US20120114768A1 - Angiogenesis regulating composition and method for regulating angiogenesis - Google Patents

Angiogenesis regulating composition and method for regulating angiogenesis Download PDF

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US20120114768A1
US20120114768A1 US13/383,805 US201013383805A US2012114768A1 US 20120114768 A1 US20120114768 A1 US 20120114768A1 US 201013383805 A US201013383805 A US 201013383805A US 2012114768 A1 US2012114768 A1 US 2012114768A1
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nitrate
nitrite
angiogenesis
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Junya Fujimori
Hidekazu Baba
Toshinori Murata
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Air Water Inc
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A61P27/06—Antiglaucoma agents or miotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system

Definitions

  • the present invention relates to a composition used to regulate angiogenesis to treat and prevent angiogenic diseases, and a method for regulating angiogenesis.
  • Angiogenesis refers to formation of a new blood vessel branch by endothelial cell proliferation and migration and remodeling from a pre-existing blood vessel of a tissue.
  • Angiogenesis is started by induced expression of VEGF (vascular endothelial growth factor) mRNA which occurs by activation of a transcription factor HIF (hypoxia-inducible factor) responding to ischemia-based hypoxia.
  • HIF hypooxia-inducible factor
  • the produced VEGF binds to a VEGF receptor (VEGFR) localized in a vascular endothelial cell, and activates an intracellular signaling pathway. This results in decomposed extracellular basement membrane, followed stepwise by vascular endothelial cell migration/proliferation, lumen formation of vascular endothelial cell, basement membrane formation, and pericyte enclosure, finally resulting in angiogenesis.
  • VEGF vascular endothelial growth factor
  • PI3K phosphatidylinositol-3-kinase
  • NPL 2 Ziche M. et al., “Nitric Oxide Promotes Proliferation and Plasminogen Activator Production by Coronary Venular Endothelium Through Endogenous bFGF”, Circulation Research, Vol. 80, pp. 845-852 (1997) (NPL 3), and Dimmeler S. et al., “Upregulation of Superoxide Dismutase and Nitric Oxide Synthase Mediates the Apoptosis-Suppressive Effects of Shear Stress on Endothelial Cells”, Arterioscler. Thromb. Vasc. Biol., Vol. 19, pp. 656-664 (1999) (NPL 4), currently it is believed that endogenous nitrogen monoxide promotes angiogenesis by its vascular endothelial cell proliferation and migration promoting effect and an apoptosis-suppressive effect.
  • angiogenesis has a large influence on wound-healing and the progress of many diseases.
  • Diseases known as involving angiogenesis include retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration, neovascular glaucoma and other ophthalmologic diseases. rheumatoid arthritis and other inflammatory diseases, solid tumors and other malignant neoplasms, and the like, and these diseases present pathologic neovascularization lacking a regulation mechanism.
  • pathologic neovascularization has been treated strategically consistently by its suppression and inhibition, generally categorized into surgical treatment and medication, as disclosed in Japanese Patent Laying-Open No. 2002-284685 (PTL 1) and Japanese Patent Laying-Open No. 2008-110950 (PTL 2).
  • VEGF antibody Bevacizumab
  • nucleic acid Pegaptanib sodium
  • the anti-VEGF antibody inhibits physiological neovascularization and causes systemic wound-healing retardation, cerebral hemorrhage, cerebrovascular accidents, myocardial infarction, angina pectoris, and other serious side effects.
  • VEGF inhibitor and other similar ophthalmovascular therapeutic agents are administered to a patient through intravitreous injection to alleviate a systemic side effect, this treatment imposes a large burden on the patients, and the intravitreous injection not only injures the patients' ocular tissues but will also constantly expose their lenses and retinal tissues to a risk of bacterial infection.
  • NPL 1 Murohara T. et al., “Nitric Oxide and Angiogenesis in Cardiovascular Disease”, Antioxidants & Redox Signaling, Vol. 4, pp. 825-831 (2002)
  • NPL 2 Noiri E. et al., “Podokinesis in endothelial cell migration: role of nitric oxide”, The American Physiological Society, Vol. 274, pp. 236-244 (1998)
  • NPL 3 Ziche M. et al., “Nitric Oxide Promotes Proliferation and Plasminogen Activator Production by Coronary Venular Endothelium Through Endogenous bFGF”, Circulation Research, Vol. 80, pp. 845-852 (1997)
  • NPL 4 Dimmeler S. et al., “Upregulation of Superoxide Dismutase and Nitric Oxide Synthase Mediates the Apoptosis-Suppressive Effects of Shear Stress on Endothelial Cells”, Arterioscler. Thromb. Vasc. Biol., Vol. 19, pp. 656-664 (1999)
  • NPL 5 Eyal B. et al., “T2-TrpRS Inhibits Preretinal Neovascularization and Enhances Physiological Vascular Regrowth in OIR as Assessed by a New Method of Quantification”, Investigative Ophthalmology & Visual Science, Vol. 47, pp. 2125-2134 (2006)
  • NPL 6 Katsumi Y. et al., “Metabolic Fate of Nitric Oxide”, Int ArchOccup Environ Health, Vol. 46, pp. 71-77 (1980)
  • NPL 7 Zhi H. et al., “Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control”, The Journal of Clinical Investigation, Vol. 115, pp. 2099-2107 (2005)
  • NPL 8 Lois E. H. Smith et al., “Oxygen-Induced Retinopathy in the Mouse”, Investigative Ophthalmology Visual Science, Vol. 35, pp. 101-111 (1994)
  • ophthalmologic angiogenic diseases have conventionally been treated mainly by surgical treatment accompanied by compensatory tissue destruction, or intravitreous medication with a high risk of inhibited physiological neovascularization, a critical systemic side effect, and bacterial infection, and there has been no established satisfactory surgical or medical treatment methodology.
  • the present invention has been made to address the above issue, and it contemplates a composition effective in treating and preventing angiogenic diseases, that can medically control angiogenic diseases, and medically regulate angiogenesis in ophthalmologic diseases in particular, and allows treatment without inhibiting physiological neovascularization, with a limited side effect and significantly safely, and an angiogenesis regulation method using the same.
  • nitrate/nitrite administered in a pharmaceutically acceptable amount has an effect regulating a process of angiogenesis.
  • the present inventors have revealed that nitrate/nitrite can regulate angiogenesis in ophthalmologic disease and suppress pathologic neovascularization without inhibiting physiological neovascularization, found out that these compounds are useful as a therapeutic and preventive medicine for ophthalmologic diseases such as retinopathy of prematurity, diabetic retinopathy (proliferative diabetic retinopathy, in particular), age-related macular degeneration, and neovascular glaucoma, and thus completed the present invention. That is, the present invention is as follows:
  • the present invention provides an angiogenesis regulating composition used to treat/prevent an angiogenic disease in a subject, characterized by containing in an effective amount at least one of nitrate, nitrite, and a compound convertible into one of nitrate and nitrite after it is absorbed into the subject.
  • the present composition regulates angiogenesis by maintaining nitrate ions in an effective concentration range in the subject.
  • the present composition contains at least one of nitrate, nitrite, and the compound in an amount providing a plasma concentration of nitrate ions within a range of 8 ⁇ mol/L to 1000 ⁇ mol/L when the composition is administered to the subject.
  • the angiogenic disease is at least one ophthalmologic disease selected from retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration, and neovascular glaucoma.
  • the present composition is a pharmaceutical composition or at least one selected from health food, functional food, food for specified health use, and dietary supplement.
  • the present invention also provides an angiogenesis regulation method administering to a subject a composition containing as an active ingredient at least one of nitrate, nitrite, and a compound convertible into one of nitrate and nitrite after it is absorbed into the subject.
  • the present method regulates angiogenesis by maintaining nitrate ions in an effective concentration range in the subject.
  • the present method provides a plasma concentration of nitrate ions in the subject within a range of 8 ⁇ mol/L to 1000 ⁇ mol/L.
  • the present invention can thus provide a composition effective in treating and preventing angiogenic diseases, that can medically control angiogenic diseases, and medically regulate angiogenesis in ophthalmologic diseases in particular, and allows treatment without inhibiting physiological neovascularization, with a limited side effect and significantly safely, and an angiogenesis regulation method using the same.
  • FIG. 1 shows a fluorescence imaging of retinal vessel obtained as a result of an exemplary experiment 1
  • FIGS. 1( a ), 1 ( b ), 1 ( c ) and 1 ( d ) are indicated in the case of administration of 15 ⁇ mol/kg of sodium nitrite, 75 ⁇ mol/kg of sodium nitrite, 75 ⁇ mol/kg of sodium nitrate, and physiological saline, respectively.
  • FIG. 2 is a graph which shows the comparison of the number of pathological vessels entered into the vitreous body in the case of administration of physiological saline, 15 ⁇ mol/kg of sodium nitrate, 15 ⁇ mol/kg of sodium nitrite and 75 ⁇ mol/kg of sodium nitrite, with the ordinate showing the number of pathological vessels.
  • FIG. 3 shows an image of retina in cross section when physiological saline is administered as a control.
  • FIG. 4 shows a fluorescence imaging of retinal vessel obtained as a result of an exemplary experiment 3
  • FIGS. 4( a ), 4 ( b ), and 4 ( c ) are indicated in the case of administration of 15 ⁇ mol/kg of sodium nitrate, 15 ⁇ mol/kg of sodium nitrite, and physiological saline, respectively.
  • FIG. 5 shows a fluorescence imaging of retinal vessel obtained as a result of an exemplary experiment 4, and FIGS. 5( a ), 5 ( b ), and 5 ( c ) are indicated in the case of administration of sodium nitroprusside (SNP), nitroglycerin (NTG), and 15 ⁇ mol/kg of sodium nitrite, respectively.
  • SNP sodium nitroprusside
  • NTG nitroglycerin
  • 15 ⁇ mol/kg of sodium nitrite respectively.
  • FIG. 6 shows a fluorescence imaging of retinal vessel obtained as a result of an exemplary experiment 5
  • FIGS. 6( a ) and 6 ( b ) are indicated in the case of administration of anti-VEGF antibody, and 15 ⁇ mol/kg of sodium nitrite, respectively.
  • FIG. 7 shows transition of plasma concentration of nitrate ions with time when 15 ⁇ mol/kg of sodium nitrite is administered to a mouse subcutaneously in a cervical region, as obtained as a result of an exemplary experiment 6.
  • nitrate is widely distributed in the nature including soil, and is incorporated into human body mainly through intake of leaf vegetables. Nitrate is absorbed via a digestive tract satisfactorily and approximately 25% of nitrate having entered systemic circulation is secreted in saliva. A portion (approximately 30%) of nitrate in saliva is reduced to nitrite by dental bacteria and is re-absorbed via a digestive tract or is reduced to nitrogen monoxide by stomach acid and thus re-absorbed via a digestive tract. It has been reported that a healthy adult has a plasma concentration of nitrate ions of 10 ⁇ mol/L to 71 ⁇ mol/L and a plasma concentration of nitrite ions of 0.15 ⁇ mol/L to 1 ⁇ mol/L.
  • the present inventors have found that the plasma concentration of nitrate and nitrite ions is commonly low in premature babies, subjects undergoing a high-concentration of oxygen therapy, diabetics, hypertensives and the like, and that angiogenesis can be regulated by recovering these ion concentrations in plasma to a normal value or increasing them to a therapeutically effective concentration. That is, the present composition supplements nitrate ions and nitrite ions that should inherently be present in the body to maintain homeostasis of angiogenesis, and the present composition is thus also applicable to newborns significantly safely.
  • the present composition is a composition which regulates angiogenesis that is used to treat/prevent diseases of subjects that are accompanied by angiogenesis, and it is characterized by containing at least one of nitrate, nitrite, and a compound convertible into one of nitrate and nitrite after it is absorbed into the subject (hereinafter generally referred to as an “active ingredient”) in an amount effective in treating/preventing angiogenic diseases.
  • nitrate/nitrite is only required to be a pharmaceutically acceptable salt formed of inorganic anions (nitrate ion: NO 3 , nitrite ion: NO 2 ) and cations, and for example the cation can be of an alkaline metal, an alkaline earth metal, or an organic base.
  • preferable alkaline metals are sodium and potassium
  • preferable alkaline earth metals are calcium and magnesium
  • preferable organic bases are arginine and lysine.
  • the present compound convertible into one of nitrate and nitrite after it is absorbed into a subject means a compound which is absorbed through oral/parenteral administration, rapidly metabolized, and circulates systemically in the form of nitrate or nitrite.
  • a compound is exemplified by nitric oxide, such as nitrogen monoxide, as is also described in Katsumi Y. et al., “Metabolic Fate of Nitric Oxide”, Int ArchOccup Environ Health, Vol. 46, pp. 71-77 (1980) (NPL 6).
  • nitrate has not been reported to have particular serious side effect, however nitrite has been reported to cause methemoglobinemia as acute and subacute toxicity.
  • the amount of nitrite administered to exhibit the effect of the present invention is approximately 1 ⁇ 8 or smaller of a maximal no-effect dose for methemoglobinemia reported for Rodentia (84 mg/kg/day for sodium nitrite) and thus also provides a safe treatment for newborns.
  • endogenous nitrogen monoxide has an effect to promote proliferation and migration of vascular endothelial cells and plays an important role in angiogenesis, and it is believed that the angiogenesis regulation mechanism by nitrate and nitrite in the present invention involves a mechanism of conversion of nitrite ions to nitrogen monoxide that proceeds in a condition with tissue ischemia, such as described for example in Zhi H. et al., “Enzymatic function of hemoglobin as a nitrite reductase that produces NO under allosteric control”, The Journal of Clinical Investigation, Vol. 115, pp. 2099-2107 (2005) (NPL 7).
  • sodium nitroprusside (SNP), nitroglycerin (NTG), and other similar general-purpose nitrogen monoxide donor drugs are not recognized to effectively suppress aggravation of retinopathy or have an angiogenesis regulation effect, and accordingly, the effect of the present invention is a significantly specific pharmacological action limited to nitrate and nitrite.
  • VEGF vascular endothelial growth factor
  • the present composition preferably regulates angiogenesis by maintaining concentration of nitrate ions in a subject within an effective range. It enables to regulate angiogenesis in ophthalmologic diseases, inflammatory disease and malignant neoplasm, and in particular, it is effective in regulating angiogenesis in at least one ophthalmologic disease selected from retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration and neovascular glaucoma.
  • the present composition as described above can reduce retinal avascular area by suppressing pathologic neovascularization without inhibiting physiological revascularization, and it can suppress intravitreous pathologic neovascularization.
  • nitrite salt is rapidly oxidized by hemoglobin and present in blood mostly as nitrate salt (nitrite ions are 5% or less than nitrate ions), and, as described above, both nitrite and nitrate salts are mutually convertible in subject's body via intestine-salivary-gland circulation and so on, and for example, therapeutically effective concentration of present composition can be defined by plasma concentration of nitrate ions.
  • the present composition preferably contains an active ingredient in such an amount that the composition provides a plasma concentration of nitrate ions within a range of 8 ⁇ mol/L to 1000 ⁇ mol/L (suitably, 16 ⁇ mol/L to 400 ⁇ mol/L, more suitably 32 ⁇ mol/L to 200 ⁇ mol/L) when it is administered to a subject.
  • the present composition administered to a subject provides a plasma concentration of nitrate ions of less than 8 ⁇ mol/L, it is possible that disease treatment/prevention is not enough because of insufficient angiogenesis regulation.
  • the present composition administered to subject provides a plasma concentration of nitrate ions exceeding 1000 ⁇ mol/L, bleeding tendency is rarely increased in retinopathy and so on.
  • plasma concentration of nitrate ions can be determined by separating from admixture in plasma with high performance liquid chromatography followed by direct spectrophotometric measurement, or by the Griess method and so on.
  • the present composition may be prepared as a pharmaceutical preparation, or may be applied as a so-called supplement or something as at least one selected from health food, functional food, food for specified health use and dietary supplement.
  • the present composition is susceptible of various dosage forms regardless of oral or parenteral administration.
  • the composition can be administered through various routes such as intravenously, endoperitoneally, intramuscularly, subcutaneously, intrapulmonarily, nasally, and externally and locally, and its dosage form includes an injectable form, an inhalable form, and the like.
  • the oral dosage form is exemplified by tablets, capsules, granules, powder, peroral liquid preparations and so on, and nitrate and nitrite are released at desired site in digestive tract by preparing these pharmaceutical preparations for a gastric disintegrating system, an enteric coated system or a controlled release type.
  • These dosage forms can be prepared by common methods for pharmaceutical preparations with attention to incompatibility and with the use of pharmaceutically acceptable excipients.
  • controlled-release form is suitable for bringing out the effect of the present invention.
  • the amount of the present composition administered varies with age, body weight, the degree of progress of the disease, the manner of administration, or the administration route, it is not limited to a particular amount as long as it can exhibit the above pharmacological action and its side effect is within an acceptable range.
  • the specific amount of administration is described below: For example, when 15 ⁇ mol/kg/day of sodium nitrite is administrated to the model mice of oxygen-induced retinal angiogenesis, maximal plasma concentration of nitrate ions reaches around 100 ⁇ mol/L, and pathologic neovascularization is suppressed without inhibition of physiological retinal neovascularization.
  • administration period of the present composition is not limited, but preferably the composition is administrated as soon as pathologic neovascularization occurred because the present composition can regulate angiogenesis without inhibition of physiological neovascularization as described hereinafter in exemplary experiment 3.
  • an angiogenic disease treated or prevented by the present invention is at least one ophthalmologic disease selected from retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration and neovascular glaucoma
  • administration of the composition is preferably carried out from when a symptom of pathologic neovascularization accompanying the ophthalmologic disease is observed, until physiological neovascularization is sufficiently progressed and tissue ischemia is alleviated.
  • retinopathy of prematurity can be prevented from developing into aggravated symptoms by starting to administer the present medicament during high concentration of oxygen therapy, and diabetic retinopathy can be prevented from developing into aggravated symptoms by starting to administer the present medicament when it is observed that a retinal blood vessel bleeds/has edema.
  • the present invention also provides a method for regulating angiogenesis by administration of the present composition including an active ingredient which is at least one of nitrate, nitrite, and a compound convertible into nitrate or nitrite after it is absorbed into the subject.
  • an active ingredient which is at least one of nitrate, nitrite, and a compound convertible into nitrate or nitrite after it is absorbed into the subject.
  • the details of the present method are similar to description for the invention above.
  • angiogenesis by maintaining concentration of nitrate ions in a subject within an effective concentration range, and the range is preferably within 8-1000 ⁇ mol/L in plasma of a subject.
  • the present method is effective for treatment of wounds and angiogenetic disease which is in particular at least one ophthalmologic disease selected from retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration and neovascular glaucoma, and in the case of these ophthalmologic diseases, administration is preferably carried out from when a symptom of pathologic neovascularization accompanying the ophthalmologic disease is observed, until physiological neovascularization is sufficiently progressed and tissue ischemia is alleviated.
  • angiogenetic disease which is in particular at least one ophthalmologic disease selected from retinopathy of prematurity, diabetic retinopathy, age-related macular degeneration and neovascular glaucoma
  • administration is preferably carried out from when a symptom of pathologic neovascularization accompanying the ophthalmologic disease is observed, until physiological neovascularization is sufficiently progressed and tissue ischemia is alleviated.
  • the effect of sodium nitrate and sodium nitrite for reducing retinal avascular area was evaluated by the model mice of oxygen-induced retinal angiogenesis which were generally used for in vivo pathologic neovascularization model in a retinal angiogenic disease.
  • Sodium nitrate or sodium nitrite as a test compound was dissolved in physiological saline for injection to prepare a test compound injection of 3-15 ⁇ mol/L.
  • Neonatal mice were bred from the 7th day (P7) to the 12th day (P12) after birth together with a parent mouse in a cage of high levels of oxygen (75 ⁇ 1% of 0 2 ) controlled by oxygen control equipment (PRO-OX110, produced by Reming Bioinstruments Co) to provide high levels of oxygen (75 ⁇ 1% of O 2 ).
  • Retinal angiogenesis was induced by breeding under normoxic condition (21% of O 2 ) from the 12th day to the 17th day after birth.
  • Sodium nitrate or sodium nitrite was injected to a cervical region hypodermically once per day in amount of 15 or 75 ⁇ mol/kg for ten days from the 7th day (P7) to the 16th day (P16) after birth.
  • physiological saline as controls was similarly injected hypodermically instead of test compound.
  • FITC-dextran fluorescein-conjugated dextran
  • M.W. 2 ⁇ 10 6 fluorescein-conjugated dextran
  • SIGMA fluorescein-conjugated dextran
  • FIG. 1 showed a fluorescence imaging of retinal vessel obtained as a result of an exemplary experiment 1, and FIGS. 1( a ), 1 ( b ), 1 ( c ) and 1 ( d ) were indicated in the case of administration of 15 ⁇ mol/kg of sodium nitrite. 75 ⁇ mol/kg of sodium nitrite, 75 ⁇ mol/kg of sodium nitrate, and physiological saline, respectively. Also the average of values of retinal avascular area, the average of values of total retinal area and the average of values of calculated areal ratio of retinal avascular area were shown in Table
  • retinal avascular area was reduced by approximately 20% and approximately 40-60% as compared to control group by administration of nitrate and nitrite, respectively.
  • Sodium nitrate or sodium nitrite as a test compound was dissolved in physiological saline for injection to prepare a test compound injection of 3-15 ⁇ mol/L.
  • C57BL/6J mouse (SLC) was used as an experimental animal.
  • the model mice of oxygen-induced retinal angiogenesis were prepared according to exemplary experiment 1.
  • Sodium nitrate or sodium nitrite was injected to a cervical region hypodermically once per day in amount of 15 or 75 ⁇ mol/kg for ten days from the 7th day (P7) to the 16th day (P16) after birth.
  • physiological saline as controls was similarly injected hypodermically instead of test compound.
  • mice On the 17th day after birth (P17), the mice were euthanised by overdose of pentobarbital, and eyes were removed together with optic nerve. The eyes were washed in physiological saline, excessive tissues were excised, and the eyes were thus fixed in a 4% paraformaldehyde phosphate buffer for 12 hours. After the fixation, the eyes were dehydrated by immersing in a graded ethanol series of 70-100% sequentially and were embedded in paraffin.
  • paraffin blocks were sliced parallel to the optic nerve with 4- ⁇ m thickness at 50- ⁇ m intervals and stained with hematoxylin eosin (HE-stain), and subsequently, retinal sectioning specimens were observed with an inverted microscope (Olympus IX71) to count the number of pathological vessels breaking the inner limiting membrane and extending into the vitreum.
  • HE-stain hematoxylin eosin
  • FIG. 2 is a graph which showed the comparison of the number of pathological vessels entered into the vitreous body in the case of administration of physiological saline, 15 ⁇ mol/kg of sodium nitrate, 15 ⁇ mol/kg of sodium nitrite and 75 ⁇ mol/kg of sodium nitrite, with the ordinate showing the number of pathological vessels.
  • FIG. 3 showed an image of retina in cross section when physiological saline was administered as a control. At the part indicated by an arrow, it was found that pathological vessel broke the inner limiting membrane and extended to the vitreum. From graph in FIG. 2 , the number of pathological vessels breaking the inner limiting membrane and extending into the vitreum was decreased by administration of sodium nitrate or sodium nitrite.
  • retinal angiogenesis is induced by providing 75% oxygen followed by breeding in room air during P12-P17. Therefore the effect of angiogenesis regulation of the invention was clearly examined by administration of sodium nitrate or sodium nitrite from the beginning of retinal angiogenesis (or from P12).
  • Sodium nitrate or sodium nitrite as a test compound was dissolved in physiological saline for injection to prepare a test compound injection of 3 ⁇ mol/L.
  • FIG. 4 showed a fluorescence imaging of retinal vessel obtained as a result of an exemplary experiment 3, and FIGS. 4( a ), 4 ( b ), and 4 ( c ) were indicated in the case of administration of 15 ⁇ mol/kg of sodium nitrate, 15 ⁇ mol/kg of sodium nitrite, and physiological saline, respectively. Also the average of values of retinal avascular area, the average of values of total retinal area and the average of values of calculated areal ratio of retinal avascular area were shown in Table 2.
  • mice of oxygen-induced retinal angiogenesis presence/absence of an effect to reduce retinal avascular area was examined for a nitrogen monoxide donor drug generally used for angina pectoris, as an antihypertensive, and so on, to clarify that the present invention has an effect with specificity.
  • nitroglycerin as a test compound was dissolved in physiological saline for injection to prepare a test compound injection of 3 ⁇ mol/L.
  • SNP sodium nitroprusside
  • NTG nitroglycerin
  • SNP dehydrate as a comparative example was dissolved in physiological saline for injection to prepare a comparative example injection of 0.4 ⁇ mol/L.
  • 50 mg of nitroglycerin injection “HK” HAKARI PHARMACEUTICAL
  • FIG. 5 showed a fluorescence imaging of retinal vessel obtained as a result of an exemplary experiment 4, and FIGS. 5( a ), 5 ( b ), and 5 ( c ) were indicated in the case of administration of sodium nitroprusside (SNP), nitroglycerin (NTG), and 15 ⁇ mol/kg of sodium nitrite, respectively. Also the average of values of retinal avascular area, the average of values of total retinal area and the average of values of calculated areal ratio of retinal avascular area were shown in Table 3.
  • retinal avascular area was increased by approximately 2.5 times and approximately 3.3 times with respect to SNP and NTG, respectively, in comparison with a group administered with nitrite.
  • SNP nor NTG was observed to reduce a typical symptom of retinopathy, or avascular area, and it has been clarified that the effect of the present invention is specific to nitrate and nitrite.
  • retinopathy treatment effect between nitrite and an anti-VEGF antibody used for treatment of progressive colorectal cancer, age-related macular degeneration and so on.
  • Sodium nitrite as a test compound was dissolved in physiological saline for injection to prepare a test compound injection of 3 ⁇ mol/ml.
  • a murine monoclonal anti-VEGF antibody produced by SANTACRUZ BIOTECHNOLOGY, INC. was condensed to prepare 500 ⁇ g IgG/ml of an injection for the comparative example.
  • Sodium nitrite was injected to a cervical region hypodermically once per day in amount of 15 ⁇ mol/kg for ten days from the 7th day (P7) to the 16th day (P16) after birth.
  • the anti-VEGF antibody was injected through the corneal limbus to the vitreous body with a microsyringe equipped with a 33G hypodermic needle, by cutting mice eyelids open when oxygenation ended (on the 12th day after birth).
  • FIG. 6 showed a fluorescence imaging of retinal vessel obtained as a result of an exemplary experiment 5, and FIGS. 6( a ) and 6 ( b ) were indicated in the case of administration of anti-VEGF antibody, and 15 ⁇ mol/kg of sodium nitrite, respectively. Also the average of values of retinal avascular area, the average of values of total retinal area and the average of values of calculated areal ratio of retinal avascular area were shown in Table 4.
  • mice of oxygen-induced retinal angiogenesis an optimal plasma concentration of nitrate ions was estimated in the case where a significant therapeutic/preventive effect was observed for an ophthalmologic angiogenic disease by administration of the present composition.
  • Sodium nitrate or sodium nitrite as a test compound was dissolved in physiological saline for injection to prepare a test compound injection of 3-15 ⁇ mol/L.
  • C57BL/6J mouse (SLC) was used as an experimental animal.
  • the model mice of oxygen-induced retinal angiogenesis were prepared according to exemplary experiment 1.
  • Sodium nitrate or sodium nitrite was injected by single dose administration in amounts of 15, 75 and 150 ⁇ mol/kg, which were dosages observed as having a significant therapeutic/preventive effect in the present invention, to a cervical region hypodermically on the 12th day (P12) after birth when 75% oxygenation ended. Thereafter, isoflurane was used to anesthetize the mice, and 150 ⁇ l of blood was collected from the abdominal aorta over time. Plasma concentration of nitrate ions was measured by ion exchange chromatography using a normal phase column under the following conditions:
  • FIG. 7 showed transition of plasma concentration of nitrate ions with time when 15 ⁇ mol/kg of sodium nitrite was administered to a mouse subcutaneously in a cervical region, as obtained as a result of exemplary experiment 6. Furthermore, when sodium nitrate was administered in amounts of 15 ⁇ mol/kg, 75 ⁇ mol/kg, and 150 ⁇ mol/kg in a cervical region subcutaneously, it provided maximum plasma concentration of nitrate ions for each amount, averaged as shown in Table 5.
  • nitrite was absorbed within 1 hour after administration, and most thereof was rapidly oxidized by hemoglobin to nitrate, and systemically circulated. That is, in the present invention, it is also clear that when the present composition containing nitrite is administered to a subject, the present invention's therapeutic/preventive effect can be monitored by measuring and managing plasma concentration of nitrate ions, rather than measuring a significantly low plasma concentration of nitrite ions.
  • the table 5 result has revealed that a maximum plasma concentration of nitrate ions by which the present invention's therapeutic/preventive effect is achieved was provided when 150 ⁇ mol/kg of sodium nitrate was administered, and that it had a value of approximately 400 ⁇ mol/L.

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