JP2018157809A - Aging macular degeneration model animal of nonhuman primate and its creation method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method of creating an AMD model animal of a nonhuman primate, and a method of evaluating an effect of a test substance for the prevention or the treatment of the AMD and a method of screening the substance having the effect of the prevention or the treatment of the AMD by using the AMD model animal created by the method.SOLUTION: A method of creating an AMD model animal is characterized by administering a sodium Iodate in a vitreous body of an animal individual of a nonhuman primate. Also, the AMD model animal of the nonhuman primate is created by the method of creating the AMD model animal. By using the created AMD model animal, a method of evaluating an effect of the test substance for prevention or treatment of the AMD is provided for evaluating the prevention or treatment effect of the AMD of the test substance.SELECTED DRAWING: None

Description

The present invention relates to a method for producing a model animal for non-human primate age-related macular degeneration (AMD), and a method for evaluating the effect of a test substance on the prevention or treatment of AMD using the AMD model animal produced by the method. And a method for screening a substance having an effect of preventing or treating AMD using the AMD model animal.
This application claims priority on March 23, 2017 based on Japanese Patent Application No. 2017-57515 for which it applied to Japan, and uses the content here.

  AMD is a disease in which macular retinal photoreceptor cells in the center of the fundus are impaired, and is one of the causes of premature blindness. AMD is mainly divided into an atrophy type (dry type) and an exudation type (wet type). In the atrophic type, the photoreceptor cells, retinal pigment epithelial cells adjacent to the photoreceptor cells, and choroidal capillaries gradually degenerately atrophy. In the exudative type, new blood vessels are generated from the choroid and the disease progresses relatively rapidly due to bleeding and edema. In human atrophic AMD, retinal pigment epithelium (RPE) degeneration (thinning and shrinking) and outer granular layer (ONL) thinning and disappearance are observed in the retina.

  A photoretinal disorder model is known as an atrophic AMD-like model animal (see, for example, Non-Patent Document 1). Intense light causes retinal damage. In fact, in the mouse photoretinal disorder model, it has been confirmed that degeneration has occurred in the retina. In addition, as AMD-like model animals, rodent and rabbit model animals in which RPE is denatured by systemic administration of sodium iodate or administration to the vitreous are known (for example, Non-Patent Document 2 or 3). reference.).

  In general, in the development of a therapeutic drug for a disease, a model animal for the disease is used to examine the therapeutic effect of a test substance. In order to obtain an appropriate evaluation of drug efficacy by a model animal, it is important that the model animal to be used accurately reflects the pathology of the target disease. Non-human primates are genetically, neuroanatomically, and pharmacokinetically closer to humans than rodents, so they are more effective than humans. You can expect to get it. For this reason, in order to develop a therapeutic agent effective for humans, it is preferable to use primates other than humans as rodents rather than rodents. In particular, since only primates have macula, rodent or rabbit AMD model animals cannot reflect the pathology of human AMD. Therefore, in order to develop preventive and therapeutic drugs for AMD, non-human primate AMD model animals that more accurately reflect the pathology of AMD are needed.

Tsutomu Okuno, 3 others, Occupational Safety and Health Research Institute Special Research Report, 2014, Vol. 44, 67-70. Junko Ishida, two others, Journal of Kawasaki Medical Society, 2016, Vol. 42, No. 2, pages 85-93. Cho et al., Japanese Journal of Ophthalmology, 2016, vol.60, p.226-237.

  The present invention relates to a method for producing a non-human primate AMD model animal, a non-human primate AMD model animal, and an effect of the test substance on the prevention or treatment of AMD using the AMD model animal produced by the method. It is an object of the present invention to provide a method for evaluating the above and a method for screening a substance having an effect of preventing or treating AMD.

  As a result of earnest research to solve the above problems, the present inventor has administered sodium iodate directly into the vitreous body of a non-human primate, thereby suppressing the effect of sodium iodate on the whole body, and human aging macular The inventors have found that retinal ONL thinning and RPE degeneration similar to degeneration can be caused around the central macular fovea, which is responsible for the majority of visual functions.

That is, a method for producing an AMD model animal according to the present invention, a non-human primate AMD model animal, a method for evaluating the effect of a test substance on the prevention or treatment of AMD using an AMD model animal produced by the method, and AMD The methods for screening for substances having the preventive or therapeutic effects are [1] to [11] below.
[1] A method for producing an AMD model animal, comprising administering sodium iodate into a vitreous body of a non-human primate animal individual.
[2] The method for producing an AMD model animal according to [1], wherein the dose of sodium iodate per vitreous is 0.5 to 2.5 mg.
[3] The method for producing an AMD model animal according to [1] or [2] above, wherein a sodium iodate solution in which sodium iodate is dissolved in water or phosphate physiological saline is injected into the vitreous body.
[4] The method for producing an AMD model animal according to any one of [1] to [3], wherein the animal individual is a cynomolgus monkey or a rhesus monkey.
[5] The method for producing an AMD model animal according to any one of the above [1] to [4], wherein in the produced AMD model animal, thinning of the ONL of the retina and degeneration of RPE are observed around the macular fovea .
[6] The method for producing an AMD model animal according to any one of [1] to [4], wherein map-like atrophy occurs in the fundus of the produced AMD model animal.
[7] The method for producing an AMD model animal according to any one of [1] to [6], wherein the produced AMD model animal is an atrophic AMD model animal.
[8] A non-human primate AMD model animal having retinal ONL thinning and RPE degeneration around the macular fovea.
[9] A non-human primate AMD model animal having a map-like atrophy in the fundus.
[10] A non-human primate AMD model animal is produced by the method for producing an AMD model animal of any one of [1] to [7],
The evaluation method of the effect with respect to the prevention or the treatment of AMD of a test substance which evaluates the prevention or the treatment effect of the test substance with AMD using the produced AMD model animal.
[11] A non-human primate AMD model animal is produced by the method for producing an AMD mole animal according to any one of [1] to [7],
Using the produced AMD model animal, evaluate the preventive or therapeutic effect of test substance AMD,
When at least one selected from the group consisting of thinning of the retina ONL and degeneration of RPE is improved after taking the test substance compared to before taking the test substance, A screening method for a substance having a preventive or therapeutic effect for AMD, wherein a test substance is selected as a substance having a preventive or therapeutic effect for AMD.

By the method for producing an AMD model animal according to the present invention, a non-human primate model animal having ONL thinning and RPE degeneration in the retina, which is a characteristic pathology of human AMD, can be produced.
By using the AMD model animal produced by the production method, it is possible to evaluate the effect of the test substance on the prevention or treatment of AMD and to screen the substance having the prevention or treatment effect of AMD with higher accuracy.

In Example 1, the OCT image of the retina of the model animal administered with sodium iodate intravitreally before administration of the sodium iodate solution (day 0 of administration), 10 days after administration, and 26 days after administration is shown. In Example 1, the OCT image of the retina of a model animal with sodium iodate intravitreal administration before administration of the sodium iodate solution (day 0 of administration) and 66 days after administration is shown. In Example 2, the fundus findings (upper stage) and spontaneous fundus of the model animal with sodium iodate intravitreal administration before administration of the sodium iodate solution (day 0 of administration), 38 days after administration, and 66 days after administration Fluorescence findings (bottom) are shown. In Example 2, the fluorescein fluorescence fundus findings of the model animal administered sodium iodate intravitreally on the 66th day after administration of the sodium iodate solution are shown. In Example 2, the HE dyeing | staining image of the structure | tissue of the foveal periphery site | part of the sodium iodate intravitreal administration model animal of the 66th day after administration of a sodium iodate solution is shown. In Reference Example 1, an HE-stained image of the tissue around the fovea of a photoretinal disorder model animal is shown.

  In the method for producing an AMD model animal according to the present invention, sodium iodate is administered into the vitreous body of a non-human primate animal individual. By administering sodium iodate directly into the vitreous, the effects of sodium iodate on the whole body are suppressed, while retinal degeneration similar to that of human AMD, particularly human atrophic AMD, that is, thinning of the retinal ONL Can cause degeneration of RPE and map-like atrophy.

  In addition, degeneration of the retina of an AMD model animal produced by the method for producing an AMD model animal according to the present invention is similar to the observation in human AMD patients, such as optical coherence tomography (OCT), fundus examination, and fundus autofluorescence. Observation can be made by combining various examinations for examining the state of the retina, such as examination, fluorescein fluorescence fundus angiography, indocyanine green fluorescence fundus angiography. Specifically, ONL thinning and RPE degeneration in the retina of AMD model animals produced by the method for producing AMD model animals according to the present invention can be observed in the same manner as in human AMD patients. . For example, the optical retina tomography (OCT) can directly observe the ONL, macular and RPE of the retina of the eyeball. In the OCT image of the eyeball of an AMD model animal according to the present invention, as in human atrophic AMD, ONL thinning, outer boundary membrane disruption, choroidal signal enhancement, and photoreceptor inner / outer layer line (Ellipsoid zone), cone outer segment tip line (interdigitation zone), and disappearance of outer granule layer are observed. Further, the map-like atrophy of the fundus of the AMD model animal is observed by a fundus examination in the same manner as human atrophic AMD. At the atrophic site of this map-like atrophy, the fundus autofluorescence examination shows low fluorescence, and the fluorescein fluorescence fundus findings show strong hyperfluorescence.

  In the present invention, the amount of sodium iodate administered into the vitreous body may be an amount sufficient to cause degeneration of the retina, and is appropriately determined in consideration of the type and age of the non-human primate to be administered. be able to. When the dose of sodium iodate is too large, damage to the retina and the like is too great, and it may be difficult to use as an AMD model animal. Therefore, in the present invention, the dose of sodium iodate per vitreous body is preferably 0.5 to 2.5 mg, more preferably 1.0 to 1.5 mg. In addition, the administration of sodium iodate into the vitreous may be a single dose or may be divided into a plurality of times at an appropriate interval.

  The method for administering sodium iodate into the vitreous is not particularly limited. For example, a sodium iodate solution in which sodium iodate is dissolved in a suitable solvent can be directly injected into the vitreous. In the present invention, the solvent for dissolving the sodium iodate solution is preferably water, phosphate physiological saline (PBS), or an appropriate buffer because the influence of the solvent itself on the vitreous body can be reduced. Water or phosphate physiological saline is more preferable.

  In the method for producing an AMD model animal according to the present invention, the animal individual causing the AMD-like retina degeneration may be a non-human primate, may be a primate, or may be a monkey. . Examples of the original monkeys include lemurs, loris, galagos and tarsiers, and examples of the true monkeys include spider monkeys, capuchin monkeys, marmosets, rhododendrons, colobus, and apes. In the method for producing an AMD model animal according to the present invention, the non-human primate for producing the AMD model animal is preferably a monkey since the mechanism of AMD onset and the response to the drug are closer to humans. More preferably, it is a cynomolgus, a colobus, or an ape, more preferably a cynomolgus monkey, a rhesus monkey, a Japanese monkey, a white gibbon, a gorilla, an orangutan, a chimpanzee, or a bonobo, and even more preferably a cynomolgus monkey or a rhesus monkey.

  Administration of sodium iodate into the vitreous can cause retinal ONL thinning, RPE degeneration, and fundus map atrophy. The animal individual before and after administration of sodium iodate can be bred by ingesting the same feed as that given during normal breeding.

  In human AMD, particularly human atrophic AMD, one or more findings selected from the group consisting of ONL thinning and RPE degeneration are observed in the retina. In the non-human primate AMD model animal produced by the method for producing an AMD model animal according to the present invention, one or more findings selected from the group consisting of ONL thinning and RPE degeneration are also observed in the retina. The

  In human AMD, particularly human atrophic AMD, map-like atrophy is observed in the fundus. Non-human primate AMD model animals produced by the method for producing AMD model animals according to the present invention also have map-like atrophy observed on the fundus. Judgment of map-like atrophy can be performed according to the same standard as human atrophic AMD (for example, Koji Takahashi, “Guidelines for Diagnosis of Atrophic Age-Related Macular Degeneration”, Jinkai, October 10, 2015) 119, No. 10, pp. 671-677). Specifically, the map-like atrophy is mainly atrophy that occurs in a region within a diameter of 6000 mm centered on the fovea of the fundus. Geographic atrophy is generally in the form of a circle, oval, tuft, or map with a sharp boundary and a diameter of 250 μm (approximately 2 times the diameter of the retinal vein diameter at the optic disc edge). Times) or more. In addition, since low pigment or depigmentation changes of the retinal pigment epithelium are observed, the choroidal middle and large blood vessels can be clearly seen.Further, the fundus autofluorescence shows clear low fluorescence. Shows hyperfluorescence.

  The produced AMD model animal is very useful for analyzing the pathophysiology of AMD, evaluating the effect of the test substance on the prevention or treatment of AMD, and screening for a substance having an effect of preventing or treating AMD. Since the AMD model animal has a pathological condition observed in human atrophic AMD, in particular, pathological analysis of atrophic AMD, evaluation of the effect of the test substance on the prevention or treatment of atrophic AMD, and atrophic type It is very useful for screening for substances having preventive or therapeutic effects for AMD.

  Specifically, a test substance which is a candidate compound for a therapeutic agent for AMD is administered to an AMD model animal produced by the method for producing an AMD model animal according to the present invention, and the tissue peculiar to AMD in the eye, particularly the retina. The influence on the clinical characteristics is examined, and the therapeutic effect of the test substance on AMD is evaluated. One or more selected from the group consisting of retinal ONL thinning, RPE degeneration, and fundus map-like atrophy were improved by the administered test substance compared to before taking the test substance. In some cases, the test substance is evaluated as having a therapeutic effect on AMD. The method of administering the test substance to the AMD model animal is not particularly limited and may be oral administration, enema administration, intravenous administration, or nasal administration. Alternatively, it may be directly injected into an eye tissue such as a vitreous body.

  Similarly, a plurality of test substances are evaluated as to whether they have a therapeutic effect or a preventive effect on AMD, and a test substance evaluated as having a therapeutic effect or a preventive effect is defined as a substance having a preventive or therapeutic effect on AMD. By selecting, it is possible to screen for substances having an effect of preventing or treating AMD.

  EXAMPLES Next, although an Example is shown and this invention is demonstrated further in detail, this invention is not limited to a following example.

  The animal experiments conducted in the following examples and the breeding of the used animals were conducted in Japan under the Act on the Protection and Management of Animals (Act No. 105 of 1973), “Standards for Storage and Pain Reduction” (2006 Ministry of the Environment Notification No. 88), “Basic Guidelines for Conducting Animal Experiments at Executing Organizations Controlled by the Ministry of Health, Labor and Welfare” (2006 Notification by the Ministry of Health, Labor and Welfare), and “Animals” It was conducted in compliance with the “Guidelines for Proper Implementation of Experiments” (Formed by the 2006 Science Council of Japan).

[Example 1]
Cynomolgus monkey AMD model animals were prepared and examined for histological features unique to human AMD in the retina.

<Production of cynomolgus monkey sodium iodate intravitreal administration model animal>
50 μL of 20 mg / mL or 30 mg / mL sodium iodate solution (the dose of sodium iodate per vitreous is 1 mg or 1.5 mg) in the vitreous body of one eye of a cynomolgus monkey did. The sodium iodate solution was a solution in which sodium iodate was dissolved in water for injection, and was sterilized by passing through a filter having a pore size of 0.22 μm in advance.

  About the produced sodium iodate intravitreal administration model animal, the retinal thickness was measured before administration of the sodium iodate solution (day 0 of administration), 10 days after administration, and 26 days after administration. The OCT image of the retina near the macular fovea obtained by the measurement is shown in FIG. The upper part is a diagram expressing the thickness of the retina in shades, and the lower part is a sectional view of the retina. The thickness of the retina was measured by tracing the portion of the OCT image that hits the outer retina and measuring the thickness. The model animal is further bred, and an OCT image of the retina 66 days after administration of the sodium iodate solution is shown in FIG. In FIG. 2, the upper part is a cross-sectional view of the retina before administration of the sodium iodate solution, and the lower part is a cross-sectional view of the retina on the 66th day after administration of the sodium iodate solution.

  In FIG. 1 and FIG. 2, the region where the thickness of the central portion is thin is the macular fovea. As shown in FIG. 1, on the 10th day after administration of the sodium iodate solution, the retinal thickness is thin centered on the macular fovea, and this thin portion is 26 days after administration of the sodium iodate solution. It was more spread to the eyes. This thinning of the thickness of the macular fovea and the vicinity thereof mainly indicates that the ONL is thinned. Furthermore, from the cross-sectional view of the retina shown in FIG. 2, on the 66th day after administration, in addition to the thinning of the ONL, in the fovea, RPE, photoreceptor layer (photocell inner / outer segment layer line, cone outer segment) The tip line) disappeared, and disruption of the outer boundary membrane and enhancement of the choroidal signal were observed. From these results, administration of sodium iodate into the vitreous caused ONL thinning and RPE degeneration around the foveal fovea, and non-human primate sodium iodate intravitreal administration model animals It was found to show typical clinical findings seen in human atrophic AMD.

[Example 2]
Cynomolgus monkey AMD model animals were prepared and examined for the presence of histological features peculiar to human AMD in the fundus and macular fovea.

<Fundation examination, fundus autofluorescence examination, and fluorescein fluorescence fundus imaging>
Specifically, in the same manner as in Example 1, a sodium iodate intravitreal administration model animal prepared by administering 1.5 mg of sodium iodate per vitreous body of the right eye of a 9-year-old cynomolgus monkey The fundus was examined by fundus examination and fundus autofluorescence before administration of the sodium acid solution (day 0 of administration), 38 days after administration, and 66 days after administration. The fundus examination and the fundus autofluorescence examination were performed by a conventional method using a fundus camera equipped with an autofluorescence imaging function. In addition, fluorescein fluorescence fundus angiography was also performed on the model animals on day 66 after administration. Fluorescein fluorescence fundus angiography was performed by a conventional method using a fundus camera capable of fluorescence observation after intravenous injection of fluorescein to a model animal.

  The results of the fundus examination and the fundus autofluorescence examination are shown in FIG. In the figure, the upper row shows the fundus findings, and the lower row shows the fundus autofluorescence findings. No atrophy was observed before administration (“Day 0” in the figure), but 38 days after administration (“Day 38” in the figure) and 66 days after administration (“Day 66” in the figure). In the center of the macular fovea, the diameter of the atrophy is about 2 to 2.5 times the diameter of the nipple (the diameter of the retinal vein at the edge of the optic nerve head) (the area indicated by the arrow in the figure). Was recognized. The atrophy lesion showed markedly low fluorescence in the fundus autofluorescence. This atrophic lesion was determined to be map-like atrophy because it had a clear border and a low fluorescence that was evident in the fundus autofluorescence, in addition to the map-like morphology and the size of its diameter. It was. This map-like atrophy was classified as central map-like atrophy clinically because it had atrophy in the fovea.

  Fluorescein fluorescent fundus findings are shown in FIG. As a result, similar to human atrophic AMD, the atrophic lesion area showed strong hyperfluorescence due to window defects (a state in which RPE is abnormal and choroidal fluorescence is transmitted). In addition, drusen-like hyperfluorescent sites were scattered around the atrophy lesion area. From these findings, it was possible to determine that this atrophy lesion area was map-like atrophy.

<HE staining of tissue around the fovea>
A model animal of sodium iodate intravitreal administration on the 66th day after administration of the sodium iodate solution was subjected to HE (hematoxylin and eosin) staining of the tissue around the fovea to examine the morphology of the RPE layer. After euthanizing the animal, the eyeball was removed, fixed with 2.5% glutaraldehyde solution, and then stained with HE by a conventional method.

  An HE-stained image of the tissue around the fovea is shown in FIG. Denaturation and disappearance of the RPE layer (in the black frame in the figure) could be confirmed. In addition, the entire retina was degenerated, and the photoreceptor cells were lost.

  Based on these findings, the produced model animal with sodium iodate intravitreal administration had the map-like atrophy, degeneration of RPE, and photoreceptor damage, which are characteristic findings in human atrophic AMD. It was confirmed that it is suitable as an AMD model animal.

[Reference Example 1]
A model animal of photoretinal injury was prepared from cynomolgus monkeys, and the morphology of the retinal tissue was examined by HE staining.

<Production of cynomolgus monkey photoretinopathy model animal>
Under ketamine anesthesia, one eye of a cynomolgus monkey fixed in an upward state was irradiated with blue light having a wavelength of 460 nm and an illuminance of 1800 lux for 30 minutes vertically from above 5 cm of the eye using a variable wavelength light source. By performing this irradiation for 3 days, a photoretinal disorder model animal was produced.

<HE staining of tissue around the fovea>
The produced photoretinal disorder model animal was subjected to HE staining of the tissue around the fovea and examined the morphology of the RPE layer. HE staining was performed in the same manner as in Example 2.

  FIG. 6 shows an HE-stained image of the tissue around the fovea. No significant modification was observed in the RPE layer. The image in which the photoreceptor layer is detached from the RPE layer is an artifact at the time of specimen preparation. In the figure, long and narrow cells in the photoreceptor layer are photoreceptors. That is, the photoretinal disorder model animal has a disorder in the retina, but its pathological condition is different from the observation observed in human atrophic AMD, and is not suitable as a human atrophic AMD model animal. It has been found.

Claims (11)

  A method for producing an age-related macular degeneration model animal, comprising administering sodium iodate into a vitreous body of a non-human primate animal individual.   The method for producing an age-related macular degeneration model animal according to claim 1, wherein the dose of sodium iodate per vitreous is 0.5 to 2.5 mg.   The method for producing an age-related macular degeneration model animal according to claim 1 or 2, wherein a sodium iodate solution obtained by dissolving sodium iodate in water or phosphate physiological saline is injected into the vitreous.   The method for producing an age-related macular degeneration model animal according to any one of claims 1 to 3, wherein the animal individual is a cynomolgus monkey or a rhesus monkey.   In the produced age-related macular degeneration model animal, thinning of the outer granular layer of the retina and degeneration of the retinal pigment epithelium are observed in the periphery of the macular fovea. A method for producing an age-related macular degeneration model animal.   The method for producing an age-related macular degeneration model animal according to any one of claims 1 to 4, wherein map-like atrophy has occurred on the fundus of the produced age-related macular degeneration model animal.   The method for producing an age-related macular degeneration model animal according to any one of claims 1 to 6, wherein the produced age-related macular degeneration model animal is an atrophic age-related macular degeneration model animal.   A non-human primate age-related macular degeneration model animal having retinal ONL thinning and RPE degeneration around the macular fovea.   A non-human primate age-related macular degeneration model animal having a map-like atrophy in the fundus. A non-human primate age-related macular degeneration model animal is produced by the method for producing an age-related macular degeneration model animal according to any one of claims 1 to 7,
A method for evaluating the effect of a test substance on the prevention or treatment of age-related macular degeneration, which evaluates the effect of the test substance on age-related macular degeneration using the produced age-related macular degeneration model animal. A non-human primate age-related macular degeneration model animal is produced by the method for producing an age-related macular degeneration model animal according to any one of claims 1 to 7,
Using the produced age-related macular degeneration model animal, evaluate the preventive or therapeutic effect of age-related macular degeneration of the test substance,
One or more selected from the group consisting of thinning of the outer granular layer of the retina and degeneration of the retinal pigment epithelium are improved after ingesting the test substance compared to before ingesting the test substance In this case, a screening method for a substance having a preventive or therapeutic effect on age-related macular degeneration, wherein the test substance is selected as a substance having a preventive or therapeutic effect on age-related macular degeneration.