WO2023211999A1

WO2023211999A1 - Compositions and methods for treating eye disorders

Info

Publication number: WO2023211999A1
Application number: PCT/US2023/019902
Authority: WO
Inventors: Thomas J. WUBBEN; Cagri Besirli; Jason Miller; Jason Rech; Moloy GOSWAMI
Original assignee: The Regents Of The University Of Michigan
Priority date: 2022-04-29
Filing date: 2023-04-26
Publication date: 2023-11-02

Abstract

Provided herein are compositions and methods for treating eye disorders. In particular, provided herein are compositions and methods for treating and preventing retinal pigment epithelium (RPE) epithelial-to-mesenchymal transition (EMT), epiretinal membrane (ERM), and/or proliferative vitreoretinopathy.

Description

COMPOSITIONS AND METHODS FOR TREATING EYE DISORDERS

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with government support under AT011652 and HL156989 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

BACKGROUND

Proliferative vitreoretinopathy (PVR) represents the greatest risk of failure of retinal detachment repair surgery. PVR is the most common cause for failure of rhegmatogenous retinal detachment repair and is characterized by the growth and contraction of cellular membranes within the vitreous cavity and on both sides of the retinal surface as well as intraretinal fibrosis. Epiretinal membrane (ERM) is in the spectrum of PVR, causing retinal traction, anatomic distortion, and vision loss. Both PVR and ERM are common sequalae of ocular trauma. Currently, PVR and ERM are thought to be an abnormal wound healing response that is primarily driven by inflammatory, retinal, and RPE cells. At this time, surgery is the only management option for PVR and ERM as there is no proven pharmacologic agent for the treatment or prevention of PVR. Laboratory research to better understand PVR pathophysiology and clinical trials of various agents to prevent PVR formation are ongoing.

No pharmacotherapies have prevented the formation of PVR or ERM, so a significant unmet need exists.

SUMMARY

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SUBSTITUTE SHEET ( RULE 26 ) The epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is critical in PVR pathogenesis. Increased glycolysis is a hallmark of RPE EMT. Pyruvate kinase M2 (PKM2) is a key regulator of glycolysis that exists in different oligomeric states, dimer and tetramer, and has been implicated in glycolytic reprogramming of cells. Experiments described herein demonstrated that pharmacologically inducing PKM2 tetramerization is a therapeutic strategy for PVR. Accordingly, provided herein are compositions and methods for treating and preventing retinal pigment epithelium (RPE) epithelial-to-mesenchymal transition (EMT), ERM, and/or PVR using PKM2 activators.

For example, in some embodiments, provided herein is a method of treating or preventing proliferative vitreoretinopathy (PVR) or ERM in a subject, comprising administering a PKM2 activator to the eye of a subject, wherein the administering prevents, treats or reduces one or more signs or symptoms of PVR or ERM in the subject.

Also provided herein is a method of treating or preventing retinal pigment epithelium (RPE), ERMM, and/or epithelial-to-mesenchymal transition (EMT) in a subject, comprising administering a PKM2 activator to the eye of a subject, wherein the administering prevents, treats or reduces one or more signs or symptoms of EMT in the subject.

Further embodiments provide a method of treating proliferative vitreoretinopathy (PVR) and/or ERM in a subject, comprising a) diagnosing the subject with PVR and/or ERM; and b) administering a PKM2 activator to the eye of a subject, wherein the administering treats or reduces symptoms of PVR and/or ERM in the subject.

Other embodiments provide the use of a composition comprising an activator of PKM2 to prevent, treat or reduce symptoms of PVR in a subject.

In some embodiments, the subject has been treated for retinal detachment (e.g., with cryotherapy, laser surgery, and/ or surgery). In some embodiments, the subject has not been treated with a PKM2 activator prior to said treatment for retinal detachment. In some embodiments, the subject is under the age of 50 (e.g., 50 or less, 49 or less, 48 or less, 47 or less, 46 or less, 45 or less, or 40 or less). In some embodiments, the retinal detachment is caused by trauma. In some embodiments, the retinal detachment is not caused by aging. In some embodiments, the PKM2 activator is delivered to retinal pigment epithelium (RPE) cells.

The present disclosure is not limited to particular PKM2 activators. In some embodiments, the activator is a small molecule. Examples include but are not limited to

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SUBSTITUTE SHEET ( RULE 26 )

those shown in Table 1. In some embodiments, the activator is formulated for injection (e.g., intravitreal injection, suprachoroidal injection, subretinal injection, peribulbar injection, subconjunctival injection intracameral injection), for oral delivery, or as an eye drop or suspension. In some embodiments, the method or use further comprises administering an additional agent. For example, in some embodiments, the additional agent is methotrexate or other antiproliferative agents.

Additional embodiments are described herein.

DESCRIPTION OF THE FIGURES

FIG. 1 shows human fetal RPE (hfRPE) EMT. (a) Cultures demonstrating well- differentiated hfRPE cells (top row) that have undergone EMT with a more fibroblastic phenotype (hfRPE-F) 3, 7, and 12 days after plating at 10% density. DAPI-blue, Vimentin- yellow, a-smooth muscle actin (SMA)-green and Phalloidin-red. Merge=a-SMA and phalloidin. (b) Ratio of fluorescent intensities obtained from EMT (hfRPE-F) cultures at 3, 7, and 12 days. ****, pcO.OOOl; ns=non- significant, (c) qRT-PCR of EMT markers vimentin (VIM) and smooth muscle actin (ACTA2) as well as RPE-specific markers CRALBP, RPE65, and BEST1. *, p<0.05. (d) Western blot showing increased levels of EMT markers N-Cadherin (NCAD) and aSMA and decreased levels of epithelial marker E-cadherin (ECAD), (e) Quantitative analysis of Western blot. *, p<0.05

FIG. 2 shows that hfRPE EMT increases PKM2 S37 phosphorylation. qRT-PCR (a) showing increased expression of PKM2 in hfRPE-F. Western blot (b) showing increased levels of PKM2 S37 phosphorylation and decreased levels of PKM2 Y 105 phosphorylation in hfRPE-F 7 days after plating at low density. Quantitative analysis of Western blot (c) depicted for phosphorylated PKM2 levels normalized to total PKM2.

FIG. 3 shows that PK activity is reduced in hfRPE EMT and ML- 265 increases PK activity safely, (a) Pyruvate kinase activity was measured in hfRPE and hfRPE undergoing EMT (hfRPE-F) using a LDH-coupled enzyme assay, (b) hfRPE or hfRPE-F cells treated with ML-265 show increased PK activity compared to vehicle treatment, (c) ML-265 or

SUBSTITUTE SHEET ( RULE 26 ) DMSO replaced every 2 days with TEER measurement immediately prior to drug replacement.

FIG. 4 shows that ML- 265 treatment reduces hfRPE proliferation, contraction, and migration after induction of EMT. (a) hfRPE cells were plated at low density in the presence or absence of ML-265 and cell numbers quantified by CYQUANTTM Cell Proliferation Assay over time, (b) Inhibitory effect of ML-265 on hfRPE-F cell-mediated collagen gel contraction, (c) Representative images of hfRPE migration in pilot wound closure assay after 48hours in the presence of DMSO or 25 pM ML-265. hfRPE cells were plated at low density in the presence or absence of ML-265 for 7 days and harvested for qRT-PCR (d) and Western blot (e).

FIG. 5 shows that induction of PKM2 tetramerization blocks essential signaling pathways necessary for aerobic glycolysis and nucleotide biosynthesis in EMT-induced hfRPE. (a) HfRPE-F cells were plated at low density and treated with ML-265 or vehicle for 7 days at which time they were collected, crosslinked with DSS, and analyzed for PKM2 expression, (b) Cells were collected after 7 days in the presence of ML-265 or vehicle, (c) hfRPE cells were plated at low density in the presence or absence of ML-265 for 7 days and harvested for qRT-PCR.

FIG. 6 shows that induction of PKM2 tetramerization alters the metabolic profile of hfRPE undergoing EMT. (a) HfRPE-F cells were treated with 25 pM ML-265 or DMSO for 7 days and normalized signal intensities were determined by targeted LC-MS/MS. ML-265 treatment altered central glucose metabolism (b) and nucleotide biosynthesis (c) in hfRPE induced to undergo EMT. *, p<0.05, **; p<0.01; ***, p<0.005. F6P: fructose 6-phosphate, 2- PG: 2-phosphoglycerate, Pyr: pyruvate, Lac: lactate, a-KG: alpha-ketoglutarate, Succ: succinate, Mai: malate, Gin: glutamine, Glu: glutamate, Asp: Aspartate, OA: orotic acid, UTP: uridine triphosphate, TDP: thymidine diphosphate, UA: uric acid, ATP: adenosine triphosphate.

FIG. 7 shows that PKM2 expression is necessary for survival during initiation of hfRPE EMT. (a) TEER measurements show that lentivirus transfection and knockdown of PKM2 is not toxic to highly differentiated, hfRPE cells. n=3 (b) Lentivirus transfection was monitored by expression of Green fluorescent protein (GFP) in highly differentiated, hfRPE over a period of 2 weeks, (c) Western blot showing specific reduction of PKM2 expression (and not PKM1) in hfRPE when transfected with lentiviruses with shRNA against PKM2. (d) hfRPE-F show reduced proliferation with PKM2 knockdown. n=3, *, p<0.05. (e) Fold change

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SUBSTITUTE SHEET ( RULE 26 ) in early apoptosis, caspase 3/7 activity, and necrosis following PKM2 knockdown in hfRPE- F. n=3; **, p<0.01. hfRPE-F: fibroblastic like (EMT) human fetal RPE; NT: non-targeted.

FIG. 8 shows that PKM2 expression is required for maintenance of hfRPE EMT. (a) Western blot shows an increase in PKM2 protein level in hfRPE EMT and complete knockdown with siRNA, (b) hfRPE EMT cell proliferation following PKM2 knockdown. n=3; ****, p<0.0001. (c) Fold change in early apoptosis, caspase 3/7 activity, and necrosis following PKM2 knockdown in hfRPE EMT. n-3; **, p<0.01. hfRPE-F: fibroblastic-like (EMT) human fetal RPE; NT: non-targeted.

DEFINITIONS

To facilitate an understanding of the present disclosure, a number of terms and phrases are defined below:

The term "derivative" of a compound, as used herein, refers to a chemically modified compound wherein the chemical modification takes place either at a functional group of the compound or backbone.

As used herein, the term “subject” refers to organisms to be treated by the methods of the present disclosure. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and most preferably includes humans. In the context of the disclosure, the term “subject” generally refers to an individual who will receive or who has received treatment (e.g. , administration of a compound of the present disclosure and optionally one or more other agents) for a condition characterized by an eye disorder.

The term “diagnosed,” as used herein, refers to the recognition of a disease by its signs and symptoms (e.g., resistance to conventional therapies), or genetic analysis, pathological analysis, histological analysis, and the like.

As used herein, the term “effective amount” refers to the amount of a compound (e.g., a compound of the present disclosure) sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations, applications or dosages and is not intended to be limited to a particular formulation or administration route.

As used herein, the term “co-administration” refers to the administration of at least two agent(s) (e.g., a compound of the present disclosure) or therapies to a subject. In some embodiments, the co-administration of two or more agents/therapies is concurrent. In some embodiments, a first agent/therapy is administered prior to a second agent/therapy. Those of skill in the art understand that the formulations and/or routes of administration of the various

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SUBSTITUTE SHEET ( RULE 26 ) agents/therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents/therapies are coadministered, the respective agents/therapies are administered at lower dosages than appropriate for their administration alone. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents/therapies lowers the requisite dosage of a known potentially harmful (e.g., toxic) agent(s).

As used herein, the term “toxic” refers to any detrimental or harmful effects on a cell or tissue as compared to the same cell or tissue prior to the administration of the toxicant.

As used herein, the term “pharmaceutical composition” refers to the combination of an active agent with a carrier, inert or active, making the composition especially suitable for diagnostic or therapeutic use in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier” refers to any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions {e.g. , such as an oil/water or water/oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants. See e.g., Martin, Remington's Pharmaceutical Sciences, 15th Ed, Mack Publ. Co, Easton, PA [1975]).

As used herein, the term “pharmaceutically acceptable salt” refers to any pharmaceutically acceptable salt (e.g., acid or base) of a compound of the present disclosure which, upon administration to a subject, is capable of providing a compound of this disclosure or an active metabolite or residue thereof. As is known to those of skill in the art, “salts” of the compounds of the present disclosure may be derived from inorganic or organic acids and bases. Examples of acids include, but are not limited to, hydrochloric, hydrobromic, sulfuric, nitric, perchloric, fumaric, maleic, phosphoric, glycolic, lactic, salicylic, succinic, toluene-p-sulfonic, tartaric, acetic, citric, methanesulfonic, ethanesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic acid, and the like. Other acids, such as oxalic, while not in themselves pharmaceutically acceptable, may be employed in the preparation of salts useful as intermediates in obtaining the compounds of the disclosure and their pharmaceutically acceptable acid addition salts.

Examples of bases include, but are not limited to, alkali metals {e.g., sodium) hydroxides, alkaline earth metals (e.g. , magnesium), hydroxides, ammonia, and compounds of formula NW wherein W is Cw alkyl, and the like.

Examples of salts include, but are not limited to: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate,

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SUBSTITUTE SHEET ( RULE 26 ) cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, flucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2- naphthalenesulfonate, nicotinate, oxalate, palmoate, pectinate, persulfate, phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate, undecanoate, and the like. Other examples of salts include anions of the compounds of the present disclosure compounded with a suitable cation such as Na⁺, NH4L and NW4⁺ (wherein W is a C alkyl group), and the like.

For therapeutic use, salts of the compounds of the present disclosure are contemplated as being pharmaceutically acceptable. However, salts of acids and bases that are non- pharmaceutically acceptable may also find use, for example, in the preparation or purification of a pharmaceutically acceptable compound.

As used herein, the term "sample" is used in its broadest sense. In one sense, it is meant to include a specimen or culture obtained from any source, as well as biological and environmental samples. Biological samples may be obtained from animals (including humans) and encompass fluids, solids, tissues, and gases. Biological samples include blood products, such as plasma, serum and the like. Environmental samples include environmental material such as surface matter, soil, water, and industrial samples. Such examples are not however to be construed as limiting the sample types applicable to the present disclosure.

As used herein, the terms "purified" or "to purify" refer, to the removal of undesired components from a sample. As used herein, the term "substantially purified" refers to molecules that are at least 60% free, preferably 75% free, and most preferably 90%, or more, free from other components with which they usually associated.

The term “test compound” refers to any chemical entity, pharmaceutical, drug, and the like, that can be used to treat or prevent a disease, illness, sickness, or disorder of bodily function, or otherwise alter the physiological or cellular status of a sample (e.g. , PKM2 levels). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by using the screening methods of the present disclosure. A “known therapeutic compound” refers to a therapeutic compound that has been shown (e.g., through animal trials or prior experience with administration to humans) to be effective in such treatment or prevention.

DETAILED DESCRIPTION OF THE DISCLOSURE

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SUBSTITUTE SHEET ( RULE 26 ) Provided herein are compositions and methods for treating eye disorders. In particular, provided herein are compositions and methods for treating and preventing retinal pigment epithelium (RPE) epithelial-to-mesenchymal transition (EMT), epiretinal membrane (ERM), and/or proliferative vitreoretinopathy.

Proliferative vitreoretinopathy (PVR) represents a major cause of failure of retinal detachment repair surgery. Epiretinal membrane (ERM) is in PVR spectrum and occurs after retinal tear or retinal detachment repair surgery alone or as part of PVR. No pharmacotherapies have prevented the formation of PVR or ERM to date.¹ The epithelial-to- mesenchymal transition (EMT) of retinal pigment epithelial (RPE) cells is important in PVR pathogenesis.² Increased glycolysis is a hallmark of RPE EMT.³⁴ PKM2 is the tightly regulated enzyme that controls the rate-limiting final step of glycolysis, and it exists in different oligomeric states.^5,6 The tetramer has high catalytic activity and reduces lactate production.⁷ The non-tetrameric state, or monomeric/dimeric form, has low catalytic activity, favors accumulation of glycolytic intermediates, and is driven by post-translational phosphorylation.⁸ Unlike the tetramer, the monomeric/dimeric PKM2 has moonlighting activities beyond its enzymatic function.⁶ It can translocate to the nucleus, primarily via serine (S37) phosphorylation^{9 10}, to mediate the expression of numerous proglycolytic genes via transcriptional co-activation of b-catenin or hypoxia-inducible factor 1-alpha (Hif-la).^9-12 Of note, small molecule modulators of PKM2, like ML-265, induce tetramerization, reducing nuclear PKM2 and glycolytic reprogramming in certain cells.^7,11,12

Experiments described herein demonstrated that small molecule PKM2 activation induces PKM2 tetramerization, increases PK activity, and reduces PKM2 nuclear translocation upon EMT induction in hfRPE. The resultant downstream transcriptional and metabolic changes then lead to inhibition of EMT-induced hfRPE cell proliferation, migration, and contraction as well as markers of EMT.

Accordingly, provided herein are compositions and methods for activating PKM2 to treat and/or preventing PVR.

I. Activators

Provided herein are PKM2 activators. Exemplary, non-limiting examples are provided below. Examples include, but are not limited to, small molecules, biologies, or genetic therapies.

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SUBSTITUTE SHEET ( RULE 26 ) In some embodiments, the activators are small molecules. In some exemplary embodiments, the small molecule is ML-265

Fructose 1,6-bisphosphate trisodium salt octahydrate,

In some embodiments, the small molecule is a molecule from Table 1 or an additional molecule described in WO 2022/020424; herein incorporated by reference in its entirety.

Table 1

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SUBSTITUTE SHEET ( RULE 26 )

In some embodiments, compounds are targeted to retinal pigment epithelial (RPE) cells (e.g., via an antibody or other targeting moiety).

The present disclosure contemplates the use of any genetic manipulation for use in modulating the expression of PKM2. Examples of genetic manipulation include, but are not limited to, gene knockout e.g. , removing a pathway component (e.g., inhibitor of PKM2)) gene from the chromosome using, for example, recombination), CRISPR, expression of antisense constructs with or without inducible promoters, and the like. Delivery of nucleic acid construct to cells in vitro or in vivo may be conducted using any suitable method. A suitable method is one that introduces the nucleic acid construct into the cell such that the desired event occurs (e.g., expression of PKM2).

In some embodiments, stem cells (e.g., induced pluripotent stem cells, pluripotent stem cells, embryonic stem cells, adult stem cells, etc.) are reprogrammed (e.g., in vitro, ex vivo, or in vivo) to modulate the expression or function of PKM2. Such stem cells are then

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SUBSTITUTE SHEET ( RULE 26 ) introduced to a subject in need of treatment (e.g., to the eye of a subject). In some embodiments, the metabolic reprogramming of stem cells increases survival, differentiation, and integration into the retinal architecture.

Introduction of molecules carrying genetic information into cells is achieved by any of various methods including, but not limited to, directed injection of naked DNA constructs, bombardment with gold particles loaded with said constructs, and macromolecule mediated gene transfer using, for example, liposomes, biopolymers, and the like. Exemplary methods use gene delivery vehicles derived from viruses, including, but not limited to, adenoviruses, retroviruses, vaccinia viruses, and adeno-associated viruses. Because of the higher efficiency as compared to retroviruses, vectors derived from adenoviruses are the preferred gene delivery vehicles for transferring nucleic acid molecules into host cells in vivo.

In some embodiments, candidate PKM2 activators are screened for activity (e.g., using the methods described herein or another suitable assay).

The present disclosure further provides pharmaceutical compositions (e.g., comprising the compounds described above). The pharmaceutical compositions of the present disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic). In some embodiments, compositions are formulated for topical delivery (e.g., to the eye). In some embodiments, compositions are formulated for injection into the eye (e.g., intravitreal injection).

The pharmaceutical formulations of the present disclosure, which may conveniently be presented in unit dosage form, may be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present disclosure may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present disclosure, such as dyes, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly

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SUBSTITUTE SHEET ( RULE 26 ) interfere with the biological activities of the components of the compositions of the present disclosure. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Dosing is dependent on severity and responsiveness of the disease state to be treated or prevented, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of the disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. The administering physician can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual oligonucleotides, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models or based on the examples described herein. In general, dosage is from 0.01 pg to 100 g per kg of body weight, and may be given once or more daily, weekly, monthly or yearly. The treating physician can estimate repetition rates for dosing based on measured residence times and concentrations of the drug in bodily fluids or tissues. Following successful treatment, it may be desirable to have the subject undergo maintenance therapy to prevent the recurrence of the disease state, wherein the oligonucleotide is administered in maintenance doses, ranging from 0.01 pg to 100 g per kg of body weight, once or more daily, to once every 20 years.

II. Methods of treating and/or preventing PVR and ERM

In some embodiments, methods and uses of PKM2 activators in the treatment and/or prevention of PVR and/or ERM are provided.

For example, in some embodiments, provided herein is a method of treating or preventing proliferative vitreoretinopathy (PVR) or ERM in a subject, comprising administering a PKM2 activator to the eye of a subject, wherein the administering prevents, treats or reduces one or more signs or symptoms of PVR and/or ERM in the subject.

In some embodiments, the subject has been treated for retinal detachment (e.g., with cryotherapy, laser surgery, and/ or surgery). In some embodiments, the subject has not been treated with a PKM2 activator prior to said treatment for retinal detachment. In some embodiments, the subject is under the age of 50 (e.g., 50 or less, 49 or less, 48 or less, 47 or less, 46 or less, 45 or less, or 40 or less). In some embodiments, the retinal detachment is caused by trauma. In some embodiments, the retinal detachment is not caused by aging. In

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SUBSTITUTE SHEET ( RULE 26 ) some embodiments, the PKM2 activator is delivered to retinal pigment epithelium (RPE) cells.

In some embodiments, prior to treatment, the subject is screened for one or more signs or symptoms of PVR. In some embodiments, subjects are identified as having an increased risk of developing PVR prior to treatment.

The composition can be formulated for local (e.g., ocular; intraocular space; etc.), parenteral, oral, or topical administration. For example, a parenteral formulation could comprise or consist of a prompt or sustained release liquid preparation, dry powder, emulsion, suspension, or any other standard formulation. Compositions can be delivered via eye drops or other topical eye delivery method. Compositions may be delivered intraocularly, anywhere in the eye including, for example, the vitreous cavity, the anterior chamber, suprachoroidal space etc. Compositions may be delivered intravitreally as is commonly done with intravitreal injections of Lucentis (ranibizumab), Avastin (bevacizumab), triamcinolone acetonide, antibiotics, etc. Compositions may be delivered periocularly (e.g. to the tissue around the eyeball (globe) but within the bony orbit). Compositions may be delivered via intraocular implant (e.g. gancyclovir implant, fluocinolone implant, etc.). In intraocular implant delivery, devices containing compositions of the present invention are surgically implanted (e.g. within the vitreous cavity), and the drug is released into the eye (e.g. at a predetermined rate). Compositions may be administered using encapsulated cell technology (e.g. by Neurotech) in which genetically modified cells are engineered to produce and secrete compositions of the present disclosure. Compositions may be delivered via transcleral drug delivery using a device sutured or placed next to the globe that would slowly elute the drug, which would then diffuse into the eye.

In some embodiments, compositions of the present disclosure (e.g., small molecule PKM2 activator) are administered optically, for example, using the techniques described herein, and/or other techniques (e.g. injection, topical administration, etc.) (See, e.g., Janoria et al. Expert Opinion on Drug Delivery. July 2007, Vol. 4, No. 4, Pages 371-388; Ghate & Edelhauser. Expert Opin Drag Deliv. 2006 March; 3(2):275-87 ; Bourges et al. Adv Drug Deliv Rev. 2006 November 15; 58(11):1182-202. Epub 2006 Sep. 22; Gomes Dos Santos et al. Curr Pharm Biotechnol. 2005 February; 6(1):7- 15 ; herein incorporated by reference in their entireties).

In some embodiments, PKM2 activators are co-administered with another treatment for PVR (e.g., laser or other surgery, methotrexate, vitamins, or nutritional supplements).

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SUBSTITUTE SHEET ( RULE 26 ) In some embodiments, one or more PKM2 activators described herein is administered in combination with an anti-proliferative agent (e.g., intravitreal methotrexate, 5-FU, etc). In some embodiments, a reduced dose of methotrexate is used that still provides for a therapeutic benefit and reduces adverse effects of methotrexate.¹³

In some embodiments, the present disclosure provides a method for treating patients suffering from or at risk of PVR or ERM. In some embodiments, a pharmaceutical composition comprising at least one PKM2 activator is delivered to such a patient in an amount and at a location sufficient to treat the disorder or disease. In some embodiments, activators are delivered to the patient systemically or locally, and it will be within the ordinary skill of the medical professional treating such patient to ascertain the most appropriate delivery route, time course, and dosage for treatment. It will be appreciated that application of the method of treating a patient most preferably substantially alleviates or even eliminates such symptoms; however, as with many medical treatments, application of the method is deemed successful if, during, following, or otherwise as a result of the method, the symptoms of the disease or disorder in the patient subside to a degree ascertainable.

In some embodiments, compositions (e.g., small molecule PKM2 activators) are provided as part of a kit. In some embodiments, a kit of the present disclosure comprises one or more photoreceptor protective compositions and/or photoreceptor protective pharmaceutical compositions. In some embodiments, a kit is configured for co-administration with one or more additional compositions (e.g. pharmaceutical compositions).

EXPERIMENTAL

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present disclosure and are not to be construed as limiting the scope thereof.

Example 1

To mimic the loss of cell contact observed in the PVR process and stimulate EMT, primary human fetal RPE (hfRPE) were seeded at 10% density (Figure 1). Cultured hfRPE have similar gene expression and biologic functionality as native RPE. They are the most well-characterized model of RPE metabolism and disease.⁴ HfRPE seeded at 10% density (hfRPE-F) demonstrated a fibroblastic-like phenotype in this cell culture model of PVR with induction of EMT markers SMA and NCAD (Figure 1). PKM2 expression was increased and the phosphorylation state (Ser37) of PKM2 was also increased, indicating that PKM2 is in the

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SUBSTITUTE SHEET ( RULE 26 ) low activity, dimeric state (Figure 2). A continuous, enzyme-coupled assay that measures the depletion of NADH determined PK activity, and PK activity was found to be significantly lower in hfRPE cells undergoing EMT (Figure 3a). Treatment with the small molecule PKM2-specific activator, ML- 265, increased PK activity >3-fold in hfRPE EMT without demonstrating a change in the transepithelial electrical resistance (TEER) of well- differentiated hfRPE (Figure 3b and c). TEER is a non-invasive measure of tight-junction integrity in RPE cells and requires many key cellular processes to be well coordinated.

The ability of ML-265 to inhibit EMT-induced hfRPE cell proliferation, contraction, and migration as well as markers of EMT was assessed in a multitude of in vitro assays. ML- 265 treatment reduced EMT-induced hfRPE proliferation, contraction, and migration. Additionally, treatment with ML-265 reduced EMT markers SMA and NCAD (Figure 4). To assess if ML-265 alters PKM2’s oligomeric ratio, hfRPE-F were treated with ML-265 or vehicle and cross-linked. PKM2 oligomeric state was assessed by Western Blot. As depicted in Figure 5a, ML-265 treatment induced PKM2 tetramerization in hfPRE induced to undergo EMT. Considering the tetrameric form does not translocate to the nucleus to function as a transcriptional coactivator, the subcellular localization of PKM2 in hfRPE and hfRPE-F treated with ML-265 or vehicle was assessed by Western blot. HfRPE that have undergone EMT demonstrate an increased accumulation of PKM2 in the nuclear fraction suggesting a potential role for PKM2 moonlighting functions (Figure 5b). ML-265 reduced the nuclear levels of PKM2 in hfRPE-F cells (Figure 5b). Accordingly, the expression of HIF1A and potential downstream target genes such as MYC, CCND1, GLUT1, and PDK1 were reduced with ML-265 treatment (Figure 5 c), and the metabolic profile of hfRPE undergoing EMT, as determined from targeted metabolomics, was altered significantly, including those metabolites in nucleotide biosynthesis and glycolysis (Figure 6).

Example 2

The role of PKM2 in EMT was validated via genetic methods. Lentiviral vectors carrying short-hairpin RNAs (shRNA) targeted against Pkm2 efficiently transduced highly differentiated hfRPE and knocked down PKM2 (Fig. 7a-c). EMT was then induced in these hfRPE cultures by plating at 10% density. Knockdown of PKM2 resulted in decreased cell proliferation upon plating with increased markers of cell death (Fig. 7d and e).

Knockdown of PKM2 via siRNA (Fig. 8a) in hfRPE cells that had already undergone EMT (hfRPE-F) similarly resulted in decreased cell proliferation (Fig. 8b) with increased markers of cell death, including markers of early apoptosis, caspase activity, and necrosis

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SUBSTITUTE SHEET ( RULE 26 ) (Fig. 8c). This data indicates that PKM2 expression is required for both the induction and maintenance of hfRPE EMT.

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SUBSTITUTE SHEET ( RULE 26 ) REFERENCES

1. Wubben, T. J., Besirli, C. G. & Zacks, D. N. Pharmacotherapies for Retinal Detachment. Ophthalmology 123, 1553-1562 (2016).

2. Tamiya, S. & Kaplan, H. J. Role of epithelial-mesenchymal transition in proliferative vitreoretinopathy. Exp. Eye Res. 142, 26-31 (2016).

3. Zhao, C. et al. mTOR- mediated dedifferentiation of the retinal pigment epithelium initiates photoreceptor degeneration in mice. J. Clin. Invest. 121, 369-383 (2011).

4. Adijanto, J. & Philp, N. J. Cultured primary human fetal retinal pigment epithelium (hfRPE) as a model for evaluating RPE metabolism. Exp. Eye Res. 126, 77-84 (2014).

5. Wubben, T. J. et al. Photoreceptor metabolic reprogramming provides survival advantage in acute stress while causing chronic degeneration. Sci Rep 7, 17863 (2017).

6. Prakasam, G., Iqbal, M. A., Bamezai, R. N. K. & Mazurek, S. Posttranslational Modifications of Pyruvate Kinase M2: Tweaks that Benefit Cancer. Front Oncol 8, 22 (2018).

7. Anastasiou, D. et al. Pyruvate kinase M2 activators promote tetramer formation and suppress tumorigenesis. Nat. Chem. Biol. 8, 839-847 (2012).

8. Wubben, T. J. et al. Small molecule activation of metabolic enzyme pyruvate kinase muscle isozyme 2, PKM2, circumvents photoreceptor apoptosis. Sci Rep 10, 2990 (2020).

9. Yang, W. et al. Nuclear PKM2 regulates -catenin transactivation upon EGFR activation. Nature 480, 118-122 (2011).

10. Yang, W. et al. ERKl/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat. Cell Biol. 14, 1295-1304 (2012).

11. Palsson-McDermott, E. M. et al. Pyruvate kinase M2 regulates Hif- lot activity and IL- ip induction and is a critical determinant of the warburg effect in LPS-activated macrophages. Cell Metab. 21, 65-80 (2015).

12. Angiari, S. et al. Pharmacological Activation of Pyruvate Kinase M2 Inhibits CD4+ T Cell Pathogenicity and Suppresses Autoimmunity. Cell Metab. 31, 391-405.e8 (2020).

13. Abdi, F., Mohammadi, S. S. & Falavarjani, K. G. Intravitreal Methotrexate. J Ophthalmic Vis Res 16, 657-669 (2021).

All publications and patents mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the described method and system of the disclosure will be apparent to those skilled in the art without departing from the

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SUBSTITUTE SHEET ( RULE 26 ) scope and spirit of the disclosure. Although the disclosure has been described in connection with specific preferred embodiments, it should be understood that the disclosure as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the disclosure that are obvious to those skilled relevant fields are intended to be within the scope of the following claims.

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SUBSTITUTE SHEET ( RULE 26 )

Claims

We claim:

1. A method of treating or preventing proliferative vitreoretinopathy (PVR) in a subject, comprising administering a PKM2 activator to the eye of a subject, wherein said administering prevents, treats or reduces symptoms of PVR in said subject.

2. A method of treating or preventing retinal pigment epithelium (RPE) epithelial-to-mesenchymal transition (EMT) in a subject, comprising administering a PKM2 activator to the eye of a subject, wherein said administering prevents, treats or reduces symptoms of EMT in said subject.

3. A method of treating or preventing epiretinal membrane (ERM) in a subject, comprising administering a PKM2 activator to the eye of a subject, wherein said administering prevents, treats or reduces symptoms of ERM in said subject.

4. The method of any of the preceding claims, wherein said subject has been treated for retinal detachment.

5. The method of claim 4, wherein said treatment is cryotherapy, laser surgery, or surgery.

6. The method of any of the preceding claims, wherein said subject has not been treated with a PKM2 activator prior to said treatment for retinal detachment.

7. The method of any of the preceding claims, wherein said subject is under the age of 50.

8. The method of claim 4, wherein said retinal detachment is caused by trauma.

9. The method of claim 4, wherein said retinal detachment is not caused by aging.

10. The method of any of the preceding claims, wherein said PKM2 activator is delivered to retinal pigment epithelium (RPE) cells.

11. A method of treating proliferative vitreoretinopathy (PVR) in a subject, comprising a) diagnosing said subject with PVR; and b) administering a PKM2 activator to the eye of a subject, wherein said administering treats or reduces symptoms of PVR in said subject.

12. The method of any of the preceding claims, wherein said activator is a small molecule.

13. The method of claim 11, wherein said small molecule is

15. The method of any of the preceding claims, wherein said activator is formulated for injection, for oral delivery, or as an eye drop.

16. The method of claim 15, wherein said injection is intravitreal injection.

17. The method of any of the preceding claims, further comprising administering an additional agent.

18. The method of claim 17, wherein said additional agent is methotrexate.

19. The use of a composition comprising an activator of PKM2 to prevent, treat or reduce symptoms of, EMT, ERM, and/or PVR in a subject.