JP6153838B2 - Vascular permeability inhibitor - Google Patents

Description

  The present invention relates to a vascular permeability inhibitor containing nitrate.

  In the modified Davis classification, diabetic retinopathy has three stages: (i) simple retinopathy with increased vascular permeability, (ii) preproliferative retinopathy with vascular occlusion, and (iii) proliferative retinopathy with angiogenesis. (For example, see Hideyo Funatsu, “Findings and severity classification of diabetic retinopathy”, Monthly Diabetes Vol. 2, No. 3, 49-50, 2010 (Non-Patent Document 1)).

  The increase in vascular permeability that occurs in the first stage of diabetic retinopathy is caused by non-enzymatic glycation of proteins in the body due to the continuing hyperglycemia caused by diabetes, which is the primary disease, and the advanced glycation end product (Advanced Glycation End product: AGEs) is produced. AGEs induce vascular endothelial growth factor (VEGF) expression in retinal glial cells (Lu, M., “Advanced Glycation End Products Increase Retinal Vascular Endothelial Growth Factor Expression”, J Clin Invest 101 (6 ): 1219-1224, 1998 (Non-Patent Document 2)), and then VEGF induces the expression of adhesion molecules (InterCellular Adhesion Molecule-1: ICAM-1) in vascular endothelial cells. Leukocytes in the blood adhere and aggregate on the vascular endothelium due to ICAM-1 expression, and release cytokines and free radicals. For this reason, the tight junction, which is an adhesion device between vascular endothelial cells, is decomposed, the vascular permeability is enhanced by the breakdown of the retinal blood barrier, the blood component leaks, and edema / bleeding occurs.

  In diabetes, when edema occurs near the macula, it is called diabetic macular edema and causes severe visual impairment. As other ophthalmic diseases with edema, wet age-related macular degeneration and myopic macular degeneration are known, but both diseases result in edema / bleeding in the retina due to the development and proliferation of choroidal neovascularization. It is a serious disease that causes atrophy and scarring on the macula, resulting in decreased visual acuity.

  In the treatment of edema in the ophthalmic field, steroids for suppressing inflammatory reactions, anti-VEGF antibodies for invalidating VEGF in the eye, VEGF antagonists, pseudo-VEGF receptors, etc. are used to reduce systemic side effects. Therefore, these drugs are administered directly into the eye. Intraocular injection of drugs is invasive and painful to the patient, but also carries the risk of intraocular infection. Furthermore, although intraocular injection is local, the administered drug enters the blood and enters the systemic circulation, so the risk of systemic side effects cannot be eliminated. There is a risk of exacerbation, adrenal cortex dysfunction, diabetes deterioration due to promotion of gluconeogenesis, etc. In addition, anti-VEGF drugs need to be administered intraocularly once a month for 3 months (Lucentis packaged document (revised March 2013) (Non-patent Document 3)), so that the physical burden on the patient is reduced. In addition, since it is also necessary to refrain from washing and washing the face for several days after intraocular administration, the patient's QOL decreases.

  In addition to ophthalmic diseases, acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) are known as diseases associated with edema and bleeding due to increased vascular permeability. In these diseases, inflammation of the lungs triggers production of inflammatory molecules such as cytokines, mobilization of leukocytes (alveolar macrophages, neutrophils) in the lungs, damages the capillary endothelium and alveolar epithelium, and capillaries And the air space is destroyed, blood vessel permeability is increased and edema occurs. Regarding the treatment of ALI and ARDS, respiratory management therapy and drug therapy have been tried, but no drug therapy that can directly improve the symptoms has been established.

JP 2011-037830 A

Hideyo Funatsu, “Findings and severity classification of diabetic retinopathy”, monthly diabetes vol. 2 No. 3, 49-50, 2010 Lu, M., `` Advanced Glycation End Products Increase Retinal Vascular Endothelial Growth Factor Expression '', J Clin Invest 101 (6): 1219-1224, 1998 Lucentis Attached Document (revised March 2013)

  As described above, the conventional treatment of ophthalmic diseases accompanied by edema / bleeding due to increased vascular permeability mainly involves intraocular administration of invasive drugs, and suffers from patient physical distress and QOL reduction, Is associated with the risk of systemic side effects of the administered drug and no satisfactory medical treatment has been established. In addition, no pharmacological treatment has been found that can directly improve symptoms in pulmonary diseases with edema / bleeding.

  The object of the present invention is to develop ophthalmic diseases with edema / bleeding such as diabetic macular edema, wet age-related macular degeneration, myopic macular degeneration, acute lung injury, lung with edema / bleeding such as acute respiratory distress syndrome, etc. To provide a vascular permeability inhibitor with few side effects and high safety, which can treat diseases and the like medically, reduce physical pain of patients, and improve QOL of patients.

  The vascular permeability inhibitor of the present invention is a drug that suppresses vascular permeability used for the prevention or treatment of diseases associated with at least one of edema and hemorrhage caused by increased vascular permeability, and contains a nitrate. It is characterized by.

  The vascular permeability inhibitor of the present invention is preferably administered within a range of 5 μmol / kg to 1300 μmol / kg per day in terms of nitrate ion.

  The vascular permeability inhibitor of the present invention is preferably administered frequently so as to suppress fluctuations in nitrate ion concentration in plasma.

  Diseases accompanied by at least one of edema and bleeding due to increased vascular permeability in the present invention include diabetic macular edema, wet age-related macular degeneration, myopic macular degeneration, acute lung injury and acute respiratory distress syndrome More preferably, it is at least one selected from the group consisting of:

  According to the present invention, vascular permeability in ophthalmic diseases and pulmonary diseases is suppressed, and diabetic macular edema, wet age-related macular degeneration, myopic macular degeneration, acute lung injury, acute respiratory distress syndrome, etc. Prevention and treatment of ophthalmic diseases and pulmonary diseases with edema / bleeding that can prevent or treat diseases with bleeding without causing physical pain or reduction in QOL to patients with high safety and low side effects Medicine is provided.

FIG. 1A is a fluorescence contrast photograph of a retinal blood vessel showing the results of Experimental Example 1. FIG. 1A shows the case where control 1 is administered, and FIG. 1B shows the case where test compound 1 is administered. It is a graph which shows the number of edema generation | occurrence | production when test compound 1 and control 1 are each administered to STZ induction diabetes model mouse for 7 months. It is a graph which compares and shows the leakage amount of Evans blue at the time of administering each of the test compound 2 and the control 2 to the STZ-induced diabetes model mouse. It is a fluorescence contrast photograph which shows a mode that leukocytes have adhered to the blood vessel wall as a result of Experimental example 3. It is a graph which compares the number of leukocytes adhering to the retinal blood vessel at the time of administering each of the test compound 3 and the control 3 to STZ-induced diabetes model mice together with healthy mice. It is a graph which compares and shows the VEGF expression level in a retinal tissue at the time of administering each of the test compound 4 and the control 4 to STZ induction diabetes model mouse. FIG. 7A is an optical micrograph of a HE-stained specimen of the middle lobe of the lung shown as a result of Experimental Example 5 comparing the test compound administration group 5 and the control group 5, and FIG. b) shows the control group 5 respectively. FIG. 8A is an optical micrograph of a HE-stained specimen of the middle lobe of the lung shown as a result of Experimental Example 6 in comparison with the test compound administration group 6 and the control group 6. FIG. Shows the test compound administration group 6 respectively.

  In ophthalmological and pulmonary diseases, edema and hemorrhage are caused by blood cell adhesion and blood vessel permeability increased due to breakdown of tight junctions between vascular endothelial cells due to leukocyte adhesion to vascular endothelial cells and expression of cytokines such as vascular endothelial growth factor (VEGF). Generated by leakage of components. The inventor has an influence on the suppression of intravascular adhesion / aggregation of leukocytes involved in the enhancement of vascular permeability and the expression of cytokines such as VEGF, at a safe pharmaceutically acceptable dose of nitrate, It has been found that it has an action of suppressing vascular permeability. In particular, by increasing the nitrate concentration in the body, it suppresses vascular permeability in ophthalmic and pulmonary diseases, diabetic macular edema, wet age-related macular degeneration, myopic macular degeneration, acute lung injury, acute breathing It is possible to prevent and treat diseases accompanied by edema and bleeding such as impulsive syndrome.

Nitrate in the present invention include inorganic anions (nitrate ion: N0 3 ^_-) and may be a pharmaceutically acceptable salt formed from a cation, e.g., an alkali metal as the cation, alkaline earth metal, or an organic base Can be used. For example, sodium and potassium are preferable as the alkali metal, calcium and magnesium are preferable as the alkaline earth metal, and arginine and lysine are preferable as the organic base.

  Originally, nitrate is widely distributed in the natural world including soil, and is taken into the human body mainly by ingestion of leafy vegetables. For example, raw spinach leaves contain about 0.2 g of nitrate in 100 g of nitrate (converted by the Ministry of Education, Culture, Sports, Science and Technology, “Fiveth Amendment Japanese Food Standard Ingredients Table”). However, it is empirically known that it is a safe compound with no problems. The only evidence of nitrate toxicity is the development of methemoglobinemia due to overdose. As a result of detailed investigations by the inventor, it was confirmed that blood nitrate until 2000 μmol (about 170 mg) of sodium nitrate was administered per kg of body weight. It has been found that no increase in methemoglobin occurs.

  As a conventional technique in the ophthalmic field using nitrate, a method for controlling pathological angiogenesis of proliferative retinopathy has been disclosed (Patent Document 1), but the method is suitably applied to the end stage of diabetic retinopathy. Rather, studies and inventions have not yet been made on the effectiveness and application of nitrate to edema and bleeding caused by increased vascular permeability as in the present invention, which is preferably applied in the early stage of diabetic retinopathy. Absent.

  The preventive / therapeutic effect of the present invention for ophthalmic diseases and pulmonary diseases accompanied by edema / bleeding is defined by the daily nitrate ion dose, and the effective dose is 5 μmol / kg (body weight) to 1300 μmol / kg (body weight) per day. ), Preferably 10 μmol / kg (body weight) to 100 μmol / kg (body weight), more preferably 15 μmol / kg (body weight) to 75 μmol / kg (body weight).

  The vascular permeability inhibitor of the present invention is divided into effective daily doses multiple times (particularly preferably 3 times or more per day) as will be apparent from the results of Experimental Examples 1 and 2 described later. Are preferably administered. That is, the vascular permeability inhibitor of the present invention makes it easier to obtain this effect by reducing fluctuations in plasma nitrate ion concentration by frequent administration and constantly maintaining the nitrate ion concentration at a high level.

  Furthermore, as the dosage form of nitrate in the present invention, various dosage forms can be used regardless of parenteral or oral, since nitrate is easily dissolved in water and absorbability from the digestive tract is good. Examples of parenteral dosage forms include injections (not including injection into the vitreous) and inhalants, while oral dosage forms include tablets, capsules, granules, powders, and liquids for internal use. Etc. These preparations can be prepared using pharmaceutically acceptable pharmaceutical additives while paying attention to the change in formulation by using widely used pharmaceutical techniques. In addition, as described above, in order to obtain this effect more effectively, it is necessary to constantly increase the blood nitrate ion concentration, so that the preparation with controlled release brings out the effect of the present invention. It is a suitable dosage form. Moreover, the vascular permeability inhibitor of the present invention may be provided in the form of food for specified health use, health food, supplement, and the like.

  Hereinafter, although an example of an experiment is given and the present invention is explained in detail, the present invention is not limited to these.

<Experimental example 1: Evaluation of prevention and treatment effects of nitrate ophthalmic disease edema>
The effect of nitrate on the prevention and treatment of edema was evaluated using drug-induced diabetes model mice that are widely used as diabetes model animals.

(Preparation of test compound 1)
Sodium nitrate (Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water (Wako Pure Chemical Industries, Ltd.) as a test compound to prepare a sodium oral administration solution having a concentration of 3 mmol / L (Test Compound 1). For comparison of the effect, distilled water was used as a control solution (Control 1).

(experimental method)
1) Preparation of STZ-induced diabetes model mouse C57BL / 6J mice (Japan SLC, Inc.) were used as experimental animals. At 5 weeks after birth, 100 mg / kg body weight of streptozotocin (SIGMA, hereinafter abbreviated as “STZ”) injection was administered once a day for 2 days into the abdominal cavity of mice to induce diabetes. One week after STZ administration, the blood glucose level of all the heads was measured with Antsense Rose (Horiba, Ltd.), and 300-500 mg / dL was used for the experiment.

2) Evaluation test STZ-induced diabetes model mice were divided into two groups, a test compound 1 administration group (test compound administration group 1) and a control 1 administration group (control group 1), once a day for 7 months, sodium nitrate. Test compound 1 or control 1 corresponding to 15 μmol / kg (body weight) was intragastrically administered using an oral sonde.

  After 7 months from the start of administration of test compound 1 and control 1, the blood glucose level of each mouse was measured with Antsense Rose (Horiba, Ltd.). Subsequently, thoracotomy was performed under deep anesthesia, and Evans Blue (SIGMA) physiological saline solution (30 mg / mL) was perfused from 2 mL of the left ventricle over 2 minutes. Thereafter, the eyeball was removed and fixed with 4% paraformaldehyde phosphate buffer (Wako Pure Chemical Industries, Ltd.) for 4 hours. After fixation, the retina was detached and a retinal extension specimen was prepared. Microscopic observation was carried out using a fluorescent inverted microscope (Olympus IX71), and a site where Evans blue leaked from the retinal blood vessel was regarded as an edema occurrence site and evaluated.

(Results and effects)
FIG. 1 is a fluorescence contrast photograph of a retinal blood vessel showing the results of Experimental Example 1. FIG. 1 (a) shows the case where control 1 is administered, and FIG. 1 (b) shows the case where test compound 1 is administered. Yes. FIG. 2 shows the results of Experimental Example 1, when test compound 1 and control 1 were each administered to STZ-induced diabetes model mice for 7 months (control group 1: N = 4, test compound administration group 1: N = 6). ) Is a graph showing the number of edema occurrences, and the vertical axis shows the number of edema (pieces). Prior to the evaluation of edema by Evans Blue, blood glucose levels of test compound administration group 1 and control group 1 were measured. As a result, test compound administration group 1 had an average of 755.3 mg / dL and control group 1 had an average of 752.5 mg / dL. No effect on the primary disease (diabetes mellitus) by administration of test compound 1 was observed. However, the number of edema caused by increased blood vessel permeability due to hyperglycemia was reduced to about 1/3 in the test compound administration group 1 compared to the control group 1. From this result, it became clear that nitrate has the effect of preventing or treating edema that develops in ophthalmic diseases such as diabetic retinopathy.

<Experimental Example 2: Evaluation of inhibitory effect of nitrate on ocular disease vascular permeability>
In ophthalmological and pulmonary diseases, edema and hemorrhage are caused by blood cell adhesion and blood vessel permeability increased due to breakdown of tight junctions between vascular endothelial cells due to leukocyte adhesion to vascular endothelial cells and expression of cytokines such as vascular endothelial growth factor (VEGF). Generated by leakage of components. Regarding the inhibitory effect of nitrate on vascular permeability, a pigment (Evans blue) that specifically binds to albumin, a blood component, was administered into blood vessels using STZ-induced diabetic model mice. Evaluation was made by measuring the amount of leaked Evans Blue.

(Preparation of test compound 2)
Sodium nitrate (Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water (Wako Pure Chemical Industries, Ltd.) to prepare an aqueous sodium nitrate solution having a concentration of 150 mg / L (test compound 2). For comparison of the effect, distilled water was used as a control administration liquid (Control 2).

(experimental method)
1) Preparation of STZ-induced diabetes model mouse STZ-induced diabetes model mouse was prepared according to Experimental Example 1.

2) Evaluation test STZ-induced diabetes model mice were divided into two groups, a test compound 2 administration group (test compound administration group 2) and a control 2 administration group (control group 2), and each group was tested for 12 months. The solution was placed in a water bottle for mice (Natsume Seisakusho Co., Ltd.) and allowed to freely ingest. The amount of water consumed per day was calculated from the change in the weight of the water supply bottle containing the test solution. The average amount of sodium nitrate administered to the test compound administration group 2 during the evaluation test period was 1300 μmol / kg (body weight) per day.

  The vascular permeability evaluation test was performed after measuring the blood glucose level of each group of STZ-induced diabetes model mice using Antsense Rose (Horiba, Ltd.). First, Evans Blue saline with a concentration of 30 mg / mL was administered from the tail vein (dose: Evans Blue 45 mg / kg (body weight)), returned to the breeding gauge, and allowed to live as usual for 2 hours. Waited for leakage of blood components. Two hours later, blood was collected from the mouse jaw vein and used as a sample for measuring Evans blue concentration in plasma. Subsequently, deep anesthesia was performed, and 1% paraformaldehyde-citric acid aqueous solution (paraformaldehyde (Wako Pure Chemical Industries, Ltd.) 0.05M citric acid (Wako Pure Chemical Industries, Ltd.) at a dose of 1 mL / 5 g (mouse body weight) from the left ventricle. Kogyo Co., Ltd.) was dissolved in aqueous solution) to wash the blood. Thereafter, the eyeball was quickly removed, and the retina was peeled off and dried at 4 ° C. until a constant weight was obtained, and the dry weight of the retina was measured. Evans blue leaked into the retinal tissue was extracted by adding 30 μL of formamide (Wako Pure Chemical Industries, Ltd.) to one retina and heating at 70 ° C. for 18 hours. The extracted solution was centrifuged at 10,000 G for 20 minutes at 4 ° C., and the supernatant was used as a sample for measuring Evans blue concentration in retinal tissue. The Evans blue concentration in plasma and retinal tissue was calculated by measuring absorbance at a wavelength of 620 nm using an absorptiometer (JASCO Corporation, V-670). The amount of Evans Blue leakage was calculated using the following formula.

Evans blue leakage = (Evans blue concentration in retinal tissue / retinal weight) / (Evans blue concentration in plasma x perfusion time)
(Results and effects)
FIG. 3 shows the results of Experimental Example 2 in which Evans Blue when test compound 2 and control 2 were each administered to STZ-induced diabetes model mice (control group 2: N = 6, test compound administration group 2: N = 4). Is a graph showing the amount of leakage of Evans blue, and the vertical axis represents the amount of Evans blue leakage (μg · μL / g · μg · h). Prior to the evaluation of vascular permeability with Evans Blue, the blood glucose levels of test compound administration group 2 and control group 2 were measured. As a result, test compound administration group 2 averaged 397.0 mg / dL, and control group 2 averaged 412.3 mg / dL. There was no significant difference, and there was no effect on the primary disease (diabetes mellitus) by administration of test compound 2. However, the amount of Evans blue leaked into the retinal tissue, which is an indicator of increased vascular permeability, was reduced to about 1/12 in the test compound administration group 2 compared to the control group 2. From this result, it became clear that nitrate has the effect of suppressing the increase in vascular permeability caused by hyperglycemia.

<Experimental example 3: Evaluation of leukocyte adhesion / aggregation inhibitory effect of nitrate on intraocular capillaries>
The inhibitory effect of nitrate on the adhesion / aggregation of leukocytes to the vascular endothelium causing hypervascular permeability was evaluated using STZ-induced diabetes model mice.

(Preparation of test compound 3)
Sodium nitrate (Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water (Wako Pure Chemical Industries, Ltd.) to prepare an aqueous sodium nitrate solution having a concentration of 150 mg / L (test compound 3). For comparison of the effect, distilled water was used as a control solution (Control 3).

(experimental method)
1) Preparation of STZ-induced diabetes model mouse STZ-induced diabetes model mouse was prepared according to Experimental Example 1.

2) Evaluation test STZ-induced diabetes model mice were divided into two groups, a test compound 3 administration group (test compound administration group 3) and a control 3 administration group (control group 3), and each group was tested for 6 months. The solution was placed in a water bottle for mice (Natsume Seisakusho Co., Ltd.) and allowed to freely ingest. The amount of water consumed per day was calculated from the change in the weight of the water supply bottle containing the test solution. The average amount of sodium nitrate administered to the test compound administration group 3 during the evaluation test period was 1300 μmol / kg (body weight) per day.

  The test for evaluating adhesion / aggregation of leukocytes to the vascular endothelium was conducted by utilizing the adsorption property of leukocytes of concanavalin A (ConA) labeled with fluorescence (FITC). First, STZ-induced diabetic mice were opened under deep anesthesia, and after right atrial appendage incision, FITC-ConA solution with a concentration of 40 μg / mL (FITC-ConA (Vector Laboratories) dissolved in phosphate buffer (Life Technologies Japan))) 2.5 mL was perfused from the left ventricle at a rate of 1 mL / min. Next, 10 mL of phosphate buffer (Life Technologies Japan) was perfused systemically to wash away leukocytes that did not adhere to the vascular endothelium. Thereafter, the eyeball was quickly removed, fixed with 4% paraformaldehyde phosphate buffer (Wako Pure Chemical Industries, Ltd.) for 4 hours, and the retina was detached to prepare a retinal extension specimen. For microscopic observation, a fluorescent inverted microscope (Olympus IX71) was used, and the number of white blood cells adhered to the vascular endothelium per retina was counted and evaluated.

(Results and effects)
FIG. 4 is a fluorescence contrast photograph showing a state in which leukocytes are adhered to the blood vessel wall as a result of Experimental Example 3, and the tip of the white arrow in the photograph indicates the leukocytes adhered to the blood vessel wall. FIG. 5 shows, as a result of Experimental Example 3, the number of leukocytes adhering to the retinal blood vessels when the test compound 3 and the control 3 were administered to STZ-induced diabetes model mice, respectively, compared with healthy mice (control group 3: N = 4, test compound administration group 3: N = 4, healthy mice: N = 2) graph, and the vertical axis represents the number of adherent leukocytes per retina (number). From the onset of diabetes, the number of retinal vascular endothelial adhesion leukocytes in control group 3, which had been administered distilled water for 6 months, was about 10 times higher than that in healthy mice. However, in the test compound administration group 3 which continued to administer the aqueous sodium nitrate solution for 6 months, the number of adherent leukocytes decreased to about 5/9 of the control group 3. From this result, it became clear that nitrate has an effect of suppressing leukocyte adhesion and aggregation to intraocular capillaries caused by hyperglycemia.

<Experimental Example 4: Evaluation of the Effect of Nitrate on Vascular Endothelial Growth Factor Expression>
The effect of nitrate on the expression of vascular endothelial growth factor (VEGF), a protein involved in increased vascular permeability, was evaluated using STZ-induced diabetes model mice.

(Preparation of test compound 4)
Sodium nitrate (Wako Pure Chemical Industries, Ltd.) was dissolved in distilled water (Wako Pure Chemical Industries, Ltd.) to prepare an aqueous sodium nitrate solution having a concentration of 150 mg / L (test compound 4). For comparison of the effect, distilled water was used as a control administration liquid (Control 4).

(experimental method)
1) Preparation of STZ-induced diabetes model mouse STZ-induced diabetes model mouse was prepared according to Experimental Example 1.

2) Evaluation test STZ-induced diabetes model mice were divided into two groups, a test compound 4 administration group (test compound administration group 4) and a control 4 administration group (control group 4), and each group was tested for 7 months. The solution was placed in a water bottle for mice (Natsume Seisakusho Co., Ltd.) and allowed to freely ingest. The amount of water consumed per day was calculated from the change in the weight of the water supply bottle containing the test solution. The average amount of sodium nitrate administered to the test compound administration group 4 during the evaluation test period was 1300 μmol / kg (body weight) per day.

  Quantification of VEGF in retinal tissue was performed by SDS-PAGE and Western blotting. First, the eyeballs of STZ-induced diabetic mice were removed under deep anesthesia, the retina was detached and dissolved with M-PER (Thermo Scientific). About the obtained retinal solution, VEGF was isolate | separated by the measurement of a total protein amount, and SDS-PAGE. The amount of VEGF expressed was detected by Western blotting using an anti-VEGF antibody (abcam). Immunostar LD (Wako Pure Chemical Industries) was used as a detection reagent. The detection results were quantified by a densitometry method using ImageJ (NIH).

(Results and effects)
FIG. 6 shows a comparison of VEGF expression levels in retinal tissues when test compound 4 and control 4 were each administered to STZ-induced diabetes model mice as a result of Experimental Example 4 (control group 4: N = 7, Test compound administration group 4: N = 3) graph, the vertical axis is the relative value of VEGF expression level (control group 4 is 1). The VEGF expression level in the retinal tissue of the test compound administration group 4 which continued to administer the sodium nitrate aqueous solution for 7 months from the onset of diabetes decreased by about 60% compared to the control group 4. From this result, it became clear that nitrate influences the expression of VEGF, which is a protein involved in vascular permeability enhancement.

<Experimental Example 5: Evaluation of therapeutic effect on lung injury accompanied by nitrate edema and bleeding>
The effect of nitrate on pulmonary disease with edema / bleeding was evaluated using a lung injury model mouse in which pulmonary injury with edema / bleeding was induced by administration of high-concentration oxygen.

(Preparation of test compound 5)
Sodium nitrate (Wako Pure Chemical Industries, Ltd.) was dissolved in physiological saline for injection (Otsuka Pharmaceutical Co., Ltd.) to prepare a sodium nitrate injection solution having a concentration of 20 mmol / L (test compound 5). For comparison of effects, physiological saline for injection was used as a control administration solution (Control 5).

(experimental method)
1) Preparation of lung injury model mouse C57BL / 6J mice (Japan SLC Co., Ltd.) were used as experimental animals. In a cage in which a newborn mouse was controlled to a high concentration oxygen (75 ± 1% O ₂ ) state by an oxygen control apparatus (Biosphere, Ltd., model number: PRO-OX 110) together with a parent mouse from 1 to 12 days after birth Reared in From the 12th day onward, it was returned to atmospheric pressure conditions (21% O ₂ ) and reared until the 17th day after birth.

2) Evaluation test The lung injury model mice were divided into two groups, a test compound 5 administration group (test compound administration group 5) and a control 5 administration group (control group 5), once a day, from 12 days to 17 days after birth. The test compound 5 or the control 5 corresponding to 100 μmol / kg (body weight) of sodium nitrate was subcutaneously injected into the neck for 6 days.

  On day 17 after birth, the mice were euthanized by deep anesthesia, the lungs were removed, and fixed in 4% paraformaldehyde phosphate buffer (Wako Pure Chemical Industries, Ltd.) for 48 hours. After fixation, each lung lobe was cut out, dehydrated with an ethanol series, and embedded in paraffin. Thereafter, the lung lobe was sliced 4 μm thick, and after deparaffinization, hematoxylin-eosin staining (HE staining) was performed to obtain a light microscope specimen. For the microscopic observation, an inverted microscope (Olympus IX71) was used to evaluate sites of injury such as edema / bleeding sites and alveolar wall damage.

(Results and effects)
FIG. 7 is an optical micrograph of a HE-stained specimen of the middle lobe of the lung showing the test compound administration group 5 and the control group 5 as a result of Experimental Example 5, and FIG. 7 (a) shows the test compound administration group 5 FIG. 7B shows the control group 5 respectively. In the middle lobe of the control group 5 (FIG. 7 (b)), it was observed that alveolar walls were damaged and inflamed due to high-concentration oxygen, and edema / bleeding occurred over a wide area (FIG. 7 (b)). ) Inside, circle). On the other hand, in the test compound administration group 5, edema / bleeding was suppressed and the alveolar wall was repaired (FIG. 7 (a)). From these results, it was suggested that nitrate has an effect of treating edema / bleeding that occurs in inflammatory lung diseases.

<Experimental Example 6: Evaluation of inhibitory effect of nitrate on leukocyte aggregation / infiltration in lung injury tissue>
Using a high-concentration oxygen-induced lung injury model mouse, the inhibitory effect of nitrate on the aggregation / wetting of leukocytes to the inflamed site involved in increased vascular permeability was evaluated.

(Preparation of test compound 6)
Sodium nitrate (Wako Pure Chemical Industries, Ltd.) was dissolved in physiological saline for injection (Otsuka Pharmaceutical Co., Ltd.) to prepare a sodium nitrate injection solution (test compound 6) having a concentration of 20 mmol / L. For comparison of effects, physiological saline for injection was used as a control administration solution (Control 6).

(experimental method)
1) Preparation of a lung injury model mouse A lung injury model mouse was prepared according to Experimental Example 5.

2) Evaluation test The lung injury model mice were divided into two groups, a test compound 6 administration group (test compound administration group 6) and a control 6 administration group (control group 6), once a day, from 12 days to 17 days after birth. Test compound 6 or control 6 corresponding to 100 μmol / kg (body weight) of sodium nitrate was subcutaneously injected into the neck for 6 days.

  On day 17 after birth, the mice were euthanized by deep anesthesia, the lungs were removed, and fixed in 4% paraformaldehyde phosphate buffer (Wako Pure Chemical Industries, Ltd.) for 48 hours. After fixation, each lung lobe was cut out, dehydrated with an ethanol series, and embedded in paraffin. Thereafter, the lung lobe was sliced 4 μm thick, and after deparaffinization, hematoxylin-eosin staining (HE staining) was performed to obtain a light microscope specimen. For microscopic observation, an inverted microscope (Olympus IX71) was used to examine leukocytes that were aggregated and wetted at the alveolar inflammation site.

(Results and effects)
FIG. 8 is an optical micrograph of a HE-stained specimen of the middle lobe of the lung showing the test compound administration group 6 and the control group 6 as a result of Experimental Example 6, and FIG. 8 (a) shows the control group 6 and FIG. 8 (b) shows the test compound administration group 6 respectively. In the control group 6 and the test compound administration group 6, the site where the alveolar wall was damaged or inflamed due to the high concentration of oxygen and the blood component leaked into the alveoli was scrutinized. (The tip of the white arrow in FIG. 8A) was occasionally found, but was hardly confirmed in the test compound administration group 6 (FIG. 8B). From these results, it was suggested that nitrate has an effect of suppressing the aggregation and infiltration of leukocytes in lung injury tissues.

  The embodiments and experimental examples disclosed this time should be considered as illustrative in all points and not restrictive. The scope of the present invention is defined by the terms of the claims, rather than the description above, and is intended to include any modifications within the scope and meaning equivalent to the terms of the claims.

Claims (4)

A drug that suppresses vascular permeability used for the prevention or treatment of a disease associated with at least one of edema and bleeding caused by increased vascular permeability, comprising nitrate ion (NO ₃ ⁻ ) as an anion , sodium, potassium An inhibitor of vascular permeability, comprising nitrate containing calcium, magnesium, arginine or lysine as a cation .   The vascular permeability inhibitor according to claim 1, which is administered within a range of 5 µmol / kg to 1300 µmol / kg per day in terms of nitrate ion.   The vascular permeability inhibitor according to claim 1 or 2, which is administered frequently so as to suppress fluctuations in nitrate ion concentration in plasma.   A disease with at least one of edema and bleeding caused by increased vascular permeability is from the group consisting of diabetic macular edema, wet age-related macular degeneration, myopic macular degeneration, acute lung injury and acute respiratory distress syndrome The vascular permeability inhibitor according to any one of claims 1 to 3, which is at least one selected.