JP6811983B2 - Oral composition having retinal ganglion cell death inhibitory activity - Google Patents

Description

The present invention relates to an oral composition containing ollotic acid or a salt of ollotic acid and having retinal ganglion cell death inhibitory activity, a retinal ganglion cell death inhibitor for administration to a glaucoma patient, and the like.

In Japan, the prevalence of various eye diseases is rising due to the fastest growing proportion of the elderly population in the world. In particular, glaucoma has increased dramatically since 1990, and is currently the number one cause of blindness in a survey by the Ministry of Health, Labor and Welfare. Glaucoma is also defined as glaucoma optic neuropathy, and is a disease that causes visual field impairment due to optic nerve damage. Increased intraocular pressure, loss of retinal ganglion cells, etc. are observed as symptoms, but at present, instillation is performed. Drug-induced reduction in intraocular pressure is the treatment of choice. For example, an 8-azaprostaglandin analog as an intraocular pressure-lowering agent (see, for example, Patent Document 1) and a pharmaceutical composition for treating or preventing glaucoma having an adenosine A2a receptor-activating action (for example, Patent Document 1). There is also a report on (see 2). Therefore, with respect to the increase in intraocular pressure, it has become possible to obtain fairly good intraocular pressure control due to the progress of various eye drops, but the effect of lowering intraocular pressure alone causes retinal ganglion cell loss. It is said that the progression of accompanying glaucoma cannot be stopped sufficiently. In addition, it is said that one of the causes of glaucomatous optic neuropathy is apoptosis of retinal ganglion cells (RGC), and it is known that many patients in Japan develop glaucoma even with normal intraocular pressure. In addition, it has been reported that blood flow promotion is considered to be effective for normal tension glaucoma (NTG), and cassis anthocyanin is effective (see Patent Document 3 and the like). Especially in Japan, since there are many normal-tension glaucoma, protection and activation treatment of retinal ganglion cells that do not depend on the decrease in intraocular pressure are required.

On the other hand, ollotic acid has attracted attention as a growth-promoting factor for microorganisms and rats, but based on the finding that the combined use of amino acids with ollotic acid can significantly lower the uric acid level compared to ollotic acid alone. , Orot acid or a salt thereof, and an amino acid as an active ingredient for lowering the uric acid level (see, for example, Patent Document 4 and the like) have been proposed.

In addition, a composition for preventing or treating stomatitis containing glutathione and / or olottic acid (see, for example, Patent Document 5), an endurance enhancer containing olottic acid or a salt thereof as an active ingredient, and such endurance improving. Pharmaceuticals and foods containing agents (see, for example, Patent Document 6 and the like) and compositions for treating diseases caused by inhaling tobacco smoke containing ollotic acid (see, for example, Patent Document 7 and the like) have been proposed. ing.

Japanese Unexamined Patent Publication No. 2014-122250 Japanese Unexamined Patent Publication No. 2012-046513 JP-A-2007-055903 Japanese Unexamined Patent Publication No. 2011-098896 Japanese Patent Application Laid-Open No. 2008-512381 Japanese Unexamined Patent Publication No. 2011-136907 Japanese Unexamined Patent Publication No. 2011-026204

An object of the present invention is to provide a composition having a retinal ganglion cell death inhibitory activity, and an application of the composition as a retinal ganglion cell death inhibitor or a preventive agent for glaucoma.

The present inventors have been studying the usefulness of ollotic acid for a long period of time, and when ollotic acid is administered enterally, intravenously, by suction or orally, it exerts various effects. I have confirmed that. On the other hand, the present inventors have continued to search for components for treating glaucoma and prepared various components, but have not been able to find a component that produces a satisfactory effect. Meanwhile, when ollotic acid, which had not been reported to be effective against glaucoma by the above administration method, was orally administered to rats, ollotic acid showed an inhibitory effect on retinal ganglion cell death due to optic neuropathy. After confirmation, the present invention was completed.

That is, the present invention is shown below.
(1) An oral composition containing ollotic acid or a salt of olottic acid and having retinal ganglion cell death inhibitory activity.
(2) The oral composition according to (1) above, wherein retinal ganglion cell death is caused by optic nerve damage.
(3) A retinal ganglion cell death inhibitor, which comprises the oral composition according to (1) or (2) above, for administration to a glaucoma patient.
(4) A retinal ganglion cell death preventive agent comprising the oral composition according to the above (1) or (2) for administration to a non-glaucoma patient.
Other forms include an oral retinal ganglion cell death inhibitor or a retinal ganglion cell death preventive agent containing ollotic acid or a salt of ollotic acid as an active ingredient.

According to the present invention, glaucoma can be treated, ameliorated and / or prevented.

It is a figure which shows the protective action of the retinal cell by ollotic acid. In the figure, the horizontal axis shows the ollotic acid concentration in the antioxidant-free medium (AO (-)) and the antioxidant-containing medium (AO (+)), and the vertical axis shows rfu (relative) in the cell proliferation assay. Fluorescence units; relative fluorescence units). The number of living cells per mm ² in the retinal ganglion cell layer in rats treated with orot acid solution and rats treated with water is shown.

The oral composition having the retinal ganglion cell death inhibitory activity of the present invention is not particularly limited as long as it is an oral composition containing olottic acid or a salt thereof, but an oral composition containing olottic acid or a salt thereof as an active ingredient. The forms of the retinal ganglion cell death inhibitor and the retinal ganglion cell death preventive agent for use can be preferably exemplified. The above-mentioned ollotic acid is also called uracil-6-carboxylic acid, but according to the IUPAC nomenclature, it is a heteroaromatic ring represented by "1,2,3,6-tetrahydro-2,6-dioxo-4-pyrimidinecarboxylic acid". It is a kind of compound.

The above-mentioned ollotic acid can be used in, for example, a production method in which a bacterium belonging to the genus Corinebacterium having an ollottic acid-producing ability is cultured, ollotic acid is produced and accumulated in the culture, and the ollotic acid is collected (see Japanese Patent Publication No. 7-10235). It can be produced and accumulated in the culture broth by a fermentation method using microorganisms, and from the above culture, a conventional purification means already known, for example, a precipitation method, an ion exchange resin, a chromatograph using activated charcoal, or the like can be used. It can be purified and collected by using a separation and purification method such as a imaging method. It can also be prepared by using a known chemical synthesis method or the like. Further, a commercially available product can be used.

The salt of the ollotic acid is not particularly limited as long as it is a pharmaceutically acceptable salt, but when the ollotate is dissolved in water, the aqueous solution shows neutral to weakly acidic, and precipitates or precipitates during storage. Examples thereof include choline salts, lysine salts, arginine salts, and ornithine salts, which are less likely to occur, and in the case of beverages, these water-soluble salts are preferable. Further, since it does not need to be water-soluble when taken as a supplement such as capsules and tablets, metal salts such as poorly soluble sodium salt, potassium salt, magnesium salt, calcium salt and ammonium salt can be mentioned. In addition, ollotic acid can also form a salt with carnitine and be solubilized, and carnitine salt (L-carnitine orotic acid) has good solubility, but the aqueous solution has a low pH, so purine bases such as guanosine are required. The pH can be increased to weak acidity by adding or basic amino acids. The water-soluble salt of ollotic acid in the present invention has the same embodiment as that of the above-mentioned water-soluble salt of ollotic acid when the ollotic acid and the base are dissolved in water alone, that is, For convenience, a combination of olottic acid and a base, which exhibits an embodiment in which the ion of olottic acid and the counter cation coexist by ionization in the aqueous solution, can be included. Examples of the combination of olottic acid and base include a combination of ollotic acid hydrate such as an ollotic acid-free form or ollotic acid monohydrate and lysine, arginine, or ornithine; specifically, ollotic acid. Preferable combinations include free form and lysine, ollotic acid free form and arginine, ollotic acid free form and ornitine, ollotic acid monohydrate and lysine, ollotic acid monohydrate and arginine, and olottic acid monohydrate and arginine. Can be listed in.

The effects of the oral composition of the present invention include the ability to suppress cell death (apopulation) of retinal ganglion cells due to optic nerve damage, the ability to maintain the number of viable cells in the retinal ganglion cell layer, or glaucoma associated with these actions. The ability to prevent, improve glaucoma, or treat glaucoma; the ability to treat intractable diseases caused by abnormalities of optic nerve cells such as weak vision; can be mentioned. The retinal ganglion cell death inhibitor and the retinal ganglion cell death preventive agent of the present invention are not particularly limited as long as they exhibit the effects of the above-mentioned oral composition of the present invention, and the oral composition of the present invention is not particularly limited. When used as a retinal ganglion cell death inhibitor or a retinal ganglion cell death preventive agent, it is formulated into tablets, capsules, fine granules, powders, granules, syrups, suspensions, etc. For elderly people who have difficulty swallowing, disintegrating tablets that show rapid disintegration in the mouth, and liquid preparations such as syrups and suspensions are preferable. .. Further, the oral composition of the present invention can be used not only for pharmaceutical purposes but also in the form of supplements such as liquid preparations and capsules.

In formulating the above-mentioned retinal ganglion cell death inhibitor and retinal ganglion cell death preventive agent, ordinary pharmaceutically acceptable carriers, binders, stabilizers, excipients, diluents, pH buffers, etc. Various compounding ingredients for preparation such as a disintegrant, a solubilizer, a solubilizing agent, and an isotonic agent can be added.

In addition, the subjects (persons) to whom the oral composition of the present invention is administered include patients with glaucoma who cause optic neuropathy such as primary open-angle glaucoma, normal tension glaucoma, mixed glaucoma, and secondary glaucoma, and weak vision. Even subjects who do not suffer from intractable optic neuropathy or glaucoma (non-glaucoma patients) can be mentioned as those who need / desire to prevent such glaucoma.

The administration mode of the retinal ganglion cell death inhibitor or the retinal ganglion cell death preventive agent of the present invention is appropriately set according to the health condition of the subject to be administered, the severity of symptoms, age, weight, judgment of a doctor, and the like. However, in order to obtain the cell death inhibitory effect, it is necessary to administer for a certain period of time, and it is necessary to administer the same amount in several divided doses instead of a single high-concentration administration. From the viewpoint, in the case of oral administration to humans, administration of 1 mg to 6 g / day, preferably 10 mg to 3 g / day, more preferably 50 mg to 1 g / day, still more preferably 100 mg to 600 mg / day in terms of ollotic acid. An embodiment in which the amount is administered once, twice, three times, four times or five times a day can be exemplified. Specifically, a mode in which a supplement containing 100 to 200 mg is administered 1 to 3 times a day can be preferably mentioned. The administration period for treatment is, for example, 1 week, 2 weeks, 1 month, 3 months, 6 months, 9 months, 1 year, 1 year and 3 months, 1 year and 6 months, 1 year and 9 months, 2 years or Two years or more can be mentioned, and the administration period for prevention includes, for example, 1 month, 3 months, 6 months, 9 months, 1 year, 1 year and 3 months, 1 year and 6 months, 1 year and 9 months. Two years or more than two years can be mentioned.

Hereinafter, the present invention will be described in more detail with reference to Examples, but the technical scope of the present invention is not limited to these examples.

[Protective action of retinal cells]
The cytoprotective effect of ollotic acid on retinal cells including retinal ganglion cells was confirmed using primary cultured retinal neurons. Oxidative stress is known to be involved in glaucoma, and it has been suggested that ingestion of antioxidant components improves glaucoma. Therefore, we investigated the cytoprotective effect of ollotic acid on oxidative stress loading by stressing the retinal cells by making them single cells and culturing the retinal cells in a medium to which no antioxidant was added. In addition, the cytoprotective effect of ollotic acid was also examined in a medium in which an antioxidant and ollotic acid were used in combination.

The retina was isolated from the eyeballs excised from an 8-week-old C57BL / 6J mouse (male), and the cells were separated using Gentl MACS to obtain a single cell state. Retinal cells in the state of single-cell 1.4 × 10 ⁵ cells / 50 [mu] L / well were seeded into 96-well plates and incubated for 15 minutes at 37 ° C.. B-27 supplement (gibco, # 17504-001) (manufactured by Life Technology Co., Ltd.), which is a medium containing an antioxidant component suitable for culturing and proliferating nerve cells (hereinafter, also referred to as "B-27 medium"). To each well, an ollotic acid preparation solution prepared by dissolving 50 μL of ollotic acid in an arginine aqueous solution was added to each well so that the final concentrations of ollotic acid were 0, 25 ppm and 50 ppm, respectively, and the cells were cultured at 37 ° C. for another 2 hours. Similarly, if the above B27 supplement is removed from the five antioxidant components (vitamin E, vitamin E acetate, superoxide dismutase, catalase and glutathione) that are said to suppress apoptosis due to oxidative stress damage in cells, apoptosis is enhanced. In a possible medium (gibco, # 10889-038) (manufactured by Life Technology Co., Ltd.) (hereinafter, also referred to as "B-27-AO medium"), so that the final concentrations of ollotic acid are 0, 25 ppm, and 50 ppm, respectively. An ollotic acid preparation solution prepared by dissolving 50 μL of ollotic acid in an aqueous arginine solution was added to each well, and the cells were cultured at 37 ° C. for another 2 hours. Then, 10 μL of Amalar Blue (manufactured by Invitrogen), a reagent for measuring cell proliferation, was added to each well, and the cells were cultured at 37 ° C. for 18 hours. The viability of cells was quantitatively evaluated by measuring the fluorescence intensity (Ex = 560 nm, Em = 590 nm) with a high-sensitivity fluorescent microplate reader MACS GEMINE. The results are shown in FIG.

(result)
As is clear from FIG. 1, in the B-27-AO medium, retinal cells were significantly increased when 25 ppm or 50 ppm of boron acid was added as compared with the case where no boron acid was added (B-27-AO control). It was confirmed that the Viability of the retina was improved. In addition, it was confirmed that the Viability of retinal cells was significantly improved when 25 ppm or 50 ppm of ollotic acid was added in the B-27 medium as compared with the case where ollotic acid was not added (B-27control). .. Therefore, it was confirmed that the addition of ollotic acid at a very low concentration of 25 ppm brought about the effect of improving the Viability of cells regardless of the addition of antioxidants, and that ollotic acid had a cytoprotective effect on retinal cells. ..

[Examination using optic nerve amputation model]
(Measurement of rat drinking water)
Nine rats (SD male rats, manufactured by Charles River Laboratories, Japan, 4 to 5 weeks old, average body weight of about 100 g) were acclimatized by normal breeding for 1 week after arrival, and the test was started. The daily drinking amount of one rat was measured in advance for several days, and it was confirmed that the daily drinking amount of 250 g of body weight, which is the average of the experimental period, is about 30 mL / animal. Since the administration of drinking water was carried out for 2 months, the body weight and the amount of drinking water were measured every 2 weeks in consideration of the change in the amount of drinking water with the growth of the rat.

(Preparation of ollotic acid solution)
In order to orally administer ollotic acid-free monohydrate (manufactured by Kyowa Hakko Bio Co., Ltd.) to animals at a dose of 500 mg / kg / day, an ollotic acid solution was prepared as follows. Carnitine was added to increase the solubility of ollotic acid. The preparation method is as follows.
(1) Dissolve 8.4 g of carnitine (manufactured by Lonza Japan) in 900 mL of tap water.
(2) Stir with a stirrer to completely dissolve, and then add 4.2 g of ollotic acid-free monohydrate (manufactured by Kyowa Hakko Bio Co., Ltd.) to dissolve.
(3) Transfer to a measuring cylinder and adjust to 1 L with tap water.
The ollotic acid solution prepared by this method was stored at 4 ° C.

(Orotic acid solution administration)
The above ollotic acid solution was freely ingested in a water bottle, and the body weight and the amount of drinking water were measured every two weeks. The control rats were given tap water. The ollotic acid solution was changed twice a week. Orot acid solution administration was carried out for 2 months.

(Preparation of optic nerve amputation model rat)
After administration of the ollotic acid solution for 2 months, after light inhalation anesthesia with sevoflurane, 40 mg / kg of Nembutal was intramuscularly administered. The rat was fixed on a retention table and a partial incision was made in the upper conjunctiva of the eyeball. With both hands, two 45-degree bends were used to avoid tissue and expose the optic nerve (pain category C). By cutting only the optic nerve with a micro-scissors and placing a 2 mm ³ hemostatic gelatin sponge (Sponzel, manufactured by Astellas Pharmaceutical Co., Ltd.) impregnated with 1% fluorogold (FG) on the optic nerve stump, retinal ganglion cells FG labeling was performed. The conjunctiva was reduced and an antibacterial eye ointment was instilled. As described above, only one eye was treated for optic nerve stump, and the companion eye was left untreated. After the operation, administration of ollotic acid solution was continued.

(Evaluation by counting retinal ganglion cells)
Ten days after the optic nerve stump treatment, the rat was dislocated from the cervical spine under sevoflurane anesthesia, the eyeball was removed, the retina was fixed with 4% paraformaldehyde (PFA), a flat mount specimen was prepared, and then the retinal nerve. Nodal cells were counted and quantified. Photographs of FG-labeled retinal ganglion cells under a fluorescence microscope in a total of 12 regions, 4 locations per retina, moving 90 degrees each, and 3 locations concentrically from the center to the periphery of the 4 locations. Was taken. In each photograph, the number of cells in the portion showing the average cell body distribution in the range of 500 pixels square was counted, and the cell density of the retinal ganglion cells was calculated. Awakening was confirmed by heat insulation treatment, and the gauge was returned to the gauge.

(result)
As is clear from FIG. 2, the number of living cells in the retinal ganglion cell layer was significantly higher in all of the orot acid solution-administered group (4 animals) than in the control group (5 animals). Therefore, it was confirmed that administration of the ollotic acid solution for 2 months before the treatment of the optic nerve stump showed a remarkable inhibitory effect on retinal ganglion cell death due to optic nerve damage (see FIG. 1).

(Summary)
In the previous experiment, when the same amount of ollotic acid solution as in this experiment was given as a single dose in a sonde for 1 week, no significant result was obtained, so the following two points were considered.
1) In order to obtain the cell death inhibitory effect, long-term administration is required to some extent.
2) It is necessary to inoculate the same amount in several divided doses instead of a single high-concentration dose.

The present invention is useful in the medical field in that it can prevent, improve, and treat eye diseases caused by damage to retinal ganglion cells such as glaucoma.

Claims (4)

Orotic acid or a salt of orotic acid (excluding lithium orotic acid) containing oral compositions for inhibition or prevention of retinal ganglion cell death. The oral composition according to claim 1, wherein retinal ganglion cell death is caused by optic nerve damage. A retinal ganglion cell death inhibitor, which comprises the oral composition according to claim 1 or 2, for administration to a glaucoma patient. A retinal ganglion cell death preventive agent comprising the oral composition according to claim 1 or 2, for administration to a non-glaucoma patient.