WO2023149420A1

WO2023149420A1 - Method and composition for preventing or treating myopia

Info

Publication number: WO2023149420A1
Application number: PCT/JP2023/003017
Authority: WO
Inventors: 一男坪田; 俊英栗原; ホヌクジョン; エンチョウ; 紀和子森
Original assignee: 株式会社坪田ラボ
Priority date: 2022-02-01
Filing date: 2023-01-31
Publication date: 2023-08-10

Abstract

The present disclosure relates to a method for preventing or treating myopia and a composition to be used in this method. The present inventors found that VEGF deficiency induces loss of the choroidal capillary plate and choroidal thinning to thereby cause myopia of the eye. To prevent or treat myopia, therefore, provided are a method and a composition for delivering VEGF or a VEGF agonist to the choroid to retain the choroid. Retaining of the choroid by maintaining the expression, secretion and reception of VEGF is effective in preventing or treating myopia.

Description

Methods and compositions for preventing or treating myopia

The present invention relates to methods and compositions for preventing or treating myopia, and more particularly to methods and compositions using VEGF for choroidal preservation.

Myopia refers to the state in which the focus is focused in front of the retina due to the elongation of the axial length of the eye, and it can be said that the longer the axial length of the eye, the stronger the myopia. As the prevalence of myopia increases, there is growing interest in the factors involved in the development of myopia and methods of slowing or preventing myopia progression. Recent studies have suggested that the choroid is important in regulating eye growth and in the development of myopia, and have shown that choroidal thinning is a structural feature of myopia. The negative correlation between choroidal thickness and axial length suggests that changes in choroidal thickness may be predictive biomarkers of axial length elongation. However, the detailed mechanism by which the choroid is involved in the onset and progression of myopia is still unclear.

The choroid is a fine blood vessel-rich outer lining of the retina. In addition to its function of supplying oxygen and nutrients to retinal cells, the choroid is involved in tissue remodeling of the sclera outside the eyeball and regulation of eye growth. It has the function of supplying factors. A decrease in choroidal thickness, or a decrease in blood flow, contributes to scleral ischemia and hypoxia, and is thought to influence changes in scleral structure that increase axial length of the eye. Therefore, maintaining and increasing choroidal thickness and blood flow are attracting attention as new targets in the prevention and treatment of myopia.

Methods for suppressing the progression of myopia by maintaining or increasing the thickness of the choroid have been proposed. It is

The present disclosure aims to provide a method for preventing or treating myopia, and a composition used therefor.

The present inventors have clarified that VEGF deficiency induces the disappearance of the choriocapillaris plate and the thinning of the choroid, causing myopia of the eye. proved to be effective. The present invention is based on such findings and includes the following aspects.

[Embodiment 1] A method for inhibiting or treating myopia in a subject in need thereof, comprising delivering VEGF or a VEGF agonist to the choroid of the subject.
[Aspect 2] The method of aspect 1, wherein the VEGF or VEGF agonist is administered to the subject in the form of DNA, RNA or protein.
[Embodiment 3] The method of embodiment 1 or 2, wherein the VEGF or VEGF agonist is administered to the subject using a viral vector.
[Aspect 4] The method according to aspect 1, which comprises transplanting VEGF-secreting cells or VEGF agonist-secreting cells into a subject.
[Aspect 5] The method according to aspect 1, comprising administering to the subject an agent that promotes VEGF expression.
[Aspect 6] The method according to any one of aspects 1 to 5, wherein the choroid is maintained.
[Aspect 7] The method according to any one of aspects 1 to 6, wherein VEGF expression, secretion or reception in the choroid is maintained.
[Embodiment 8] A therapeutically effective amount of DNA or RNA encoding VEGF or a VEGF agonist, VEGF protein, VEGF agonist, VEGF secreting cells, VEGF agonist secreting cells, or a VEGF expression promoter for suppressing or treating myopia composition.
[Embodiment 9] The composition of Embodiment 8 for delivering VEGF or a VEGF agonist to the choroid of a subject.
[Embodiment 10] The composition according to

embodiment

9 or 10, wherein VEGF or a VEGF agonist is incorporated into a viral vector.
[Embodiment 11] The composition according to any one of Embodiments 9 to 10 for maintaining choroid.
[Embodiment 12] The composition according to any one of Embodiments 9 to 11 for maintaining VEGF expression, secretion or reception in the choroid.
[Embodiment 13] Use of DNA or RNA encoding VEGF or a VEGF agonist, VEGF protein, VEGF agonist, VEGF-secreting cells, VEGF agonist-secreting cells, or a VEGF expression promoter for suppressing or treating myopia.
[Embodiment 14] Use of DNA or RNA encoding VEGF or a VEGF agonist, VEGF protein, VEGF agonist, VEGF-secreting cells, VEGF agonist-secreting cells, or a VEGF expression promoter in the manufacture of a medicament used for suppressing or treating myopia.
[Embodiment 15] Use according to embodiment 13 or 14 for delivering VEGF or a VEGF agonist to the choroid of a subject.
[Aspect 16] Use according to any one of aspects 13 to 15, wherein the VEGF or VEGF agonist is incorporated into a viral vector.
[Aspect 17] Use according to any one of aspects 13 to 16 for maintaining the choroid.
[Embodiment 18] Use according to any one of Embodiments 13 to 17 for maintaining VEGF expression, secretion or reception in the choroid.

According to the present disclosure, a new method for myopia prevention and treatment can be provided by maintaining the choroid with VEGF.

Fig. 3 is a graph showing changes in axial length of mice due to RPE-specific VEGF deficiency. Vertical axis: axial length, horizontal axis: age, black circles: control, white circles: VEGF ^RPE knockout. FIG. 10 is a graph showing changes in choroidal thickness in mice due to RPE-specific VEGF deficiency. Vertical axis: choroidal thickness, horizontal axis: age, black circles: control, white circles: VEGF ^RPE knockout. Graph showing changes in refraction in mice with RPE-specific VEGF deficiency. Vertical axis: refractivity, horizontal axis: age, black circles: control, white circles: VEGF ^RPE knockout. Electron microscope images of the choroid of RPE-specific VEGF-deficient mice. FIG. 10 is a graph showing changes in axial length of mice due to RPE-specific VEGF overexpression. FIG. Vertical axis: axial length, horizontal axis: age, black circles: control, white circles: VEGF overexpression. FIG. 10 is a graph showing changes in choroidal thickness in mice due to RPE-specific VEGF overexpression. FIG. Vertical axis: choroidal thickness, horizontal axis: age, black circles: control, white circles: VEGF overexpression. FIG. 10 is a graph showing changes in refractive power of mice with RPE-specific VEGF overexpression. FIG. Vertical axis: refractivity, horizontal axis: age, black circles: control, white circles: VEGF overexpression. Electron microscopy images of the choroid of RPE-specific VEGF overexpressing mice.

The details of the present invention will be explained. The present invention is not limited to the contents of the following embodiments and examples, and includes various modifications and applications within the scope of the gist of the present invention.

[Method for suppressing or treating myopia]
One aspect of the present disclosure relates to methods for inhibiting or treating myopia using vascular endothelial growth factor (VEGF). Vascular endothelial growth factor (VEGF) is a growth factor that can induce angiogenesis and promote angiogenesis in vivo. VEGF is supplied to the choroid by secretion from the retinal pigment epithelium (RPE). The present inventors focused on the fact that VEGF is effective in maintaining the choroid, and confirmed the effect of VEGF deficiency or overexpression on the maintenance of the choroid, axial length, and refractive power. The present inventors used transgenic mice as a model for RPE-specific VEGF deficiency or overexpression, observed morphological changes in the choroid, and measured axial length, choroid thickness, and refractive power. . Consequently, it has been found that myopia can be inhibited or treated in a subject in need of treatment by delivering VEGF or a VEGF agonist to the choroid of the subject. Accordingly, one aspect of the present disclosure relates to a method for inhibiting or treating myopia in a subject in need thereof comprising delivering VEGF or a VEGF agonist to the choroid of the subject. As disclosed herein, the importance of VEGF in choroidal maintenance was discovered using transgenic mice deficient or overexpressing RPE-specifically VEGF.

The subject can be a mammal, including humans, or a non-human mammal, including dogs, cats, cows, and horses, but is preferably a human.

The gene sequence of human VEGF (also called VEGF-A) is known, and a person skilled in the art can easily obtain sequence information from, for example, the NCBI database. VEGF is a protein with a dimer structure in which two subunits with a molecular weight of about 20,000 are bound, and in humans, subtypes such as VEGF121, VEGF165, VEGF189, and VEGF206 are known due to differences in splicing. The subtype of VEGF that can be used in the present invention is not particularly limited.

As used herein, a VEGF agonist refers to any substance that activates a VEGF receptor like VEGF. In some embodiments, the VEGF agonist comprises a fragment or variant of VEGF. Also, in some embodiments, the VEGF agonist is a VEGF protein other than VEGF-A belonging to the VEGF family, such as VEGF-B, VEGF-C, VEGF-D, VEGF-E, PlG-1, or PlGF- 2, or fragments or variants thereof. Additionally, in some embodiments, the VEGF agonist comprises an anti-VEGF receptor antibody capable of activating the VEGF receptor.

　VEGF or a VEGF agonist can be administered to a subject in the form of DNA, RNA or protein. A nucleic acid encoding VEGF or a VEGF agonist may be administered to a subject using a plasmid or expression vector. The expression vector can be, for example, but not limited to, a viral vector, especially an adenoviral vector. Other viral vectors that can be used include, for example, retrovirus, adeno-associated virus, pox, baculovirus, vaccinia, herpes simplex, Epstein-Barr, geminivirus, and calymovirus vectors. Nucleic acids encoding VEGF or VEGF agonists may include regulatory elements for choroid- or RPE-specific expression of the protein.

DNA or RNA (messenger RNA) encoding VEGF or a VEGF agonist may be administered to a subject using DDS such as liposomes. Other available DDS include charged lipids, nucleic acid-protein complexes, and biopolymers. VEGF or a VEGF agonist may also be administered to a subject in the form of a protein. Administration can be performed, for example, by topical administration to the choroid.

　The delivery of VEGF or a VEGF agonist to the choroid of a subject may be performed by transplanting VEGF-secreting cells or VEGF agonist-secreting cells into the subject. A VEGF-secreting cell can be any VEGF-secreting cell, such as, for example, a retinal pigment epithelial cell (RPE). Alternatively, cells that have been genetically engineered to secrete VEGF or a VEGF agonist may be used.

　The delivery of VEGF to the choroid of the subject may be achieved by administering to the subject an agent that promotes the expression of VEGF. For example, the von Hippel-Gendau (VHL) protein is a tumor suppressor that functions to negatively regulate the expression of hypoxia-inducible genes that are regulated by hypoxia-inducible transcription factors (HIFs). Loss of VHL activity results in increased HIF-1α and upregulation of angiogenic factors including VEGF and PDGF. Therefore, VHL inhibitors can be used as VEGF expression promoters.

As mentioned above, the thickness of the choroid is maintained by VEGF. Accordingly, one aspect of the present disclosure also relates to methods for maintaining the choroid in a subject in need of treatment comprising delivering VEGF or a VEGF agonist to the choroid of the subject. One aspect of the present disclosure also relates to a method for inhibiting or treating myopia in a subject in need of treatment comprising maintaining VEGF expression, secretion or reception in the choroid of the subject.

[Composition for suppressing or treating myopia]
One aspect of the present disclosure relates to compositions for inhibiting or treating myopia using VEGF or VEGF agonists. More specifically, one embodiment of the present disclosure contains an effective amount of DNA or RNA encoding VEGF or a VEGF agonist, VEGF protein, VEGF agonist, VEGF-secreting cells, VEGF agonist-secreting cells, or a VEGF expression promoter and compositions for preventing or treating myopia. Such compositions can be used to deliver VEGF or VEGF agonists to the choroid of a subject.

The composition according to the present disclosure is, for example, topically administered to the eye. Examples of administration forms of the present composition include ophthalmic administration (including instillation of ocular ointment and eye wash), subconjunctival administration, intraconjunctival administration, and subtenon administration.

The dosage form of the present composition is not particularly limited, but includes, for example, eye drops, eye ointments, injections, patches, gels, inserts, etc. Eye drops are preferred, for example. In addition, these can be prepared using the usual technique widely used in the said field.

Eye drops include tonicity agents such as sodium chloride, potassium chloride and concentrated glycerin; buffering agents such as sodium phosphate, sodium acetate and epsilon-aminocaproic acid; Surfactants such as ethylene hydrogenated castor oil; stabilizers such as sodium citrate and sodium edetate; However, the range of 4 to 8 is generally preferred. Eye ointments can be prepared using commonly used bases such as white petrolatum and liquid paraffin.

The composition according to the present disclosure may contain an appropriate amount of other components within a range that does not impair the effects of the present invention. Other ingredients include water, oily ingredients, surfactants, preservatives, sugars, buffers, pH adjusters, tonicity agents, stabilizers, cooling agents, polyhydric alcohols, excipients, lipids, Proteins, ingredients for DDS, thickening agents, and the like. These components can be blended singly or in an appropriate combination of two or more. The amount of water to be blended can be the balance of the composition.

One aspect of the present disclosure relates to the use of VEGF or VEGF agonists to suppress or treat myopia. Yet another aspect of the disclosure relates to the use of VEGF or a VEGF agonist in the manufacture of a medicament for use in preventing or treating myopia. VEGF or VEGF agonists may be used in DNA or RNA, or protein form. VEGF or VEGF agonists may also be used in the form of cells that secrete VEGF or VEGF agonists. Additionally, agents that promote the expression of VEGF can be used for these purposes as well.

While preferred embodiments of the invention have been described herein, it will be apparent to those skilled in the art that such embodiments are provided for purposes of illustration only, and those skilled in the art will Various modifications, changes and substitutions could be made without departing from the invention. It should be understood that various alternative embodiments of the invention described herein may be used in practicing the invention. Also, the contents of all publications, including patents and patent applications, referenced herein are deemed to be incorporated by reference as if expressly set forth herein. should be interpreted.

The present invention will be described in more detail below with experimental examples.

[Experiment 1]
In this experiment, mice expressing Cre recombinase under the RPE-specific Best1 promoter were crossed with Vegf-floxed mice and used as an RPE-specific VEGF-deficient model. Also, littermate floxed mice that do not express the Cre transgene were used as a control group.

(Measurement of axial length, choroid thickness, and refractive index)
Axial length, choroid thickness, and refractive power of each group of mice were measured from 3 weeks old to 10 weeks old. Spectral domain optical coherence tomography (Envisu R4310, manufactured by Leica) was used to measure axial length and choroidal thickness. Refractive power was measured using an infrared photorefractor for mice (Professor Schaeffel, Tubingen University).

(Observation of choroid by electron microscope)
Eyes of 10-week-old mice were harvested, fixed with 2.5% glutaraldehyde in PBS (Phosphate Buffered Saline) overnight at 4° C., and rinsed with 0.1 M sodium cacodylate buffer for 1 hour. They were then fixed with 1% OsO4 in 0.1 M cacodylate buffer for 2 hours followed by dehydration with graded ethanol solutions. In addition, the eyes were infiltrated overnight in a 1:2 mixture of propylene oxide and epon-araldite and embedded in 100% resin. The blocks were cut and observed at an acceleration voltage of 100 kV using a transmission electron microscope (JEM1400 plus; JEOL).

(result)
Figure 1 shows the results of measuring the axial length of RPE-specific VEGF-deficient mice. During the entire period up to 10 weeks of age, the axial length of the VEGF-deficient mice was elongated compared to the control group. FIG. 2 shows the measurement results of the choroidal thickness. The choroid of VEGF-deficient mice was thinner than that of the control group. Figure 3 shows the measurement results of the refractive index. VEGF-deficient mice exhibited myopic refraction compared to emmetropic refraction in controls.

Fig. 4 is an image of the morphology of the choroid observed with an electron microscope. It was confirmed that the choriocapillary lamina of the VEGF-deficient mice was thinner and disappeared compared to the control group.

From the above results, it was found that VEGF deficiency in RPE causes axial lengthening and myopia.

[Experiment 2]
In this experiment, mice obtained by mating mice expressing Cre recombinase under the RPE-specific Best1 promoter with Vhl-floxed mice were used as an RPE-specific VEGF overexpression model. Also, littermate floxed mice that do not express the Cre transgene were used as a control group. Axial length, thickness of choroid, measurement of refraction and observation of choroid are the same as Experiment 1.

(result)
FIG. 5 shows the measurement results of the axial length of RPE-specific VEGF overexpressing mice. During the entire period up to 10 weeks of age, elongation of the axial length of the VEGF-overexpressing mice was suppressed compared to the control group. FIG. 6 shows the measurement results of the choroidal thickness. We confirmed that the choroid of VEGF-overexpressing mice was thickened during periods other than 3 and 6 weeks of age. Fig. 7 shows the measurement results of the refractive index. Both control and VEGF-overexpressing mice exhibited emmetropia.

Fig. 8 is an image of the morphology of the choroid observed with an electron microscope. It was confirmed that the choriocapillary lamina of the VEGF-overexpressing mice was thicker than that of the control group.

From the above results, it was found that VEGF overexpression from RPE suppresses axial length elongation.

In summary, loss of the choriocapillaris due to RPE-derived VEGF deficiency results in choroidal thinning, axial length elongation, and myopic refractive power, and VEGF overexpression suppresses axial length elongation. was found, suggesting that VEGF is important for the maintenance of the choroid and the suppression of myopia progression.

Claims

A method for inhibiting or treating myopia in a subject in need of treatment comprising delivering VEGF or a VEGF agonist to the choroid of the subject.
The method of claim 1, wherein the VEGF or VEGF agonist is administered to the subject in the form of DNA, RNA or protein.
The method of claim 1 or 2, wherein the VEGF or VEGF agonist is administered to the subject using a viral vector.
The method according to claim 1, comprising transplanting VEGF-secreting cells or VEGF agonist-secreting cells to the subject.
The method according to claim 1, comprising administering to the subject an agent that promotes expression of VEGF.
The method according to any one of claims 1 to 5, wherein the choroid is maintained.
The method according to any one of claims 1 to 6, wherein VEGF expression, secretion or reception in the choroid is maintained.
A composition for suppressing or treating myopia, containing a therapeutically effective amount of DNA or RNA encoding VEGF or a VEGF agonist, a VEGF protein, a VEGF agonist, a VEGF-secreting cell, a VEGF agonist-secreting cell, or a VEGF expression promoter.
9. The composition of claim 8, for delivering VEGF or a VEGF agonist to the choroid of a subject.