TW201945022A - Use of inhibiting S100A16 in treating degenerative retinal diseases capable of protecting the photoreceptors and restoring the retinal function - Google Patents

Abstract

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

Description

Inhibition of S100A16 in the treatment of retinal degenerative 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 particularly to the use of a drug for treating degenerative retinal diseases. The present invention also relates to a composition, in particular to a composition for treating retinal degenerative diseases with a 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 pigmented retinitis or age-related macular degeneration each year, leading to blindness. Age-related macular degeneration is a degenerative retinal disease that occurs in elderly people over the age of 60. It gradually causes the degradation of retinal cells and eventually leads to blindness. Currently, no effective treatments 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 to be applied to degenerative diseases. According to previous studies, animal models of retinal degeneration caused by continuous light can be used to explore the treatment effect of age-related macular degeneration. Although the results show that multiple complexes and treatments can delay the degree of retinal degradation caused by continuous light, there is no treatment effect. In recent years, some treatments, including gene therapy and growth factor injection, have also been used as a means to delay the loss of retinal cells, however, these patients still have no vision restoration treatment.

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

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

In order to achieve the above-mentioned object of the present invention, the present invention provides a use of a ribonucleic acid that inhibits the expression of S100A16 for preparing a medicine for treating degenerative retinal diseases.

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

Preferably, the pharmaceutical product comprises a pharmaceutically acceptable carrier.

The present invention further 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 ribonucleic acid for inhibiting the expression of S100A16 and HSP27 for the preparation of a medicine for treating degenerative retinal diseases.

Preferably, the ribonucleic acid expressing S100A16 and the ribonucleic acid expressing HSP27 are used separately, simultaneously or sequentially.

The advantage of the present invention is that by inhibiting the expression of S100A16 gene alone, the number of apoptosis of retinal cells is significantly reduced by the present invention. Even when the expression of S100A16 and HSP27 genes are inhibited at the same time, the number of retinal cells is the same as the normal state to achieve protection Photoreceptors and restore retinal function.

The "S100A16" referred to in the present invention has the full name of S100 calcium-binding protein A16.

As used herein, the term "combination", when applied to two or more RNAs, is intended to define a material in which the two or more RNAs are bound.

The pharmaceutical system 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, hydrocarbons [such as petroleum gum ( 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, odor absorbers, Fragrances, pH adjusting agents, occlusive agents, emollients, thickeners, solubilizing agents, penetration enhancers, anti- Bowel agents (anti-irritants), coloring agents (colorants), propellant (PROPELLANTS) a surface active agent (surfactant), or other similar or suitable vehicle according to the present invention.

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

The medicinal composition is an ophthalmic agent for topical administration to the eye.

The "local administration of the eye" in the present invention refers to, for example, subocular fascia cavity, subconjunctival, intraocular, intravitreal, anterior chamber, subretinal, suprachoroid, or post-eyeball, and the like.

The ophthalmic agent is formulated as eye drops or ointment.

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

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

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

Ophthalmic formulations further include sustained release formulations, such as: gel formulations, microlipid formulations, lipid microemulsion formulations, microparticle formulations, nanoparticle formulations and implant formulations so that RNA can be continuously accessed Behind the eyes.

Among them, the ribonucleic acid expressing S100A16 and the ribonucleic acid expressing HSP27 are used separately, simultaneously, or sequentially.

The ribonucleic acid that inhibits the expression of S100A16 and the ribonucleic acid that expresses 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 administrations, preferably simultaneous administrations can achieve the additive effect.

According to some embodiments, the medicine or composition of the present disclosure is used to treat degenerative retinal diseases such as Retinitis Pigmentosa, macular degeneration, Macular dystrophy, and senile macular degeneration. Lesions (age-related macular degeneration, AMD), diabetic retinopathy, cone-rod dystrophy, optic neuritis, etc.

In the following, with reference to the drawings and the preferred embodiments of the present invention, the technical measures adopted by the present invention to achieve the purpose are further explained.

Preparation Example 1

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

Example 1

SD male rats (Sprague-Dawley rats) weighing 200-225 grams were used and the experiment was divided into six groups of 9 animals each with (1) normal light (100 to 200 lux, lux); (2) intense light; ( 3) Strong light and sham; (4) Strong light and shHSP27; (5) Strong light and shS100A16; (6) Strong light, shS100A16 and shHSP27. Before the experiment, the retinal potential spectroscopy (ERG) measurement signal was used as the basis for judging retinal function. Animals in group (1) were cycled for 12 days in the dark for 12 hours under normal light at 100 to 200 lux, and animals in groups (2) to (6) were cycled for 12 hours in the dark for 12 hours under intense light For 7 days to induce retinal degeneration, the ERG signal was measured again on day 7 (week 1). After the strong light irradiation, the animals were restored to the normal light environment (100 to 200 lux) from the 8th day. The animals in groups (3) to (6) were anesthetized and taken from the lRNA containing shRNA in Preparation Example 1 mL ( 20,000 IU) were injected into the space outside the eyes of the infected rats near the retina's serrated margin (ora serrata). Group (3) was infected with lentivirus containing shLUC, group (4) was infected with lentivirus containing shHSP27, and (5 The group) was infected with lentivirus containing shS100A16, and the group (6) infection was simultaneously given with lentivirus containing shS100A16 and shHSP27 (1 mL each of 20,000 IU, mixed and administered together). On the 14th day (week 2), the ERG signal was measured again. After the ERG signal was measured again on the 21st day (week 2), the animal was sacrificed, and tissue sections (about 4 to 5 mm thick) of the retina and TUNEL (Terminal deoxynucleotidyl) transferase dUTP nick end labeling) analysis of cell death.

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

Please refer to Figure 1 and Figure 2. Groups that inhibit the expression of S100A16 gene (5), or groups that simultaneously inhibit the expression of S100A1 and HSP27 gene (6), will make ERG two weeks after intense light (third week) Both the a-wave and b-wave signals were recovered. The a-wave signals mainly originated from the inner part of the photoreceptors, and the b-wave signals originated from Müller cells or bipolar cells. In addition, in the group (5) that inhibited the expression of the S100A16 gene, the b-wave signal began to recover after one week (second week) of intense light. Therefore, no matter whether the S100A16 gene is inhibited alone, or the S100A16 and HSP27 genes are inhibited simultaneously, the signal reception and transmission of the optic nerve can be significantly restored, and then the retinal function can be restored.

(2) Relationship between inhibition of S100A16 and HSP27 gene expression and retinal cell apoptosis

Counting the number of apoptotic cells on retinal sections, it can be found that compared to the group with strong light (2), the number of cells that inhibited the expression of HSP27 gene (4) and the group that inhibited the expression of S100A16 gene (5) Significantly decreased; more notably, compared with the group with intense light and sham (3), the number of apoptosis that was inhibited by the S100A16 and HSP27 genes (6) was significantly lower than that in the group (6) The lighting group (1) is similar.

Therefore, long-term light exposure can cause retinal cell apoptosis and loss of retinal function. Inhibiting S100A16 alone, or inhibiting both S100A16 and HSP27 gene expression, reduces the number of retinal apoptosis and restores retinal function.

It will be apparent to those skilled in the art that various modifications and variations can be made in accordance with the present invention without departing from the scope and spirit of the invention. Although the invention has been described with 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 mode of the present invention, different modifications obvious to those skilled in the art are also included in the scope of the following patent applications.

Figure 1 is a line chart of a-wave signals for detecting ERG; the data are mean ± standard deviation, and the statistical analysis is multiple comparison of one-way ANOVA and LSD. Figure 2 is a line chart of b-wave signals for detecting ERG; the data are mean ± standard deviation, and the statistical analysis is multiple comparison of one-way ANOVA and LSD. Figure 3 is a histogram of the number of apoptotic cells per retinal slice; the data are mean ± standard deviation, and the statistical analysis is a multiple comparison of one-way ANOVA and LSD.

Claims (10)

A ribonucleic acid for inhibiting the expression of S100A16 is used for preparing a medicine for treating degenerative retinal diseases. The use according to claim 1, wherein the ribonucleic acid comprises small interfering RNA (siRNA), small molecular ribonucleic acid (microRNA), small hairpin ribonucleic acid (shRNA) or double-stranded ribonucleic acid (dsRNA). The use according to claim 1 or 2, wherein the pharmaceutical product comprises a pharmaceutically acceptable carrier. Use according to claim 3, wherein the carrier comprises liposomes. A composition for treating degenerative retinal diseases, comprising a ribonucleic acid that inhibits the expression of S100A16 and heat shock protein 27. The composition according to claim 5, wherein the ribonucleic acid comprises small interfering ribonucleic acid, small molecule ribonucleic acid, small hairpin ribonucleic acid, or double-stranded ribonucleic acid. A pharmaceutical composition for treating degenerative retinal diseases, comprising the composition according to claim 5 or 6 and a pharmaceutically acceptable carrier. The pharmaceutical composition according to claim 7, wherein the pharmaceutical composition is an ophthalmic agent for topical administration to the eye. A ribonucleic acid for inhibiting the expression of S100A16 and heat shock protein 27 is used for preparing a medicine for treating degenerative retinal diseases. The use according to claim 9, wherein the ribonucleic acid system that inhibits the expression of S100A16 and heat shock protein 27 is used separately, simultaneously, or sequentially.