TWI696467B - Inhibition of S100A16 in the treatment of degenerative retinal diseases - Google Patents

Abstract

The invention provides a use of a ribonucleic acid that inhibits the expression of S100A16, or inhibits the expression of S100A16 and HSP27, for the preparation of a medicinal product for treating retinal degenerative diseases. The present invention also provides a composition for treating degenerative retinal diseases, which comprises a ribonucleic acid that inhibits the expression of S100A16 and HSP27. The present invention also provides a pharmaceutical composition and use including the aforementioned composition and a pharmaceutically acceptable carrier thereof. By inhibiting the S100A16 gene alone, or simultaneously suppressing the gene expression of S100A16 and HSP27 according to the present invention, it can effectively protect photoreceptors and restore retinal function.

Description

Inhibition of S100A16 in the treatment of degenerative retinal diseases

The present invention relates to the use of a ribonucleic acid that inhibits the expression of S100A16 or S100A16 and heat shock protein 27 (HSP27), and in particular refers to the use of drugs for treating retinal degenerative diseases. The invention also relates to a composition, in particular to a composition for treating retinal degenerative diseases with ribonucleic acid that inhibits the expression of S100A16 and HSP27.

Many people lose their vision due to degenerative retinal diseases, which often cause irreversible damage to retinal cells and cause blindness. Epidemiological statistics show that about 17% of people suffer from retinitis pigmentosa or age-related macular degeneration, which eventually leads to blindness. Age-related macular degeneration is a degenerative disease of the retina, which occurs in elderly people over the age of sixty. It gradually causes the degradation of retinal cells and eventually leads to blindness. Currently, no effective treatment and drugs have been developed to restore vision.

In recent years, it has been found that the use of neural precursor cells or stem cells to replace damaged cells has considerable potential for use in degenerative diseases. According to previous studies, the animal model of retinal degeneration caused by continuous light can be used to explore the therapeutic effect of age-related macular degeneration. Although the results of the study show that various compounds and therapies can delay the degree of retinal degeneration caused by continuous light, there is no therapeutic effect. In recent years, some treatments including gene therapy and growth factor injection have also been used as delaying loss of retinal cells. However, these patients still have no vision recovery treatment.

In view of this, how to develop drugs to restore retinal function, the existing technology really needs to be improved.

In order to overcome the shortcomings of the prior art, the object of the present invention is to provide a use of ribonucleic acid that inhibits the expression of S100A16 or S100A16 and HSP27 to achieve the effect of restoring retinal function and reducing retinal cell apoptosis.

In order to achieve the above-mentioned object of the invention, the present invention provides a use of ribonucleic acid that inhibits the expression of S100A16 for the preparation of a medicinal product for the treatment of degenerative retinal diseases.

Preferably, the ribonucleic acid comprises small interfering RNA (siRNA), small molecule ribonucleic acid (microRNA), small hairpin ribonucleic acid (shRNA), double stranded ribonucleic acid (dsRNA) or the like .

Preferably, the pharmaceutical product includes a pharmaceutically acceptable carrier.

The present invention also provides a composition for treating degenerative retinal diseases, which comprises a ribonucleic acid that inhibits the expression of S100A16 and HSP27.

The present invention also provides a pharmaceutical composition for treating degenerative retinal diseases, which comprises the composition as described above and a pharmaceutically acceptable carrier.

The invention also provides a use of a ribonucleic acid that inhibits the expression of S100A16 and HSP27 for the preparation of a medicinal product for treating degenerative diseases of the retina.

Preferably, the ribonucleic acid that inhibits the expression of S100A16 and the ribonucleic acid that is expressed by HSP27 are used separately, simultaneously or sequentially.

The advantage of the present invention is that, by inhibiting the expression of S100A16 gene alone, this invention significantly reduces the amount of retinal cell apoptosis. Even when the gene expression of S100A16 and HSP27 is simultaneously suppressed, the amount of retinal cell apoptosis is the same as the normal state to achieve protection Photoreceptors and restore retinal function.

The full name of "S100A16" in the present invention is S100 calcium-binding protein A16 (S100 calcium-binding protein A16).

As used herein, the term "combination", when applied to two or more ribonucleic acids, is intended to define materials in which the two or more ribonucleic acids bind.

The pharmaceutical product of the present invention can use the techniques well known to those skilled in the art to prepare the above-mentioned ribonucleic acid and a pharmaceutically acceptable carrier into a dosage form suitable for the present invention. The "pharmaceutically acceptable carrier" according to the present invention includes, but is not limited to, liposomes, water, alcohols, glycols, and hydrocarbons [such as petroleum jelly ( petroleum jelly and white petrolatum], wax [such as paraffin and yellow wax], preserving agents, antioxidants, solvents, emulsifiers (emulsifier), suspending agent, decomposer, binding agent, excipient, stabilizing agent, chelating agent, diluent , Gelling agent, preservative, lubricant, absorption enhancers, active agents, humectants, odor absorbers, Fragrances, pH adjusting agents, occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, resistance Anti-irritants, colorants, propellants, surfactants, or other similar or suitable carriers of the present invention.

The pharmaceutical composition of the present invention may include, but is not limited to oral administration, intravenous injection, topical eye administration, or the like.

Among them, the pharmaceutical composition is an ophthalmic agent for local administration to the eye.

The "local eye administration" in the present invention refers to the administration methods such as subfascial cavity, subconjunctival, intraocular, intravitreal, anterior chamber, subretinal, suprachoroidal or posterior eyeball and similar administration methods.

Wherein, the ophthalmic agent is formulated as eye drops or eye ointment.

The "eye drops" described in the present invention are made by dissolving active ingredients (such as ribonucleic acid) in sterile aqueous solutions, such as physiological saline and buffer solutions. The eye drops can be provided in the form of a powder composition for dissolution before use, or in combination with the powder composition before use.

The "eye ointment" described in the present invention is prepared by mixing active ingredients (such as ribonucleic acid) and ointment base and according to a common method.

When preparing the composition of the present invention to form an ophthalmic ointment, the above ointment bases include, but are not limited to: oil bases [such as petroleum jelly, liquid paraffin, polyethylene, selen 50, viscoelastic glue (plastibase), polyethylene glycol (macrogol) Or a combination thereof], an emulsion having an oil phase and an aqueous phase emulsified by a surfactant, and a water-soluble matrix (such as hydroxypropyl methyl cellulose, carboxypropyl methyl cellulose and polyethylene glycol).

The ophthalmic agent further includes sustained-release dosage forms, such as: gel formulations, liposome formulations, lipid microemulsion formulations, particulate formulations, nanoparticle formulations and implant formulations, so that ribonucleic acid can enter continuously Behind the eyes.

Among them, the ribonucleic acid that inhibits the expression of S100A16 and the ribonucleic acid that is expressed by HSP27 are used separately, simultaneously or sequentially.

The ribonucleic acid that inhibits the expression of S100A16 and the ribonucleic acid that is expressed by HSP27 can be formulated together in a single dosage form; alternatively, the two can be separately formulated and packaged together; or, the two can be administered independently. In the case of separate administration, it is preferably simultaneous administration, which can achieve an additive effect.

According to some embodiments, the pharmaceuticals or compositions of the present disclosure are used to treat degenerative retinal diseases such as Retinitis Pigmentosa, macular degeneration, macular dystrophy, and age-related macular degeneration Disease (age-related macular degeneration, AMD), diabetic retinopathy, rod-cell dystrophy (cone-rod dystrophy), optic neuritis, etc.

In the following, in conjunction with the drawings and the preferred embodiments of the present invention, the technical means adopted by the present invention to achieve the objectives will be further described.

Preparation Example 1

Obtained pLKO.1-puro plasmid-based shRNA from the National RNAi Core Facility Platform (Central Research Institute, Taipei, Taiwan), including shLUC (Clone ID: TRCN231719 sham group, shS100A16 (Clone ID: TRCN0000340011) and shHSP27 (Clone ID: TRCN0000321339). purified shLUC, shS100A16, shRNA plasmids are shHSP27 of liposomes (Lipofectamine ^® available from Invitrogen / Life Technologies, Carlsbad, CA , USA), expression plasmid (pMD2.G) and packaging The vector (psPAX2) was transfected into H293T cells together to produce a lentivirus containing shRNA.

Example 1

Take SD male rats (Sprague-Dawley rat) weighing 200-225 grams, and the experiment is divided into six groups of 9 animals, each of which is (1) normal light (100 to 200 lux, lux); (2) strong light; ( 3) Strong light and sham; (4) Strong light and shHSP27; (5) Strong light and shS100A16; (6) Strong light and shS100A16 and shHSP27. Before the experiment began, the measurement signal of electroretinography (ERG) was used as the basis for judging retinal function. Animals in group (1) were cycled for 12 hours in the dark, with normal light from 100 to 200 lux for 12 days, and animals in groups (2) to (6) were cycled for 12 hours in the dark, with 5000 lux for 12 hours A total of 7 days to induce retinal degeneration, the ERG signal was measured again on the 7th day (Week 1). After the strong light irradiation, the animals were restored to normal light environment (100 to 200 lux) from the 8th day. After the animals in groups (3) to (6) were anesthetized, 1 mL of shRNA-containing lentivirus from Preparation Example 1 ( 20,000 IU) were injected into the space under the ora serrata outside the eyes of the infected rats. Group (3) was infected with shLUC-containing lentivirus, group (4) was infected with shHSP27-containing lentivirus, and (5) ) Group infected with lentivirus containing shS100A16, group (6) infection was administered simultaneously with lentivirus containing shS100A16 and shHSP27 (both 1 mL of 20,000 IU each, mixed and administered together). On the 14th day (Week 2), the ERG signal was measured again. On the 21st day (Week 2), the ERG signal was measured again. The animals were sacrificed. Tissue section (thickness about 4 to 5 mm) and TUNEL (Terminal deoxynucleotidyl) were performed on the retina. transferase dUTP nick end labeling) analysis of cell death.

(1) Relationship between S100A16 and HSP27 gene expression and retinal function

Please refer to Figure 1 and Figure 2, the group that inhibits the expression of S100A16 gene (5), or the group that simultaneously suppresses the expression of S100A1 and HSP27 gene (6), will cause ERG after two weeks of intense light (third week) Both the a-wave and b-wave signals are recovered. The a-wave signal mainly originates from the inner section of the photoreceptor, and the b-wave signal originates from Müller cells or bipolar cells. In addition, in the group (5) that suppressed the expression of the S100A16 gene, the b-wave signal started to recover after a week of strong light (second week). Therefore, whether it inhibits the S100A16 gene alone, or inhibits the S100A16 and HSP27 genes at the same time, it can significantly restore the signal reception and transmission of the optic nerve, and then restore retinal function.

(2) The relationship between the inhibition of S100A16 and HSP27 gene expression and retinal cell apoptosis

Counting the number of apoptosis on retinal slices shows that compared with the group with strong light (2), the group with HSP27 gene suppression (4) and the group with S100A16 gene suppression (5) Significantly decreased; more notably, compared with the group with strong light and sham (3), the group that simultaneously inhibited the expression of S100A16 and HSP27 genes (6) The number of apoptosis was more normal than the group with a significant decrease The lighting groups (1) are similar.

Therefore, long-term light exposure will lead to retinal cell apoptosis and loss of retinal function. Suppressing the gene expression of S100A16 alone or simultaneously inhibiting the expression of S100A16 and HSP27 reduces the number of retinal cell apoptosis and restores retinal function.

The different modifications and changes that can be made according to the present invention will obviously not deviate from the scope and spirit of the present invention for those skilled in the art. Although the invention has described specific preferred specific facts, it must be understood that the invention should not be unduly limited to such specific specific facts. In fact, in implementing the described modes of the present invention, different amendments that are obvious to those skilled in the art are also covered by the following patent applications.

Figure 1 is a line chart of the a-wave signal detected by ERG; the data is the mean ± standard deviation, and the statistical analysis is a one-way ANOVA and LSD multiple comparison. Figure 2 is a line chart of the b-wave signal detected by ERG; the data is the mean ± standard deviation, and the statistical analysis is a one-way ANOVA and LSD multiple comparison. Figure 3 is a histogram of the number of apoptosis in each retinal slice; the data is the mean ± standard deviation, and the statistical analysis is a one-way ANOVA and LSD multiple comparison.

Claims (8)

A ribonucleic acid that inhibits the performance of S100A16 for the preparation of a medicament for treating retinal degenerative diseases, wherein the ribonucleic acid is small interfering RNA (siRNA), small molecule ribonucleic acid (microRNA), small hairpin ribose Nucleic acid (shRNA) or double stranded ribonucleic acid (dsRNA). The use as claimed in claim 1, wherein the pharmaceutical product comprises a pharmaceutically acceptable carrier. The use as claimed in claim 2, wherein the carrier comprises liposomes. A composition for treating degenerative retinal diseases, which comprises a ribonucleic acid that inhibits the expression of S100A16 and heat shock protein 27, wherein the ribonucleic acid is a small interfering ribonucleic acid, a small molecule ribonucleic acid, a small hairpin ribonucleic acid, or a double-stranded ribose Nucleic acid. A pharmaceutical composition for treating degenerative retinal diseases, which comprises the composition according to claim 4 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 5, wherein the pharmaceutical composition is an ophthalmic agent for topical administration to the eye. A ribonucleic acid that inhibits the expression of S100A16 and heat shock protein 27 for the preparation of a medicament for treating retinal degenerative diseases, wherein the ribonucleic acid is a small interfering ribonucleic acid, a small molecule ribonucleic acid, a small hairpin ribonucleic acid, or a double-stranded ribose Nucleic acid. The use according to claim 7, wherein the ribonucleic acid that inhibits the expression of S100A16 and heat shock protein 27 is used separately, simultaneously or sequentially.

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