JP6505408B2 - In vivo diagnostic and therapeutic composition for hypoxia related eye disease - Google Patents

Description

  The present invention relates to in vivo diagnostic and therapeutic compositions for hypoxia-related eye diseases.

  The presence of hypoxic regions in living tissues is known to be involved in various eye diseases, malignant transformation of cancer and the like.

  Pimonidazole (pimonidazole), which is a 2-nitroimidazole compound, is widely used as a reagent for detecting the hypoxic region in living tissue. Detection of the hypoxic region using pimonidazole is carried out by using pimonidazole binding to hypoxic cells. More specifically, first, pimonidazole is administered to a living body. Then, pimonidazole binds to hypoxic cells. Subsequently, a sample in which the target tissue is fixed by formalin fixation or the like is prepared, and stained with an anti-pimonidazole antibody to detect pimonidazole bound to hypoxic cells. This makes it possible to observe the hypoxic region. However, in order to detect the hypoxic region using pimonidazole, it is necessary to fix the target tissue, and the hypoxic region can not be detected in vivo.

  Therefore, development of a fluorescent probe capable of detecting a hypoxic region in vivo has been conducted. For example, Patent Document 1 is characterized by comprising a near infrared fluorescent dye, a hypoxic binding site that is activated by reductive metabolism in a hypoxic region to bind to a nucleophilic biomolecule, and a linker bond. A near infrared fluorescent probe for low oxygen region imaging has been described.

  Indirect evaluation by fluorescence fundus angiography is currently performed as a method of estimating hypoxia in hypoxia-related ocular diseases. Fluorescent fundus imaging is a method in which fluorescein, which is a fluorescent substance, is administered to a patient by drip infusion, and the blood vessels of the fundus are photographed with a fundus camera. This method is an old examination method with a history of over 50 years, but its usefulness makes it indispensable in ophthalmology medical care.

  As a similar fluorescence fundus imaging method, a method is known in which a fluorescent substance, indocyanine green (ICG), is administered to a patient by drip infusion, and a blood vessel of the fundus is photographed with a fundus camera. Since indocyanine green is a fluorescent substance that emits fluorescence in the near infrared region, it is easy to distinguish between the fluorescence of indocyanine green and the autofluorescence emitted by the living body. In addition, since near-infrared light has high permeability to living tissues, it is possible to observe deeper regions of tissues such as choroids with indocyanine green.

Unexamined-Japanese-Patent No. 2010-203966

  However, in fluorescein fundus angiography, hypoxia-related ocular diseases progress to the final stage, and capillary occlusion is not observed until avascular area and new blood vessels are observed, and the existence of hypoxic regions can be estimated and evaluated. It becomes. For this reason, it is not possible to directly evaluate the early to middle hypoxia area of hypoxia-related eye disease. In addition, in the case of fluorescent fundus imaging, there is a serious problem that there is a serious side effect of dying by anaphylactic shock at a rate of 1 in about 200,000.

  Further, the near-infrared fluorescent probe for low-oxygen region imaging described in Patent Document 1 has a high background and takes a long time to detect a signal because it always emits light without having an on-off function of fluorescence. There was a case.

  Under such circumstances, the present invention aims to provide a diagnostic agent capable of directly detecting the hypoxic region of the eye in vivo. Another object of the present invention is to provide a therapeutic composition capable of simultaneously performing diagnosis and treatment of hypoxia-related eye diseases.

The present invention is as follows.
[1] A diagnostic agent for in vivo hypoxia-related ocular disease, which comprises a hypoxic fluorescent probe as an active ingredient.
[2] The diagnostic agent according to [1], which is for intravitreal injection.
[3] The diagnostic agent according to claim 1 or 2, wherein the hypoxic fluorescent probe is a compound represented by the following formula (1).
[In Formula (1), R ¹ each independently represents H, COOH, SO ₃ ⁻ , or an alkoxy group having 1 to 3 carbon atoms, and R ² is absent or a linear or branched C 1 to 10 carbon atom] It represents a chain alkyl group, ^{R 3} represents an electron withdrawing group, ^{R 4} is an alkyl ^{_{group, - (CH 2) a COO}} -, - (CH 2) a SO 3 -, or - _(CH 2) _a O ^- represents, X ^- or absent ^{Cl -, Br -, I -} , or represents a ^{PF 6-,} n represents an integer of 1 to 4, a is an integer of 1-6. ]
[4] The diagnostic agent according to any one of claims 1 to 3, wherein the hypoxic fluorescent probe is a compound represented by the following formula (2).
[5] A composition for treating hypoxia-related eye disease, comprising the diagnostic agent according to any one of [1] to [4] and an angiogenesis inhibitor as active ingredients.
[6] The therapeutic composition according to [5], wherein the angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) capture substance.
[7] The therapeutic composition according to [6], wherein the VEGF capture agent is an anti-VEGF antibody.

  The present invention can provide a diagnostic agent capable of directly detecting the hypoxic region of the eye in vivo. In addition, it is possible to provide a therapeutic composition capable of simultaneously performing diagnosis and treatment of hypoxia-related eye disease.

(A) is a blood vessel cultured in the presence of a final concentration of 0.5, 1.0 and 1.5 μM of a hypoxic fluorescent probe (GPU-327) and under an environment of oxygen concentration of 1%, 5% and 20% It is the photograph which detected the fluorescence in endothelial progenitor cells (EPC). (B) to (d) are graphs of the fluorescence intensity of (a). (E) Umbilical vein endothelial cells (HUVEC) cultured in the presence of GPU-327 at a final concentration of 0.5, 1.0 and 1.5 μM and in an environment of oxygen concentration of 1%, 5% and 20% It is a photograph which detected fluorescence of (F) to (h) are graphs of the fluorescence intensity of (e). (A) is a graph showing the expression level of VEGF mRNA. (B) is a photograph which shows the result of the western blotting which detected expression of HIF-1 alpha protein and lamin B protein. (C) is a graph showing the amount of protein expression of (b). (A) and (b) is a photograph which shows a skin flap ischemia mouse model. (C) is a photograph showing the result of fluorescence observation. (A) is a photograph which shows the result of having observed fluorescence of GPU-327 in vivo. (B) is a photograph which shows the result of having observed in vitro the vascular structure of the retina of the eye observed by (a). (C) is a photograph which shows the result of having observed fluorescence of GPU-327 in the sample same as (b). (D) is a photograph in which the fluorescence images of (b) and (c) are integrated. (E) is a photograph which shows the result of having observed the cross section of the eye of the model produced by the same experimental method as (a)-(d) in vitro. (F) is a photograph which shows the result of having observed fluorescence of GPU-327 in the sample same as (e). (G) is the photograph which integrated the fluorescence image of (e) and (f). (A) is a photograph which shows the result of having observed fluorescence of GPU-327 in vivo. (B) is a photograph which shows the result of having observed in vitro the vascular structure of the retina of the eye observed by (a). (C) is a photograph which shows the result of having observed fluorescence of GPU-327 in the sample same as (b). (D) is a photograph in which the fluorescence images of (b) and (c) are integrated. (A) is a photograph which shows the result of having observed the cross section of the eye in vitro. (B) is a photograph which shows the result of having observed fluorescence of GPU-327 in the sample same as (a). (C) is the photograph which integrated the fluorescence image of (a) and (b). (A) and (b) is a photograph which shows the result of fluorescence observation. (C) is a graph of the fluorescence intensity detected in (b). (A), (b) and (c) are photographs of the fundus taken using a fundus camera two days, one week and two weeks after retinal vein occlusion. (D), (e) and (f) are photographs showing the results of observing fluorescence of GPU-327 in vivo using a fundus camera two days, one week and two weeks after retinal vein occlusion. It is a photograph which shows the result of fluorescence fundus imaging. (A) is the photograph which detected fluorescence of GPU-327 in vivo. (B) is a photograph of the fundus of the eye observed in (a). (C) is a photograph which shows the result of having observed in vitro the vascular structure of the retina of the eye observed by (a). (D) is a photograph showing the result of staining with pimonidazole in the same sample as (c). (E) is a photograph in which the fluorescence images of (c) and (d) are integrated. The arrows in (c), (d) and (e) indicate the occlusion site of the retinal vein. (A) is a graph showing an electroretinogram 60 days after administration of PBS or GPU-327. (B) is a graph showing the ratio of the amplitude of the right eye to the amplitude of the left eye for the a-wave measured at each stimulus intensity. (C) is a graph showing the ratio of the amplitude of the right eye to the amplitude of the left eye for the b-wave measured at each stimulus intensity. (D) is an optical micrograph of an eye of the GPU-327 administration group. (E) is a light micrograph of an eye of the PBS administration group.

[Diagnoses for hypoxia-related eye diseases]
In one embodiment, the present invention provides an in vivo diagnostic agent for hypoxia-related ocular disease, which comprises a hypoxic fluorescent probe as an active ingredient.

  Heretofore, there has been no method for visualizing hypoxic regions of the eye in vivo. On the other hand, the inventors can detect hypoxic-related eye disease by detecting the hypoxic region of the eye both in vitro and in vivo by applying the hypoxic fluorescent probe to the eye. It was revealed for the first time that there was something.

  According to the diagnostic agent of the present embodiment, the hypoxic region of the eye can be directly detected not only in vitro but also in vivo. This makes it possible to realize the direct evaluation of the early to middle hypoxic regions of hypoxia-related eye diseases, which could not be achieved conventionally.

  For example, in the case of young people with diabetic retinopathy, the activity of retinopathy is high and the progression is fast, so if the timing of treatment is missed, serious complications such as neovascular glaucoma occur and the vision prognosis becomes extremely poor. In such diseases, appropriate activity assessment, ie assessment of hypoxia, is very important. By directly evaluating the condition of the hypoxic region in the early to middle stages of the disease, less invasive early treatment can be performed.

  Moreover, in recent years, a relationship between diseases such as glaucoma and macular degeneration due to intense myopia, which are the main causes of blindness, and a decrease in blood flow has been pointed out. By observing the hypoxic region of the eye in vivo using the diagnostic agent of this embodiment, it is expected that it can also contribute to the investigation of the cause of these diseases.

(Hypoxia fluorescent probe)
A hypoxic fluorescent probe is a compound whose fluorescence characteristics change in an ordinary oxygen environment and a low oxygen environment. The hypoxic fluorescent probe is not particularly limited as long as its toxicity to humans and animals is within an acceptable range, and examples thereof include compounds whose fluorescent properties are largely changed by reductive metabolism in vivo under hypoxic conditions.

  The wavelength of fluorescence emitted by the low-oxygen fluorescent probe is not particularly limited, but is preferably fluorescence in the near infrared region (wavelength: 650 to 1000 nm). Near infrared fluorescence is easy to distinguish from autofluorescence emitted by a living body. In addition, near infrared light has high permeability to living tissues, and absorption by in vivo substances is also limited.

  Examples of hypoxic fluorescent probes that emit near-infrared fluorescence include cyanine dye derivatives and BODIPY dye derivatives.

  The change in fluorescence properties under hypoxic conditions of the hypoxic fluorescent probe may be reversible or irreversible. If changes in the fluorescent properties of the hypoxic fluorescent probe are reversible, the hypoxic environment is improved, and the fluorescent properties change when approaching the normal oxygen environment, so monitoring the temporal change of oxygen concentration Will also be possible.

As a more specific hypoxic fluorescent probe, for example, a compound represented by the following formula (1) can be mentioned.
[In Formula (1), R ¹ each independently represents H, COOH, SO ₃ ⁻ , or an alkoxy group having 1 to 3 carbon atoms, and R ² is absent or a linear or branched C 1 to 10 carbon atom] It represents a chain alkyl group, ^{R 3} represents an electron withdrawing group, ^{R 4} is an alkyl ^{_{group, - (CH 2) a COO}} -, - (CH 2) a SO 3 -, or - _(CH 2) _a O ^- represents, X ^- or absent ^{Cl -, Br -, I -} , or represents a ^{PF 6-,} n represents an integer of 1 to 4, a is an integer of 1-6. ]

  As the above-mentioned electron-withdrawing substituent, a nitro group, a cyano group, a halogen atom, a halogenated alkyl group, a halogenated aryl group, a halogenated heteroaryl group, a nitrogen-containing heteroaryl group, a carbonyl group, a sulfonyl group, a phosphoryl group, And alkynyl groups and the like.

The hypoxic fluorescent probe may be, for example, a compound represented by the following formula (2) (hereinafter sometimes referred to as “GPU-327”).

  GPU-327 shows almost no fluorescence, but when administered to a living body, it undergoes reductive metabolism in the hypoxic region, is converted to a compound shown in the following formula (3), is activated, and emits fluorescence in the near infrared region It will be. Moreover, since the compound shown to following formula (3) couple | bonds with nucleophilic biomolecules, such as a protein, and it stays in a low oxygen area for a while, a low oxygen area can be visualized. The compound represented by the following formula (3) is excreted from the body by metabolism after a certain period of time.

[In the formula (3) represents a _NO 2, _NH 2, NO or NHOH each ^{R 5} is independently. However, all of R ⁵ do not simultaneously become NO ₂ . ]

  As described below, the inventors revealed that GPU-327 enables visualizing the hypoxic region of the eye in vivo. In addition, it was confirmed that it is safe to administer GPU-327 to a living body.

  The dose for topical administration of GPU-327 may be, for example, 0.1 to 1000 μg. In particular, when administering GPU-327 by vitreous injection, the dose may be, for example, 0.1 to 50 μg, for example, 0.1 to 10 μg. In addition, the dose for systemic administration of GPU-327 may be, for example, 1 to 500 mg / 60 kg body weight, for example, 1 to 200 mg / 60 kg.

(Normal oxygen region and low oxygen region)
In the present specification, the normal oxygen region means a region where oxygen is present at a concentration corresponding to the concentration of oxygen with which cells in culture are in contact in a normal atmosphere having an oxygen concentration of about 20%. . The low oxygen region means a region in which the concentration of oxygen is lower than that of the ordinary oxygen region. In other words, "hypoxia area" means an area where the amount of oxygen present is less than that required for cell survival or cell healthy function.

(Oxygen related eye disease)
Examples of hypoxia-related eye diseases that can be diagnosed by the diagnostic agent of the present embodiment include diabetic retinopathy, retinopathy of prematurity, retinal vascular occlusion, macular degeneration, glaucoma and the like.

(Formulation and administration method)
The diagnostic agent of the present embodiment may be administered systemically or locally. In the case of topical administration, only a small amount of diagnostic agent is required, and the risk of side effects such as anaphylactic shock can also be reduced. Examples of topical administration include, for example, intravitreal injection. That is, the diagnostic agent of the present embodiment may be for intravitreal injection. As described later, the inventors revealed that the hypoxic region of the eye can be detected by administering the diagnostic agent by vitreous injection.

  The diagnostic agent of the present embodiment may be a diagnostic composition in which a pharmaceutically acceptable carrier is mixed. The diagnostic agent or the diagnostic composition may be, for example, orally in the form of a tablet, a coated tablet, a pill, a powder, a granule, a capsule, a solution, a suspension, an emulsion or the like, or an injection, a suppository, It can be administered parenterally in the form of a skin external preparation and the like.

  Pharmaceutically acceptable carriers (additives) include excipients, binders, disintegrants, lubricants, emulsifiers, stabilizers, diluents, thickeners, wetting agents, pH adjusters, for injections Solvent etc. are mentioned.

  Examples of the excipient include organic excipients, inorganic excipients and the like. Examples of organic excipients include sugar derivatives such as lactose and sucrose; starch derivatives such as corn starch and potato starch; cellulose derivatives such as crystalline cellulose; and gum arabic. Examples of inorganic excipients include sulfates such as calcium sulfate.

  As the binder, the above-mentioned excipients, gelatin, polyvinyl pyrrolidone, polyethylene glycol and the like can be mentioned.

  As the disintegrant, the above-mentioned excipients; starch or derivatives of cellulose such as croscarmellose sodium, sodium carboxymethyl starch etc .; crosslinked polyvinyl pyrrolidone etc. may be mentioned.

  Lubricants include talc, stearic acid, colloidal silica, waxes such as beeswax and geewol, sulfates such as sodium sulfate, lauryl sulfates such as sodium lauryl sulfate, starch derivatives in the above-mentioned excipients, etc. Be

  Emulsifying agents include colloidal clays such as bentonite and veegum; anionic surfactants such as sodium lauryl sulfate; cationic surfactants such as benzalkonium chloride; nonionic surfactants such as polyoxyethylene alkyl ether; Examples include (poly) glyceryl fatty acid ester surfactants such as polyglyceryl acid-10, polyglyceryl distearate-10, polyglyceryl tristearate-10, and polyglyceryl pentastearate-10.

  As the stabilizer, parahydroxybenzoic acid esters such as methylparaben and propylparaben; alcohols such as chlorobutanol; and phenols such as phenol and cresol.

  Examples of diluents include water, ethanol, propylene glycol and the like.

  As the thickener, xanthan gum, sodium alginate, polyvinyl alcohol, hydroxyethyl cellulose, sodium polyacrylate, carrageenan, sodium carboxymethylcellulose, polyvinyl pyrrolidone and the like can be mentioned.

  The wetting agent may, for example, be Sorbit, glycerin, concentrated glycerin, ethylene glycol, propylene glycol, 1,3-butylene glycol, polyethylene glycol, polypropylene glycol or the like.

  Examples of pH adjusters include phthalic acid, phosphoric acid, citric acid, succinic acid, acetic acid, fumaric acid, malic acid, carbonic acid and their potassium salts, sodium salts or ammonium salts, sodium hydroxide and the like.

  Examples of solvents for injection include water, ethanol, glycerin and the like.

  The diagnostic agent or diagnostic composition of the present embodiment contains a hypoxic fluorescent probe as an active ingredient. Here, "containing as an active ingredient" is not particularly limited as long as the diagnostic agent or diagnostic composition of the present embodiment contains a hypoxic fluorescent probe to such an extent that the effect of the present invention is exhibited. The content of the hypoxic fluorescent probe contained in the diagnostic agent or the diagnostic composition means 0.001 to 20% by mass, for example 0.01 to 10% by mass.

  The administration method of the diagnostic agent or the diagnostic composition is not particularly limited, and may be appropriately determined according to the condition, body weight, age, sex, etc. of the patient. For example, tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions and the like are orally administered. In addition, the injections are intravenously administered alone or mixed with ordinary fluid replacements such as glucose and amino acids, and further, if necessary, intraarterial, intramuscular, intradermal, subcutaneous, intraperitoneal or vitreous administration. Be done. Suppositories are administered rectally. The external skin preparation is applied, affixed or sprayed to the affected area.

  The dose of the diagnostic agent or composition for diagnosis varies depending on the patient's condition, body weight, age, sex, etc. and can not be determined generally, but in the case of oral administration, 1 to 500 mg / 60 kg body weight per dosage unit form An active ingredient (hypoxia fluorescent probe) may be administered. In the case of systemic administration by injection, for example, an active ingredient of 1 to 500 mg / 60 kg body weight may be administered per dosage unit form. In the case of administration by intravitreal injection, for example, 1 to 50 μg of the active ingredient may be administered per dosage unit form, and for example, 1 to 10 μg of the active ingredient may be administered. In the case of suppositories, for example, an active ingredient of 1 to 500 mg / 60 kg body weight per dosage unit form may be administered. In the case of a skin external preparation, for example, an active ingredient of 1 to 500 mg / 60 kg body weight may be administered per dosage unit form.

[Composition for treatment of hypoxia related eye disease]
In one embodiment, the present invention provides a composition for treating hypoxia-related eye disease, which comprises the above-mentioned diagnostic agent or composition for diagnostic and an angiogenesis inhibitor as active ingredients.

  By administering the therapeutic composition of this embodiment to a patient, diagnosis and treatment of hypoxia-related ocular diseases can be simultaneously performed.

  In addition, as described above, the change in the fluorescence properties of the hypoxic fluorescent probe contained in the therapeutic composition of the present embodiment under hypoxic conditions may be reversible or irreversible. If changes in the fluorescent properties of the hypoxic fluorescent probe are reversible, the hypoxic environment is improved, and the fluorescent properties change when approaching the normal oxygen environment, so monitoring the temporal change of oxygen concentration Can. Therefore, by observing the fluorescence of the hypoxic fluorescent probe, it can also be evaluated whether the hypoxic environment of the patient's eye has improved, that is, whether a therapeutic effect has been achieved.

(Angiogenesis inhibitor)
Angiogenesis inhibitors include VEGF capture agents, VEGF receptor antagonists, VEGF receptor kinase inhibitors, angiostatin, endostatin, fumagillin, fumagillol derivatives, anti-angiogenic steroids and the like.

  Examples of the VEGF capture substance include anti-VEGF antibodies such as lucentis (ranibizumab) and avastin (bevacizumab); aptamers such as macugen (pegaptanib sodium); fusion proteins such as airia and the like.

  Examples of fumagillol derivatives include O-monochloroacetylcarbamoyl fumagillol, O-dichloroacetylcarbamoyl fumagillol and the like.

(Formulation and administration method)
The therapeutic composition of the present embodiment may be administered systemically or locally. In the case of topical administration, the amount of therapeutic composition required may be small and the risk of side effects such as anaphylactic shock may also be reduced. Examples of topical administration include, for example, intravitreal injection. That is, the therapeutic composition of the present embodiment may be for vitreous injection.

  As a dosage form and administration method of the therapeutic composition of this embodiment, the thing similar to the diagnostic agent or diagnostic composition of the said embodiment is mentioned. In addition, the dose of the therapeutic composition of the present embodiment can be appropriately set in the range in which the effects of the contained hypoxic fluorescent probe and the angiogenesis inhibitor are exhibited.

  In one embodiment, the present invention provides a method of detecting the hypoxic region of the eye in vivo comprising administering a hypoxic fluorescent probe to the eye. In the method of this embodiment, administration of the hypoxic fluorescent probe may be performed by vitreous injection. Also, as the hypoxic fluorescent probe, the same compounds as those described above can be used.

  In one embodiment, the present invention provides a method of diagnosing hypoxia-related eye disease in vivo comprising administering a hypoxic fluorescent probe to the eye. In the method of this embodiment, administration of the hypoxic fluorescent probe may be performed by vitreous injection. Also, as the hypoxic fluorescent probe, the same compounds as those described above can be used.

  In one embodiment, the present invention provides a method of treating hypoxia-related eye disease, comprising administering a hypoxic fluorescent probe and an angiogenesis inhibitor to the eye. In the treatment method of this embodiment, the same compounds as those described above can be used as a hypoxic fluorescent probe. Moreover, as the angiogenesis inhibitor, the same as those described above can be used.

  In one embodiment, the invention provides a hypoxic fluorescent probe for the diagnosis of hypoxia-related eye disease. In the present embodiment, as the hypoxic fluorescent probe, the same compounds as those described above can be used.

  In one embodiment, the present invention provides a composition for the treatment of hypoxia-related eye disease, comprising a hypoxic fluorescent probe and an angiogenesis inhibitor as active ingredients. In the composition of this embodiment, the same compounds as those described above can be used as the hypoxic fluorescent probe. Moreover, as the angiogenesis inhibitor, the same as those described above can be used.

  In one embodiment, the present invention provides the use of a hypoxic fluorescent probe for the manufacture of a diagnostic for hypoxia related eye disease. In the present embodiment, as the hypoxic fluorescent probe, the same compounds as those described above can be used.

  In one embodiment, the present invention provides the use of a hypoxic fluorescent probe and an angiogenesis inhibitor for producing a composition for the treatment of hypoxia-related eye disease. In the present embodiment, as the hypoxic fluorescent probe, the same compounds as those described above can be used. Moreover, as the angiogenesis inhibitor, the same as those described above can be used.

  Hereinafter, the present invention will be described by way of experimental examples, but the present invention is not limited to the following experimental examples.

[Experimental Example 1]
(Synthesis of hypoxic fluorescent probe)
The hypoxic fluorescent probe (hereinafter sometimes referred to as "GPU-327") represented by the following formula (2) was synthesized.

  GPU-327 was synthesized according to the following reaction scheme (I). The details of each synthesis step will be described below. In addition, each compound which attached the number of 1-9 in the following description respond | corresponds to each compound which attached the number of 1-9 in reaction scheme (I).

(Synthesis of 3,5-Dinitrobenzyl alcohol (2))
A solution of 3,5-dinitrobenzoic acid (1,3.19 g, 15.0 mmol) in anhydrous THF (25 mL) under nitrogen atmosphere with stirring under ice-cooling with 0.95 M borane-THF complex in THF (20.0 mL) (19.0 mmol) was carefully added dropwise over 20 minutes. The mixture was further stirred for 90 minutes under ice cooling, and further stirred at room temperature for 22 hours. The reaction was quenched by carefully adding water: acetic acid (1: 1, 1.0 mL) to the reaction mixture, and after adding a saturated aqueous solution of sodium hydrogen carbonate (20 mL) and water (10 mL), chloroform (30 mL × 3) Extraction was done. After distilling off the solvent under reduced pressure, an orange solid is obtained, which is further purified by silica gel column chromatography (ethyl acetate: n-hexane = 3: 7) to obtain light yellow solid compound 2 (2.47 g, 83%) I got ¹ H-NMR (CDCl ₃ , 400 MHz) δ: 8.97 (s, 1 H), 8.59 (s, 2 H), 4.97 (d, J = 5.3 Hz, 2 H), 2.18 (t , J = 5.3 Hz, 1 H).

(Synthesis of 3,5-Dinitrobenzyl bromide (3))
A solution of phosphorus tribromide (1.7 mL, 17.9 mmol) in chloroform (7.3 mL) in a solution of 3,5-dinitrobenzyl alcohol (2, 1.97 g, 9.94 mmol) in chloroform (20 mL) for 15 minutes The reaction solution was dropwise added, and then heated under reflux for 2 hours under the protection of calcium chloride. After further stirring at room temperature for 16.5 hours, water (30 mL) was carefully added to the reaction mixture, and extraction was performed with chloroform (35 mL × 6). The organic layers were combined, washed with saturated aqueous sodium hydrogen carbonate solution (200 mL) and saturated brine (200 mL), dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. The obtained yellow solid was purified by silica gel column chromatography (ethyl acetate: n-hexane = 5: 95 to 20:80) to obtain compound 3 (2.25 g, 87%) as pale yellow crystals. m. p. 94.0-95.5 ° C .; ¹ H-NMR (CDCl ₃ , 400 MHz) δ: 8.99 (t, J = 2.0 Hz, ¹ H), 8.59 (d, J = 2.0 Hz, 2 H ), 4.61 (s, 2H).

(Synthesis of 3,5-Dinitrobenzyl iodide (4))
Stir a solution of 10% (w / w) sodium iodide in acetone (43.9 mmol, 75.0 mL) in a solution of 3,5-dinitrobenzyl bromide (3, 2.92 g, 11.2 mmol) in acetone (75 mL) The mixture was then added over 75 minutes and stirred at room temperature for additional 4 hours. Water (80 mL) was added to the reaction mixture and extraction was further performed with chloroform (80 mL × 3). The combined organic layer was washed with saturated aqueous sodium hydrogen carbonate solution (100 mL) and then dried over anhydrous magnesium sulfate. Furthermore, the solvent was distilled off under reduced pressure to obtain Compound 4 (3.39 g, 98%) as a pale yellow solid. m. p. 90.0-93.0 ° C .; ¹ H-NMR (CDCl ₃ , 400 MHz) δ: 8.93 (t, J = 2.1 Hz, 1 H), 8.55 (d, J = 2.2 Hz, 2 H ), 4.57 (s, 2H).

(Synthesis of 5-Methoxy-2,3,3-trimethyl-3H-indole (6))
A solution of isopropyl methyl ketone (0.64 mL, 5.97 mmol) and 4-methoxyphenylhydrazine hydrochloride (5, 1.05 g, 6.01 mmol) in ethanol (25.7 mL) was heated to reflux for 3 hours. After cooling to room temperature, the solvent was evaporated under reduced pressure. The residue was dissolved in chloroform (40 mL), washed with water (30 mL × 2), the organic layer was dried over anhydrous magnesium sulfate, and the solvent was evaporated under reduced pressure. Brown semisolid Compound 6 (1.05 g, 93%) was obtained. ¹ H-NMR (CDCl ₃ , 400 MHz,) δ: 7.43 (d, J = 8.2 Hz, 1 H), 6.85-6.80 (m, 2 H), 3.83 (s, 3 H), 2.24 (s, 3 H), 1. 29 (6 H, s).

(Synthesis of 1- (3,5-Dinitrobenzyl) -5-methoxy-2,3,3-trimethyl-3H-indolium iodide (7))
5-methoxy-2,3,3-trimethyl-3H-indole (6, 189.3 mg, 1.00 mmol) and 3,5-dinitrobenzyl iodide (4, 462.0 mg, 1.50 mmol) in anhydrous THF 6.0 mL) The solution was heated to reflux under a nitrogen atmosphere for 22 hours. After cooling to room temperature, the resulting precipitate was collected by filtration, and the obtained solid was washed with acetone (3 mL × 3) and diethyl ether (3 mL). Compound 7 (375.4 mg, 75%) was obtained as an orange solid. ¹ H-NMR (CD ₃ CN, 400 MHz) δ: 8.92 (t, J = 2.2 Hz, 1 H), 8.47 (d, J = 1.9 Hz, 2 H), 7.54 (d, J = 9.2 Hz, 1 H), 7.34 (d, J = 2.4 Hz, 1 H), 7.05 (dd, J = 8.9, 2.7 Hz, 1 H), 5. 84 (s, 2 H) , 3.88 (s, 3 H), 2. 80 (s, 3 H), 1.62 (s, 6 H); ¹³ C-NMR (CD ₃ CN: CD ₃ OD: CDCl ₃ , 125 MHz) δ: 196 ., 160.9, 148.0, 143.2, 134.5, 132.9, 126.8, 118.2, 115.7, 114.2, 108.4, 55.0, 54.2 ^{, 49.0,21.2; HRMS (ESI +)} : Calcd for C 19 H 20 N 3 O 5 [M-I] + = 3 0.1403, Found, 370.1384.

(Synthesis of 5-Methoxy-1,2,3,3-tetramethyl-3H-indolium iodide (8))
Dissolve 5-methoxy-2,3,3-trimethyl-3H-indole (6, 193.2 mg, 1.02 mmol) in methyl iodide (4.0 mL) and heat to reflux for 2 hours, then release to room temperature It was cold. The resulting precipitate is treated with n-hexane and collected by filtration, and the obtained solid is washed with n-hexane (3 mL × 2) and dried under reduced pressure to obtain brown powder 8 (319.0 mg, 94%) I got ¹ H-NMR (CDCl ₃ , 400 MHz) δ: 7.59 (d, J = 9.2 Hz, 1 H), 7.06-7.02 (m, 2 H), 4.23 (s, 3 H), 3 .90 (s, 3 H), 3.04 (s, 3 H), 1. 65 (s, 6 H).

(Synthesis of 5-Methoxy-1,3,3-trimethyl-2- {6- (N-phenylacetamido) hexa-1,3,5-trienyl} -3H-indolium iodide (9))
A mixture of glutaconaldehyde dianneal hydrochloride (57.1 mg, 0.201 mmol), acetyl chloride (56.6 μL, 0.797 mmol) and acetic anhydride (0.2 mL) was heated at 110 ° C. A solution of methoxy-1,2,3,3-tetramethyl-3H-indolium iodide (8, 30.1 mg, 0.0909 mmol) in acetic anhydride (2.0 mL) was added dropwise, and the mixture was further stirred for 30 minutes. The solvent was evaporated under reduced pressure below 50 ° C., and the resulting residue was dissolved in methylene chloride (0.4 mL) and added to diethyl ether (100 mL) with vigorous stirring. The resulting precipitate is collected by filtration and purified by silica gel column chromatography (methylene chloride: n-hexane: methanol = 20: 4: 0.5 to 20: 4: 4) to give Compound 9 (109.2 mg) as a red solid. Obtained quantitatively. ¹ H-NMR (CDCl ₃ , 400 MHz) δ: 8.14 (d, J = 13.8 Hz, 1 H), 7.73 (dd, J = 15.1, 11.4 Hz, 1 H), 7.63- 7.50 (m, 3 H), 7.44 (s, 1 H), 7.41 (d, J = 7.6 Hz, 1 H), 7.19 (dd, J = 8.0, 1.5 Hz, 2 H ), 7.11 (br t, J = 12.7 Hz, 1 H), 7.01 (dd, J = 8.8, 2.4 Hz, 1 H), 6.98 (d, J = 2.4 Hz, 1 H) ), 6.81 (dd, J = 14.5, 11.2 Hz, 1 H), 5.38 (dd, J = 13.7, 11.4 Hz, 1 H), 4.18 (s, 3 H), 3 .89 (s, 3H), 1.97 (s, 3H), 1.71 (s, 6H); 13 C-NMR (CDCl 3, 100MHz) δ: 178.4,169 2, 160.9, 153.7, 149.8, 144.6, 140.1, 137.9, 134.8, 130.6, 129.9, 129.7, 128.1, 115.2, 114.5, 113.5, 113.4, 108.6, 56.1, 51.4, 35.6, 27.0, 23.3; HRMS (ESI ⁺ ): Calcd for C ₂₆ H ₂₉ N ₂ O ₂ [M−I] ⁺ = 401.2229, Found, 401.2248.

(2- [7- {1- (3,5-Dinitrobenzyl) -5-methoxy-3,3-dimethylindol-2-ylidene} hepta-1,3,5-trienyl] -5-methoxy-1,3, Synthesis of 3-trimethyl-3H-indolium iodide (GPU-327))
Compound 9 (69.6 mg, 0.132 mmol), anhydrous sodium acetate (16.4 mg, 0.200 mmol) and 1- (3,5-dinitrobenzyl) -5-methoxy-2,3,3-trimethyl-3H- The indolenium iodide (7,49.7 mg, 0.0999 mmol) was added to ethanol (10 mL) and heated to reflux for 45 minutes under an argon atmosphere. After distilling off the solvent under reduced pressure, silica gel column chromatography (methylene chloride: n-hexane: methanol = 20: 4: 20 to 4: 4: 0.8) and ODS flash chromatography (water: acetonitrile = 60: 40 to 36) : 64) to obtain GPU-327 (13.8 mg, 18%) as blue powder. ¹ H-NMR (CD ₃ OD, 400 MHz) δ: 8.92 (t, J = 2.0 Hz, 1 H), 8.44 (d, J = 1.9 Hz, 2 H), 7.97 (br s, br s, 1H), 7.66 (br s, 1 H), 7.42 (br s, 1 H), 7.39 (d, J = 8.1 Hz, 2 H), 7.19 (d, J = 2.2 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1 H), 7.03 (dd, J = 8.8, 2.1 Hz, 1 H), 6.93 (d, J = 8.2 Hz, 1 H) ), 6.83 (dd, J = 8.6, 2.5 Hz, 1 H), 6.51 (br t, J = 14.4 Hz, 2 H), 6.31 (br s, 1 H), 5.87 (D, J = 13.0 Hz, 1 H), 5.38 (br s, 2 H), 3.87 (s, 3 H), 3.81 (s, 3 H), 3.73 (s, 3 H), 1 .76 (s ^{6H), 1.70 (s, 6H} ); 13 C-NMR (CD 3 OD, 125MHz) δ: 177.1,167.0,161.2,158.5,155.6,154.6,150 .3, 146.9, 145.3, 142.0, 137.6, 137.1, 128.0, 127.7, 126.8, 118.9, 115.3, 114.4 , 114.3, 110.6, 110.2, 109.7, 108.9, 101.6, 79.5, 56.5, 56.4, 51.8, 46.1, 32.6, 28 HRMS (ESI ⁺ ): Calcd for C ₃₇ H ₃₉ N ₄ O ₆ [M−I] ⁺ = 635.2864 Found, 635.2875.

[Experimental Example 2]
(Visualization of hypoxic regions of EPC and HUVEC by GPU-327)
Vascular endothelial precursor cells (EPCs) and umbilical vein endothelial cells (HUVECs) were cultured in the presence of GPU-327, and the hypoxia selectivity of GPU-327 was examined.

  EPCs were isolated from umbilical cord blood and cultured in Iscove's modified Dulbecco's medium (IMDM medium) containing 10% fetal bovine serum (FBS) and basic fibroblast growth factor (b-FGF). HUVEC were purchased from Cambrex and cultured according to the instruction.

  A final concentration of 0.5, 1.0 and 1.5 μM of GPU-327 was added to the EPC and HUVEC medium, and the cells were cultured for 6 hours in an environment of oxygen concentration of 1%, 5% and 20%. Subsequently, each cell was washed with phosphate buffer (PBS) to remove unbound GPU-327.

  Subsequently, the fluorescence of GPU-327 was detected using a fluorescence microscope (type “BX51”, Olympus Co., Ltd.) attached with a CCD camera (type “H6741CE”, Tucsen) and a fluorescent filter for ICG (Omega Optical Co.).

  The results are shown in FIG. FIG. 1 (a) is a photograph of fluorescence detected in EPC under each condition. FIG.1 (b)-(d) make the fluorescence intensity of FIG. 1 (a) a graph. FIG. 1 (e) is a photograph of fluorescence detected in HUVEC under each condition. FIGS. 1 (f) to 1 (h) are graphs of the fluorescence intensities of FIG. 1 (e). “*” In FIGS. 1 (b) to 1 (d) and (f) to (h) indicates that there is a significant difference at a risk rate of less than 5%. Data were analyzed using Student's t test and Paired t test. One-way analysis of variance (one-way ANOVA) and Bonferroni's multiple comparisons were used when comparing multiple groups. Hereinafter, in all the experimental examples, statistical analysis was performed using software (trade name “StatView ver. 5.0”, SAS). In the graph, the y-axis error bar indicates the standard deviation.

  As a result, it was confirmed that there is a significant difference in the fluorescence intensity of GPU-327 under normal oxygen conditions (oxygen concentration 20%) and low oxygen conditions (oxygen concentration 5% or 1%). This result indicates that GPU-327 can detect hypoxic regions.

[Experimental Example 3]
(Measurement of expression levels of VEGF and HIF-1α in EPC and HUVEC)
The expression levels of vascular endothelial growth factor (VEGF) and hypoxia inducible factor (HIF) -1α in EPC and HUVEC cultured under each oxygen condition of Experimental Example 2 were measured. The expression level of VEGF was measured by quantitative RT-PCR. More specifically, first, total RNA was extracted from each cell using RNeasy Mini Kit (Qiagen). Subsequently, cDNA was synthesized from total RNA using ReverTra-Plus (trademark) (Toyobo Co., Ltd.) and used as a template for quantitative RT-PCR. As a primer for VEGF gene amplification, a sense primer (5'-AGATGAGCTTCTCACAGCACAAC-3 ', SEQ ID NO: 1) and an antisense primer (5'-AGGACTTATACCGGGATTTCTTG-3', SEQ ID NO: 2) were used. The expression level of HIF-1α was measured by western blotting.

  30 μg of nuclear extract of each cell was separated by SDS polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. Subsequently, the transfer membrane is stained using rabbit anti-HIF-1α antibody (Novus) as primary antibody and horseradish peroxidase (HRP) -labeled goat anti-rabbit IgG (Invitrogen) as secondary antibody, Detection was performed with an enhanced chemiluminescence (ECL) system (GE Healthcare). As a control, lamin B protein was detected. The detection of lamin B protein was performed using goat anti-lamin B antibody (Santa Cruz) as the primary antibody and HRP-labeled rabbit anti-goat IgG (Invitrogen) as the secondary antibody.

  The results are shown in FIG. FIG. 2 (a) is a graph showing the expression level of VEGF mRNA in each cell. FIG. 2 (b) is a photograph showing the results of Western blotting that detected the expression of HIF-1α protein and Lamin B protein in each cell. FIG. 2 (c) is a graph showing the amount of protein expression in FIG. 2 (b). “*” In FIG. 2 (a) and FIG. 2 (c) indicates that there is a significant difference at a risk rate of less than 5%.

  As a result, compared to HUVEC, EPC responded strongly to hypoxic conditions, and it was revealed that the amount of VEGF gene expression and the level of HIF-1α protein expression were significantly increased under hypoxic conditions.

  On the other hand, although HUVEC had a weak response to hypoxic conditions, in Experimental Example 2, a significant increase in the fluorescence intensity of GPU-327 was observed under hypoxic conditions. This result indicates that GPU-327 does not directly detect the expression of VEGF or HIF-1α but detects hypoxic conditions.

[Experimental Example 4]
(Application of GPU-327 to a flap ischemia mouse model)
We confirmed that GPU-327 can accurately detect hypoxic regions, and conducted a study using a flap ischemia mouse model for the purpose of examining the applicability of GPU-327 to regions other than the eye. The

  FIGS. 3 (a) and 3 (b) are photographs showing a skin flap ischemia mouse model. As shown in FIG. 3 (a), a peninsular shaped incision (3 cm × 2 cm) was made on the back surface of the mouse. Thereby, blood circulation exists only on one side of the incision and an ischemic gradient is generated. Two days after incision preparation, GPU-327 was administered by tail vein administration, and the fluorescence of GPU-327 was observed using a human fundus camera (model “RC-2”, Kowa Co., Ltd.).

  In this model, an oxygen tension gradient occurs in the ischemic tissue. The caudal region of the flap is one-fifth the oxygen pressure of the caudal region of the flap. Therefore, it is expected that the fluorescence of GPU-327 will be the strongest since the area of the caudal edge of the flap is the lowest in oxygen pressure.

  FIG. 3 (b) is a photograph taken without a fluorescence filter, and FIG. 3 (c) is a photograph showing a fluorescence image using the fluorescence filter. As a result, it was confirmed that GPU-327 can accurately detect the hypoxic region. However, contrary to the above-mentioned expectation, it was the central part of the flap that the fluorescence of GPU-327 was the strongest. This result was considered to be due to the fact that GPU-327 did not reach the area of the caudal edge of the flap.

[Experimental Example 5]
(Application of GPU-327 to Retinal Artery Occlusion Mouse Model)
We applied GPU-327 to a retinal artery occlusion mouse model and examined whether it could accurately detect the hypoxic region.

  By administering rose bengal to the mouse tail vein and irradiating the retinal artery with laser light, the retinal artery can be occluded to produce local hypoxia in the retina. A retinal artery occlusion mouse model was created by this method.

  More specifically, first, the mouse was anesthetized to expand the pupil. Subsequently, after administering 40 mg / kg of Rose Bengal (Nacalai Tesque) into the tail vein, a glass cover is placed on the cornea and a slit lamp krypton laser delivery system (type "MC-7000", Nidek) is used. Laser light was irradiated to occlude the retinal artery. The maximum peak absorption wavelength of Rose Bengal is 550 nm. The size of the laser spot was about 50 μm. The laser power was about 50 mW. The laser pulse interval was 3 seconds. In order to stop the blood flow, 2-3 times of laser beam irradiation was required. In each eye, one artery was occluded.

  Immediately after preparation of the retinal artery occlusion mouse model, 1 μL of GPU-327 at a concentration of 0.2 μg / μL was administered by vitreous injection. Two days later, the fluorescence of GPU-327 was observed in vivo using a fundus lens (type “28D”, volk) and a fundus camera for humans (type “RC-2”, Kowa). The eyes were then removed and observed in vitro. More specifically, the retinal vasculature of the removed eye was stained with fluorescently labeled isolectin B4, flat-mounted and observed with a fluorescence microscope. Moreover, the nucleus of the removed eye cell was stained with 4 ', 6'-diamino-2-phenylindole (DAPI), and the cross section was observed with a fluorescence microscope (BZX700, KEYENCE). Moreover, the fluorescence of GPU-327 was observed using a fluorescence microscope (BZX700, KEYENCE company).

  FIG. 4 (a) is a photograph showing the results of observing the fluorescence of GPU-327 in vivo. An increase in fluorescence was observed only around the occluded artery, and it became clear by this method that hypoxic regions of the eye can be detected directly in vivo.

  FIG.4 (b) is a photograph which shows the result of having observed the vascular structure of the retina of the eye observed in FIG. 4 (a) in vitro. FIG.4 (c) is a photograph which shows the result of having observed fluorescence of GPU-327 in the sample same as FIG.4 (b). FIG.4 (d) is the photograph which integrated the fluorescence image of FIG.4 (b) and FIG.4 (c). FIG.4 (e) is a photograph which shows the result of having observed in vitro the cross section of the model eye produced by the same experimental method as (a)-(d). FIG.4 (f) is a photograph which shows the result of having observed fluorescence of GPU-327 in the sample same as FIG.4 (e). FIG.4 (g) is the photograph which integrated the fluorescence image of FIG.4 (e) and FIG.4 (f). From this result, it was confirmed that the result of detection of the hypoxic region of the eye in vivo is consistent with the result of detection in vitro.

  From the above results, it was shown that GPU-327 can accurately detect the hypoxic region in the eye of the retinal artery occlusion mouse model.

[Experimental Example 6]
(Application of GPU-327 to a retinal vein occlusion mouse model)
GPU-327 was applied to a retinal vein occlusion mouse model to investigate whether it could accurately detect hypoxic regions.

  First, in the same manner as in Experimental Example 5, a retinal vein was irradiated with laser light to occlude the retinal vein to prepare a retinal vein occlusion mouse model. Three to five veins were occluded in each eye. Immediately after creating the retinal vein occlusion mouse model, GPU-327 was administered by vitreous injection. Two days later, the fluorescence of GPU-327 was observed in vivo using a fundus lens (type “28D”, volk) and a fundus camera for humans (type “RC-2”, Kowa). The eyes were then removed and observed in vitro. More specifically, the retinal vasculature of the removed eye was stained with fluorescently labeled isolectin B4, flat-mounted and observed with a fluorescence microscope.

  FIG. 5 (a) is a photograph showing the results of observing the fluorescence of GPU-327 in vivo. As compared with the arterial occlusion model in which only one artery was blocked, the vein occlusion model in which 3 to 5 veins were blocked in each eye showed a broader hypoxic region. The method has revealed that the hypoxic region of the eye can be detected directly in vivo.

  FIG.5 (b) is a photograph which shows the result of having observed in vitro the vascular structure of the retina of the eye observed by FIG. 5 (a). FIG.5 (c) is a photograph which shows the result of having observed fluorescence of GPU-327 in the sample same as FIG.5 (b). FIG.5 (d) is the photograph which integrated the fluorescence image of FIG.5 (b) and FIG.5 (c). From this result, it was confirmed that the result of detection of the hypoxic region of the eye in vivo is consistent with the result of detection in vitro.

  From the above results, it was shown that GPU-327 can accurately detect the hypoxic region in the eye of the retinal vein occlusion mouse model.

[Experimental Example 7]
(Application of GPU-327 to choroidal ischemia mouse model)
GPU-327 was applied to a choroidal ischemia mouse model to examine whether GPU-327 administered by vitreous injection can pass through the retina and reach the choroid. In the choroidal ischemia mouse model, the internal carotid artery of the mouse is ligated to cause ischemia not only in the choroid but also in the entire eye.

  After the mice were anesthetized, one carotid artery was exposed and the internal carotid artery was ligated in two places with 8-0 silk thread. The ocular artery is branched from the internal carotid artery, and ligation causes the entire eye to undergo ischemia. Clinically, this model is an eye ischemia syndrome model, in which the entire eyeball is ischemic, and therefore, ischemia occurs not only in the surface layer of the retina but also in the deep layer of the choroid. Immediately after internal carotid artery ligation, 1 μL of GPU-327 at a concentration of 0.2 μg / μL was administered to both eyes by vitreous injection. Two days later, the fluorescence of GPU-327 was observed in vivo using a fundus camera.

  FIG. 6 (a) is a photograph showing the results of in vitro observation of a cross section of the eye. (B) is a photograph which shows the result of having observed fluorescence of GPU-327 in the sample same as (a). (C) is the photograph which integrated the fluorescence image of (a) and (b).

  The eye on the side where the internal carotid artery was ligated showed an increase in fluorescence intensity, but the eye on the side not ligated did not show an increase in fluorescence. In the cross section, the whole eye was faint and fluorescence was enhanced. In particular, strong enhancement of fluorescence was observed in the optic nerve. The vitreous injected GPU-327 was found to reach the deep part of the eye such as choroid and optic nerve.

[Experimental Example 8]
(Application of GPU-327 to mouse model of retinopathy of prematurity)
GPU-327 was applied to the retinopathy of prematurity mouse model. In the retinopathy of prematurity mouse model, retinal blood vessel growth is arrested by rearing a 7-day-old young mouse whose retinal blood vessels have not been completed yet under conditions of 75% oxygen concentration until 12 days after birth. This is a model in which relative ischemia occurs by returning to the normal oxygen condition again after 12 days after birth, and then retinal neovascularization is observed.

  In the mouse model of retinopathy of prematurity 6 hours after return to normal oxygen condition on the 12th day of birth to examine the optimal concentration of GPU-327, 0.832, 1.664, 3.328 μg / g Body weight GPU-327 was injected tail vein. The dose of 0.832 μg / g body weight corresponds to twice the dose of 25 mg / 60 kg body weight, which is the dose of ICG in the human fluorescein fundus imaging method. Similarly, the doses of 1.664 and 3.328 μg / g body weight correspond to four and eight times the dose of ICG in the human fluorescein angiography method, respectively.

  One hour after administration of GPU-327, observe the fluorescence of GPU-327 in the eye using a fundus lens (type "28D", volk) and a fundus camera for humans (type "RC-2", Kowa) did.

  FIG. 7 (a) is a photograph showing the result of fluorescence observation. As a result, an increase in fluorescence intensity was observed depending on the dose of GPU-327.

  In addition, in the retinopathy mouse model of retinopathy 2 hours, 6 hours, 1 day and 5 days after return to normal oxygen condition on the 12th day of birth, 1.664 μg / g body weight of GPU-327 tail vein It injected and observed the time-dependent change (n = 6). One hour after administration of GPU-327, observe the fluorescence of GPU-327 in the eye using a fundus lens (type "28D", volk) and a fundus camera for humans (type "RC-2", Kowa) did. As a control, the fluorescence of GPU-327 was observed in the eyes of mice injected with tail vein injection of 1.664 μg / g body weight of GPU-327 in normal mice at 12 days of age.

  FIG. 7 (b) is a photograph showing the result of fluorescence observation. FIG. 7 (c) is a graph showing the fluorescence intensity detected in FIG. 7 (b). “*” In FIG. 7 (c) indicates that there is a significant difference at a risk factor of less than 1%.

  As a result, strong fluorescence was observed in a mouse model of retinopathy of prematurity two hours and six hours after return to the normal oxygen condition.

  From the above results, it was shown that GPU-327 can accurately detect the hypoxic region in the eye of the retinopathy of prematurity mouse model.

[Experimental Example 9]
(Application of GPU-327 to retinal vein occlusion rabbit model)
In the retinopathy of prematurity mouse model, the entire retina is hypoxic, which may not be suitable for detection of local hypoxic regions in the retina. Therefore, GPU-327 was applied to a retinal vein occlusion rabbit model to examine whether or not the hypoxic region could be accurately detected.

  More specifically, a contact lens (trade name "transequator lens", volk) is used in place of the cover glass, the laser spot size is about 125 μm, the laser power is 150 to 300 mW, and the laser pulse interval is A retinal vein occlusion rabbit model was prepared in the same manner as the above-described retinal artery occlusion mouse model except that one main vein was occluded for 0.5 seconds.

  Immediately after preparation of the retinal vein occlusion rabbit model, 50 μL of GPU-327 at a concentration of 0.2 μg / μL was administered by vitreous injection. Subsequently, the fundus was photographed using a fundus camera two days, one week and two weeks after retinal vein occlusion, and the fluorescence of GPU-327 was observed in vivo using a fundus camera.

  8 (a), 8 (b) and 8 (c) are photographs of the fundus taken using a fundus camera two days, one week and two weeks after retinal vein occlusion. Moreover, FIG. 8 (d), FIG. 8 (e), and FIG. 8 (f) observed fluorescence of GPU-327 in vivo using a fundus camera two days, one week and two weeks after retinal vein occlusion. It is a photograph which shows a result.

  Immediately after the laser irradiation, an upset of the retinal vein occurred, and as shown in FIG. 8 (a), a slight retinal hemorrhage was observed two days after retinal vein occlusion as in human retinal vein occlusion. It was considered that the preparation was successful. In addition, as shown in FIG. 8 (b), whitening of the retina on the laser irradiated side in the vicinity of the optic nerve was observed one week after the retinal vein occlusion. The increase in fluorescence of GPU-327 was observed only on the laser-irradiated side of the eye, and the fluorescence of GPU-327 was observable up to 2 weeks after retinal vein occlusion. Also, in the vitreous cavity, no increase in GPU-327 fluorescence was observed. In rabbits, unlike humans, most of the retina is perfused from the choroidal blood flow, and only a part of the retina around the optic papilla is perfused from the retinal blood flow. Also in the model in which the retinal vein is blocked this time, the choroidal blood flow return region is not hypoxic, and the increase in the fluorescence of GPU-327 is not observed. An increase in GPU-327 fluorescence was observed only in the area of reflux from the retinal artery.

  The above results show that GPU-327 has sufficient detection performance in the low oxygen region. Also, it has been shown that GPU-327 changes to a form that emits fluorescence after being taken into the retina.

[Experimental Example 10]
(Fluorescent fundus angiography of retinal vein occlusion rabbit model)
The fluorescein fundus angiography of the retinal vein occlusion rabbit model 2 weeks after the retinal vein occlusion in Experimental Example 9 was performed using fluorescein. FIG. 9 is a photograph showing the results of fluorescent fundus imaging. As a result, neovascularization, leakage of fluorescence and avascular area were observed.

[Experimental Example 11]
(Comparison with GPU-327 and pimonidazole in detection of retinal hypoxia area in retinal vein occlusion rabbit model)
Hypoxic regions in eyes of a retinal vein occlusion rabbit model 2 days after retinal vein occlusion in Experimental Example 9 were stained with GPU-327 and pimonidazole and compared.

  Staining with pimonidazole was performed as follows. First, in a rabbit 90 minutes before sacrifice, 50 μL (60 mg / kg body weight) of 400 μM pimonidazole hydrochloride (Millipore) was injected by vitreous injection. Subsequently, after killing the rabbit, the eyeball was removed and fixed with 4% paraformaldehyde. Subsequently, the eyeball whole mount was stained at room temperature for 1 hour using FITC-labeled anti-pimonidazole antibody diluted 50-fold with blocking solution. In addition, counter staining was also performed using TRITC-labeled isolectin B4.

  FIG. 10 (a) is a photograph showing fluorescence of GPU-327 detected in vivo. FIG. 10 (b) is a photograph of the fundus of the eye observed in FIG. 10 (a). FIG. 10 (c) is a photograph of a fluorescence image showing the result of in vitro observation of the vascular structure of the retina of the eye observed in FIG. 10 (a) by staining with isolectin B4. FIG. 10 (d) is a photograph of a fluorescence image showing the result of staining with pimonidazole in the same sample as FIG. 10 (c). FIG.10 (e) is the photograph which integrated the fluorescence image of FIG.10 (c) and FIG.10 (d). Arrows in FIGS. 10 (c), (d) and (e) indicate the occlusion site of the retinal vein.

  As shown in FIG. 10 (a), the fluorescence of GPU-327 was observed not only around the retinal vein occlusion site but in a wider range. On the other hand, as shown in FIG. 10 (d), in the staining with pimonidazole, only the periphery of the retinal vein occlusion site was stained.

  Pimonidazole is said to be capable of detecting a low oxygen region with an oxygen concentration of about 1%. Taken together with the results of Experimental Example 2 in which the fluorescence of GPU-327 is significantly increased at an oxygen concentration of 5%, the results of this experimental example show that GPU-327 can detect a milder hypoxic region than pimonidazole. Indicates that there is.

[Experimental Example 12]
(Examination of the safety of GPU-327)
Phosphate buffered saline (PBS) or GPU-327 was administered to rabbit eyes, and electroretinogram measurement and retinal pathological evaluation were performed to examine the safety of GPU-327.

First, before administration of PBS or GPU-327, the pupil was dilated, and under general anesthesia and local anesthesia, dark adaptation was performed for at least 1 hour, and electroretinograms of rabbits were measured in a dark room. The measurement of the electroretinogram was performed as follows. First, a white light emitting diode built-in contact lens electrode (type “LW-102”, Mayo Co., Ltd.) was attached to the cornea of both eyes, and a ground electrode was attached to the ear. Subsequently, the electroretinograms of both eyes were simultaneously measured using an electroretinogram measurement device (type “LE-3000”, Tomey Inc.). The frequency band was set to 0.3 to 300 Hz. The stimulation intensity was set to 360, 3000 and 20000 cd / m ² and the duration was set to 1 millisecond. Electroretinogram measurements began with the weakest stimulus after at least 1 hour of dark adaptation. Background light was not used. Four stimulations were performed at each light intensity, and the average value was calculated.

  As a result, no significant difference was observed between the right eye and the left eye of the rabbit in terms of amplitude and latency in the electroretinogram before administration of PBS or GPU-327.

  Subsequently, PBS or GPU-327 was administered by vitreous injection to only one eye of the rabbit. The dose of PBS was 50 μL. In addition, the dose of GPU-327 was 50 μL of a solution of 0.2 μg / μL. Nothing was administered to the other eye and served as a control.

  Electroretinograms are measured 60 days after administration of PBS or GPU-327 as described above, and amplitude and latency for right and left eyes of rabbits (PBS administration vs. control, or GPU-327 administration vs. control) Was calculated. Moreover, in order to investigate the relevance of the influence by vitreous administration of PBS and GPU-327, the unpaired t test (Unpaired t test) was performed.

  FIG. 11 (a) is a graph showing an electroretinogram 60 days after administration of PBS or GPU-327. As a result, in the PBS administration group and the GPU-327 administration group, patterns of normal electroretinograms such as a wave and b wave were observed. FIG. 11 (b) is a graph showing the ratio of PBS administration to control or GPU-327 administration to control for the a-wave measured at each stimulus luminance. Further, FIG. 11 (c) is a graph showing the ratio of the amplitude of the right eye to the amplitude of the left eye for the b-wave measured at each stimulus luminance. No significant difference was observed in the electroretinogram between the PBS administration group and the GPU-327 administration group.

  Subsequently, retinal pathological evaluation of eyes in the PBS administration group and the GPU-327 administration group was performed using a light microscope. Fig. 11 (d) is an optical micrograph of an eye of the GPU-327 administration group, and Fig. 11 (e) is an optical micrograph of an eye of the PBS administration group.

  As a result, no abnormality such as atrophy of the photoreceptor layer was observed in any of the groups, and no inflammation of the retina, the pigment epithelial layer, and the optic nerve was observed.

  From the above results, it was shown that GPU-327 is safe.

  The present invention can provide a diagnostic agent capable of directly detecting the hypoxic region of the eye in vivo. In addition, it is possible to provide a therapeutic composition capable of simultaneously performing diagnosis and treatment of hypoxia-related eye disease.

Claims (4)

A hypoxic fluorescent probe is contained as an active ingredient, and the hypoxic fluorescent probe is a compound represented by the following formula ( 2 ), which is used to administer 0.1 to 50 μg by vitreous injection, for vitreous injection Diagnostic agent for in vivo hypoxia related eye disease.
A composition for treating hypoxia-related eye disease, comprising the diagnostic agent according to claim 1 and an angiogenesis inhibitor as active ingredients. The therapeutic composition according to claim 2 , wherein the angiogenesis inhibitor is a vascular endothelial growth factor (VEGF) capture agent. The therapeutic composition according to claim 3 , wherein the VEGF capture agent is an anti-VEGF antibody.