of autoimmune cystitis.Sugino Y, Nishikawa N, Yoshimura K, Kuno S, Hayashi Y, Yoshimura N, Okazaki T, Kanematsu A, Ogawa O.

  Accordingly, an object of the present invention is to provide a method for easily producing a model non-human animal exhibiting symptoms of chronic cystitis.

  In order to achieve the object, the method for producing a chronic cystitis model non-human animal of the present invention includes a step of administering hydrogen peroxide to the non-human animal.

  The chronic cystitis model non-human animal of the present invention is obtained by the method for producing a chronic cystitis model non-human animal of the present invention.

The screening method for a therapeutic agent for chronic cystitis of the present invention comprises the following steps (A) to (C).
(A) A step of administering the test substance to the chronic cystitis model non-human animal of the present invention (B) A step of confirming improvement of the symptoms of chronic cystitis in the model non-human animal administered with the test substance ( C) A step of selecting a test substance that improves symptoms of chronic cystitis based on the result obtained in the step (B)

  According to the method for producing a chronic cystitis model non-human animal of the present invention, a model animal that appropriately represents the pathological condition of chronic cystitis can be provided by simple treatment. In the model non-human animal obtained by the above-mentioned production method, for example, chronic cystitis cycle of inflammatory cell infiltration and stromal layer hyperplasia due to damage and rupture of bladder mucosa caused by increased mucosal permeability caused thereby Can be said to appropriately represent the pathology of chronic cystitis. For this reason, it can be said that the model non-human animal is extremely useful, for example, for screening for a therapeutic candidate agent for chronic cystitis. Furthermore, according to the method of producing a chronic cystitis model non-human animal of the present invention, the model non-human animal can be obtained by administering hydrogen peroxide, which is generally available at a low cost, to the non-human animal. This is also advantageous from the viewpoint of the labor and cost required to manufacture the device.

In Example 1, it is a graph which shows the result of having compared the frequency | count of urination and the increase in the bladder weight of the chronic cystitis model non-human animal of this invention, and the conventional cystitis model animal. In Example 1, it is a graph which shows the mucosa permeability and blood-vessel permeability in the chronic cystitis model non-human animal of this invention. In Example 1, it is a photograph which shows the bladder tissue in the chronic cystitis model non-human animal of this invention. In Example 1, it is a photograph which shows the bladder tissue in the chronic cystitis model non-human animal of this invention. In Example 1, it is a graph which shows the efficacy evaluation of the existing therapeutic agent with respect to the chronic cystitis model non-human animal of this invention. In Example 1, it is a graph which shows the expression induction of the inflammatory marker and the fibrosis marker in the chronic cystitis model non-human animal of this invention. In Example 2, it is a graph which shows the evaluation of the bladder pain in the chronic cystitis model non-human animal of this invention. In Example 2, it is a graph which shows the efficacy evaluation of the existing therapeutic agent with respect to the evaluation of the bladder pain in the chronic cystitis model non-human animal of this invention.

(1) Method for Producing Chronic Cystitis Model Non-Human Animal The method for producing a chronic cystitis model non-human animal of the present invention includes the step of administering hydrogen peroxide to the non-human animal as described above. To do.

  In the present invention, any active oxygen generator that generates active oxygen can be used in place of the hydrogen peroxide, but it is preferable to use hydrogen peroxide. In the present invention, the hydrogen peroxide can also be said to be a chronic cystitis inducer.

  In the present invention, the dosage of the hydrogen peroxide is not particularly limited. The upper limit of the hydrogen peroxide dose is, for example, 10 mL per kg body weight, preferably 7 mL per kg body weight, more preferably 5 mL per kg body weight, and the lower limit is, for example, per kg body weight. 1 mL, preferably 1.5 mL per kg body weight, more preferably 2 mL per kg body weight, and the range is, for example, 1 to 10 mL per kg body weight, preferably 1.5 to 7 mL per kg body weight And more preferably 2 to 5 mL per kg body weight.

  In the present invention, the form of the hydrogen peroxide is not particularly limited. The hydrogen peroxide may be administered as it is, for example, but is preferably administered as a hydrogen peroxide solution mixed with a solvent. Examples of the solvent include water, physiological saline, and buffer solution. When the hydrogen peroxide is administered as a hydrogen peroxide solution, the upper limit of the hydrogen peroxide concentration in the hydrogen peroxide solution is, for example, 5.0 v / v%, preferably 3.0 v / v%. More preferably 2.0 v / v%, and the lower limit thereof is, for example, 0.1 v / v%, preferably 0.3 v / v%, more preferably 1.0 v / v%. And the range is, for example, 0.1 to 5.0 v / v%, preferably 0.3 to 3.0 v / v%, more preferably 1.0 to 2.0 v / v%. is there. The hydrogen peroxide concentration in the hydrogen peroxide solution is preferably 1.5 v / v%.

  In the present invention, the method for administering hydrogen peroxide is not particularly limited, and for example, a method capable of administering hydrogen peroxide to the bladder directly or indirectly is preferable. Examples of the administration method include intravenous administration and intravesical administration, and preferably intravesical administration by inserting a catheter from the urethra.

  In the present invention, the non-human animal is not particularly limited. Examples of the non-human animal include rodents, rabbits, cats, cows, monkeys, dogs, weasels, wild boars, and primates. Examples of the rodents include mice, rats, hamsters, and guinea pigs. Other non-human animals include, for example, rabbits, dogs, cats, ferrets, goats, sheep, cows, pigs, cynomolgus monkeys, rhesus monkeys, and marmosets. The non-human animals are preferably rodents, more preferably mice and rats, and particularly preferably mice. As the non-human animal to which hydrogen peroxide is administered, for example, a general animal used for producing a model animal can be used.

  By such a method for producing a chronic cystitis model non-human animal of the present invention, a model non-human animal exhibiting symptoms of chronic cystitis can be obtained. The characteristics of the chronic cystitis model non-human animal of the present invention will be described later.

(2) Chronic cystitis model non-human animal The chronic cystitis model non-human animal of the present invention is obtained by the method for producing a chronic cystitis model non-human animal of the present invention as described above.

The chronic cystitis model non-human animal of the present invention exhibits at least one characteristic selected from the group consisting of (a) to (h) below for at least one week, for example, in a state where the hydrogen peroxide has not been administered.
(A) at least two urinations per 15 minutes (b) bladder fibrosis (c) bladder wall hypertrophy (d) bladder inflammation (e) compared to normal non-human animals relative to the model non-human animals, Increased bladder weight (f) Increased vascular permeability compared to normal non-human animals for the model non-human animals (g) Increased white blood cell count in the bladder compared to normal non-human animals for the model non-human animals Increase (h) Increased time of licking action of the lower abdomen during bladder dilatation compared to normal non-human animals for the model non-human animals

  The unadministered state means a state in which no further hydrogen peroxide is administered after the administration of hydrogen peroxide at the time of production.

  The chronic cystitis model non-human animal of the present invention can maintain cystitis symptoms for a long time in the non-administered state. The period showing the characteristics (a) to (h) is, for example, at least 1 week, preferably at least 2 weeks, more preferably at least 1 month.

  In (a), the number of urinations per 15 minutes is, for example, at least 2 to 3 times, preferably at least 4 to 5 times, more preferably at least 5 times.

  In (b), the fibrosis of the bladder means that the extracellular matrix such as collagen fibers (collagen) increases, and as a result, the bladder hardens. The fibrosis of the bladder can be indirectly detected by, for example, a fibrosis marker. As a specific example, in the bladder of the chronic cystitis model non-human animal, when the expression level of the fibrosis marker is significantly higher than that of a normal non-human animal with respect to the chronic cystitis model non-human animal, the chronic bladder It can be judged that the fibrosis of the bladder in the non-human animal model of inflammation has occurred. The fibrosis marker is not particularly limited. Examples of the fibrosis markers include transforming growth factor β (TGF) -β, fibroblast growth factor-2 (FGF) -2, and platelet-derived growth factor (platelet-derived). growth factors such as growth factor (PDGF)), insulin-like growth factor-1 (IGF-1), connective tissue growth factor (CTGF), and collagen and its Examples thereof include hydroxyproline, which is a constituent amino acid. The fibrosis marker is preferably TGF-β and collagen, and more preferably collagen. Whether or not the model non-human animal is significantly higher than the normal non-human animal can be determined, for example, based on a general statistical significance difference (the same applies hereinafter).

  In (c), the enlargement of the bladder wall means that the thickness of the bladder wall increases as a result of inflammation or fibrosis. The enlargement of the bladder wall can be determined by anatomical findings such as dissecting the bladder taken out of the non-human animal and measuring the thickness of the bladder wall. As a specific example, in the bladder of the chronic cystitis model non-human animal, when the thickness of the bladder wall is significantly increased compared to the normal non-human animal for the chronic cystitis model non-human animal, the chronic bladder It can be judged that the thickness of the bladder wall of the flame model non-human animal is increased. Whether or not the model non-human animal is significantly increased compared to the normal non-human animal can be determined based on, for example, a general statistical significance difference (hereinafter the same).

  In the above (d), the inflammation of the bladder means a biological defense reaction against harmful stimuli such as infection, damage and injury to the bladder tissue. The inflammation of the bladder can be indirectly detected by, for example, an inflammation marker. As a specific example, when the expression level of the inflammatory marker in the bladder of the chronic cystitis model non-human animal is significantly higher than that of a normal non-human animal relative to the chronic cystitis model non-human animal, the chronic cystitis It can be judged that inflammation of the bladder of the model non-human animal occurs. The inflammation marker is not particularly limited. Examples of the inflammatory marker include inflammatory cytokines such as IL-1β, TFN-α and IL-6, and chemokines such as CCL1, CCL2, CXCL1 and CXCL2. The inflammatory marker is preferably the chemokines and the inflammatory cytokines, and more preferably the inflammatory cytokines.

  In (e), the increase in the weight of the bladder is, for example, that the weight of the bladder of the chronic cystitis model non-human animal is significantly increased compared to a normal non-human animal with respect to the chronic cystitis model non-human animal. If it is, it can be determined that the bladder weight of the chronic cystitis model non-human animal has increased.

  In (f) above, vascular permeability means the mobility of substances present in blood to the bladder tissue. Therefore, vascular permeability means, for example, the degree to which a high molecular weight protein or blood cell component passes through the blood vessel wall from within the blood vessel. When inflammation occurs, vascular permeability increases and leukocyte infiltration and edema occur. As shown in the Examples described later, in the chronic cystitis model non-human animal, the increase in vascular permeability continues even after the following mucosal permeability (i) returns to the original state. Therefore, the period in which the increase in vascular permeability lasts is preferably longer than the period in which the increase in mucosal permeability persists. The blood vessel permeability is measured by, for example, injecting a dye for staining such as Evans Blue into the vein of the non-human animal, then removing the bladder from the non-human animal and measuring the concentration of the dye for staining in the bladder tissue. Can be measured. As a specific example, in the bladder of the chronic cystitis model non-human animal, when vascular permeability is significantly increased as compared with a normal non-human animal with respect to the chronic cystitis model non-human animal, the chronic cystitis It can be judged that the vascular permeability of the model non-human animal is increased.

  In (g), the white blood cells are not particularly limited. Examples of the leukocytes include neutrophils, eosinophils, basophils, lymphocytes, macrophages and monocytes. As shown in the examples described later, in the non-human animal model of chronic cystitis, leukocytes were removed after the loss and thinning of the bladder wall due to the increase in mucosal permeability described below (i) returned to the original state. The infiltration of inflammatory cells such as is sustained. Therefore, the period in which the increase in the white blood cell count is preferably longer than the period in which the increase in mucosal permeability continues. As a specific example, in the bladder of the chronic cystitis model non-human animal, when leukocytes are significantly increased compared to the normal non-human animal relative to the chronic cystitis model non-human animal, the chronic cystitis model non-human animal It can be determined that leukocytes in human animals are increasing.

  In (h) above, the lower abdominal licking behavior (licking behavior) means a stress response to bladder pain in the chronic cystitis model non-human animal. Specifically, the licking action means an action of bending the upper body and licking the urethral opening or its periphery with the tongue. As shown in the examples described later, in the chronic cystitis model non-human animal, the time of licking action of the lower abdomen continues even after the mucosal permeability (i) below returns to the original state. . The time for the licking behavior is, for example, the time during which the chronic cystitis model non-human animal is performing the licking behavior at 1 to 30 minutes, preferably 0 to 60 minutes with reference to the time of bladder expansion. . Examples of the method of expanding the bladder include a method of injecting the solvent into the bladder of the chronic cystitis model non-human animal. As a specific example, in the chronic cystitis model non-human animal, on the basis of the time when physiological saline is injected into the bladder of the chronic cystitis model non-human animal, for example, the time of the licking action in 1 to 30 minutes When the chronic cystitis model non-human animal is significantly increased compared to the normal non-human animal, it can be determined that the licking time of the chronic cystitis model non-human animal is increased.

  Other symptoms include, for example, (i) increased mucosal permeability. Mucosal permeability is an indicator of the barrier mechanism of the bladder mucosal layer, and an increase means barrier mechanism failure and failure. The mucosal permeability can be measured by, for example, injecting a dye for staining such as Evans Blue into the non-human animal, then removing the bladder from the non-human animal and measuring the concentration of the dye for staining.

  When the symptom of (a) to (h) or (i) is compared with the chronic non-human animal of the chronic cystitis model of the present invention and a normal non-human animal with respect to the model non-human animal, for example, a statistically significant difference Can be judged based on As the statistical analysis method, a known statistical analysis method can be selected and used. The statistical analysis method is not particularly limited, and examples thereof include one-way ANOVA, one-way repeated measurement ANOVA, Dunnett's test, Student's t test, and Wilcoxon test, and these can be combined. For example, in the above test, it can be determined that a significant difference was observed when the P value was less than 0.05, while it was determined that no significant difference was observed when the P value was 0.05 or more. . Examples of the normal non-human animal include animals that have not developed chronic cystitis.

(3) Screening method for candidate treatment for chronic cystitis The screening method for a candidate treatment for chronic cystitis of the present invention comprises the following steps (A) to (C) as described above. .
(A) A step of administering the test substance to the chronic cystitis model non-human animal of the present invention (B) A step of confirming improvement of the symptoms of chronic cystitis in the model non-human animal administered with the test substance ( C) A step of selecting a test substance that improves symptoms of chronic cystitis based on the result obtained in the step (B)

  In the step (A), the test substance is not particularly limited. Examples of the test substance include natural compounds, organic compounds, inorganic compounds, nucleic acid molecules, single compounds such as proteins and peptides, compound libraries, gene library expression products, cell extracts, cell culture supernatants, fermentations, and the like. Examples include microbial products, marine organism extracts and plant extracts.

  In the step (A), the method for administering the test substance to the chronic cystitis model non-human animal of the present invention is not particularly limited. Examples of the administration method include oral administration and parenteral administration. Examples of the parenteral administration include intravenous administration, intramuscular administration, intraperitoneal administration, subcutaneous administration, nasal administration, pulmonary administration, and transdermal administration. When the test substance is a protein or a peptide, for example, a viral vector having a gene encoding the protein or the like is constructed, and the gene is converted to the chronic cystitis model non-human animal of the present invention using its infectivity. And may be expressed in the body.

  In the step (B), confirmation of improvement against chronic cystitis can be performed based on symptoms exhibited by the model non-human animal. Examples of the symptoms used for confirming improvement for chronic cystitis include the above (a) to (h). In the present invention, “improvement” means, for example, that a symptom of chronic cystitis is restored to a normal state, or a symptom of chronic cystitis is alleviated. A person skilled in the art can determine whether or not the symptoms of chronic cystitis have improved in the model non-human animal based on the description herein. Specific examples include, for example, comparison of symptoms between the model non-human animal administered with the test substance and normal non-human animals relative to the model non-human animal, or symptoms before or after administration of the test substance. It can be determined based on the comparison.

  And in the said (C) process, the treatment candidate agent of chronic cystitis can be obtained by selecting the test substance which improves the symptom of chronic cystitis from the result of the said (B) process. In the present invention, treatment includes, for example, the meaning of improvement and prevention of symptoms of chronic cystitis.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, this invention is not limited to the aspect described in the Example.

[Example 1]
Hydrogen peroxide was administered from the mouse urethra to produce a chronic cystitis model mouse, and its properties were confirmed.

(1) Method for Producing Chronic Cystitis Model Mice As mice for injecting hydrogen peroxide, 5 to 6 week-old female C57BL / 6J mice (Japan SLC, Inc.) were used. The growth of the mice was carried out using a cage maintained at 20-26 ° C., a relative humidity of 35% -75% and a light / dark cycle of 12 hours, and the light period was started automatically from 8 am. Groups of 4-6 animals per animal were used. Mice were given food and water ad libitum.

The administration of of H ₂ O ₂ to the mice was carried out as follows. First, a 24-G polypropylene catheter was inserted into the bladder under isoflurane anesthesia until urine was observed through the catheter, and the lower abdomen was slightly compressed until urine was drained. Subsequently, H ₂ O ₂ (Santoku Chemical Co., Ltd.) is dissolved in physiological saline (Otsuka Pharmaceutical Co., Ltd.) to prepare a H ₂ O ₂ solution having a predetermined concentration (1.5 v / v%). 50 μL of ₂ O ₂ solution was injected into the bladder through a catheter. After 30 minutes, the H ₂ O ₂ solution was injected again into the bladder. Thus, the chronic cystitis model mouse of the Example was obtained.

In Comparative Example, cyclophosphamide (Wako Pure Chemical Industries, Ltd.) was used as a drug instead of the H ₂ O ₂ . Specifically, cyclophosphamide (Wako Pure Chemical Industries, Ltd.) is dissolved in physiological saline (Otsuka Pharmaceutical Co., Ltd.) to prepare a 15 mg / mL cyclophosphamide solution. The mice were intraperitoneally administered to give 300 mg of cyclophosphamide. As a control, no drug was added, and 50 μL of physiological saline (Otsuka Pharmaceutical Co., Ltd.) was intraperitoneally administered to the mice instead of H ₂ O ₂ . Thus, a comparative model mouse and a control model mouse were obtained.

  The following characteristics were confirmed for the Example, Comparative Example and control model mice. The results of each characteristic were statistically analyzed using the SAS program (Ver. 8.2, SAS Institute), and the mean ± S. E. Shown as M. The results of each characteristic were statistically analyzed by performing a one-way ANOVA or one-way repeated measurement ANOVA followed by a post hoc Dunnett comparison test. For all the results, the P value was less than 0.05. Cases were considered statistically significant.

(2) Measurement of urination frequency and bladder weight In the preparation method of (1), 3 hours, 1 day and 7 days after the administration of H ₂ O ₂ (1.5 v / v%) or cyclophosphamide The number of micturitions and bladder weight were measured for the eyes mice (n = 6 each). As a control, the number of urinations and bladder weight were also measured for mice (n = 6) administered with physiological saline. The number of urinations was measured as follows. First, the model mouse was grown in a transparent plastic cage (20 cm × 25 cm × 14 cm: width × length × height) covered with filter paper (Advantech Chromatography Filter No. 50, Toyo Roshi Kaisha, Ltd.). Conditioned for 30 minutes. Next, the filter paper was replaced with a new one, the mouse was allowed to move freely for 15 minutes, and the behavior of the mouse was recorded. Thereafter, the recorded content was confirmed, and the number of urinations in 15 minutes was measured by counting the number of urination spots (stains) on the filter paper. The bladder weight was measured as the wet weight of the mouse taken out of the mouse. The urination frequency and bladder weight were measured by arbitrarily selecting 6 mice from the group of mice treated under the same conditions.

These results are shown in FIG. FIG. 1 is a graph showing the number of urinations per 15 minutes and the weight of the bladder in a model mouse. In FIG. 1, the upper and lower graphs on the left are the number of urinations, the upper and lower graphs are the results of bladder weight, and the upper two graphs are the model mice of the comparative example to which cyclophosphamide was administered, the lower two graphs is the result of a model mouse in example administered with H ₂ O _2. In FIG. 1, “Control” is the result of no drug administration, and “3h, 1d, and 7d” are the results of 3 hours, 1 day, and 7 days after drug administration, respectively.

As shown in FIG. 1, the number of urinations in the model mouse of the comparative example increased at 3 hours after administration, but then decreased rapidly, and almost the same state as when no drug was administered on the 7th day after administration. became. On the other hand, the number of urinations of the model mice of the examples gradually increased after administration, and it was confirmed that the number of urinations increased further on the seventh day after administration than on the first day after administration. The bladder weight of the model mouse of the comparative example increased at 3 hours after the administration, and the state was maintained on the first day after the administration, but almost no drug administration was observed on the seventh day after the administration. It became the same state. On the other hand, the bladder weight of the model mice of the examples increased 3 hours after administration, and the state was maintained even on the 7th day after administration. These results indicate that the model mice of the examples administered with H ₂ O ₂ have a very long duration of cystitis symptoms compared to the model mice of the comparative examples administered with cyclophosphamide. ing.

(3) Measurement of mucosal permeability and vascular permeability of bladder The mucosal permeability and vascular permeability of the bladder were measured. Specifically, in order to evaluate mucosal permeability, in the preparation method of (1), H ₂ O _{2 was} not administered (n = 6), or the H ₂ O ₂ (1.5 v / v%) was administered. Three hours later, mice on day 1 and day 7 (n = 6, 4 and 10 respectively) were dissolved in sterile physiological saline using a catheter, and 5% Evans Blue (Nacalai Tesque, Inc.) 0. 05 mL was injected into the bladder. Further, in order to evaluate vascular _{permeability,} H 2 _{O 2} untreated, or the _{H 2 O 2 (1.5v / v} %) 3 hours after administration, 1 and 7 days of mice (each n = For 6), 0.1 mL of 5% Evans Blue dissolved in sterile saline was injected into the tail vein. At 30 minutes after injection, the bladder was removed from the model mouse. The bladder was then heated overnight (56 ° C.) in 1 mL of formamide to extract Evans blue. The dye (Evans blue) concentration (μg / mL) in the obtained extract was quantified with a spectrophotometer (Bio-Rad) to evaluate the mucosal permeability and vascular permeability of the bladder.

  These results are shown in FIG. FIG. 2 is a graph showing mucosal permeability and vascular permeability in the bladder of a model mouse by pigment concentration. In FIG. 2, the left graph is the result of mucosal permeability, the right graph is the result of vascular permeability, “Control” is the result of no drug administration, and “3h, 1d and 7d” are the results, respectively. The results are 3 hours, 1 day and 7 days after drug administration.

As shown in FIG. 2, the mucosal permeability of the model mouse of the example administered with H ₂ O ₂ increased after the administration, and the state was maintained even on the first day after the administration, but it was 7 days after the administration. In the eyes, the condition was almost the same as when no drug was administered. In addition, the vascular permeability of the model mice of the above examples increased after administration, and the state was maintained even on the seventh day after administration. These results show that, in the model mice of Examples administered with H ₂ O ₂ , inflammation was caused by the failure and failure of the barrier mechanism in the bladder mucosa layer, but the failure and failure of the barrier mechanism was cured within a few days. Later, it has been shown that the symptoms of cystitis with increased vascular permeability persist.

(4) Histological examination of urinary bladder Under the pentobarbital sodium anesthesia for the model mouse of the example administered with H ₂ O ₂ prepared by the method of (1) or the control model mouse administered with physiological saline, Potassium-free phosphate buffered saline was transcardially perfused. Subsequently, the bladder was removed from the model mouse, and the bladder tissue divided in the longitudinal direction was post-fixed overnight with 4% paraformaldehyde and embedded in paraffin. The obtained paraffin-embedded tissue was cut into 5 μm sections and stained with hematoxylin and eosin. And the structure | tissue after dyeing | staining was observed with the microscope system (BZ-8100; Keyence Corporation).

These results are shown in FIG. 3 and FIG. FIG. 3 is a micrograph of the stained bladder tissue, and FIG. 4 is an enlarged photo of FIG. 3 and 4, the upper photograph shows the results of the control model mouse administered with physiological saline, and the lower photograph shows the results of the model mouse of the example administered with H ₂ O ₂ .

As shown in FIG. 3 and FIG. 4, the bladder of the model mouse of the Example was compared with the control model mouse at 3 hours or 1 day after administration, as shown in FIG. 3 hours later, the arrow portion in the photograph), thinning and interstitial enlargement occurred. On the 7th day after administration, the bladder wall deletion and thinning were restored to the original state, but the stromal layer hypertrophy was maintained. In addition, in the bladder of the model mouse of the example, infiltration of inflammatory cells such as leukocytes was confirmed in the interstitium as compared with the control model mouse, and the state was maintained even 7 days after administration. (For example, the arrow part in the 7th day photograph in FIG. 4). These results show that in the model mice of Examples administered with H ₂ O ₂ , bladder wall deletion and thinning first occurred, causing cystitis symptoms, but bladder wall deletion and thinning. It shows that the symptoms of cystitis with infiltration of inflammatory cells persist even after the stratification has healed within a few days.

(5) Confirmation of efficacy of existing therapeutic agent Intraperitoneal administration of existing therapeutic agent was carried out on the model mouse of the example 7 days after administration of H ₂ O ₂ produced by the method of (1) above. It was. As the existing therapeutic agent, a non-applicable drug that was reported to be insufficient as a therapeutic agent for chronic cystitis was used. Specifically, amitriptyline (AMT, 1 mg / kg), a tricyclic antidepressant, indomethacin (IND, 3 mg / kg), a nonsteroidal anti-inflammatory drug (NSAID), oxybutynin (OXY), an anticholinergic drug 3 mg / kg), morphine (MOR, 3 mg / kg), an opioid analgesic was used. The dose per body weight is shown in parentheses. Amitriptyline (n = 7), indomethacin (n = 7), oxybutynin (n = 7) and morphine (n = 6) include 5% DMSO and 2% Tween 80 (Nacalai Tesque, Inc.) immediately before use. Used by dissolving in physiological saline (n = 7). As a control, physiological saline (n = 6) was similarly administered intraperitoneally (V) instead of the existing therapeutic agent. Then, 30 minutes after the administration of the existing therapeutic agent, the number of urinations and the bladder weight were measured by the same method as in (2) above. Further, the number of urinations and the weight of the bladder were similarly measured for the control model mice prepared by the method (1) without administering the existing therapeutic agent.

  These results are shown in FIG. FIG. 5 is a graph showing the number of urinations per 15 minutes in the model mouse on the left, and a graph showing the bladder weight on the right. In both graphs of FIG. 5, each bar represents, from the left, a control model mouse (saline), a physiological saline-administered example model mouse (V), an AMT-administered example model mouse, an IND-administered example model mouse, It is a result of the example model mouse of OXY administration, and the example model mouse of MOR administration.

As shown in FIG. 5, the number of urinations in the example model mice administered with these existing therapeutic agents was significantly reduced as compared with the example model mice (V) not administered with the existing therapeutic agents. On the other hand, there was no significant difference in the increase in bladder weight between Example model mice administered with these existing therapeutic agents and Example model mice not administered with the existing therapeutic agents. These results did not indicate that the existing therapeutic agent was able to treat cystitis, although the number of urinations was reduced. That is, it is shown that the model mouse of the example administered with H ₂ O ₂ appropriately represents the symptoms of chronic cystitis and can be used for screening and evaluation of therapeutic agents for chronic cystitis.

(6) Expression of inflammatory cytokine and fibrosis marker Example of the method of (1), 3 hours, 1 day and 7 days after the H ₂ O ₂ administration (1.5 v / v%) For model mice (n = 4 each), inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor (TNF) -α and interleukin-6 (IL-6), and Then, the expression of transforming growth factor (TGF) -β, which is a fibrosis marker, was confirmed. Specifically, the bladder was taken out from the mouse, total RNA was extracted based on a conventional method, and the expression levels of mRNA for each inflammatory cytokine and fibrosis marker were measured by Real-Time RT-PCR method. Similarly, the expression levels of inflammatory cytokines and fibrosis marker mRNA were also measured in control model mice not administered with the drug (n = 3).

These results are shown in FIG. FIG. 6 is a graph showing mRNA expression levels of each inflammatory cytokine (IL-1β, TNF-α and IL-6) and fibrosis marker (TGF-β) in the bladder of a model mouse. In each graph of FIG. 6, “Control” is the result of a control model mouse not administered with a drug, and “3h, 1d, and 7d” are 3 hours, 1st day, and 7th day after H ₂ O ₂ administration, respectively. It is a result of the Example model mouse | mouth of an eye.

As shown in FIG. 6, in Example model mice, IL-6 gradually decreased with a peak at 3 hours after H ₂ O ₂ administration, whereas IL-1β and TNF-α were expressed from 3 hours after administration. Induction was observed and was almost sustained on day 7. In addition, as shown in FIG. 6, in the example model mice, the expression of TGF-β, which is a fibrosis marker, gradually increased. These results show that in the model mice of the Examples administered with H ₂ O ₂ , the period of cystitis symptoms lasts for a very long time, and furthermore, fibrosis of the bladder occurs. Bladder fibrosis is not a finding seen in acute cystitis with a short duration of symptoms, but a finding seen in chronic cystitis with a long duration of symptoms. This also shows that the model mouse of the example administered with H ₂ O ₂ appropriately represents the symptoms of chronic cystitis.

[Example 2]
Hydrogen peroxide was administered from the mouse urethra to produce a chronic cystitis model mouse, and bladder pain was evaluated.

(1) Evaluation of bladder pain Bladder pain was evaluated. Specifically, in the preparation method of Example (1), mice on day 1 and day 7 after physiological saline administration (n = 7 and 12 respectively), or H ₂ O ₂ (1.5 v / v %) Mice on day 1 and day 7 after administration (n = 6 and 15 respectively) were injected with 50 μL of saline into the bladder under isoflurane anesthesia using a catheter. After the injection, the physiological saline injected into the bladder was not drained, and the catheter was removed. Next, the mice were allowed to recover from anesthesia, and 20 minutes after the recovery, video recording was performed, and bladder pain was evaluated by measuring the time for licking (licking) to the lower abdomen.

These results are shown in FIG. FIG. 7 is a graph showing the time of licking behavior of the model mouse. In FIG. 7, the horizontal axis indicates the number of days after drug administration, and the vertical axis indicates the time of licking action. In FIG. 7, “Saline” is the result of the control model mouse administered with physiological saline, and “1.5% H ₂ O ₂ ” is the result of the model mouse of the example.

As shown in FIG. 7, in the control model mice administered with physiological saline, the time of licking behavior did not change in any number of days. On the other hand, the licking time of the model mouse of the example administered with H ₂ O ₂ increased on the 7th day after the administration, and the state was maintained on the 14th day (not shown) after the administration. It was. These results show that late and persistent bladder pain occurs in the model mice of the Examples administered with H ₂ O ₂ .

(2) Confirmation of efficacy of existing therapeutic agent For model mouse of Example used for evaluation of bladder pain on day 7 after administration of H ₂ O ₂ prepared by the method of Example 2 (1) The existing therapeutic agent was intraperitoneally administered 30 minutes before the physiological saline was injected into the bladder. The dosage per body weight of the existing therapeutic agent and the existing therapeutic agent was the same as in Example 1 (5). Amitriptyline (n = 12), indomethacin (n = 6), oxybutynin (n = 5) and morphine (n = 11) include 5% DMSO and 2% Tween 80 (Nacalai Tesque) immediately before use. Used by dissolving in physiological saline. As a control, physiological saline containing 5% DMSO and 2% Tween 80 (n = 15) was similarly administered intraperitoneally (Veh) instead of the existing therapeutic agent. And the time of the licking action was measured in the same manner as in Example 2 (1). In addition, the control model mouse prepared by the method of Example 2 (1) was similarly measured for the time of licking without administering the existing therapeutic agent (n = 12).

  These results are shown in FIG. FIG. 8 is a graph showing the time of licking behavior of the model mouse. In FIG. 8, each bar shows, from the left, a control model mouse (saline), a model mouse (Veh) of an example of physiological saline administration, a model mouse of an example of MOR administration, and a model mouse of an example of AMT administration. FIG. 5 shows the results of the model mouse of the example administered with OXY and the model mouse of the example administered with IND. FIG.

As shown in FIG. 8, the time of licking behavior of the model mouse of the example administered with MOR or AMT was significantly reduced as compared with the model mouse of the non-administered example (Veh). On the other hand, there was no significant difference in the time of licking behavior of the model mice of the examples administered with OXY or IND compared to the model mice of the examples not administered (Veh). In human chronic cystitis, MOR and AMT are used to relieve symptoms of bladder pain, while OXY and IND are known to have little effect on relieving symptoms of bladder pain. For this reason, the result of administering the existing therapeutic agent to the model mouse of the Example is consistent with the result in human chronic cystitis. That is, the model mouse of the example administered with H ₂ O ₂ appropriately represents the symptoms of bladder pain in chronic cystitis, indicating that it can be used for screening and evaluation of therapeutic agents for chronic cystitis.

  Although the present invention has been described with reference to the exemplary embodiments and examples, the present invention is not limited to the above exemplary embodiments and examples. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

  This application claims the priority on the basis of Japanese application Japanese Patent Application No. 2012-238528 for which it applied on October 30, 2012, and takes in those the indications of all here.

  As described above, according to the method for producing a chronic cystitis model non-human animal of the present invention, it is possible to provide a model animal that appropriately represents a pathological condition of chronic cystitis. In the model non-human animal obtained by the above-mentioned production method, for example, chronic cystitis cycle of inflammatory cell infiltration and stromal layer hyperplasia due to damage and rupture of bladder mucosa caused by increased mucosal permeability caused thereby Can be said to appropriately represent the pathology of chronic cystitis. For this reason, it can be said that the model non-human animal is extremely useful, for example, for screening for a therapeutic candidate agent for chronic cystitis. Furthermore, according to the method for producing a chronic cystitis model non-human animal of the present invention, the model non-human animal can be obtained by administering generally available hydrogen peroxide to the non-human animal. This is advantageous from the viewpoint of labor and cost required for the operation.

Claims (11)

Administering hydrogen peroxide to the non-human animal,
A chronic cystitis model non-human animal, wherein the hydrogen peroxide is administered as a hydrogen peroxide solution, and the hydrogen peroxide concentration in the hydrogen peroxide solution is 1.0 to 2.0 v / v% Manufacturing method. The manufacturing method of Claim 1 which administers 1-10 mL of said hydrogen peroxide per kg of body weight. The method according to claim 1 or 2, wherein the hydrogen peroxide concentration in the hydrogen peroxide solution is 1.5 v / v%. The production method according to any one of claims 1 to 3 , wherein the hydrogen peroxide is administered from the urethra. The non-human animal is at least one selected from the group consisting of rodents, rabbits, cats, cows, monkeys, dogs, weasels, boars, and primates. 5. The production method according to any one of 4 to 4 . The non-human animal is at least one selected from the group consisting of a mouse, rat, guinea pig, hamster, rabbit, dog, cat, ferret, goat, sheep, cow, pig, cynomolgus monkey, rhesus monkey, and marmoset. 6. The production method according to any one of 1 to 5 . A chronic cystitis model non-human animal obtained by the production method according to any one of claims 1 to 6 . The chronic cystitis model non-human animal according to claim 7 , which exhibits at least one characteristic selected from the group consisting of the following (a) to (h) in an unadministered state of hydrogen peroxide for at least one week :
(A) at least two urinations per 15 minutes (b) bladder fibrosis (c) bladder wall hypertrophy (d) bladder inflammation (e) compared to normal non-human animals relative to the model non-human animals, Increased bladder weight (f) Increased vascular permeability compared to normal non-human animals for the model non-human animals (g) Increased white blood cell count in the bladder compared to normal non-human animals for the model non-human animals Increase (h) Increased time of licking behavior of the lower abdomen during bladder dilation compared to normal non-human animals relative to the model non-human animals . The chronic cystitis model non-human animal according to claim 7 , wherein the expression level of a fibrosis marker is significantly higher than that of a normal non-human animal relative to the chronic cystitis model non-human animal. The chronic cystitis model non-human according to claim 9 , wherein the fibrosis marker is at least one selected from the group consisting of TGF-β, FGF-2, PDGF, IGF-1, CTGF, collagen and hydroxypurine. animal. A screening method for a therapeutic agent for chronic cystitis comprising the following steps (A) to (C) :
(A) A step of administering the test substance to the chronic cystitis model non-human animal according to any one of claims 7 to 10 (B) Chronic in the model non-human animal administered with the test substance. Step of confirming improvement of cystitis symptoms (C) A step of selecting a test substance that improves symptoms of chronic cystitis based on the result obtained in the step (B) .