WO2022196609A1 - Composition for preventing retinal degeneration - Google Patents
Composition for preventing retinal degeneration Download PDFInfo
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- WO2022196609A1 WO2022196609A1 PCT/JP2022/011209 JP2022011209W WO2022196609A1 WO 2022196609 A1 WO2022196609 A1 WO 2022196609A1 JP 2022011209 W JP2022011209 W JP 2022011209W WO 2022196609 A1 WO2022196609 A1 WO 2022196609A1
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- pharmaceutical composition
- phenylbutyric acid
- salt
- retina
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
Definitions
- the present invention relates to a pharmaceutical composition for suppressing retinal degeneration including retinitis pigmentosa.
- Retinitis pigmentosa is a hereditary retinal disease in which rod photoreceptors, which are photoreceptors that recognize the brightness of light, are degenerated due to genetic mutations. is a disease that can lead to blindness. At present, there is no effective treatment, and clinically, vitamin preparations have been administered for the purpose of neuroprotection, but the effects are poor. In addition, research on gene transfer therapy using viruses has been advanced so far, and clinical trials are being conducted worldwide. In addition, visual cycle modulator (Visual Cycle Modulator) is being researched as drug therapy (Non-Patent Document 1), which protects the retina by not using visual function, and maintains visual function.
- Visual Cycle Modulator Visual Cycle Modulator
- PBA phenylbutyric acid
- Patent Documents 1 and 2 phenylbutyric acid
- an object of the present invention is to provide a pharmaceutical composition for suppressing retinal degeneration including retinitis pigmentosa.
- the present inventors conducted studies to solve the above problems, found that PBA suppresses progressive retinal photoreceptor death and suppresses deterioration of visual function, and completed the present invention.
- a pharmaceutical composition for suppressing retinal degeneration containing phenylbutyric acid (PBA), a salt thereof, or a derivative thereof.
- a pharmaceutical composition for treating or preventing retinal degenerative diseases containing phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
- PBA phenylbutyric acid
- a pharmaceutical composition for treating or preventing retinal degenerative diseases containing phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
- the pharmaceutical composition of [2] wherein the retinal degenerative disease is a disease in which retinal photoreceptors are degenerated.
- the retinal degenerative disease is one or more 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)
- the pharmaceutical composition according to any one of [1] to [5] which is a tablet, granule, suspension, 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 biosynthesis in the retina and/or for improving metabolic function of mitochondria in the retina comprising phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
- a method of inhibiting retinal degeneration in a subject comprising administering phenylbutyric acid (PBA) or a salt thereof or a derivative thereof to the subject.
- a method of treating or preventing retinal degeneration in a subject comprising administering phenylbutyric acid (PBA) or a salt or derivative thereof to the subject.
- PBA phenylbutyric acid
- a method of promoting endoplasmic reticulum stress-related degradation in the retina in a subject comprising administering phenylbutyric acid (PBA) or a salt or derivative thereof to the subject.
- a method of activating mitochondrial biogenesis in the retina and/or improving metabolic function of mitochondria in the retina in a subject comprising administering phenylbutyric acid (PBA) or a salt thereof or a derivative thereof to the subject
- PBA phenylbutyric acid
- a method comprising: [13] Phenylbutyric acid (PBA) or a salt or derivative thereof for use in inhibiting retinal degeneration.
- PBA phenylbutyric acid
- phenylbutyric acid (PBA) or a salt thereof or a derivative thereof in the manufacture of a medicament for use in promoting endoplasmic reticulum stress-related degradation in the retina.
- PBA phenylbutyric acid
- PBA phenylbutyric acid
- a salt thereof or a derivative thereof in the manufacture of a medicament for use in activating mitochondrial biogenesis in the retina and/or improving metabolic function of mitochondria in the retina.
- the present invention has found that PBA promotes the maintenance of visual function in retinitis pigmentosa models, and can provide an agent for suppressing retinal degeneration including retinitis pigmentosa, which is effective even with a diagnosis.
- Fig. 10 shows the maintenance and promotion of visual function in P23H rhodopsin knock-in heterozygous retinitis pigmentosa model mice (also referred to simply as P23H knock-in heterozygous mice and P23H RP model mice in the present disclosure) by PBA.
- WT indicates wild-type mice
- Hetero indicates P23H knock-in heterozygous mice.
- A is a micrograph of a mouse retina stained with hematoxylin and eosin.
- B is a graph showing the number of nuclei in the outer nuclear layer (ONL).
- * indicates P ⁇ 0.05 and ** indicates P ⁇ 0.01.
- FIG. 10 shows the maintenance and promotion of visual function in P23H rhodopsin knock-in heterozygous retinitis pigmentosa model mice (also referred to simply as P23H knock-in heterozygous mice and P23H RP model mice in the present disclosure) by PBA.
- WT indicates wild-type mice
- FIG. 10 shows the maintenance and promotion of visual function maintenance in P23H knock-in heterozygous mice by PBA.
- a to E are dark adaptation ERG result diagrams, A shows the waveform of electroretinogram, B to C show the amplitude and latency of a-wave, respectively, and D to E show the amplitude and latency of b-wave, respectively. indicate.
- F to H are the results of photopic ERG, F shows the waveform of the electroretinogram, and G to H show the amplitude and latency, respectively. In the figure, * indicates P ⁇ 0.05.
- FIG. 2 shows PBA-induced endoplasmic reticulum stress-related degradation and increased expression of mitochondrial markers in the retina of P23H knock-in heterozygous mice.
- FIG. 2 shows improvement of mitochondrial metabolism in cell lines by PBA.
- AB respectively show that the mRNA expression levels of Pgc1- ⁇ and Tfam are increased in a PBA dose-dependent manner.
- C is an electron micrograph showing improvement in membrane potential before and after administration of BAM15, a mitochondrial protonophore uncoupler.
- D is a graph showing mitochondrial membrane potential improvement.
- E to F are graphs showing enhancement of the electron transport chain complex IV (CoX IV) activity by PBA and an increase in ATP expression level, respectively.
- CoX IV electron transport chain complex IV
- Fig. 10 shows changes in body weight of P23H knock-in heterozygous mice by oral administration of PBA.
- Fig. 10 shows elevation of mitochondrial marker expression in the retina of P23H knock-in heterozygous mice by oral administration of PBA.
- Graphs AB respectively show changes in the expression level of each gene marker by real-time reverse transcription-polymerase chain reaction (RT-PCR).
- * indicates P ⁇ 0.05 and ** indicates P ⁇ 0.01.
- One embodiment of the present invention is a pharmaceutical composition for suppressing retinal degeneration or a pharmaceutical composition for treating or preventing retinal degenerative diseases, containing phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
- PBA phenylbutyric acid
- phenylbutyric acid is not limited as long as it does not interfere with the effects of the present invention, and includes 4-phenylbutyric acid, 3-phenylbutyric acid, 2-phenylbutyric acid, 2-phenylisobutyric acid, 3-phenylisobutyric acid and the like. , preferably 4-phenylbutyric acid. It can be obtained as a reagent, an industrial raw material, etc., and can also be synthesized according to a conventional method. Phenylbutyric acid salts are not limited as long as they do not interfere with the effects of the present invention, and examples thereof include sodium salts, potassium salts, magnesium salts, calcium salts and the like, preferably sodium salts.
- the derivative of phenylbutyric acid or phenylbutyrate is not limited as long as it does not interfere with the effects of the present invention.
- saturated hydrocarbon group with 3 or less carbon atoms such as an alkyl group of 3, unsaturated hydrocarbon group with 3 or less carbon atoms, halogen, etc.
- Any hydrogen on the hydrocarbon chain of is any substituent (e.g., a saturated hydrocarbon group having 3 or less carbon atoms such as an alkyl group having 1 to 3 carbon atoms, an unsaturated hydrocarbon group having 3 or less carbon atoms, a halogen etc.).
- the derivative of phenylbutyrate may be an ester or ether of phenylbutyrate, such as methyl ester, ethyl ester, n-propyl ester, isopropyl ester, or the like.
- retinal degeneration is not particularly limited to diseases, and refers to a state in which degeneration of retinal cells has occurred. Preferably, it is a state in which degeneration of retinal photoreceptors has occurred. Degeneration of retinal photoreceptors may be degeneration of rod photoreceptors or degeneration of cone photoreceptors.
- inhibition of retinal degeneration means that administration of the pharmaceutical composition of the present embodiment, for example, inhibits the progress of degeneration, delays the progress of degeneration, degenerates is improved, and the denatured state is completely eliminated.
- the degradation of abnormal proteins caused by degeneration in the retina is promoted, the mitochondrial function is improved or enhanced, and/or the protective function of retinal photoreceptors is improved. Or, retinal degeneration is suppressed by being improved or the like.
- the index for judging suppression of retinal degeneration is not particularly limited. It may be determined that retinal degeneration is suppressed by increasing the number of retinal cells, particularly the number of retinal photoreceptors, and by increasing the amplitude and / or shortening the latency in the electroretinogram You may judge that retinal degeneration was suppressed. A statistically significant difference is preferred, but not necessarily a significant difference.
- the retinal degenerative disease to be treated by the present sample is not particularly limited as long as it causes degeneration of retinal cells, regardless of the cause of retinal degeneration. is preferred. Specifically, but not particularly limited, for example, retinitis pigmentosa, diabetic retinopathy, age-related macular degeneration, and retinal degeneration associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) One or more diseases are included.
- treatment also includes prevention of disease occurrence.
- 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 particularly limited as long as the therapeutic effect of the pharmaceutical composition can be obtained. However, it can be appropriately determined according to the type of disease, degree of disease, symptoms, patient's age, body weight, and the like.
- the content of phenylbutyric acid or a salt thereof or a derivative thereof in the pharmaceutical composition may be 10 to 1000 mg, preferably 100 to 1000 mg, per 1 g of the pharmaceutical composition. good.
- the content of phenylbutyric acid or a salt thereof or a derivative thereof in the pharmaceutical composition may be, for example, 0.001 ⁇ g/mL to 1000 mg/mL, and may be 0.001 ⁇ M to 1000 mM. .
- the dosage form of the pharmaceutical composition of this embodiment can be appropriately selected according to the administration method.
- solid preparations such as powders, granules, tablets and capsules
- liquid preparations such as solutions, syrups, suspensions and emulsions
- 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, topical administration, or the like.
- it may be administered orally in dosage forms such as tablets, granules, or suspensions, may be administered topically to the eye as an injection, or may be administered to the eye as eye drops.
- the dosage of the pharmaceutical composition of the present embodiment is 0 per day for a human weighing 60 kg, for example, when oral administration, intravenous administration, or the like is performed.
- .001-100.0 g/m 2 (body surface area), 0.01-50.0 g/m 2 (body surface area), 0.1-30.0 g/m 2 (body surface area) ), may be 1.0 to 20.0 g/m 2 (body surface area), may be 9.9 to 13.0 g/m 2 (body surface area), may be 0.1 to 10 0 g/m 2 (body surface area), 0.1 to 1.0 g/m 2 (body surface area).
- the above dosage of the pharmaceutical composition may be administered once a day, or may be administered in several divided doses per day.
- a pharmaceutical composition having a concentration of 0.001 ⁇ g/mL to 1000 mg/mL or 0.001 ⁇ M to 1000 mM is applied once per eye, once a day.
- multiple doses may be administered.
- the pharmaceutical composition of this embodiment may contain additional active ingredients as long as they do not interfere with the effects of the present invention. Additionally, it may be used in combination with additional disease treatment methods.
- the pharmaceutical composition of this embodiment may contain carriers and additives.
- carriers include pharmacologically acceptable solvents, diluents, excipients, binders, etc.
- examples include water, physiological saline, buffers, etc. is used.
- Additives include stabilizers, pH adjusters, thickeners, antioxidants, tonicity agents, buffers, solubilizers, suspending agents, preservatives, antifreeze agents, cryoprotectants, and lyophilization.
- Protective agents, bacteriostatic agents and the like can be mentioned.
- 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 or derivative thereof.
- PBA phenylbutyric acid
- promotion of endoplasmic reticulum stress-related degradation in the retina is not particularly limited, but for example, endoplasmic reticulum stress-related degradation of XBP1s (XBP1 spliced form), VCP (valosin-containing protein), Derlin1 (degradation in endoplasmic reticulum protein 1), etc. is that the gene expression level or protein expression level of a molecular marker associated with is enhanced. By enhancing the expression level of this molecular marker, it is possible to promote the degradation of abnormal proteins occurring in the retina. In particular, promotion of endoplasmic reticulum stress-related degradation promotes degradation of abnormal P23H rhodopsin, thereby suppressing retinal degeneration.
- the enhancement of the expression level of various molecular markers is not particularly limited, but in a subject administered the pharmaceutical composition of the present embodiment, compared with a control subject not administered the pharmaceutical composition, the marker It may mean that the gene or protein expression level of is increased. Alternatively, it may be determined that the expression level is enhanced when the gene or protein expression level of the marker exceeds a preset cutoff value in subjects administered with the pharmaceutical composition of the present embodiment.
- the type of phenylbutyric acid salt or derivative, the content of phenylbutyric acid or its salt or its derivative in the pharmaceutical composition, or the dosage, administration method, dosage form, additional ingredients, etc. of the pharmaceutical composition are It is not particularly limited, and the description of the pharmaceutical composition for suppressing retinal degeneration 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 the metabolic function of mitochondria in the retina, comprising phenylbutyric acid or a salt thereof or a derivative thereof. be.
- Activating mitochondrial biogenesis in the retina is not particularly limited, but for example, mitochondrial fission markers such as Fis1 (Mitochondrial fission 1 protein), autophagy markers such as LC3 (microtubule-associated protein light chain 3), or Mfn1 (mitofusin 1), Mfn2 (mitofusin 2), or the like, or Pgc1- ⁇ (peroxisome proliferators-activated receptor- ⁇ co-activator), which is a regulator of mitochondrial biogenesis -1 ⁇ ) or Tfam (mitochondrial transcription factor A), which is a mitochondrial transcription factor, or the expression level of genes or proteins is enhanced.
- mitochondrial fission markers such as Fis1 (Mitochondrial fission 1 protein), autophagy markers such as LC3 (microtubule-associated protein light chain 3), or Mfn1 (mitofusin 1), Mfn2 (mitofusin 2), or the like
- Pgc1- ⁇ peroxisome proliferators-activated
- Enhanced gene or protein expression levels of these molecules can activate mitochondrial biogenesis in the retina and promote mitophagy of damaged mitochondria. Improving the metabolic function of mitochondria in the retina is not particularly limited, but includes, for example, enhancement of mitochondrial membrane potential and enhancement of cytochrome c oxidase IV (COX IV) activity. Activation of mitochondrial biogenesis and improved mitochondrial metabolic function can result in increased ATP levels in mitochondria, leading to protection of photoreceptors. That is, retinal degeneration can be suppressed by using the pharmaceutical composition of this embodiment.
- enhancement of expression levels of various molecular markers, enhancement of mitochondrial membrane potential, and enhancement of COX IV activity are not particularly limited. It may mean increased gene or protein expression of the marker, enhanced mitochondrial membrane potential, or enhanced COX IV activity compared to a control subject without the marker. Alternatively, in subjects administered the pharmaceutical composition of the present embodiment, the gene or protein expression level of the marker, the measured value of mitochondrial membrane potential, or the measured value of COX IV activity exceeds a preset cutoff value In some cases, it may be determined that the gene or protein expression level of the marker is increased, the mitochondrial membrane potential is enhanced, or the activity of COX IV is enhanced.
- Kinds of phenylbutyric acid salts and derivatives, content of phenylbutyric acid or its salts or derivatives thereof in the pharmaceutical composition, dosage, administration method, dosage form, additional ingredients, etc. of the pharmaceutical composition in the present embodiment is not particularly limited, and the description of the pharmaceutical composition for suppressing retinal degeneration can be applied mutatis mutandis.
- mice 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 placed in a temperature-controlled room at the animal experiment facility of Keio University School of Medicine. The mice were reared at (22° C.) with a 12-hour light-dark cycle (lights on from 8:00 am to 8:00 pm) under an environment in which food and water were available ad libitum. All animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Keio University Animal Care and Use Committee.
- RNA concentration was measured using a NanoDrop 1000 (Thermo Fisher Scientific) and 1 ⁇ g of RNA was reverse transcribed using SuperScript VILO master mix (Life Technologies, Carlsbad, Calif., USA) according to the manufacturer's instructions. Primers with the following sequences were used.
- Glyceraldehyde 3-phosphate dehydrogenase forward primer 5′-ACTTTCGGCCCATCTCTCA-3′ (SEQ ID NO: 1) and reverse primer 5′-GATGACCCCTTTTGGCTCTCCAC-3′ (SEQ ID NO: 2).
- Pgc1- ⁇ forward primer 5′-GATGATACCGCAAAGAGCA-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'-ACCCTGCTCTCTCTCCGTGTTGTAAAC-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'-AGCCGATGGATTTGTCTGCCTC-3' (SEQ ID NO: 14).
- Derl1 forward primer 5′-CGCGATTTAAGGCCTGTTAC-3′ (SEQ ID NO: 15) and reverse primer 5′-GGTAGCCAGCGGTACAAAAAA-3′ (SEQ ID NO: 16).
- Tfam forward primer 5′-AGTCAGCTGATGGGTATGGAGAA-3′ (SEQ ID NO: 17) and reverse primer 5′-TGCTGAACGAGGTCTTTTTG-3′ (SEQ ID NO: 18).
- LC3b Mm00782868 (Taqman).
- Taqman probes (Applied Biosystems, Thermo Fisher Scientific) were combined with Kir2.1 (also known as Kcnj2, potassium inward rectifying channel, subfamily J, member 2; Mm00434616), Aqp4 (Mm00802131), Kir4.1 (as Kcnj10 Potassium inward rectifying channel, subfamily J, member 10; Mm00445028), Kcnv2 (potassium channel, subfamily V, member 2 (Mm00807577), and BCL2-associated X protein (Bax; Mm00432050), also known as BCL2, were used.
- Real-time PCR was performed using the StepOnePlus TM PCR system (Applied Biosystems, Thermo Fisher Scientific) and gene expression was quantified using the ⁇ CT method All mRNA levels were normalized to Gapdh.
- mice were dark adapted for at least 12 hours and then placed under dim red light before performing ERGs (7-8).
- Mice were injected intraperitoneally with a compound anesthetic [Midazolam 4 mg/kg body weight (Sand Japan Co., Ltd., Tokyo, Japan), medetomidine 0.75 mg/kg body weight (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), and butorphanol tartrate 5 mg/kg. kg body weight (Meiji Seika Pharma Co., Ltd., Tokyo, Japan)] and remained on a heating pad during the experiment.
- a compound anesthetic [Midazolam 4 mg/kg body weight (Sand Japan Co., Ltd., Tokyo, Japan), medetomidine 0.75 mg/kg body weight (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), and butorphanol tartrate 5 mg/kg. kg body weight (Meiji Seika Pharma Co., Ltd
- 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 performed using a PowerLab system 2/25 (AD Instruments, New South Wales, Australia).
- the full-field scotopic ERG measured responses to flash stimuli at stimulus intensities ranging from ⁇ 2.1 to 2.9 log cd s/m 2 . Photopic ERG was measured 10 minutes after light adaptation.
- HEK293 cells ATCC CRL-1573 were added to Dulbecco's modified Eagle's medium (#08456-65; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Life technologies, Carlsbad, CA, USA), and maintained with 100 units/ml penicillin and 100 ⁇ g/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA).
- Phenylbutyric acid (PBA) suppressed photoreceptor loss in P23H knock-in heterozygous mice (P23H RP model mice)> Previously, in P23H knock-in heterozygous mice, there was no change in the thickness of the photoreceptor layer until postnatal day 12, and the inner segment (IS) and outer segment (OS) lengths differed from wild-type (WT). It is reported that retinitis pigmentosa progresses gradually thereafter (Non-Patent Document 9). In this study, continuous administration of PBA was started from 2 weeks of age.
- sodium phenylbutyrate sodium 4-phenylbutyrate (the same applies to each example below); Sigma-Aldrich, 10 mg/kg body weight) is administered intraperitoneally every day from 2 weeks of age five times a week. gone.
- PBA was prepared in an amount of body weight (g) ⁇ 10 ⁇ L and administered in one dose for one day.
- the number of remaining photoreceptors (photoreceptor nuclei) in the retinas of P23H RP model mice administered with PBA was significantly higher than that of mice administered with vehicle ( 1A, B).
- PBA was prepared in an amount of body weight (g) ⁇ 10 ⁇ L and administered in one dose for one day.
- body weight g
- 10-week-old PBA-treated P23H RP model mice showed greater amplitude of a-waves, which reflects the function of rod photoreceptors, compared to vehicle-treated mice.
- B-wave amplitudes, which reflect function, were shown to be greater and latency to be shorter.
- continued PBA treatment was shown to preserve the visual function of the rod system.
- the latency of the b-wave which mainly indicates the function of cone photoreceptors, was shortened after PBA treatment. was done.
- ERAD is also activated by the conversion of IRE1-associated XBP1 to XBP1s, leading to the induction of VCP (also known as Cdc48 or p97) that interacts with Derlin 1, exploiting its ATPase activity to It has been previously shown that certain misfolded proteins are transported from the ER to the cytoplasm (Non-Patent Document 18), and that proteins are degraded via the ubiquitin proteasome system (UPS) ( Non-Patent Documents 16-17).
- UPS ubiquitin proteasome system
- Non-Patent Documents 18-19 (19-20), while the fusion-associated protein mitofusin protein has been shown to induce degradation via the UPS (18-19).
- This system maintains cellular homeostasis (Non-Patent Document 21) and has also been shown to maintain neuronal plasticity and survival (Non-Patent Document 22).
- intraperitoneal administration of PBA described in Examples 1 and 2 resulted in mitochondrial fission marker Fis1 (Fig. 3D) and autophagy marker LC3 (Fig. 3E).
- Non-Patent Documents 19, 23 mRNA levels of Pgc1- ⁇ , which is known to regulate mitochondrial biogenesis (Non-Patent Document 24), and Tfam (Non-Patent Document 25), a transcription factor that induces mitochondrial DNA encoding molecules involved in respiration. was shown to be increased by PBA as described in Examples 1 and 2 ( Figures 3H and I). These results indicated that PBA treatment activates mitochondrial biogenesis.
- Example 4 PBA activated oxidative phosphorylation (OXPHOS) in mitochondria in vitro> Furthermore, in order to analyze the voltage effect caused by PBA, a study using HEK293 cell line was performed. Cells were treated with 0-2.5 ⁇ M PBA 12 or 24 hours before each measurement. Mitochondrial membrane potential was determined by incubating cells with tetramethylrhodamine methyl ester (TMRE) (10 ⁇ M) at 37° C.
- TMRE tetramethylrhodamine methyl ester
- the luminescence signal was analyzed by Cytation 5 system (BioTek, Winooski, VT, USA).
- Cytation 5 system BioTek, Winooski, VT, USA.
- ATP ATP Bioluminescence Assay Kit CLSII (Sigma-Aldrich)
- the luminescence signal was analyzed using the Cytation 5 system (BioTek). was measured using As a result, PBA dose-dependently expressed Pgc1- ⁇ (FIG. 4A) and Tfam (FIG. 4B) in the HEK293 cell line.
- the mitochondrial membrane potential is essential for ATP synthesis in mitochondria, and BAM15, a mitochondrial protonophore uncoupler, counteracts this potential.
Abstract
It is found that phenylbutyric acid (PBA) can prevent progressive retinal photoreceptor cell death and can prevent the deterioration in a visual function. On the basis of these findings, a pharmaceutical composition for preventing retinal degeneration including retinitis pigmentosa can be provided.
Description
本発明は、網膜色素変性をはじめとする網膜変性を抑制するための医薬組成物に関する。
The present invention relates to a pharmaceutical composition for suppressing retinal degeneration including retinitis pigmentosa.
網膜色素変性は遺伝性の網膜疾患で、光の明暗を認識する視細胞である杆体視細胞が遺伝子変異により変性することで、初期症状として夜盲症や視野狭窄、視力低下などを呈し、最終的には失明をきたす恐れがある疾患である。
現時点では有効な治療法が無く、これまで臨床的には神経保護目的でビタミン剤を投与することがあったが、効果は乏しい。またこれまでにウイルスを用いた遺伝子導入治療に関する研究が進められており、世界的に臨床試験が行われている。また、薬物療法として視覚サイクルモジュレータ（Ｖｉｓｕａｌ Ｃｙｃｌｅ Ｍｏｄｕｌａｔｏｒ）の研究が行われているが（非特許文献１）、これは視機能を使わないようにして網膜を保護するものであり、視機能を維持しながら網膜を保護する治療が求められている。
網膜色素変性のほか、糖尿病網膜症、加齢黄斑変性、もしくはそれ以外の原因に伴う網膜変性に対する網膜を保護する治療法もなく、同様に治療法が求められている。 Retinitis pigmentosa is a hereditary retinal disease in which rod photoreceptors, which are photoreceptors that recognize the brightness of light, are degenerated due to genetic mutations. is a disease that can lead to blindness.
At present, there is no effective treatment, and clinically, vitamin preparations have been administered for the purpose of neuroprotection, but the effects are poor. In addition, research on gene transfer therapy using viruses has been advanced so far, and clinical trials are being conducted worldwide. In addition, visual cycle modulator (Visual Cycle Modulator) is being researched as drug therapy (Non-Patent Document 1), which protects the retina by not using visual function, and maintains visual function. There is a demand for a treatment that protects the retina while
There is no treatment to protect the retina against retinal degeneration associated with retinitis pigmentosa, diabetic retinopathy, age-related macular degeneration, or other causes, and treatments are similarly sought after.
現時点では有効な治療法が無く、これまで臨床的には神経保護目的でビタミン剤を投与することがあったが、効果は乏しい。またこれまでにウイルスを用いた遺伝子導入治療に関する研究が進められており、世界的に臨床試験が行われている。また、薬物療法として視覚サイクルモジュレータ（Ｖｉｓｕａｌ Ｃｙｃｌｅ Ｍｏｄｕｌａｔｏｒ）の研究が行われているが（非特許文献１）、これは視機能を使わないようにして網膜を保護するものであり、視機能を維持しながら網膜を保護する治療が求められている。
網膜色素変性のほか、糖尿病網膜症、加齢黄斑変性、もしくはそれ以外の原因に伴う網膜変性に対する網膜を保護する治療法もなく、同様に治療法が求められている。 Retinitis pigmentosa is a hereditary retinal disease in which rod photoreceptors, which are photoreceptors that recognize the brightness of light, are degenerated due to genetic mutations. is a disease that can lead to blindness.
At present, there is no effective treatment, and clinically, vitamin preparations have been administered for the purpose of neuroprotection, but the effects are poor. In addition, research on gene transfer therapy using viruses has been advanced so far, and clinical trials are being conducted worldwide. In addition, visual cycle modulator (Visual Cycle Modulator) is being researched as drug therapy (Non-Patent Document 1), which protects the retina by not using visual function, and maintains visual function. There is a demand for a treatment that protects the retina while
There is no treatment to protect the retina against retinal degeneration associated with retinitis pigmentosa, diabetic retinopathy, age-related macular degeneration, or other causes, and treatments are similarly sought after.
また尿素サイクル異常症の特効薬として既に使用されているフェニル酪酸（ｐｈｅｎｙｌｂｕｔｙｒｉｃ ａｃｉｄ（以下、ＰＢＡとも言う））が、角膜内皮における小胞体ストレスを抑制する作用があることがこれまでに示されている（特許文献１、２）。しかしながら、眼領域において、角膜内皮以外でのＰＢＡの作用については不明であり、特に網膜におけるＰＢＡの作用は不明であった。
In addition, phenylbutyric acid (hereinafter also referred to as PBA), which has already been 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, in the ocular region, the action of PBA outside the corneal endothelium was unclear, and in particular the action of PBA on the retina was unknown.
すなわち、本発明は、網膜色素変性をはじめとする網膜変性を抑制するための医薬組成物を提供することを課題とする。
That is, an object of the present invention is to provide a pharmaceutical composition for suppressing retinal degeneration including retinitis pigmentosa.
本発明者らは、上記課題を解決すべく検討を進め、ＰＢＡが進行性の網膜視細胞死を抑制すること、視機能低下を抑制することを見出し、本発明を完成させた。
The present inventors conducted studies to solve the above problems, found that PBA suppresses progressive retinal photoreceptor death and suppresses deterioration of visual function, and completed the present invention.
すなわち、この出願は、以下の発明を提供するものである。
［１］フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜変性抑制用医薬組成物。
［２］フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜変性疾患を治療又は予防するための、医薬組成物。
［３］前記網膜変性疾患が、網膜視細胞が変性した疾患である、［２］に記載の医薬組成物。
［４］前記網膜変性疾患が、網膜色素変性、糖尿病網膜症、加齢黄斑変性、及びＭＥＬＡＳ（ｍｉｔｏｃｈｏｎｄｒｉａｌ ｍｙｏｐａｔｈｙ，ｅｎｃｅｐｈａｌｏｐａｔｈｙ，ｌａｃｔｉｃ ａｃｉｄｏｓｉｓ，ａｎｄ ｓｔｒｏｋｅ－ｌｉｋｅ ｅｐｉｓｏｄｅｓ）に伴う網膜変性から選択される１つ以上の疾患である、［２］又は［３］に記載の医薬組成物。
［５］フェニル酪酸の塩が、ナトリウム塩である、［１］～［４］のいずれかに記載の医薬組成物。
［６］錠剤、顆粒剤、懸濁剤、注射剤又は点眼剤である、［１］～［５］のいずれかに記載の医薬組成物。
［７］フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜における小胞体ストレス関連分解を促進するための医薬組成物。
［８］フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜におけるミトコンドリア生合成を活性化するための及び／又は網膜におけるミトコンドリアの代謝機能を改善するための医薬組成物。
［９］対象において、網膜変性を抑制する方法であって、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を、該対象に投与することを含む、方法。
［１０］対象において、網膜変性を治療又は予防する方法であって、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を、該対象に投与することを含む、方法。
［１１］対象において、網膜における小胞体ストレス関連分解を促進する方法であって、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を、該対象に投与することを含む、方法。
［１２］対象において、網膜におけるミトコンドリア生合成を活性化する及び／又は網膜におけるミトコンドリアの代謝機能を改善する方法であって、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を、該対象に投与することを含む、方法。
［１３］網膜変性の抑制において使用するための、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体。
［１４］網膜変性の治療又は予防において使用するための、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体。
［１５］網膜における小胞体ストレス関連分解の促進において使用するための、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体。
［１６］網膜におけるミトコンドリア生合成の活性化及び／又は網膜におけるミトコンドリアの代謝機能の改善において使用するための、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体。
［１７］網膜変性の抑制において使用するための医薬の製造における、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体の使用。
［１８］網膜変性の治療又は予防において使用するための医薬の製造における、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体の使用。
［１９］網膜における小胞体ストレス関連分解の促進において使用するための医薬の製造における、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体の使用。
［２０］網膜におけるミトコンドリア生合成の活性化及び／又は網膜におけるミトコンドリアの代謝機能の改善において使用するための医薬の製造における、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体の使用。 That is, this application provides the following inventions.
[1] A pharmaceutical composition for suppressing retinal degeneration, containing phenylbutyric acid (PBA), a salt thereof, or a derivative thereof.
[2] A pharmaceutical composition for treating or preventing retinal degenerative diseases, containing phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
[3] The pharmaceutical composition of [2], wherein the retinal degenerative disease is a disease in which retinal photoreceptors are degenerated.
[4] The retinal degenerative disease is one or more 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) The pharmaceutical composition according to [2] or [3], which is a disease of
[5] The pharmaceutical composition according to any one of [1] to [4], wherein the salt of phenylbutyric acid is sodium salt.
[6] The pharmaceutical composition according to any one of [1] to [5], which is a tablet, granule, suspension, 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 biosynthesis in the retina and/or for improving metabolic function of mitochondria in the retina, comprising phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
[9] A method of inhibiting retinal degeneration in a subject, comprising administering phenylbutyric acid (PBA) or a salt thereof or a derivative thereof to the subject.
[10] A method of treating or preventing retinal degeneration in a subject, comprising administering phenylbutyric acid (PBA) or a salt or derivative thereof to the subject.
[11] A method of promoting endoplasmic reticulum stress-related degradation in the retina in a subject, comprising administering phenylbutyric acid (PBA) or a salt or derivative thereof to the subject.
[12] A method of activating mitochondrial biogenesis in the retina and/or improving metabolic function of mitochondria in the retina in a subject, comprising administering phenylbutyric acid (PBA) or a salt thereof or a derivative thereof to the subject A method comprising:
[13] Phenylbutyric acid (PBA) or a salt or derivative thereof for use in inhibiting retinal degeneration.
[14] Phenylbutyric acid (PBA) or a salt thereof or a derivative thereof for use in treating or preventing retinal degeneration.
[15] Phenylbutyric acid (PBA) or a salt or derivative thereof for use in promoting endoplasmic reticulum stress-related degradation in the retina.
[16] Phenylbutyric acid (PBA) or a salt thereof or a derivative thereof for use in activating mitochondrial biogenesis in the retina and/or improving metabolic function of mitochondria in the retina.
[17] Use of phenylbutyric acid (PBA) or a salt thereof or a derivative thereof in the manufacture of a medicament for use in inhibiting retinal degeneration.
[18] Use of phenylbutyric acid (PBA) or a salt thereof or a derivative thereof in the manufacture of a medicament for use in treating or preventing retinal degeneration.
[19] Use of phenylbutyric acid (PBA) or a salt thereof or a derivative thereof in the manufacture of a medicament for use in promoting endoplasmic reticulum stress-related degradation in the retina.
[20] Use of phenylbutyric acid (PBA) or a salt thereof or a derivative thereof in the manufacture of a medicament for use in activating mitochondrial biogenesis in the retina and/or improving metabolic function of mitochondria in the retina.
［１］フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜変性抑制用医薬組成物。
［２］フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜変性疾患を治療又は予防するための、医薬組成物。
［３］前記網膜変性疾患が、網膜視細胞が変性した疾患である、［２］に記載の医薬組成物。
［４］前記網膜変性疾患が、網膜色素変性、糖尿病網膜症、加齢黄斑変性、及びＭＥＬＡＳ（ｍｉｔｏｃｈｏｎｄｒｉａｌ ｍｙｏｐａｔｈｙ，ｅｎｃｅｐｈａｌｏｐａｔｈｙ，ｌａｃｔｉｃ ａｃｉｄｏｓｉｓ，ａｎｄ ｓｔｒｏｋｅ－ｌｉｋｅ ｅｐｉｓｏｄｅｓ）に伴う網膜変性から選択される１つ以上の疾患である、［２］又は［３］に記載の医薬組成物。
［５］フェニル酪酸の塩が、ナトリウム塩である、［１］～［４］のいずれかに記載の医薬組成物。
［６］錠剤、顆粒剤、懸濁剤、注射剤又は点眼剤である、［１］～［５］のいずれかに記載の医薬組成物。
［７］フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜における小胞体ストレス関連分解を促進するための医薬組成物。
［８］フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜におけるミトコンドリア生合成を活性化するための及び／又は網膜におけるミトコンドリアの代謝機能を改善するための医薬組成物。
［９］対象において、網膜変性を抑制する方法であって、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を、該対象に投与することを含む、方法。
［１０］対象において、網膜変性を治療又は予防する方法であって、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を、該対象に投与することを含む、方法。
［１１］対象において、網膜における小胞体ストレス関連分解を促進する方法であって、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を、該対象に投与することを含む、方法。
［１２］対象において、網膜におけるミトコンドリア生合成を活性化する及び／又は網膜におけるミトコンドリアの代謝機能を改善する方法であって、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を、該対象に投与することを含む、方法。
［１３］網膜変性の抑制において使用するための、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体。
［１４］網膜変性の治療又は予防において使用するための、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体。
［１５］網膜における小胞体ストレス関連分解の促進において使用するための、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体。
［１６］網膜におけるミトコンドリア生合成の活性化及び／又は網膜におけるミトコンドリアの代謝機能の改善において使用するための、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体。
［１７］網膜変性の抑制において使用するための医薬の製造における、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体の使用。
［１８］網膜変性の治療又は予防において使用するための医薬の製造における、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体の使用。
［１９］網膜における小胞体ストレス関連分解の促進において使用するための医薬の製造における、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体の使用。
［２０］網膜におけるミトコンドリア生合成の活性化及び／又は網膜におけるミトコンドリアの代謝機能の改善において使用するための医薬の製造における、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体の使用。 That is, this application provides the following inventions.
[1] A pharmaceutical composition for suppressing retinal degeneration, containing phenylbutyric acid (PBA), a salt thereof, or a derivative thereof.
[2] A pharmaceutical composition for treating or preventing retinal degenerative diseases, containing phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
[3] The pharmaceutical composition of [2], wherein the retinal degenerative disease is a disease in which retinal photoreceptors are degenerated.
[4] The retinal degenerative disease is one or more 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) The pharmaceutical composition according to [2] or [3], which is a disease of
[5] The pharmaceutical composition according to any one of [1] to [4], wherein the salt of phenylbutyric acid is sodium salt.
[6] The pharmaceutical composition according to any one of [1] to [5], which is a tablet, granule, suspension, 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 biosynthesis in the retina and/or for improving metabolic function of mitochondria in the retina, comprising phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
[9] A method of inhibiting retinal degeneration in a subject, comprising administering phenylbutyric acid (PBA) or a salt thereof or a derivative thereof to the subject.
[10] A method of treating or preventing retinal degeneration in a subject, comprising administering phenylbutyric acid (PBA) or a salt or derivative thereof to the subject.
[11] A method of promoting endoplasmic reticulum stress-related degradation in the retina in a subject, comprising administering phenylbutyric acid (PBA) or a salt or derivative thereof to the subject.
[12] A method of activating mitochondrial biogenesis in the retina and/or improving metabolic function of mitochondria in the retina in a subject, comprising administering phenylbutyric acid (PBA) or a salt thereof or a derivative thereof to the subject A method comprising:
[13] Phenylbutyric acid (PBA) or a salt or derivative thereof for use in inhibiting retinal degeneration.
[14] Phenylbutyric acid (PBA) or a salt thereof or a derivative thereof for use in treating or preventing retinal degeneration.
[15] Phenylbutyric acid (PBA) or a salt or derivative thereof for use in promoting endoplasmic reticulum stress-related degradation in the retina.
[16] Phenylbutyric acid (PBA) or a salt thereof or a derivative thereof for use in activating mitochondrial biogenesis in the retina and/or improving metabolic function of mitochondria in the retina.
[17] Use of phenylbutyric acid (PBA) or a salt thereof or a derivative thereof in the manufacture of a medicament for use in inhibiting retinal degeneration.
[18] Use of phenylbutyric acid (PBA) or a salt thereof or a derivative thereof in the manufacture of a medicament for use in treating or preventing retinal degeneration.
[19] Use of phenylbutyric acid (PBA) or a salt thereof or a derivative thereof in the manufacture of a medicament for use in promoting endoplasmic reticulum stress-related degradation in the retina.
[20] Use of phenylbutyric acid (PBA) or a salt thereof or a derivative thereof in the manufacture of a medicament for use in activating mitochondrial biogenesis in the retina and/or improving metabolic function of mitochondria in the retina.
本発明は、ＰＢＡが網膜色素変性モデルにおいて視機能維持を促進することを見出したことにより、網膜色素変性をはじめとする網膜変性を抑制するための剤を提供でき、これまで診断がついても有効な治療法が無かった網膜変性患者に対する新規医薬組成物を提供することができた。
The present invention has found that PBA promotes the maintenance of visual function in retinitis pigmentosa models, and can provide an agent for suppressing retinal degeneration including retinitis pigmentosa, which is effective even with a diagnosis. We were able to provide a novel pharmaceutical composition for patients with retinal degeneration for whom there was no effective treatment.
本発明の一実施形態は、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜変性抑制用医薬組成物又は網膜変性疾患を治療若しくは予防するための医薬組成物である。
One embodiment of the present invention is a pharmaceutical composition for suppressing retinal degeneration or a pharmaceutical composition for treating or preventing retinal degenerative diseases, containing phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
本実施形態において、フェニル酪酸は、本発明の効果を妨げない限り限定されず、４－フェニル酪酸、３－フェニル酪酸、２－フェニル酪酸、２－フェニルイソ酪酸、３－フェニルイソ酪酸等が挙げられるが、好ましくは４－フェニル酪酸である。試薬・工業原料等として入手することもできるし、常法に従って合成することもできる。
また、フェニル酪酸塩は、本発明の効果を妨げない限り限定されず、例えば、ナトリウム塩、カリウム塩、マグネシウム塩、カルシウム塩等が挙げられるが、好ましくはナトリウム塩である。 In the present embodiment, phenylbutyric acid is not limited as long as it does not interfere with the effects of the present invention, and includes 4-phenylbutyric acid, 3-phenylbutyric acid, 2-phenylbutyric acid, 2-phenylisobutyric acid, 3-phenylisobutyric acid and the like. , preferably 4-phenylbutyric acid. It can be obtained as a reagent, an industrial raw material, etc., and can also be synthesized according to a conventional method.
Phenylbutyric acid salts are not limited as long as they do not interfere with the effects of the present invention, and examples thereof include sodium salts, potassium salts, magnesium salts, calcium salts and the like, preferably sodium salts.
また、フェニル酪酸塩は、本発明の効果を妨げない限り限定されず、例えば、ナトリウム塩、カリウム塩、マグネシウム塩、カルシウム塩等が挙げられるが、好ましくはナトリウム塩である。 In the present embodiment, phenylbutyric acid is not limited as long as it does not interfere with the effects of the present invention, and includes 4-phenylbutyric acid, 3-phenylbutyric acid, 2-phenylbutyric acid, 2-phenylisobutyric acid, 3-phenylisobutyric acid and the like. , preferably 4-phenylbutyric acid. It can be obtained as a reagent, an industrial raw material, etc., and can also be synthesized according to a conventional method.
Phenylbutyric acid salts are not limited as long as they do not interfere with the effects of the present invention, and examples thereof include sodium salts, potassium salts, magnesium salts, calcium salts and the like, preferably sodium salts.
本実施形態において、フェニル酪酸若しくはフェニル酪酸塩の誘導体とは、本発明の効果を妨げない限り限定されず、例えば、フェニル基上の任意の水素が、任意の置換基（例えば、炭素数１～３のアルキル基等の炭素数３以下の飽和炭化水素基、炭素数３以下の不飽和炭化水素基、ハロゲンなど）で１または複数置換されたものであってもよく、フェニル酪酸又はフェニル酪酸塩の炭化水素鎖上の任意の水素が、任意の置換基（例えば、炭素数１～３のアルキル基等の炭素数３以下の飽和炭化水素基、炭素数３以下の不飽和炭化水素基、ハロゲンなど）で１または複数置換されたものであってもよい。また、フェニル酪酸塩の誘導体は、フェニル酪酸塩のエステルやエーテル等であってもよく、例えば、メチルエステル、エチルエステル、ｎ－プロピルエステル、イソプロピルエステル等であってもよい。
In the present 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. saturated hydrocarbon group with 3 or less carbon atoms such as an alkyl group of 3, unsaturated hydrocarbon group with 3 or less carbon atoms, halogen, etc.) may be substituted with one or more, phenylbutyric acid or phenylbutyrate. Any hydrogen on the hydrocarbon chain of is any substituent (e.g., a saturated hydrocarbon group having 3 or less carbon atoms such as an alkyl group having 1 to 3 carbon atoms, an unsaturated hydrocarbon group having 3 or less carbon atoms, a halogen etc.). Further, the derivative of phenylbutyrate may be an ester or ether of phenylbutyrate, such as methyl ester, ethyl ester, n-propyl ester, isopropyl ester, or the like.
本実施形態において、網膜変性とは、疾患については特に限定されず、網膜細胞の変性が生じた状態のことである。好ましくは網膜視細胞の変性が生じた状態のことである。網膜視細胞の変性は、杆体視細胞の変性又は錐体視細胞の変性のいずれでもよい。
In the present embodiment, retinal degeneration is not particularly limited to diseases, and refers to a state in which degeneration of retinal cells has occurred. Preferably, it is a state in which degeneration of retinal photoreceptors has occurred. Degeneration of retinal photoreceptors may be degeneration of rod photoreceptors or degeneration of cone photoreceptors.
本実施形態において、網膜変性が抑制されるとは、本実施形態の医薬組成物を投与することにより、例えば、変性の進行が阻害されることや、変性の進行が遅延されること、変性状態が改善されること、変性状態が完全に消失することなどが挙げられる。
In the present embodiment, inhibition of retinal degeneration means that administration of the pharmaceutical composition of the present embodiment, for example, inhibits the progress of degeneration, delays the progress of degeneration, degenerates is improved, and the denatured state is completely eliminated.
本実施形態の医薬組成物を投与することにより、網膜における変性が生じた異常タンパク質の分解が促進されることや、ミトコンドリア機能が改善若しくは向上すること、及び／又は網膜視細胞の保護機能が改善若しくは向上すること等によって、網膜変性が抑制される。
By administering the pharmaceutical composition of the present embodiment, the degradation of abnormal proteins caused by degeneration in the retina is promoted, the mitochondrial function is improved or enhanced, and/or the protective function of retinal photoreceptors is improved. Or, retinal degeneration is suppressed by being improved or the like.
網膜変性の抑制を判断する指標は特に限定されることはないが、例えば、本実施形態の医薬組成物投与後の検体において、本実施形態の医薬組成物を投与していない対照検体と比較して、網膜細胞の数、特に網膜視細胞の数が増加することにより網膜変性が抑制されたと判断してもよく、網膜電図にて振幅が増加すること及び／又は潜時が短くなることにより網膜変性が抑制されたと判断してもよい。統計学的に有意差があることが好ましいが、必ずしも有意差があることは必要とはしない。
The index for judging suppression of retinal degeneration is not particularly limited. It may be determined that retinal degeneration is suppressed by increasing the number of retinal cells, particularly the number of retinal photoreceptors, and by increasing the amplitude and / or shortening the latency in the electroretinogram You may judge that retinal degeneration was suppressed. A statistically significant difference is preferred, but not necessarily a significant difference.
本実施検体の治療対象となる網膜変性疾患は、網膜変性を引き起こす原因によらず、網膜細胞の変性が生じた疾患であれば特に限定されないが、網膜視細胞の変性が生じた疾患であることが好ましい。
具体的には、特に限定されないが、例えば、網膜色素変性、糖尿病網膜症、加齢黄斑変性、及びＭＥＬＡＳ（ｍｉｔｏｃｈｏｎｄｒｉａｌ ｍｙｏｐａｔｈｙ，ｅｎｃｅｐｈａｌｏｐａｔｈｙ，ｌａｃｔｉｃ ａｃｉｄｏｓｉｓ，ａｎｄ ｓｔｒｏｋｅ－ｌｉｋｅ ｅｐｉｓｏｄｅｓ）に伴う網膜変性から選択される１つ以上の疾患が挙げられる。 The retinal degenerative disease to be treated by the present sample is not particularly limited as long as it causes degeneration of retinal cells, regardless of the cause of retinal degeneration. is preferred.
Specifically, but not particularly limited, for example, retinitis pigmentosa, diabetic retinopathy, age-related macular degeneration, and retinal degeneration associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) One or more diseases are included.
具体的には、特に限定されないが、例えば、網膜色素変性、糖尿病網膜症、加齢黄斑変性、及びＭＥＬＡＳ（ｍｉｔｏｃｈｏｎｄｒｉａｌ ｍｙｏｐａｔｈｙ，ｅｎｃｅｐｈａｌｏｐａｔｈｙ，ｌａｃｔｉｃ ａｃｉｄｏｓｉｓ，ａｎｄ ｓｔｒｏｋｅ－ｌｉｋｅ ｅｐｉｓｏｄｅｓ）に伴う網膜変性から選択される１つ以上の疾患が挙げられる。 The retinal degenerative disease to be treated by the present sample is not particularly limited as long as it causes degeneration of retinal cells, regardless of the cause of retinal degeneration. is preferred.
Specifically, but not particularly limited, for example, retinitis pigmentosa, diabetic retinopathy, age-related macular degeneration, and retinal degeneration associated with MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes) One or more diseases are included.
本実施形態において、治療とは疾患の発生を予防することも含む。
In this embodiment, treatment also includes prevention of disease occurrence.
本実施形態において、医薬組成物中のフェニル酪酸若しくはその塩又はそれらの誘導体の含有量又はそれを含む医薬組成物の投与量若しくは投与方法は、該医薬組成物による治療効果が得られる限り特に限定されず、疾患の種類、疾患の程度、症状、患者の年齢、体重などによって適宜定めることができる。
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 particularly limited as long as the therapeutic effect of the pharmaceutical composition can be obtained. However, it can be appropriately determined according to the type of disease, degree of disease, symptoms, patient's age, body weight, and the like.
例えば、錠剤又は顆粒剤である場合、該医薬組成物中のフェニル酪酸若しくはその塩又はそれらの誘導体の含有量は、医薬組成物１ｇあたり、１０～１０００ｍｇであってよく、１００～１０００ｍｇであってよい。液剤の場合、該医薬組成物中のフェニル酪酸若しくはその塩又はそれらの誘導体の含有量は、例えば、０．００１μｇ／ｍＬ～１０００ｍｇ／ｍＬであってよく、０．００１μＭ～１０００ｍＭであってもよい。
For example, in the case of tablets or granules, the content of phenylbutyric acid or a salt thereof or a derivative thereof in the pharmaceutical composition may be 10 to 1000 mg, preferably 100 to 1000 mg, per 1 g of the pharmaceutical composition. good. In the case of a liquid formulation, 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, and may be 0.001 μM to 1000 mM. .
本実施形態の医薬組成物の剤型は、投与方法に応じて適宜選択できる。例えば、経口投与の場合は、散剤、顆粒剤、錠剤、カプセル剤等の固形製剤；溶液剤、シロップ剤、懸濁剤、乳剤等の液剤等に製剤化することができる。また、非経口投与の場合は、注射剤、液剤、懸濁剤等に製剤化することができる。
The dosage form of the pharmaceutical composition of this embodiment can be appropriately selected according to the administration method. For example, in the case of oral administration, solid preparations such as powders, granules, tablets and capsules; and liquid preparations such as solutions, syrups, suspensions and emulsions can be formulated. In the case of parenteral administration, it can be formulated into injections, liquids, suspensions, and the like.
投与経路は、治療効果が得られる限り特に限定されず、経口投与、眼投与、静脈内投与、局所投与などであってよい。例えば、錠剤、顆粒剤、又は懸濁剤などの剤型で経口投与してもよく、注射剤として眼内などに局所投与してもよく、点眼剤として眼投与してもよい。
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, topical administration, or the like. For example, it may be administered orally in dosage forms such as tablets, granules, or suspensions, may be administered topically to the eye as an injection, or may be administered to the eye as eye drops.
本実施形態の医薬組成物の投与量は、フェニル酪酸若しくはその塩又はそれらの誘導体に関しては、経口投与や静脈内投与等を行う場合は、例えば、体重６０ｋｇのヒトであれば、１日あたり０．００１～１００．０ｇ／ｍ２（体表面積）であってよく、０．０１～５０．０ｇ／ｍ２（体表面積）であってよく、０．１～３０．０ｇ／ｍ２（体表面積）であってよく、１．０～２０．０ｇ／ｍ２（体表面積）であってよく、９．９～１３．０ｇ／ｍ２（体表面積）であってもよく、０．１～１０．０ｇ／ｍ２（体表面積）、０．１～１．０ｇ／ｍ２（体表面積）であってもよい。上記投与量の医薬組成物は、１日１回で投与してもよく、１日数回に分けて投与してもよい。また、点眼剤として眼投与等を行う場合は、例えば、０．００１μｇ／ｍＬ～１０００ｍｇ／ｍＬまたは０．００１μＭ～１０００ｍＭの濃度の医薬組成物を片眼あたり１滴又は複数滴、１日１回又は複数回投与してもよい。
Regarding phenylbutyric acid, salts thereof, or derivatives thereof, the dosage of the pharmaceutical composition of the present embodiment is 0 per day for a human weighing 60 kg, for example, when oral administration, intravenous administration, or the like is performed. .001-100.0 g/m 2 (body surface area), 0.01-50.0 g/m 2 (body surface area), 0.1-30.0 g/m 2 (body surface area) ), may be 1.0 to 20.0 g/m 2 (body surface area), may be 9.9 to 13.0 g/m 2 (body surface area), may be 0.1 to 10 0 g/m 2 (body surface area), 0.1 to 1.0 g/m 2 (body surface area). The above dosage of the pharmaceutical composition may be administered once a day, or may be administered in several divided doses per day. In addition, when eye administration is performed 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 is applied once per eye, once a day. Alternatively, multiple doses may be administered.
本実施形態の医薬組成物は、本発明の効果を妨げない限り、追加の有効成分を含んでもよい。さらに、追加の疾患治療方法と組み合わせて使用してもよい。
The pharmaceutical composition of this embodiment may contain additional active ingredients as long as they do not interfere with the effects of the present invention. Additionally, it may be used in combination with additional disease treatment methods.
本実施形態の医薬組成物は、担体や添加剤が含まれていてもよい。ここで、担体としては、薬理的に許容される溶媒、希釈剤、賦形剤、結合剤などが挙げられ、液体状の医薬組成物の調製には例えば、水、生理食塩水、緩衝液などが用いられる。添加剤としては、安定剤、ｐＨ調整剤、増粘剤、抗酸化剤、等張化剤、緩衝剤、溶解補助剤、懸濁化剤、保存剤、凍害防止剤、凍結保護剤、凍結乾燥保護剤、制菌剤などが挙げられる。
The pharmaceutical composition of this embodiment may contain carriers and additives. Here, examples of carriers include pharmacologically acceptable solvents, diluents, excipients, binders, etc. For preparation of liquid pharmaceutical compositions, examples include water, physiological saline, buffers, etc. is used. Additives include stabilizers, pH adjusters, thickeners, antioxidants, tonicity agents, buffers, solubilizers, suspending agents, preservatives, antifreeze agents, cryoprotectants, and lyophilization. Protective agents, bacteriostatic agents and the like can be mentioned.
本発明の他の実施形態は、フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜における小胞体ストレス関連分解を促進するための医薬組成物である。
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 or derivative thereof.
網膜における小胞体ストレス関連分解を促進するとは、特に限定されないが、例えば、ＸＢＰ１ｓ（ＸＢＰ１ ｓｐｌｉｃｅｄ ｆｏｒｍ）、ＶＣＰ（ｖａｌｏｓｉｎ－ｃｏｎｔａｉｎｉｎｇ ｐｒｏｔｅｉｎ）、Ｄｅｒｌｉｎ１（ｄｅｇｒａｄａｔｉｏｎ ｉｎ ｅｎｄｏｐｌａｓｍｉｃ ｒｅｔｉｃｕｌｕｍ ｐｒｏｔｅｉｎ １）などの小胞体ストレス関連分解に関連する分子マーカーの遺伝子発現量又はタンパク質発現量が増強されることである。この分子マーカーの発現量増強により、網膜に発生した異常タンパク質の分解を促進することができる。特に小胞体ストレス関連分解の促進により、異常なＰ２３Ｈロドプシンに分解が促進されることにより、網膜変性を抑制することができる。
Promotion of endoplasmic reticulum stress-related degradation in the retina is not particularly limited, but for example, endoplasmic reticulum stress-related degradation of XBP1s (XBP1 spliced form), VCP (valosin-containing protein), Derlin1 (degradation in endoplasmic reticulum protein 1), etc. is that the gene expression level or protein expression level of a molecular marker associated with is enhanced. By enhancing the expression level of this molecular marker, it is possible to promote the degradation of abnormal proteins occurring in the retina. In particular, promotion of endoplasmic reticulum stress-related degradation promotes degradation of abnormal P23H rhodopsin, thereby suppressing retinal degeneration.
ここで各種分子マーカーの発現量増強とは、特に限定されないが、本実施形態の医薬組成物を投与した対象において、該医薬組成物を投与していない対照となる対象と比較して、該マーカーの遺伝子又はタンパク質発現量が増えたことを意味してもよい。
または、本実施形態の医薬組成物を投与した対象において、該マーカーの遺伝子又はタンパク質発現量が、予め設定されたカットオフ値を超えた場合に発現量が増強されたと判断してもよい。 Here, the enhancement of the expression level of various molecular markers is not particularly limited, but in a subject administered the pharmaceutical composition of the present embodiment, compared with a control subject not administered the pharmaceutical composition, the marker It may mean that the gene or protein expression level of is increased.
Alternatively, it may be determined that the expression level is enhanced when the gene or protein expression level of the marker exceeds a preset cutoff value in subjects administered with the pharmaceutical composition of the present embodiment.
または、本実施形態の医薬組成物を投与した対象において、該マーカーの遺伝子又はタンパク質発現量が、予め設定されたカットオフ値を超えた場合に発現量が増強されたと判断してもよい。 Here, the enhancement of the expression level of various molecular markers is not particularly limited, but in a subject administered the pharmaceutical composition of the present embodiment, compared with a control subject not administered the pharmaceutical composition, the marker It may mean that the gene or protein expression level of is increased.
Alternatively, it may be determined that the expression level is enhanced when the gene or protein expression level of the marker exceeds a preset cutoff value in subjects administered with the pharmaceutical composition of the present embodiment.
本実施形態における、フェニル酪酸の塩や誘導体の種類、医薬組成物中のフェニル酪酸若しくはその塩又はそれらの誘導体の含有量又は医薬組成物の投与量、投与方法、剤形、追加の成分などは特に限定されず、上述の網膜変性抑制用医薬組成物における記載を準用できる。
In the present embodiment, the type of phenylbutyric acid salt or derivative, the content of phenylbutyric acid or its salt or its derivative in the pharmaceutical composition, or the dosage, administration method, dosage form, additional ingredients, etc. of the pharmaceutical composition are It is not particularly limited, and the description of the pharmaceutical composition for suppressing retinal degeneration can be applied mutatis mutandis.
本発明の他の実施形態は、フェニル酪酸若しくはその塩又はそれらの誘導体を含む、網膜におけるミトコンドリア生合成を活性化するための及び／又は網膜におけるミトコンドリアの代謝機能を改善するための医薬組成物である。
Another embodiment of the present invention is a pharmaceutical composition for activating mitochondrial biogenesis in the retina and/or improving the metabolic function of mitochondria in the retina, comprising phenylbutyric acid or a salt thereof or a derivative thereof. be.
網膜におけるミトコンドリア生合成を活性化するとは、特に限定されないが、例えば、Ｆｉｓ１（Ｍｉｔｏｃｈｏｎｄｒｉａｌ ｆｉｓｓｉｏｎ １ ｐｒｏｔｅｉｎ）などのミトコンドリア分裂マーカーや、ＬＣ３（ｍｉｃｒｏｔｕｂｕｌｅ－ａｓｓｏｃｉａｔｅｄ ｐｒｏｔｅｉｎ ｌｉｇｈｔ ｃｈａｉｎ ３）などのオートファジーマーカー、若しくはＭｆｎ１（ｍｉｔｏｆｕｓｉｎ １）、Ｍｆｎ２（ｍｉｔｏｆｕｓｉｎ ２）などの融合マーカーの遺伝子発現量又はタンパク質発現量が増強されること、またはミトコンドリア生合成調節因子であるＰｇｃ１－α（ｐｅｒｏｘｉｓｏｍｅ ｐｒｏｌｉｆｅｒａｔｏｒｓ－ａｃｔｉｖａｔｅｄ ｒｅｃｅｐｔｏｒ－γ ｃｏ－ａｃｔｉｖａｔｏｒ－１α）若しくはミトコンドリア転写因子であるＴｆａｍ（ｍｉｔｏｃｈｏｎｄｒｉａｌ ｔｒａｎｓｃｒｉｐｔｉｏｎ ｆａｃｔｏｒ Ａ）などの遺伝子発現量又はタンパク質発現量が増強されることである。これらの分子の遺伝子発現量又はタンパク質発現量の増強により、網膜においてミトコンドリアの生合成を活性化させ、かつ損傷を受けたミトコンドリアのマイトファジーを促進することができる。
また、網膜におけるミトコンドリアの代謝機能を改善するとは、特に限定されないが、例えば、ミトコンドリア膜電位の増強や、シトクロムc酸化酵素ＩＶ（ＣＯＸ ＩＶ）の活性が増強されることである。
ミトコンドリア生合成が活性化され、ミトコンドリアの代謝機能が改善された結果、ミトコンドリアにおけるＡＴＰレベルが増加し、視細胞の保護を導くことができる。すなわち、本実施形態の医薬組成物を使用することにより、網膜変性を抑制することができる。 Activating mitochondrial biogenesis in the retina is not particularly limited, but for example, mitochondrial fission markers such as Fis1 (Mitochondrial fission 1 protein), autophagy markers such as LC3 (microtubule-associated protein light chain 3), or Mfn1 (mitofusin 1), Mfn2 (mitofusin 2), or the like, or Pgc1-α (peroxisome proliferators-activated receptor-γ co-activator), which is a regulator of mitochondrial biogenesis -1α) or Tfam (mitochondrial transcription factor A), which is a mitochondrial transcription factor, or the expression level of genes or proteins is enhanced. Enhanced gene or protein expression levels of these molecules can activate mitochondrial biogenesis in the retina and promote mitophagy of damaged mitochondria.
Improving the metabolic function of mitochondria in the retina is not particularly limited, but includes, for example, enhancement of mitochondrial membrane potential and enhancement of cytochrome c oxidase IV (COX IV) activity.
Activation of mitochondrial biogenesis and improved mitochondrial metabolic function can result in increased ATP levels in mitochondria, leading to protection of photoreceptors. That is, retinal degeneration can be suppressed by using the pharmaceutical composition of this embodiment.
また、網膜におけるミトコンドリアの代謝機能を改善するとは、特に限定されないが、例えば、ミトコンドリア膜電位の増強や、シトクロムc酸化酵素ＩＶ（ＣＯＸ ＩＶ）の活性が増強されることである。
ミトコンドリア生合成が活性化され、ミトコンドリアの代謝機能が改善された結果、ミトコンドリアにおけるＡＴＰレベルが増加し、視細胞の保護を導くことができる。すなわち、本実施形態の医薬組成物を使用することにより、網膜変性を抑制することができる。 Activating mitochondrial biogenesis in the retina is not particularly limited, but for example, mitochondrial fission markers such as Fis1 (
Improving the metabolic function of mitochondria in the retina is not particularly limited, but includes, for example, enhancement of mitochondrial membrane potential and enhancement of cytochrome c oxidase IV (COX IV) activity.
Activation of mitochondrial biogenesis and improved mitochondrial metabolic function can result in increased ATP levels in mitochondria, leading to protection of photoreceptors. That is, retinal degeneration can be suppressed by using the pharmaceutical composition of this embodiment.
ここで各種分子マーカーの発現量増強やミトコンドリア膜電位の増強、ＣＯＸ ＩＶの活性増強とは、特に限定されないが、本実施形態の医薬組成物を投与した対象において、該医薬組成物を投与していない対照となる対象と比較して、該マーカーの遺伝子又はタンパク質発現量が増えたことや、ミトコンドリア膜電位が増強したこと、ＣＯＸ ＩＶの活性が増強したことを意味してもよい。
または、本実施形態の医薬組成物を投与した対象において、該マーカーの遺伝子又はタンパク質発現量、ミトコンドリア膜電位の測定値、若しくはＣＯＸ ＩＶ活性の測定値が、予め設定されたカットオフ値を超えた場合に、該マーカーの遺伝子又はタンパク質発現量が増えた、ミトコンドリア膜電位が増強した、若しくはＣＯＸ ＩＶの活性が増強したと判断してもよい。 Here, enhancement of expression levels of various molecular markers, enhancement of mitochondrial membrane potential, and enhancement of COX IV activity are not particularly limited. It may mean increased gene or protein expression of the marker, enhanced mitochondrial membrane potential, or enhanced COX IV activity compared to a control subject without the marker.
Alternatively, in subjects administered the pharmaceutical composition of the present embodiment, the gene or protein expression level of the marker, the measured value of mitochondrial membrane potential, or the measured value of COX IV activity exceeds a preset cutoff value In some cases, it may be determined that the gene or protein expression level of the marker is increased, the mitochondrial membrane potential is enhanced, or the activity of COX IV is enhanced.
または、本実施形態の医薬組成物を投与した対象において、該マーカーの遺伝子又はタンパク質発現量、ミトコンドリア膜電位の測定値、若しくはＣＯＸ ＩＶ活性の測定値が、予め設定されたカットオフ値を超えた場合に、該マーカーの遺伝子又はタンパク質発現量が増えた、ミトコンドリア膜電位が増強した、若しくはＣＯＸ ＩＶの活性が増強したと判断してもよい。 Here, enhancement of expression levels of various molecular markers, enhancement of mitochondrial membrane potential, and enhancement of COX IV activity are not particularly limited. It may mean increased gene or protein expression of the marker, enhanced mitochondrial membrane potential, or enhanced COX IV activity compared to a control subject without the marker.
Alternatively, in subjects administered the pharmaceutical composition of the present embodiment, the gene or protein expression level of the marker, the measured value of mitochondrial membrane potential, or the measured value of COX IV activity exceeds a preset cutoff value In some cases, it may be determined that the gene or protein expression level of the marker is increased, the mitochondrial membrane potential is enhanced, or the activity of COX IV is enhanced.
本実施形態における、フェニル酪酸の塩や誘導体の種類、医薬組成物中のフェニル酪酸若しくはその塩又はそれらの誘導体の含有量、又は医薬組成物の投与量、投与方法、剤形、追加の成分などは特に限定されず、上述の網膜変性抑制用医薬組成物における記載を準用できる。
Kinds of phenylbutyric acid salts and derivatives, content of phenylbutyric acid or its salts or derivatives thereof in the pharmaceutical composition, dosage, administration method, dosage form, additional ingredients, etc. of the pharmaceutical composition in the present embodiment is not particularly limited, and the description of the pharmaceutical composition for suppressing retinal degeneration can be applied mutatis mutandis.
実施例は、開示する目的のために記載されており、本発明の範囲を制限する意図はない。
The examples are described for the purpose of disclosure and are not intended to limit the scope of the invention.
＜実験動物＞
当業者に既知の方法により得られたＰ２３Ｈノックインヘテロ接合型マウス（＋／－、２週齢、オス）を（非特許文献２）、慶應義塾大学医学部の動物実験施設で、温度管理された部屋（２２℃）で１２時間の明暗サイクル（午前８時から午後８時まで点灯）で、餌と水を自由に摂取できる環境下で飼育した。すべての動物実験は、ＡＲＶＯ Ｓｔａｔｅｍｅｎｔ ｆｏｒ ｔｈｅ Ｕｓｅ ｏｆ Ａｎｉｍａｌｓ ｉｎ Ｏｐｈｔｈａｌｍｉｃ ａｎｄ Ｖｉｓｉｏｎ Ｒｅｓｅａｒｃｈ及び慶應義塾動物実験委員会のガイドラインに準拠して実施した。 <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 placed in a temperature-controlled room at the animal experiment facility of Keio University School of Medicine. The mice were reared at (22° C.) with a 12-hour light-dark cycle (lights on from 8:00 am to 8:00 pm) under an environment in which food and water were available ad libitum. All animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Keio University Animal Care and Use Committee.
当業者に既知の方法により得られたＰ２３Ｈノックインヘテロ接合型マウス（＋／－、２週齢、オス）を（非特許文献２）、慶應義塾大学医学部の動物実験施設で、温度管理された部屋（２２℃）で１２時間の明暗サイクル（午前８時から午後８時まで点灯）で、餌と水を自由に摂取できる環境下で飼育した。すべての動物実験は、ＡＲＶＯ Ｓｔａｔｅｍｅｎｔ ｆｏｒ ｔｈｅ Ｕｓｅ ｏｆ Ａｎｉｍａｌｓ ｉｎ Ｏｐｈｔｈａｌｍｉｃ ａｎｄ Ｖｉｓｉｏｎ Ｒｅｓｅａｒｃｈ及び慶應義塾動物実験委員会のガイドラインに準拠して実施した。 <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 placed in a temperature-controlled room at the animal experiment facility of Keio University School of Medicine. The mice were reared at (22° C.) with a 12-hour light-dark cycle (lights on from 8:00 am to 8:00 pm) under an environment in which food and water were available ad libitum. All animal experiments were conducted in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines of the Keio University Animal Care and Use Committee.
＜組織学的解析＞
マウスの眼球を摘出し、４％パラホルムアルデヒド中で一晩、４℃で固定した。固定後、パラフィン包埋し（サクラファインテックジャパン、東京、日本）、視神経乳頭から網膜における最も離れた領域までを含む厚さ６～８μｍの切片を作製し、脱パラフィン化した後、ヘマトキシリンとエオジンで染色した。切片は、デジタルカメラ（オリンパス株式会社、東京、日本）を装着した顕微鏡で観察した。当業者に既知の方法により、視細胞層である外顆粒層（ｏｕｔｅｒ ｎｕｃｌｅａｒ ｌａｙｅｒ：ＯＮＬ）の厚さを、ＯＮＬの上部から下部までの距離を測定することによって評価し、杆体視細胞の外節（ｏｕｔｅｒ ｓｅｇｍｅｎｔ：ＯＳ）の長さを、後上部網膜において視神経から５００μｍのところで、ＩｍａｇｅＪソフトウェア（Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅｓ ｏｆ Ｈｅａｌｔｈ，Ｂｅｔｈｅｓｄａ，ＭＤ，ＵＳＡ；ａｖａｉｌａｂｌｅ ａｔ ｈｔｔｐ：／／ｒｓｂ．ｉｎｆｏ．ｎｉｈ．ｇｏｖ／ｉｊ／ｉｎｄｅｘ．ｈｔｍｌ）を用いて、ヘマトキシリンおよびエオジン染色を観察することによって決定し、平均化した（非特許文献２～６）。 <Histological analysis>
Mouse eyeballs were enucleated and fixed in 4% paraformaldehyde overnight at 4°C. After fixation, paraffin embedding (Sakura Finetech Japan, Tokyo, Japan), 6-8 μm thick sections from the optic nerve head to the most distant region of the retina were prepared, deparaffinized, followed by hematoxylin and eosin. stained with Sections were observed under a microscope equipped with a digital camera (Olympus Co., Tokyo, Japan). By 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 outer segment of the rod photoreceptor. (outer segment: OS) length was measured in the posterior superior retina at 500 μm from the optic nerve using ImageJ software (National Institutes of Health, Bethesda, MD, USA; available http://rsb.info.nih.gov/ij /index.html) were used and averaged by observing hematoxylin and eosin staining (2-6).
マウスの眼球を摘出し、４％パラホルムアルデヒド中で一晩、４℃で固定した。固定後、パラフィン包埋し（サクラファインテックジャパン、東京、日本）、視神経乳頭から網膜における最も離れた領域までを含む厚さ６～８μｍの切片を作製し、脱パラフィン化した後、ヘマトキシリンとエオジンで染色した。切片は、デジタルカメラ（オリンパス株式会社、東京、日本）を装着した顕微鏡で観察した。当業者に既知の方法により、視細胞層である外顆粒層（ｏｕｔｅｒ ｎｕｃｌｅａｒ ｌａｙｅｒ：ＯＮＬ）の厚さを、ＯＮＬの上部から下部までの距離を測定することによって評価し、杆体視細胞の外節（ｏｕｔｅｒ ｓｅｇｍｅｎｔ：ＯＳ）の長さを、後上部網膜において視神経から５００μｍのところで、ＩｍａｇｅＪソフトウェア（Ｎａｔｉｏｎａｌ Ｉｎｓｔｉｔｕｔｅｓ ｏｆ Ｈｅａｌｔｈ，Ｂｅｔｈｅｓｄａ，ＭＤ，ＵＳＡ；ａｖａｉｌａｂｌｅ ａｔ ｈｔｔｐ：／／ｒｓｂ．ｉｎｆｏ．ｎｉｈ．ｇｏｖ／ｉｊ／ｉｎｄｅｘ．ｈｔｍｌ）を用いて、ヘマトキシリンおよびエオジン染色を観察することによって決定し、平均化した（非特許文献２～６）。 <Histological analysis>
Mouse eyeballs were enucleated and fixed in 4% paraformaldehyde overnight at 4°C. After fixation, paraffin embedding (Sakura Finetech Japan, Tokyo, Japan), 6-8 μm thick sections from the optic nerve head to the most distant region of the retina were prepared, deparaffinized, followed by hematoxylin and eosin. stained with Sections were observed under a microscope equipped with a digital camera (Olympus Co., Tokyo, Japan). By 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 outer segment of the rod photoreceptor. (outer segment: OS) length was measured in the posterior superior retina at 500 μm from the optic nerve using ImageJ software (National Institutes of Health, Bethesda, MD, USA; available http://rsb.info.nih.gov/ij /index.html) were used and averaged by observing hematoxylin and eosin staining (2-6).
＜リアルタイム逆転写ポリメラーゼ連鎖反応（ＲＴ－ＰＣＲ）＞
ＴＲＩｚｏｌ試薬（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｃａｒｌｓｂａｄ，ＣＡ，ＵＳＡ）を用いて、生後４週齢のマウス網膜から全ＲＮＡを単離した。ＲＮＡ濃度をＮａｎｏＤｒｏｐ １０００（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）を用いて測定し、１μｇのＲＮＡをＳｕｐｅｒＳｃｒｉｐｔ ＶＩＬＯマスターミックス（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｃａｒｌｓｂａｄ，ＣＡ，ＵＳＡ）を用いて、製造者の指示に従って逆転写した。以下の配列のプライマーを使用した。
グリセルアルデヒド３－リン酸デヒドロゲナーゼ（Ｇａｐｄｈ）：フォワードプライマー５’－ＡＣＴＴＴＣＧＧＣＣＣＡＴＣＴＣＴＣＡ－３’（配列番号１）及びリバースプライマー５’－ＧＡＴＧＡＣＣＣＣＴＴＴＴＧＧＣＴＣＴＣＣＡＣ－３’（配列番号２）。
Ｐｇｃ１－α：フォワードプライマー５’－ＧＡＴＧＡＡＴＡＣＣＧＣＡＡＡＧＡＧＣＡ－３’（配列番号３）及びリバースプライマー５’－ＡＧＡＴＴＴＡＣＧＧＴＧＣＡＴＴＣＣＴ－３’（配列番号４）。
Ｆｉｓ１：フォワードプライマー５’－ＡＴＡＴＧＣＣＴＧＧＴＧＣＣＴＧＧＴＴＣ－３’（配列番号５）及びリバースプライマー５’－ＡＧＴＣＣＣＧＣＴＧＴＴＣＣＴＣＴＴＴＧ－３’（配列番号６）。
Ｍｆｎ１：フォワードプライマー５’－ＧＡＴＧＴＴＣＣＡＣＣＡＧＡＧＣＴＧＧＧＡ－３’（配列番号７）及びリバースプライマー５’－ＡＧＧＡＧＣＣＧＣＴＣＡＴＴＣＡＣＣＣＴＴＴＴＡ－３’（配列番号８）。
Ｍｆｎ２：フォワードプライマー５’－ＣＣＣＣＴＣＣＴＣＡＡＧＣＡＣＴＴＴＧＧＴＴＣ－３’（配列番号９）及びリバースプライマー５’－ＡＣＣＣＴＧＣＴＣＴＣＴＣＴＣＣＧＴＧＴＴＧＴＡＡＡＣ－３’（配列番号１０）。
Ｘｂｐ１ｓ：フォワードプライマー５’－ＣＴＧＡＧＴＣＣＧＣＡＧＣＡＧＧＴＧ－３’（配列番号１１）及びリバースプライマー５’－ＴＧＣＣＣＡＡＡＡＧＧＡＴＡＴＣＡＧＡＣＴ－３’（配列番号１２）。
ＶＣＰ：フォワードプライマー５’－ＡＡＧＴＣＣＣＣＡＧＴＴＧＣＣＡＡＧＧＡＴＧ－３’（配列番号１３）及びリバースプライマー５’－ＡＧＣＣＧＡＴＧＧＡＴＴＴＧＴＣＴＧＣＣＴＣ－３’（配列番号１４）。
Ｄｅｒｌ１：フォワードプライマー５’－ＣＧＣＧＡＴＴＴＡＡＧＧＣＣＴＧＴＴＡＣ－３’（配列番号１５）及びリバースプライマー５’－ＧＧＴＡＧＣＣＡＧＣＧＧＴＡＣＡＡＡＡＡ－３’（配列番号１６）。
Ｔｆａｍ：フォワードプライマー５’－ＡＧＴＣＡＧＣＴＧＡＴＧＧＧＴＡＴＧＧＡＧＡＡ－３’（配列番号１７）及びリバースプライマー５’－ＴＧＣＴＧＡＡＣＧＡＧＧＴＣＴＴＴＴＴＧＧ－３’（配列番号１８）。
ＬＣ３ｂ：Ｍｍ００７８２８６８（Ｔａｑｍａｎ）。
Ｔａｑｍａｎプローブ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ、Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）を、Ｋｉｒ２．１（Ｋｃｎｊ２としても知られる、カリウム内向き整流性チャネル、サブファミリーJ、メンバー２；Ｍｍ００４３４６１６）、Ａｑｐ４（Ｍｍ００８０２１３１）、Ｋｉｒ４．１（Ｋｃｎｊ１０としても知られる、カリウム内向き整流性チャネル、サブファミリーJ、メンバー１０；Ｍｍ００４４５０２８）、Ｋｃｎｖ２（カリウムチャネル、サブファミリーＶ、メンバー２（Ｍｍ００８０７５７７）、およびＢＣＬ２関連Ｘタンパク質（Ｂａｘ；Ｍｍ００４３２０５０）を用いた。リアルタイムＰＣＲをＳｔｅｐＯｎｅＰｌｕｓＴＭ ＰＣＲシステム（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ、Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）を用いて行い、ΔΔＣＴ法を用いて遺伝子発現を定量した。すべてのｍＲＮＡレベルはＧａｐｄｈに正規化した。 <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, Calif., USA). RNA concentration was measured using a NanoDrop 1000 (Thermo Fisher Scientific) and 1 μg of RNA was reverse transcribed using SuperScript VILO master mix (Life Technologies, Carlsbad, Calif., USA) according to the manufacturer's instructions. Primers with the following sequences 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′-GATGATACCGCAAAGAGCA-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'-ACCCTGCTCTCTCTCCGTGTTGTAAAC-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'-AGCCGATGGATTTGTCTGCCTC-3' (SEQ ID NO: 14).
Derl1:forward primer 5′-CGCGATTTAAGGCCTGTTAC-3′ (SEQ ID NO: 15) and reverse primer 5′-GGTAGCCAGCGGTACAAAAAA-3′ (SEQ ID NO: 16).
Tfam:forward primer 5′-AGTCAGCTGATGGGTATGGAGAA-3′ (SEQ ID NO: 17) and reverse primer 5′-TGCTGAACGAGGTCTTTTTG-3′ (SEQ ID NO: 18).
LC3b: Mm00782868 (Taqman).
Taqman probes (Applied Biosystems, Thermo Fisher Scientific) were combined with Kir2.1 (also known as Kcnj2, potassium inward rectifying channel, subfamily J,member 2; Mm00434616), Aqp4 (Mm00802131), Kir4.1 (as Kcnj10 Potassium inward rectifying channel, subfamily J, member 10; Mm00445028), Kcnv2 (potassium channel, subfamily V, member 2 (Mm00807577), and BCL2-associated X protein (Bax; Mm00432050), also known as BCL2, were used. Real-time PCR was performed using the StepOnePlus ™ PCR system (Applied Biosystems, Thermo Fisher Scientific) and gene expression was quantified using the ΔΔCT method All mRNA levels were normalized to Gapdh.
ＴＲＩｚｏｌ試薬（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｃａｒｌｓｂａｄ，ＣＡ，ＵＳＡ）を用いて、生後４週齢のマウス網膜から全ＲＮＡを単離した。ＲＮＡ濃度をＮａｎｏＤｒｏｐ １０００（Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）を用いて測定し、１μｇのＲＮＡをＳｕｐｅｒＳｃｒｉｐｔ ＶＩＬＯマスターミックス（Ｌｉｆｅ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｃａｒｌｓｂａｄ，ＣＡ，ＵＳＡ）を用いて、製造者の指示に従って逆転写した。以下の配列のプライマーを使用した。
グリセルアルデヒド３－リン酸デヒドロゲナーゼ（Ｇａｐｄｈ）：フォワードプライマー５’－ＡＣＴＴＴＣＧＧＣＣＣＡＴＣＴＣＴＣＡ－３’（配列番号１）及びリバースプライマー５’－ＧＡＴＧＡＣＣＣＣＴＴＴＴＧＧＣＴＣＴＣＣＡＣ－３’（配列番号２）。
Ｐｇｃ１－α：フォワードプライマー５’－ＧＡＴＧＡＡＴＡＣＣＧＣＡＡＡＧＡＧＣＡ－３’（配列番号３）及びリバースプライマー５’－ＡＧＡＴＴＴＡＣＧＧＴＧＣＡＴＴＣＣＴ－３’（配列番号４）。
Ｆｉｓ１：フォワードプライマー５’－ＡＴＡＴＧＣＣＴＧＧＴＧＣＣＴＧＧＴＴＣ－３’（配列番号５）及びリバースプライマー５’－ＡＧＴＣＣＣＧＣＴＧＴＴＣＣＴＣＴＴＴＧ－３’（配列番号６）。
Ｍｆｎ１：フォワードプライマー５’－ＧＡＴＧＴＴＣＣＡＣＣＡＧＡＧＣＴＧＧＧＡ－３’（配列番号７）及びリバースプライマー５’－ＡＧＧＡＧＣＣＧＣＴＣＡＴＴＣＡＣＣＣＴＴＴＴＡ－３’（配列番号８）。
Ｍｆｎ２：フォワードプライマー５’－ＣＣＣＣＴＣＣＴＣＡＡＧＣＡＣＴＴＴＧＧＴＴＣ－３’（配列番号９）及びリバースプライマー５’－ＡＣＣＣＴＧＣＴＣＴＣＴＣＴＣＣＧＴＧＴＴＧＴＡＡＡＣ－３’（配列番号１０）。
Ｘｂｐ１ｓ：フォワードプライマー５’－ＣＴＧＡＧＴＣＣＧＣＡＧＣＡＧＧＴＧ－３’（配列番号１１）及びリバースプライマー５’－ＴＧＣＣＣＡＡＡＡＧＧＡＴＡＴＣＡＧＡＣＴ－３’（配列番号１２）。
ＶＣＰ：フォワードプライマー５’－ＡＡＧＴＣＣＣＣＡＧＴＴＧＣＣＡＡＧＧＡＴＧ－３’（配列番号１３）及びリバースプライマー５’－ＡＧＣＣＧＡＴＧＧＡＴＴＴＧＴＣＴＧＣＣＴＣ－３’（配列番号１４）。
Ｄｅｒｌ１：フォワードプライマー５’－ＣＧＣＧＡＴＴＴＡＡＧＧＣＣＴＧＴＴＡＣ－３’（配列番号１５）及びリバースプライマー５’－ＧＧＴＡＧＣＣＡＧＣＧＧＴＡＣＡＡＡＡＡ－３’（配列番号１６）。
Ｔｆａｍ：フォワードプライマー５’－ＡＧＴＣＡＧＣＴＧＡＴＧＧＧＴＡＴＧＧＡＧＡＡ－３’（配列番号１７）及びリバースプライマー５’－ＴＧＣＴＧＡＡＣＧＡＧＧＴＣＴＴＴＴＴＧＧ－３’（配列番号１８）。
ＬＣ３ｂ：Ｍｍ００７８２８６８（Ｔａｑｍａｎ）。
Ｔａｑｍａｎプローブ（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ、Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）を、Ｋｉｒ２．１（Ｋｃｎｊ２としても知られる、カリウム内向き整流性チャネル、サブファミリーJ、メンバー２；Ｍｍ００４３４６１６）、Ａｑｐ４（Ｍｍ００８０２１３１）、Ｋｉｒ４．１（Ｋｃｎｊ１０としても知られる、カリウム内向き整流性チャネル、サブファミリーJ、メンバー１０；Ｍｍ００４４５０２８）、Ｋｃｎｖ２（カリウムチャネル、サブファミリーＶ、メンバー２（Ｍｍ００８０７５７７）、およびＢＣＬ２関連Ｘタンパク質（Ｂａｘ；Ｍｍ００４３２０５０）を用いた。リアルタイムＰＣＲをＳｔｅｐＯｎｅＰｌｕｓＴＭ ＰＣＲシステム（Ａｐｐｌｉｅｄ Ｂｉｏｓｙｓｔｅｍｓ、Ｔｈｅｒｍｏ Ｆｉｓｈｅｒ Ｓｃｉｅｎｔｉｆｉｃ）を用いて行い、ΔΔＣＴ法を用いて遺伝子発現を定量した。すべてのｍＲＮＡレベルはＧａｐｄｈに正規化した。 <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, Calif., USA). RNA concentration was measured using a NanoDrop 1000 (Thermo Fisher Scientific) and 1 μg of RNA was reverse transcribed using SuperScript VILO master mix (Life Technologies, Carlsbad, Calif., USA) according to the manufacturer's instructions. Primers with the following sequences were used.
Glyceraldehyde 3-phosphate dehydrogenase (Gapdh):
Pgc1-α:
Fis1:
Mfn1:
Mfn2: forward primer 5'-CCCCTCCTCAAGCACTTTGGTTC-3' (SEQ ID NO: 9) and reverse primer 5'-ACCCTGCTCTCTCTCCGTGTTGTAAAC-3' (SEQ ID NO: 10).
Xbp1s:
VCP: forward primer 5'-AAGTCCCCAGTTGCCAAGGATG-3' (SEQ ID NO: 13) and reverse primer 5'-AGCCGATGGATTTGTCTGCCTC-3' (SEQ ID NO: 14).
Derl1:
Tfam:
LC3b: Mm00782868 (Taqman).
Taqman probes (Applied Biosystems, Thermo Fisher Scientific) were combined with Kir2.1 (also known as Kcnj2, potassium inward rectifying channel, subfamily J,
＜網膜電図（ｅｌｅｃｔｒｏｒｅｔｉｎｏｇｒａｍ（ＥＲＧ））のレコーディング＞
当業者に既知の方法に基づき、マウスを少なくとも１２時間暗順応させ、その後、ＥＲＧを実施する前に暗赤色灯下に置いた（非特許文献７～８）。マウスを腹腔内複合麻酔薬［ミダゾラム４ｍｇ／ｋｇ体重（サンドジャパン株式会社、東京、日本）、メデトミジン０．７５ｍｇ／ｋｇ体重（日本全薬工業株式会社、福島、日本）、及びブトルファノール酒石酸塩５ｍｇ／ｋｇ体重（Ｍｅｉｊｉ Ｓｅｉｋａ ファルマ株式会社、東京、日本）］で麻酔し、実験中は加熱パッドの上に置いたままにした。トロピカミドとフェニルフリンの混合物（各０．５％；Ｍｙｄｒｉｎ－Ｐ（登録商標）；参天製薬、大阪、日本）を一滴使用して、マウスの瞳孔を拡張させた。活性金線電極を角膜上に配置し、設置電極と基準電極をそれぞれ尾部上と口内に配置した。
網膜電図のレコーディングは、ＰｏｗｅｒＬａｂシステム２／２５（ＡＤインスツルメンツ、ニューサウスウェールズ、オーストラリア）を用いて行った。全視野暗順応ＥＲＧは、－２．１から２．９ｌｏｇ ｃｄ ｓ／ｍ２の範囲の刺激強度でのフラッシュ刺激への応答を測定した。明順応ＥＲＧは光適応１０分後に測定した。０．４から１．４ ｌｏｇ ｃｄ ｓ／ｍ２の範囲のフラッシュ刺激への応答を、３０ｃｄ ｓ／ｍ２のバックグラウンドにて測定した。応答は差動増幅され、０．３から１０００Ｈｚの範囲のデジタルバンドパスフィルタを介してフィルタリングされた。各刺激は、市販の刺激装置（Ｇａｎｚｆｅｌｄ Ｓｙｓｔｅｍ ＳＧ－２００２；ＬＫＣ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｉｎｃ．）を用いて行った。a波の振幅は、ベースラインからトラフまでを測定し、b波の振幅は、a波のトラフからb波のピークまでを測定した。a波とb波の潜時は、それぞれ刺激の開始から各波のピークまでを測定した。ピークポイントはシステムによって自動的に算出され、試験者がその算出結果を確認した。 <Recording of electroretinogram (ERG)>
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 performing ERGs (7-8). Mice were injected intraperitoneally with a compound anesthetic [Midazolam 4 mg/kg body weight (Sand Japan Co., Ltd., Tokyo, Japan), medetomidine 0.75 mg/kg body weight (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), andbutorphanol tartrate 5 mg/kg. kg body weight (Meiji Seika Pharma Co., Ltd., Tokyo, Japan)] and remained on a heating pad during the experiment. A drop of a mixture of tropicamide and phenylfurine (0.5% each; Mydrin-P®; Santen Pharmaceutical, Osaka, Japan) was used to dilate the pupils of mice. 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 performed using aPowerLab system 2/25 (AD Instruments, New South Wales, Australia). The full-field scotopic ERG measured responses to flash stimuli at stimulus intensities ranging from −2.1 to 2.9 log cd s/m 2 . Photopic ERG was measured 10 minutes after light adaptation. Responses to flash stimuli ranging from 0.4 to 1.4 log cd s/ m2 were measured in a background of 30 cd s/ m2 . Responses were differentially amplified and filtered through a digital bandpass filter ranging from 0.3 to 1000 Hz. Each stimulation was performed 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 a-wave trough to b-wave peak. The a-wave and b-wave latencies were measured from the onset of stimulation to the peak of each wave, respectively. The peak point was automatically calculated by the system, and the tester confirmed the calculation result.
当業者に既知の方法に基づき、マウスを少なくとも１２時間暗順応させ、その後、ＥＲＧを実施する前に暗赤色灯下に置いた（非特許文献７～８）。マウスを腹腔内複合麻酔薬［ミダゾラム４ｍｇ／ｋｇ体重（サンドジャパン株式会社、東京、日本）、メデトミジン０．７５ｍｇ／ｋｇ体重（日本全薬工業株式会社、福島、日本）、及びブトルファノール酒石酸塩５ｍｇ／ｋｇ体重（Ｍｅｉｊｉ Ｓｅｉｋａ ファルマ株式会社、東京、日本）］で麻酔し、実験中は加熱パッドの上に置いたままにした。トロピカミドとフェニルフリンの混合物（各０．５％；Ｍｙｄｒｉｎ－Ｐ（登録商標）；参天製薬、大阪、日本）を一滴使用して、マウスの瞳孔を拡張させた。活性金線電極を角膜上に配置し、設置電極と基準電極をそれぞれ尾部上と口内に配置した。
網膜電図のレコーディングは、ＰｏｗｅｒＬａｂシステム２／２５（ＡＤインスツルメンツ、ニューサウスウェールズ、オーストラリア）を用いて行った。全視野暗順応ＥＲＧは、－２．１から２．９ｌｏｇ ｃｄ ｓ／ｍ２の範囲の刺激強度でのフラッシュ刺激への応答を測定した。明順応ＥＲＧは光適応１０分後に測定した。０．４から１．４ ｌｏｇ ｃｄ ｓ／ｍ２の範囲のフラッシュ刺激への応答を、３０ｃｄ ｓ／ｍ２のバックグラウンドにて測定した。応答は差動増幅され、０．３から１０００Ｈｚの範囲のデジタルバンドパスフィルタを介してフィルタリングされた。各刺激は、市販の刺激装置（Ｇａｎｚｆｅｌｄ Ｓｙｓｔｅｍ ＳＧ－２００２；ＬＫＣ Ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｉｎｃ．）を用いて行った。a波の振幅は、ベースラインからトラフまでを測定し、b波の振幅は、a波のトラフからb波のピークまでを測定した。a波とb波の潜時は、それぞれ刺激の開始から各波のピークまでを測定した。ピークポイントはシステムによって自動的に算出され、試験者がその算出結果を確認した。 <Recording of electroretinogram (ERG)>
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 performing ERGs (7-8). Mice were injected intraperitoneally with a compound anesthetic [Midazolam 4 mg/kg body weight (Sand Japan Co., Ltd., Tokyo, Japan), medetomidine 0.75 mg/kg body weight (Nippon Zenyaku Kogyo Co., Ltd., Fukushima, Japan), and
Electroretinogram recordings were performed using a
＜細胞培養＞
ＨＥＫ２９３細胞（ＡＴＣＣ ＣＲＬ－１５７３）を、１０％ウシ胎児血清（Ｌｉｆｅ ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｃａｒｌｓｂａｄ，ＣＡ，ＵＳＡ）を添加したＤｕｌｂｅｃｃｏ’ｓ ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ ｍｅｄｉｕｍ（＃０８４５６－６５；ナカライテスク、京都、日本）、及び１００ｕｎｉｔ／ｍｌペニシリン及び１００μｇ／ｍｌストレプトマイシン（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ，Ｓｔ．Ｌｏｕｉｓ，ＭＯ，ＵＳＡ）で維持した。 <Cell culture>
HEK293 cells (ATCC CRL-1573) were added to Dulbecco's modified Eagle's medium (#08456-65; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Life technologies, Carlsbad, CA, USA), and maintained with 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA).
ＨＥＫ２９３細胞（ＡＴＣＣ ＣＲＬ－１５７３）を、１０％ウシ胎児血清（Ｌｉｆｅ ｔｅｃｈｎｏｌｏｇｉｅｓ，Ｃａｒｌｓｂａｄ，ＣＡ，ＵＳＡ）を添加したＤｕｌｂｅｃｃｏ’ｓ ｍｏｄｉｆｉｅｄ Ｅａｇｌｅ’ｓ ｍｅｄｉｕｍ（＃０８４５６－６５；ナカライテスク、京都、日本）、及び１００ｕｎｉｔ／ｍｌペニシリン及び１００μｇ／ｍｌストレプトマイシン（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ，Ｓｔ．Ｌｏｕｉｓ，ＭＯ，ＵＳＡ）で維持した。 <Cell culture>
HEK293 cells (ATCC CRL-1573) were added to Dulbecco's modified Eagle's medium (#08456-65; Nacalai Tesque, Kyoto, Japan) supplemented with 10% fetal bovine serum (Life technologies, Carlsbad, CA, USA), and maintained with 100 units/ml penicillin and 100 μg/ml streptomycin (Sigma-Aldrich, St. Louis, MO, USA).
＜統計解析＞
すべての結果は、平均±標準偏差として表される。その結果値は、３群間の比較についてはＴｕｋｅｙのポストホック検定を用いた一方向ＡＮＯＶＡにより解析し、２群間の比較についてはＳＰＳＳ Ｓｔａｔｉｓｔｉｃｓ ２４（ＩＢＭ，Ａｒｍｏｎｋ，ＮＹ，ＵＳＡ）ソフトウェアを用いたスチューデントの両側t検定により解析した。Ｐ＜０．０５で統計的に有意差があると判断した。 <Statistical analysis>
All results are expressed as mean ± standard deviation. The resulting values were analyzed by one-way ANOVA with Tukey's post-hoc test for comparison between three groups, and SPSS Statistics 24 (IBM, Armonk, NY, USA) software for comparison between two groups. Analyzed by Student's two-tailed t-test. Differences were considered statistically significant at P<0.05.
すべての結果は、平均±標準偏差として表される。その結果値は、３群間の比較についてはＴｕｋｅｙのポストホック検定を用いた一方向ＡＮＯＶＡにより解析し、２群間の比較についてはＳＰＳＳ Ｓｔａｔｉｓｔｉｃｓ ２４（ＩＢＭ，Ａｒｍｏｎｋ，ＮＹ，ＵＳＡ）ソフトウェアを用いたスチューデントの両側t検定により解析した。Ｐ＜０．０５で統計的に有意差があると判断した。 <Statistical analysis>
All results are expressed as mean ± standard deviation. The resulting values were analyzed by one-way ANOVA with Tukey's post-hoc test for comparison between three groups, and SPSS Statistics 24 (IBM, Armonk, NY, USA) software for comparison between two groups. Analyzed by Student's two-tailed t-test. Differences were considered statistically significant at P<0.05.
＜実施例１ フェニル酪酸（ＰＢＡ）がＰ２３Ｈノックインヘテロ接合型マウス（Ｐ２３Ｈ ＲＰモデルマウス）における視細胞喪失を抑制した＞
これまでに、Ｐ２３Ｈノックインヘテロ接合型マウスでは、生後１２日目までは視細胞層の厚さに変化はなく、内節（ＩＳ）と外節（ＯＳ）の長さは野生型（ＷＴ）と同程度であり、その後、徐々に網膜色素変性が進行することが報告されている（非特許文献９）。
本検討において、生後２週齢からＰＢＡの連続投与を開始した。連続投与は、フェニル酪酸ナトリウム（４－フェニル酪酸ナトリウム（以下の各実施例でも同様）；Ｓｉｇｍａ－Ａｌｄｒｉｃｈ、１０ｍｇ／ｋｇ体重）を、生後２週齢から週５回、毎日腹腔内投与することで行った。なお、腹腔内投与において、ＰＢＡは、体重（ｇ）×１０μLの量に調製して１日分の投与量を１回で投与した。その結果、ＰＢＡを投与したＰ２３Ｈ ＲＰモデルマウスの網膜では、１０週齢になると、残存する視細胞数（視細胞核数）がビヒクルを投与したマウスよりも有意に多くなったことが示された（図１Ａ、Ｂ）。この結果から、Ｐ２３Ｈ ＲＰモデルマウスにおいて、ＰＢＡ投与によって視細胞の生存が促進され、網膜変性が抑制されることが示された。 <Example 1 Phenylbutyric acid (PBA) suppressed photoreceptor loss in P23H knock-in heterozygous mice (P23H RP model mice)>
Previously, in P23H knock-in heterozygous mice, there was no change in the thickness of the photoreceptor layer until postnatal day 12, and the inner segment (IS) and outer segment (OS) lengths differed from wild-type (WT). It is reported that retinitis pigmentosa progresses gradually thereafter (Non-Patent Document 9).
In this study, continuous administration of PBA was started from 2 weeks of age. For continuous administration, sodium phenylbutyrate (sodium 4-phenylbutyrate (the same applies to each example below); Sigma-Aldrich, 10 mg/kg body weight) is administered intraperitoneally every day from 2 weeks of age five times a week. gone. In the intraperitoneal administration, PBA was prepared in an amount of body weight (g)×10 μL and administered in one dose for one day. As a result, at 10 weeks of age, the number of remaining photoreceptors (photoreceptor nuclei) in the retinas of P23H RP model mice administered with PBA was significantly higher than that of mice administered with vehicle ( 1A, B). These results indicate that PBA administration promotes photoreceptor cell survival and suppresses retinal degeneration in P23H RP model mice.
これまでに、Ｐ２３Ｈノックインヘテロ接合型マウスでは、生後１２日目までは視細胞層の厚さに変化はなく、内節（ＩＳ）と外節（ＯＳ）の長さは野生型（ＷＴ）と同程度であり、その後、徐々に網膜色素変性が進行することが報告されている（非特許文献９）。
本検討において、生後２週齢からＰＢＡの連続投与を開始した。連続投与は、フェニル酪酸ナトリウム（４－フェニル酪酸ナトリウム（以下の各実施例でも同様）；Ｓｉｇｍａ－Ａｌｄｒｉｃｈ、１０ｍｇ／ｋｇ体重）を、生後２週齢から週５回、毎日腹腔内投与することで行った。なお、腹腔内投与において、ＰＢＡは、体重（ｇ）×１０μLの量に調製して１日分の投与量を１回で投与した。その結果、ＰＢＡを投与したＰ２３Ｈ ＲＰモデルマウスの網膜では、１０週齢になると、残存する視細胞数（視細胞核数）がビヒクルを投与したマウスよりも有意に多くなったことが示された（図１Ａ、Ｂ）。この結果から、Ｐ２３Ｈ ＲＰモデルマウスにおいて、ＰＢＡ投与によって視細胞の生存が促進され、網膜変性が抑制されることが示された。 <Example 1 Phenylbutyric acid (PBA) suppressed photoreceptor loss in P23H knock-in heterozygous mice (P23H RP model mice)>
Previously, in P23H knock-in heterozygous mice, there was no change in the thickness of the photoreceptor layer until postnatal day 12, and the inner segment (IS) and outer segment (OS) lengths differed from wild-type (WT). It is reported that retinitis pigmentosa progresses gradually thereafter (Non-Patent Document 9).
In this study, continuous administration of PBA was started from 2 weeks of age. For continuous administration, sodium phenylbutyrate (sodium 4-phenylbutyrate (the same applies to each example below); Sigma-Aldrich, 10 mg/kg body weight) is administered intraperitoneally every day from 2 weeks of age five times a week. gone. In the intraperitoneal administration, PBA was prepared in an amount of body weight (g)×10 μL and administered in one dose for one day. As a result, at 10 weeks of age, the number of remaining photoreceptors (photoreceptor nuclei) in the retinas of P23H RP model mice administered with PBA was significantly higher than that of mice administered with vehicle ( 1A, B). These results indicate that PBA administration promotes photoreceptor cell survival and suppresses retinal degeneration in P23H RP model mice.
＜実施例２ ＰＢＡはＰ２３Ｈ ＲＰモデルマウスの視覚機能を回復させた＞
これまでに、Ｐ２３Ｈ ＲＰモデルマウスでは、６週齢で暗順応ＥＲＧによる杆体視細胞機能の障害が認められ、１０週齢で明順応ＥＲＧによる錐体視細胞機能の障害が認められ、いずれも徐々に進行することが報告されている（非特許文献２）。
そのため、ＰＢＡ治療による組織学的な改善が、より良い視覚機能の保持に寄与しているかどうかを調べるために、暗順応ＥＲＧ（図２Ａ－Ｅ）と明順応ＥＲＧ（図２Ｆ－Ｈ）を測定した。Ｐ２３Ｈ ＲＰモデルマウスに、ＰＢＡ（１０ｍｇ／ｋｇ体重）を、生後２週齢から週５回、毎日腹腔内投与することで行った。なお、腹腔内投与において、ＰＢＡは、体重（ｇ）×１０μLの量に調製して１日分の投与量を１回で投与した。その結果、暗順応ＥＲＧにおいて、１０週齢のＰＢＡ処理したＰ２３Ｈ ＲＰモデルマウスではビヒクル処理したマウスと比較して、杆体視細胞の機能を反映するa波の振幅が大きくなり、さらにその後の網膜神経機能を反映するb波の振幅が大きくなり、潜時が短くなることが示された。つまり、ＰＢＡ処理の継続によって、杆体系の視覚機能が保護されることが示された。また、明順応ＥＲＧにおいて、主に錐体視細胞の機能を示すb波の潜時がＰＢＡ処理後に短くなり、Ｐ２３Ｈ ＲＰモデルマウスではＰＢＡ処理によって、錐体系の視覚機能も保護されることが示された。 <Example 2 PBA restored the visual function of P23H RP model mice>
So far, in P23H RP model mice, impairment of rod photoreceptor function by dark-adapted ERG was observed at 6 weeks of age, and impairment of cone photoreceptor function by light-adapted ERG was observed at 10 weeks of age. has been reported to progress to (Non-Patent Document 2).
Therefore, we measured dark-adapted ERGs (Fig. 2A-E) and light-adapted ERGs (Figs. 2F-H) to investigate whether the histological improvement with PBA treatment contributed to the preservation of better visual function. did. PBA (10 mg/kg body weight) was intraperitoneally administered to P23HRP model mice 5 times a week every day from 2 weeks of age. In the intraperitoneal administration, PBA was prepared in an amount of body weight (g)×10 μL and administered in one dose for one day. As a result, in the dark-adapted ERG, 10-week-old PBA-treated P23H RP model mice showed greater amplitude of a-waves, which reflects the function of rod photoreceptors, compared to vehicle-treated mice. B-wave amplitudes, which reflect function, were shown to be greater and latency to be shorter. In other words, continued PBA treatment was shown to preserve the visual function of the rod system. In addition, in the light-adapted ERG, the latency of the b-wave, which mainly indicates the function of cone photoreceptors, was shortened after PBA treatment. was done.
これまでに、Ｐ２３Ｈ ＲＰモデルマウスでは、６週齢で暗順応ＥＲＧによる杆体視細胞機能の障害が認められ、１０週齢で明順応ＥＲＧによる錐体視細胞機能の障害が認められ、いずれも徐々に進行することが報告されている（非特許文献２）。
そのため、ＰＢＡ治療による組織学的な改善が、より良い視覚機能の保持に寄与しているかどうかを調べるために、暗順応ＥＲＧ（図２Ａ－Ｅ）と明順応ＥＲＧ（図２Ｆ－Ｈ）を測定した。Ｐ２３Ｈ ＲＰモデルマウスに、ＰＢＡ（１０ｍｇ／ｋｇ体重）を、生後２週齢から週５回、毎日腹腔内投与することで行った。なお、腹腔内投与において、ＰＢＡは、体重（ｇ）×１０μLの量に調製して１日分の投与量を１回で投与した。その結果、暗順応ＥＲＧにおいて、１０週齢のＰＢＡ処理したＰ２３Ｈ ＲＰモデルマウスではビヒクル処理したマウスと比較して、杆体視細胞の機能を反映するa波の振幅が大きくなり、さらにその後の網膜神経機能を反映するb波の振幅が大きくなり、潜時が短くなることが示された。つまり、ＰＢＡ処理の継続によって、杆体系の視覚機能が保護されることが示された。また、明順応ＥＲＧにおいて、主に錐体視細胞の機能を示すb波の潜時がＰＢＡ処理後に短くなり、Ｐ２３Ｈ ＲＰモデルマウスではＰＢＡ処理によって、錐体系の視覚機能も保護されることが示された。 <Example 2 PBA restored the visual function of P23H RP model mice>
So far, in P23H RP model mice, impairment of rod photoreceptor function by dark-adapted ERG was observed at 6 weeks of age, and impairment of cone photoreceptor function by light-adapted ERG was observed at 10 weeks of age. has been reported to progress to (Non-Patent Document 2).
Therefore, we measured dark-adapted ERGs (Fig. 2A-E) and light-adapted ERGs (Figs. 2F-H) to investigate whether the histological improvement with PBA treatment contributed to the preservation of better visual function. did. PBA (10 mg/kg body weight) was intraperitoneally administered to P23H
＜実施例３ ＰＢＡはＰ２３Ｈ ＲＰモデルマウスの網膜における小胞体ストレス関連分解（ＥＲＡＤ）及びミトコンドリアマーカーの発現上昇を誘導した＞
これまでにＰ２３Ｈ変異ロドプシンがＥＲストレスを引き起こす一方（非特許文献９～１５）、異常タンパク質を分解するＥＲＡＤシステム（非特許文献１６～１７）がＰ２３Ｈ ＲＰモデルマウスで誘導されることも明らかになっている（非特許文献９）。また、ＥＲＡＤはＩＲＥ１に関連したＸＢＰ１のＸＢＰ１ｓへの変換によって活性化され、Ｄｅｒｌｉｎ １と相互作用するＶＣＰ（Ｃｄｃ４８またはｐ９７としても知られている）の誘導につながり、そのＡＴＰａｓｅ活性を利用して、特定のミスフォールドされたタンパク質がＥＲから細胞質へと輸送されること（非特許文献１８）、さらにユビキチンプロテアソームシステム（ＵＰＳ）を介してタンパク質が分解されることがこれまでに明らかになっている（非特許文献１６～１７）。
Ｐ２３Ｈ ＲＰモデルマウスの網膜における小胞体ストレス関連分解に関する検討を行った結果、実施例１及び２に記載するＰＢＡの腹腔内投与により、ＸＢＰ１ｓ（図３Ａ）、ＶＣＰ（図３Ｂ）、Ｄｅｒｌｉｎ１（図３Ｃ）の発現が亢進していることが示された。これらの結果から、ＰＢＡはＥＲＡＤの誘導を促進し、異常なＰ２３Ｈロドプシンを除去することが示唆された。 <Example 3 PBA induced increased expression of endoplasmic reticulum stress-associated degradation (ERAD) and mitochondrial markers in the retina of P23H RP model mice>
So far, while P23H mutant rhodopsin causes ER stress (Non-Patent Documents 9-15), it has also been revealed that the ERAD system (Non-Patent Documents 16-17) that degrades abnormal proteins is induced in P23H RP model mice. (Non-Patent Document 9). ERAD is also activated by the conversion of IRE1-associated XBP1 to XBP1s, leading to the induction of VCP (also known as Cdc48 or p97) that interacts withDerlin 1, exploiting its ATPase activity to It has been previously shown that certain misfolded proteins are transported from the ER to the cytoplasm (Non-Patent Document 18), and that proteins are degraded via the ubiquitin proteasome system (UPS) ( Non-Patent Documents 16-17).
As a result of examining the endoplasmic reticulum stress-related degradation in the retina of P23H RP model mice, intraperitoneal administration of PBA described in Examples 1 and 2 reduced XBP1s (Fig. 3A), VCP (Fig. 3B), and Derlin1 (Fig. 3C). ) was shown to be upregulated. These results suggested that PBA promotes the induction of ERAD and removes abnormal P23H rhodopsin.
これまでにＰ２３Ｈ変異ロドプシンがＥＲストレスを引き起こす一方（非特許文献９～１５）、異常タンパク質を分解するＥＲＡＤシステム（非特許文献１６～１７）がＰ２３Ｈ ＲＰモデルマウスで誘導されることも明らかになっている（非特許文献９）。また、ＥＲＡＤはＩＲＥ１に関連したＸＢＰ１のＸＢＰ１ｓへの変換によって活性化され、Ｄｅｒｌｉｎ １と相互作用するＶＣＰ（Ｃｄｃ４８またはｐ９７としても知られている）の誘導につながり、そのＡＴＰａｓｅ活性を利用して、特定のミスフォールドされたタンパク質がＥＲから細胞質へと輸送されること（非特許文献１８）、さらにユビキチンプロテアソームシステム（ＵＰＳ）を介してタンパク質が分解されることがこれまでに明らかになっている（非特許文献１６～１７）。
Ｐ２３Ｈ ＲＰモデルマウスの網膜における小胞体ストレス関連分解に関する検討を行った結果、実施例１及び２に記載するＰＢＡの腹腔内投与により、ＸＢＰ１ｓ（図３Ａ）、ＶＣＰ（図３Ｂ）、Ｄｅｒｌｉｎ１（図３Ｃ）の発現が亢進していることが示された。これらの結果から、ＰＢＡはＥＲＡＤの誘導を促進し、異常なＰ２３Ｈロドプシンを除去することが示唆された。 <Example 3 PBA induced increased expression of endoplasmic reticulum stress-associated degradation (ERAD) and mitochondrial markers in the retina of P23H RP model mice>
So far, while P23H mutant rhodopsin causes ER stress (Non-Patent Documents 9-15), it has also been revealed that the ERAD system (Non-Patent Documents 16-17) that degrades abnormal proteins is induced in P23H RP model mice. (Non-Patent Document 9). ERAD is also activated by the conversion of IRE1-associated XBP1 to XBP1s, leading to the induction of VCP (also known as Cdc48 or p97) that interacts with
As a result of examining the endoplasmic reticulum stress-related degradation in the retina of P23H RP model mice, intraperitoneal administration of PBA described in Examples 1 and 2 reduced XBP1s (Fig. 3A), VCP (Fig. 3B), and Derlin1 (Fig. 3C). ) was shown to be upregulated. These results suggested that PBA promotes the induction of ERAD and removes abnormal P23H rhodopsin.
これまでにＥＲストレスはＶＣＰによるミトコンドリアの分裂及び融合を制御することでミトコンドリアの品質管理にも影響を与えること（非特許文献１８～１９）、ＶＣＰはオートファジーにより異常なミトコンドリアを排除するために分裂を誘導すること（非特許文献１９～２０）、一方で融合関連タンパク質であるミトフシンタンパク質はＵＰＳを介して分解を誘導することが明らかになっている（非特許文献１８～１９）。このシステムは細胞の恒常性を維持し（非特許文献２１）、神経の可塑性と生存を維持することも明らかになっている（非特許文献２２）。
Ｐ２３Ｈ ＲＰモデルマウスの網膜におけるミトコンドリアの分裂及び融合に関する検討を行った結果、実施例１及び２に記載するＰＢＡの腹腔内投与により、ミトコンドリア分裂マーカーＦｉｓ１（図３Ｄ）とオートファジーマーカーＬＣ３（図３Ｅ）のｍＲＮＡレベルが上昇することが示された。また、融合マーカーであるＭｆｎ１（図３Ｆ）とＭｆｎ２（図３Ｇ）のｍＲＮＡレベルもＰＢＡによって上昇することが示された。これらの結果から、ＶＣＰがミトフシンタンパク質の分解を誘導することが示唆された。 So far, ER stress also affects mitochondrial quality control by controlling mitochondrial fission and fusion by VCP (Non-Patent Documents 18-19). (19-20), while the fusion-associated protein mitofusin protein has been shown to induce degradation via the UPS (18-19). This system maintains cellular homeostasis (Non-Patent Document 21) and has also been shown to maintain neuronal plasticity and survival (Non-Patent Document 22).
As a result of studying mitochondrial fission and fusion in the retina of P23H RP model mice, intraperitoneal administration of PBA described in Examples 1 and 2 resulted in mitochondrial fission marker Fis1 (Fig. 3D) and autophagy marker LC3 (Fig. 3E). ) was shown to increase mRNA levels. It was also shown that the mRNA levels of fusion markers Mfn1 (Fig. 3F) and Mfn2 (Fig. 3G) were also elevated by PBA. These results suggested that VCP induces degradation of mitofusin protein.
Ｐ２３Ｈ ＲＰモデルマウスの網膜におけるミトコンドリアの分裂及び融合に関する検討を行った結果、実施例１及び２に記載するＰＢＡの腹腔内投与により、ミトコンドリア分裂マーカーＦｉｓ１（図３Ｄ）とオートファジーマーカーＬＣ３（図３Ｅ）のｍＲＮＡレベルが上昇することが示された。また、融合マーカーであるＭｆｎ１（図３Ｆ）とＭｆｎ２（図３Ｇ）のｍＲＮＡレベルもＰＢＡによって上昇することが示された。これらの結果から、ＶＣＰがミトフシンタンパク質の分解を誘導することが示唆された。 So far, ER stress also affects mitochondrial quality control by controlling mitochondrial fission and fusion by VCP (Non-Patent Documents 18-19). (19-20), while the fusion-associated protein mitofusin protein has been shown to induce degradation via the UPS (18-19). This system maintains cellular homeostasis (Non-Patent Document 21) and has also been shown to maintain neuronal plasticity and survival (Non-Patent Document 22).
As a result of studying mitochondrial fission and fusion in the retina of P23H RP model mice, intraperitoneal administration of PBA described in Examples 1 and 2 resulted in mitochondrial fission marker Fis1 (Fig. 3D) and autophagy marker LC3 (Fig. 3E). ) was shown to increase mRNA levels. It was also shown that the mRNA levels of fusion markers Mfn1 (Fig. 3F) and Mfn2 (Fig. 3G) were also elevated by PBA. These results suggested that VCP induces degradation of mitofusin protein.
ミトコンドリアの品質管理の間、損傷したミトコンドリアを新しい健康なミトコンドリアに置き換えるミトコンドリア生合成が行われる（非特許文献１９、２３）。ミトコンドリア生合成を調節することが知られているＰｇｃ１－αと（非特許文献２４）、呼吸に関連する分子をコードするミトコンドリアＤＮＡを誘導する転写因子であるＴｆａｍ（非特許文献２５）のｍＲＮＡレベルが実施例１及び２に記載するＰＢＡによって上昇することが示された（図３Ｈ及びＩ）。これらの結果から、ＰＢＡ処理により、ミトコンドリア生合成が活性化することが示された。
During mitochondrial quality control, mitochondrial biogenesis occurs to replace damaged mitochondria with new, healthy mitochondria (Non-Patent Documents 19, 23). mRNA levels of Pgc1-α, which is known to regulate mitochondrial biogenesis (Non-Patent Document 24), and Tfam (Non-Patent Document 25), a transcription factor that induces mitochondrial DNA encoding molecules involved in respiration. was shown to be increased by PBA as described in Examples 1 and 2 (Figures 3H and I). These results indicated that PBA treatment activates mitochondrial biogenesis.
＜実施例４ ＰＢＡはミトコンドリアにおける酸化的リン酸化（ＯＸＰＨＯＳ）をｉｎ ｖｉｔｒｏで活性化した＞
さらにＰＢＡにより生じる電位効果を解析するために、ＨＥＫ２９３細胞株を用いた検討を行った。各測定の１２又は２４時間前に、細胞を０～２．５μＭのＰＢＡで処理した。ミトコンドリア膜電位は、細胞をテトラメチルローダミンメチルエステル（ＴＭＲＥ）（１０μＭ）で、３７℃、３０分間インキュベートし、ＢＡＭ１５（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）投与前後の平均輝度を、共焦点顕微鏡（ＴＣＳ－ＳＰ５；ライカ、東京、日本）を用いて測定し、次の計算式により算出した：膜電位＝ＢＡＭ１５添加前の平均輝度１４秒間(７枚×２秒おき)／ＢＡＭ１５添加直後の平均輝度１４秒間(７枚×２秒おき)。シトクロムc酸化酵素（ＣＯＸ ＩＶ）活性の測定は、保存用液体窒素で細胞を瞬間凍結した後、キットに付属の溶解バッファーに入れてから、製造業者の指示に従い、Ｃｏｍｐｌｅｘ ＩＶ Ｒｏｄｅｎｔ Ｅｎｚｙｍｅ Ａｃｔｉｖｉｔｙ Ｍｉｃｒｏｐｌａｔｅ Ａｓｓａｙ Ｋｉｔ（Ａｂｃａｍ）を用いて行った。発光シグナルを、Ｃｙｔａｔｉｏｎ
５システム（ＢｉｏＴｅｋ，Ｗｉｎｏｏｓｋｉ，ＶＴ，ＵＳＡ）を用いて測定した。ＡＴＰ測定のために、瞬間凍結サンプルは、ＡＴＰ Ｂｉｏｌｕｍｉｎｅｓｃｅｎｃｅ Ａｓｓａｙ Ｋｉｔ ＣＬＳＩＩ（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）を用いてＡＴＰ含有量を測定する前に、溶解緩衝液に入れ、発光シグナルを、Ｃｙｔａｔｉｏｎ ５システム（ＢｉｏＴｅｋ）を用いて測定した。
この結果、ＨＥＫ２９３細胞株において、ＰＢＡはＰｇｃ１－α（図４Ａ）とＴｆａｍ（図４Ｂ）を用量依存的に発現させた。ミトコンドリアの膜電位はミトコンドリアにおけるＡＴＰ合成に不可欠であり、ミトコンドリアのプロトノフォア脱共役剤であるＢＡＭ１５はこの電位を打ち消す。ＢＡＭ１５投与前後の電位の減算法を用いて、ＰＢＡで処理した細胞では膜電位が上昇した（図４Ｃ、Ｄ）。さらに、ＰＢＡによって、シトクロムc酸化酵素ＩＶ（ＣＯＸ ＩＶ）の活性が増加し（図４Ｅ）、その結果、ＡＴＰレベルが増加したことが示された（図４Ｆ）。これらの結果から、ＰＢＡがミトコンドリアの機能を増加させて、細胞保護に利用できるＡＴＰレベルを獲得できることが示され、これにより病態改善につながることが示唆された（非特許文献３、２６～２７）。 <Example 4 PBA activated oxidative phosphorylation (OXPHOS) in mitochondria in vitro>
Furthermore, in order to analyze the voltage effect caused by PBA, a study using HEK293 cell line was performed. Cells were treated with 0-2.5 μM PBA 12 or 24 hours before each measurement. Mitochondrial membrane potential was determined by incubating cells with tetramethylrhodamine methyl ester (TMRE) (10 μM) at 37° C. for 30 minutes, and measuring the average brightness before and after administration of BAM15 (Sigma-Aldrich) using a confocal microscope (TCS-SP5; Leica , Tokyo, Japan), and calculated by the following formula: Membrane potential = average luminance for 14 seconds before addition of BAM15 (7 sheets x 2 seconds) / average luminance for 14 seconds immediately after addition of BAM15 (7 sheets) × every 2 seconds). Cytochrome c oxidase (COX IV) activity was measured by flash freezing cells in liquid nitrogen for storage, placing them in the lysis buffer provided with the kit, and then using the Complex IV Rodent Enzyme Activity Microplate Assay Kit according to the manufacturer's instructions. (Abcam). The luminescence signal was analyzed by Cytation
5 system (BioTek, Winooski, VT, USA). For ATP measurements, snap-frozen samples were placed in lysis buffer before ATP content was determined using the ATP Bioluminescence Assay Kit CLSII (Sigma-Aldrich), and the luminescence signal was analyzed using theCytation 5 system (BioTek). was measured using
As a result, PBA dose-dependently expressed Pgc1-α (FIG. 4A) and Tfam (FIG. 4B) in the HEK293 cell line. The mitochondrial membrane potential is essential for ATP synthesis in mitochondria, and BAM15, a mitochondrial protonophore uncoupler, counteracts this potential. Using the subtraction method of potentials before and after administration of BAM15, membrane potential was elevated in cells treated with PBA (Fig. 4C,D). Furthermore, PBA was shown to increase the activity of cytochrome c oxidase IV (COX IV) (Fig. 4E), resulting in increased ATP levels (Fig. 4F). These results indicate that PBA can increase mitochondrial function and acquire ATP levels that can be used for cell protection, suggesting that this leads to improvement of pathological conditions (Non-Patent Documents 3, 26-27). .
さらにＰＢＡにより生じる電位効果を解析するために、ＨＥＫ２９３細胞株を用いた検討を行った。各測定の１２又は２４時間前に、細胞を０～２．５μＭのＰＢＡで処理した。ミトコンドリア膜電位は、細胞をテトラメチルローダミンメチルエステル（ＴＭＲＥ）（１０μＭ）で、３７℃、３０分間インキュベートし、ＢＡＭ１５（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）投与前後の平均輝度を、共焦点顕微鏡（ＴＣＳ－ＳＰ５；ライカ、東京、日本）を用いて測定し、次の計算式により算出した：膜電位＝ＢＡＭ１５添加前の平均輝度１４秒間(７枚×２秒おき)／ＢＡＭ１５添加直後の平均輝度１４秒間(７枚×２秒おき)。シトクロムc酸化酵素（ＣＯＸ ＩＶ）活性の測定は、保存用液体窒素で細胞を瞬間凍結した後、キットに付属の溶解バッファーに入れてから、製造業者の指示に従い、Ｃｏｍｐｌｅｘ ＩＶ Ｒｏｄｅｎｔ Ｅｎｚｙｍｅ Ａｃｔｉｖｉｔｙ Ｍｉｃｒｏｐｌａｔｅ Ａｓｓａｙ Ｋｉｔ（Ａｂｃａｍ）を用いて行った。発光シグナルを、Ｃｙｔａｔｉｏｎ
５システム（ＢｉｏＴｅｋ，Ｗｉｎｏｏｓｋｉ，ＶＴ，ＵＳＡ）を用いて測定した。ＡＴＰ測定のために、瞬間凍結サンプルは、ＡＴＰ Ｂｉｏｌｕｍｉｎｅｓｃｅｎｃｅ Ａｓｓａｙ Ｋｉｔ ＣＬＳＩＩ（Ｓｉｇｍａ－Ａｌｄｒｉｃｈ）を用いてＡＴＰ含有量を測定する前に、溶解緩衝液に入れ、発光シグナルを、Ｃｙｔａｔｉｏｎ ５システム（ＢｉｏＴｅｋ）を用いて測定した。
この結果、ＨＥＫ２９３細胞株において、ＰＢＡはＰｇｃ１－α（図４Ａ）とＴｆａｍ（図４Ｂ）を用量依存的に発現させた。ミトコンドリアの膜電位はミトコンドリアにおけるＡＴＰ合成に不可欠であり、ミトコンドリアのプロトノフォア脱共役剤であるＢＡＭ１５はこの電位を打ち消す。ＢＡＭ１５投与前後の電位の減算法を用いて、ＰＢＡで処理した細胞では膜電位が上昇した（図４Ｃ、Ｄ）。さらに、ＰＢＡによって、シトクロムc酸化酵素ＩＶ（ＣＯＸ ＩＶ）の活性が増加し（図４Ｅ）、その結果、ＡＴＰレベルが増加したことが示された（図４Ｆ）。これらの結果から、ＰＢＡがミトコンドリアの機能を増加させて、細胞保護に利用できるＡＴＰレベルを獲得できることが示され、これにより病態改善につながることが示唆された（非特許文献３、２６～２７）。 <Example 4 PBA activated oxidative phosphorylation (OXPHOS) in mitochondria in vitro>
Furthermore, in order to analyze the voltage effect caused by PBA, a study using HEK293 cell line was performed. Cells were treated with 0-2.5 μM PBA 12 or 24 hours before each measurement. Mitochondrial membrane potential was determined by incubating cells with tetramethylrhodamine methyl ester (TMRE) (10 μM) at 37° C. for 30 minutes, and measuring the average brightness before and after administration of BAM15 (Sigma-Aldrich) using a confocal microscope (TCS-SP5; Leica , Tokyo, Japan), and calculated by the following formula: Membrane potential = average luminance for 14 seconds before addition of BAM15 (7 sheets x 2 seconds) / average luminance for 14 seconds immediately after addition of BAM15 (7 sheets) × every 2 seconds). Cytochrome c oxidase (COX IV) activity was measured by flash freezing cells in liquid nitrogen for storage, placing them in the lysis buffer provided with the kit, and then using the Complex IV Rodent Enzyme Activity Microplate Assay Kit according to the manufacturer's instructions. (Abcam). The luminescence signal was analyzed by Cytation
5 system (BioTek, Winooski, VT, USA). For ATP measurements, snap-frozen samples were placed in lysis buffer before ATP content was determined using the ATP Bioluminescence Assay Kit CLSII (Sigma-Aldrich), and the luminescence signal was analyzed using the
As a result, PBA dose-dependently expressed Pgc1-α (FIG. 4A) and Tfam (FIG. 4B) in the HEK293 cell line. The mitochondrial membrane potential is essential for ATP synthesis in mitochondria, and BAM15, a mitochondrial protonophore uncoupler, counteracts this potential. Using the subtraction method of potentials before and after administration of BAM15, membrane potential was elevated in cells treated with PBA (Fig. 4C,D). Furthermore, PBA was shown to increase the activity of cytochrome c oxidase IV (COX IV) (Fig. 4E), resulting in increased ATP levels (Fig. 4F). These results indicate that PBA can increase mitochondrial function and acquire ATP levels that can be used for cell protection, suggesting that this leads to improvement of pathological conditions (
＜実施例５ ＰＢＡの経口投与により、Ｐ２３Ｈ ＲＰモデルマウスのミトコンドリアマーカーの発現上昇を誘導した＞
ＰＢＡの経口投与による、Ｐ２３Ｈ ＲＰモデルマウスの網膜におけるミトコンドリア生合成関連因子の発現への影響に関する検討を行った。Ｐ２３Ｈ ＲＰモデルマウスに、ＰＢＡ（２００ｍｇ／ｋｇ体重）を、生後２週齢から生後４週齢まで、１４日間毎日経口投与することで行った。なお、経口投与において、ＰＢＡは、体重（ｇ）×２０μLの量に調製して１日分の投与量を２回に分けて投与した。すなわち、ＰＢＡを２００ｍｇ／ｋｇ体重で投与する場合、例えば体重が２０ｇのマウスあたりＰＢＡを１日４ｍｇ投与することが必要であるため、１００ｍｇ／１０ｍＬ（ＰＢＳ）の濃度となるように調製したＰＢＡの懸濁液を１日４００μＬ（２００μＬ×２回）投与した。
この結果、ＰＢＡを投与した群（ＰＢＡ２００ｍｇ／ｋｇ）と対照群（ＰＢＳを投与した群）とでは、投与初日から投与最終日に至るまで、体重に大きな差は生じず、また体重減少は見られなかった（図５）。その一方で、網膜内ｍＲＮＡ発現をリアルタイムＰＣＲで確認したところ、ＰＢＡを投与した群は、対照群と比較して、ミトコンドリアマーカーであるｐｇｃ１－α及びｔｆａｍの発現が上昇した（図６）。このことはＰＢＡの腹腔内投与のときと同様の結果を示すものである。 <Example 5 Oral administration of PBA induced increased expression of mitochondrial markers in P23H RP model mice>
We investigated the effect of oral administration of PBA on the expression of mitochondrial biosynthesis-related factors in the retina of P23H RP model mice. PBA (200 mg/kg body weight) was orally administered to P23H RP model mice every day for 14 days from 2 weeks old to 4 weeks old. In oral administration, PBA was prepared in an amount of body weight (g)×20 μL and administered in two doses for one day. That is, when PBA is administered at 200 mg/kg body weight, for example, it is necessary to administer 4 mg of PBA per day per mouse weighing 20 g. 400 μL (200 μL×2 times) of the suspension was administered per day.
As a result, between the PBA-administered group (PBA 200 mg/kg) and the control group (PBS-administered group), there was no significant difference in body weight from the first day of administration to the last day of administration, and no weight loss was observed. No (Fig. 5). On the other hand, when retinal mRNA expression was confirmed by real-time PCR, the PBA-administered group showed increased expression of mitochondrial markers pgc1-α and tfam compared to the control group (FIG. 6). This shows similar results to the intraperitoneal administration of PBA.
ＰＢＡの経口投与による、Ｐ２３Ｈ ＲＰモデルマウスの網膜におけるミトコンドリア生合成関連因子の発現への影響に関する検討を行った。Ｐ２３Ｈ ＲＰモデルマウスに、ＰＢＡ（２００ｍｇ／ｋｇ体重）を、生後２週齢から生後４週齢まで、１４日間毎日経口投与することで行った。なお、経口投与において、ＰＢＡは、体重（ｇ）×２０μLの量に調製して１日分の投与量を２回に分けて投与した。すなわち、ＰＢＡを２００ｍｇ／ｋｇ体重で投与する場合、例えば体重が２０ｇのマウスあたりＰＢＡを１日４ｍｇ投与することが必要であるため、１００ｍｇ／１０ｍＬ（ＰＢＳ）の濃度となるように調製したＰＢＡの懸濁液を１日４００μＬ（２００μＬ×２回）投与した。
この結果、ＰＢＡを投与した群（ＰＢＡ２００ｍｇ／ｋｇ）と対照群（ＰＢＳを投与した群）とでは、投与初日から投与最終日に至るまで、体重に大きな差は生じず、また体重減少は見られなかった（図５）。その一方で、網膜内ｍＲＮＡ発現をリアルタイムＰＣＲで確認したところ、ＰＢＡを投与した群は、対照群と比較して、ミトコンドリアマーカーであるｐｇｃ１－α及びｔｆａｍの発現が上昇した（図６）。このことはＰＢＡの腹腔内投与のときと同様の結果を示すものである。 <Example 5 Oral administration of PBA induced increased expression of mitochondrial markers in P23H RP model mice>
We investigated the effect of oral administration of PBA on the expression of mitochondrial biosynthesis-related factors in the retina of P23H RP model mice. PBA (200 mg/kg body weight) was orally administered to P23H RP model mice every day for 14 days from 2 weeks old to 4 weeks old. In oral administration, PBA was prepared in an amount of body weight (g)×20 μL and administered in two doses for one day. That is, when PBA is administered at 200 mg/kg body weight, for example, it is necessary to administer 4 mg of PBA per day per mouse weighing 20 g. 400 μL (200 μL×2 times) of the suspension was administered per day.
As a result, between the PBA-administered group (
Claims (8)
- フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜変性抑制用医薬組成物。 A pharmaceutical composition for suppressing retinal degeneration, containing phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
- フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜変性疾患を治療又は予防するための、医薬組成物。 A pharmaceutical composition for treating or preventing retinal degenerative diseases, containing phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
- 前記網膜変性疾患が、網膜視細胞が変性した疾患である、請求項２に記載の医薬組成物。 The pharmaceutical composition according to claim 2, wherein the retinal degenerative disease is a disease in which retinal photoreceptors are degenerated.
- 前記網膜変性疾患が、網膜色素変性、糖尿病網膜症、加齢黄斑変性、及びＭＥＬＡＳ（ｍｉｔｏｃｈｏｎｄｒｉａｌ ｍｙｏｐａｔｈｙ，ｅｎｃｅｐｈａｌｏｐａｔｈｙ，ｌａｃｔｉｃ ａｃｉｄｏｓｉｓ，ａｎｄ ｓｔｒｏｋｅ－ｌｉｋｅ ｅｐｉｓｏｄｅｓ）に伴う網膜変性から選択される１つ以上の疾患である、請求項２又は３に記載の医薬組成物。 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 epiisodes) 4. The pharmaceutical composition of claim 2 or 3, wherein
- フェニル酪酸の塩が、ナトリウム塩である、請求項１～４のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 4, wherein the salt of phenylbutyric acid is the sodium salt.
- 錠剤、顆粒剤、懸濁剤、注射剤又は点眼剤である、請求項１～５のいずれか一項に記載の医薬組成物。 The pharmaceutical composition according to any one of claims 1 to 5, which is a tablet, granule, suspension, injection or eye drop.
- フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜における小胞体ストレス関連分解を促進するための医薬組成物。 A pharmaceutical composition for promoting endoplasmic reticulum stress-related degradation in the retina, containing phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
- フェニル酪酸（ＰＢＡ）若しくはその塩又はそれらの誘導体を含む、網膜におけるミトコンドリア生合成を活性化するための及び／又は網膜におけるミトコンドリアの代謝機能を改善するための医薬組成物。 A pharmaceutical composition for activating mitochondrial biosynthesis in the retina and/or for improving the metabolic function of mitochondria in the retina, containing phenylbutyric acid (PBA) or a salt thereof or a derivative thereof.
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