WO2019093262A1 - Inhibition of myopia by controlling intestinal environment - Google Patents

Abstract

It is clear that changes in intestinal bacterial flora are correlated with the onset of myopia, and as a result, it was found that it is possible to inhibit myopia by improving intestinal bacterial flora. It was also found that it is possible to screen for myopia inhibitors using changes in intestinal bacterial flora as an indicator and that lactoferrin acts as a myopia inhibitor.

Description

Myopia suppression by intestinal environment control

The present invention relates to drugs for the prevention and treatment of myopia. In particular, the present invention relates to a method of screening a myopia inhibitor which suppresses the progression of myopia by controlling the intestinal environment, and a myopia inhibitor.

In recent years, the number of myopic patients continues to increase. For example, nearly 90% of young people in China and 96.5% of 19-year-old men in Seoul are said to be nearsighted. Not only in Asia, but in the United States and Europe, myopia patients have doubled compared to 50 years ago, and it is estimated that about one third of the world's population, or 2.5 billion myopia patients (Non-Patent Document 1) ).

Despite the large number of patients, myopia is not so serious as it can be corrected with eyeglasses and contact lenses. However, intense myopia, which is severe myopia, is the fourth leading cause of blindness in Japan, and is also known to be a risk factor for various eye diseases such as myopic traction macular disease.

It is expected that the incidence of myopia will increase from 22.9% to 49.8% from 2000 to 2050 worldwide, and the number of people suffering from severe myopia will increase from 163 million to 938 million. (Non-Patent Document 2). As mentioned above, since strong myopia is known to significantly increase the risk of various eye diseases including blindness, an increase in patients with strong myopia may lead to an increase in eye disease patients such as blindness. is expected. Therefore, the need for myopia prevention is increasing.

Recent studies have reported that myopia is associated with inflammation, inflammation at the local eye causes myopia to progress, and systemic inflammatory diseases increase the risk of myopia (Non-Patent Document 3) ). On the other hand, it has been clarified that the intestinal bacterial flora is correlated with various diseases, and application of the intestinal bacterial flora to treatment of diseases is attempted by changing the intestinal bacterial flora. For example, not only for intestinal inflammatory diseases such as inflammatory bowel disease, but also for inflammatory diseases that occur in sites distant from the intestine such as multiple sclerosis, and systemic inflammatory diseases such as diabetes, It has been revealed that the improvement of the internal bacterial flora has an effect (Non-patent Documents 4 and 5).

In Japan and other Asian countries where myopia has increased significantly, eating habits have been changing significantly since the 1950s. It is thought that the intestinal bacterial flora is also greatly changed along with it. As the proportion of myopic patients in Asia is increasing significantly as if responding to changes in diet, it is possible that changes in intestinal flora contribute to the increase in myopic patients. However, studies on the correlation between changes in intestinal microbiota and myopia have not been conducted yet.

Dolgin, E., 2015, Nature, Vol. 519, p. Holden, B. A. et al., 2016, Ophthalmology, Vol. 123 (5), p. 1036-1042. Lin, H-J. Et al., 2016, EBioMedicine, Vol. 10, p. 269-281. Honda, K. and Littman, D. L., 2012, Annu. Rev. Immunol., Vol. 30, pp. 759-795. Miyake, S. et al., 2015, PLoS One, 10 (9): e0137429 .. doi: 10.0.1371 / journal.pone.0137429 Jiang X. et al., 2017, The Association for Research in Vision and Ophthalmology 2017 Annual Meeting Jang Y. S. et al., 2015, Mucosal Immunol. Vol. 8 (4), p. 906-917.

If the intestinal flora is involved in myopia development, it may improve the intestinal flora and prevent myopia or suppress the progression of myopia. An object of the present invention is to develop a method for suppressing the onset of myopia while examining the possibility that a change in intestinal microflora is involved in the onset of myopia. If changes in the intestinal flora are involved in myopia development, then capturing the changes in the intestinal flora and improving them can not only reduce the number of myopia patients that increase but also cause myopia Blindness and various eye diseases can be reduced.

In particular, since it is anticipated that myopia prevention methods will be applied mainly to underage children, simpler and less invasive prevention methods are required. An object of the present invention is to provide a medicine and a supplement that prevent and suppress myopia by improving intestinal microflora. Another object of the present invention is to provide a screening method of medicines and supplements for improving myopia.

The present invention relates to the following methods for screening a myopia inhibitor, and a myopia inhibitor.
(1) A method of screening a myopia inhibitor using changes in intestinal bacterial flora as an index.
(2) A method for screening a myopia inhibitor, which comprises administering a candidate substance that changes intestinal microbiota to a myopia model animal and measuring an index of myopia to determine the presence or absence of myopia suppression effect.
(3) The method for screening a myopia inhibitor according to (2), wherein the myopia model animal wears a minus lens and induces myopia.
(4) The method for screening a myopia inhibitor according to (2) or (3), wherein the index of myopia is at least one of axial length, refractive value, thickness of sclera, inflammation of sclera.
(5) A myopia inhibitor containing an active ingredient that improves the intestinal bacterial flora.
(6) The myopia inhibitor according to (5), wherein the active ingredient is lactoferrin.
(7) A method for treating or preventing myopia by administering a myopia-suppressing agent containing an active ingredient that improves the intestinal bacterial flora.
(8) The method for treating or preventing myopia according to (7), wherein the active ingredient is lactoferrin.

As it became clear that intestinal microbiota is involved in myopia development, it became possible to provide a medicine that suppresses myopia progression. It is possible to develop a myopia inhibitor with a new concept of improving the intestinal environment and suppressing inflammation.

The figure which shows the influence on the myopia induction of antibiotic administration. The figure which shows the influence with respect to myopia induction of lactoferrin administration. FIG. 3 (A) shows the effect of lactoferrin administration on myopia induction. FIG. 3 (B) is a diagram showing the difference between the amounts of change in FIG. 3 (A). The figure which shows the anti-inflammatory effect in the sclera of lactoferrin administration.

The screening method of the myopia inhibitor of the present invention is a method in which a candidate substance is administered to a myopia model animal in which myopia has been induced, and screening is performed using a change in intestinal bacterial flora as an index. The animal inducing myopia is not limited to conventionally used animals such as chicks, mice, rats and monkeys, and myopia can be induced in various animals and used as a model animal. In particular, the myopia model mouse shown below can be suitably used because it exhibits symptoms closely resembling those of human strong myopia.

Specifically, the candidate substance is administered to a myopia model animal, and the effects of the candidate substance are examined by measuring and comparing the axial length and the refraction value, which are indices of myopia, before and after myopia induction by minus lens wearing. Good. As shown below, it is clear that changes in intestinal flora are involved in myopia development, so substances that improve intestinal flora can be administered as candidate substances and their effects can be seen .

In addition, it becomes possible to analyze the intestinal flora and analyze bacteria correlated with myopia suppression. The intestinal flora before and after induction of myopia can be analyzed to identify bacteria that are greatly reduced or increased, and candidate bacteria can be screened using these bacteria as an indicator. Furthermore, it becomes possible to administer a compound etc. which targets these bacteria. With respect to the further decreasing bacteria, it is also possible to administer the cultured bacteria and use it as a myopia inhibitor.

A substance having an effect of suppressing myopia by screening can be used for suppressing or preventing myopia as a pharmaceutical composition or a supplement, a specified health and nutrition food, a nutritive function food, or a functional display food containing the compound as an active ingredient. Pharmaceutical compositions and supplements can contain, in addition to the active ingredient, acceptable carriers, adjuvants, excipients, buffers, diluents and the like that can be generally contained in pharmaceutical compositions and supplements.

As shown below, the present inventors improved the intestinal flora and found that lactoferrin has an effect as a myopia inhibitor. Lactoferrin is an iron-binding glycoprotein with a molecular weight of about 80,000 that has a similar structure to transferrin, and has a high concentration in colostrum and is present in exocrine fluids such as tears and saliva, and neutrophils, It is believed to play an important role in the protection of infants (infants).

As the physiological functions of lactoferrin, various actions such as anti-inflammatory action, antibacterial / antiviral activity, immunoregulatory action, antioxidant action, iron absorption regulating action, bifidobacteria growth promoting action are known, but they are effective for myopia suppression There have been no reports of having a

Hereinafter, the present invention will be described in detail with reference to examples. It was analyzed whether the intestinal bacterial flora was changed using a myopia model mouse induced with a minus lens (Lens-indcued Myopia, hereinafter sometimes referred to as LIM). The myopia model mouse developed by the present inventors is a mouse model having symptoms closely resembling those of human myopia in which the degree of axial length extension, refraction value, and sclera become thinner than normal (non-patented) Literature 6). Myopia with refractive properties, axial length, sclera thickness, all with properties similar to human myopia by wearing a lens of -30 diopter (diopter, D) in mice for at least 3 weeks and performing myopia induction It is confirmed that the model can be derived.

The method for producing the myopia model mouse is briefly described. As a young mouse is easier to induce myopia, it is desirable to wear a minus lens as soon as possible after weaning. Here, a 3-week-old C57BL6J male mouse is used. The mice are anesthetized with a mixture of Domitol (registered trademark, Nippon Zenyaku Kogyo), Betorfahr (registered trademark, Meiji Seika Pharma) and midazolam (sand), and the skull is exposed with scissors. Prepare a column with a screw thread on the skull and fix it with dental cement (Super-Bond, Sun Medical). One week later (at 4 weeks of age), in order to induce myopia, a -30 D negative lens (Rainbow Optical Laboratory) is worn on one side and only the frame is worn on the other as a control. The frame passes through a hole provided at the top of the frame through a post erected on the skull and is fixed by a nut. A lens having a shape projecting laterally is adhered to a frame portion on the lower side of the lens so that the lens is not scratched by the front leg or the like when the lens is worn on the mouse. The protector prevents the mouse from touching the lens and the lens is not scratched. In addition, since the lens is fixed to the support using a nut, it can be adjusted in position or removed to clean the dirt as the mouse grows.

<Correlation between changes in intestinal flora and myopia onset>
Example 1 Induction of Changes in Intestinal Bacterial Flora by Antibiotics and Myopia Inhibitory Effect Antibiotics were administered to myopia model mice to kill enteric bacteria, and the effects on myopia induction were analyzed. Four weeks from 3 to 7 weeks of age, mice were given normal water for the control group (n = 5) and antibiotic water for the antibiotic administration group (n = 5) to affect myopia induction Was analyzed. Antibiotic water was prepared by dissolving 1.0 g / L of ampicillin, 0.5 g / L of vancomycin and 1.0 g / L of neomycin in ultrapure water. It is believed that the administration of these three antibiotics kills most enterobacteria.

At 4 weeks of age, refractive value and axial length were measured using a refractometer and SD-OCT (Spectral Domain-Optical Coherence Tomography) as myopic evaluation items, respectively, and then a right lens of -30 diopter minus lens, left eye I wore only a frame without a lens and performed myopia induction for 3 weeks. During that time, the lens was removed from the mouse once every two days, and the lens was cleaned and then worn again. After 3 weeks myopia induction (7 weeks old), measure axial length, refraction value again, change with myopia induction before (refractive value after LIM, or axial length-refractive value before LIM, or The axial length) was calculated (FIG. 1). Data are shown as mean ± standard error. The difference between the groups is tested for significance by one-way analysis of variance, and a p value of less than 0.05 is statistically significant and indicated as * in the figure. Unless there is particular notice, when there is a significant difference in the specification, the p value is less than 0.05. Moreover, when p value is less than 0.01, it has shown by **. The same applies to the following data.

FIG. 1 left is the result of calculating the difference in axial length before and after myopia induction. The change in the axial extension of the frame with only frame in the antibiotic administration group and the control group, and the eye with myopia induction (LIM) is shown. In the control group, the eye length of myopia-induced eyes is significantly (P <0.01), whereas in the antibiotic administration group, only the frame-induced myopia-induced eye is There was no difference in axial length.

In addition, regarding the influence of the administration of the antibiotic on the refraction value, a significant difference (P <0.01) was generated between the control group and the antibiotic administration group (FIG. 1 right). When the refraction values of the control group and the antibiotic administration group were compared in the myopia-inducing eyes, the change in the myopia induction-guided eye was significantly smaller in the antibiotic administration group compared to the control group. These results suggest that the antibiotics administered reduce enteric bacteria and that the enteric environment has been modified to suppress myopia progression. That is, it was shown for the first time that changes in the intestinal flora are directly involved in myopia induction.

<Example of myopia suppressant by improving intestinal microbiota>
Example 2 Induction of Changes in Intestinal Bacterial Flora by Lactoferrin Administration The results of Example 1 suggested that myopia suppression can be achieved by improving the intestinal flora. Therefore, it was examined whether myopia suppression occurs by administering a substance that improves the intestinal bacterial flora. Specifically, lactoferrin, which has been reported to have anti-inflammatory activity, was administered to myopia model mice to examine its effect.

Bovine lactoferrin (Sigma- Aldrich, purity 95 95%) was adjusted to 50 or 160 mg / ml with Ringer solution (Otsuka Pharmaceutical) and stored at -20 ° C until use. Three-week-old male C57BL6J mice were orally administered lactoferrin 6 days a week or Ringer's solution as a control with a gastric tube. Administration was performed for 4 weeks until 7 weeks of age. The dose of lactoferrin was administered at 500 mg / kg / day or 1600 mg / kg / day. The control group was dosed with Ringer's solution equivalent to the lactoferrin administration group. One week after the start of lactoferrin administration, that is, at 4 weeks of age, the refractive index and axial length of mice were measured as myopic evaluation items using a refractometer and SD-OCT, respectively. Thereafter, in the same manner as in Example 1, myopia guidance was performed by wearing a minus lens of -30 diopters for the right eye and only a frame without lenses for the left eye, and wearing the minus lens for 3 weeks. After induction of myopia for 3 weeks (ie, 7 weeks after administration of lactoferrin for 4 weeks), the refraction value and axial length were measured again to calculate the change from that before induction of myopia.

FIG. 2 shows the results of administration of lactoferrin at 500 mg / kg / day. The experiment was started with 5 mice in each group, but the mouse died due to the operation of the gastric sound, etc., so when measuring 7 weeks, the control group, the lactoferrin administration group (referred to as LF in the figure), n = 2, n = 3.

Since the number of analyzes (n) is small, both the change in axial length and the change in refractive value are significant between the eye wearing the frame alone and the eye performing myopia induction in both the control group and the lactoferrin-administered group There was no difference. However, there was a tendency that the change in axial length due to lactoferrin administration (FIG. 2 left) and the change in refraction value (FIG. 2 right) decrease.

Next, lactoferrin is administered at 1600 mg / kg / day, and the results of analyzing the myopia suppression effect are shown. The experiment was conducted in the same manner as when 500 mg / kg / day was administered except for the dose of lactoferrin. The axial length of the control group was significantly different between the eye wearing only the frame and the myopia-inducing eye in the control group, but no significant difference was seen in the lactoferrin administration group (Fig. 3 (A) left). That is, it was shown that lactoferrin administration suppresses the extension of the axial length.

On the other hand, the refraction value was significantly different between the control group and the lactoferrin-administered group, and the eye wearing only the frame and the myopia-inducing eye (control: P <0.01, lactoferrin-administered group: P <0.05, FIG. 3 (A) right). In both groups, myopia induced by the minus lens showed a decrease in the refraction value, but a change in the lactoferrin administration group tended to decrease.

In order to see the difference in change between the eye with myopia induction and the eye without the myopia induction with only the eye wearing the frame, the amount of change with myopia induction (LIM) and without (frame) The difference was calculated (FIG. 3 (B)). The axial length showed a significant difference between the control group and the lactoferrin administration group (Fig. 3 (B) left), but the refraction value tended to decrease in the lactoferrin administration group compared to the control group. However, no significant difference was observed (FIG. 3 (B) right).

<Verification of anti-inflammatory effect of lactoferrin>
[Example 3] Gene expression analysis in sclera From the above analysis results, it was shown that changes in intestinal bacterial flora are deeply involved in myopia development. In addition, lactoferrin is known to have various physiological activities, but as an action associated with myopia suppression, it is known to have an anti-inflammatory action (Non-patent Document 7). If the myopia inhibitory effect of lactoferrin is due to anti-inflammatory action, inflammation in the sclera may be suppressed. Therefore, in myopic model mice, it was analyzed whether lactoferrin suppresses the inflammatory response of sclera.

In the same manner as in Example 2, mice were administered lactoferrin at a dose of 1600 mg / kg / day to the lactoferrin administration group, and Ringer's solution was administered to the control group from 3 weeks to 7 weeks, 4 weeks to 7 weeks Myopia was guided by a negative lens to the age. Physically isolate the sclera from a control eye wearing only the frames of each group (n = 3) and myopia-inducing eyes wearing the lens under a stereomicroscope, and extract RNA with each three sclera as one sample did.

RNA extraction was performed using miRNeasy Micro Kit (QIAGEN). Reverse transcription was performed using ReverTra Ace (Toyobo Co., Ltd.) and realtime-PCR using THUNDERBIRD SYBR qPCR Mix (Toyobo Co., Ltd.) in StepOnePlus (Applied Biosystems) (amplification conditions: 95 ° C., 15 sec, 60 ° C., 60 sec) 40 cycles).

IL-6 was used as a primer for detecting an inflammatory response, and GAPDH was used as a control. The sequences of the primers used are as follows.
IL-6
forward: 5'-CTACCCCAATTTCCATGC-3 '(SEQ ID NO: 1)
reverse: 5'-ACCACAGTGAGGAATTGTCCA-3 '(SEQ ID NO: 2)
GAPDH
forward: 5'-AGGAGCGA GACC CC ACT AAC-3 '(SEQ ID NO: 3)
reverse: 5'-GATGACCCTTTTGGCTCCAC-3 '(SEQ ID NO: 4)

As shown in FIG. 4, in the control group, IL-6 expression level was significantly increased in myopia-induced eyes, whereas in lactoferrin administration group, IL-6 expression level was increased in myopia-induced eyes. It was not. Therefore, it is clear that lactoferrin administration suppresses inflammation of sclera. It is suggested that lactoferrin suppresses scleral inflammation and improves myopia by improving changes in intestinal flora.

The present inventors have found for the first time that substances that improve the intestinal environment and suppress inflammation exhibit a myopia suppression effect. The same effect can be expected from substances having the effect of improving the intestinal environment and suppressing inflammation, and it has become possible to search for a new concept of myopia inhibitor. In addition, since it has been shown that changes in intestinal microflora are involved in myopia development, it is also possible to identify bacteria that suppress myopia development in the future and to suppress myopia progression.

It is possible to provide a myopia inhibitor which suppresses myopia for which there has been no effective treatment until now. As a result, it also leads to the prevention of blindness due to intense myopia and other eye diseases. In addition, since lactoferrin contained in milk shows a myopia suppression effect, there is a possibility that progression of myopia can be suppressed by administering it as a supplement. In addition, it has become possible to screen myopia inhibitors with the new concept of acting on the intestinal flora.

Claims (6)

A method of screening a myopia inhibitor using changes in intestinal microflora as an indicator. In myopic model animals,
Administer a candidate substance that changes the intestinal flora,
A method for screening a myopia inhibitor which determines the presence or absence of myopia suppression effect by measuring an index of myopia. The myopia model animal is
Wear a negative lens,
The method for screening a myopia inhibitor according to claim 2, which induces myopia. The index of myopia is
4. The method for screening a myopia-suppressing agent according to claim 2, wherein the axial length, refractive value, scleral thickness, scleral inflammation is at least one or more. An agent for suppressing myopia, which comprises an active ingredient that improves intestinal microbiota. The myopia inhibitor according to claim 5, wherein the active ingredient is lactoferrin.

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