JP2024069730A - Composition for preventing retinal degeneration - Google Patents

Abstract

To provide a pharmaceutical composition for preventing retinal degeneration including retinitis pigmentosa.SOLUTION: It has been found that phenylbutyric acid (PBA) prevents progressive retinal photoreceptor cell death and also prevents a deterioration in visual function. On the basis of these findings, a pharmaceutical composition for preventing retinal degeneration including retinitis pigmentosa can be provided.SELECTED DRAWING: Figure 1

Description

The present invention relates to a pharmaceutical composition for suppressing retinal degeneration, including retinitis pigmentosa.

Retinitis pigmentosa is a hereditary retinal disease in which genetic mutations cause the rod photoreceptor cells, which detect light brightness, to degenerate. Initial symptoms include night blindness, narrowing of the visual field, and decreased vision, and can ultimately lead to blindness.
At present, there is no effective treatment, and vitamins have been administered clinically for neuroprotection, but they are not very effective. Research on gene transfer therapy using viruses has been conducted, and clinical trials are being conducted worldwide. Research on visual cycle modulators as a drug therapy has been conducted (Non-Patent Document 1), but this protects the retina by preventing the use of visual function, and there is a need for a treatment that protects the retina while maintaining visual function.
There is a similar need for treatments that protect the retina from retinitis pigmentosa, diabetic retinopathy, age-related macular degeneration, or other causes of retinal degeneration.

Furthermore, phenylbutyric acid (hereinafter, also referred to as PBA), which is already used as a specific drug for urea cycle disorders, has been shown to have the effect of suppressing endoplasmic reticulum stress in the corneal endothelium (Patent Documents 1 and 2). However, the effect of PBA in the ocular region other than the corneal endothelium is unknown, and the effect of PBA in the retina in particular is unknown.

International Publication No. 2015/064768 International Publication No. 2018/164113

PLoS One, 2015 May 13;10(5):e0124940 J Biol Chem., 2011;286(12):10551-10567 FASEB J., 2020;34(4):5016-5026 PLoS One, 2017;12(6):e0178627 Free Radic Biol Med., 2014;71:176-185 Clin Exp Ophthalmol., 2014;42(6):555-563 Commun Biol., 2020;3(1):767 Mol Neurobiol., 2019;56(12):8124-8135 Mol Neurobiol., 2015;52(1):679-695 Bioorg Med Chem., 2010;18(19):7022-7028 Invest Ophthalmol Vis Sci., 2012;53(12):7590-7599 Adv Exp Med Biol., 2012;723:191-197 Biochim Biophys Acta Mol Basis Dis., 1864(9 Pt B):2938-2948 Front Cell Neurosci., 2019;13:535 Cell Death Dis, 2014;5:e1236 Nat Struct Mol Biol., 2014;21(4):325-335 Trends Cell Biol., 2012;22(1):22-32 eLife, 2020;9:e57306 Front Cell Dev Biol., 2020;8:270 Curr Opin Cell Biol, 2015;37:18-27 Traffic, 2018;19(8):569-577 Neurobiol Dis, 2016;90:3-19 Ageing Res Rev., 2020;66:101250 J Neurosci Res., 2017;95(10):2025-2029 FEBS Lett., 2018;592(5):793-811 Redox Biol., 2020;37:101779 Neural Regen Res., 2017;12(8):1252-1255

That is, the objective of the present invention is to provide a pharmaceutical composition for suppressing retinal degeneration, including retinitis pigmentosa.

The inventors conducted research to solve the above problems and discovered that PBA inhibits progressive retinal photoreceptor cell death and inhibits visual function decline, leading to the completion of the present invention.

That is, this application provides the following inventions.
[1] A pharmaceutical composition for inhibiting retinal degeneration, comprising phenylbutyric acid (PBA) or a salt thereof, or a derivative thereof.
[2] A pharmaceutical composition for treating or preventing a retinal degenerative disease, comprising phenylbutyric acid (PBA) or a salt thereof, or a derivative thereof.
[3] The pharmaceutical composition described in [2], wherein the retinal degenerative disease is a disease in which retinal photoreceptor cells are degenerated.
[4] The pharmaceutical composition according to [2] or [3], wherein the retinal degenerative disease is one or more diseases selected from retinitis pigmentosa, diabetic retinopathy, age-related macular degeneration, and retinal degeneration associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes).
[5] The pharmaceutical composition according to any one of [1] to [4], wherein the salt of phenylbutyric acid is a sodium salt.
[6] The pharmaceutical composition according to any one of [1] to [5], which is a tablet, granule, injection or eye drop.
[7] A pharmaceutical composition for promoting endoplasmic reticulum stress-related degradation in the retina, comprising phenylbutyric acid (PBA) or a salt thereof, or a derivative thereof.
[8] A pharmaceutical composition for activating mitochondrial biogenesis in the retina and/or improving mitochondrial metabolic function in the retina, comprising phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.

By discovering that PBA promotes the maintenance of visual function in a retinitis pigmentosa model, the present invention provides an agent for suppressing retinal degeneration, including retinitis pigmentosa, and a novel pharmaceutical composition for patients with retinal degeneration who have been diagnosed but have had no effective treatment until now.

FIG. 1 shows the promotion and maintenance of visual function in P23H rhodopsin knock-in heterozygous retinitis pigmentosa model mice (also referred to simply as P23H knock-in heterozygous mice or P23H RP model mice in this disclosure) by PBA. In the figure, WT indicates wild-type mice, and Hetero indicates P23H knock-in heterozygous mice. A is a micrograph of mouse retina stained with hematoxylin and eosin. B is a graph showing the number of nuclei in the outer nuclear layer (ONL). In the figure, * indicates P<0.05, and ** indicates P<0.01. This is a diagram showing the maintenance and promotion of visual function in P23H knock-in heterozygous mice by PBA. A to E are diagrams showing the results of dark-adapted ERG, A shows the waveform of electroretinogram, B to C show the amplitude and latency of the a-wave, respectively, and D to E show the amplitude and latency of the b-wave, respectively. F to H are diagrams showing the results of light-adapted ERG, F shows the waveform of electroretinogram, and G to H show the amplitude and latency, respectively. In the diagram, * indicates P<0.05. This figure shows the upregulation of endoplasmic reticulum stress-related degradation and mitochondrial markers in the retina of P23H knock-in heterozygous mice by PBA. Graphs A to I show the change in the expression level of each gene marker by real-time reverse transcription polymerase chain reaction (RT-PCR). In the figure, * indicates P<0.05, and ** indicates P<0.01. FIG. 1 shows the improvement of mitochondrial metabolism in cell lines by PBA. A to B show that the mRNA expression levels of Pgc1-α and Tfam increase in a dose-dependent manner with PBA. C shows an electron micrograph showing the improvement of membrane potential before and after administration of BAM15, a mitochondrial protonophore uncoupler. D shows a graph showing the improvement of mitochondrial membrane potential. E to F show the enhancement of activity of complex IV (CoX IV) of the electron transport chain and the increase in ATP expression level by PBA. In the figure, * indicates P<0.05, and ** indicates P<0.01.

One embodiment of the present invention is a pharmaceutical composition for inhibiting retinal degeneration or for treating or preventing a retinal degenerative disease, comprising phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.

In this embodiment, the phenylbutyrate salt is not limited as long as it does not interfere with the effects of the present invention, and examples include sodium salts.

In this embodiment, the derivative of phenylbutyric acid or phenylbutyrate is not limited as long as it does not interfere with the effects of the present invention. For example, any hydrogen on the phenyl group may be replaced with any substituent (e.g., an alkyl group having 1 to 3 carbon atoms, a halogen, etc.), or any hydrogen on the hydrocarbon chain of phenylbutyric acid or phenylbutyrate may be replaced with any substituent (e.g., an alkyl group having 1 to 3 carbon atoms, a halogen, etc.).

In this embodiment, the retinal degeneration is not particularly limited to a disease, and refers to a state in which retinal cells have degenerated. Preferably, it refers to a state in which retinal photoreceptor cells have degenerated. The degeneration of retinal photoreceptor cells may be either rod photoreceptor cell degeneration or cone photoreceptor cell degeneration.

In this embodiment, suppression of retinal degeneration means, for example, that the progression of degeneration is inhibited, the progression of degeneration is delayed, the degenerative state is improved, or the degenerative state is completely eliminated, by administering the pharmaceutical composition of this embodiment.

By administering the pharmaceutical composition of this embodiment, retinal degeneration is suppressed by promoting the decomposition of abnormal proteins that have caused degeneration in the retina, improving or enhancing mitochondrial function, and/or improving or enhancing the protective function of retinal photoreceptor cells.

The indicator for determining whether retinal degeneration has been suppressed is not particularly limited, but for example, it may be determined that retinal degeneration has been suppressed by an increase in the number of retinal cells, particularly the number of retinal photoreceptor cells, in a sample after administration of the pharmaceutical composition of this embodiment, compared to a control sample to which the pharmaceutical composition of this embodiment has not been administered, or by an increase in the amplitude and/or a shortened latency in an electroretinogram. It is preferable that there is a statistically significant difference, but it is not necessarily necessary that there is a significant difference.

The retinal degenerative disease to be treated in this embodiment is not particularly limited as long as it is a disease in which degeneration of retinal cells occurs, regardless of the cause of retinal degeneration, but is preferably a disease in which degeneration of retinal photoreceptor cells occurs.
Specific examples of the disease include, but are not limited to, one or more diseases selected from retinitis pigmentosa, diabetic retinopathy, age-related macular degeneration, and retinal degeneration associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes).

In this embodiment, treatment also includes preventing the onset of disease.

In this embodiment, the content of phenylbutyric acid or a salt thereof or a derivative thereof in the pharmaceutical composition, or the dosage or administration method of the pharmaceutical composition containing it, is not particularly limited as long as the therapeutic effect of the pharmaceutical composition is obtained, and can be appropriately determined depending on the type of disease, the severity of the disease, symptoms, age, weight, etc. of the patient.

For example, in the case of a tablet or granule, the content of phenylbutyric acid or a salt thereof, or a derivative thereof in the pharmaceutical composition may be, for example, 10 to 1000 mg, or 100 to 1000 mg, per 1 g of pharmaceutical composition. In the case of a liquid, the content of phenylbutyric acid or a salt thereof, or a derivative thereof in the pharmaceutical composition may be, for example, 0.001 μg/mL to 1000 mg/mL, or 0.001 μM to 1000 mM.

The dosage form of the pharmaceutical composition of this embodiment can be appropriately selected depending on the administration method. For example, in the case of oral administration, it can be formulated into solid preparations such as powders, granules, tablets, and capsules; or liquid preparations such as solutions, syrups, suspensions, and emulsions. In addition, in the case of parenteral administration, it can be formulated into injections, liquids, suspensions, and the like.

The route of administration is not particularly limited as long as a therapeutic effect is obtained, and may be oral administration, ocular administration, intravenous administration, local administration, etc. For example, it may be administered orally as a tablet or granule, locally into the eye as an injection, or ocular administration as eye drops.

The dosage of the pharmaceutical composition of this embodiment, in the case of oral administration, intravenous administration, etc., for phenylbutyric acid or a salt thereof, or a derivative thereof, may be, for example, 1.0 to 20.0 g/m ² (body surface area) per day, or may be 9.9 to 13.0 g/m ² (body surface area). The pharmaceutical composition of the above dosage may be administered once a day, or may be administered in divided doses several times a day. In addition, when administering to the eye as an eye drop, for example, a pharmaceutical composition having a concentration of 0.001 μg/mL to 1000 mg/mL or 0.001 μM to 1000 mM may be administered as one or more drops per eye, once or several times a day.

The pharmaceutical composition of this embodiment may contain additional active ingredients as long as the effects of the present invention are not hindered. Furthermore, it may be used in combination with additional disease treatment methods.

The pharmaceutical composition of the present embodiment may contain a carrier or an additive. Here, examples of the carrier include pharmacologically acceptable solvents, diluents, excipients, binders, etc., and for example, water, physiological saline, buffer solutions, etc. are used to prepare liquid pharmaceutical compositions. Examples of the additives include stabilizers, pH adjusters, thickeners, antioxidants, isotonicity agents, buffers, dissolution aids, suspending agents, preservatives, cryoprotectants, cryoprotectants, lyophilization protectants, bacteriostatic agents, etc.

Another embodiment of the present invention is a pharmaceutical composition for promoting endoplasmic reticulum stress-related degradation in the retina, comprising phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.

Promoting endoplasmic reticulum stress-related degradation in the retina means, but is not limited to, enhancing the gene expression level or protein expression level of molecular markers associated with endoplasmic reticulum stress-related degradation, such as XBP1s (XBP1 spliced form), VCP (valosin-containing protein), and Derlin1 (degradation in endoplasmic reticulum protein 1). By enhancing the expression level of these molecular markers, it is possible to promote the degradation of abnormal proteins that have developed in the retina. In particular, promoting endoplasmic reticulum stress-related degradation promotes the degradation of abnormal P23H rhodopsin, thereby suppressing retinal degeneration.

Here, the enhancement of the expression level of various molecular markers is not particularly limited, but may mean that the gene or protein expression level of the marker is increased in a subject administered with the pharmaceutical composition of the present embodiment, compared to a control subject not administered with the pharmaceutical composition.
Alternatively, when the gene or protein expression level of the marker in a subject administered with the pharmaceutical composition of this embodiment exceeds a preset cutoff value, it may be determined that the expression level is enhanced.

In this embodiment, the type of salt or derivative of phenylbutyric acid, the content of phenylbutyric acid or its salt or derivative in the pharmaceutical composition, the dosage of the pharmaceutical composition, the administration method, dosage form, additional ingredients, etc. are not particularly limited, and the descriptions of the pharmaceutical composition for inhibiting retinal degeneration described above can be applied mutatis mutandis.

Another embodiment of the present invention is a pharmaceutical composition for activating mitochondrial biogenesis in the retina and/or improving mitochondrial metabolic function in the retina, comprising phenylbutyric acid or a salt thereof or a derivative thereof.

Activation of mitochondrial biogenesis in the retina includes, but is not limited to, enhancement of gene expression or protein expression of a mitochondrial fission marker such as Fis1 (mitochondrial fission 1 protein), an autophagy marker such as LC3 (microtubule-associated protein light chain 3), or a fusion marker such as Mfn1 (mitofusin 1) or Mfn2 (mitofusin 2), or enhancement of gene expression or protein expression of a mitochondrial biogenesis regulator Pgc1-α (peroxisome proliferators-activated receptor-γ co-activator-1α) or a mitochondrial transcription factor Tfam (mitochondrial transcription factor A) and the like. Increasing the gene expression or protein expression of these molecules can activate mitochondrial biogenesis in the retina and promote mitophagy of damaged mitochondria.
Furthermore, improving the metabolic function of mitochondria in the retina includes, but is not limited to, for example, enhancing mitochondrial membrane potential and enhancing the activity of cytochrome c oxidase IV (COX IV).
As a result of activating mitochondrial biogenesis and improving mitochondrial metabolic function, ATP levels in mitochondria can be increased, leading to protection of photoreceptor cells. In other words, retinal degeneration can be suppressed by using the pharmaceutical composition of the present embodiment.

Here, the enhancement of the expression level of various molecular markers, the enhancement of mitochondrial membrane potential, and the enhancement of COX IV activity are not particularly limited, and may mean that the gene or protein expression level of the marker has increased, the mitochondrial membrane potential has been enhanced, or the activity of COX IV has been enhanced in a subject to which the pharmaceutical composition of the present embodiment has been administered, compared to a control subject to which the pharmaceutical composition has not been administered.
Alternatively, when the expression level of the gene or protein of the marker, the measured value of the mitochondrial membrane potential, or the measured value of COX IV activity in a subject administered the pharmaceutical composition of the present embodiment exceeds a preset cutoff value, it may be determined that the expression level of the gene or protein of the marker has increased, the mitochondrial membrane potential has been enhanced, or the activity of COX IV has been enhanced.

In this embodiment, the type of salt or derivative of phenylbutyric acid, the content of phenylbutyric acid or its salt or derivative in the pharmaceutical composition, the dosage of the pharmaceutical composition, the administration method, dosage form, additional ingredients, etc. are not particularly limited, and the descriptions of the pharmaceutical composition for inhibiting retinal degeneration described above can be applied mutatis mutandis.

The examples are provided for disclosure purposes and are not intended to limit the scope of the invention.

<Experimental animals>
P23H knock-in heterozygous mice (+/-, 2 weeks old, male) obtained by a method known to those skilled in the art (Non-Patent Document 2) were kept in the animal experiment facility of Keio University School of Medicine in a temperature-controlled room (22°C) with a 12-hour light/dark cycle (lights on from 8 am to 8 pm) and with free access to food and water. All animal experiments were performed in accordance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Keio University Animal Experimentation Committee.

<Histological analysis>
Mouse eyes were enucleated and fixed overnight at 4°C in 4% paraformaldehyde. After fixation, the eyes were embedded in paraffin (Sakura Finetech Japan, Tokyo, Japan) and 6-8 μm-thick sections were prepared from the optic disc to the most distant region of the retina, deparaffinized, and stained with hematoxylin and eosin. The sections were observed under a microscope equipped with a digital camera (Olympus Corporation, Tokyo, Japan). Using methods known to those skilled in the art, the thickness of the photoreceptor layer, the outer nuclear layer (ONL), was assessed by measuring the distance from the top to the bottom of the ONL, and the length of the rod outer segment (OS) was determined in the posterior superior retina, 500 μm from the optic nerve, by observing hematoxylin and eosin staining using ImageJ software (National Institutes of Health, Bethesda, MD, USA; available at http://rsb.info.nih.gov/ij/index.html), and averaged (Non-Patent Documents 2 to 6).

<Real-time reverse transcription polymerase chain reaction (RT-PCR)>
Total RNA was isolated from 4-week-old mouse retinas using TRIzol reagent (Life Technologies, Carlsbad, CA, USA).
The RNA was measured using noDrop 1000 (Thermo Fisher Scientific), and 1 μg of RNA was reverse transcribed using SuperScript VILO Master Mix (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. The following primers were used:
Glyceraldehyde 3-phosphate dehydrogenase (Gapdh): forward primer 5'-ACTTTCGGCCCATCTCTCA-3' (SEQ ID NO:1) and reverse primer 5'-GATGACCCCTTTTGGCTCTCCAC-3' (SEQ ID NO:2).
Pgc1-α: forward primer 5′-GATGAATACCGCAAAGAGCA-3′ (SEQ ID NO: 3) and reverse primer 5′-AGATTTACGGTGCATTCCT-3′ (SEQ ID NO: 4).
Fis1: forward primer 5'-ATATGCCTGGTGCCTGGTTC-3' (SEQ ID NO:5) and reverse primer 5'-AGTCCCGCTGTTCCTCTTTG-3' (SEQ ID NO:6).
Mfn1: forward primer 5'-GATGTTCCACCAGAGCTGGGA-3' (SEQ ID NO: 7) and reverse primer 5'-AGGAGCCGCTCATTCACCCTTTTA-3' (SEQ ID NO: 8).
Mfn2: forward primer 5'-CCCCTCCTCAAGCACTTTGGTTC-3' (SEQ ID NO: 9) and reverse primer 5'-ACCCTGCTCTCTCTCTCCGTGTTGTAAAC-3' (SEQ ID NO: 10).
Xbp1s: forward primer 5'-CTGAGTCCGCAGCAGGTG-3' (SEQ ID NO: 11) and reverse primer 5'-TGCCCAAAAGGATATCAGACT-3' (SEQ ID NO: 12).
VCP: forward primer 5'-AAGTCCCCAGTTGCCAAGGATG-3' (SEQ ID NO: 13) and reverse primer 5'-AGCCGATGGATTGTCTGCCTC-3' (SEQ ID NO: 14).
Derl1: forward primer 5'-CGCGATTTAAGGCCTGTTAC-3' (SEQ ID NO: 15) and reverse primer 5'-GGTAGCCAGCGGTACAAAA-3' (SEQ ID NO: 16).
Tfam: forward primer 5'-AGTCAGCTGATGGGTATGGAGAA-3' (SEQ ID NO: 17) and reverse primer 5'-TGCTGAACGAGGTCTTTTTTGG-3' (SEQ ID NO: 18).
LC3b: Mm00782868 (Taqman).
Taqman probes (Applied Biosystems, Thermo Fisher Scientific) were used for Kir2.1 (also known as Kcnj2, potassium inward rectifier channel, subfamily J, member 2; Mm00434616), Aqp4 (Mm00802131), Kir4.1 (also known as Kcnj10, potassium inward rectifier channel, subfamily J, member 10; Mm00445028), Kcnv2 (potassium channel, subfamily V, member 2 (Mm00807577), and BCL2-associated X protein (Bax; Mm00432050). Real-time PCR was performed using the StepOnePlus ^™ PCR system (Applied Biosystems, Thermo Fisher Scientific). Analysis was performed using the Fisher Scientific software and gene expression was quantified using the ΔΔCT method. All mRNA levels were normalized to Gapdh.

<Electroretinogram (ERG) Recording>
Based on methods known to those skilled in the art, mice were dark-adapted for at least 12 hours and then placed under dim red light before ERG was performed (Non-Patent Documents 7-8). Mice were anesthetized with intraperitoneal compound anesthetic [midazolam 4 mg/kg body weight (Sandoz Japan Co., Ltd., Tokyo, Japan), medetomidine 0.75 mg/kg body weight (Nippon Zenyaku Kogyo Co., Fukushima, Japan), and butorphanol tartrate 5 mg/kg body weight (Meiji Seika Pharma Co., Ltd., Tokyo, Japan)] and kept on a heating pad during the experiment. Mice pupils were dilated using a drop of a mixture of tropicamide and phenylphrine (0.5% each; Mydrin-P®; Santen Pharmaceutical Co., Osaka, Japan). An active gold wire electrode was placed on the cornea, and ground and reference electrodes were placed on the tail and in the mouth, respectively.
Electroretinogram recordings were made using a PowerLab System 2/25 (AD Instruments, New South Wales, Australia). Full-field dark-adapted ERGs were measured in response to flash stimuli at stimulus intensities ranging from -2.1 to 2.9 log cd s/ ^m2 . Light-adapted ERGs were measured 10 min after light adaptation. Responses to flash stimuli ranging from 0.4 to 1.4 log cd s/ ^m2 were measured against a background of 30 cd s/ ^m2 . Responses were differentially amplified and filtered through a digital band-pass filter ranging from 0.3 to 1000 Hz. Each stimulus was delivered using a commercially available stimulator (Ganzfeld System SG-2002; LKC Technologies, Inc.). A-wave amplitude was measured from baseline to trough, and b-wave amplitude was measured from the a-wave trough to the b-wave peak. The a-wave and b-wave latencies were measured from the onset of stimulation to the peak of each wave, which was automatically calculated by the system and confirmed by the examiner.

<Cell culture>
HEK293 cells (ATCC CRL-1573) were cultured in 10% fetal bovine serum (Life
The cells were maintained in Dulbecco's modified Eagle's medium (#08456-65; Nacalai Tesque, Kyoto, Japan) supplemented with 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA).

Statistical analysis
All results are expressed as mean ± standard deviation. Comparisons between three groups were analyzed by one-way ANOVA with Tukey's post-hoc test, and comparisons between two groups were analyzed by two-tailed Student's t test using SPSS Statistics 24 (IBM, Armonk, NY, USA) software. P < 0.05 was considered statistically significant.

Example 1: Phenylbutyric acid (PBA) suppressed photoreceptor cell loss in P23H knock-in heterozygous mice (P23H RP model mice)
It has been reported that in P23H knock-in heterozygous mice, there is no change in the thickness of the photoreceptor layer until postnatal day 12, and the length of the inner segment (IS) and outer segment (OS) is similar to that of wild-type (WT) mice, after which retinitis pigmentosa gradually progresses (Non-Patent Document 9).
In this study, continuous administration of PBA was started from 2 weeks of age. Continuous administration was performed by intraperitoneally administering sodium phenylbutyrate (Sigma-Aldrich, 10 mg/kg body weight) 5 times a week every day from 2 weeks of age. As a result, it was shown that the number of remaining photoreceptor cells (photoreceptor nuclei) in the retina of P23H RP model mice administered PBA was significantly higher than that of mice administered the vehicle at 10 weeks of age (Figure 1A, B). This result showed that PBA administration promotes the survival of photoreceptor cells and inhibits retinal degeneration in P23H RP model mice.

Example 2: PBA restored visual function in P23H RP model mice.
It has been reported that in P23H RP model mice, impairment of rod photoreceptor function was observed at 6 weeks of age based on dark-adapted ERG, and impairment of cone photoreceptor function was observed at 10 weeks of age, both of which progress gradually (Non-Patent Document 2).
Therefore, to examine whether the histological improvement by PBA treatment contributes to the preservation of better visual function, dark-adapted ERG (Fig. 2A-E) and light-adapted ERG (Fig. 2F-H) were measured. P23H RP model mice were intraperitoneally administered PBA (10 mg/kg body weight) daily, 5 times a week, from 2 weeks of age. As a result, dark-adapted ERG showed a larger amplitude of a-wave, which reflects the function of rod photoreceptors, and a larger amplitude and shorter latency of b-wave, which reflects the function of retinal nerve cells, in 10-week-old PBA-treated P23H RP model mice compared with vehicle-treated mice. In other words, it was shown that continued PBA treatment protects the visual function of the rod system. Furthermore, in light-adapted ERG, the latency of the b-wave, which mainly indicates the function of the cone photoreceptor cells, was shortened after PBA treatment, indicating that PBA treatment also protects the visual function of the cone system in P23H RP model mice.

Example 3: PBA induced increased expression of endoplasmic reticulum stress-associated degradation (ERAD) and mitochondrial markers in the retina of P23H RP model mice.
It has been shown that P23H mutant rhodopsin induces ER stress (Non-Patent Documents 9 to 15), while the ERAD system that degrades abnormal proteins (Non-Patent Documents 16 to 17) is induced in P23H RP model mice (Non-Patent Document 9). It has also been shown that ERAD is activated by the conversion of IRE1-associated XBP1 to XBP1s, leading to the induction of VCP (also known as Cdc48 or p97), which interacts with Derlin 1, and that specific misfolded proteins are transported from the ER to the cytoplasm using its ATPase activity (Non-Patent Document 18), and that proteins are further degraded via the ubiquitin proteasome system (UPS) (Non-Patent Documents 16 to 17).
As a result of examining ER stress-related degradation in the retina of P23H RP model mice, it was shown that the expression of XBP1s (Fig. 3A), VCP (Fig. 3B), and Derlin1 (Fig. 3C) was enhanced by the PBA treatment described in Examples 1 and 2. These results suggest that PBA promotes the induction of ERAD and removes abnormal P23H rhodopsin.

It has been revealed that ER stress also affects mitochondrial quality control by controlling mitochondrial division and fusion by VCP (Non-Patent Documents 18-19), that VCP induces division to eliminate abnormal mitochondria by autophagy (Non-Patent Documents 19-20), and that mitofusin proteins, which are fusion-related proteins, induce degradation via the UPS (Non-Patent Documents 18-19). It has also been revealed that this system maintains cellular homeostasis (Non-Patent Document 21) and neuronal plasticity and survival (Non-Patent Document 22).
As a result of investigating mitochondrial fission and fusion in the retina of P23H RP model mice, it was shown that the PBA treatment described in Examples 1 and 2 increased the mRNA levels of the mitochondrial fission marker Fis1 (Figure 3D) and the autophagy marker LC3 (Figure 3E). It was also shown that the mRNA levels of the fusion markers Mfn1 (Figure 3F) and Mfn2 (Figure 3G) were increased by PBA. These results suggest that VCP induces the degradation of mitofusin proteins.

During mitochondrial quality control, mitochondrial biogenesis is carried out to replace damaged mitochondria with new healthy mitochondria (Non-Patent Documents 19, 23). It was shown that the mRNA levels of Pgc1-α, which is known to regulate mitochondrial biogenesis (Non-Patent Document 24), and Tfam, a transcription factor that induces mitochondrial DNA that encodes molecules related to respiration (Non-Patent Document 25), were increased by PBA as described in Examples 1 and 2 (Figures 3H and I). These results indicate that PBA treatment activates mitochondrial biogenesis.

Example 4: PBA activates mitochondrial oxidative phosphorylation (OXPHOS) in vitro
To further analyze the potential effect caused by PBA, a study was carried out using HEK293 cell line. The cells were treated with 0-2.5 μM PBA 12 or 24 hours before each measurement. The mitochondrial membrane potential was measured by incubating the cells with tetramethylrhodamine methyl ester (TMRE) (10 μM) at 37°C for 30 minutes, measuring the average brightness before and after administration of BAM15 (Sigma-Aldrich) using a confocal microscope (TCS-SP5; Leica, Tokyo, Japan), and calculating the potential using the following formula: membrane potential = average brightness for 14 seconds before addition of BAM15 (7 images x 2 seconds apart) / average brightness for 14 seconds immediately after addition of BAM15 (7 images x 2 seconds apart). Measurement of cytochrome c oxidase (COX IV) activity was performed using the Complex IV Rodent Enzyme Activity Microplate Assay Kit (Abcam) according to the manufacturer's instructions after flash freezing the cells in liquid nitrogen and then placing them in the lysis buffer provided with the kit. Luminescence signals were measured using a Cytation 5 system (BioTek, Winooski, VT, USA). For ATP measurements, flash frozen samples were placed in lysis buffer before measuring ATP content using the ATP Bioluminescence Assay Kit CLSII (Sigma-Aldrich), and luminescence signals were measured using a Cytation 5 system (BioTek).
As a result, PBA induced dose-dependent expression of Pgc1-α (Figure 4A) and Tfam (Figure 4B) in HEK293 cell lines. Mitochondrial membrane potential is essential for mitochondrial ATP synthesis, and BAM15, a mitochondrial protonophore uncoupler, counteracts this potential. Using the subtraction method of potential before and after BAM15 administration, the membrane potential was increased in cells treated with PBA (Figure 4C, D). Furthermore, it was shown that PBA increased the activity of cytochrome c oxidase IV (COX IV) (Figure 4E), which resulted in increased ATP levels (Figure 4F). These results indicate that PBA can increase mitochondrial function and obtain ATP levels that can be used for cytoprotection, which may lead to improvement of pathological conditions (Non-Patent Documents 3, 26-27).

Claims (8)

A pharmaceutical composition for inhibiting retinal degeneration, comprising phenylbutyric acid (PBA) or a salt thereof, or a derivative thereof. A pharmaceutical composition for treating or preventing a retinal degenerative disease, comprising phenylbutyric acid (PBA) or a salt thereof or a derivative thereof. The pharmaceutical composition according to claim 2, wherein the retinal degenerative disease is a disease in which retinal photoreceptor cells are degenerated. The pharmaceutical composition according to claim 2 or 3, wherein the retinal degenerative disease is one or more diseases selected from retinitis pigmentosa, diabetic retinopathy, age-related macular degeneration, and retinal degeneration associated with MELAS (mitochondrial myopathy, encephalopathies, lactic acidosis, and stroke-like episodes). The pharmaceutical composition according to any one of claims 1 to 4, wherein the salt of phenylbutyric acid is a sodium salt. The pharmaceutical composition according to any one of claims 1 to 5, which is in the form of a tablet, granule, injection or eye drop. A pharmaceutical composition for promoting endoplasmic reticulum stress-related degradation in the retina, comprising phenylbutyric acid (PBA) or a salt thereof or a derivative thereof. A pharmaceutical composition for activating mitochondrial biogenesis in the retina and/or improving mitochondrial metabolic function in the retina, comprising phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.