Microparticle compositions and methods of use thereof

Abstract

Microparticulate (MP) formulations formed from one or more poly(hydroxyacid) polymers having a molecular weight ranging from 5kD to 60kD, and one or more active agents are injected into the eye of a subject to address eye disorders. The MP formulations assure high drug loading and extended delivery of the active agent of six to twelve months. The active agents may include peroxisome proliferator-activated receptor alpha (PPARα) signaling agonists such as PPARα agonist A190 (IUPAC name 3-((4-((4-fluorobenzyl)oxy)- 3- methylbenzyl)amino)benzoic acid). The formulations have therapeutic and protective effects against retinal degeneration diseases such as age-related macular degeneration (AMD), but may also be used for treating or relieving the symptoms of retinal inflammation, retinal neovascularization, retinal vascular leakage, retinopathy of prematurity (ROP), diabetic retinopathy (DR), and diabetic macular edema (DME).

Description

[0001] MICROPARTICLE COMPOSITIONS AND

[0002] METHODS OF USE THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of United States Provisional patent application 63 / 733,159, filed December 12, 2024.

[0003] STATEMENT OF FEDERALLY SPONSORED RESEARCH AND DEVELOPMENT

[0004] This invention was made with government support under grant numbers R01EY033477, R21EY028279 and R01EY030472 awarded by the National Institutes of Health. The United States government has certain rights in the invention.

[0005] BACKGROUND OF THE INVENTION

[0006] Technical Field

[0007] The invention generally relates to microparticle (MP) formulations of agonists of peroxisome proliferator- activated receptor alpha (PPARa) signaling designed for high drug loading and sustained release. In particular, the invention provides MP formulations of PPARa agonists such as A190 (IUPAC name 3-((4-((4-fluorobenzyl)oxy)-3- methylbenzyl) amino)benzoic acid) suitable for intraocular injection and having therapeutic and protective effects against retinal degeneration diseases such as age-related macular degeneration (AMD).

[0008] Description of Related Art

[0009] Age-related macular degeneration (AMD) remains a significant global health challenge, leading to severe central vision impairment and compromised quality of life in the elderly population. There are two forms of AMD, geographic atrophy (dry) AMD and neovascular (wet) AMD. Dry AMD involves the progressive degeneration of retinal pigment epithelium (RPE) and photoreceptors, along with drusen accumulation. Wet AMD is characterized by choroidal neovascularization, vascular leakage, and subretinal fibrosis, leading to rapid vision loss. Presently, the prevailing therapeutic strategy for neovascular AMD involves the intravitreal administration of anti- vascular endothelial growth factor (VEGF) agents. Despite its effectiveness, this approach requires frequent injections due to the short half-life of the drugs, imposing substantial financial and logistical burdens on patients and healthcare systems. Further, there is no effective treatment for retinal degeneration in AMD.

[0010] To address this medical need, innovative therapeutic approaches are being investigated, with a focus on targeting lipid metabolism and modulating inflammation pathways. Peroxisome proliferator- activated receptor alpha (PPARa) has emerged as an intriguing drug target, owing to its regulatory roles in energy homeostasis, inflammation, angiogenesis, and neuroprotection. Two independent and prospective clinical studies FIELD and ACCORD reported that fenofibrate, a PPARa agonist, conferred promising therapeutic effects on diabetic retinopathy (DR) in type 2 diabetic patients.

[0011] While fibrates support the clinical efficacy of PPARa agonists in treating retinal diseases, new formulations of PPARa agonists are needed with improved pharmacokinetics and pharmacodynamics, enhanced selectivity and potency.

[0012] SUMMARY OF THE INVENTION

[0013] Other features and advantages of the present invention will be set forth in the description of invention that follows, and in part will be apparent from the description or may be learned by practice of the invention. The invention will be realized and attained by the compositions and methods particularly pointed out in the written description and claims hereof.

[0014] This disclosure describes novel MP formulations of PPARa agonists, such as the exemplary PPARa agonist A190 (IUPAC name 3-((4-((4-fluorobenzyl)oxy)-3-methylbenzyl)amino)benzoic acid). The MP formulations exhibit high drug loading and sustained release within the eye and have therapeutic and protective effects against retinal degeneration diseases such as AMD. In some aspects, the delivery of the formulations is via injection into the eye, such as via intravitreal injection.

[0015] It is an object of this invention to provide a method of treating at least one symptom of an eye disorder in a subject in need thereof, comprising administering to an eye of the subject a formulation comprising microparticles distributed within a vehicle, wherein the microparticles in the formulation comprise: a polymer or copolymer comprising one or more poly(hydroxyacid) polymers, wherein the one or more poly(hydroxyacid) polymers have a molecular weight ranging from 5kD to 60kD, and an agonist of PPARa signaling at a loading of 5-50% by weight, wherein the agonist of PPARa signaling has a general chemical structure

[0016]

[0017] wherein:

[0018] k is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms;

[0019] m is 0, 1, 2, 3, 4, or 5 carbon atoms;

[0020] n is 0, 1, 2, 3, 4, or 5 carbon atoms;

[0021] R2is selected from the group: hydrogen (H), chlorine (Cl), fluorine (F), bromine (Br), iodine (I), nitro (NO2), CH3, CH2CH3, branched or unbranched alkyl chains with 3-10 carbon atoms, OCH3, OCH2CH3, branched or unbranched alkoxy chains with 3-10 carbon atoms, haloalkyls, haloalkoxyls, cycloalkyls, halocycloalkyls, O-para-alkylbenzyls, O-para-alkyloxybenzyls, and O-para-halobenzyls, wherein the benzene ring comprising R2comprises one, two, three, or four of said R2substituents substituted in any combination of said R2substituents and arranged in any pattern in the ring including ortho, meta, mono, di, tri, and tetrasubstituted;

[0022] R3is selected from the group: H, Cl, F, Br, I, NO2, CH3, CH2CH3, branched or unbranched alkyl chains with 3-10 carbon atoms, OCH3, OCH2CH3, branched or unbranched alkoxy chains with 3-10 carbon atoms, haloalkyls, haloalkoxyls, cycloalkyls, halocycloalkyls, O-para-alkylbenzyls, O-para-alkyloxybenzyls, and O-para-halobenzyls, wherein the benzene ring comprising R3comprises one, two, three, four, or five of said R3substituents substituted in any combination of said R3substituents and arranged in any pattern in the ring including ortho, meta, para, mono, di, tri, tetra, and pentasubstituted;

[0023] R4is selected from the group: H, alkyl, and acyl; R5is selected from the group: H, Cl, F, Br, I, NO2, CH3, CH2CH3, branched or unbranched alkyl chains with 3-10 carbon atoms, OCH3, OCH2CH3, branched or unbranched alkoxy chains with 3-10 carbon atoms, haloalkyls, haloalkoxyls, cycloalkyls, halocycloalkyls, O-para-alkylbenzyls, O-para-alkyloxybenzyls, and O-para-halobenzyls, wherein the benzene ring comprising R5comprises one, two, three, or four of said R5substituents substituted in any combination of said R5substituents and arranged in any pattern in the ring including ortho, meta, para, mono, di, tri, and tetrasubstituted;

[0024] R1is selected from the group consisting of carboxylic acids, carboxylic acid isosteres, hydroxamic acids, hydroxamic esters, phosphonic acids, phosphinic acids, sulfonic acids, sulfinic acids, sulfonamides, acyl sulfonamides, sulfonylureas, acylureas, tetrazoles, thiazolidine diones, oxazolidine diones, oxadiazol-5(4H)-ones, thiadiazol-5(4H)-ones, oxathiadiazole-2-oxides, oxadiazol-5(4H)-thiones, isoxazoles, tetramic acids, cyclopentane 1,3-diones, cyclopentane 1,2-diones, squaric acids, substituted phenols, heteroarenes, amidines, hydroxy amides, alkyl hydroxy amidines, and

[0025] ^Ol L

[0026] o

[0027] II

[0028] , Sv

[0029] N \'

[0030] H \

[0031]

[0032] and salts thereof,

[0033] R8and R9are independently selected from the group consisting of H, F, Cl, Br, I, alkyl, alkoxy, and cycloalkyl comprising R8is linked to R9;

[0034] R10and R11are independently selected from the group consisting of H, alkyl, and cycloalkyl wherein R10is linked to R11); and

[0035] X is O, NH, S, or CH2.

[0036] In some aspects, the one or more poly(hydroxyacid) are selected from the group consisting of poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and poly (caprolactone). In further aspects, the one or more poly(hydroxyacid) polymers is or includes PLGA. In yet additional aspects, the one or more poly(hydroxyacid) polymers are present at two different molecular weight ranges, such as, for example PLGA at 54kD and PLGA at 45kD. In some aspects, the one or more poly(hydroxyacid) polymers are two different poly(hydroxyacid) polymers. In additional aspects, the microparticles have an average diameter ranging from 1 to 100 pm. In yet other aspects, the microparticles have an average diameter ranging from 5 to 25pm. In alternative aspects, the one or more poly(hydroxyacid) polymers are formulated as a co-polymer with polyethylene glycol (PEG) wherein the PEG is present at IkD to lOkD. In other aspects, the co-polymer with PEG is selected from the group consisting of PLGA-PEG, PLA-PEG, PGA-PEG, and polycaprolactone-PEG. In other aspects, the co-polymer with PEG comprises PLGA at 40-60kD. In still further aspects, additional polymers can be included in the microparticle to further enhance sustained delivery, and additional biologically active agents may be included in the microparticle to assist in treatment (e.g., anti-inflammatories, etc.)

[0037] In further aspects, the administration is performed by injection into the eye. In additional aspects, the vehicle in the formulation is a liquid (e.g., aqueous). In yet further aspects, the injection is performed no more than once every six months. In alternative aspect, the injection is performed no more than once every twelve months.

[0038] BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Fig. 1A-D. A190-MP exhibited spherical shape and sustained drug release profile. (A) Structure of A190. (B) Representative scanning electron microscopy images of A190-MP. Scale bar: 10 pm. (C) In vitro drug release profile of A190-MP made of different polymers under sink conditions at 37°C (n = 3). (D-E) A190-MP provided sustained release of A190 to the retina (D) and vitreous humor (E) for at least 6 months, following intravitreal injection of 1 pl of A190-MP-F5 (30 pg A190) (n = 4). Data represented mean ± SEM. Fig.2A-O. Intravitreal injection of A190-MP prevented ERG decline and retinal degeneration and improved mitochondrial function in Vldlr'~ mice. (A-C) Quantification of photopic (cone) and scotopic (rod) ERG a-wave amplitudes (n = 4-7). (D-G) Representative images of PNA labeling of cones in whole-mounted retinas of Vldlr ' mice at 6 months post-injection. Scale bar: 20 pm. PNA-positive cones were quantified in both central (E) and peripheral (G) regions of the retina (n = 5). (H-M) OCT measurements of total retinal thickness (H, I), outer retinal (OR) thickness (J, K) and outer nuclear layer (ONL) thickness (L, M) at 0.18 mm intervals from the optic nerve head of Vldl1' mice at 6 months after A190-MP injection and Blank-MP injection. The upper panels (H, J, L) represent measurements from the inferior to superior, while the lower panels (I, K, M) depict measurements from the nasal to temporal (n = 6-7). (N, O) Representative Western blots of PPARa, PGCla, TOMM20 and P-actin in retinal tissues of Vldl '~ mice at 6 months post-injection, and quantification by densitometry and normalization by P-actin levels (n = 5). Data represented mean ± SEM and analyzed by unpaired Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0040] Fig. 3A-N. Intravitreal injection of A190-MP reduced retinal vascular leakage and neovascularization in Vldl '~ mice. (A-C) Representative images of fluorescein angiography in Blank-MP- and A190-MP-treated Vldl '~ mice at 6 months post-injection, at 5 min postfluorescein injection. White arrows indicate vascular leakage. Quantification of the number (B) and area (C) of leakage (n = 6). (D-I) Representative images of subretinal neovascularization (SRNV, D) and intraretinal neovascularization (IRNV, G) visualized with isolectin GS-IB4 staining in RPE / choroidal and retinal flat mounts, respectively. White arrows indicate neovascular lesions. Scale bar: 1000 pm. Quantification of SRNV and IRNV numbers (E, H) and areas (F, I) (n = 4-5). (J-L) Representative OCT images displaying subretinal neovascular lesions (ILM, internal limiting membrane; INL, inner nuclear layer; ONL, outer nuclear layer; BM, Bruch's membrane). Quantification of subretinal lesion number (K) and average lesion volume (L), from serial OCT images (n = 5-6). (M, N) Representative Western blots of VEGF, ICAM-1, IL-ip, and P-actin in retinal tissues of Vldlr'~ mice at 6 months postinjection, and quantification by densitometry and normalization by P-actin levels (n = 5). Data represented mean ± SEM and analyzed by unpaired Student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001.

[0041] Fig.4A-M. Intravitreal injection of A190-MP alleviated retinal degeneration nAbca4~'~ / Rdh8~ / _mice. (A, B) Quantification of photopic (cone) and scotopic (rod) ERG a-wave amplitudes -1-in Blank-MP- and A190-MP-treated Abca4~,~IRdh8~,~ mice (n = 6-9). (C, D) PNA-positive cones were quantified in both central (C) and peripheral (D) regions of the flat-mounted retina (n = 5). (E) Representative PNA staining in retinal sections of Ahead ' / Rdh8'7' mice at 6 months post-injection (GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; IS, photoreceptor inner segment; OS, photoreceptor outer segment). Scale bar: 50 pm. (F) Average OS length of Ahead ' / Rdh8'7' mice at 6 months post-injection (n = 5). (G) OCT measurements of ONL thickness at 0.18 mm intervals from the optic nerve head of Ahead7' / Rdh8'7' mice at 6 months after A190-MP injection and Blank-MP injection. The left panel represents measurements from the inferior to superior, while the right panel depicts measurements from the nasal to temporal (n = 6). (H) Representative H& E staining of retinal sections of Ahead7' / Rdh8 ' mice at 6 months post-injection (ONH, optic nerve head). Scale bar: 50 pm. (I) Quantification of the ONL thickness in H& E-stained retinal sections from the inferior to superior (n = 4-5). (J) TUNEL-positive cells were quantified and compared in Blank-MP- and A190-MP-treated Ahead7' / Rdh8 ' mice (n = 5). (K) Representative 3-NT immunofluorescence staining and DAPI staining in retinal sections of Abca4'7 / Rdh8' ' mice at 6 months post-injection. Scale bar: 50 pm. (L, M) Representative Western blots of pNF-KB, NOX4 and P-actin in retinal tissues of Ahead7' / Rdh8 ' mice at 6 months post-injection, and quantification by densitometry and normalization by P-actin levels (n = 4). Data represented mean ± SEM and analyzed by unpaired Student’s t-test. * P < 0.05, ** P < 0.01.

[0042] Fig. 5A-L Intravitreal injection of A190-MP improved mitochondrial function in the retina of Ahead'1' / Rdh87' mice. (A-D) Mitochondrial stress test in the retinal biopsy punches from wildtype (129S) and Ahead7' / Rdh8 ' mice at 4 months of age. Quantification of basal respiration (B), maximal respiration (C), and spare respiration capacity (D) (OCR, oxygen consumption rate; FCCP, carbonyl cyanide-4- (trifluoromethoxy) phenylhydrazone; RAA, rotenone / antimycin A) (n = 5). (E, F) Quantification of maximal respiration (E) and spare respiration capacity (F) of mitochondrial stress test in the retinal biopsy punches from Blank-MP- and A190-MP-treated Abca4'77Rdh8'7' mice at 6 months post-injection (n = 5-6). (G) Representative TOMM20 immunofluorescence staining in retinal sections with nuclei counterstained by DAPI of Blank-MP- and A190-MP-treated Abca4'77Rdh8'7' mice at 6 months post-injection. Scale bar: 50 pm. (H, I) Representative Western blots of PPARa, PGCla, TOMM20, CPT1A and P-actin in retinal tissues ot Abca4'77Rdh8'7' mice at 6 months post-injection, and quantification by densitometry and normalization by P-actin levels (n = 5). Data represented mean ± SEM and analyzed by unpaired Student’s t-test. * P < 0.05, ** P < 0.01.

[0043] Fig. 6A-H. A 190 treatment ameliorated oxidative stress-induced damage in photoreceptor-derived cells. 661W cells were pretreated with A190 for 2 hr, and then 4-HNE (10 pM) was added and incubated for another 24 hr. Vehicle-treated cells were used as control (Ctrl). (A) The cell viability was measured by trypan blue exclusion assay and normalized to the control (n = 3). (B) Cytotoxicity was determined by lactate dehydrogenase (LDH) release and normalized to the control (n = 5). (C) The TUNEL positive cells (%) (n = 3). (D, E) Representative Western blots of BCL-2, BAX and P-actin, and quantification by densitometry and normalization by P-actin levels (n = 3). (F) ROS production was measured using H2DCFDA and normalized by total protein concentration (n = 6). (G) Representative 3-NT (green) immunofluorescence staining images. Nuclei were stained with DAPI (blue). Scale bar: 20 pm. (H) Quantification of 3-NT fluorescence intensity (n = 3). Data represented mean ± SEM and analyzed by one-way ANOVA with Bonferroni post hoc test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0044] Fig. 7A-L. A 190 treatment mitigated oxidative stress-induced mitochondrial dysfunction.

[0045] 661W cells were pretreated with A190 (10 pM) for 2 hr, and then 4-HNE (10 pM) was added and incubated for another 24 hr. Vehicle-treated cells were used as control (Ctrl). (A) ATP production rates from mitochondria and glycolysis were measured using real-time ATP rate assay in 661W cells in the presence or absence of 4-HNE (10 pM) for 24 hr. Left panel presented the values of ATP production rate, while the right panel presents the ratio of ATP production rate (n = 4). (B, C) Comparison of mitochondrial ATP production rate (B) and glycolysis ATP production rate (C) (n = 4). (D-H) Mitochondrial stress test in 661 W cells treated with 4-HNE (10 pM) and A190 (10 pM) for 24 hr. Quantification of ATP production (E), basal respiration (F), maximal respiration (G), and spare respiration capacity (H) (OCR, oxygen consumption rate; FCCP, carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone; RAA, rotenone / antimycin A) (n = 8-10). (I) Representative immunofluorescence images of the mitochondrial morphology by TOMM20 staining with the nuclei counterstained by DAPI. Scale bar: 10 pm. (J) Quantification of cells with fragmented mitochondria (n = 3). (K, L) Representative Western blots of PPARa, PGCla, TOMM20 and P-actin, and quantification by densitometry and normalization by P-actin levels (n = 3). Data represented mean ± SEM, analyzed by unpaired Student’s t-test in Fig. B& C and one-way ANOVA with Bonferroni post hoc test in Fig. D-L. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

[0046] Fig. 8A-H. A 190 exerts neuroprotective and antioxidant effects via a PPARa-dependent mechanism. 661W cells were transfected with scrambled siRNA or PPARa siRNA for 48 hr, then treated with A190 (10 pM) for 2 hr, followed by exposure to 4-HNE (10 pM) for 24 hr. (A, B) Representative Western blots of PPARa, PGCla, TOMM20 and P-actin, and quantification by densitometry and normalization by P-actin levels (n = 4). (C) The cell viability was measured by trypan blue exclusion assay and normalized to the control (n = 4). (D) ROS production in 661W cells was measured using H2DCFDA and normalized by total protein concentration (n = 5-6). Primary Muller cells from wild-type and PPARα- / -mice were pretreated with A 190 (10 pM) for 2 hr, and then 4-HNE (10 pM) was added and incubated for another 24 hr. (E, F) Representative Western blots of PGCla, TOMM20 and P-actin, and quantification by densitometry and normalization by P-actin levels (n = 3). (G) The cell viability in wild-type and PPARα- / -Muller cells was measured by trypan blue exclusion assay and normalized to the control (n = 3-4). (H) ROS production in wild-type and PPARα- / -Muller cells was measured using H2DCFDA and normalized by total protein concentration (n = 6). Data represented mean ± SEM, analyzed by unpaired Student’s t-test in Fig. B& F and oneway ANOVA with Bonferroni post hoc test in Fig. C-D and G-H. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, non- significant.

[0047] Fig. 9A-G. Intravitreal injection of Blank- MP and A190-MP in Vldlr7' mice. (A-C) Quantification of photopic (cone) and scotopic (rod) ERG b-wave amplitudes of Vldlr1' mice (n = 4-7). (D) Representative PNA staining in retinal sections of Vldlr- / -retina at 6 months post-injection (INL, inner nuclear layer; ONL, outer nuclear layer; IS, photoreceptor inner segment; OS, photoreceptor outer segment). Scale bar: 50 pm. (E) OCT measurements of the inner retinal (IR) thickness at 0.18 mm intervals from the optic nerve at 6 months postinjection of A190-MP and Blank-MP. The left panel represents measurements from the inferior to superior, while the right panel depicts measurements from the nasal to temporal (n = 6-7). (F) Representative fundus photographs of Vldlr- / -mice at 6 months post-injection. (G) Representative H& E staining of Vldlr- / -retinal sections at 6 months post-injection, with subretinal lesions indicated by black stars (GCL, ganglion cell layer; IPL, inner plexiform layer; OPL, outer plexiform layer; RPE, retinal pigment epithelium). Scale bar: 200 pm (upper panel); 50 pm (lower panel). Data represented mean ± SEM and analyzed by unpaired Student’s t-test. * P < 0.05, *** P < 0.001. Fig. 10. Concentration of A 190 in the vitreo-retina tissues following intravitreal injection of free A190 solution (30 pg A190) in C57BL6 / 7 mice (n = 4). Data represented mean ± SEM.

[0048] Fig. 11A-I. Intravitreal injection of Blank-MP and A190-MP in Abca^'IRdhS'1' mice. (A, B) Quantification of photopic (cone) and scotopic (rod) ERG b-wave amplitudes of Abca4'7' / Rdh8'7' mic (n = 6-9). (C) Representative images of PNA cone labeling in whole-mounted retinas of Abca4'77Rdh8'' mice at 6 months post-injection. Scale bar: 20 pm. (D) OCT measurements of total retinal thickness at 0.18 mm intervals from the optic nerve head of Abca4' 7Rdh8' ' mice at 6 months after A190-MP injection and Blank-MP injection. The left panel represents measurements from the inferior to superior, while the right panel depicts measurements from the nasal to temporal (n = 6). (E) Quantification of the total retinal thickness in Abca4' / Rdh8' ' mice at 6 months post-injection in H& E-stained retinal sections (n = 4-5). (F) Representative TUNEL and DAPI staining in retinal sections of Abca4~7Rdh8~ / _mice at 6 months post-injection. Scale bar: 50 pm. (G, H) Representative fundus photographs of Abca4' / Rdh8'' mice at 2 and 6 months post-injection. (I) The intraocular pressure at 2, 4 and 6 months post-injection (n = 9-11). Data represented mean ± SEM and analyzed by unpaired Student’s t-test. * P < 0.05, ns, nonsignificant.

[0049] Fig. 12 A- J. Intravitreal injection of A190-MP had no detectable toxic effects in normal C57BL / 6J mice. (A, B) Quantification of photopic and scotopic ERG a- and b-wave amplitudes prior to and 4 weeks after injection (n = 4). (C) Representative H& E staining in retinal sections of untreated, Blank-MP and A190-MP groups at 6 months post- injection (GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; RPE, retinal pigment epithelium). Scale bar: 50 pm. (D) Quantification of the total retinal thickness in H& E-stained retinal sections (n = 4). (E, F) Representative images and quantification of TUNEL-positive cells in retinal sections of untreated, Blank-MP and A190-MP groups at 6 months post-injection (n = 4). Scale bar: 50 pm. (G) The intraocular pressure (IOP) both before and six months after the injection (n = 4). (H, I) Serum levels of liver enzymes (ALP, AST, ALT) and kidney function (BUN, creatinine) in mice at 6 months post-injection (n = 2). (J) Representative H& E staining of heart, kidney, liver and spleen sections of untreated, Blank-MP and A190-MP groups at 6 months postinjection. Scale bar: 100 μm. Data represented mean ± SEM and analyzed by one-way ANOVA with Turkey's post-hoc comparison, ns, nonsignificant.

[0050] Fig. 13A-H. A190 treatment in 661W cells. (A) The effect of 4-hydroxynonenal (4-HNE) on 661W cells for 24 hr. EtOH was used as a negative control. Cell viability was measured by MTT assay and normalized to the Ctrl (n = 4). (B) The effect of A190 on 661W cells for 24 hr. DMSO was used as vehicle control. Cell viability was measured by trypan blue assay and normalized to the Ctrl (n = 3). (C) Representative phase-contrast images of 661W cells, untreated (Ctrl) or treated with different concentrations of A 190 for 24 hr. Scale bar: 200 μm. (D) Representative images of TUNEL and DAPI staining of 661W cells exposed to 4-HNE (10 pM) and treated with A190 (10 pM). Scale bar: 100 μm. (E-H) Glycolysis stress test in 661W cells treated with 4-HNE (10 pM) and A190 (10 pM). Quantification of glycolysis (F), glycolytic capacity (G), and glycolytic reserve (H) (ECAR, extracellular acidification rate; 2-DG, 2-deoxy-glucose) (n = 7-8). Data represented mean ± SEM, analyzed by one-way ANOVA with Dunnett’s post hoc test in Fig. A& B and one-way ANOVA with Bonferroni post hoc test in Fig. F-H. * P < 0.05, ** P < 0.01, *** P < 0.001 **** P < 0.0001.

[0051] Figure 14A-C. A190-MP formulations with higher drug loading (DL) and sustained release profile. Representative SEM images of (A) A190-MP-F6 and (B) A190-MP-F7. (C) In-vitro drug release profiles of A190-MP-F6 and A190-MP-F7 exhibited sustained release behavior for >5 months (Mean+SD, n=3 repeats).

[0052] DETAILED DESCRIPTION

[0053] A 190, a non-fibrate PPARa agonist from the 4-benzyloxy-benzylamino chemotype, exhibits an EC50 (half maximal effective concentration) of ~40 nM and >2700-fold selectivity for the PPARa transcription factor over other isoforms of PPAR. Despite this selectivity, small molecules like A 190 are typically not amenable to e.g., intravitreal injection due to their rapid clearance from the vitreous humor, which limits their retention time in the eye.

[0054] As disclosed herein, to prolong the bioavailability of A 190 in the retina, A 190 was encapsulated in polymeric microparticles (MPs), enabling controlled (delayed, extended, sustained etc.) release of the drug release via a single intravitreal injection. The polymeric MPs remained in the vitreous for an extended duration post-injection, maintaining effective drug concentrations at the target site over a prolonged period (at least 6 months). For patients suffering from diseases caused by PPARa inactivity or low activity, such as AMD, intravitreal injection of the present formulations avoids frequent injections (e.g., every 6 months instead of monthly), thereby improving patient compliance, minimizing associated side effects, and prolonging efficacy. The polymers in the MPs disclosed herein include poly (lactic-co-glycolic acid) (PLGA), an FDA-approved biodegradable and biocompatible polymer with broad applications in ocular settings.

[0055] DEFINITIONS PLGA54kDa refers to a specific grade of the synthetic, biodegradable poly(lactic-co-glycolic acid) (PLGA) copolymer that has an average molecular weight of approximately 54 kilodaltons (kDa).

[0056] PLGA45kDa refers to a specific type of poly(lactic-co-glycolic acid) (PLGA) copolymer with an average molecular weight of approximately 45,000 Daltons (45 kDa).

[0057] PEG5kDa is a type of polyethylene glycol (PEG) with an average molecular weight of approximately 5,000 Daltons.

[0058] PLGA45kDa-PEG5kDa refers to an amphiphilic block copolymer made of PLGA45kDa and PEG5kDa. The components of such block copolymers are generally covalently bonded or linked. This copolymer is used to create nanoparticles for drug delivery because the PLGA part degrades to release a drug, while the PEG part provides stealth properties (e.g. prolonged circulation time and reduced immune response) by forming a protective layer on the nanoparticle surface.

[0059] The term “w / w" stands for weight-by-weight or mass percentage, which expresses the proportion of a substance in a mixture as a percentage of the total mass.

[0060] PPARa refers to peroxisome proliferator- activated receptor alpha.

[0061] Intraocular injection refers to an injection administered to an eye, including but not limited to intravitreal, subvitreal, subretinal, suprachoroidal, retrobulbar, subconjunctival, peribulbar and epischelera.

[0062] Dv50 refers to the volume-weighted median diameter of microparticles.

[0063] The "span value" of a microparticle is a measurement of its particle size distribution width, calculated as the ratio of the difference between the 90th and 10th percentile particle diameters to the 50th percentile diameter: A lower span value indicates a narrower distribution and greater uniformity in particle size, while a higher value signifies a wider distribution. Span value was used to analyze the polydispersity of microparticles given by Span = (D90 -D10) / D50, wherein Dv10, Dv50, and Dv90 are particle size distribution measurements representing the diameters at which 10%, 50%, and 90% of the total volume of particles in a sample are smaller than the respective values. As used herein, the term “variant” refers to, for example, congeners, analogs, homologs and / or prodrugs of a chemical such as A 190. Such variants are also agonists of PPARa and have activity as agonists that is the same as that of A190 or is at least about 50% of the activity of A190, and can be greater than the activity of A190, e.g. about 1.5 to 10 times greater.

[0064] An analog is a compound with a structurally similar but not identical chemical composition to another compound. This similarity may arise from replacing one or more atoms, functional groups, or substructures with others, while the core structure remains largely the same.

[0065] The term “congener” as used herein refers to one of many variants or configurations of a common chemical structure that are related to each other by origin, structure, and / or function. A chemical congener is a specific member of a group of related chemical substances that share a similar core structure but have slightly different compositions. These differences can be in the number of atoms, the arrangement of atoms, or the oxidation state of an element. Congeners have a common base structure, which gives them similar chemical properties. The differences between congeners are minor but distinct. In other words, a congener is a closely related chemical compound that shares a common structural skeleton but differs in the nature or position of substituents.

[0066] In chemistry, "homolog" refers to a member of a homologous series, which is a group of organic compounds with the same functional group(s) and similar chemical properties, where each successive member differs by a methylene.

[0067] A prodrug is an inactive or less active form of a compound (e.g., a medication) that becomes active after being converted by enzymes or other chemical processes within the body. This conversion is a metabolic process that transforms the administered compound into the active drug, which can improve factors like solubility, bioavailability, and targeted delivery, or reduce side effects. Prodrugs are administered in a non-active or "dormant" state. Once in the body, they undergo a chemical change, often through hydrolysis or enzymatic action, to release the active drug. This strategy is used to overcome limitations of the active drug, such as poor solubility, low absorption, or undesirable side effects. Common mechanisms include carrier-linked prodrugs, where a chemical "carrier" is cleaved off, or bioprecursor prodrugs, where the molecule is transformed by enzymes. Benefits include: Improved efficacy (a prodrug can be designed to deliver the active drug more effectively to a target site); reduced toxicity (by masking certain features, prodrugs can decrease side effects and toxicity); enhanced solubility that can make the drug easier to administer and absorb; better delivery can improve the drug's ability to cross biological membranes and / or prolong its time in the body.

[0068] A “drug depot” generally refers to an injectable medication that releases slowly over time. A depot injection is a form of long-acting medication that is injected and stored at the site of injection (e.g., in the vitreous of the eye) in a "depot" from which it is slowly released into the bloodstream over weeks or months.

[0069] An agonist is a molecule that binds to another biological molecule and activates it. THE MICROPARTICLES (MPs)

[0070] In some aspects, the invention provides MPs comprising one or more polymeric materials useful in controlling the duration of action of active agents incorporated therein. MPs comprising these polymeric materials allow for controlled release of the active agents, thus increasing their half-lives.

[0071] In some aspects, the MPs in the formulation comprise: a polymer or copolymer comprising one or more poly(hydroxyacid) polymers, for example 1, 2, 3 or more. The one or more poly(hydroxyacid) polymers have a molecular weight or average molecular weight ranging from about 5kD to 60kD, such as about 5, 10, 15, 20,,25, 30, 35, 40, 45, 50, 55 or 60 kD, including all decimal fractions in between these values.

[0072] In some aspects, the one or more poly(hydroxyacid) polymers are poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and

[0073] poly (caprolactone). In preferred aspects, the one or more poly(hydroxyacid) polymers is or includes PLGA. In yet additional aspects, the one or more poly (hydroxy acid) polymers are present at two different molecular weight ranges, for example, PLGA at 54kD and PLGA at 45kD.

[0074] In some aspects, the one or more poly(hydroxyacid) polymers include two or more different poly (hydroxy acid) polymers. For example, the one or more poly (hydroxy acid) polymers may be formulated as a co-polymer with polyethylene glycol (PEG) such as PLGA-PEG, PLA-PEG, PGA-PEG, and polycaprolactone-PEG. Tri-polymers such as PLA-PEG-PLA may also be employed. In certain aspects, the PLGA that is present in the MPs has an average molecular weight of from about 25 to 100 kilodaltons (kDa), such as about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 kDa. In some aspects, the PLGA has an average molecular weight of from about 40 to 60 kDa, or from about 50 to about 60 kDa, such as about 51, 52, 53, 54 55, 56, 57, 58 59 or 60 kDa. In some aspects, the PLGA has an average molecular weight of about 54 kilodaltons (kDa) and when this is the case, the PLGA is referred to herein as “PLGA54kDa”.

[0075] In some aspects, the MPS comprise i) poly(lactic-co-glycolic acid) (PLGA) copolymer (also known as poly(lactic acid-glycolic acid)), and ii) an amphiphilic block copolymer comprising PLGA copolymer and polyethylene glycol (PEG). In some aspects, the MPs also comprise iii) a PPARa signaling agonist or prodrug thereof. In some aspects, the PPARa agonist is the drug A190 or a variant (e.g. a congener, analog, or homolog thereof that has at least 50% of the activity of A190, as described elsewhere herein).

[0076] Alternatively, the PPARa agonist may be present in the MPs as a prodrug of the active agent, such as A190, that becomes active to the extent described below after processing within the body of a subject.

[0077] In further aspects, the PLGA is present in an amphiphilic copolymer comprising both PLGA and polyethylene glycol (PEG). In general, the PLGA of the amphiphilic copolymer has a molecular weight ranging from about 25 to 100 kDa, such as about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 kDa. In some aspects, the PLGA has an average molecular weight of from about 30 to 60 kDa, or from about 40 to about 50 kDa, such as about 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 kDa. In other aspects, the co-polymer with PEG comprises PLGA at 40-60kD. In some aspects, the PLGA has an average molecular weight of about 45 kDa.

[0078] In general, the PEG that is present in the amphiphilic copolymer has an average molecular weight of about 1-10 kDa, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kDa. In some aspects, the average molecular weight of the PEG is about 5 kD.

[0079] In some embodiments, the MP is a blend of two polymers, e.g., PLGA54kD and PLGA-PEG. In these embodiments, the PLGA and the block copolymer can be present in the MPs in a ratio of from about 5 / 1 or 1 / 5 (w / w), such as about 5 / 1, 4 / 1, 3 / 1, 2 / 1, 1 / 1, 1 / 2, 1 / 3, 1 / 4 or 1 / 5. In some aspects, the ratio is 1 / 1 (w / w).

[0080] The MPs have an average diameter ranging from about 1 to about 100 pm, such as from about from 1, 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 pm. In some aspects, the average MP size generally ranges from about 5-25 pm ± 1.0-1.5 pm, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 pm, including all decimal fractions in between these values (e.g. 5.0, 5.1, 5.2, 5.3....etc.). In some aspects, the MP size is from about 10-15 pm, such as about 10, 11, 12, 13, 14, or 15 pm, including all decimal fractions in between these values (e.g. 10.0, 10.1, 10.2, 10.3...14.7, 14.8, 14.9 and 15.0). In some aspects, the average MP size is from about 12-13 pm, such as about 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9 or 13.0 pm, ± 1.0-1.5 pm, such as ± 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5. In some aspects, the MP size is about 12.4 pm ± 1.2 pm. These sizes are suitable for delivery e.g. using fine gauge needles such as those used for injecting drugs into the eye.

[0081] The span value of the MPs generally ranges from about 0.5 to 5.0, such as about 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 or 5.0. In some aspects, the span value ranges from about 0.5 to about 2.0, such as about 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or 2.0. In further aspects, the span value ranges from about 0.9 to to 1.5, and in some aspects, the span value is about 1.1.

[0082] Before administration, the MPs described herein are typically loaded with a drug, such as a small molecule drug, although this is not always the case. The MPs may be provided to a user without preloading and the user can then load a drug of choice. Thus, the MPs themselves are encompassed herein.

[0083] In some aspects disclosed herein, what is provided is the MPs comprising a drug as an active agent, especially a drug suitable for treating a disease or condition of the eye. Such “loaded” MPs are suitable for delivery to the eye, e.g. via intraocular delivery, such as by intraocular injection. In some aspects, the drug is a non-fibrate PPARa agonist. In further aspects, the drug is A 190 and / or a variant thereof as described herein.

[0084] Drug loading into the MPs is typically in the range of from about 1-50%, such as about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50% including all decimal fractions in between these values such as about 1.1, 1.2, 1.3... up to about 49.8, 49.9 and 50%. In some aspects, the drug loading ranges from about 10 to 20%, such as about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20%, including all decimal fractions in between these values (e.g. 10.0, 10.1, 10.2, 10.3....19.7, 19.8, 19.09, 20.0%). In some aspects, the drug loading is from about 13-20%, such as about 14% or about 20%.

[0085] The average MP size generally ranges from about 5-20 pm ± 1.0- 1.5 pm, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 pm, including all decimal fractions in between these values (e.g. 5.0, 5.1, 5.2, 5.3....19.7, 19.8, 19.09, 20.0). In some aspects, the MP size is from about 10-15 pm, such as about 10, 11, 12, 13, 14, or 15 pm, including all decimal fractions in between these values (e.g. 10.0, 10.1, 10.2, 10.3...14.7, 14.8, 14.9 and 15.0). In some aspects, the average MP size is from about 12-13 pm, such as about 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9 or 13.0 pm, ± 1.0-1.5 pm, such as ± 1.0, 1.1, 1.2, 1.3, 1.4 or 1.5. In some aspects, the MP size is about 12.4 pm ± 1.2 pm. This size is suitable for delivery e.g. using fine gauge needles such as those used for injecting drugs into the eye.

[0086] A190

[0087] In some aspects of the present disclosure, the drug which is incorporated into the MPs described herein is A 190 and / or one or more variants thereof, such as a congener or other variant having at least about 50% of the physiological (biological) activity of the A190. Variants are further defined in the definitions section above and exemplary aspects of such variants are described below. A variant typically has at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 130, 140, 150, etc....up to about 950, 960, 970, 980, 990 or 1000 percent of the activity of A 190 (including all decimal fractions and / or percentages between and within these ranges and values), when compared using a standard art-recognized test of activity such as that described in the Example herein.

[0088] In additional aspects, the form of A 190 that is present in the MPs is a prodrug as defined elsewhere herein. Such prodrugs are typically inactive when administered, or at least have very low activity (e.g., less than 10% of the activity of A190), until they are processed within the body. After processing, the activity of the processed A190 or variant thereof is at least about 50% of the PPARa agonist activity of A190, as described above for A190.

[0089] “A190” refers to the family of drugs described in issued US patent US11447452B2, the complete contents of which is hereby incorporated by reference in entirety. A 190 and exemplary variants thereof have the following chemical structure: II

[0090]

[0091] wherein:

[0092] k is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms;

[0093] m is 0, 1, 2, 3, 4, or 5 carbon atoms;

[0094] n is 0, 1, 2, 3, 4, or 5 carbon atoms;

[0095] R2is selected from the group: hydrogen (H), chlorine (Cl), fluorine (F), bromine (Br), iodine (I), nitro (NO2), CH3, CH2CH3, branched or unbranched alkyl chains with 3-10 carbon atoms, OCH3, OCH2CH3, branched or unbranched alkoxy chains with 3-10 carbon atoms, haloalkyls, haloalkoxyls, cycloalkyls, halocycloalkyls, O-para-alkylbenzyls, O-para-alkyloxybenzyls, and O-para-halobenzyls, wherein the benzene ring comprising R2comprises one, two, three, or four of said R2substituents substituted in any combination of said R2substituents and arranged in any pattern in the ring including ortho, meta, mono, di, tri, and tetrasubstituted;

[0096] R3is selected from the group: H, Cl, F, Br, I, NO2, CH3, CH2CH3, branched or unbranched alkyl chains with 3-10 carbon atoms, OCH3, OCH2CH3, branched or unbranched alkoxy chains with 3-10 carbon atoms, haloalkyls, haloalkoxyls, cycloalkyls, halocycloalkyls, O-para-alkylbenzyls, O-para-alkyloxybenzyls, and O-para-halobenzyls, wherein the benzene ring comprising R3comprises one, two, three, four, or five of said R3substituents substituted in any combination of said R3substituents and arranged in any pattern in the ring including ortho, meta, para, mono, di, tri, tetra, and pentasubstituted;

[0097] R4is selected from the group: H, alkyl, and acyl;

[0098] R5is selected from the group: H, Cl, F, Br, I, NO2, CH3, CH2CH3, branched or unbranched alkyl chains with 3-10 carbon atoms, OCH3, OCH2CH3, branched or unbranched alkoxy chains with 3-10 carbon atoms, haloalkyls, haloalkoxyls, cycloalkyls, halocycloalkyls, O-para-alkylbenzyls, O-para-alkyloxybenzyls, and O-para-halobenzyls, wherein the benzene ring comprising R5comprises one, two, three, or four of said R5substituents substituted in any combination of said R5substituents and arranged in any pattern in the ring including ortho, meta, para, mono, di, tri, and tetrasubstituted;

[0099] R1is selected from the group consisting of carboxylic acids, carboxylic acid isosteres, hydroxamic acids, hydroxamic esters, phosphonic acids, phosphinic acids, sulfonic acids, sulfinic acids, sulfonamides, acyl sulfonamides, sulfonylureas, acylureas, tetrazoles, thiazolidine diones, oxazolidine diones, oxadiazol-5(4H)-ones, thiadiazol-5(4H)-ones, oxathiadiazole-2-oxides, oxadiazol-5(4H)-thiones, isoxazoles, tetramic acids, cyclopentane 1,3-diones, cyclopentane 1,2-diones, squaric acids, substituted phenols, heteroarenes, amidines, hydroxy amides, alkyl hydroxy amidines, and

[0100] ^,01 f

[0101] o

[0102] II N V

[0103] H \

[0104]

[0105] and salts thereof,

[0106] R8and R9are independently selected from the group consisting of H, F, Cl, Br, I, alkyl, alkoxy, and cycloalkyl comprising R8is linked to R9; R10and R11are independently selected from the group consisting of H, alkyl, and cycloalkyl wherein R10is linked to R11); and

[0107] X is O, NH, S, or CH2.

[0108] Exemplary R1 substituents are shown in Table 1 below.

[0109] Table 1. R1 substituents

[0110] Number Chemical structure

[0111] vm

[0112] " i''N:

[0113] H

[0114] Oli11

[0115]

[0116] In non-limiting embodiments, R8and R9of structure ix of Table 1 are selected from the group: hydrogen (H), halogens (F, Cl, Br, I), alkyl (e.g., branched or unbranched, Ci to Cio), alkoxy (e.g., branched or unbranched, Ci to Cio), and cyclo (R8linked to R9). In structure ix of Table 1, X can be O, NH, S, or CH2. In non-limiting embodiments, R1and R11of structure x of Table 1 are selected from the group: H, alkyl (e.g., branched or unbranched, Ci to Cio), and cyclo (R10linked to R11).

[0117] In other non-limiting examples, R1is selected from the group: carboxylic acids, and carboxylic acid isosteres including hydroxamic acids, hydroxamic esters, phosphonic acids, phosphinic acids, sulfonic acids, sulfinic acids, sulfonamides, acyl sulfonamides, sulfonylureas, acylureas, tetrazoles, thiazolidine diones, oxazolidine diones, oxadiazol-5(4H)-ones, thiadiazol-5(4H)-ones, oxathiadiazole-2-oxides, oxadiazol-5(4H)-thiones, isoxazoles, tetramic acids, cyclopentane 1,3-diones, cyclopentane 1,2-diones, squaric acids, substituted phenols, heteroarenes, amidines, hydroxyamides, alkyl hydroxyamidines, including the exemplary structures shown in Table 2, and further including salts of any of the above.

[0118] TABLE 2. Other examples of R1

[0119] Generic Group n = 0 - 4

[0120] o

[0121] Carboxylic acids

[0122]

[0123] Hydroxamic acids

[0124]

[0125] Hydroxamic esters

[0126]

[0127]

[0128] Phosphonic acids

[0129]

[0130] Phosphinic acids

[0131]

[0132] Sulfonic acids

[0133]

[0134] Sulfinic acids

[0135]

[0136] Sulfonamides

[0137]

[0138]

[0139] Acyl sulfonamides

[0140]

[0141] Acyl sulfonamides

[0142] Sulfonylureas

[0143]

[0144] Acylureas

[0145] Tetrazoles

[0146]

[0147] Thiazolidine diones

[0148]

[0149] Oxazolidine diones

[0150]

[0151] Oxadiazol-5(4H)-ones

[0152]

[0153] Thiadiazol-5(4H)-ones

[0154]

[0155] Oxathiadiazole-2-oxides

[0156]

[0157] Oxadiazole-5(4H)- thiones

[0158]

[0159] Isoxazoles

[0160]

[0161] Tetramic acids

[0162]

[0163] Cyclopentane 1,3-diones

[0164]

[0165]

[0166] Cyclopentane 1,2-diones

[0167]

[0168]

[0169] Squaric acids

[0170]

[0171]

[0172] Substituted phenols

[0173]

[0174]

[0175] Heteroarenes

[0176]

[0177]

[0178] Amidines

[0179]

[0180] Hydroxyamides

[0181]

[0182] Alkyl hydroxyamidines

[0183]

[0184] In at least certain non-limiting embodiments, the R2substituent of chemical structure I is selected from the group: H, F, Cl, Br, I, nitro (NO2), alkyl (e.g., CH3, CH2CH3, or any alkyl chain with 3-10 carbon atoms, branched or unbranched), alkoxy (e.g., OCH3, OCH2CH3, or any alkoxy chain with 3-10 carbon atoms, branched or unbranched), haloalkyl (e.g., CH2Cl, CHBr2, CF3), haloalkoxyl, (e.g., OCH2Cl, OCHBr2, OCF3), a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon haloalkyl having e.g., 1 to 3 halogen atoms, a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon haloalkoxyl having, e.g., 1 to 3 halogen atoms, cycloalkyl, halocycloalkyl, O-para-alkylbenzyl (e.g., where alkyl is methyl, ethyl, or propyl); O-para-alkyloxybenzyl (e.g., where alkyl is methyl, ethyl, or propyl); and O-para-halobenzyl (wherein halo=Cl, F, Br, or I).

[0185] In at least certain non-limiting embodiments, R4and R5together form a cycloalkyl comprising two to 10 carbon atoms, or a halocycloalkyl comprising two to 10 carbon atoms and substituted with one or more halogen (Cl, F, Br, I) atoms.

[0186] In at least certain embodiments, when one of R4and R5is an alkyl or cycloalkyl (as defined herein), the other of R4and R5is H.

[0187] In at least certain non-limiting embodiments, each R6and R7substituent of chemical structure I can selected from the group: H, F, Cl, Br, I, alkyl (e.g., branched or unbranched, Ci to Cio), and haloalkyl (e.g., CH2Cl, CHBr2, CF3). R6and R7may together consist of a double bonded O.

[0188] In at least certain non-limiting embodiments, R6and R7together form a cycloalkyl comprising two to 10 carbon atoms, or a halocycloalkyl comprising two to 10 carbon atoms and substituted with one or more halogen (Cl, F, Br, I) atoms.

[0189] In at least certain embodiments, when one of R6and R7is an alkyl or cycloalkyl (as defined herein), the other of R6and R7is H.

[0190] In certain non-limiting aspects of the present disclosure, examples of the compounds utilized in the present MPs include:

[0191]

[0192]

[0193]

[0194] OMe

[0195]

[0196] ASD 181

[0197] ASD 203

[0198] ASD 200

[0199]

[0200] COMPOSITIONS

[0201] The MPs described herein are generally delivered (administered) in a pharmaceutical composition and the present invention encompasses such formulations / compositions. The compositions generally include purified MPs as described herein (i.e., comprising at least one drug such as a PPARa agonist), and a pharmacologically suitable (physiologically compatible) carrier. Generally, such compositions are prepared as liquid solutions or suspensions. However, solid forms suitable for solution in, or suspension in, liquids prior to administration are also contemplated (e.g., lyophilized forms of the MPs), as are emulsified preparations. In some aspects, the formulations are liquid and are aqueous or oil-based suspensions or solutions.

[0202] In some aspects, the drug-loaded MPs are mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredients, e.g., pharmaceutically acceptable salts. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol and the like, or combinations thereof. In addition, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, preservatives, and the like. The compositions of the present invention may contain any such additional ingredients so as to provide the composition in a form suitable for administration, particularly administration into the eye. The final amount of drug-loaded MPs in the formulations varies but is generally from about 1-99%. Still other suitable formulations for use in the present invention are found, for example, in Remington's Pharmaceutical Sciences, 22nd ed. (2012; eds. Allen, Adejarem Desselle and Felton).

[0203] Some examples of materials which can serve as pharmaceutically acceptable carriers include, but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins (such as human serum albumin), buffer substances (such as Tween® 80, phosphates, glycine, sorbic acid, or potassium sorbate), partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes (such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, or zinc salts), colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, polyacrylates, sugars such as lactose, glucose and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil and soybean oil; glycols; such a propylene glycol or polyethylene glycol; esters such as ethyl oleate and ethyl laurate; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as sodium lauryl sulfate and magnesium stearate. Releasing agents, coating agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0204] " Pharmaceutically acceptable salts" of the compounds refers to the relatively nontoxic, inorganic and organic acid addition salts and base addition salts of compounds of the present disclosure. In some aspects, these salts are prepared in situ during the final isolation and purification of the compounds. In particular, acid addition salts can be prepared by separately reacting the purified compound in its free base form with a suitable organic or inorganic acid and isolating the salt thus formed. Exemplary acid addition salts include the hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, oxalate, valerate, oleate, palmitate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactiobionate, sulfamates, malonates, salicylates, propionates, methylene-bis-P-hydroxynaphthoates, gentisates, isethionates, di-p-toluoyltartrates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, cyclohexylsulfamates and laurylsulfonate salts, and the like. See, for example S. M. Berge, et al., " Pharmaceutical Salts," J. Pharm. Sci., 66, 1-19 (1977) which is incorporated herein by reference. Base addition salts can also be prepared by separately reacting the purified compound in its acid form with a suitable organic or inorganic base and isolating the salt thus formed. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. Suitable inorganic base addition salts are prepared from metal bases which include sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, zinc hydroxide and the like. Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include those amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use. ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N, N'-dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, and dicyclohexylamine, and the like. In some aspects, the salt is an HC1 (hydrochloride) salt.

[0205] The pharmaceutical compositions of the present invention are preferably packaged in a unit dosage form. The term "unit dosage form" refers to a form that is suitable for dosing a patient, i.e., such that each unit produces the desired therapeutic effect, either alone or in combination with one or more additional units. For example, a unit dosage form may be a unit package comprising a needle prefilled with a dose of a pharmaceutical composition suitable for intraocular administration; or a unit dosage form may be a unit package comprising an ampule or other container prefilled with a single dose or multiple doses of a pharmaceutical composition suitable for intraocular administration and ready to be loaded into a syringe. Such units may be available e.g. in a blister pack or other packaging suitable for shipping and storage. All such packaged doses of the pharmaceutical compositions are encompassed herein.

[0206] METHODS

[0207] The compositions disclosed herein are generally administered intraocularly, such as by intraocular injection, generally by an ophthalmologist. In some aspects, the compositions are administered by intravitreal injection or by another intraocular route as discussed elsewhere herein.

[0208] Typically, a volume of 1 to 100 pL, e.g. 25 pL, 50 pL, or 100 pL, and usually no more than 100 pL, of the subject composition is delivered to the eye by intravitreal injection without removing the vitreous. For intravitreal administration, the vector can be delivered in the form of a suspension. Typically, the process involves numbing the eye, cleaning the surface with an antiseptic (e.g., povidone-iodine) and using a sterile speculum to hold the eyelids open. In other words, a topical anesthetic is applied to the surface of the eye followed by a topical antiseptic solution. The eye is held open, with or without instrumentation, and the composition is injected through the sclera with a short, narrow needle, for example a 30-gauge needle, into the vitreous cavity of the eye of a subject, under direct observation of a medical professional, e.g., an ophthalmologist. The ophthalmologist carefully injects a suitable amount of the composition comprising the drug-loaded MPs through the eye and into the vitreous humor. An antibiotic drop is typically administered after the injection.

[0209] Intravitreal administration is generally well tolerated. At the conclusion of the procedure, there is sometimes mild redness at the injection site. There is occasional tenderness, but most patients do not report any pain. No eye patch or eye shield is necessary after this procedure, and activities are not restricted. Sometimes, an antibiotic eye drop is prescribed for several days to help prevent infection.

[0210] Alternatively, in some aspects, a vitrectomy is performed, and the entire volume of vitreous gel is replaced by an infusion of the subject composition. In such cases, up to about 4 mL of the subject composition is delivered, e.g., to a human eye.

[0211] As a further alternative, the compositions are delivered by subretinal injection, a surgical procedure in which a composition comprising the MPs is injected into the space between the retina and the retinal pigment epithelium (RPE). This technique is used primarily for treating submacular hemorrhages and managing conditions like full-thickness macular holes. The subretinal space is an immune-privileged location, which allows for a reduced concentration of drug and minimizes systemic side effects.

[0212] In some aspects, the deposition of a composition containing the microparticles as described herein is in effect the placement or formation of a “depot” for slow, extended release of the drug (e.g. A190) that is in the MPs, The slow or extended release generally occurs for at least weeks and generally for months, such as for 1-12 months, e.g. about 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11 or 12 months. In some aspects, release of A190 persists for up to 6 months or for at least 6 months.

[0213] Since the conditions that are treated with the MPs described herein are long-term conditions, the injections are repeated for as long as the condition persists, which may be for the lifetime of the patient, or until the patient and / or the medical professional (e.g., an ophthalmologist) decide that further treatment is not necessary or would not be helpful, e.g., if blindness occurs and there is no hope of restoring sight. If injections are conducted longterm, they generally and advantageously are performed only about once every 6 months, which greatly increases patient compliance and decreases inconvenience. However, other dosing schedules may be observed, as determined by a medical professional. For example, the injections may be every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. Generally, the injections are scheduled every 3, 4, 5, 6, 7, 8, or 9 months, with 5, 6 or 7 months being usual, and every 6 months generally being preferred. The frequency of injections may be decreased if the condition stabilizes.

[0214] The compositions disclosed herein may be administered with other medicinal agents. For example, treatments for macular degeneration depend on its type and stage, with options including oral vitamin supplements for the dry form and eye injections and / or laser therapy for the more advanced "wet" form. For dry macular degeneration, lifestyle changes like a healthy diet and quitting smoking are recommended, while supplements may help slow progression. Wet macular degeneration treatments like anti-VEGF (vascular endothelial growth factor) injections are the main approach to stop or slow vision loss. Such treatments may be combined with and / or used in conjunction with the present methods. For example, anti-VEGF agents may be administered at the same time or even in the same compositions. Common anti-VEGF medications include but are not limited to: ranibizumab (Lucentis®), a fragment of a monoclonal antibody designed to bind to and inhibit VEGF; aflibercept (Eylea®), a fusion protein that acts as a decoy receptor to trap VEGF; bevacizumab (Avastin®), a full-length monoclonal antibody that was initially approved for cancer treatment but is now used off-label for eye conditions; brolucizumab (Beovu®), a monoclonal antibody that binds to and inhibits the different forms of VEGF-A, suppressing the formation of new blood vessels and reducing vascular permeability, which is used for treating wet AMD and diabetic macular edema; and faricimab (Vabysmo®), a bispecific antibody that targets both VEGF and angiopoietin-2 proteins. The compositions disclosed herein may also be administered in conjunction with gene therapies.

[0215] DISEASES THAT ARE TREATED

[0216] The compositions disclosed herein are used to treat and / or prevent or inhibit and / or prevent progression of (arrest progression of) at least one symptom of an eye disease or condition. The eye disease or condition may be caused by inactivity or low activity of the protein PPARa. Inactivity or low activity of the protein PPARa in humans is linked to a range of eye diseases, most notably those with underlying metabolic and inflammatory components, including diabetic keratopathy, diabetic retinopathy, and age-related macular degeneration (AMG). Inactivity can also contribute to other conditions affecting the retina and cornea by disrupting energy metabolism, leading to comeal nerve degeneration, epithelial erosion, impaired comeal sensitivity, and increased oxidative stress and inflammation. Any and / or all of these diseases / conditions can be treated by the methods disclosed herein.

[0217] Dry AMD is characterized by a buildup of yellow deposits called drusen between the retinal pigment epithelium and the underlying choroid of the macula, which may be observed by fundus photography. This results in a slowly progressive loss of vision. The condition typically affects vision in both eyes, although vision loss often occurs in one eye before the other. Other changes may include pigment changes and RPE atrophy (the thinning and loss of the retinal pigment epithelium). For example, in certain cases called central geographic atrophy, or “GA”, atrophy of the retinal pigment epithelial and subsequent loss of photoreceptors in the central part of the eye is observed. Dry AMD has been associated with mutations in CD59 and genes in the complement cascade.

[0218] Wet AMD is a progressed state of dry AMD and occurs in about 10% of dry AMD patients. Pathological changes include retinal pigment epithelial cells (RPE) dysfunction, fluid collecting under the RPE, and choroidal neovascularization (CNV) in the macular area. Fluid leakage, RPE or neural retinal detachment and bleeding from ruptured blood vessels can occur in severe cases. Symptoms of wet AMD may include visual distortions, such as straight lines appearing wavy or crooked, a doorway or street sign looking lopsided, or objects appearing smaller or farther away than they really are; decreased central vision; decreased intensity or brightness of colors; and well-defined blurry spot or blind spot in the field of vision. Onset may be abrupt and worsen rapidly. Diagnosis may include the use of an Amsler grid to test for defects in the subject's central vision (macular degeneration may cause the straight lines in the grid to appear faded, broken or distorted), fluorescein angiogram to observe blood vessel or retinal abnormalities, and optical coherence tomography to detect retina swelling or leaking blood vessels. A number of cellular factors have been implicated in the generation of CNV, among which are vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), pigment epithelium-derived factor (PEDF), hypoxia inducible factor (HIF), angiopoietin (Ang), and other cytokines, mitogen-activated protein kinases (MAPK) and others.

[0219] In at least certain alternate embodiments, the active agent compositions and methods for treating ocular disorders and conditions, and particularly retinal conditions and disorders, which in certain non-limiting embodiments, include retinal inflammation, retinal neovascularization, retinal vascular leakage, retinopathy of prematurity (ROP), diabetic retinopathy (DR), age-related macular degeneration (e.g., wet AMD), and diabetic macular edema (DME) (or others disorders or conditions described elsewhere herein)

[0220] It is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0221] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0222] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Representative illustrative methods and materials are herein described; methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

[0223] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual dates of public availability and may need to be independently confirmed.

[0224] It is noted that, as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as support for the recitation in the claims of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitations, such as "wherein [a particular feature or element] is absent", or "except for [a particular feature or element]", or "wherein [a particular feature or element] is not present (included, etc.)...".

[0225] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0226] The invention is further described by the following non-limiting examples which further illustrate the invention, and are not intended, nor should they be interpreted to, limit the scope of the invention.

[0227] EXAMPLES

[0228] Example 1. Sustained Release of a Novel Non-fibrate PPARa Agonist from Microparticles for Neuroprotection in Murine Models of Age-related Macular Degeneration

[0229] The present study utilized two representative models of AMD to evaluate the therapeutic potential of A190-loaded polymeric microparticles (A190-MP). The very low-density lipoprotein receptor (Vldlr) gene knockout mouse model, associated with retinal angiomatous proliferation, a subtype of wet AMD, shows key AMD features, including retinal vascular leakage, neovascularization, electroretinogram (ERG) decline, and photoreceptor degeneration. The ATP-binding cassette transporter 4 (Abca4) and retinol dehydrogenase 8 (Rdh ) double-knockout Abcad^ / RdhS7' ) mouse model, which recapitulates Stargardt disease and some dry AMD pathologies, exhibits ERG decline, photoreceptor loss, RPE atrophy, and lipofuscin accumulation. Both models capture the progressive, chronic nature of AMD, which is essential for evaluating the long-term effects of A190-MP.

[0230] Material and Methods

[0231] Materials

[0232] PLGA (LA: GA 50:50, acid terminated) with molecular weight (MW) 7, 18, 34, 54 kDa were purchased from Evonik Corporation (Birmingham, AL). PLGA45kDa-PEG5kDa was custom- synthesized. A190 was made synthetically. Poly (vinyl alcohol) (PVA; MW -30,000-70,000, 87-90% hydrolysis), dichloromethane, acetonitrile, methanol and trifluoroacetic acid were obtained from Sigma-Aldrich (St. Louis, MO).

[0233] Preparation of A190 microparticles (A190-MP)

[0234] A190-MP were prepared by using a solvent evaporation method. All glassware, centrifuge tubes, and solvents used in the microparticle preparation were autoclaved / filtered beforehand. Briefly, 100 mg of PLGA with different MW and 25 mg A 190 (target drug loading 20 wt%) were dissolved in 1 ml dichloromethane. The oil phase was then added into 40 ml 1% PVA solution under homogenization at 8000 rpm using a high-shear L5M-A mixer (Sil verson Laboratory Mixers, MA) for 2 min. The emulsion was then poured into 60 ml of 0.3% PVA solution under magnetic stirring at 700 rpm and allowed to stir for 2 hr. Then the system was moved to a vacuum chamber under magnetic stirring for another 2 hr to allow complete evaporation of the organic solvent residue. The solidified A190-MP was filtered through a 40-pm cell strainer to eradicate large, aggregated particles. The filtered suspension was centrifuged at 500 xg for 10 min, followed by three cycles of washing with autoclaved water. The final A190-MP were resuspended in ultrapure water, and a small amount of this suspension was placed on a glass slide to inspect for any residual drug crystals using an optical microscope. Blank microparticles (Blank-MP) were produced using the same procedure, with no A190 dissolved in the PLGA solution. For subsequent animal studies, A190-MP and Blank-MP were freshly prepared.

[0235] Characterization of A190-MP

[0236] The average particle size of A190-MP was measured by laser diffraction using Malvern Mastersizer 3000 (Malvern Instruments, England). A190-MP suspension (20 pl) was added to the wet dispersion media (water) until the obscuration reached 5-10%. “Dv50” denotes the volume- weighted median diameter of microparticles, and unless otherwise specified, the average particle size of A190-MP is expressed as Dv50. Span value was used to analyze the poly dispersity of microparticles given by Span = (D90 - D10) / D50, where Dv10, Dv50, and Dv90 are particle size distribution measurements representing the diameters at which 10%, 50%, and 90% of the total volume of particles in a sample are smaller than the respective values.

[0237] The surface morphology of A190-MP was evaluated using scanning electron microscopy (SEM; Hitachi SU-70 FE-SEM, Hitachi High-Tech Inc, CA). Briefly, lyophilized A190-MP underwent platinum sputtering for 90 sec under vacuum, followed by SEM images taken under an accelerated voltage of 5 kV and 15 mm working distance. To calculate the drug loading of A190-MP, ~30 pl of A190-MP were lyophilized in preweighed Eppendorf tubes. After lyophilization, the final weights of the tubes were taken, and the lyophilized particles were dissolved in 1 ml acetonitrile. The drug content was measured using HPLC-UV (Shimadzu Prominence LC system) with a Pursuit 5 C18 column, and the mobile phase was comprised of acetonitrile and water (80:20 v / v) containing 0.1% trifluoro acetic acid (flow rate of 1 ml / min). The drug was detected at a wavelength of 227 nm. The drug loading (DL) was calculated using the following equation. DL (%) = (amount of Al 90 in particles) / (weight of particles)

[0238] The in vitro drugrelease profiles of A190-MP were evaluated under sink conditions using phosphate-buffered saline (PBS) with 0.2% Tween 80 (pH 7.4) as the release media. The setup was held at 37°C on a platform shaker operating at 120 rpm. A190-MP (containing -800 pg A 190) were suspended in 12 ml of release media in triplicate inside 15 ml Falcon® tubes. At predetermined time points, the tubes were centrifuged at 1500 rpm for 10 min, the released media was collected and the tubes were refilled with fresh media. The A 190 concentration in the collected released media was analyzed using HPLC-UV.

[0239] Animals and Ethics Approval

[0240] VI dlr'7' mice on the C57BL / 6J background were purchased from Jackson Laboratories (Bar Harbor, ME). Ahead ' / Rdh8'7' mice were kindly provided by a colleague and bred into the 129 Sv / Ev background in our lab. VI dlr7' mice and Abca4'7 / Rdh8' ' mice were genotyped as described previously. Age-matched wild-type (C57BL / 6J) mice were used for the safety study. Our study examined male and female mice, and similar findings are reported for both sexes.

[0241] All animal experiments were performed in compliance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and all protocols were approved by the Institute Animal Care and Use Committee at Wake Forest University and Virginia Commonwealth University.

[0242] Ocular Pharmacokinetic Study of A190-MP

[0243] Ocular pharmacokinetics were evaluated for A190-MP and A 190 solution (in DMSO) in C57BL / 6J mice (8 weeks old) following a single intravitreal injection. Mice were deeply anesthetized following injection of a mixture of 50 mg / kg ketamine and 5 mg / kg xylazine (Vedco, Saint Joseph, MO). One pl of A190-MP or A190 solution (30 pg A190) was administered via intravitreal injection into both eyes using a 31-gauge needle under a surgical ophthalmic microscope. At day 7, and at 1, 2, 4 and 6 months post-injection, the mice were euthanized, and the eyeballs were enucleated. Retina tissues, vitreous humor and blood plasma were collected. A 190 levels in the collected ocular tissues were analyzed using a Liquid Chromatography Mass Spectrometer (LC-MS-2020; Shimadzu Corporation). In brief, vitreous humor and retina were homogenized in 200 pl of water before extraction using Next Advance bullet blender (Stellar Scientific, Owings Mills, MD) for 5 min. 150 pl of tissue homogenate / blood plasma was spiked with 150 pl of internal standard (Fenofibric acid D6, Toronto Research Chemicals, Toronto, Canada) followed by 800 pl of cold acetonitrile (for retina and plasma) / cold methanol (for vitreous humor) to allow protein precipitation. The samples were vortex-mixed and centrifuged for 10 min at 20,000 rpm at 4°C. After centrifugation, the supernatant was transferred to glass tubes and evaporated with nitrogen gas at 45 °C, ensuring the organic solvents were completely evaporated. Dry residues were reconstituted in 150 pl of a mixture containing acetonitrile / water (1:1 v / v) with 0.1% formic acid, vortex mixed for 10 min and then filtered using a 0.22 pm hydrophobic filter. The samples were then injected into the LC-MS with Phenomenex LC column (Luna Omega 3 pm Polar C18 100 A, with dimensions of 100 mm x 2.1 mm); the mobile phase consisted of LCMS acetonitrile / water (60:40 v / v) containing 0.1% formic acid. The separation was achieved at a 0.3 ml / min flow rate over 9 min. The concentration of A190 in the samples was analyzed against a 9-point standard curve (1 ng / g-1000 ng / g). Pharmacokinetic parameters were calculated using mean concentration vs time data using non-compartmental methods with PK Solver software.

[0244] Intravitreal Injection of Microparticles

[0245] At the age of 8 weeks, mice were randomly assigned into the A190-MP group and Blank-MP control group. After mice were deeply anesthetized and pupils were dilated with tropicamide ophthalmic solution (Bausch & Lomb, Bridgewater, NJ), a small hole was punctured through the inferior sclera with a 31-gauge beveled needle, ensuring blood vessels were unaffected. Subsequently, a 31-gauge needle, attached to a microliter syringe, was penetrated through the opening to deliver 1 pl of A190-MP (30 pg A190) or the same dose of Blank-MP (125 pg) slowly into the vitreous cavity. Following the intravitreal injection, antibiotic ointment was applied to the eyes to prevent infection.

[0246] Electroretinogram (ERG) Recording

[0247] Retinal function was measured with ERG, as described previously. The photopic and scotopic ERG were recorded at different time points post-injection using the Espion Visual Electrophysiology System (Diagnosys, Orlando, FL). The data in both eyes were recorded, and the average of the left and right eyes were used for the analysis.

[0248] Spectral-domain Optical Coherence Tomography (SD-OCT) and Quantification of Lesion Volume

[0249] SD-OCT (Envisu ™ R-series SDOIS system, Leica, Morrisville, NC) images were captured using the following parameters: rectangular scan: 1000 A-scans per B-scan, 100 B-scans per frame, and the meridian crossing through the center of the optic nerve, and then cross-sectional images of the retina were produced, from which the thickness of various retinal layers can be measured. The thickness of each retinal layer was measured using the SPECTIVE DIVER 9X9 SPIDERGRAM mode (InVivoVU, Bioptigen). Measurements were taken along both the vertical meridian from superior to inferior and the horizontal meridian from nasal to temporal, with the optic nerve as the central reference point, at intervals of 0.18 mm. Lesion volume (mm3) was calculated following a published protocol (R. S. Sulaiman, et al., A Simple Optical Coherence Tomography Quantification Method for Choroidal Neovascularization, J Ocul Pharmacol Ther, 31 (2015) 447-454). Data represents the average of both eyes of each subject when binocular data was available.

[0250] Fundus Images and Fundus Fluorescein Angiography (FFA)

[0251] Fundus images and FFA were performed with the retinal imaging microscope (Micron IV, Phoenix Research Laboratories, Bend, OR) as described previously. Fluorescent fundus images were captured after intraperitoneal injection with 5% fluorescein sodium. The numbers of fluorescein leakage spots and the lesion area were measured by Image J software (National Institutes of Health, Bethesda, MD). Data from both eyes were averaged.

[0252] Retinal Flat Mounts and RPE / Choroidal Flat Mounts Preparation

[0253] Retinas and RPE / choroids were dissected as described previously. After euthanasia, mice were perfused with warm PBS. The eyeballs were then isolated and fixed in 4% paraformaldehyde in PBS for 10 min. After cleaning off the surrounding muscle and fat, the cornea and lens were removed. The detached retina was carefully separated from the posterior segment and fixed in 4% formaldehyde for another 30 min to prepare retinal flat mounts. The remaining posterior eyecups, consisting of the RPE, choroid, and sclera, were used to prepare RPE / choroid flat mounts.

[0254] Histology

[0255] Enucleated eyeballs were fixed in Davidson fixative for 24 hr. After dehydration with ethanol and subsequent embedding in paraffin, serial sections with a thickness of 5 pm were cut and stained using hematoxylin and eosin (H& E). The images were captured under Olympus Microscope (BX43, Olympus, Tokyo, Japan) attached to a digital camera (U-TV0.5XC-3, Olympus). The thickness of the retinal layers was measured in sections from the optic nerve head (ONH) along the vertical meridian toward the superior and inferior regions at 200 pm intervals using cellSens™ imaging software (Olympus).

[0256] Immunofluorescence Staining

[0257] For mouse retinal paraffin sections were stained, following standard de-paraffinization and antigen retrieval. After blocking, sections were incubated overnight with primary antibodies for 3-NT (1:100, ab61392, Abeam, Waltham, Boston) and TOMM20 (1:100, ab186735, Abeam), followed by incubation with secondary antibodies (Table 3) for 1 hr. Nuclei were counterstained with DAPI (1:1000, D9542, Sigma) and mounted on slides with antifade mounting medium (Vector Laboratories, Newark, CA).

[0258] For immunostaining using mouse RPE / choroidal and retinal flat mounts, flat mounts were blocked and permeabilized. Retinal vasculature and lesions were stained with fluoresceinated isolectin GS-IB4 (10 pg / mL; 121413, Invitrogen™, Waltham, Massachusetts). Stacks of images covering the entire thickness of the retinal vasculature of IB4 in retinal and RPE / choroidal flat mounts were captured with Cytation 5 Cell Imaging Multi-Mode Reader (BioTek, Clinton, NC). The IB4 positive lesion number and area were analyzed by Image J software.

[0259] For 661W cells, immunofluorescence analysis was performed on cells grown on collagen I-coated glass coverslips (Corning, Corning, NY). Cells were fixed in 4% paraformaldehyde for 15 min and washed in PBS. After blocking with 5% BSA and 0.3% Triton X-100 in PBS for 1 hr. Fixed cells were incubated overnight at 4°C with primary antibodies against 3-NT (1:100, ab61392, Abeam) and TOMM20 (1:100, ab186735, Abeam). TOMM20, a translocase of the outer mitochondrial membrane, correlates with mitochondrial mass, and is utilized as a marker for mitochondrial morphology. Mitochondria were classified as "fragmented" if over 50% of the mitochondrial area consisted of punctiform mitochondria. Following incubation with primary antibodies, cells were incubated with secondary antibodies (Table 1) and DAPI (1:1000) for 2 hr. For each experiment, 3-4 immunofluorescent images per group were captured randomly using a Zeiss Axio Observer Z1 microscope with an Apotome 3 (Carl Zeiss, Oberkochen, Germany). Cone Staining and Quantification Analysis

[0260] Fluorescent peanut agglutinin (PNA; 1:1000, FL-1071, Vector Laboratories), was applied to stain retinal flat mounts and paraffin sections. On the retinal flat mounts, cones were counted and the number of cells was counted and averaged by a masked observer. For retinal paraffin sections, the length of the cone outer segments (OS) was quantified over a 500 pm area extending from the ONH in each region of the retina.

[0261] Terminal Deoxynucleotidyl Transferase-mediated dUTP nick-end-labeling (TUNEL) Assay

[0262] TUNEL staining was performed according to the manufacturer’s protocol (In situ Cell Death Detection Kit, 11684795910, Roche, Basel, Switzerland), and DAPI was applied for nuclear counterstaining. For mouse retinal sections, TUNEL-positive nuclei were counted automatically using the TUNEL cell counter plugin for ImageJ software.

[0263] Bioenergetic Profiling and Real-Time ATP Rate

[0264] Seahorse XF Pro analyzer (Agilent Technologies, Charlotte, NC) was used to measure the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Briefly, the mouse retina was isolated, 1-mm in diameter biopsy punches were obtained at 1.5 mm distance from the optic nerve and then loaded in the spheroid microplate with the photoreceptor side up. Retinal punches were incubated in Seahorse XF DMEM (103575-100, Agilent) supplemented with 12 mM glucose, 5 mM HEPES, 5 mM pyruvate, and 2 mM glutamine. OCR was measured following the protocol for the Seahorse XF Cell Mito Stress Test Kit with sequential injections of a final concentration of oligomycin (an inhibitor of ATP synthase, 2 pM), carbonyl cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP, an uncoupler of mitochondrial oxidative phosphorylation, 1 pM), and rotenone / antimycin A (RAA, a complex I & III inhibitor, 2 pM).

[0265] 661W cells were cultured at a density of 6 × 103cells / well in XF Pro cell culture microplates. For real-time ATP rate assay, 1.5 pM oligomycin and 0.5 pM RAA were added to measure cellular ATP production. For cell Mito Stress test, OCR was recorded after injection of oligomycin (1.5 pM), FCCP (1.5 pM), and RAA (0.5 pM) sequentially. These results were normalized to cell number. All the Seahorse data were automatically calculated and analyzed using the Wave software and XF Report Generators (Agilent Technologies).

[0266] Cell Culture and Treatments

[0267] The 661W cell line, derived from mouse cone photoreceptors, was kindly provided by a colleague. 661W cells were cultured in Dulbecco's Modified Eagle Medium (DMEM; 10-027-CV, Corning) supplemented with 10% fetal bovine serum (FBS; S11150, Atalanta, Elizabeth, NJ) and 1% penicillin / streptomycin (30-002-CI, Corning). Subsequently, cells were pre-treated with A 190 for 2 hr prior to the addition of 4-hydroxynonenal (4-HNE; 393204, Millipore, Burlington, MA) for an additional 24 hr incubation, with ethanol as the vehicle control. Cells of passages 6-15 were used and then harvested in accordance with the respective assays. Phase contrast images were taken with a Cytation 5 Cell Imaging System (BioTek).

[0268] PPARa expression was knocked down in 661W cells by transfection with ON-TARGETplus mouse Ppara siRNA SMARTpool (Dharmacon, Lafayette, CO) or with nontargeting siRNA SMARTpool as a negative control, using HiPerFect transfection reagent (301704, QIAGEN, Germantown, MD) according to the manufacturer’s instructions.

[0269] Primary mouse Muller cells were cultured as described previously. The retinas of WT and PPARα- / -mice were isolated, minced into fragments and cultured in DMEM with 10% FBS and 1% penicillin / streptomycin.

[0270] Cell Viability

[0271] Cell viability was determined by either MTT or trypan blue exclusion assays. MTT assay was performed according to the manufacturer’s protocol (11465007001, Sigma-Aldrich). Trypan blue exclusion assay was employed following the treatments as previously described. The viable cells were counted using an automated cell counter (Nexcelom Bioscience, Lawrence, MA).

[0272] Lactate Dehydrogenase (LDH) Assay

[0273] The CyQUANT™ LDH cytotoxicity assay (C20300, Invitrogen™) was used to assess the amount of LDH released following the manufacturer’s instructions. In brief, the supernatants from 661W cells were mixed with a substrate reaction for 30 min and the absorbances at 490 nm were measured. To eliminate the contributions of endogenous LDH, the absorbance readings (background OD value) from media incubated without any treatment were subtracted. The degree of cell death was regarded as a percentage of the total cell death measured in parallel wells with cells lysis solution (maximum OD value).

[0274] Cytotoxicity (%) was determined using this formula: Cytotoxicity (%) = (Experimental - background) A490 value / (Maximum - background) A490 value.

[0275] Intracellular Reactive Oxygen Species (ROS) Production

[0276] A general oxidative stress indicator, fluorescent probe 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA), was used to determine the ROS levels in 661W cells, according to the manufacturer’s instruction (C6827, Invitrogen™). CM-H2DCFDA was added at a final concentration of 10 pM for 45 min at 37°C and then washed in PBS. ROS production was measured with a fluorescence plate reader and normalized to the protein concentration.

[0277] Western Blot Analysis

[0278] Equal amounts of total protein from each sample were used for immunoblotting analysis. Antibodies are listed in the antibody table (Table 3). Band intensity was semiquantified with ImageJ, normalized to P-actin levels.

[0279] Table 3. Antibodies.

[0280] Antibody Manufacturer Catalog Dilution Applications Number

[0281] AlexaFluor 488 Jackson ImmunoResearch, 711-545-152 1:300 IF donkey anti-rabbit West Grove, PA

[0282] AlexaFluor 488 Jackson ImmunoResearch 715-545-150 1:300 IF donkey anti-mouse

[0283] Alexa Fluor 594 Jackson ImmunoResearch 715-585-152 1:300 IF donkey anti-rabbit

[0284] Alexa Fluor 594 Jackson ImmunoResearch 715-585-150 1:300 IF donkey anti-mouse

[0285] Rabbit Anti- Novus Biologicals, NB600-636 1:1000 WB PPARa Centennial, CO

[0286] Rabbit Anti- Novus Biologicals NBP1-04676 1:1000 WB PGCla

[0287] Rabbit Anti- Abeam, Waltham, Boston ab186735 1:1000 WB TOMM20

[0288] Rabbit Anti- VEGF Abeam ab46154 1:1000 WB Mouse Anti- Abeam ab171123 1:1000 WB ICAM-1

[0289] Goat Anti-IL-i R& D Systems, Minneapolis, AF-401-NA 1:1000 WB MN

[0290] Rabbit Anti- Proteintech, Rosemont, IL 15184-1-AP 1:1000 WB CPT1A

[0291] Rabbit Anti-pNF- Cell Signaling, Danvers, 3033 1:1000 WB

[0292] KB MA

[0293] Rabbit Anti-NOX4 Santa Cruz Biotechnology, sc-30141 1:1000 WB Santa Cruz, CA

[0294] Rabbit Anti-BCL- Cell Signaling 3498 1:1000 WB

[0295] 2

[0296] Rabbit Anti-B AX Cell Signaling 2772 1:1000 WB HRP conjugated Santa Cruz Biotechnology sc-47778 1:5000 WB Mouse Anti-P- actin

[0297] Peroxidase Vector Laboratory, Newark, PI-1000 1:5000 WB conjugated Goat CA

[0298] Anti-rabbit

[0299] Peroxidase Vector Laboratory PI-2000 1:5000 WB conjugated Horse

[0300] Anti-mouse

[0301] Peroxidase Vector Laboratory PI-9500 1:5000 WB conjugated Horse

[0302] Anti-goat

[0303] Abbreviations: IF, immunofluorescence; WB, western blot

[0304] Statistical Analysis

[0305] All the measurements were confirmed by an independent, blinded researcher. Data are presented as mean ± standard error (SEM). Quantitative data analyses were analyzed using unpaired Student's t test for two groups, and analysis of variance (ANOVA) following a post hoc test for more than two groups. P value < 0.05 was regarded as statistical significance.

[0306] Intraocular Pressure (IOP) Measurement

[0307] IOP was measured with a non-invasive rebound Icare tonometer (Icare Finland Oy, Finland) in awake, unanesthetized mice. The probe made a gentle and brief contact with the eye while taking the measurement. The values were averaged in six independent measurements. Mean values of both eyes were calculated and used as an individual data point for analysis. For Abca4- / - / Rdh8- / -mice, IOP was monitored at 2, 4, and 6 months post-injection. For C57BL / 6J mice, IOP was measured both before and six months after the injection.

[0308] Systemic Toxicities of Blank-MP and A190-MP

[0309] To evaluate any systemic toxicity of the microparticles, blood serum chemistry and histological analyses of major body organs, including the heart, kidney, liver, and spleen, were performed at 6 months post-injection.

[0310] Blood Serum Chemistry

[0311] Aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), creatinine, and blood urea nitrogen (BUN) in the serum were assessed as indicators of liver and renal function.

[0312] Organ Histology

[0313] At the terminal point, major organs, including heart, kidney, liver, and spleen were collected for histological analysis. The organs were embedded in paraffin blocks and sectioned into 5-pm-thick sections. The sections were stained with hematoxylin and eosin (H& E). The images were captured using Nikon E800 light microscope (Tokyo, Japan) equipped with digital camera.

[0314] Glycolytic Stress Test

[0315] 661W cells were cultured at a density of 6 × 103cells / well in Seahorse XF Pro cell culture microplates. For glycolysis stress test, extracellular acidification rate (ECAR) was measured after sequentially injection of 10 mM glucose, 1 pM oligomycin, and 50 mM 2-deoxyglucose (2-DG, a glucose analogue). These results were normalized to cell number. All the Seahorse data were automatically calculated and analyzed using the Wave software and XF Report Generators (Agilent Technologies).

[0316] Results

[0317] Characteristics of A190-MP

[0318] A190 (structure shown in Fig. 1A) was successfully loaded into PEGA microparticles, and the physicochemical characteristics of A190-MP are listed in Table 4. A190-MP were spherical in shape with smooth surfaces (Fig. IB). The average particle size of the microparticles ranged from 8.7 ± 0.7 pm to 16.7 ± 0.6 pm, suitable for passing through the fine gauge needle used for intravitreal injection. The particle size distribution as indicated by span value, shows that PEGA54kDa / PEGA45kDa-PEG5kDa (F5) had the lowest span value of 1.1, and PLGA7kDa (F1) had the highest span value of 3.6. To confirm the absence of drug crystals in the formulations, visual inspection was performed under a phase contrast microscope after washing the microparticles. We did not observe drug crystals in the final MP formulations except for PLGA7kDa (F1) indicating poor drug encapsulation of A190 into PEGA polymers with lower MW. Intravitreal injection of A190-MP prevented ERG decline and retinal degeneration, and improved mitochondrial function in Vldlr- / -mice.

[0319] Previous studies revealed an age-dependent decline of photoreceptor function in Vldlr / _mice compared to age-matched wild-type (WT) mice. ERG responses were recorded at 1, 3, and 5 months post-administration of A190-MP. A190-MP significantly increased photopic ERG a-wave amplitudes at all time points compared to the Blank-MP group, with 35.03%, 73.25%, and 35.66% higher amplitudes at 1, 3, and 5 months, respectively (Fig. 2A-C), indicating preserved cone function. Additionally, A190-MP enhanced scotopic a-wave amplitudes at 3 months (Fig. 2B). Photopic b-wave amplitudes were increased in the A190-MP group at 3 months, while scotopic b-wave amplitudes were significantly higher at 1, 3, and 5 months (Figure 9A-C). Collectively, these findings suggest that A190-MP treatment ameliorates ERG decline in Vldlr ' mice.

[0320] Table 4. Physicochemical properties of A190-MP

[0321] Formulation Polymer Average Span Drug Particle Eoading Size (pm) (wt%) A190-MP-F1 PLGA7kDa 8.7 ± 0.7 3.6 7.4 % A190-MP-F2 PLGA18kDa 11 ± 1.0 1.3 10.0 % A190-MP-F3 PLGA34kDa 14.3 ± 1.6 1.3 9.3 % A190-MP-F4 PEGAs4|<|)a 16.7 + 0.6 1.2 11.0 % PLGA54kDa / PLGA45kDa- 12.4 ± 1.2 1.1 14.0 % A190-MP-F5

[0322] PEGs|<|)a

[0323]

[0324] Abbreviations: PLGA, Poly (lactic-co-glycolic acid); PEG, polyethylene glycol.

[0325] The drug release profiles for A190-MP formulations demonstrated a wide range of controlled release behaviors under in vitro sink conditions (Fig. 1C). The drug release rate from A190-MP was significantly affected by the MW of PEGA. A190-MP-F1 (PLGA7kDa) exhibited the fastest release, reaching nearly complete drug release within 20 days. A190-MP-F2 (PLGA18kDa) and F3 (PEGA34kDa) showed intermediate release profiles over 3-4 months, with cumulative release reaching approximately 80-90% by 100 days. A190-MP-F4 (PLGA54kDa) and F5 (PEGA54kDa / PEGA45kDa-PEG5kDa) displayed the slowest release, extending beyond 6 months and reaching only approximately 80-85% release by 180 days. These slower-releasing A190-MP formulations exhibited a triphasic release pattern. The distinctive in vitro release profile of A190-MP-F5 (PLGA54kDa / PLGA45kDa-PEG5kDa), extending over approximately 6 months, aligns optimally with the planned animal study duration. Consequently, A190-MP-F5 was selected for subsequent ocular pharmacokinetic, efficacy, and safety studies. Henceforth, the term 'A190-MP' will specifically denote A 190-MP-F5 here.

[0326] Ocular pharmacokinetics of A190-MP

[0327] A190-MP provided sustained drug release for at least 6 months in the vitreo-retina tissues following an intravitreal injection (Fig. ID and E). The calculated AUC0-180days for the retina and vitreous humor were 3074.4 ± 481.9 ng*d / g and 5308.3 ± 1267.4 ng*d / g, respectively. The maximum drug concentrations (Cmax) achieved were 81.0 ± 23.7 ng / g for the retina and 203.7 ± 88.8 ng / g for the vitreous humor. The time to peak drug concentration (Tmax) was 26 ± 9.6 days for the retina and 15.6 ± 4.2 days for the vitreous humor.

[0328] At 6 months post-injection, 3.1 ± 1.04 ng / g and 15.1 ± 0.89 ng / g of A190 were still detected in the retina and vitreous humor, respectively. Analysis of plasma samples following A190-MP injection at each timepoint had A 190 concentration below the detection limit (1 ng / g). As a comparison, we injected free A 190 solution (in dimethyl sulfoxide) at the same dose into the vitreous of C57BL / 6 mice. However, drug levels in the vitreo-retina tissue were significantly diminished to 1.9 ± 0.45 ng / g in two weeks after injection (Fig. 10). Hence the sustained release of A190-MP over 6 months underscores the potential of A190-MP for longterm drug delivery, reducing the frequency of injections.

[0329] Intravitreal injection of A190-MP prevented ERG decline and retinal degeneration and improved mitochondrial function in Vldlr- / -mice.

[0330] Previous studies revealed an age-dependent decline of photoreceptor function in Vldlr / _mice compared to age-matched wild-type (WT) mice. ERG responses were recorded at 1, 3, and 5 months post-administration of A190-MP. A190-MP significantly increased photopic ERG a-wave amplitudes at all time points compared to the Blank-MP group, with 35.03%, 73.25%, and 35.66% higher amplitudes at 1, 3, and 5 months, respectively (Fig. 2A-C), indicating preserved cone function. Additionally, A190-MP enhanced scotopic a-wave amplitudes at 3 months (Fig. 2B). Photopic b-wave amplitudes were increased in the A190-MP group at 3 months, while scotopic b-wave amplitudes were significantly higher at 1, 3, and 5 months (Fig. 9A-C). Collectively, these findings suggest that A190-MP treatment ameliorates ERG decline in VI dlr'7' mice.

[0331] Fluorescent PNA staining, which labels cone photoreceptors, was used to evaluate the effect of A190-MP on cone density at 6 months post-injection. PNA staining showed higher intensity and abundance in the outer segments of cones in the A190-MP group (Fig. 9D). Quantification of PNA-labeled cones in whole-mounted retinas revealed significantly higher cone densities in both the central and peripheral retina of the A190-MP group compared to the Blank-MP group at 6 months (Fig. 2D-G), indicating that A190-MP effectively ameliorates cone degeneration in Vldlr ' mice.

[0332] Previous studies have consistently reported a noticeable thinning in the outer retina (outer plexiform layer [OPL] to RPE) and outer nuclear layer (ONL) in Vldlr'7' mice. To quantify this, SD-OCT measured the thickness of the total retina, outer retina, and ONE from inferior to superior and nasal to temporal at 6 months post-injection (Fig. 2H-M). A190-MP significantly increased the thickness of the outer retina (Fig. 2J and K) and ONE (Fig. 2E and M) compared to Blank-MP controls, demonstrating effective alleviation of photoreceptor loss in Vldlr'7' mice. No significant difference was observed in the inner retinal thickness (inner limiting membrane [ILM] to OPL) (Fig. 9E).

[0333] We measured the expression levels of mitochondria-related molecules (Fig. 2N). Western blot analysis revealed that the expression of PPARa, PGCla and TOMM20, were significantly increased in the A190-MP group compared to the Blank-MP group in Vldlr'7' retinas (Fig. 2O). Collectively, our results revealed that A190-MP protected photoreceptors and retinal mitochondria in Vldlr'7' retinas.

[0334] Intravitreal injection of A190-MP reduced retinal vascular leakage and neovascularization in Vldlr7' mice.

[0335] We further investigated the anti-angiogenic effects of A190-MP in Vldlr ' mice. Fundus examination showed depigmented patches in the retina (Fig. 9F). FFA revealed numerous intense hyperfluorescent spots, indicating vascular leakage, in the Blank-MP-treated mice (Fig. 3 A). In contrast, the A190-MP-treated group exhibited fewer leakage spots and reduced leakage areas (Fig. 3B and C). In addition, isolectin- stained flat-mounted RPE / choroid and retina were used to evaluate the effects of A190-MP on subretinal neovascularization (SRNV) and intraretinal neovascularization (IRNV), respectively (Fig. 3D and G). A190-MP treatment reduced both the number and area of SRNV per RPE / choroid compared to the Blank-MP group (Fig. 3E and F). Similarly, the number and area of IRNV per retina were also decreased in the A190-MP group (Fig. 3H and I).

[0336] In OCT images, subretinal lesions appear as hyperreflective clumps in the outer retina of Vldlr ' mice, consistent with findings from H& E-stained retinal sections (Fig. 9G). Reflectivity B -scans showed these lesions as irregularities within the outer retina (Fig. 3 J). The Blank-MP group had an average of 14.7 lesions per image, while the A190-MP group had 10.9 lesions per image (Fig. 3K), aligning with isolectin staining results. The average lesion volumes decreased in the A190-MP group compared to the Blank-MP group (Fig. 3L).

[0337] Prior studies have demonstrated elevated proinflammatory factors VEGF, ICAM-1, and IL-1β in Vldlr7' retinas, indicating retinal inflammation. Six months after injection, levels of VEGF, ICAM-1, and IL-1β were significantly reduced in A190-MP-treated Vldlr7' retinas compared to those treated with Blank-MP (Fig. 3M and N).

[0338] The above results suggested that a single intravitreal injection of A190-MP ameliorated retinal vascular leakage, neovascularization and inflammation by suppressing the release of pro-inflammatory and pro-angiogenic cytokines in Vldlr ' mice for at least 6 months.

[0339] Intravitreal injection of 190-MP alleviated retinal degeneration in. \bca4 '~ / Rdh8 '~ mice.

[0340] Previous studies have shown progressive photoreceptor degeneration Abca4~ ~ / Rdh8~ / _mice. To assess the protective effect of A190-MP, we examined ERG responses in these mice. As shown in Fig. 4A and B, photopic and scotopic a- wave responses were significantly higher in A190-MP-treated mice compared to Blank-MP controls at 4 and 6 months postinjection. No significant differences were noted in b-wave amplitudes between the groups, except for a higher scotopic b-wave in the A190-MP group at 6 months (Fig. 11A and B).

[0341] To assess A190-MP's protective effect against retinal degeneration in Abca4'7 / Rdh8' ' mice, PNA immunostaining was also used to view cone photoreceptors (Fig. 11C). The average number of PNA-positive cells in the A190-MP group was significantly higher than that in the Blank-MP group, in both central and peripheral retina (Fig. 4C and D). Consistently, PNA-stained cone outer segments were longer in A190-MP-treated mice compared to those treated with Blank-MP (Fig. 4E and F).

[0342] Previous research has demonstrated progressive thinning of ONL in Abca4'7 / Rdh8'' mice from 3 to 6 months of age. OCT analysis revealed increased ONL thickness of A 190-MP-treated mice in both vertical and horizontal retinal directions (Fig. 4G). Histological analysis of retinal paraffin sections corroborated these findings (Fig. 4H and I). However, no significant difference in total retinal thickness was observed by OCT or retinal histology (Fig. 11D and E).

[0343] To evaluate whether increased ONL thickness in A190-MP-treated mice were related to photoreceptor protection, TUNEL was performed in retinal sections at 6 months postinjection (Fig. 11F). The A190-MP group exhibited fewer TUNEL-positive cells in the ONL compared to the Blank-MP group (Fig. 4J). These findings further confirmed that intravitreal administration of A190-MP attenuated the photoreceptor death in Abca4'77Rdh8'' mice.

[0344] To assess the anti-oxidant effect, we performed immunolabeling of 3-nitrotyrosine (3-NT), a marker of oxidative stress. Intense 3-NT labeling was observed in the photoreceptor layer of Abca4' / Rdh8'' mice with Blank-MP, which was markedly reduced by A190-MP treatment (Fig. 4K). Given that the NOX4 / ROS / NF-κB pathway is known to drive oxidative stress and inflammation, we further examined the effects on oxidative stress-related targets. Western blotting revealed significant reductions in pNF-κB and NOX4 levels following A190-MP treatment in Abca4'7Rdh8'' mice (Fig. 4L and M).

[0345] Taken together, these observations suggested that intravitreal administration of A 190-MP conferred neuroprotection of photoreceptors in Abca4'7Rdh8'' mice.

[0346] During the observation period, fundus examinations revealed no signs of hemorrhages or exudates, and the retinal blood vessels appeared normal in both the Blank-MP and A190-MP groups (Fig. 11G and H).

[0347] Intravitreal injection of A190-MP improved mitochondrial function in the retina of Abca4⁻ / Rdh8⁻ / ⁻ mice.

[0348] To assess mitochondrial function alterations in Abca4' 7Rdh8' ' mice, retinal biopsy punches were used to measure OCR in Abca4' 7Rdh8' ' mice and the age- and genetic background-matched WT mice at 4 months of age. Compared to WT retinas, Abca4' / Rdh8'' retinas displayed reductions of basal respiration, maximal respiration and spare respiration capability (Fig. 5A-D), suggesting impaired mitochondrial function in Abca4' / Rdh8'' retinas. Six months post-intravitreal injection, A190-MP treatment significantly increased maximal respiration and spare respiratory capacity in Abca4' / Rdh8'' retinas (Fig. 5E and F), suggesting an enhancement of mitochondrial function and improved retinal energy flux with A190-MP treatment.

[0349] To further investigate whether A190-MP regulate mitochondria integrity, we assessed mitochondrial content by immunostaining of TOMM20 on retinal sections. Higher intensity of TOMM20 immunoreactivity was observed in the outer and inner segments of A190-MP- treated Abca4⁻ / Rdh8⁻ / ⁻ mice with moderate labeling in other retinal layers, compared to the Blank-MP-treated group at 6 months post-injection (Fig. 5G). Additionally, Western blot analysis also revealed increased levels of mitochondrial-related proteins PPARa, PGCla, TOMM20, and CPT1A in Abca4⁻ / Rdh8⁻ / ⁻ retinas of A190-MP-treated mice (Fig. 5H and I).

[0350] Taken together, these findings indicated that intravitreal injection of A190-MP protected mitochondrial function and integrity in Abca4⁻ / Rdh8⁻ / ⁻ mice through activating PPARα.

[0351] Intravitreal injection of A190-MP had no detectable toxic effects in normal C57BL / 6J mice.

[0352] ERG results showed no significant differences in photopic or scotopic a- or b-wave amplitudes between untreated C57BL / 6J mice and those treated with either Blank-MP or A190-MP at 4 weeks post-injection, indicating that neither microparticle affects retinal function (Fig. 12 A and B). H& E staining revealed no changes in retinal morphology or total retinal thickness in the microparticle-treated groups compared to the untreated controls at 6 months (Fig. 12C and D). Similarly, TUNEL staining also showed no significant difference in ONL between treated and untreated eyes (Fig. 12E and F). Furthermore, no significant changes in intraocular pressure (IOP) were observed in both A190-MP-treated C57BL / 6J mice (Fig. 12G) and Abca4⁻ / Rdh8⁻ / ⁻ mice (Fig. 12I). Taken together, these results suggested that A190-MP did not cause any severe ocular toxicity during the experimental period in mice.

[0353] To assess systemic toxicity of the intravitreal microparticle injection, we measured serum levels of liver enzymes (ALP, AST, ALT) and kidney function indicators (BUN, creatinine) (Fig. 12 H and I). No significant differences were found between microparticle-treated and untreated C57BL / 6J mice. Histopathological examination also showed no abnormalities in the heart, kidneys, liver, or spleen of the microparticle-treated group (Fig. 12 J).

[0354] A190 treatment ameliorated oxidative stress-induced damage in photoreceptor-derived cells.

[0355] To explore the mechanism of A190-MP, we utilized free A190 for in vitro studies. Given the role of oxidative stress in AMD pathogenesis, we used 4-HNE, a lipid peroxidation product, as an oxidative stress inducer. To determine the optimal 4-HNE dosage, exposure of 661W cells to 0-20 pM 4-HNE for 24 hr showed a -50% reduction in cell viability at 10 pM (Fig. 13A). Concurrently, A190 treatment at different concentrations for 24 hr demonstrated safety in 661W cells from 2.5 to 20 pM (Fig. 13B and C).

[0356] As shown in Fig. 6A, 10 pM 4-HNE significantly reduced cell viability compared to control groups. A 190 mitigated this decrease in a concentration-dependent manner. Similarly, 4-HNE treatment markedly increased LDH release, which was significantly reduced by A 190 in a dose-dependent manner (Fig. 6B). These results indicated that A190 at 10 pM protected against 4-HNE-induced cellular damage, and this concentration was therefore selected for subsequent experiments.

[0357] TUNEL staining revealed that 4-HNE exposure significantly increased apoptosis in 661W cells, whereas A 190 effectively attenuated the apoptosis (Fig. 13D and Fig. 6C). Western blot analyses showed that 4-HNE treatment increased the pro-apoptotic factor BAX and decreased the anti-apoptotic factor BCL-2 (Fig. 6D). A190 treatment significantly suppressed 4-HNE-induced BAX expression and reversed the BCL-2 / BAX ratio (Fig. 6E), aligning with the TUNEL results.

[0358] Furthermore, 4-HNE-stressed 661W cells exhibited a marked increase in ROS production, which was alleviated by A 190 (Fig. 6F). Immuno staining revealed elevated levels of 3-NT in 4-HNE- treated cells, which were restored by A 190 treatment (Fig. 6G and H).

[0359] These results showed that A 190 exerted a protective effect against 4-HNE-induced oxidative stress in a mouse photoreceptor-derived cell line, consistent with our in vivo data.

[0360] A190 treatment mitigated oxidative stress-induced mitochondrial dysfunction.

[0361] Cells under oxidative stress exhibit impaired mitochondrial homeostasis. In 4-HNE-treated 661W cells, real-time ATP assays revealed decreased mitochondrial ATP production and increased glycolytic ATP, indicating a metabolic shift from mitochondrial to glycolytic ATP production under oxidative stress (Fig. 7A-C).

[0362] As measured by OCR (Fig. 7D), 4-HNE-stressed 661W cells exhibited significant decreases in ATP production, basal and maximal respiration, and spare respiratory capacity, compared to those in control groups. A 190 treatment improved OCR compared to those in 4-HNE groups (Fig. 7E-H). Additionally, A190 mitigated the increased glycolysis, glycolytic capacity, and glycolytic reserve induced by 4-HNE, as shown by the ECAR measurements (Fig. 13E-H). These data demonstrate that A 190 promotes oxidative phosphorylation and attenuates metabolic disturbances induced by oxidative stress.

[0363] We also evaluated the effects of A 190 on the structure of mitochondria. In 661W cells exposed to 4-HNE, TOMM20 immunostaining revealed fragmented mitochondria, which was mitigated by A190 treatment (Fig. 71 and J). Additionally, 4-HNE exposure significantly reduced the expression of PPARa, PGCla, and TOMM20, which was attenuated by A 190 (Fig. 7K and L), consistent with our observations in vivo, further suggesting that A 190 improves mitochondrial metabolism in 4-HNE-stimulated 661W cells.

[0364] Overall, the above results showed that A 190, as a PPARa agonist, exerted its neuroprotection primarily through improving mitochondrial metabolism under oxidative stress.

[0365] A190 exerts neuroprotective and antioxidant effects via a PPARa-dependent mechanism.

[0366] To further investigate whether the effects of A 190 were mediated through PPARa, we used PPARa-specific siRNA to knock down PPARa in 661W cells. Western blot analysis confirmed reduced PPARa levels and decreased PGCla and TOMM20 in these cells compared to controls (Fig. 8A and B). A190 significantly reduced 4-HNE-induced cellular damage and ROS production in cells with scrambled siRNA but had diminished effects in PPARa knockdown cells (Fig. 8C and D). In addition, we used primary Muller cells from WT and PPARa knockout (PPARa1) mice. In primary Muller cells from PPARα- / -mice, the reductions in PGCla and TOMM20 were more pronounced compared to their WT counterparts (Fig. 8E and F). A 190 effectively attenuated 4-HNE-induced cellular damage and ROS production in WT Muller cells, while this effect was abolished in PPARα- / -Muller cells (Fig. 8G and H). These findings collectively suggested that the antioxidative and neuroprotective effects of A 190 were PPARa-dependent.

[0367] Discussion

[0368] AMD, a common condition in the elderly, is primarily treated with frequent intravitreal anti-VEGF injections. However, this approach carries risks such as pain, infection, vitreous hemorrhage, retinal detachment, and endophthalmitis. Long-term VEGF suppression may lead to neurodegeneration, fibrosis, and scar formation, and there is growing concern over anti-VEGF resistance. Moreover, the lack of effective treatments for dry AMD underscores the urgent need for novel therapeutic strategies.

[0369] In the present study, we evaluated the efficacy of A 190, a novel non-fibrate PPARa agonist, for the first time in animal models manifesting some pathologies of wet and dry AMD. A 190 has been shown to have superior potency and selectivity for PPARa over other PPAR isoforms Employing a polymeric microparticle-based drug delivery system, a single intravitreal injection of A190-MP exhibited sustained and controlled release of A 190 over 6 months. In the Vldlr7' mouse model, A190-MP injection effectively protected mitochondrial function, thereby protecting photoreceptors and reducing vascular leakage and neovascularization. In the Abca4'7Rdh8'' mouse model, A190-MP alleviated mitochondrial dysfunction, thereby mitigating oxidative stress and retinal degeneration. Moreover, in vitro studies demonstrated that A 190 provided similar protective effects by enhancing mitochondrial function in a photoreceptor-derived cell line against oxidative stress. These results highlight the potential of A190-MP as a multifaceted therapeutic approach for the treatment of AMD.

[0370] To address the issue of sustained release, we optimized A190-MP formulations utilizing the different molecular weights of PLGA with similar end terminal groups and LA: GA ratios. The variation in drug loading and drug release profile of different A190-MP formulations can be attributed to the different MWs of polymers, crystallinity and physicochemical properties of microparticles (e.g. porosity, particle size, morphology etc.). High burst release can lead to elevated initial drug concentrations, which may increase the risk of toxicity. While beneficial in acute cases, a controlled release is preferred for chronic retinal diseases. We selected PLGA54kDa / PLGA45kDa-PEG5kDa blend microparticles for our subsequent in vivo studies. The selected A190-MP released -15% of the total dose within 24 hr in vitro, ensuring therapeutic levels during the critical early stages without risking excessive concentrations. Along with sustained release behavior of PLGA, the addition of PEG into the formulation has added benefits. PEGylation masks particle surfaces, reducing immune recognition and minimizing inflammation in the sensitive ocular environment.

[0371] The microparticles were formulated using a scalable emulsification method, suitable for large-scale manufacturing. A190-MP likely formed a depot in the vitreous chamber away from the visual axis after intravitreal injection due to lowering of the glass transition temperature (Tg) of PLGA, facilitated by water absorption. This causes microparticles to settle at the inferior vitreous, reducing the risk of vision interference.

[0372] Clinical studies emphasize the pivotal role of cone dysfunction in AMD progression, with documented cone degeneration and impaired function in AMD patients. Our findings showed that A190-MP injection significantly mitigated cone ERG decline, cone density loss, and cone outer segment shortening. Additionally, OCT results further indicated that A 190-MP preserved ONL thickness. Taken together, these results show that A190-MPs alleviate photoreceptor degeneration in AMD models, underscoring the therapeutical potential for dry AMD.

[0373] Vascular abnormalities play a critical role in wet AMD progression. Our previous research showed that Vldlr ' mice exhibited vascular leakage and both subretinal and intraretinal neovascularization. This present study revealed that A190-MP reduced retinal vascular leakage. OCT analysis and vascular staining in flat mounted retina and RPE / choroid demonstrated a reduction in neovascularization in Vldlr ' mice. Furthermore, A190-MP significantly reduced expression of inflammatory factors in retinal tissues, suggesting that its anti-angiogenic effects may be mediated through inflammation suppression.

[0374] In this study, we employed real-time measurements of mitochondrial function using live retinal biopsies and demonstrated significant metabolic impairment in Abca4⁻ / Rdh8⁻ / ⁻ retinas. A190-MP ameliorated mitochondrial dysfunction and enhanced energy flux, with similar effects observed in an in vitro photoreceptor-derived cell line under oxidative stress. Furthermore, the expression of PPARa, PGCla and TOMM20 was upregulated after the treatment with A 190 both in vivo and in vitro. PGCla is a key regulator of mitochondrial activity and biogenesis, and TOMM20 is a commonly used marker to assess mitochondrial content. A 190 also ameliorated oxidative stress-induced mitochondrial fragmentation. Moreover, A190-MP increased CPT1A in Abca4⁻ / Rdh8⁻ / ⁻ retinas, indicating enhanced mitochondrial fatty acid oxidation. Taken together, these results indicate that A190 protects mitochondrial function and integrity.

[0375] To elucidate the underlying mechanism, we investigated whether A 190 exerts its protective effects through PPARa activation. To clarify mechanism underlying the protective effects of A 190, we demonstrated that the beneficial effects of A 190 on cell viability and ROS production were diminished by PPARa knockout and siRNA knockdown, indicating that A 190 protects photoreceptors through a PPARa-dependent pathway.

[0376] In summary, A190-MP were successfully formulated with a high drug loading capacity and sustained drug release in the vitreo-retina for at least 6 months following a single intravitreal injection. A190-MP conferred promising therapeutic effects in both wet AMD and dry AMD models with no detectable side effects. Our findings indicate that A190-MP enhances mitochondrial function and prevents photoreceptor degeneration, thereby improving retinal function and ONL structure in AMD models. Moreover, in vitro studies indicate that the beneficial effect of A 190 is mediated through the activation of PPARa. Collectively, these findings provide valuable insights into the multifaceted potential of A190-MP as a therapeutic intervention targeting photoreceptor preservation for AMD.

[0377] Conclusions

[0378] These studies demonstrate that activation of PPARa is a successful therapeutic strategy for treating AMD. In an exemplary aspect, microparticle-mediated drug delivery of PPARa agonist A 190 achieved sustained therapeutic effects for the long-term treatment of AMD, providing an example of the use of microparticle mediated drug delivery of small molecules for the treatment of retinal disorders.

[0379] Example 2. Additional formulations

[0380] Newer formulations exhibited higher drug loadings (DLs) compared to previous batches; A190-MP-F6 made of PLGA54k / PLGA45k-PEG5k (1:1 ratio) with 6.8 pm size and 21.5 wt% DL; A190-MP-F7 made of PLGAs4k with 7.9 pm size and 20.3 wt% DL. These two new formulations exhibited >20% drug loading and sustained drug release for at least 160 days.

[0381] We have further developed other promising A190-MP formulations through polymer blends and increased the target drug loading to at least 30 wt %.

[0382] While the invention has been described in terms of its several exemplary embodiments, those skilled in the art will recognize that the invention can be practiced with modification within the spirit and scope of the appended claims. Accordingly, the present invention should not be limited to the embodiments and aspects described above, but should further include all modifications and equivalents thereof within the spirit and scope of the description provided herein.

Claims

CLAIMSWe claim:

1. A method of treating at least one symptom of an eye disorder in a subject in need thereof, comprising administering to an eye of the subject a formulation comprising microparticles distributed within a vehicle, wherein the microparticles in the formulation comprisea polymer or copolymer comprising one or more poly(hydroxyacid) polymers, wherein the one or more poly(hydroxyacid) polymers have a molecular weight ranging from 5kD to 60kD, andan agonist of PPARa signaling at a loading of 5-50% by weight, wherein the agonist of PPARa signaling has a general chemical structureIIwherein:k is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms;m is 0, 1, 2, 3, 4, or 5 carbon atoms;n is 0, 1, 2, 3, 4, or 5 carbon atoms;R2is selected from the group: hydrogen (H), chlorine (Cl), fluorine (F), bromine (Br), iodine (I), nitro (NO2), CH3, CH2CH3, branched or unbranched alkyl chains with 3-10 carbon atoms, OCH3, OCH2CH3, branched or unbranched alkoxy chains with 3-10 carbon atoms, haloalkyls, haloalkoxyls, cycloalkyls, halocycloalkyls, O-para-alkylbenzyls, O-para-alkyloxybenzyls, and O-para-halobenzyls, wherein the benzene ring comprising R2comprises one, two, three, or four of said R2substituents substituted in any combination ofsaid R2substituents and arranged in any pattern in the ring including ortho, meta, mono, di, tri, and tetrasubstituted;R3is selected from the group: H, Cl, F, Br, I, NO2, CH3, CH2CH3, branched or unbranched alkyl chains with 3-10 carbon atoms, OCH3, OCH2CH3, branched or unbranched alkoxy chains with 3-10 carbon atoms, haloalkyls, haloalkoxyls, cycloalkyls, halocycloalkyls, O-para-alkylbenzyls, O-para-alkyloxybenzyls, and O-para-halobenzyls, wherein the benzene ring comprising R3comprises one, two, three, four, or five of said R3substituents substituted in any combination of said R3substituents and arranged in any pattern in the ring including ortho, meta, para, mono, di, tri, tetra, and pentasubstituted;R4is selected from the group: H, alkyl, and acyl;R5is selected from the group: H, Cl, F, Br, I, NO2, CH3, CH2CH3, branched or unbranched alkyl chains with 3-10 carbon atoms, OCH3, OCH2CH3, branched or unbranched alkoxy chains with 3-10 carbon atoms, haloalkyls, haloalkoxyls, cycloalkyls, halocycloalkyls, O-para-alkylbenzyls, O-para-alkyloxybenzyls, and O-para-halobenzyls, wherein the benzene ring comprising R5comprises one, two, three, or four of said R5substituents substituted in any combination of said R5substituents and arranged in any pattern in the ring including ortho, meta, para, mono, di, tri, and tetrasubstituted;R1is selected from the group consisting of carboxylic acids, carboxylic acid isosteres, hydroxamic acids, hydroxamic esters, phosphonic acids, phosphinic acids, sulfonic acids, sulfinic acids, sulfonamides, acyl sulfonamides, sulfonylureas, acylureas, tetrazoles, thiazolidine diones, oxazolidine diones, oxadiazol-5(4H)-ones, thiadiazol-5(4H)-ones, oxathiadiazole-2-oxides, oxadiazol-5(4H)-thiones, isoxazoles, tetramic acids, cyclopentane 1,3-diones, cyclopentane 1,2-diones, squaric acids, substituted phenols, heteroarenes, amidines, hydroxy amides, alkyl hydroxy amidines, andoo OR1'1RsR’and salts thereof,R8and R9are independently selected from the group consisting of H, F, Cl, Br, I, alkyl, alkoxy, and cycloalkyl comprising R8is linked to R9;R10and R11are independently selected from the group consisting of H, alkyl, and cycloalkyl wherein R10is linked to R11); andX is O, NH, S, or CH2.

2. The method of claim 1 wherein the one or more poly(hydroxyacid) are selected from the group consisting of poly (lactic acid) (PLA), poly (glycolic acid) (PGA), poly(lactic-co-glycolic acid) (PLGA), and poly (caprolactone).

3. The method of claim 2 wherein the one or more poly(hydroxyacid) polymers is or includes PLGA.

4. The method of claim 2 wherein the one or more poly(hydroxyacid) polymers are present at two different molecular weight ranges.

5. The method of claim 4 wherein the at least two different poly (hydroxy acid) polymers are PLGA at 54kD and PLGA at 45kD.

6. The method of claim 4 wherein the one or more poly(hydroxyacid) polymers are two different poly (hydroxy acid) polymers.

7. The method of claim 1 wherein the microparticles have an average diameter ranging from 1 to 100 pm.

8. The method of claim 8 wherein the microparticles have an average diameter ranging from 5 to 25pm.

9. The method of claim 1 wherein the one or more poly(hydroxyacid) polymers are formulated as a co-polymer with polyethylene glycol (PEG) wherein the PEG is present at IkD to lOkD.

10. The method of claim 9 wherein the co-polymer with PEG is selected from the group consisting of PLGA-PEG, PLA-PEG, PGA-PEG, and polycaprolactone-PEG.

11. The method of claim 10 wherein the co-polymer with PEG comprises PLGA at 40- 60kD.

12. The method of claim 1 wherein the administration is performed by injection into the eye.

13. The method of claim 12 wherein the vehicle in the formulation is a liquid.

14. The method of claim 12 wherein the injection is performed no more than once every six months.

15. The method of claim 12 wherein the injection is performed no more than once every twelve months.

16. A method of treating at least one symptom of an eye disorder in a subject in need thereof, comprising injecting into an eye of the subject a formulation comprising microparticles containing at least on active agent distributed within a liquid vehicle, wherein the microparticles in the formulation comprise a polymer or copolymer comprising one or more poly(hydroxyacid) polymers, wherein the one or more poly(hydroxyacid) polymers have a molecular weight ranging from 5kD to 60kD.

17. The method of claim 16 wherein the eye disorder is selected from the group consisting of retinal inflammation, retinal neovascularization, retinal vascular leakage, retinopathy of prematurity (ROP), diabetic retinopathy (DR), age-related macular degeneration (e.g., wet AMD), and diabetic macular edema (DME).

18. The method of claim 16 wherein the active agent is agonist of PPARa signaling.

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