Pharmaceutical composition for treating retinitis pigmentosa

Abstract

This invention includes, e.g., a pharmaceutical composition serving to treat retinitis pigmentosa and containing nanoparticles in which statin is encapsulated.

Description

Technical Field

[0001] This application claims priority from Japanese Patent Application No. 2022-012146, the entire disclosure of which is incorporated herein by reference in its entirety. The present disclosure relates to the treatment of retinitis pigmentosa.

Background Art

[0002] Retinitis pigmentosa is a disease in which photoreceptor cells and pigment epithelial cells of the retina degenerate due to gene mutations, and typical symptoms include night blindness and visual field constriction associated with photoreceptor cell disorders. The frequency of retinitis pigmentosa is said to be 1 in 4,000 to 8,000 people, but currently there is no established treatment method for restoring the function of the retina or suppressing the progression of the disease, and it remains a symptomatic treatment.

[0003] Statins have the function of reducing blood cholesterol by inhibiting HMG-CoA reductase and are widely used as therapeutic agents for hypercholesterolemia. Statins are also known to have an angiogenesis-promoting effect. This effect was initially observed only at high doses far exceeding clinical doses in model animals, but PLGA nanoparticles encapsulating pitavastatin are selectively delivered to vascular endothelial cells in a lower limb ischemia model and induce angiogenesis at low doses, and it has been reported that they improve the symptoms of pulmonary hypertension at low doses in a pulmonary hypertension model, and clinical trials have been conducted for severe ischemic limbs and pulmonary hypertension, respectively. PLGA nanoparticles encapsulating pitavastatin have also been reported to suppress tissue damage in an atherosclerosis model.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Non-Patent Documents

[0005] Non - Patent Document 1 K. Ichimura et al., Int Heart J 59, 1432 - 1444 (2018). Non - Patent Document 2 S. Katsuki et al., Circulation 129, 896 - 906 (2014). Summary of the Invention Problems to be Solved by the Invention

[0006] The present disclosure aims to provide a pharmaceutical composition for treating retinitis pigmentosa. Means for Solving the Problems

[0007] The inventors have found that in retinitis pigmentosa, peripheral blood inflammatory monocytes and retinal peripheral - derived macrophages increase, and statin - encapsulated nanoparticles reduce peripheral blood inflammatory monocytes and peripheral - derived retinal macrophages in retinitis pigmentosa model animals and suppress cone cell death, thereby completing the present invention.

[0008] In one aspect, the present disclosure provides a pharmaceutical composition comprising statin - encapsulated nanoparticles for treating retinitis pigmentosa. Effects of the Invention

[0009] The present disclosure provides a pharmaceutical composition comprising statin - encapsulated nanoparticles for treating retinitis pigmentosa. Brief Description of the Drawings

[0010] [Figure 1]Figure 1 shows the ratio of inflammatory monocytes (IMo) in wild-type (WT) and rd10 mice (postnatal day 21 (P21): WT n=7, rd10 n=7; P31: WT n=8, rd10 n=6; P42: WT n=5, rd10 n=5). The central horizontal bar represents the median, the boxes represent the 25th–75th percentiles, and the whiskers represent 1.5 times the interquartile range from the bottom and top of the boxes. Outliers are indicated by dots. Wilcoxon rank-sum test results: *P < 0.05, **P < 0.01. (The same applies to the following figures.) [Figure 2] Figure 2 shows the proportion of monocyte subsets in patients with retinitis pigmentosa (RP) (n=31) and controls (n=16). [Figure 3] Figure 3 shows the correlation between the proportion of monocyte subsets and the rate of decline in retinal sensitivity (MD gradient) in patients with retinitis pigmentosa (RP). [Figure 4] Figure 4 shows the percentage of microglia (P21:WT n=8, rd10 n=8; P31:WT n=9, rd10 n=6; P42:WT n=7, rd10 n=9) (left) and macrophages (P21:WT n=8, rd10 n=8; P31:WT n=9, rd10 n=6; P42:WT n=7, rd10 n=9) (right) in viable cells of the retina of WT and rd10 mice. [Figure 5] Figure 5 shows the percentage of IMo in peripheral blood in rd10 mice and rd10;Ccl2- / - mice (P21: rd10 n=6, rd10;Ccl2- / - n=5; P31: rd10 n=7, rd10;Ccl2- / - n=7; P42: rd10 n=5, rd10;Ccl2- / - n=7). [Figure 6] Figure 6 shows the proportion of resident microglia (left) and macrophages (right) in surviving retinal cells in rd10 mice and rd10;Ccl2- / - mice (P21: rd10 n=6, rd10;Ccl2- / - n=6; P31: rd10 n=5, rd10;Ccl2- / - n=5; P42: rd10 n=5, rd10;Ccl2- / - n=5). [Figure 7]Figure 7 shows TUNEL staining of the retina of rd10 (rd10;Ccl2+ / +) mice and rd10;Ccl2- / - mice (P21) (left), and the number of TUNEL-positive cells in the central, mid-peripheral, and peripheral regions of the nasal and temporal hemispheres of the retina (rd10 n=8; rd10;Ccl2- / - n=8) (right). Scale bar: 50 μm. [Figure 8] Figure 8 shows hematoxylin-eosin (HE) staining of the retinas of rd10 (rd10;Ccl2+ / +) mice and rd10;Ccl2- / - mice (P26) (left), and the number of photoreceptor cells in the central, mid-peripheral, and peripheral regions of the nasal and temporal hemispheres of the retina (rd10 n=20; rd10;Ccl2- / - n=10) (right). Scale bar: 50 μm. [Figure 9] Figure 9 shows peanut agglutinin (PNA) staining of the retina of rd10 (rd10;Ccl2+ / +) mice and rd10;Ccl2- / - mice (P42) (left), and the number of PNA-positive cone cells (rd10 n=21; rd10;Ccl2- / - n=15) in regions 200 μm and 500 μm from the optic disc (right). Scale bar: 50 μm. [Figure 10] Figure 10 shows the photopic electroretinogram (ERG) (left) and ERGb wave amplitude (rd10 n=6;rd10;Ccl2- / - n=8) (right) of rd10 (rd10;Ccl2+ / +) mice and rd10;Ccl2- / - mice (P35). [Figure 11] Figure 11 shows the uptake of FITC into peripheral blood IMo in rd10 mice (P17) intravenously administered with PBS or FITC-loaded nanoparticles (FITC-NP) (PBS n=4; FITC-NP n=4). The left side shows representative data, and the right side shows the quantification results (percentage exceeding the threshold). [Figure 12]Figure 12 shows the uptake of FITC in retinal samples from rd10 mice (P17) administered intravenously with PBS or FITC-NP (PBS n=4; FITC-NP n=4). The left side shows representative data for microglia and macrophages, and the right side shows the quantification results (percentage exceeding the threshold). [Figure 13] Figure 13 shows the percentage of peripheral blood IMo (PBS n=10; FITC-NP n=11; PVS-NP n=14) (top) and the percentage of macrophages and microglia in viable retinal cells (PBS n=14; FITC-NP n=12; PVS-NP n=14) (bottom) of rd10 mice (P31) administered intravenously with PBS, FITC-NP, or pitavastatin-encapsulated nanoparticles (PVS-NP). [Figure 14] Figure 14 shows PNA staining of the retina of rd10 mice (P52) administered intravenously with PBS, FITC-NP, or PVS-NP (left), and the number of PNA-positive cone cells in the 200 μm and 500 μm regions from the optic nerve head (PBS n=9; FITC-NP n=10; PVS-NP n=9) (right). Scale bar: 50 μm. [Figure 15] Figure 15 shows the photopic ERG (left) and ERGb wave amplitude (PBS n=6; PVS-NP n=8) (right) of rd10 mice (P35) administered intravenously with PBS or PVS-NP. [Figure 16] Figure 16 shows the photopic ERGb wave amplitudes (PBS n=8; PVS-NP low n=10; PVS-NP middle n=20; PVS-NP high n=20) of rd10 mice (P49) that were intravenously administered PBS (100 μl PBS), PVS-NP low (0.1 mg PVS / kg), PVS-NP middle (0.3 mg PVS / kg), and PVS-NP high (1.0 mg PVS / kg). [Figure 17]Figure 17 shows the number of PNA-positive cone cells (PBS n=5; PVS-NP low n=6; PVS-NP middle n=7; PVS-NP high n=8) in rd10 mice (P49) that were intravenously administered PBS (100 μl PBS), PVS-NP low (0.1 mg PVS / kg), PVS-NP middle (0.3 mg PVS / kg), and PVS-NP high (1.0 mg PVS / kg). The left graph shows the measurement results at 250 μm from the optic nerve head, and the right graph shows the measurement results at 750 μm from the optic nerve head. [Figure 18] Figure 18 shows the average number of PNA-positive cone cells at the two measurement points in Figure 17 for each mouse. [Figure 19] Figure 19 shows the number of PNA-positive pyramidal cells (PBS 2 / M n=15; PVS-NP 1 / M n=14; PVS-NP 2 / M n=16) (left) and photopic ERGb wave amplitude (PBS 2 / M n=24; PVS-NP 1 / M n=14; PVS-NP 2 / M n=16) (right) of rd10 mice (P49) that were intravenously administered PBS (100 μl PBS) every two weeks (PBS 2 / M), PVS-NP (0.75 mg PVS / kg) every four weeks (PVS-NP 1 / M), or PVS-NP (0.5 mg PVS / kg) every two weeks (PVS-NP 2 / M). [Modes for carrying out the invention]

[0011] Unless otherwise specified, terms used herein have the meanings generally understood by those skilled in the art in the fields of organic chemistry, medicine, pharmacy, molecular biology, microbiology, etc. Some definitions of terms used herein are given below, but these definitions take precedence over general understanding.

[0012] In one embodiment, the present disclosure relates to a pharmaceutical composition comprising statin-encapsulated nanoparticles for the treatment of retinitis pigmentosa.

[0013] In this specification, the term "statin" means a compound having HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase inhibitory activity. Examples of statins include pitavastatin, atorvastatin, pravastatin, simvastatin, fluvastatin, and rosuvastatin. In one embodiment, the statin is pitavastatin. In this specification, when referring to statins, the term is used to include the free form and its pharmaceutically acceptable salts and solvates thereof. Examples of pharmaceutically acceptable salts include alkali metal salts such as sodium salts and potassium salts, alkaline earth metal salts such as calcium salts and magnesium salts, organic amine salts such as phenethylamine salts, and ammonium salts, and examples of solvates include solvates with water or alcohol. For example, an example of a pharmaceutically acceptable salt of pitavastatin is pitavastatin calcium, and an example of a solvate of pitavastatin or a pharmaceutically acceptable salt thereof is pitavastatin calcium hydrate (e.g., pentahydrate). Statin-encapsulated nanoparticles may contain one or more statins, or other drugs in addition to statins.

[0014] The statin-encapsulated nanoparticles of this disclosure contain statins inside nanoparticles composed of a biocompatible polymer. The biocompatible polymer may be a polymer obtained by polymerizing one or more monomers selected from, for example, D,L-lactide, D-lactide, L-lactide, D,L-lactic acid, D-lactic acid, L-lactic acid, glycolide, glycolic acid, ε-caprolactone, ε-hydroxyhexanoic acid, γ-butyrolactone, γ-hydroxybutyric acid, δ-valerolactone, δ-hydroxyvaleric acid, hydroxybutyric acid, and malic acid. Examples of biocompatible polymers include polylactic acid, polyglycolic acid, lactic acid-glycolic acid copolymers (also known as poly lactide-co-glycolide, PLGA), and lactic acid-aspartic acid copolymers. In one embodiment, the biocompatible polymer is PLGA.

[0015] The term PLGA includes polymers containing lactic acid or lactide and glycolic acid or glycolide in various proportions. The ratio of lactic acid or lactide to glycolic acid or glycolide may be, for example, 1:99 to 99:1, preferably 3:1. The weight-average molecular weight of PLGA may be, for example, 5,000 to 200,000, or 15,000 to 25,000. PLGA can be synthesized by known methods, or commercially available PLGA may be used.

[0016] The particle size of statin-encapsulated nanoparticles may be, for example, 1-1000 nm, 2-500 nm, 3-300 nm, 10-300 nm, or 50-300 nm. The particle size may also be 100-300 nm or 200-300 nm. In one embodiment, the particle size is 50-300 nm. In this specification, particle size refers to the equivalent spherical diameter measured by dynamic light scattering, and the median diameter (D 50 It is expressed as (D). 50 D50 is the particle size (50% diameter) at the point where the cumulative curve reaches 50% of the total volume of the particle collection, with the total volume of the particle collection being 100%. The cumulative curve and D50 can be determined using a particle size analyzer such as the Nanotrac Wave-EX150 (manufactured by Mictotrac BEL Corp).

[0017] Statin-encapsulated nanoparticles may have their surfaces modified with polyethylene glycol (PEG). For example, nanoparticles with PEG-modified surfaces can be obtained by using PEG-modified biocompatible polymers in the production of nanoparticles. Modifying the particle surface with PEG can improve the blood stability of the nanoparticles.

[0018] Statin-encapsulated nanoparticles may be manufactured by any method. For example, statin-encapsulated nanoparticles can be manufactured by the emulsion method in water. In the emulsion method in water, two types of solvents are used: a good solvent in which the biocompatible polymer dissolves and a poor solvent in which the biocompatible polymer does not dissolve. The good solvent and the poor solvent can be appropriately selected by a person skilled in the art depending on the nanoparticles to be manufactured.

[0019] Examples of poor solvents include water. When water is used as the poor solvent, a surfactant may be added to the water. Examples of surfactants include polyvinyl alcohol (PVA), lecithin, hydroxymethylcellulose, and hydroxypropylcellulose. In one embodiment, the poor solvent is water and the surfactant is PVA.

[0020] Suitable solvents include low-boiling, poorly water-soluble organic solvents such as alkanes, acetone, methanol, ethanol, ethyl acetate, diethyl ether, cyclohexane, benzene, toluene, or mixtures thereof. In one embodiment, the suitable solvent is acetone or a mixture of acetone and ethanol in a 2:1 ratio.

[0021] In the aqueous emulsion method, a biocompatible polymer is first dissolved in a good solvent, and a drug solvent is added and mixed to it. When this good solvent containing the polymer and drug is added dropwise to a poor solvent under stirring, the good solvent rapidly diffuses into the poor solvent. As a result, emulsification of the good solvent occurs in the poor solvent, and emulsion droplets of the good solvent are formed. Next, due to interdiffusion between the good and poor solvents, the organic solvent continuously diffuses from within the emulsion into the poor solvent, reducing the solubility of the biocompatible polymer and drug within the emulsion droplets, and generating nanoparticles of spherical crystalline particles containing the drug. Subsequently, the good solvent, the organic solvent, is removed by centrifugation or reduced-pressure distillation. The resulting nanoparticles can be used as is, or after being powdered by operations such as freeze-drying.

[0022] Statin-encapsulated nanoparticles may be manufactured using a forced thin-film microreactor. When using a forced thin-film microreactor, first, a good solvent containing the polymer and the drug, and a poor solvent are introduced between processing surfaces that are arranged opposite each other and at least one of which rotates relative to the other. The good solvent and the poor solvent are mixed in the thin-film fluid formed by this process, and drug-encapsulating nanoparticles are deposited in the thin-film fluid. As a forced thin-film microreactor, for example, ULREA SS-11 (M Technique Co. Ltd.) can be used.

[0023] Statin-encapsulated nanoparticles may contain, for example, 0.01 to 99% by weight, 0.1 to 30% by weight, 0.5 to 20% by weight, or 1 to 15% by weight of statin. In one embodiment, the statin-encapsulated nanoparticles contain 1 to 15% by weight of statin. In this specification, the statin content is expressed as the ratio of the weight of statin to the weight of the statin-encapsulated nanoparticles. The statin content can be determined by measuring the weight of statin extracted from a predetermined weight of statin-encapsulated nanoparticles and calculating the ratio of the weight of statin to the weight of the statin-encapsulated nanoparticles.

[0024] Statin-encapsulated nanoparticles can be compounded into redispersible aggregated particles (nanocomposites) when powdered by operations such as freeze-drying. For example, nanoparticles can be compounded in a redispersible manner by drying them with organic or inorganic substances. This compounding allows the nanoparticles to aggregate into easily handleable aggregated particles, which then disperse upon contact with moisture during use to exhibit their properties. In one embodiment, statin-encapsulated nanoparticles are compounded with a sugar alcohol or sucrose. Using substances such as sugar alcohols can reduce variations in encapsulation rates, and these substances act as excipients, improving the handling of the nanoparticles. Examples of sugar alcohols include mannitol, trehalose, sorbitol, erythritol, maltose, and xylitol. In one embodiment, the sugar alcohol is trehalose. Compounding can also be performed by fluidized bed drying granulation instead of freeze-drying.

[0025] Examples in this specification have shown that peripheral blood inflammatory monocytes and peripherally derived macrophages in the retina are increased in retinitis pigmentosa, and that statin-encapsulated nanoparticles reduce peripheral blood inflammatory monocytes and peripherally derived retinal macrophages and suppress cone cell death in animal models of retinitis pigmentosa. Therefore, statin-encapsulated nanoparticles can be used in the treatment of retinitis pigmentosa.

[0026] Retinitis pigmentosa is a disease in which photoreceptor cells and / or pigment epithelial cells of the retina degenerate due to gene mutations. Retinitis pigmentosa includes conditions in which rod cells, cone cells, and pigment epithelial cells, or two or more selected cell types from these, degenerate. In retinitis pigmentosa, degeneration of rod cells often precedes the degeneration of cone cells. Conditions in which only rod cells degenerate are called rod dystrophy, and conditions in which both rod and cone cells degenerate are called rod-cone dystrophy; both are included in the definition of retinitis pigmentosa in this disclosure. Retinitis pigmentosa may be caused by any of the gene mutations, and there may be one or more causative gene mutations.

[0027] In this specification, treatment of retinitis pigmentosa includes improvement, suppression and delay of exacerbation, and suppression and delay of disease progression, as well as improvement of one or more symptoms or findings of retinitis pigmentosa. Symptoms of retinitis pigmentosa include night blindness, visual field constriction, decreased visual acuity, photophobia, diurnal blindness, color vision deficiency, and photopsia. Findings of retinitis pigmentosa include (1) fundus findings (retinal vascular narrowing, rough retinal color, osteocotyl-like pigmentation, multiple white spots, optic nerve atrophy, macular degeneration), (2) abnormalities in electroretinography (attenuated, negative, or absent), (3) hyperfluorescence or hypofluorescence due to retinal pigment epithelial atrophy in fundus autofluorescence findings, and (4) abnormalities (discontinuity or absence) of the ellipsoid zone (IS / OS) in the fovea on optical coherence tomography.

[0028] Statin-encapsulated nanoparticles may be included in pharmaceutical compositions. For example, a pharmaceutical composition may contain statin-encapsulated nanoparticles in amounts of 0.000001-99.9% by weight, 0.00001-99.8% by weight, 0.0001-99.7% by weight, 0.001-99.6% by weight, 0.01-99.5% by weight, 0.1-99% by weight, 1-50% by weight, 1-40% by weight, 1-30% by weight, 1-20% by weight, or 1-15% by weight. The pharmaceutical composition may further contain pharmaceutically acceptable additives as needed. Examples of pharmaceutically acceptable additives include excipients, lubricants, binders, disintegrants, solubilizers, suspending agents, isotonic agents, buffers, analgesics, preservatives, antioxidants, colorants, and sweeteners.

[0029] Pharmaceutical compositions may be in dosage forms such as tablets, capsules, powders, granules, solutions, suspensions, emulsions, inhalants, injections, eye drops, and eye ointments. Injectable preparations include solution-type injections, suspension-type injections, emulsion-type injections, and injections prepared at the time of use. In one embodiment, the pharmaceutical composition is an injection. These preparations can be prepared by conventional methods.

[0030] Statin-encapsulated nanoparticles or pharmaceutical compositions containing them are administered to a subject in an amount capable of producing the desired effect (hereinafter referred to as the effective dose). The dosage and duration of administration can be appropriately determined by a person skilled in the art depending on the subject's age, weight, health condition, and other conditions. For example, the daily dose of statin in statin-encapsulated nanoparticles or pharmaceutical compositions containing them may be 0.001 mg / kg to 100 mg / kg, 0.003 mg / kg to 10 mg / kg, 0.005 mg / kg to 5 mg / kg, 0.01 mg / kg to 3 mg / kg, 0.01 mg / kg to 1 mg / kg, 0.01 mg / kg to 0.75 mg / kg, 0.01 mg / kg to 0.5 mg / kg, or 0.01 mg / kg. mg / kg~0.3mg, 0.01mg / kg~0.25mg, 0.01mg / kg~0.1mg, 0.01mg / kg~0.09mg, 0.01mg / kg~0.08mg, 0.01mg / kg~ 0.07mg, 0.01mg / kg~0.06mg, 0.01mg / kg~0.05mg, 0.01mg / kg~0.04mg, 0.01mg / kg~0.03mg, 0.03mg / kg~1mg / k g, 0.03mg / kg~0.75mg / kg, 0.03mg / kg~0.5mg / kg, 0.03mg / kg~0.3mg, 0.03mg / kg~0.25mg, 0.03mg / kg~0.1mg , 0.03mg / kg~0.09mg, 0.03mg / kg~0.08mg, 0.03mg / kg~0.07mg, 0.03mg / kg~0.06mg, 0.03mg / kg~0.05mg, 0.1m The statin can be administered in amounts of g / kg to 1 mg / kg, 0.1 mg / kg to 0.75 mg / kg, 0.1 mg / kg to 0.5 mg / kg, 0.1 mg / kg to 0.3 mg / kg, 0.3 mg / kg to 1 mg / kg, 0.3 mg / kg to 0.75 mg / kg, or 0.3 mg / kg to 0.5 mg / kg, and can be administered once a day or in multiple divided doses (e.g., two, three, or four times). In one embodiment, statin-encapsulated nanoparticles or a pharmaceutical composition containing the same are administered per day in an amount of statin (e.g., pitavastatin calcium) of 0.01 mg / kg to 0.3 mg / kg or 0.03 mg / kg to 0.1 mg / kg.Statin-encapsulated nanoparticles or pharmaceutical compositions containing them may be administered to adults at a dose of 1 to 10 mg / body (e.g., 1, 2, 4, 8, or 10 mg / body) or 1 to 8 mg / body (e.g., 1, 2, 4, or 8 mg / body) per day, as a statin (e.g., pitavastatin calcium). Administration may be a single dose or multiple doses. In the case of multiple doses, administration may be daily, every few days (e.g., 2, 3, 4, 5, or 6 days), every week or every few weeks (e.g., 2, 3, 4, 5, or 6 weeks), every month or every few months (e.g., 2, 3, 4, 5, or 6 months). In one embodiment, administration is twice a week (e.g., once every 3 or 4 days) or once every 1 to 4 weeks (e.g., every 1, 2, 3, or 4 weeks). The duration of administration may be, for example, one day or several days (e.g., 2, 3, 4, 5, or 6 days), one week or several weeks (e.g., 2, 3, 4, 5, or 6 weeks), one month or several months (e.g., 2, 3, 4, 5, or 6 months), or longer. Administration may be systemic or topical, and may be oral or parenteral (e.g., intravenous, intramuscular, intrabronchial, intranasal, intraocular). In one embodiment, statin-encapsulated nanoparticles or a pharmaceutical composition containing them are administered intravenously.

[0031] In this disclosure, the subject is a mammal (e.g., human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.), preferably a human. In one embodiment, the subject is a human subject suffering from retinitis pigmentosa (also referred to as a patient with retinitis pigmentosa).

[0032] In one embodiment, this disclosure provides a method for treating retinitis pigmentosa, comprising administering an effective amount of statin-encapsulated nanoparticles to a subject in need of treatment. In one embodiment, this disclosure provides statin-encapsulated nanoparticles for the treatment of retinitis pigmentosa. In one aspect, this disclosure provides the use of statin-encapsulated nanoparticles for the manufacture of a pharmaceutical product for the treatment of retinitis pigmentosa. This disclosure provides, in one embodiment, the use of statin-encapsulated nanoparticles for the treatment of retinitis pigmentosa.

[0033] Exemplary embodiments of this disclosure are described below. [1] A pharmaceutical composition containing statin-encapsulated nanoparticles for the treatment of retinitis pigmentosa. [2] The pharmaceutical composition according to claim 1, wherein the statin is pitavastatin or a pharmaceutically acceptable salt thereof. [3] The pharmaceutical composition according to claim 1 or 2, wherein the statin is pitavastatin calcium. [4] A pharmaceutical composition according to any one of the above 1 to 3, wherein the statin-encapsulated nanoparticles contain 1 to 15% by weight of statin. [5] A pharmaceutical composition according to any one of the above 1 to 4, wherein the statin-encapsulated nanoparticles contain PLGA. [6] A pharmaceutical composition according to any one of 1 to 5, wherein the particle size of the statin-encapsulated nanoparticles is 50 to 300 nm. [7] A pharmaceutical composition according to any one of the above 1 to 6, comprising 1 to 15% by weight of statin-encapsulated nanoparticles. [8] A pharmaceutical composition according to any one of items 1 to 7 above, administered at a daily dose of 0.01 mg / kg to 0.5 mg / kg of statin. [9] A pharmaceutical composition according to any one of items 1 to 8 above, which is administered intravenously.

[10] A pharmaceutical composition according to any one of items 1 to 9 above, administered twice a week or once every 1 to 4 weeks.

[0034]

[11] A method for treating retinitis pigmentosa, comprising administering an effective amount of statin-encapsulated nanoparticles to a subject in need of treatment.

[12] Use of statin-encapsulated nanoparticles for the manufacture of pharmaceuticals for the treatment of retinitis pigmentosa.

[13] Use of statin-encapsulated nanoparticles to treat retinitis pigmentosa.

[0035] The present invention will be described in more detail below with reference to examples, but the present invention is not limited in any way to these examples. [Examples]

[0036] 1. Materials and Methods animal WT(C57BL / 6J) mouse, B6.CXB1-Pde6β rd10 / J(rd10) mouse, and B6.129S4-Ccl2 tm1Rol / J(Ccl2 - / - The mice were purchased from Jackson Laboratory (West Grove, PA). Rd10 mice were used in Ccl2 - / - Crossbreeding with mice resulted in rd10;Ccl2 - / - I made it.

[0037] Patients and control subjects Because blood monocytes can be affected by systemic diseases such as cardiovascular disease, hypertension, and diabetes, subjects were selected who were generally healthy and relatively young, under 45 years of age. Patients with retinitis pigmentosa (RP) were diagnosed based on their history of night blindness, characteristic fundus findings (e.g., osteocoelastic pigmentation in the mid-peripheral and peripheral retina), visual field constriction and / or ring scotoma, and reduced or absent a- and b-wave amplitudes on electroretinography. Patients with cone dystrophy, cone-rod dystrophy, Vietti crystallin retinopathy, uveitis, or systemic disease were excluded. Participants were recruited consecutively in 2017 and 2018, and blood samples were collected from 31 RP patients and 16 age- and sex-matched healthy subjects. RP patients were followed for more than one year and underwent at least three HFA10-2 tests to obtain mean deviation (MD) gradients. The analysis used the subjects' visual acuity at the time of blood collection, HFA10-2 test results from the time closest to blood collection, and the MD gradient. The test results for each subject's right eye were used in the analysis.

[0038] Clinical Testing The subjects' best corrected visual acuity (BCVA) was measured using a 5m Landolt decimal VA chart (CV-6000; Tomey, Nagoya, Japan) or a single-letter Landolt test card (HP-1258; Handaya, Tokyo). Values ​​were converted to the logarithm of the minimum separation threshold angle (logMAR). Refractive errors were corrected using multiple lenses of different diopters at each visit to ensure that visual acuity was best corrected. Visual acuity was based on the smallest Landolt C letter that the subject was able to correctly answer 60% or more times (3 / 5). Automated static perimetry was performed using a Humphrey Field Analyzer (HFA; Humphrey Instruments, San Leandro, CA) with the central 10-2 Swedish Interactive Thresholding Algorithm Standard Program. Lenses were modified according to the test distance. If the reliability of the test was insufficient (i.e., poor fixation >20%, false positive >15%, or false negative >33%), the perimetry test was repeated.

[0039] Flow cytometry Immunolabeled cells were analyzed using FlowJo software on a BD FACSVerse system (BD Biosciences, Franklin Lakes, NJ). Samples were prepared and analyzed as follows:

[0040] - Mouse blood monocytes Peripheral blood was collected by cardiac puncture from mice, and red blood cells were lysed with VersaLyse Lysing solution (Becton Dickinson Biosciences, San Jose, CA) for 10 minutes at room temperature. The cells were washed twice with ice-cold FACS buffer (phosphate-buffered saline (PBS) containing 2% fetal bovine serum (FBS)). Fc receptors were blocked with anti-mouse CD16 / CD32 (eBioscience) for 5 minutes at 4°C and then incubated on ice for 20 minutes with antibodies against the following: mouse CD192 (CCR2) (Alexa Fluor 647-conjugated, clone SA203G11; Biolegend, San Diego, CA), CD11b (BV421-conjugated, clone M1 / 70; Biolegend), Ly-6C (Alexa fluor 488-conjugated, clone HK1.4; Biolegend), Ly-6G (allophycocyanin [APC]-cy7-conjugated, clone 1A8; Biolegend), and CX3CR1 (phycoerythrin [PE]-conjugated, clone SA011F11, Biolegend). Dead cells were excluded by the fluorescent marker 7-AAD (BD Pharmingen, San Diego, CA). Inflammatory monocytes were identified as CD11b + Ly-6C hi Ly-6G lo-neg cells.

[0041] - mouse microglia and macrophages The retinas of mice were extracted from the eyeballs, finely chopped, and digested in a water bath at 37°C for 30 minutes (with 1.2 mg / ml collagenase D [Roche Diagnostics, Indianapolis, IN] and 40 μg / ml DNase I [Sigma-Aldrich, St. Louis, MO]). After digestion, the tissue was separated by pipetting to obtain a single-cell suspension. The cells were washed twice with ice-cold FACS buffer (PBS containing 2% FBS). The Fc receptor was blocked with anti-mouse CD16 / CD32 (eBioscience) at 4°C for 10 minutes, and then incubated on ice for 20 minutes with antibodies against the following: mouse CD11c (PE-Cy7-conjugated, clone N418; Biolegend), CD45 (APC-conjugated, clone 30-F11; Biolegend), Ly-6C (APC-cy7-conjugated, clone HK1.4; Biolegend), Ly-6G (APC-cy7-conjugated, clone 1A8; Biolegend), CD11b (PE-conjugated, clone M1 / 70; Biolegend), CD192 (CCR2) (FITC-conjugated, clone SA203G11; Biolegend), and CX3CR1 (BV421-conjugated, clone SA011F11; Biolegend). Dead cells were excluded using 7-AAD (BD Pharmingen). Microglia were identified using CD11b. hi CD11c mid CD45 mid Ly-6G lo Ly-6C lo We define cells as macrophages, and macrophages are CD11b hi CD11c hi CD45 hi Ly-6G lo Ly-6C lo Cells were defined as such. In each experiment, wild-type retina was used as a control to gate microglia and macrophages.

[0042] - Human blood monocytes Whole blood (8 ml) was collected from the precubital vein of each subject using a BD Vacutainer CPT cell preparation tube (BD Biosciences) containing heparin sodium. The obtained samples were immediately (within 30 minutes) subjected to density gradient separation of mononuclear cells. The separated mononuclear cells were then pelleted by slow centrifugation (200 g) and dispensed into 5 ml tubes in PBS containing 2% FBS. The samples were stored in a freezer at -80°C until analysis and thawed 30 minutes prior to analysis. For immunolabeling, the cells were washed twice with ice-cold FACS buffer (PBS containing 2% FBS). Fc receptors were blocked with anti-mouse CD16 / CD32 (eBioscience) at 4°C for 5 minutes, and then incubated on ice for 20 minutes with antibodies against the following: human CD56 (NCAM) (PerCP / Cy5.5-conjugated, clone HCD56; Biolegend), CD19 (PerCP / Cy5.5-conjugated, clone HIB19; Biolegend), HLA-DR (APC-conjugated, clone L243; Biolegend), CD14 (PE-conjugated, clone M5E2; Biolegend), CD16 (BV421-conjugated, clone 3G8; Biolegend), CD192 (CCR2) (FITC-conjugated, clone K036C2; Biolegend), and CX3CR1 (PEcy7-conjugated, clone 2A9-1, Biolegend). Dead cells were excluded using 7-AAD (BD Pharmingen). Monocytes were HLA-DR + CD2 - CD19 - CD56 - The cells were identified. These cells were then classified into three subsets according to their CD14 and CD16 expression levels.

[0043] Retinal Hole-Mount Staining Mouse eyeballs were extracted and fixed in 4% paraformaldehyde at 4°C for 1 hour. After removing the cornea and lens, the retina was separated from the posterior cup. Each retina was blocked for 1 hour in PBS containing 10% skim milk and 0.3% Triton-X 100 (9002-93-1; Wako), and incubated overnight at 4°C with fluorescein isothiocyanate (FITC)-conjugated peanut agglutinin (PNA) (1:100, L7381; Sigma-Aldrich). Fluorescence images were acquired using a fluorescence microscope (BZ-X700; Keyence, Osaka, Japan). The number of PNA-positive cone cells was measured using Image J ver. 1.52a software (US National Institutes of Health [NIH]) and measured 0.015625 mm in the superior, inferior, temporal, and nasal regions located 200 μm, 250 μm, 500 μm, or 750 μm from the optic nerve head. 2 The retinal region was counted, and the values ​​for each region were averaged. The analysis was performed blindly without informing the observers of the sample name or conditions.

[0044] Histological examination Mouse eyeballs were excised, fixed in 4% paraformaldehyde (in PBS) for 24 hours, and mounted on paraffin. Sections (5 μm thick) were prepared along the horizontal meridian and stained with hematoxylin and eosin (H&E). Five sections were randomly selected from each eye. Cell counts in the outer granular layer (ONL) were measured in the central region (200 μm from the optic disc), mid-peripheral region (500 μm from the optic disc), and peripheral region (1000 μm from the optic disc) of the nasal and temporal hemisphere retina. 2 The count was performed within a square area. Numbers and letters were assigned to the tissue samples, and the analysis was performed blindly without informing the observers of the conditions.

[0045] TUNEL staining TUNEL staining was performed using the ApopTag Fluorescein In Situ Apoptosis Detection Kit (Merck Millipore, Darmstadt, Germany) according to the manufacturer's instructions. Immunofluorescence imaging was acquired using a fluorescence microscope (BZ-X700; Keyence). 10,000 μm of the central (200 μm from the optic nerve), intermediate peripheral (500 μm from the optic nerve), and peripheral (1000 μm from the optic nerve) regions of the nasal and temporal hemispheres of the retina. 2 The number of TUNEL-positive cells in each region was obtained using ImageJ software, ver. 1.52a. The ONL region was measured for each square region, and the density of TUNEL-positive cells within the ONL was calculated, resulting in cells / mm². 2 The results were presented as follows. The observers were blinded to the sample names and conditions, and the analysis was performed without informing them.

[0046] Electroretinography (ERG) Photopic ERG was recorded via an LED contact lens using a PuREC system (PC-100; Mayo Corporation, Aichi, Japan). Mice were anesthetized by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg), and their body temperature was maintained at 37°C with a heating pad. Pupils were dilated with 0.5% tropicamide and 0.5% phenylephrine hydrochloride. After topical application of oxubuprocaine, the LED contact lens was attached to the cornea of ​​the mouse. A reference electrode was placed on the tongue, and a ground electrode was clipped to the tail. Mice were then subjected to a temperature reading of 30 cd / m². 2 The image was adapted for 10 minutes against a background of white light with an intensity of 3.0 cd·s / m. 2 Sixteen photos of photopic flash were taken and averaged.

[0047] Preparation of PLGA nanoparticles For the preparation of nanoparticles (NPs), a PLGA polymer (Wako Pure Chemical Industries, Osaka, Japan) with an average molecular weight of 20,000 and a lactide-to-glycolide ratio of 75:25 was used. FITC (D Dojindo Laboratories, Kumamoto, Japan) or pitavastatin calcium (Wako, Osaka, Japan) (hereinafter simply referred to as pitavastatin (PVS)) was incorporated into the PLGA nanoparticles. The nanoparticles were prepared using ULREA SS-11 (M Technique Co. Ltd., Osaka, Japan). For FITC-NPs, a tank containing liquid A (an aqueous solution containing 2.0% polyvinyl alcohol (PVA)) was pressurized to 0.3 MPa and moved at a set temperature of 43°C (measured temperature approximately 40°C) and a speed of 120 ml / min. Next, liquid B (a solution containing PLGA, FITC, acetone, and ethanol in a weight ratio of 4.04:0.20:63.84:31.92) was moved at a rate of 100 ml / min at a set temperature of 41°C (measured temperature: approximately 30°C). Liquids A and B were reacted on a rotating disk rotating at 1000 rpm with a back pressure of 0.02 MPa. The solvent in the resulting mixture was removed by distillation using an evaporator. Next, the resulting suspension was purified to remove excess PVA and unsealed reagents, and powdered by freeze-drying. In PVS-NP, a tank containing liquid A (an aqueous solution containing 0.17% PVA) was pressurized to 0.3 MPa and moved at a rate of 156 mL / min at a set temperature of 25°C (measured temperature: approximately 24°C). Next, liquid B (a solution containing PLGA, PVS, acetone, and ethanol in a weight ratio of 0.7:0.15:66.10:33.05) was transferred at a set temperature of 25°C (measured temperature: approximately 24°C) at a rate of 100 ml / min. Liquids A and B were reacted on a rotating disk rotating at 400 rpm under a back pressure of 0.02 MPa. The solvent in the resulting mixture was removed by distillation using an evaporator, and the mixture was powdered by freeze-drying. FITC-NP and PVS-NP contained 6.8 ± 0.4% (w / v) of FITC and 2.79 ± 0.05% (w / v) of PVS, respectively.Particle size was measured using Nanotrac Wave-EX150 (MicrotracBEL Corp). The particle size was 252 nm for FITC-NP and 202 nm for PVS-NP.

[0048] In vivo distribution of nanoparticles 17-day-old (P17) rd10 mice were given a single intravenous injection of FITC-NP (0.5 mg FITC-NP / 100 μl PBS). Blood samples were collected 2 hours later, and FITC uptake into IMo was analyzed by flow cytometry. Additionally, retinal samples were collected 24 hours after intravenous injection of FITC-NP, and FITC uptake into macrophages and microglia was analyzed. Blood cells were mouse Ly-6C (APC-cy7-conjugated, clone HK1.4; Biolegend) and Ly-6G (PerCP / Cy5.5-conjugated, clone 1A8; Biolegend) labeled with the following antibodies: CD192 (CCR2) (Alexa Fluor 647-conjugated, clone SA203G11; Biolegend), CD11b (BV421-conjugated, clone M1 / 70; Biolegend), and CX3CR1 (PE-conjugated, clone SA011F11, Biolegend). FITC expression was observed in CD11b. + Ly-6C hi Ly-6G lo-negEvaluation was performed using IMo. CD192(CCR2)(FITC-conjugated, clone SA203G11; Biolegend) was not used to evaluate FITC-NP cellular uptake. Retinal cells were stained with the following antibodies: CD11c(PE-Cy7-conjugated, clone N418; Biolegend), CD45(APC-conjugated, clone 30-F11; Biolegend), Ly-6C(APC-cy7-conjugated, clone HK1.4; Biolegend), Ly-6G(APC-cy7-conjugated, clone 1A8; Biolegend), CD11b(PE-conjugated, clone M1 / 70, Biolegend), and CX3CR1(BV421-conjugated, clone SA011F11; Biolegend). Subsequently, the fluorescence of FITC in microglia and macrophages was measured.

[0049] NP treatment of rd10 mice rd10 mice were divided into three groups at P21: PBS group (100 μl PBS), FITC-NP group (0.5 mg FITC-NP / 100 μl PBS), and PVS-NP group (0.0065 mg PVS / 0.5 mg PVS-NP / 100 μl PBS; equivalent to 0.325 mg PVS / kg of body weight). PBS, FITC-NP, or PVS-NP were administered intravenously via the tail vein twice a week (once every 3 or 4 days) from P21 until the end of each experiment.

[0050] Dose-finding study To determine the optimal dose of PVS-NP, rd10 mice were divided into four groups at P21 for dose-finding studies: PBS group (100 μl PBS), PVS-NP low group (0.1 mg PVS / kg), PVS-NP middle group (0.3 mg PVS / kg), and PVS-NP high group (1.0 mg PVS / kg). Intravenous administration via the tail vein was performed once a week from P21 until the end of each experiment. Photopic ERG and PNA-positive pyramidal cell counts were analyzed at P49.

[0051] Usage setting test To determine the optimal administration method for PVS-NP, rd10 mice were divided into three groups at P21 for dose-finding studies: PBS (100 μl PBS) every two weeks, PVS-NP (0.75 mg PVS / kg) every four weeks, and PVS-NP (0.5 mg PVS / kg) every two weeks. From P21 until the end of each experiment, the drugs were administered intravenously via the tail vein at the respective intervals. At P49, the number of photopic ERGs and PNA-positive pyramidal cells were analyzed.

[0052] statistical analysis The correlation coefficient between the MD gradient in the HFA10-2 test and the proportion of monocyte subsets in patients with RP was analyzed using Spearman's rank correlation test. Comparisons between paired data were performed using the Wilcoxon rank-sum test. A p-value of ≤0.05 was considered statistical significance. Statistical analysis of the data was performed using JMP1 Pro 13.0.0 software (SAS, Cary, NC).

[0053] 2.Results Increased inflammatory monocytes in peripheral blood of rd10 mice and RP patients. To investigate whether RP is associated with increased circulating monocyte count, we analyzed the number of inflammatory monocytes (IMo) in the blood of rd10 mice, a clinically relevant model of RP with the Pde6b mutation. In rd10 mice, rod cell death begins around P18, and most rod cells are gone by P30. This is followed by gradual pyramidal degeneration. Peripheral blood from wild-type (WT) and rd10 mice was analyzed by flow cytometry at P21, P31, and P42. The results showed that CD11b in rd10 mice was higher compared to WT mice. + Ly-6C hi Ly-6G lo IMo counts showed a significant increase in P21 (p<0.01), P31 (p<0.01), and P42 (p<0.05) (Figure 1). These cells also showed high expression of CCR2 and CX3CR1 (data not shown).

[0054] Next, we evaluated the monocyte changes in RP patients. Peripheral blood samples from the subjects were analyzed by flow cytometry, and monocytes were identified by CD14 + CD16 ++ non-classical, CD14 ++ CD16 + Intermediate, and CD14 ++ CD16 - They were classified into classical monocytes. As a result, there was no significant difference in the proportion of total monocytes between RP patients and controls, but in subset analysis, CD14 in RP patients was significant. ++ CD16 + A significant increase in the proportion of intermediate monocyte subsets was observed (p=0.0098, Figure 2), and these cells also showed high expression of CCR2 and CX3CR1 (data not presented). In addition, CD14 was observed in RP patients. ++ CD16 + A higher proportion of intermediate monocyte subsets was associated with a larger MD gradient (rate of decline in retinal sensitivity) (ρ=-0.4933, p=0.0042) (Figure 3). These results suggest that RP is associated with increased peripheral blood IMo in both human and animal models.

[0055] Changes in retinal microglia and macrophages in rd10 mice To investigate the recruitment of IMo to the retina of rd10 mice, flow cytometry analysis of retinal myeloid cells was performed. Since retinal microglia and macrophages can be distinguished using the surface markers CD45 and CD11c, retinal cells were stained for CD11b, CD11c, CD45, Ly6C, Ly6G, CCR2, and CX3CR1. The retina of WT mice was stained with CD11b hi CD11c mid CD45 mid Ly-6G lo Ly-6C lo Microglia were present, but CD11b hi CD11c hi CD45 hi Ly-6G lo Ly-6C loMacrophages were absent. On the other hand, in the retina of rd10 mice, an increase in these cell populations was observed in P21, P31, and P42 (p<0.01, Figure 4).

[0056] Involvement of the CCL2 / CCR2 axis in pyramidal cell death in rd10 mice To investigate the roles of IMo and peripherally derived macrophages, we used Ccl2-deficient rd10 mice (rd10;Ccl2) in which the CCL2 / CCR2 axis, essential for the recruitment of myeloid cells to the blood or lesions, is blocked. - / - A study was conducted to create a sample. Ccl2 deficiency resulted in decreased peripheral blood IMo in P21, P31, and P42 (p<0.05, p<0.01, and p<0.01, respectively; Figure 5). rd10;Ccl2 - / - In the mouse retina, macrophages were significantly reduced in P21 and P31 (both p<0.05), and slightly reduced in P42 (p=0.06), while the proportion of resident microglia was reduced in rd10;Ccl2 - / - Mouse and rd10;Ccl2 + / + There was no significant difference compared to mice (Figure 6). These data indicate that the CCL2 / CCR2 axis is important for IMo / macrophage recruitment in rd10 mice.

[0057] Next, we evaluated the effect of Ccl2 deficiency on rod and cone degeneration in rd10 mice. TUNEL staining at P21, where rod cell death peaked, showed that rd10;Ccl2 - / - Mouse and rd10;Ccl2 + / + No significant difference was observed in the number of TUNEL-positive cells in the outer granular layer (ONL) between mice and rd10 mice (Figure 7). Consistent with this, HE staining at P26 showed no significant difference in ONL thickness in the presence or absence of Ccl2 (Figure 8), suggesting that Ccl2 deficiency may not affect rod degeneration in rd10 mice. In contrast, pyramidal cell density assessed by PNA labeling showed a difference in P52 between rd10 and Ccl2. + / + Compared to mice, rd10;Ccl2 - / -The levels were significantly higher in mice (Figure 9). Furthermore, cone function was analyzed using photopic ERG. The photopic ERGb wave was rd10;Ccl2 + / + Compared to mice, rd10;Ccl2 - / - This was significantly maintained in mice (Figure 10). These results suggest that the CCL2 / CCR2 axis promotes the recruitment and engraftment of peripherally derived macrophages to the retina, which contributes to cone degeneration in rd10 mice.

[0058] Drug delivery to peripheral blood IMo and retinal macrophages We investigated drug delivery using PLGA nanoparticles to peripheral blood IMo and macrophages migrated from the periphery. FITC-encapsulated nanoparticles were injected into the tail vein of P17 rd10 mice, and Ly-6C was administered 2 hours after injection. hi The specificity of drug delivery was evaluated by analyzing the delivery of FITC to IMo. FITC-NP efficiently delivered FITC to a large number of IMo (p<0.05, Figure 11). Drug delivery efficiency to retinal microglia and macrophages was analyzed 24 hours after intravenous injection of nanoparticles. FITC delivery was observed in 3.1±1.7% of macrophages after FITC-NP administration. In contrast, FITC was not detected in microglia even after FITC-NP administration (Figure 12). These results indicate that nanoparticles are a promising drug delivery system that targets IMo and peripherally derived macrophages but does not target microglia.

[0059] Using this drug delivery system, the efficacy of pitavastatin-encapsulated nanoparticles (PVS-NP) was evaluated in rd10 mice. rd10 mice were administered PBS, FITC-NP, or PVS-NP intravenously twice weekly starting from P21. At P31, serum IMo and retinal macrophages were significantly reduced in the PVS-NP group compared to the PBS group (Figure 13). There were no significant differences in the proportion of microglia among the three treatment groups (Figure 13). Cone density at P52 was significantly maintained in the PVS-NP group compared to the PBS and FITC-NP groups (Figure 14). In addition, photopic ERG waves at P35 were significantly maintained in the PVS-NP group compared to the PBS group (Figure 15).

[0060] To determine the optimal dose of PVS-NP, rd10 mice were divided into four groups at P21 for dose-finding studies: PBS group (100 μl PBS), PVS-NP low group (0.1 mg PVS / kg), PVS-NP middle group (0.3 mg PVS / kg), and PVS-NP high group (1.0 mg PVS / kg). PVS-NP was administered intravenously via the tail vein once a week from P21 until the end of each experiment. Photopic ERG and PNA-positive cone cell counts were analyzed at P49. Photopic ERG b waves were significantly higher in the PVS-NP middle and PVS-NP high groups compared to the PBS group (p<0.05) (Figure 16). Similarly, regarding PNA-positive cone cell counts, cone degeneration was significantly suppressed in the PVS-NP middle and PVS-NP high groups compared to the PBS group (p<0.01) (Figures 17, 18).

[0061] To determine the optimal dosage of PVS-NP, the dosage was divided into three groups based on the concentration of the PVS-NP middle group (0.3 mg PVS / kg), and a dosage setting study was conducted: PBS (100 μl PBS) administered every two weeks, PVS-NP (0.75 mg PVS / kg) administered every four weeks, and PVS-NP (0.5 mg PVS / kg) administered every two weeks. From P21 until the end of each experiment, intravenous administration was performed via the tail vein at the respective administration intervals. On P49, the number of photopic ERGs and PNA-positive pyramidal cells was analyzed. The photopic ERG b wave was significantly higher in the PVS-NP (0.75 mg PVS / kg) administered every four weeks group compared to the PBS group (p<0.01) (Figure 19). Regarding the number of PNA-positive pyramidal cells, pyramidal degeneration was significantly suppressed in the PVS-NP (0.75 mg PVS / kg) administered every 4 weeks and the PVS-NP (0.5 mg PVS / kg) administered every 2 weeks compared to the PBS group (p<0.05) (Figure 20).

[0062] These results suggest that statin-encapsulated nanoparticles are a promising treatment option for retinitis pigmentosa.

Claims

1. A pharmaceutical composition comprising statin-encapsulated nanoparticles for the treatment of retinitis pigmentosa, wherein the statin is pitavastatin or a pharmaceutically acceptable salt thereof.

2. The pharmaceutical composition according to claim 1, wherein the statin-encapsulated nanoparticles contain 1 to 15% by weight of statin.

3. The pharmaceutical composition according to claim 1, wherein the statin-encapsulated nanoparticles contain PLGA.

4. The pharmaceutical composition according to claim 1, wherein the particle size of the statin-encapsulated nanoparticles is 50 to 300 nm.

5. The pharmaceutical composition according to claim 1, comprising 1 to 15% by weight of statin-encapsulated nanoparticles.

6. The pharmaceutical composition according to claim 1, administered at a daily dose of 0.01 mg / kg to 0.5 mg / kg of statin.

7. The pharmaceutical composition according to claim 1, which is administered intravenously.

8. The pharmaceutical composition according to claim 1, wherein the statin is pitavastatin calcium.

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