High-dose statins for age-related macular degeneration

Abstract

To provide a method of treating age-related macular degeneration (AMD) in a patient.SOLUTION: The method comprises: identifying a patient as having AMD; and administering to the patient a high-dose statin, where the high-dose statin is effective in regressing drusen in the patient.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Priority claims This application claims priority under 35 U.S.C. §119(e) to U.S. Patent Application No. 62 / 241,522, filed October 14, 2015, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to materials and methods of using high-dose statins for the treatment of age-related macular degeneration. [Background technology]

[0003] Age-related macular degeneration (AMD) is a multifactorial, heterogeneous disease with at least 100 different risk genes reported in the literature and several distinct phenotypes, including the type and size of drusen (Miller, 2013 Am J Ophthalmol 155(1): 1-35.e13). AMD is the leading cause of irreversible vision loss in the developed world and is broadly classified into atrophic or "dry" and neovascular or "wet" forms. Summary of the Invention

[0004] Provided herein are materials and methods of using high-dose statins for the treatment of age-related macular degeneration. While effective antiangiogenic treatments exist for neovascular AMD, effective treatments for the more prevalent dry form are lacking.

[0005] In some embodiments, the method provided herein comprises a method for treating age-related macular degeneration (AMD) in a patient. The method comprises identifying a patient as having AMD and administering a high-dose statin to the patient. The high-dose statin is effective in causing drusen regression in the patient. The method can comprise monitoring the patient for drusen regression. Drusen regression can be at least 85% reduction in drusen size or complete regression.

[0006] In some embodiments, the methods provided herein include the method for treating age-related macular degeneration (AMD) in patients.The method includes identifying the patient as having AMD and administering high-dose statin to the patient.High-dose statin is effective in preventing the atrophy of retinal pigment epithelium (RPE) in patients.

[0007] In some embodiments, the method provided herein comprises the method for treating age-related macular degeneration (AMD) in patients.The method comprises identifying the patient as having AMD and administering high-dose statin to the patient.High-dose statin is effective in preventing the atrophy of one or more photoreceptors in patients.

[0008] In some embodiments, the methods provided herein include the method for treating age-related macular degeneration (AMD) in patients.The method includes identifying the patient as having AMD and administering high-dose statin to the patient.High-dose statin is effective in preventing geographic atrophy.

[0009] In some embodiments, the methods provided herein include the method for treating age-related macular degeneration (AMD) in patients.The method includes identifying the patient as having AMD and administering high-dose statin to the patient.High-dose statin is effective in preventing vision loss in patients.

[0010] In some embodiments, the method provided herein comprises a method for treating age-related macular degeneration (AMD) in a patient.The method comprises identifying a patient as having AMD and administering a high-dose statin to the patient, wherein the high-dose statin is effective in improving the patient's visual acuity.Visual acuity can be improved by at least 3 letters.Visual acuity can be improved by at least 12 letters.

[0011] In some embodiments, the methods provided herein include the method for treating age-related macular degeneration (AMD) in patients.The method includes identifying the patient as having AMD and administering high-dose statin to the patient.High-dose statin is effective in preventing the progression to wet AMD in patients.

[0012] In any of the methods provided herein, the patient can be a mammal.For example, the patient can be a human.In some embodiments, the human has giant soft drusen and / or drusenoid PED in one or both eyes.

[0013] In any of the methods provided herein, the high-dose statin can be administered for at least 12 months. In some embodiments, the high-dose statin can be administered for at least 36 months.

[0014] In any of the methods provided herein, the high-dose statin can be administered orally.

[0015] In any of the methods provided herein, the high-dose statin may be selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, simvastatin, and analogs thereof. In some embodiments, the high-dose statin may comprise a dose equivalent to at least 40 mg of atorvastatin. The high-dose statin may be at least 40 mg of atorvastatin. In some embodiments, the high-dose statin may comprise a dose equivalent to at least 80 mg of atorvastatin. The high-dose statin may be at least 80 mg of atorvastatin.

[0016] In any of the methods provided herein, the method may further comprise administering an additional therapeutic agent selected from the group consisting of anti-inflammatory agents, anti-angiogenic agents, antioxidants, omega-3 fatty acids, and vitamins / minerals.

[0017] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs.Methods and materials for use in this disclosure are described herein, and other suitable methods and materials known in the art can also be used.Materials, methods, and examples are only examples and are not intended to be limiting.All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety.In the case of conflict, the present specification, including definitions, will control.In addition, materials, methods, and examples are only examples and are not intended to be limiting.

[0018] The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims. [Brief explanation of the drawings]

[0019] [Figure 1] Figure 1 shows a series of fundus examination images. At the start of the study, bilateral widespread confluent giant soft drusen and pigmentary changes are seen (top row). Administration of Age-Related Eye Disease Study (AREDS) supplementation did not affect the drusen (middle row). After 6 months of atorvastatin 80 mg, resolution of the drusen is seen (bottom row). [Figure 2] Figure 2 shows spectral-domain optical coherence tomography (SD-OCT). At the start of the study, structural distortion of the overlying RPE and photoreceptors is evident (top row). After 6 months of atorvastatin 80 mg, complete disappearance of drusen (Figure 1) is observed without concomitant atrophy of the RPE (bottom row). [Figure 3-1]Figure 3 shows that lipophilic statins increase the phagocytic function of ARPE-19 cells. (A-C) Microscopic images of ARPE-19 cells incubated with carboxylate microparticles (5 × 10 beads / ml) for 6 hours and then imaged using a confocal microscope collecting a large volume of 50 images. (A) Confocal image stack showing ARPE-19 cells with fluorescein-labeled beads and DAPI-labeled nuclei. The depth of image A within the stack is indicated by the blue line in B and C. (B) View through the same stack at the location indicated by the vertical line in A. (C) View through the same stack at the location indicated by the horizontal line in A. (D) Graph showing the percent of bead-positive cells, or phagocytic cells, represented on the Y-axis, or fluorescence intensity, or phagocytic index, represented on the X-axis, determined by flow cytometry of ARPE-19 cells incubated with fluorescein-labeled carboxylate microparticles and treated with 50 μM atorvastatin (ATV), lovastatin (LOV), or simvastatin (SIMV) for 6 hours. [Figure 3-2] Figure 3 shows that lipophilic statins increase the phagocytic function of ARPE-19 cells. (E) is a graph measuring the percentage of phagocytic cells. (F) is a graph measuring the mean fluorescence intensity (or phagocytic index). Data are expressed as mean ± SE. *p<0.05 vs. control group. [Figure 4] Figure 4 contains graphs showing that atorvastatin increases the phagocytic function of ARPE-19 cells. (A) Percentage of bead-positive cells, or phagocytic cells, represented on the Y-axis, and fluorescence intensity, or phagocytic index, represented on the X-axis, were determined by flow cytometry of ARPE-19 cells incubated with fluorescein-labeled carboxylate microparticles and treated with 1, 25, 50, or 75 μM atorvastatin for 6 hours. (B) Quantification of the percentage of phagocytic cells. (C) Quantification of the mean fluorescence intensity (or phagocytic index). Each experiment was repeated three independent times. Data are presented as the mean ± SE. *p<0.05 vs. the control group. [Figure 5-1]Figure 5 includes graphs showing that atorvastatin increases fluorescence recovery in the membrane of ARPE-19 cells after photobleaching. After 3 hours of treatment with 50 μM atorvastatin, FRAP measurements of ARPE-19 cells were performed using the dye BODIPY® FL C 12. (A) Mean recovery curves of BODIPY® FL C 12 in control (n=16 cells) or atorvastatin-treated (n=16 cells) ARPE-19 cells from two independent experiments. [Figure 5-2] Figure 5 contains graphs showing that atorvastatin increases fluorescence recovery in the membrane of ARPE19 cells after photobleaching. FRAP measurements of ARPE-19 cells were performed using the dye BODIPY® FL C 12 after 3 hours of treatment with 50 μM atorvastatin. (B) Mean normalized equilibrium half-time of BODIPY® FL C 12 in control (n=16 cells) or atorvastatin-treated (n=16 cells) ARPE19 cells from two independent experiments. Data are presented as mean ± SE. *p<0.05 vs. control group. [Figure 6]Figure 6 is a series of graphs showing that atorvastatin restores the impaired phagocytic function induced by cholesterol crystals and ox-LDL in ARPE-19 cells. (A) ARPE-19 cells were incubated with carboxylate microparticles (5 x 10 beads / ml), 1 or 2 mg / ml cholesterol crystals, with or without 50 μM atorvastatin for 6 hours, and the percent of phagocytic cells represented by bead-positive cells was quantified. (B) Quantification of the mean fluorescence intensity (or phagocytic index) determined by flow cytometry of ARPE-19 cells incubated with fluorescein-labeled carboxylate microparticles and treated with 1 or 2 mg / ml CHL for 6 hours, with or without co-incubation with 50 μM atorvastatin. (C) ARPE-19 cells were treated with 300 μg / ml ox-LDL for 18 hours and then incubated with carboxylate microparticles (5 × 10 beads / ml) with or without 50 μM atorvastatin for 6 hours. Quantification of the percentage of phagocytic cells, represented by bead-positive cells. (D) Quantification of the mean fluorescence intensity (or phagocytic index) determined by flow cytometry for ARPE-19 cells incubated with fluorescein-labeled carboxylate microparticles and treated with oxLDL with or without 50 μM atorvastatin for 18 hours. Data are expressed as mean ± SE. *p<0.05 vs. control group. §p<0.05 vs. 2 mg / ml CHL or oxLDL groups. [Figure 7-1] Figure 7 is a series of graphs showing that atorvastatin inhibits cholesterol crystal- and oxLDL-induced IL-6 and IL-8 secretion in ARPE-19 cells. IL-6 (A) and IL-8 (B) secretion from ARPE-19 cells was determined by Western blot of culture medium 6 hours after CHL treatment with or without pretreatment with 0.1, 0.5, or 1 μM atorvastatin. Data are presented as mean ± SE. *p<0.05 vs. CHL-treated group. [Figure 7-2]Figure 7 is a series of graphs showing that atorvastatin inhibits cholesterol crystal- and oxLDL-induced IL-6 and IL-8 secretion in ARPE-19 cells. IL-6 (C) and IL-8 (D) secretion from ARPE-19 cells 18 hours after oxLDL treatment with or without pretreatment with 0.1, 0.5, or 1 μM atorvastatin, as determined by Western blot and ELISA, respectively. Data are presented as mean ± SE. *p<0.05 vs. CHL-treated group. DETAILED DESCRIPTION OF THE INVENTION

[0020] Age-related macular degeneration (AMD) is the leading cause of irreversible vision loss in adults in Western countries (Wong et al., 2014 Lancet Glob Health 2(2):e106-16).

[0021] AMD is broadly classified into two types. The atrophic or "dry" type is the most prevalent and is characterized by the accumulation of extracellular deposits called drusen between the retinal pigment epithelium (RPE) and the choroid. Progression to advanced AMD may involve, for example, atrophy of the RPE and / or one or more photoreceptors, and / or abnormal choroidal neovascularization (neovascular or "wet" AMD). Although it is less prevalent than the dry type, neovascular AMD is associated with rapid vision loss. However, despite effective antiangiogenic treatments for neovascular AMD, effective treatments for the more prevalent dry type are lacking.

[0022] Provided herein is a method for using high-dose statin to treat AMD in patients.For example, provided method can be used to cause drusen (for example, soft drusen) to regress, cause drusenoid pigment epithelial detachment (PED) to regress, prevent RPE atrophy, prevent one or more photoreceptor atrophy, prevent vision loss, improve vision, and / or prevent dry AMD from progressing to wet AMD.

[0023] Drusen One of the hallmarks of dry AMD is the accumulation of drusen, which are components derived from the local tissue (RPE / retina) and from the circulation (Curcio et al., 2011 Br J Ophthalmol 95(12):1638-45; Wu et al., 2010 J Neurochem 114(6):1734-44). Drusenoid pigment epithelial detachment (PED) is also associated with AMD, in which the retinal pigment epithelium separates from the underlying Bruch's membrane due to the presence of one or more drusen.

[0024] Drusen can be hard or soft. "Hard" drusen are small, clear, and far apart, and may not cause long-term vision problems, if any. "Soft" drusen have unclear margins, are large, and are tightly packed together. Lipids are the main constituents of drusen, with esterified cholesterol (EC), unesterified cholesterol (UC), and phosphatidylcholine accounting for 40% of the volume of hard drusen. Soft drusen are more fragile than hard drusen and, consistent with their high lipid composition, are oily upon excision. The presence of soft drusen is one of the major risk factors for the subsequent development of advanced dry or wet AMD. In some embodiments, the drusen treated by the methods described herein are soft drusen.

[0025] Statins The methods described herein involve the administration of high-dose statins. Statins (or HMG-CoA reductase inhibitors) are a class of cholesterol-lowering drugs that are structurally similar to HMG-CoA and are shown below. [ka]

[0026] Statins inhibit the enzyme HMG-CoA reductase by competitively binding to HMG-CoA reductase at the HMG-CoA active site. Any statin may be used in the methods described herein. Non-limiting examples of statins include atorvastatin (LIPITOR®), cerivastatin, fluvastatin (LESCOL®), lovastatin (MEVACOR®, ALTOCOR™), pitavastatin (LIVALO®), pravastatin (PRAVACHOL®, SELEKTINE®), rosuvastatin (CRESTOR®), simvastatin (ZOCOR®), analogs thereof, and combinations thereof. In some embodiments, the statin is atorvastatin.

[0027] Statins can be either lipophilic or hydrophilic. Lipophilic statins include, for example, atorvastatin, lovastatin, and simvastatin. Hydrophilic statins include, for example, fluvastatin, rosuvastatin, and pravastatin. In some embodiments, the statin is lipophilic (e.g., atorvastatin).

[0028] The term "high dose" as used herein refers to any dose that exceeds the daily dose (DDD) defined according to the World Health Organization (WHO). The 2015 ATC / DDD Index indicates the DDD as 20 mg for atorvastatin, 0.2 mg for cerivastatin, 60 mg for fluvastatin, 45 mg for lovastatin, 2 mg for pitavastatin, 30 mg for pravastatin, 10 mg for rosuvastatin, and 30 mg for simvastatin (see, e.g., whocc.no / atc_ddd_index / ). For example, in embodiments where the statin is atorvastatin (having a DDD of 20 mg), the high dose of atorvastatin can be at least 40 mg, at least 50 mg, at least 60 mg, at least 70 mg, at least 80 mg, at least 90 mg, or at least 100 mg. In some embodiments, the high-dose statin is at least 80 mg of atorvastatin.

[0029] In other embodiments, the dose equivalent of high-dose atorvastatin can be used.The equivalent dose of other statins can be easily determined by those skilled in the art.For example, based on the DDD of statins, the equivalent dose of 80mg of atorvastatin can be 0.8mg for cerivastatin, 240mg for fluvastatin, 180mg for lovastatin, 8mg for pitavastatin, 120mg for pravastatin, 40mg for rosuvastatin and 120mg for simvastatin.

[0030] Also provided herein is a method of using maintenance dose statin.For example, following the effective treatment of AMD, the amount of statin administered can be reduced from high dose statin to maintenance dose statin.As used herein, " maintenance dose " refers to the dose of statin that is less than high dose and is approximately equal to the WHO DDD of statin.For example, the maintenance dose of atorvastatin (with DDD of 20mg) can be about 15mg, about 20mg, about 25mg, about 30mg, about 35mg, about 40mg, about 45mg, about 50mg, about 55mg or about 60mg.In some embodiments, the maintenance dose of statin is about 60mg of atorvastatin.In some embodiments, the maintenance dose of statin is about 40mg of atorvastatin.

[0031] In other embodiments, the dose equivalent of maintenance dose atorvastatin can be used.The equivalent dose of other statins can be easily determined by those skilled in the art.For example, based on the DDD of statins, the equivalent maintenance dose of 40mg of atorvastatin can be 0.4mg for cerivastatin, 120mg for fluvastatin, 90mg for lovastatin, 4mg for pitavastatin, 60mg for pravastatin, 20mg for rosuvastatin and 60mg for simvastatin.

[0032] How to use Provided herein is a method of using high-dose statins to treat AMD in patients. In some embodiments, the present disclosure provides a method of causing regression of drusen (e.g., soft) and / or drusenoid PED in patients. In some embodiments, the present disclosure provides a method of preventing (i.e., reducing the risk of) atrophy of RPE and / or one or more photoreceptors in patients. In some embodiments, the present disclosure provides a method of preventing (i.e., reducing the risk of) vision loss and / or improving vision in patients. In some embodiments, the present disclosure provides a method of preventing (i.e., reducing the risk of) AMD progression (e.g., from dry AMD to wet AMD) in patients. The methods provided herein may include administering to patients a high-dose statin as described herein. The methods provided herein may also include continuously administering to patients a maintenance-dose statin as described herein. The method provided herein can also include identifying patients as having AMD (for example, dry AMD) or soft drusen, and optionally selecting patients based on the criteria that they have AMD or soft drusen.The method of diagnosing AMD and drusen in patients is known in the art.The method provided herein can also include monitoring patients for the effectiveness of administering high-dose statin described herein to patients (for example, monitoring patients for drusen regression, atrophy of RPE and / or one or more photoreceptors, vision loss and / or visual acuity, AMD progression).

[0033] The patient may include both mammals and non-mammals. Non-limiting examples of mammals include, for example, humans, non-human primates (e.g., apes and monkeys), cows, horses, sheep, rats, mice, pigs, and goats. Non-limiting examples of non-mammals include, for example, fish and birds. In some embodiments, the patient is a human. In some embodiments, the patient is a human with AMD with high-risk characteristics for progression (e.g., the presence of many large soft drusen and / or drusenoid PEDs in one or both eyes).

[0034] The administration of high-dose and / or maintenance-dose statin described herein to patients can include administering statin for an appropriate period of time.For example, the high-dose statin described herein can be administered to patients for at least 12 months, at least 15 months, at least 18 months, at least 21 months, at least 24 months, at least 30 months, at least 33 months, at least 36 months, at least 39 months, at least 42 months, at least 45 months, or at least 48 months.In some embodiments, the high-dose and / or maintenance-dose statin described herein is administered to patients for at least 36 months.

[0035] Administration of the high-dose and / or maintenance-dose statins described herein to patients can include administering the statin once or multiple times daily (e.g., once daily, twice daily, three times daily, etc.), provided that the total daily amount conforms to the daily doses described herein. For example, a high-dose statin (e.g., atorvastatin 80 mg) can be administered once daily at full high-dose statin, twice daily with two doses combined to total full high-dose statin, three times daily with three doses combined to total full high-dose statin, etc. In some embodiments, the high-dose and / or maintenance-dose statins described herein are administered to patients once daily.

[0036] The high-dose and / or maintenance dose statins described herein can be administered by any route, such as intravenous (IV), ocular (e.g., intravitreal, topical drops, or topical ointment), intramuscular, subcutaneous, oral, intranasal, inhalation, transdermal, and parenteral.In some embodiments, the statins described herein are administered orally.In some embodiments, the statins described herein are administered intravenously.

[0037] For oral administration, the high-dose and / or maintenance-dose statins described herein can be in the form of pills, tablets, powders, liquids, capsules, or other suitable oral dosage forms. Tablets or capsules can be prepared with pharmaceutically acceptable excipients such as binders, fillers, lubricants, disintegrants, or wetting agents. Liquid preparations for oral administration can take the form of, for example, solutions, syrups, or suspensions, or they can be provided as dry products to be reconstituted with saline or other suitable liquid vehicle before use.

[0038] The specific dosages of the high-dose and / or maintenance-dose statins described herein will, of course, be determined by the particular circumstances of each individual patient, including the patient's size, weight, age, and sex, the nature and stage of the disease being treated, the aggressiveness of the disease condition, and the route of administration of the compound.

[0039] A "therapeutically effective amount" of a high-dose statin (e.g., high-dose atorvastatin) described herein is typically sufficient to achieve a desired effect (e.g., causing regression of drusen and / or drusenoid PED, reducing the amount of immunogenic and / or toxic substances, preventing atrophy, preventing vision loss, improving vision, preventing progression, etc.), and may vary according to the nature and severity of AMD and the efficacy of the high-dose statin described herein.

[0040] An "effective amount" of a maintenance dose statin (e.g., maintenance dose atorvastatin) described herein is typically sufficient to prevent any further changes in the measured parameters and may vary according to the nature and severity of AMD and the efficacy of the maintenance dose statin described herein.

[0041] As used herein, " preventing " refers to reducing the risk of developing or progressing a condition in a subject, just as a therapeutic treatment does not need to cure 100% of subjects or symptoms to be effective and clinically useful, and a preventive treatment does not need to eliminate 100% of the entire risk of developing or progressing a condition in a subject.Therefore, a treatment that "prevents" RPE atrophy, one or more photoreceptor atrophy, vision loss, and / or dry AMD to wet AMD progression reduces the risk of RPE atrophy, one or more photoreceptor atrophy, vision loss, and / or dry AMD to wet AMD progression in a subject.

[0042] The method for causing regression of drusen and / or drusenoid PED per patient can comprise administering high-dose statin as described herein to the patient.The regression of drusen and / or drusenoid PED can be assessed by any suitable method, including, for example, ophthalmic examination such as biomicroscopy, three-dimensional fundus examination (e.g., color fundus photography), macular function assessment, optical coherence tomography (OCT), autofluorescence, and / or angiography (e.g., fluorescein angiography).The administration of high-dose statin as described herein can be effective in reducing the size of drusen in patients compared with patients with AMD who do not receive high-dose statin as described herein. For example, drusen regression can be at least 5%, at least 10%, at least 15%, 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. Drusen can be assessed by measuring parameters such as volume, height, diameter, and / or number. Thus, drusen regression can be assessed by determining certain parameters of drusen in a patient at a first time point (e.g., prior to administration of a high-dose statin), determining the same parameters in the same patient at a second time point (e.g., after administration of a high-dose statin), and comparing the parameters measured at the first and second time points. A reduction in the measured parameter from the first time point to the second time point is indicative of drusen regression. In some embodiments, administration of a high-dose statin can be effective in reducing drusen by at least 40%. In some embodiments, administration of a high-dose statin as described herein can be effective in causing complete regression (i.e., 100% regression) of drusen. Administration of a maintenance dose statin as described herein can be effective in preventing drusen from expanding and / or forming in patients.

[0043] Drusenoid pigment epithelial detachment (PED), in which the retinal pigment epithelium separates from the underlying Bruch's membrane due to the presence of one or more drusen, is also associated with AMD. The method for regressing drusenoid PED can result in the resolution of drusenoid PED by PED normalization and / or reattachment to Bruch's membrane. The resolution of drusenoid PED can be assessed by any suitable method, including, for example, OCT, angiography (e.g., fluorescein angiography), autofluorescence, and / or macular status. The administration of high-dose statins as described herein can be effective in regressing drusenoid PED in patients compared with patients with AMD who do not receive high-dose statins as described herein. The administration of high-dose statins as described herein can be effective in regressing PED and / or reattaching PED to Bruch's membrane. For example, PED can be normalized by at least 5%, at least 10%, at least 15%, 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. For example, separation of PED and Bruch's membrane can be reduced by at least 5%, at least 10%, at least 15%, 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%. The PED can be assessed for normalization, e.g., by measuring parameters such as volume, height, and / or diameter, and for reattachment to Bruch's membrane, e.g., by measuring parameters such as distance of separation.Thus, PED normalization and / or reattachment to Bruch's membrane can be assessed by determining certain parameters of drusen in a patient at a first time point (e.g., prior to administration of a high-dose statin), determining the same parameters in the same patient at a second time point (e.g., after administration of a high-dose statin), and comparing the parameters measured at the first and second time points. A decrease in the measured parameter from the first time point to the second time point is indicative of regression of drusenoid PED (e.g., PED normalization and / or reattachment to Bruch's membrane). In some embodiments, the separation between the PED and Bruch's membrane can be reduced by at least 30%. Administration of a maintenance dose statin as described herein can be effective in preventing PED enlargement and / or formation in a patient.

[0044] Without being bound by theory, it is believed that the regression of resulting drusen and / or drusenoid PED enhances the phagocytic function of RPE cells.Phagocytic function can be evaluated by any suitable method, including, for example, flow cytometry.The administration of high-dose statins described herein can be effective in enhancing the phagocytic function of RPE cells in patients compared with patients with AMD who have not received high-dose statins described herein.For example, the proportion of phagocytic cells can be increased by at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.The administration of maintenance-dose statins described herein can be effective in preventing PED from expanding and / or forming in patients.

[0045] Drusen are known to contain several immunogenic and toxic substances, such as complement (Crabb, 2014 Cold Spring Harb Perspect Med 4(7):a017194-4), 7-ketocholesterol (Rodriguez et al., 2014 Exp Eye Res 128:151-5), and amyloid (Luibl et al., 2006 J Clin Invest 116(2):378-85). Administration of high-dose statins as described herein can be effective in reducing the amount of immunogenic and / or toxic substances in patients compared to patients with AMD who do not receive high-dose statins as described herein. Administration of maintenance-dose statins as described herein can be effective in preventing an increase in the amount of immunogenic and / or toxic substances in patients.

[0046] A method for preventing atrophy of the RPE and / or one or more photoreceptors in a patient may include administering a high-dose statin described herein to the patient. The RPE interacts closely with and provides nutrients to photoreceptors. Drusen are located between the RPE and its vascular supply, the choriocapillaris. Without being bound by theory, drusen may deprive the RPE and photoreceptor cells of oxygen and nutrients, which may lead to atrophy of both the RPE and photoreceptors. Atrophy of both the RPE and photoreceptors is typically referred to as geographic atrophy. Prevention of atrophy can be assessed by any suitable method, including ophthalmic examinations such as biomicroscopy, three-dimensional fundus examination (e.g., color fundus photography), macular function assessment, OCT, autofluorescence, and / or angiography (e.g., fluorescein angiography). The administration of high-dose statin as described herein can be effective in reducing the amount of atrophy of RPE and / or one or more photoreceptors in patients compared with patients with AMD who do not receive high-dose statin as described herein.For example, the amount of atrophy can be reduced by at least 5%, at least 10%, at least 15%, 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99%.Atrophy can be evaluated by measuring parameters such as autofluorescence or retinal thickness, for example. Thus, the amount of atrophy can be assessed by determining a particular parameter of drusen in a patient at a first time point (e.g., prior to administration of a high-dose statin), determining the same parameter in the same patient at a second time point (e.g., after administration of a high-dose statin), and comparing the parameter measured at the first and second time points. A maintenance or reduction in the parameter measured from the first time point to the second time point is indicative of prevention of atrophy (e.g., atrophy of the RPE and / or one or more photoreceptors).In some embodiments, the methods provided herein can be used to prevent both RPE and photoreceptor atrophy in patients.Administering the maintenance dose statin described herein can be effective in preventing the increase in the amount of atrophy of RPE and / or one or more photoreceptors in patients.

[0047] In some embodiments, administration of high-dose statins described herein can be effective in causing complete disappearance of drusen (i.e., 100% regression) in patients and preventing atrophy of both the RPE and photoreceptors (i.e., geographic atrophy).

[0048] A method for preventing vision loss and / or improving vision in a patient can include administering a high-dose statin described herein to the patient. Vision can be assessed by any suitable method, including, for example, determining the smallest letter the patient can read on a standard chart (e.g., a Snellen chart) or card held at a distance of about 20 feet. In some embodiments, the methods provided herein can be used to prevent vision loss in a patient. Administration of a high-dose statin described herein can be effective in preventing vision loss in a patient compared to a patient with AMD who has not received a high-dose statin described herein. For example, administration of a high-dose statin described herein can be effective in preventing vision loss by losing 5 letters or less, 4 letters or less, 3 letters or less, 2 letters or less, or 1 letter or less. In some embodiments, administration of a high-dose statin described herein is effective in preventing vision loss by losing 2 letters or less. In some embodiments, administration of a statin described herein is effective in preventing vision loss by maintaining vision (i.e., no detectable loss of vision). Administration of a maintenance dose statin described herein can be effective in preventing vision loss in a patient.

[0049] In some embodiments, the method provided herein can be used to improve patient's visual acuity.The administration of high-dose statin as described herein can be effective in improving patient's visual acuity compared with patients with AMD who do not receive high-dose statin as described herein.For example, the administration of high-dose statin as described herein can be effective in improving visual acuity by at least 2 letters, at least 3 letters, at least 4 letters, at least 5 letters, at least 6 letters, at least 7 letters, at least 8 letters, at least 9 letters, at least 10 letters, at least 11 letters, at least 12 letters, at least 13 letters, at least 14 letters, at least 15 letters.In some embodiments, the administration of high-dose statin as described herein is effective in improving visual acuity by 3 letters.In some embodiments, the administration of high-dose statin as described herein is effective in improving visual acuity by 12 letters.

[0050] A method for preventing AMD progression (e.g., from dry AMD to wet AMD) in a patient can include administering a high-dose statin described herein to the patient. Prevention of AMD progression can be assessed by any suitable method, including, for example, eye examinations such as biomicroscopy, intraocular pressure measurement, three-dimensional fundus examination (e.g., color fundus photography), macular function assessment, OCT, and / or angiography (e.g., fluorescein angiography and OCT-based angiography (OCTA)). Administration of a high-dose statin described herein can be effective in preventing progression to wet AMD in a patient compared to patients with AMD who do not receive a high-dose statin described herein. In some embodiments, administration of a high-dose statin described herein can be effective in preventing progression from dry AMD to wet AMD. Administration of a high-dose statin described herein can be effective in preventing progression to wet AMD. For example, progression to wet AMD can include, for example, abnormal choroidal neovascularization. For example, the administration of high-dose statin described herein can be effective in reducing and / or preventing choroidal neovascularization.The progression of AMD can be evaluated by measuring parameters such as neovascularization.Therefore, the progression of AMD is evaluated by determining certain parameters of drusen in a patient at a first time point (for example, before administering high-dose statin), determining the same parameters in the same patient at a second time point (for example, after administering high-dose statin), and comparing the parameters measured at the first time point and the second time point.The maintenance or reduction of the parameters measured from the first time point to the second time point is an indication of the prevention of AMD progression (for example, from dry AMD to wet AMD).

[0051] The method provided herein can also include the administration of additional therapeutic agents in addition to statin.As used herein, additional therapeutic agents include any molecule that can have therapeutic effect on AMD.Examples of therapeutic agents include anti-inflammatory agents (for example, anti-IL-6 agents, anti-IL-8 agents, aspirin, ibuprofen and naproxen), angiogenesis inhibitors (for example, anti-VEGF agents, ranibizumab, bevacizumab, acadesine and AMPK activators), antioxidants (for example, vitamin C, vitamin E, vitamin A, glutathione, catalase, etc.), omega-3 fatty acids (for example, alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)), and vitamins / minerals (for example, vitamin C, vitamin E, vitamin A, lutein, zeaxanthin, zinc and copper).

[0052] The therapeutic agents described herein can be administered by any route (e.g., intraocular, intravenous (IV), intramuscular, subcutaneous, oral, ocular (e.g., intravitreal, topical drops, or topical ointment), intranasal, inhalation, transdermal, and parenteral), and by any method (e.g., injection, pump (e.g., implantable pump), etc.). In some embodiments, the active agents described herein are administered orally.

[0053] The high-dose statin (e.g., high-dose atorvastatin) and the therapeutic agent described herein can be administered to a patient independently or simultaneously. When the high-dose statin and the therapeutic agent are administered to a patient simultaneously, the high-dose statin and the therapeutic agent can be in a single dosage form, or the high-dose statin and the active agent can be in separate dosage forms.

[0054] The methods provided herein can also include administering a maintenance dose of statin.For example, after effective treatment of AMD (for example, regression of drusen), the administration of high-dose statin described herein can be replaced with the administration of a maintenance dose of statin.

[0055] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims. [Example]

[0056] Example 1: High-dose statins for regression of drusen and improvement of visual acuity

[0057] This example provides the first evidence that treatment with high-dose atorvastatin results in regression of drusen and improvement of visual acuity in patients with AMD who have high-risk features for progression.

[0058] method We conducted a case report and pilot multicenter prospective intervention study with IRB approval. Patients over 50 years of age were required to have a diagnosis of AMD and the presence of numerous large soft drusen / drusenoid PEDs in both eyes. Exclusion criteria included the following: the presence (or history) of significant geographic atrophy or choroidal neovascularization in either eye; other ocular diseases (except mild cataracts) that could reduce vision; a history of ophthalmic surgery (other than cataract extraction); current or recent (within 2 years) statin therapy at a dose equivalent to atorvastatin 40 mg; a history of liver disease, rhabdomyolysis, or allergy to statins; pregnancy or breastfeeding; current use of medications known to interact with statins (e.g., cyclosporine, systemic itraconazole, clarithromycin, HIV protease inhibitors); and elevated transaminase or creatinine kinase levels at baseline. Pseudophakia was not a reason for exclusion unless accompanied by significant posterior capsule opacification.

[0059] result Initial Event Report An otherwise healthy 63-year-old man with AMD presented with deteriorating visual acuity. His baseline visual acuity was 20 / 25 with significant distortion; he was already receiving Age-Related Eye Disease Study (AREDS) supplementation. Fundus examination revealed bilateral extensive confluent giant soft drusen and pigmentary changes (Figure 1, top row). Spectral-domain optical coherence tomography (SD-OCT) confirmed significant degrees of these deposits and pigment epithelial detachment, as well as architectural distortion of the overlying RPE and photoreceptors (Figure 2, top row). No subretinal or intraretinal fluid was present. Standard AREDS supplementation was continued. One year later, the patient became more symptomatic, and his visual acuity significantly decreased to 20 / 30. The patient was initiated on atorvastatin, with a planned stepwise increase in dosage from an initial daily test dose of 10 mg to a target daily dose of 80 mg over a 9-month period. After 6 months of atorvastatin 80 mg, visual acuity improved to 12 letters, 20 / 20, and fundus examination and SD-OCT revealed complete resolution of drusen (Figure 1) without concomitant RPE atrophy (Figure 2).

[0060] Pilot study Of the 26 patients enrolled in the pilot study, 23 achieved this. Three patients dropped out of the study: one due to muscle spasms, one due to muscle pain, and one because the patient felt the drug was inducing hair loss.

[0061] Ten of the 23 patients (Table 1) responded to treatment, showing regression of drusen deposits, with 8 patients showing near-complete regression. On average, responders gained 3 letters, while nonresponders lost 2.2 letters. The mean time to response was 11.7 months (range, 3-22). The mean person-years of follow-up was approximately 30. No patients converted to neovascular AMD. According to an online risk calculator (based on Klein et al., 2011 Arch Ophthalmol. 129(12):1543-50, available on the World Wide Web at caseyamdcalc.ohsu.edu), we should have expected 14% of our cases (3-4 patients out of 23) to convert to neovascular AMD. [Table 1]

[0062] Responders were older than non-responders (mean age 70.6 vs. 66.2) and had similar baseline cholesterol levels. Cholesterol level reduction did not appear to correlate with response status. Responders tended to be more likely to use multivitamins and consume less alcohol, while there were no apparent differences in aspirin use, fish oil consumption, or antihypertensive medications. Almost no smokers were present in our study, and therefore we were unable to assess the effects of smoking. High-dose atorvastatin did not appear to have a positive or negative effect on pigmentary changes or the progression of existing atrophy.

[0063] Despite the high-risk characteristics of our patient cohort, none of them progressed to neovascular or wet AMD.

[0064] Consideration These results indicate that high-dose statin treatment can be used to treat high-risk characteristics of dry AMD and prevent progression to atrophy or vision loss. Example 2: Atorvastatin promotes phagocytosis and attenuates pro-inflammatory responses in human retinal pigment epithelial cells

[0065] Methods and Materials material The human RPE cell line ARPE-19 was purchased from ATCC (Manassas, VA, USA). DMEM / F-12, HEPES medium, fetal bovine serum (FBS), and penicillin-streptomycin were obtained from Life Technologies (Grand Island, NY, USA). Recombinant human IL-1α was obtained from R&D Systems (Minneapolis, MN, USA). Cholesterol crystals were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-IL-18, anti-IL-6, and anti-β-actin antibodies were obtained from ABeam (Cambridge, MA, USA). Anti-IL-1β and anti-IL-8 antibodies were purchased from R&D Systems (Minneapolis, MN, USA). HRP-conjugated secondary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA).

[0066] Statins Atorvastatin (atorvastatin calcium trihydrate), simvastatin, and lovastatin (mevinolin from Aspergillus sp.) were purchased from Sigma-Aldrich (St. Louis, MO, USA). They were reconstituted in dimethyl sulfoxide (DMSO; ATCC, Manassas, VA, USA). A 5 mM stock solution of each statin was prepared, and further dilutions were made in culture medium. Control cultures were incubated with DMSO at a final concentration corresponding to the highest concentration added with the statin.

[0067] cell culture ARPE-19 cells were maintained in DMEM / F-12, HEPES medium supplemented with 10% FBS, 100 U / mL penicillin, and 100 μg / mL streptomycin. Cells were grown at 37°C in a humidified 5% CO atmosphere and passaged when they reached 80% confluence.

[0068] Preparation of cholesterol crystal solution Cholesterol crystals were pulverized in a grinder and then sterilized with ultraviolet light for 30 minutes. ARPE-19 culture medium was added to the cholesterol crystals to prepare a 6 mg / mL stock solution. The stock solution was sonicated until the cholesterol crystals were uniformly suspended in the culture medium.

[0069] Preparation of oxidized LDL LDL (Lee BioSolutions, Maryland Heights, MO, USA) was oxidized using CuSO4 (Sigma, St. Louis, MO, USA) as described elsewhere (Hendriks et al., 1996 Biochem J 314 (Pt 2):563-8). Briefly, LDL (600 μl, 0.25 mg / ml), CuSO4 (22.5 μl, 1.6 mM), and PBS (277.5 μl) were mixed and incubated at 37°C. After 24 h of incubation, the oxidation reaction was stopped using 1 mM EDTA. Immediately after oxidation, lipoproteins were desalted using PD-10 disposable desalting columns (GE Healthcare, Buckinghamshire, UK).

[0070] Phagocytosis analysis by flow cytometry Flow cytometry assays were used to assess phagocytosis according to the protocol described by Pranab (Mukherjee et al., 2007 Proc Natl Acad Sci USA 104:13158-63). Briefly, ARPE-19 cells were seeded in 12-well plates and cultured until 90% confluent. 5 × 10 7Cells were incubated with 1 μm diameter Fluoresbrite® YG Carboxylate Microspheres (Polysciences, PA, USA) at 1 μm / ml alone or in combination with statins (atorvastatin, lovastatin, or simvastatin), cholesterol crystals, or ox-LDL. The drug concentrations and exact duration of treatment are described separately for each experiment in the Results section. After incubation, cells were washed three times with PBS to remove all extracellular beads, then trypsinized (0.25% trypsin-EDTA, Gibco, USA) for 1 minute and neutralized with prewarmed culture medium. The cell suspension was collected and centrifuged (241 g, 5 minutes). For each sample, the cell pellet was resuspended in 0.5 ml of PBS. Cells were then analyzed for engulfed green fluorescent beads (excitation wavelength 441 nm, emission wavelength 486 nm) on a FACScalibur flow cytometer using CellQuest 3.0.1 (Becton Dickinson, Mountain View, CA, USA) and FlowJo 10.0 software. The percentage of phagocytic cells present in each group, as well as the mean fluorescence intensity of the enclosed particles, were recorded.

[0071] Fluorescence recovery after photobleaching (FRAP) for assessing cell membrane fluidity ARPE-19 cells were cultured on 35 mm glass-bottom dishes (MatTec Corporation, MA) in DMEM / F12 medium supplemented with 10% FBS and 1% penicillin / streptomycin. 70–80% confluent cells were treated with 50 μM atorvastatin for 3 hours. Thirty minutes before measuring membrane fluidity, cells were incubated with 5 μM 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diazas-indacene-3-dodecanoic acid (BODIPY® FL C12) (Thermo Fisher Scientific), a green fluorophore with a 12-carbon saturated hydrocarbon tail, dissolved in FluoroBrite™ DMEM medium (Thermo Fisher Scientific). Cells were then washed with FluoroBrite™ DMEM medium to remove any unincorporated dye and maintained at 37°C and 5% CO2 using an environmental chamber attached to the microscope stage for FRAP measurements. FRAP measurements were performed by photobleaching fine regions of the cell membrane (bleach Regions of Interest (ROIs) were drawn using a 7x7 pixel square) with short (2 s) focused pulses of light from an argon laser (458, 477, 488, and 514 nm lines simultaneously at 100% transmittance) through a 63x, 1.4 numerical aperture oil immersion lens on a Zeiss LSM 510 Axiovert 200M confocal laser scanning microscope with an additional 2x digital zoom (total magnification = [10x][63x][2x] = 1,260x). Recovery of fluorescence within the photobleached region due to lateral diffusion of adjacent intact fluorophores was assessed by repeatedly scanning the cell surface (every 500 ms) with an attenuated laser beam (488 nm line at 3% transmittance).

[0072] Western blot 1 x 10 in a 6-well plate 5ARPE-19 cells were seeded at a density of 1000 cells / well. After 24 hours, the cells were stimulated with IL-1α (5 ng / ml) for 8 hours, treated with atorvastatin (0.1, 0.5, or 5 μM) for 16 hours, and then incubated with either 2 mg / ml cholesterol crystals for 6 hours or 300 μg / ml oxLDL for 18 hours, with or without atorvastatin. After treatment, the culture medium was collected and centrifuged at 13.3 g for 15 minutes at 4°C. The supernatant was collected and stored at -80°C. Culture medium (for IL-6 and IL-8) was added to each lane, and the samples were electrophoresed. Proteins were transferred to PVDF membranes, blocked with nonfat milk, and incubated with primary antibodies against IL-6 and IL-8. The membranes were then washed and incubated with secondary antibodies. The membrane was developed with enhanced chemiluminescence, and the intensity of the protein bands was measured using the software Image Lab 4.1 (Bio-Rad, Hercules, CA, USA).

[0073] Enzyme-linked immunosorbent assay (ELISA) oxLDL-induced IL-8 was measured by analysis of conditioned medium from ARPE-19 cells using an ELISA kit (IL-8, R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions.

[0074] statistical analysis All experiments were performed in triplicate. Statistical analysis was performed using GraphPad Prism 5.0a. Results are expressed as mean ± SE. Statistically significant differences between two treatment groups were analyzed by unpaired t-test. A value of p<0.05 was set as statistically significant.

[0075] result Lipophilic statins enhance the phagocytic function of ARPE-19 cells To validate a method for reliably measuring the phagocytic function of ARPE-19 cells, cells were incubated with carboxylate microparticles for 6 hours and then imaged using a confocal microscope, collecting a large set of 50 images. The cells were able to actively absorb the particles, which were located proximal to the nucleus (Figure 3A-C), demonstrating that polystyrene microparticles can be phagocytosed by ARPE-19 cells under these established conditions and that this method can accurately assess the phagocytic function of ARPE-19 cells.

[0076] To examine the effects of lipophilic but not hydrophilic statins on the phagocytic function of human RPE cells, ARPE-19 cells were incubated with polystyrene microparticles containing 50 μM of either atorvastatin, lovastatin, or simvastatin. The percentage of phagocytosed ARPE-19 cells per group and the mean fluorescence intensity of the enclosed particles were measured by flow cytometry 6 hours after treatment. As shown in Figures 3D and 3E, treatment with atorvastatin, lovastatin, or simvastatin significantly increased the percentage of ARPE-19 phagocytic cells from 31% to 43%, 53%, and 56%, respectively (p<0.05). Similarly, the mean fluorescence intensity within ARPE-19 cells increased (p<0.05) from 332 to 455, 502, and 513, respectively (Figure 3F). This showed that all three statins increased the phagocytic function of ARPE-19 cells, with simvastatin having the most potent effect, followed by lovastatin and atorvastatin.

[0077] Atorvastatin increases the phagocytic function of ARPE-19 cells in a dose-dependent manner High-dose atorvastatin resulted in the regression of drusen deposits and improved visual acuity in selected AMD patients (Example 1; Vavvas et al., 2016 EBioMedicine 5:198-203). To further investigate the effect of high-dose atorvastatin on the phagocytic function of ARPE-9 cells, cells were incubated with both microparticles and different doses of atorvastatin for 6 hours, and the percentage of phagocytic RPE cells was assessed by flow cytometry as previously described. As shown in Figures 4A and 4B, 1, 25, 50, or 75 μM atorvastatin increased the percentage of phagocytic ARPE-9 cells to 35%, 40%, 42%, and 46%, respectively, compared to the baseline percentage (31%) of the DMSO control group (p<0.05). In addition, treatment of cells with 1, 25, 50, or 75 μM atorvastatin significantly increased the mean fluorescence intensity of the cells from 340 in the control group to 368, 443, 453, and 467, respectively (p<0.05) (Figure 4C). These data clearly demonstrate that atorvastatin increases the proportion of phagocytic cells and the phagocytic activity of ARPE-19 cells in a dose-dependent manner.

[0078] Atorvastatin increases membrane fluidity in ARPE-19 cells To test the effect of atorvastatin on RPE cell membrane fluidity, ARPE-19 cells were incubated with 50 μM atorvastatin for 3 hours and with BODIPY® FL C12 for 30 minutes. FRAP measurements were performed by photobleaching a small area of ​​the cell membrane, and the recovery of fluorescence within the bleached area was assessed by repeatedly scanning the cell surface with an attenuated laser beam, as described in the methods. The results showed that atorvastatin treatment increased the recovery of fluorescence at the membrane of ARPE-19 cells after photobleaching (Figure 5A). In addition, it reduced the half-life of fluorescence equilibration compared to the control group (p<0.05) (Figure 5B). Both findings indicate that atorvastatin increases the membrane fluidity of ARPE-19 cells. This increase could at least partially explain the increased phagocytic function of ARPE-19 cells after atorvastatin treatment.

[0079] Atorvastatin protects ARPE-19 phagocytic function from cholesterol crystal- and ox-LDL-induced impairment The effects of cholesterol crystals and ox-LDL on the phagocytic function of RPE cells were investigated, as dysfunction of this function has been linked to AMD (Nandrot et al., 2007 Adv Exp Med Biol 801:978-1). ARPE-19 cells were treated with 2 mg / ml cholesterol crystals for 6 hours or 300 μg / ml oxLDL for 18 hours. Phagocytic function was then assessed by flow cytometry as described in the methods. Cholesterol crystals significantly reduced the percentage of phagocytic cells from 31% to 22% (p<0.05) (Figure 6A) and the mean fluorescence intensity of ARPE-19 cells from 332 to 280 (p<0.05) (Figure 6B). Similarly, ox-LDL reduced the percentage of phagocytic cells from 31% to 26% (p<0.05) (Figure 6C) and the mean fluorescence intensity of cells from 325 to 290 (p<0.05) (Figure 6D).

[0080] Because atorvastatin enhances phagocytic function in ARPE-19 cells, we investigated whether it could rescue and preserve the phagocytic properties of these cells despite the impairment induced by cholesterol crystals and ox-LDL. Pretreatment of cells with 50 μM atorvastatin for 6 h completely reversed the decrease in the percentage of phagocytic cells and mean fluorescence intensity induced by cholesterol crystals (Figures 6A and B) and ox-LDL (Figures 6C and D).

[0081] Taken together, these results suggest that atorvastatin protects the phagocytic function of ARPE-19 cells from the impairment induced by cholesterol crystals and ox-LDL.

[0082] Atorvastatin inhibits cholesterol crystal- and ox-LDL-induced IL-6 and IL-8 secretion in ARPE-19 cells Cholesterol crystals induce the secretion of the proinflammatory cytokines IL-6 and IL-8 in ARPE-19 cells (Hu et al., 2014 Discovery Med. 18(97):7-14). Because these cytokines are associated with the development and progression of AMD, we investigated whether atorvastatin could inhibit this effect. ARPE-19 cells were stimulated with IL-1α, treated with different concentrations of atorvastatin, and then incubated with cholesterol crystals. IL-6 and IL-8 levels were assessed using Western blot or ELISA. As expected, cholesterol crystals increased ARPE-19 secretion of IL-6 and IL-8 by 3.2-fold and 2.5-fold, respectively, compared with control treatment (Figures 7A and B). However, pretreatment of cells with 0.1, 0.5, or 1 μM atorvastatin significantly reduced IL-6 levels by 1.7-fold, 1.1-fold, and 0.8-fold, respectively, and IL-8 levels by 1.8-fold, 1.6-fold, and 1.1-fold, respectively, compared to controls (p<0.05) (Figures 7A and 7B). Additionally, we wished to examine the effects of ox-LDL on IL-6 and IL-8 in ARPE-19 cells and the effect of atorvastatin on the resulting results. Similar to cholesterol crystals, incubation of ARPE-19 cells with ox-LDL led to a 2.6-fold and 1.3-fold increase in secreted IL-6 and IL-8, respectively. However, pretreatment of cells with 0.1, 0.5, or 1 μM atorvastatin reduced IL-6 levels by 1.9-fold, 1.3-fold, and 1.4-fold, respectively, and IL-8 levels by 1.2-fold, 1.02-fold, and 0.99-fold, respectively, compared with the control (p<0.05) (Figures 7C and 7D). Both results indicate that atorvastatin can inhibit cholesterol crystal- and ox-LDL-induced IL-6 and IL-8 secretion in ARPE-19 cells in a dose-dependent manner.

[0083] Taken together, these data highly suggest that atorvastatin has an anti-inflammatory role in human RPE cells challenged with inflammatory inducers.

[0084] Consideration Our study provides evidence that lipophilic statins enhance the phagocytic function of ARPE-19 cells and that atorvastatin can protect these cells from the dysfunction of phagocytic function and inflammatory effects induced by cholesterol crystals and ox-LDL. In addition, atorvastatin increases the membrane fluidity of ARPE-19 cells, which at least partially explains the positive effect of statins on the phagocytic function of these cells.

[0085] Since we used atorvastatin in our clinical studies, we further focused on its in vitro effects on ARPE-19 cells. Interestingly, atorvastatin not only increased the baseline phagocytic function of ARPE-19 cells, but also protected the cells against cholesterol crystal- and ox-LDL-induced phagocytic dysfunction. In addition to its preventive role against AMD, the observed effect of atorvastatin on reversing the characteristics of dry AMD may be partially explained by its ability to induce greater phagocytosis of existing drusen by RPE cells.

[0086] In summary, this study demonstrates that the lipophilic statins atorvastatin, lovastatin, and simvastatin have similar enhancing effects on the phagocytic function of ARPE-19 cells. It also demonstrates that atorvastatin is an effective drug for protecting ARPE-19 cells against cholesterol crystal- and ox-LDL-induced phagocytic dysfunction and inflammatory effects. In addition, our study suggests increased cell membrane fluidity as an important mechanism for the observed effects of statins on the phagocytic function of ARPE-19 cells. More importantly, our results introduce statins, a widely used and well-tolerated class of drugs with few serious side effects, as a potentially effective pharmaceutical for preventing and treating AMD.

[0087] Other embodiments While the present disclosure has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative and not limiting of the scope of the disclosure, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. 1. A pharmaceutical composition for treatment selected from: i) regression of drusen in said patient; ii) regression of drusenoid pigment epithelial detachment (PED); iii) prevention of progression of AMD to wet AMD; iv) prevention of atrophy of the retinal pigment epithelium (RPE); v) prevention of atrophy of one or more photoreceptors; vi) prevention of vision loss; and vii) combinations thereof in a patient with age-related macular degeneration (AMD), the pharmaceutical composition comprising a high-dose statin, wherein the high-dose statin is effective in: i) regression of drusen in said patient; ii) regression of drusenoid pigment epithelial detachment (PED) in said patient; iii) prevention of progression of AMD to wet AMD in said patient; iv) prevention of atrophy of the retinal pigment epithelium (RPE); a) preventing atrophy of the retinal pigment epithelium (RPE) in said patient; b) preventing atrophy of one or more photoreceptors in said patient; c) preventing loss of vision in said patient; or d) a combination of the above, wherein said high-dose statin is at least 40 mg per day of atorvastatin, at least 0.4 mg per day of cerivastatin, at least 120 mg per day of fluvastatin, at least 90 mg per day of lovastatin, at least 4 mg per day of pitavastatin, at least 60 mg per day of pravastatin, at least 20 mg per day of rosuvastatin, or at least 60 mg per day of simvastatin.

2. 1. A pharmaceutical composition for use in treating age-related macular degeneration (AMD) in a patient, said pharmaceutical composition for: i) regression of drusen in said patient; ii) regression of drusenoid pigment epithelial detachment (PED); iii) prevention of progression of AMD to wet AMD; iv) prevention of atrophy of the retinal pigment epithelium (RPE); v) prevention of atrophy of one or more photoreceptors; vi) prevention of vision loss; or vii) a combination of the above, wherein the pharmaceutical composition comprises a high-dose statin, and the treatment comprises administering the high-dose statin to the patient, wherein the high-dose statin induces i) regression of drusen in said patient; ii) regression of drusenoid pigment epithelial detachment (PED) in said patient; iii) prevention of progression of AMD to wet AMD in said patient. iv) preventing atrophy of the retinal pigment epithelium (RPE) in said patient; v) preventing atrophy of one or more photoreceptors in said patient; vi) preventing vision loss in said patient; or vii) a combination of the above, wherein the high-dose statin is at least 40 mg per day of atorvastatin, at least 0.4 mg per day of cerivastatin, at least 120 mg per day of fluvastatin, at least 90 mg per day of lovastatin, at least 4 mg per day of pitavastatin, at least 60 mg per day of pravastatin, at least 20 mg per day of rosuvastatin, or at least 60 mg per day of simvastatin.

3. 3. The pharmaceutical composition of claim 1 or 2, wherein the treatment further comprises identifying the patient as having AMD and selecting the patient based on the criteria that the patient has AMD.

4. 3. The pharmaceutical composition of claim 1 or 2, wherein the treating further comprises identifying the patient as having dry AMD and selecting the patient based on the criteria that the patient has dry AMD.

5. 3. The pharmaceutical composition of claim 1 or 2, wherein the treatment further comprises monitoring the patient for the effectiveness of the high-dose statin.

6. The treatment further comprises monitoring the patient for regression of drusen, regression of drusenoid PED, atrophy of the RPE, atrophy of one or more photoreceptors, visual loss, or AMD progression, wherein the monitoring comprises measuring a parameter indicative of regression of drusen, regression of drusenoid PED, atrophy of the RPE, atrophy of one or more photoreceptors, visual loss, or AMD progression in the patient at a first time point prior to administration of the high-dose statin; and measuring the same parameter in the first time point at a second time point after administration of the high-dose statin, and comparing the parameter measured at the first time point with the second time point, wherein a reduction in the parameter from the first time point to the second time point indicates regression of drusen, regression of drusenoid PED, prevention of atrophy of the RPE, prevention of atrophy of one or more photoreceptors, prevention of vision loss, or prevention of AMD progression.

7. 6. The pharmaceutical composition of claim 5, wherein the treatment further comprises monitoring the patient for visual acuity, the monitoring comprising measuring a parameter indicative of visual acuity in the patient at a first time point prior to administration of the high-dose statin, measuring the same parameter in the patient at a second time point after administration of the high-dose statin, and comparing the parameter measured at the first and second time points, wherein an increase in the parameter from the first time point to the second time point indicates an improvement in visual acuity.

8. The pharmaceutical composition of claim 1 or 2, wherein the patient is a human.

9. 3. The pharmaceutical composition of claim 1 or 2, wherein the treatment further comprises identifying the patient as having a high-risk profile for AMD progression and selecting the patient based on the criteria that the patient has a high-risk profile for AMD progression.

10. 3. The pharmaceutical composition of claim 1 or 2, wherein the patient is administered the high-dose statin for at least 12 months, at least 15 months, at least 18 months, at least 21 months, at least 24 months, at least 30 months, at least 33 months, at least 36 months, at least 39 months, at least 42 months, at least 45 months, or at least 48 months.

11. 3. The pharmaceutical composition of claim 1 or 2, wherein the high-dose statin is administered by a route selected from the group consisting of: i) intravenous administration, ii) ocular administration, iii) intramuscular administration, iv) subcutaneous administration, v) oral administration, vi) intranasal administration, vii) inhalation administration, viii) transdermal administration, ix) intravitreal administration, and x) parenteral administration.

12. 3. The pharmaceutical composition of claim 1 or 2, wherein the high-dose statin is administered by an ocular route selected from the group consisting of intravitreal, topical drops, or topical ointment.

13. 3. The pharmaceutical composition of claim 1, wherein the statin is selected from the group consisting of atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin.

14. 3. The pharmaceutical composition of claim 1, wherein the statin is a lipophilic statin.

15. 15. The pharmaceutical composition of claim 14, wherein the lipophilic statin is selected from the group consisting of atorvastatin, lovastatin, and simvastatin.

16. 3. The pharmaceutical composition of claim 1 or 2, wherein the treatment further comprises administering a maintenance dose of a statin to the patient after the patient has received the high dose of the statin.

17. 17. The pharmaceutical composition of claim 16, wherein the maintenance dose of the statin administered per day is lower than the high dose of the statin administered per day.

18. 3. The pharmaceutical composition of claim 1 or 2, wherein the high-dose statin is administered once daily, twice daily, or three times daily.

19. 3. The pharmaceutical composition of claim 1, wherein the treatment further comprises administering to the patient an additional therapeutic agent, wherein the additional therapeutic agent is selected from the group consisting of anti-inflammatory agents, anti-angiogenic agents, antioxidants, omega-3 fatty acids, and vitamins / minerals.

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