SAS:clf 8123-111807-02 04/15/25 06621 GROWTH AND DIFFERENTIATION FACTOR 15 FOR TREATMENT OF POSTERIOR CAPSULAR OPACIFICATION CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims the benefit of U.S. Application No.63/635,242, filed April 17, 2024, which is herein incorporated by reference in its entirety. FIELD [0002] This relates to the field of vision, specifically to the use of Growth and Differentiation Factor (GDF)-15 for the inhibition of posterior capsular opacification (PCO). ACKNOWLEDGMENT OF GOVERNMENT SUPPORT [0003] This invention was made with government support under EY008098 awarded by the National Institute of Health. The government has certain rights in the invention. BACKGROUND [0004] PCO, often referred to as “secondary cataract,” is the most common postoperative complication of cataract extraction. In PCO, the posterior capsule undergoes secondary opacification due to the migration, proliferation, and differentiation of lens epithelial cells (LECs). PCO can cause significant visual symptoms, particularly when it involves the central visual axis. Despite advances in surgical techniques and intraocular lens (IOL) design, this condition continues to impose a significant burden on patients. A need remains for compositions that inhibit PCO. SEQUENCE LISTING [0005] The Sequence Listing is submitted as an Extensible Markup Language (XML) file. The contents of the sequence listing (8123-111807-02_Sequence Listing.xml, Size: 17,165 bytes, Date of Creation: March 17, 2025) is herein incorporated by reference in its entirety. SUMMARY [0006] Methods are disclosed for inhibiting posterior capsular opacification in a subject. These methods include a) selecting a subject having an eye with a lens with posterior capsular opacification or an eye with a lens at risk of developing posterior capsular opacification from a cataract surgery, and b) locally delivering to the eye of the subject a composition comprising an effective amount of growth differentiation factor 15 (GDF-15) and a pharmaceutically acceptable carrier, thereby inhibiting posterior capsular opacification in the subject.
SAS:clf 8123-111807-02 04/15/25 06621 [0007] The foregoing and other objects, features, and advantages of the invention will become more apparent from the following detailed description, which proceeds with reference to the accompanying figures. BRIEF DESCRIPTION OF THE FIGURES [0008] FIGs.1A-1D. Immunofluorescence stained with fibronectin and DAPI. NPCs were pretreated with (FIG.1A) Smad2 inhibitor (SB431542) or (FIG.1B) cultured simultaneously with GDF-15. Similarly, FHL124 cells were pretreated with (FIG.1C) Smad2 inhibitor (SB431542) or (FIG.1D) co-cultured with GDF-15. Both the Smad2 inhibitor and GDF-15 inhibited GDF-11-induced upregulation of EMT marker, fibronectin, in both NPCs and FHL124. Int Den = integrated density. Scale bar = 100 μm. [0009] FIGs.2A-2C. ARPE-19 and FHL-124 were treated with TGFβ2 alone or in combination with GDF-15 for 24h. Typical Western blotting of (FIG.2A) p-Smad2, Smad2 and (FIG.2B) fibronectin, and the quantification of images. Simultaneous culture with GDF-15 reduced the protein expression of TGFβ2-induced upregulation of p-Smad2 and fibronectin, as well as (FIG.2C) the gene expression of FN1. Protein and gene expressions were normalized to GAPDH. Relative fold changes are represented as 2-ΔΔCT compared to PBS only group. N=3 per group, data were expressed as the mean ± SEM. *P < 0.05, ***P < 0.001 by Student’s t-test. [0010] FIGs.3A-3C. (FIG.3A) Mouse lenses treated with increasing concentrations of TGFβ2 showed increased formation of opacity. (FIG.3B) A significant difference in the appearance of the lenses treated with PBS, TGFβ2 (10 ng/mL), and TGFβ2 with GDF-15 pretreatment. (FIG.3C) Treatment with 1 ng/mL TGFβ2 in lenses elevated the expression levels of EMT markers, α smooth muscle actin 2 (Acta2), fibronectin 1 (Fn1), and vimentin (Vim). Pretreatment with GDF-15 for 1 h alleviated the TGFβ2-induced EMT marker elevation. Scale bar = 300 μm. Relative fold changes are represented as 2-ΔΔCT compared to PBS only group. N = 3 per group, data were expressed as the mean ± SEM. *P < 0.05, **P < 0.01 by one-way ANOVA and post hoc t-test with Tukey correction. [0011] FIG.4. GDF-15 alleviates TGFβ2-induced EMT by impeding Smad2 phosphorylation, potentially inhibiting the opacity in the lenses. SEQUENCES [0012] The nucleic and amino acid sequences listed herein are shown using standard letter abbreviations for nucleotide bases and amino acids. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. [0013] SEQ ID NOs: 1 and 2 are exemplary amino acid sequences of GDF-15. [0014] SEQ ID NOs: 3-18 are the nucleic acid sequences of primers.
SAS:clf 8123-111807-02 04/15/25 06621 DETAILED DESCRIPTION [0015] Cataracts are a major cause of vision impairment and blindness worldwide (Hashemi et al., Eye (Lond) 34:1357-1370, 2020; Heruye et al., Pharmaceuticals (Basel) 13, 2020). A 2010 study showed that cataracts lead to an estimated 51% of the blindness that affects 10.8 million people worldwide (Donatella et al., British Journal of Ophthalmology 96:614, 2012; Khairallah et al., Investigative Ophthalmology & Visual Science 56:6762-6769, 2015). Aging and diabetes are major risk factors for cataracts (Lim et al., Nutrients 12, 2020). With current long-life expectancies and a large diabetic population, the number of people blinded by cataracts is estimated to reach 40 million by 2025 (Donatella et al., British Journal of Ophthalmology 96:614, 2012; Fang et al., BMC Public Health 22:2068, 2022). [0016] Currently, the main treatment for cataracts is surgical removal of the opaque lens and replacement with a synthetic lens (Thompson et al., Prim Care 42:409-423, 2015). However, complications can still occur after cataract surgery. A common complication is secondary cataract, also known as PCO (Wang et al., Investigative Ophthalmology & Visual Science 54:323-332, 2013; Ma et al., Biochem Biophys Res Commun 447:689-695, 2014). PCO occurs in ~20%–50% of adults after cataract surgery (Chang et al., Chem Biol Interact 276:149-154, 2017; Sun et al., Life Sci 265:118741, 2021). A need remains for agents that prevent and treat PCO. It is disclosed herein that GDF-15 can be used to inhibit PCO. Summary of Terms [0017] Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:
SAS:clf 8123-111807-02 04/15/25 06621 [0018] Administration: The introduction of a composition into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravenous, the composition is administered by introducing the composition into a vein of the subject. Exemplary routes of administration include, but are not limited to, topical (such as to the surface of the eye), oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes. [0019] Animal: Living multi-cellular vertebrate organisms, a category that includes, for example, mammals and birds. The term mammal includes both human and non-human mammals. Similarly, the term “subject” includes both human and veterinary subjects. [0020] Conservative variants: "Conservative" amino acid substitutions are those substitutions that do not substantially affect or decrease an activity of GDF-15, such as the ability of the polypeptide to inhibit PCO. Specific, non-limiting examples of a conservative substitution include the following examples: Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu
SAS:clf 8123-111807-02 04/15/25 06621 [0021] The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid, provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Non-conservative substitutions are those that reduce an activity, such as the ability of a GDF-15 to inhibit PCO. [0022] Cataract: A cloudy area in the lens of the eye that leads to a decrease in vision of the eye. Symptoms may include faded colors, blurry or double vision, halos around light, trouble with bright lights, and difficulty seeing at night. Cataracts may be partial or complete, stationary or progressive, hard or soft. Histologically, the main types of age-related cataracts are nuclear sclerosis, cortical, and posterior subcapsular. Cataracts can be classified by using the lens opacities classification system LOCS III. In this system, cataracts are classified based on type as nuclear, cortical, or posterior. The cataracts are further classified based on severity on a scale from 1 to 5. [0023] Cataract Surgery: The removal of the lens of the eye that has developed a cataract, e.g., an opaque or cloudy area. During cataract surgery, the cloudy lens is removed from the posterior chamber, either by emulsification in place or by surgical removal. An artificial intraocular lens implant is usually inserted at the same location, or less frequently in front of the chamber, to restore useful focus. Cataract surgery is generally performed by an ophthalmologist in an out-patient setting, such as at surgical center, and local anesthesia is used. [0024] In phacoemulsification (phaco), the natural lens is fragmented by an ultrasonic probe and removed by suction. Femtosecond laser-assisted phacoemulsification surgery uses a laser to make the corneal incision, execute the capsulotomy, which provides access to the lens, and initiate lens fragmentation, which reduces energy requirements for phacoemulsification. The small incision size used in phacoemulsification generally allows for suture-less incision closure. In extracapsular cataract extraction (ECCE), and its variation manual small incision cataract surgery (MSICS), the lens is removed from its capsule and manually extracted from the eye, either whole or after being split into a small number of substantial pieces. Comparative trials of MSICS against phaco in dense cataracts have found no significant difference in outcomes. In most surgeries, an artificial intraocular lens is inserted. [0025] Consists Essentially Of/Consists Of: With regard to a polypeptide, a polypeptide that consists essentially of a specified amino acid sequence if it does not include any additional amino acid residues. However, the polypeptide can include additional non-peptide components, such as labels (for example, fluorescent, radioactive, or solid particle labels), sugars or lipids. With regard to a polypeptide, a polypeptide that consists of a specified amino acid sequence does not include any additional amino acid residues, nor does it include additional biological components, such as nucleic acids lipids, sugars, nor does it include labels. A polypeptide that consists or consists essential of a specified amino acid sequence can be glycosylated or have an amide modification.
SAS:clf 8123-111807-02 04/15/25 06621 [0026] Effective amount: A quantity of compound, such as GDF-15 sufficient to achieve a desired effect in a subject being treated, such as the inhibition of PCO. For instance, this can be the amount necessary to inhibit, treat or ameliorate a symptom of PCO in a lens of an eye in a subject. In some aspects, it is the amount necessary to treat a subject by a measurable amount over a period of time, or to measurably inhibit progression of PCO in a subject. [0027] An effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining an effective response. For example, an effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment lasting several days or weeks. However, the effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of an agent, such as GDF-15m can be packaged in an amount, or in multiples of the effective amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components. [0028] Epithelial-mesenchymal transition (EMT): A process by which epithelial cells lose their cell polarity and cell–cell adhesion, and gain migratory and invasive properties to become mesenchymal stem cells, which are multipotent stromal cells that can transdifferentiate into a variety of cell types. EMT is essential for numerous developmental processes including mesoderm formation and neural tube formation. EMT has also been shown to occur in wound healing, in organ fibrosis and in the initiation of metastasis in cancer progression. Several signaling pathways (TGF-β, FGF, EGF, HGF, Wnt/beta-catenin and Notch) and hypoxia may induce EMT. [0029] Growth and Differentiation Factor (GDF)-15: A protein belonging to the transforming growth factor beta superfamily that was first identified as Macrophage inhibitory cytokine-1 (MIC-1). GDF-15 is believed to play a role in regulating inflammatory pathways and to be involved in regulating apoptosis, angiogenesis, cell repair and cell growth. An exemplary amino acid sequence and mRNA sequence for human GDF-15 is provided in GENBANK® Accession No. NM_004864.4, March 17, 2024, incorporated by reference herein, and exemplary amino acid sequence and mRNA sequence for mouse GDF-15 is provided in GENBANK® Accession No. NM_011819.4, February 18, 2024, incorporated by reference herein. [0030] Inhibiting or treating a disease: Inhibiting a disease, such as, but not limited to, PCO, refers to inhibiting the full development of a disease. In several examples, inhibiting a disease refers to lessening symptoms or signs of the particular disease, such as corneal opacification. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition related to the disease. Treatment can be measured using success or indicia of success in the attenuation or amelioration of an injury, pathology or condition, including any objective or subjective parameter such as abatement, remission, diminishing of symptoms or making the condition more tolerable to the patient, slowing in the rate of degeneration or decline, making the final point of
SAS:clf 8123-111807-02 04/15/25 06621 degeneration less debilitating, improving a subject’s physical state. The treatment may be assessed by objective or subjective parameters; including the results of a physical examination or tests, such as vision tests. [0031] Peptide Modifications: GDF-15 polypeptides include synthetic aspects of polypeptides described herein. In addition, analogs (non-peptide organic molecules), derivatives (chemically functionalized polypeptide molecules obtained starting with the disclosed polypeptide sequences) and variants (homologs) of these proteins can be utilized in the methods described herein. Each polypeptide of this disclosure is comprised of a sequence of amino acids, which may be either L- and/or D- amino acids, naturally occurring and otherwise. [0032] Peptides can be modified by a variety of chemical techniques to produce derivatives having essentially the same activity as the unmodified polypeptides, and optionally having other desirable properties. For example, carboxylic acid groups of the protein, whether carboxyl-terminal or side chain, can be provided in the form of a salt of a pharmaceutically-acceptable cation or esterified to form a C1-C16 ester, or converted to an amide of formula NR1R2 wherein R1 and R2 are each independently H or C1-C16 alkyl, or combined to form a heterocyclic ring, such as a 5- or 6- membered ring. Amino groups of the polypeptide, whether amino-terminal or side chain, can be in the form of a pharmaceutically-acceptable acid addition salt, such as the HCl, HBr, acetic, benzoic, toluene sulfonic, maleic, tartaric and other organic salts, or can be modified to C1-C16 alkyl or dialkyl amino or further converted to an amide. [0033] Hydroxyl groups of the polypeptide side chains may be converted to C1-C16 alkoxy or to a C1- C16 ester using well-recognized techniques. Phenyl and phenolic rings of the polypeptide side chains may be substituted with one or more halogen atoms, such as fluorine, chlorine, bromine or iodine, or with C1-C16 alkyl, C1-C16 alkoxy, carboxylic acids and esters thereof, or amides of such carboxylic acids. Methylene groups of the polypeptide side chains can be extended to homologous C2-C4 alkylenes. Thiols can be protected with any one of a number of well-recognized protecting groups, such as acetamide groups. Those skilled in the art will also recognize methods for introducing cyclic structures into the polypeptides of this invention to select and provide conformational constraints to the structure that result in enhanced stability. [0034] Peptides can be amidated, which is a post-translational modification to form an amide that can enhance the biological activity of the polypeptide. In amidiation, the C-terminal amino acid (polypeptide-COOH) is modified to form and amide (polypeptide-CONH2). The amide may be formed by post-translational C-terminal amidation. The amino acid to be modified can be followed by a glycine, which provides the amide group. The process of post-translational amidation of a polypeptide derived from a precursor proprotein is well characterized and involves three enzymatic steps (originally described in Cuttitta, The Anatomical Record, 236:87-93, 1993). Step one involves endoproteolytic cleavage at a pair of basic amino acids near the carboxy terminus of the protein. Step
SAS:clf 8123-111807-02 04/15/25 06621 two involves carboxypeptidase-mediated removal of basic residues. Step three is the amidation reaction, which involves oxidation of a terminal glycine to form the amide of the neighboring carboxy terminal amino acid. Glycine is the only known amino acid to function as an amide donor for its neighboring amino acid. [0035] Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents. [0036] In general, the nature of the carrier will depend on the particular mode of administration being employed. For instance, parenteral and topical formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed). [0037] Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L-isomers being preferred. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. A polypeptide includes both naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A polypeptide has an amino terminal (N- terminal) end and a carboxy-terminal end. In some aspects, the polypeptide is GDF-15, or a fragment thereof. [0038] Posterior capsular opacification (PCO): A condition, often referred to as “secondary cataract,” is the most common postoperative complication of cataract extraction. In PCO, the posterior capsule undergoes secondary opacification due to the migration, proliferation, and differentiation of lens epithelial cells. PCO occurs in 20-50% of patients within 2 to 5 years of cataract surgery. Children and infants have a significantly higher incidence and earlier onset of PCO, along with the potential for associated amblyopia. In children, reported rates of PCO reach 100%. The
SAS:clf 8123-111807-02 04/15/25 06621 pathophysiology of PCO is multifactorial. During routine phacoemulsification surgery, the surgeon excises a portion of the anterior capsule (capsulorrhexis), removes the cataractous lens material, and then implants a synthetic lens into the intact capsular bag. PCO occurs when residual LECs on the residual anterior capsule undergo three phenomena: proliferation, migration toward the posterior capsule, and normal and abnormal differentiation. Multiple cytokines and growth factors, including transforming growth factor β (TGF-β), fibroblast growth factor 2 (FGF-2), and hepatocyte growth factor (HFG), and matrix metalloproteinases (MMOs) have been implicated in the pathogenesis of PCO. Exogenous hyaluronic acid (HA), a component of some viscoelastic substances used during cataract surgery, may result in increased rates of ex vivo PCO. [0039] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein (such as an antibody) is more enriched than the peptide or protein is in its natural environment within a cell. In one aspect, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation. The GDF-15polypeptides disclosed herein can be purified (and/or synthesized) by any of the means known in the art (see, e.g., Guide to Protein Purification, ed. Deutscher, Meth. Enzymol.185, Academic Press, San Diego, 1990; and Scopes, Protein Purification: Principles and Practice, Springer Verlag, New York, 1982). Substantial purification denotes purification from other proteins or cellular components. A substantially purified protein is at least about 60%, 70%, 80%, 90%, 95%, 98% or 99% pure. Thus, in one specific, non- limiting example, a substantially purified protein is 90% free of other proteins or cellular components. [0040] Subject: Living multi-cellular vertebrate organisms, a category that includes human and non- human mammals. In an example, a subject is a human. In an additional example, a subject is selected that is in need of inhibiting a dengue virus infection. For example, the subject is uninfected and at risk of dengue virus infection. [0041] Topical: Medication applied to a body surface. Exemplary formulations for topical administration to the eye include a drop, ointment or contact lens can be utilized. An ointment is a homogeneous, viscous, semi-solid preparation. An ointment is most commonly a greasy, thick water- in-oil emulsion (80% oil, 20% water) having a high viscosity, that is intended for external application to the skin or mucous membranes. Ointments have a water number that defines the maximum amount of water that they can contain. They are used as emollients or for the application of active ingredients to the skin for protective, therapeutic, or prophylactic purposes and where a degree of occlusion is desired. An eye drop is a liquid drop applied directly to the surface of the eye usually in small amounts such as a single drop or a few drops. Eye drops usually contain saline to match the salinity of the eye, and can include a lubricant. [0042] Transforming Growth Factor (TGF)-β: A molecule that interacts with the TGFβ receptor. TGF-β is a multifunctional set of peptides that controls proliferation, differentiation, and other
SAS:clf 8123-111807-02 04/15/25 06621 functions in many cell types. TGF-β acts synergistically with transforming growth factor-alpha (TGF- α) in inducing transformation. It also acts as a negative autocrine growth factor. Dysregulation of TGF-β activation and signaling may result in apoptosis. Many cells synthesize TGF-β and almost all of them have specific receptors for this peptide. TGF-β1, TGF-β2, and TGF-β3 all function through the same receptor signaling systems. [0043] TGF-β1 is a peptide of 112 amino acid residues derived by proteolytic cleavage from the C- terminal of a precursor protein. TGFs interact with a conserved family of cell surface serine/threonine-specific protein kinase receptors, and generate intracellular signals using SMADs. Proteins from the TGF-beta superfamily are only active as homo- or heterodimer; the two chains being linked by a single disulfide bond. Exemplary amino acid and mRNA sequences are disclosed in GENBANK® Accession No. NM_000660.7, February 19, 2023, incorporated herein by reference. [0044] TGF-β3 is a peptide formed as a preproprotein of 412 amino acids in length. Exemplary amino acid and mRNA sequences are disclosed in GENBANK® Accession No. NC_000014.9, February 252022, incorporated herein by reference. GDF-15 Polypeptides [0045] GDF-15 is a secreted ligand of the TGF superfamily of proteins. Ligands of this family bind various TGF receptors leading to recruitment and activation of SMAD family transcription factors that regulate gene expression. The encoded preproprotein is proteolytically processed to generate each subunit of the disulfide-linked homodimer. The protein is expressed in a broad range of cell types, acts as a pleiotropic cytokine and is involved in the stress response program of cells after cellular injury. Increased protein levels are associated with disease states such as tissue hypoxia, inflammation, acute injury, and oxidative stress. In some aspects, the GDF-15 polypeptide is a human GDF-15 polypeptide. [0046] The sequence of human GDF-15 can be accessed at GENBANK®, Accession Nos. NP_004855 (protein) and NM_004864 (mRNA), as available on March 17, 2024. An exemplary amino acid sequence of human GDF-15 protein is: MPGQELRTVNGSQMLLVLLVLSWLPHGGALSLAEASRASFPGPSELHSEDSRFRELRKRYED LLTRLRANQSWEDSNTDLVPAPAVRILTPEVRLGSGGHLHLRISRAALPEGLPEASRLHRALF RLSPTASRSWDVTRPLRRQLSLARPQAPALHLRLSPPPSQSDQLLAESSSARPQLELHLRPQAA RGRRRARARNGDHCPLGPGRCCRLHTVRASLEDLGWADWVLSPREVQVTMCIGACPSQFR AANMHAQIKTSLHRLKPDTVPAPCCVPASYNPMVLIQKTDTGVSLQTYDDLLAKDCHCI (SEQ ID NO: 1). [0047] Similar to other members of the transforming growth factor (TGF)-β superfamily, GDF-15 is formed as a full-length dimer protein and is cleaved at an RXXR site with the secretion of a mature dimeric protein. The GDF-15 consists of 308 amino acids as shown above. It undergoes dimerization
SAS:clf 8123-111807-02 04/15/25 06621 by a specific disulfide linkage to form the pro- GDF-15 dimeric precursor. The pro-protein dimer undergoes proteolytic cleavage catalyzed by furin-like protease at the amino acid target sequence RXXR to release C-terminal dimeric mature GDF-15. The mature dimer and the pro-GDF-15 are then secreted into the extracellular matrix. GDF-15 may exist in multiple forms within the cell: the pro- GDF-15 monomer (~40 kDa), the pro-GD-F15 dimer (~80 kDa), and the mature dimer (~30 kDa). In addition, the GDF-15 precursor protein contains an N-terminal signal peptide. See Baek and Eling, Pharmacol Ther.2019 Jun; 198: 46–58. [0048] A GDF-15 polypeptide of use is: ARNGDHCPLG PGRCCRLHTV RASLEDLGWA DWVLSPREVQ VTMCIGACPS QFRAANMHAQ IKTSLHRLKP DTVPAPCCVP ASYNPMVLIQ KTDTGVSLQT YDDLLAKDCH CI (SEQ ID NO: 2, which is underlined in SEQ ID NO: 1 above). [0049] Human GDF-15 is synthesized as pro-GDF-15, which then dimerizes through cysteine residues to form pro-GDF-15 dimer, it is then cleaved at an RXXR site, forming a 112 amino acid C- terminal dimeric protein and a pro-peptide. The carboxy-terminal domain of GDF- 15 contains the characteristic seven conserved cysteine residues necessary for the formation of the cysteine knot and the single interchain disulfide bond. A subject may be treated with a human GDF-15 polypeptide, or a biologically active fragment or variant thereof, including without limitation mature GDF-15 dimers. In aspects, the dimers are cross-linked. [0050] The sequence of a GDF-15 may be altered in various ways known in the art to generate targeted changes in sequence. The GDF-15 will usually be substantially similar to the sequences provided herein, i.e. will differ by at least one amino acid, and may differ by at least two but not more than about ten amino acids. The sequence alternations may be substitutions, insertions, or deletions. Scanning mutations that systematically introduce alanine, or other residues, may be used to determine key amino acids. In some aspects, the GDF-15 includes at most one to at most ten conservative substitutions, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative substitutions. The GDF-15 can include 1 or 2 conservative substitutions. In aspects these substitutions are outside of the amino acid sequence of the mature form (in the pre-pro region). [0051] The GDF-15 may be truncated at the C-terminus or the N-terminus by, for example, about 1 , about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, or more amino acids, provided that the truncated peptide retains substantially the same biological activity as the native protein, or native mature dimer. Thus, the GDF-15 can include a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the C-terminus. The GDF-15 can include a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids at the N-terminus. [0052] Modifications of interest that do not alter primary sequence include chemical derivatization of polypeptides, e.g., acetylation, or carboxylation. Also included are modifications of glycosylation, e.g. those made by modifying the glycosylation patterns of a polypeptide during its synthesis and
SAS:clf 8123-111807-02 04/15/25 06621 processing or in further processing steps; e.g. by exposing the polypeptide to enzymes which affect glycosylation, such as mammalian glycosylating or deglycosylating enzymes. Also embraced are sequences that have phosphorylated amino acid residues, e.g. phosphotyrosine, phosphoserine, or phosphothreonine. [0053] Also of use are polypeptides that have been modified using ordinary molecular biological techniques and synthetic chemistry to improve their resistance to proteolytic degradation or to optimize solubility properties or to render them more suitable as a therapeutic agent. For examples, the backbone of the peptide may be cyclized to enhance stability (see Friedler et al. (2000) J. Biol. Chem.275:23783-23789). Exemplary peptides are derivative peptides that may be one modified by glycosylation, pegylation, phosphorylation or any similar process that retains at least one biological function of the peptide from which it was derived. The GDF-15 can be amidated. Analogs of such polypeptides include those containing residues other than naturally occurring L-amino acids, e.g. D- amino acids or non-naturally occurring synthetic amino acids. [0054] For example, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into peptides. Non-classical amino acids include, but are not limited to, the D- isomers of the common amino acids, 2,4-diaminobutyric acid, alpha-amino isobutyric acid, 4- aminobutyric acid, Abu, 2-amino butyric acid, gamma-Abu, epsilon-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, beta-alanine, fluoro-amino acids, designer amino acids such as beta-methyl amino acids, Calpha-methyl amino acids, Nalpha-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary). In other specific aspects, branched versions of the peptides listed herein are provided, such as by substituting one or more amino acids within the sequence with an amino acid or amino acid analog with a free side chain capable of forming a peptide bond with one or more amino acids (and thus capable of forming a "branch"). Cyclical peptides are also contemplated. [0055] Also included are peptide derivatives which are differentially modified during or after synthesis, such as by benzylation, glycosylation, acetylation, phosphorylation, amidation, pegylation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. In specific aspects, the peptides are acetylated at the N- terminus and/or amidated at the C-terminus. In one example, the GDF-15 includes a carboxy terminal amide. [0056] Peptidomimetics are compounds based on, or derived from, peptides and proteins. Peptidomimetics can be obtained by structural modification of known peptide sequences using unnatural amino acids, conformational restraints, isosteric replacement, and the like. The subject peptidomimetics constitute the continum of structural space between peptides and non-peptide
SAS:clf 8123-111807-02 04/15/25 06621 synthetic structures; peptidomimetics may be useful, therefore, in delineating pharmacophores and in helping to translate peptides into nonpeptide compounds with the activity of the parent peptides. These are also of use in the presently disclosed methods. [0057] Peptidomimetic and organomimetic aspects are envisioned, whereby the three-dimensional arrangement of the chemical constituents of such peptido- and organomimetics mimic the three- dimensional arrangement of the polypeptide backbone and component amino acid side chains, resulting in such peptido- and organomimetics of GDF-15 having measurable or enhanced ability to treat PCO. For computer modeling applications, a pharmacophore is an idealized three-dimensional definition of the structural requirements for biological activity. Peptido- and organomimetics can be designed to fit each pharmacophore with current computer modeling software (using computer assisted drug design or CADD). Also included are mimetics prepared using such techniques. [0058] Various commercial synthetic apparatuses are available, for example, automated synthesizers by Applied Biosystems, Inc., Foster City, Calif., Beckman, etc. By using synthesizers, naturally occurring amino acids may be substituted with unnatural amino acids. The particular sequence and the manner of preparation will be determined by convenience, economics, purity required, and the like. [0059] If desired, various groups may be introduced into the peptide during synthesis or during expression, which allow for linking to other molecules or to a surface. Thus, cysteines can be used to make thioethers, histidines for linking to a metal ion complex, carboxyl groups for forming amides or esters, amino groups for forming amides, and the like. [0060] The subject GDF-15 can be prepared by in vitro synthesis, using conventional molecular biology methods as known in the art, such as cloning, transformation, and purification. The polypeptides can also be isolated and purified in accordance with conventional methods of recombinant synthesis. A lysate may be prepared of the expression host and the lysate purified using HPLC, exclusion chromatography, gel electrophoresis, affinity chromatography, or other purification technique. For the most part, the compositions which are used will comprise at least 20% by weight of the desired product, more usually at least about 75% by weight, preferably at least about 95% by weight, and for therapeutic purposes, usually at least about 99.5% by weight, in relation to contaminants related to the method of preparation of the product and its purification. Usually, the percentages will be based upon total protein. Pharmaceutical Compositions and Methods [0061] Methods are disclosed herein for inhibiting PCO in a subject, that include: a) selecting a subject with PCO or that has a cataract surgery in an eye, and b) locally delivering to the eye of the subject a composition comprising an effective amount of growth differentiation factor 15 (GDF-15) and a pharmaceutically acceptable carrier to a lens with PCO, or to a lens at risk of developing PCO resulting from the cataract surgery. In some aspects, the effective amount of GDF-15 reduces
SAS:clf 8123-111807-02 04/15/25 06621 epithelial-mesenchymal transformation of cell in the lens. In some aspects, the subject has undergone the cataract surgery, and has yet to develop PCO in the lens. In more aspects, the subject has undergone cataract surgery, and has developed PCO in the lens. The subject can be a human subject. The subject can be a veterinary subject. [0062] In some aspects the subject has, or is developing, PCO. PCO has two forms, fibrous and pearl (also referred to as proliferative). Fibrous PCO occurs due to abnormal proliferation of LECs, and presents as wrinkles and folds on the posterior capsule at the site of fusion of the anterior and posterior capsules. Histological examination reveals extracellular matrix (ECM) accumulation and elongated fibroblast cells. Pearl PCO is responsible for the majority of PCO-related visual loss. Pearl PCO is composed of normally differentiated LECs that line the equatorial lens region. Examination shows clusters of swollen, opacified, and differentiated LECs called bladder or Wedl cells. The presently disclosed methods can inhibit both of these forms. [0063] The subject can have undergone, or be undergoing, a cataract surgery. These include, but are not limited to, manual small incision cataract surgery or phacoemulsification, with implantation of an intraocular lens IIOL). After the removal of a cataract, the intraocular lens is usually implanted, such as to replace a damaged natural lens. A foldable IOL may be implanted through a 1.8 to 2.8 mm (0.071 to 0.110 in) incision, whereas a rigid poly(methyl methacrylate) (PMMA) lens requires a larger cut. Foldable IOLs are made of silicone, hydrophobic, or hydrophilic acrylic material of appropriate refractive power. In aspects, the IOL is inserted through the incision, usually into the capsular bag from which the cataract was removed (in-the-bag implantation). Sometimes, a sulcus implantation— in front of the capsular bag, but behind the iris—may be required because of posterior capsular tears or zonular dialysis (inadequate support for the capsular bag). The presently disclosed methods can be used to inhibit PCO after a cataract surgery. [0064] In some aspects, the composition is administered prior to the cataract surgery. For example, the composition can be administered within about an hour prior to the cataract surgery. The composition can be administered 60 to 30 minutes prior to the cataract surgery, such as about 55, 50, 45, 40 or 35 minutes prior to the cataract surgery. Exemplary administration includes about 30 minutes prior to the cataract surgery. Additional exemplary administration includes about 4 hours, 2 hours, or 1 hour prior to the cataract surgery. [0065] In other aspects, the composition is administered to the eye of the subject at the time of the cataract surgery. Exemplary administration includes within about 10 minutes of the cataract surgery, within about 5 minutes of the cataract surgery, or within about 2 minutes of the cataract surgery. In aspects, the GDF-15 is administered about 10 minutes after the cataract surgery, about 5 minutes after the cataract surgery, or about 2 minutes of after cataract surgery. In aspects, the composition is administered about 10 minutes before the cataract surgery, about 5 minutes after the cataract surgery, or about 2 minutes of before cataract surgery.
SAS:clf 8123-111807-02 04/15/25 06621 [0066] In further aspects, the subject has undergone the cataract surgery, and the composition is administered following the cataract surgery. In aspects, the composition is administered within about an hour following the cataract surgery. In more aspects, the composition is administered within about 2, 3, 4, or 5 hours following the cataract surgery. In more aspects, the composition is administered two times a day, daily, biweekly or weekly following the cataract surgery. [0067] In some aspects, the subject has PCO. In some aspects, a subject is identified with onset of blurry vision or visual acuity after cataract extraction. Slit lamp examination reveals a semi-opaque membrane with variable levels of fibrosis forming on the posterior capsule. Other notable signs are: a) Elschnig’s pearls: seen in pearl-type PCO, in which clusters of residual LECs appear as round, clear “pearls” that shine on retro-illumination. If these accumulate on the visual axis, they can cause decreased visual acuity. b) Soemmering rings: rings of residual LECs and cortical fibers that form between the posterior capsule and the edges of the anterior capsule remnant. These are often too peripheral to cause visual symptoms, but they can cause glare and visual loss if severe. c) Capsular wrinkling The disclosed methods can inhibit any of these signs in a subject. [0068] The composition can be administered to the subject over a period of time. The composition can be administered for a period of one week, one month, one year or two years. The composition can be administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 weeks. The composition can be administered for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months. The composition can be administered for 1, 23, 4, or 5 years. The composition can be administered indefinitely. The composition may be administered about every 96 hours, every 72 hours, every 48 hours, every 24 hours, every 12 hours, every 6 hours, every 3 hours, or every 1 hour. The composition can be administered for a duration of about1 day, 2 days, 3 days, 5 days, 7 days, 10 days, 14 days, 20 days, 30 days, 60 days, 90 days, 120 days, 180 days, or 365 days. [0069] If desired, the effective amount of the composition may be administered as one, two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms. In some aspects, the composition may be administered two or three times daily. In other aspects, the active compound will be administered once daily. [0070] The GF15 is administered at a dosage, alone or in combination with other agents, that reduces epithelial-mesenchymal transition in the eye of the subject. The effectiveness of the dosage can be evaluated by assessing vision of the subject, or the evaluation of opacification, such as with a slit lamp. The evaluation can be for maintenance of the clarity of the lens, or recovery of vision in a subject with PCO, such as at least about 5% recovery, at least about 10% recovery, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 75%, at least about 85%, at least about 95% or more, e.g. assessing by conventional measures of vision or corneal
SAS:clf 8123-111807-02 04/15/25 06621 opacity. It is contemplated that compositions will be obtained and used under the guidance of a physician for in vivo use. The dosage of the therapeutic formulation will vary widely, depending upon the nature of the disease, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like. In some aspects, the presently disclosed methods produce at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% reduction in loss of function, relative to function measured in absence treatment. In aspects, the presently disclosed methods result in reduced corneal opacity. Treatment may result in at least about a 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even 100% decrease in symptoms of PCO, compared to a subject that is not treated with the presently disclosed compositions. [0071] GDF15 can be administered at a concentration ranging from 0.1 μM to 10 mM, e.g., between 0.5 μM and 1 mM; between 1.0 μM and 500 μM; between 2.0 μM and 250 μM; at a dose of from about 0.1 ml to 1 ml/day, or any amount appropriate for human therapy. GDF15 can present in the compositions at a concentration range of 0.1-10%, with preferred ranges between 1-5% and 2-2.5% (mg/ml). Exemplary liquid formulations for eye drops contain 2-2.5% (mg/ml) of the disclosed composition. The composition can be formulated as a paste, an ointment, a gel, a liquid, an aerosol, a mist, a polymer, a film, an emulsion, or a suspension. The formulations are usually administered topically to the eye. [0072] An effective among of GDF-15 can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives or buffers that may be required. Examples of suitable aqueous and nonaqueous carriers that may be employed in the pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. such as gels, water soluble jellies, creams, lotions, suspensions, foams, powders, slurries, ointments, solutions, oils, pastes, suppositories, sprays, emulsions, saline solutions, dimethylsulfoxide (DMSO)-based solutions. In general, carriers with higher densities are capable of providing an area with a prolonged exposure to the active ingredients. In contrast, a solution formulation may provide more immediate exposure of the active ingredient to the chosen area. [0073] These compositions may also contain agents such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into the compositions. The composition can include benzalkonium chloride. [0074] The compositions described herein may be formulated for administration topically to the eye and surrounding tissues, particularly to the inner surface of the eye and the inner surface of the eyelids
SAS:clf 8123-111807-02 04/15/25 06621 (including e.g. cornea, conjunctiva and sclera). Such compositions, for example, may be formulated for instillation administration, administration into conjunctival sac and conjunctival administration. In aspects, the compositions described herein may be formulated for topical delivery to the cornea, such as an eye drop, an ointment or a contact lens. [0075] Eye drop formulations may include a liquid or semisolid pharmaceutical composition adapted to administration to the eye. A typical example of an eye drop composition is an ophthalmic solution to be administered dropwise to the eye. In certain aspects, the size of the drop is between about 10 and about 100 μL. The drop size may be greater than about 10 μL, greater than about 20 μL, greater than about 30 μL, greater than about 40 μL, greater than about 50 μL, greater than about 60 μL, greater than about 70 μL, greater than about 80 μL, greater than about 90 μL, or greater than about 100 μL. The drop size may be less than about 10 μL, less than about 20 μL, less than about 30 μL, less than about 40 μL, less than about 50 μL, less than about 60 μL, less than about 70 μL, less than about 80 μL, less than about 90 μL, or less than about 100 μL. [0076] In an aspect, the compositions described herein may be liquid formulations that may include an ophthalmic solution of GDF-15 and/or a microemulsion of GDF-15. Active pharmaceutical ingredients (APIs) for which microemulsions have been developed include cyclosporine A and flurbiprofen axetil. Successful approaches to extend the contact time of liquid dosage forms with ocular tissues and to increase the tissue uptake of the API include the use of excipients that increase viscosity, enhance penetration, or cyclodextrins. Cyclodextrins are cyclic oligosaccharides that form inclusion complexes with APIs that increase the aqueous solubility and bioavailability of hydrophobic APIs. In an aspect, the compositions described herein may include β-cyclodextrin and a therapeutically effective amount of GDF-15. [0077] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients, which are compounds that allow increased penetration of, or assist in the delivery of, therapeutic molecules across the membranes of the eye, including, but not limited to, the cornea, conjunctiva, and sclera. There are many of these penetration-enhancing molecules known to those trained in the art of topical formulation. Examples of such carriers and excipients include, but are not limited to, humectants (e.g., urea), glycols (e.g., propylene glycol), alcohols (e.g., ethanol), fatty acids (e.g., oleic acid), surfactants (e.g., isopropyl myristate and sodium lauryl sulfate), pyrrolidones, glycerol monolaurate, sulfoxides, terpenes (e.g., menthol), amines, amides, alkanes, alkanols, water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. [0078] In some aspects, the compositions described herein may include liquid formulations, semi- solid formulations, and multicompartment formulations. The composition includes an effective amount of GDF-15. The composition can include a pharmaceutically acceptable carrier. Exemplary pharmaceutically acceptable carriers for use in ocular formulations include alcohol, castor oil,
SAS:clf 8123-111807-02 04/15/25 06621 glycerin, polyoxyl 35 castor oil, Tyloxapol, polyethylene glycol 8000 (PEG-8000), ethanol, glycerin, cremaphor, propylene glycol (pG), polypropylene glycol (ppG), and polysorbate 80. In some cases, citrate buffer and sodium hydroxide are included to adjust pH. [0079] In some aspects, the compositions described herein include one or more of a solubilizing agent, an alcohol, an acid, and a preservative. In some aspects, the compositions described herein include a solubilizing agent and an alcohol. In some aspects, the compositions described herein include a solubilizing agent and an acid. In some aspects, the compositions described herein include a solubilizing agent and a preservative. In some aspects, the compositions described herein include a solubilizing agent, an alcohol, and an acid. In some aspects, the compositions described herein include a solubilizing agent, an alcohol, an acid, and a preservative. In some aspects, the solubilizing agent is vitamin E TPGS (d-α-tocopheryl polyethylene glycol 1000 succinate). In some aspects, the compositions described herein include a solubilizing agent in an amount, by weight, of about 0.5% to about 75%, or about 1% to about 70%, or about 1% to about 65%, or about 1% to about 60%, or about 1% to about 55%, or about 1% to about 50%, or about 1% to about 45%, or about 1% to about 40%, or about 1% to about 35%, or about 1% to about 30%, or about 1% to about 25%, or about 1% to about 20%, or about 1% to about 15%, or about 1% to about 10%, or about 1% to about 5%. [0080] In some aspects, the alcohol is a sugar alcohol, such as mannitol. In some aspects, the compositions described herein include an alcohol in an amount by weight, of about 0.5% to about 75%, or about 0.5% to about 70%, or about 0.5% to about 65%, or about 0.5% to about 60%, or about 0.5% to about 55%, or about 0.5% to about 50%, or about 0.5% to about 45%, or about 0.5% to about 40%, or about 0.5% to about 35%, or about 0.5% to about 30%, or about 0.5% to about 25%, or about 0.5% to about 20%, or about 0.5% to about 15%, or about 0.5% to about 10%, or about 0.5% to about 9%, or about 0.5% to about 8%, or about 0.5% to about 7%, or about 0.5% to about 6%, or about 0.5% to about 5%, or about 0.5% to about 4%, or about 0.5% to about 3%, or about 0.5% to about 2%, or about 0.5% to about 1%. In some aspects, the acid is boric acid. In some aspects, the compositions described herein include an acid in an amount, by weight, of about 0.5% to about 75%, or about 0.5% to about 70%, or about 0.5% to about 65%, or about 0.5% to about 60%, or about 0.5% to about 55%, or about 0.5% to about 50%, or about 0.5% to about 45%, or about 0.5% to about 40%, or about 0.5% to about 35%, or about 0.5% to about 30%, or about 0.5% to about 25%, or about 0.5% to about 20%, or about 0.5% to about 15%, or about 0.5% to about 10%, or about 0.5% to about 9%, or about 0.5% to about 8%, or about 0.5% to about 7%, or about 0.5% to about 6%, or about 0.5% to about 5%, or about 0.5% to about 4%, or about 0.5% to about 3%, or about 0.5% to about 2%, or about 0.5% to about 1%. In some aspects, the preservative is polyquaternium-1 (polyquad). In some aspects, the compositions described herein include a preservative in an amount, by weight, of about 0.001% to about 5%, or about 0.001% to about 4%, or about 0.001% to about 3%, or about 0.001% to about 2%, or about 0.001% to about 1%, or about 0.001% to about 0.5%, or about 0.001% to about
SAS:clf 8123-111807-02 04/15/25 06621 0.1%, or about 0.001% to about 0.009%, or about 0.001% to about 0.008%, or about 0.007%, or about 0.001% to about 0.006%, or about 0.001% to about 0.005%. [0081] In an aspect, the compositions described herein may be semi-solid formulations that include a gel or viscous excipient and GDF-15. Such semi-solid formulations include high viscosity formulations that increase bioavailability by increasing the residence time in the precorneal area. In situ gels are viscous liquids that undergo sol-to-gel transitions upon ocular application because of changes in pH, temperature or electrolyte concentration. Gelling excipients with favorable mucoadhesive properties further increase the residence time. Polymers or gelling excipients employed in developing these drug forms include gellan gum, sodium alginate, poloxamer, and cellulose acetate phthalate. In an aspect, the compositions described herein may include a thermogel using poloxamer 407 or gellan gum, and comprising a therapeutically effective amount of GDF-15. [0082] In some aspects, the composition includes a gelling excipient, such as gellan gum or sodium alginate. In some aspects, the composition includes a gelling excipient in an amount, by weight, of about 0.5% to about 20%, or about 0.1% to about 15%, or about 0.1% to about 10%, or about 0.1% to about 9%, or about 0.1% to about 8%, or about 0.1% to about 7%, or about 0.1% to about 6%, or about 0.1% to about 5%, or about 0.1% to about 4%, or about 0.1% to about 3%, or about 0.1% to about 2%, or about 0.1% to about 1%, or about 0.1% to about 0.9%, or about 0.1% to about 0.8%, or about 0.1% to about 0.7%, or about 0.1% to about 0.6%, or about 0.1% to about 0.5%. In some aspects, the composition includes a poloxamer. In some aspects, the composition includes a poloxamer in an amount, by weight, of about 1% to about 75%, or about 1% to about 70%, or about 1% to about 65%, or about 1% to about 60%, or about 1% to about 55%, or about 1% to about 50%, or about 1% to about 45%, or about 1% to about 40%, or about 1% to about 35%, or about 1% to about 30%, or about 1% to about 25%, or about 1% to about 20%, or about 1% to about 15%, or about 1% to about 10%, or about 1% to about 9%, or about 1% to about 8%, or about 1% to about 7%, or about 1% to about 6%, or about 1% to about 5%, or about 1% to about 4%, or about 1% to about 3%, or about 1% to about 2%. In some aspects, the composition includes a surfactant, such as TWEEN® 80 or polyoxyl stearate. In some aspects, the composition includes a surfactant in an amount, by weight, of about 0.01% to about 20%, or about 0.01% to about 15%, or about 0.01% to about 10%, or about 0.01% to about 9%, or about 0.01% to about 8%, or about 0.01% to about 7%, or about 0.01% to about 6%, or about 0.01% to about 5%, or about 0.01% to about 4%, or about 0.01% to about 3%, or about 0.01% to about 2%, or about 0.01% to about 1%, or about 0.01% to about 0.5%, or about 0.01% to about 0.1%, or about 0.01% to about 0.09%, or about 0.01% to about 0.08%, or about 0.07%, or about 0.01% to about 0.06%, or about 0.01% to about 0.05%. In other aspects, the composition includes a cyclodextrin, such as (2-hydroxypropyl)-β-cyclodextrin. In some aspects, the compositions described herein include a cylcodextrin in amount, by weight, of about 0.5% to about 95%, or about 0.5% to about 90%, or about 0.5% to about 85%, or about 0.5% to about 80%, or about 0.5% to about 75%, or about 0.5% to about 70%, or about 0.5% to about 65%, or about 0.5% to about
SAS:clf 8123-111807-02 04/15/25 06621 60%, or about 0.5% to about 55%, or about 0.5% to about 50%, or about 0.5% to about 45%, or about 0.5% to about 40%, or about 0.5% to about 35%, or about 0.5% to about 30%, or about 0.5% to about 25%, or about 0.5% to about 20%, or about 0.5% to about 15%, or about 0.5% to about 10%, or about 0.5% to about 9%, or about 0.5% to about 8%, or about 0.5% to about 7%, or about 0.5% to about 6%, or about 0.5% to about 5%, or about 0.5% to about 4%, or about 0.5% to about 3%, or about 0.5% to about 2%, or about 0.5% to about 1%. In an aspect, the composition includes an effective amount of GDF-15 and one or more of a gelling excipient (e.g., gellan gum or sodium alginate), a poloxamer, a solubilizing agent (e.g., vitamin E TPGS), a surfactant (e.g., TWEEN® 80 or polyoxyl stearate), a polyether (e.g., a polyethylene glycol, propylene glycol, Cremophor), and a cyclodextrin (e.g., (2-hydroxypropyl)-β-cyclodextrin). [0083] In an aspect, the composition can be a multicompartment formulation of GDF-15 such as, nanoparticles, liposomes, dendrimers, or niosomes that include GDF-15. Nanoparticles are polymeric carriers, which improve bioavailability thanks to increased corneal penetration and a larger surface area for dissolution. A relative limitation of nanoparticles is their low capacity. Liposomes are limited by their suboptimal stability, high cost and challenging technology for their large-scale production. Niosomes and discosomes are two-layered carriers, which increase bioavailability by extending its precorneal residence time. In an aspect, the compositions described herein include nanoparticles that comprise an effective amount of GDF-15. In an aspect, the composition can include poly(ethylene glycol) (PEG) nanoparticles. In some aspects, the nanoparticle formulation may include methoxy poly(ethylene glycol)-poly(lactide) (mPEG-PLA) nanoparticles. [0084] Eye drops or sprays can be provided in unit dose dispensers (such as eye drop bottles that dispense a metered unit dose. These can include, for example, wetting agents and an inert matrix. As one example of an inert matrix, liposomes may be prepared from dipalmitoyl phosphatidylcholine (DPPC), such as egg phosphatidylcholine (PC). Liposomes can be applied topically, either in the form of drops or as an aqueous based cream, or can be injected intraocularly. In a formulation for topical application, the active agent is slowly released over time as the liposome capsule degrades due to wear and tear from the eye surface. In a formulation for intraocular injection, the liposome capsule degrades due to cellular digestion. Both of these formulations provide advantages of a slow release drug delivery system, allowing the subject to be exposed to a substantially constant concentration of the active agent over time. In one example, the active agent can be dissolved in an organic solvent such as DMSO or alcohol as previously described and contain a polyanhydride, poly(glycolic) acid, poly(lactic) acid, or polycaprolactone polymer. [0085] Posterior chamber injection is also of use in the methods disclosed herein. In some aspects, the composition is formulated for intraocular injection, such as posterior chamber injection. Multiple injections into the posterior chamber are also of use. See, for example, U.S. Patent No.11,096,822. A hydrogel can be used for administration.
SAS:clf 8123-111807-02 04/15/25 06621 [0086] Implants are also of use in the methods disclosed herein. The implants can be inserted into the eye by a variety of methods, including placement by forceps or by trocar following making an incision (for example, a 2-3 mm incision) or other suitable sites. In some cases, the implant can be placed by trocar without making a separate incision, but instead by forming a hole directly into the eye with the trocar. The method of placement can influence the release kinetics. For example, implanting the device into the vitreous or the posterior chamber with a trocar may result in placement of the device deeper within the vitreous than placement by forceps, which may result in the implant being closer to the edge of the vitreous. The location of the implanted device may influence the concentration gradients of the therapeutic agent surrounding the device, and thus influence the release rates (for example, a device placed closer to the edge of the vitreous may result in a slower release rate, see U.S. Patent No.5,869,079 and U.S. Patent No.6,699,493). In one aspect, an implant is formulated with a bioerodible polymer matrix. In an aspect the implant is inserted under the artificial lens. [0087] Generally, when implants are used, the GDF-15 is homogeneously distributed through the polymeric matrix, such that it is distributed evenly enough that no detrimental fluctuations in rate of release occur because of uneven distribution of the immunosuppressive agent in the polymer matrix. The selection of the polymeric composition to be employed varies with the desired release kinetics, the location of the implant, patient tolerance, and the nature of the implant procedure. The polymer can be included as at least about 10 weight percent of the implant. In one example, the polymer is included as at least about 20 weight percent of the implant. In another aspect, the implant comprises more than one polymer, see U.S. Patent No.6,699,493. Characteristics of the polymers generally include biodegradability at the site of implantation, compatibility with the agent of interest, ease of encapsulation, and water insolubility, amongst others. Generally, the polymeric matrix is not fully degraded until the drug load has been released. [0088] For use in some of the methods of the present disclosure, active compound (e.g., GDF-15) can be given per se or as a pharmaceutical composition containing, for example, about 0.1 to about 99.5% (such as about 0.5 to about 90%) of active ingredient in combination with a pharmaceutically acceptable carrier. Actual dosage levels of the active ingredients in the pharmaceutical compositions may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the patient. [0089] The selected dosage level will depend upon a variety of factors including the route of administration, the time of administration, the rate of excretion of the particular compound(s) being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound(s) employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the
SAS:clf 8123-111807-02 04/15/25 06621 medical arts. A physician or veterinarian can readily determine and prescribe the therapeutically effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the pharmaceutical composition or compound at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of GDF-15 used will be that amount of the compound that is the lowest dose effective to produce a therapeutic effect. [0090] Additional agents that can be administered to the subject include antibacterial and antifungal antibiotics, as well as steroids and non-steroidal anti-inflammatory agents to reduce risk of infection and inflammation. Additional agents can be administered by any route. The additional agents can be formulated separately, or in the same composition as one or more miRNAs and/or exosomes. [0091] Agents of use include minoglycosides (for example, amikacin, apramycin, arbekacin, bambermycins, butirosin, dibekacin, dihydrostreptomycin, fortimicin(s), gentamicin, isepamicin, kanamycin, micronomicin, neomycin, neomycin undecylenate, netilmicin, paromomycin, ribostamycin, sisomicin, spectinomycin, streptomycin, tobramycin, trospectomycin), amphenicols (for example, azidamfenicol, chloramphenicol, florfenicol, thiamphenicol), ansamycins (for example, rifamide, rifampin, rifamycin sv, rifapentine, rifaximin), β-lactams (for example, carbacephems (e.g., loracarbef), carbapenems (for example, biapenem, imipenem, meropenem, panipenem), cephalosporins (for example, cefaclor, cefadroxil, cefamandole, cefatrizine, cefazedone, cefazolin, cefcapene pivoxil, cefclidin, cefdinir, cefditoren, cefepime, cefetamet, cefixime, cefmenoxime, cefodizime, cefonicid, cefoperazone, ceforanide, cefotaxime, cefotiam, cefozopran, cefpimizole, cefpiramide, cefpirome, cefpodoxime proxetil, cefprozil, cefroxadine, cefsulodin, ceftazidime, cefteram, ceftezole, ceftibuten, ceftizoxime, ceftriaxone, cefuroxime, cefuzonam, cephacetrile sodium, cephalexin, cephaloglycin, cephaloridine, cephalosporin, cephalothin, cephapirin sodium, cephradine, pivcefalexin), cephamycins (for example, cefbuperazone, cefmetazole, cefininox, cefotetan, cefoxitin), monobactams (for example, aztreonam, carumonam, tigemonam), oxacephems, flomoxef, moxalactam), penicillins (for example, amdinocillin, amdinocillin pivoxil, amoxicillin, ampicillin, apalcillin, aspoxicillin, azidocillin, azlocillin, bacampicillin, benzylpenicillinic acid, benzylpenicillin sodium, carbenicillin, carindacillin, clometocillin, cloxacillin, cyclacillin, dicloxacillin, epicillin, fenbenicillin, floxacillin, hetacillin, lenampicillin, metampicillin, methicillin sodium, mezlocillin, nafcillin sodium, oxacillin, penamecillin, penethamate hydriodide, penicillin G benethamine, penicillin g benzathine, penicillin g benzhydrylamine, penicillin G calcium, penicillin G hydrabamine, penicillin G potassium, penicillin G procaine, penicillin N, penicillin O, penicillin V, penicillin V benzathine, penicillin V hydrabamine, penimepicycline, phenethicillin potassium, piperacillin, pivampicillin, propicillin, quinacillin, sulbenicillin, sultamicillin, talampicillin, temocillin, ticarcillin), other (for example, ritipenem), lincosamides (for example, clindamycin, lincomycin), macrolides (for example, azithromycin, carbomycin, clarithromycin, dirithromycin, erythromycin, erythromycin acistrate, erythromycin estolate, erythromycin glucoheptonate, erythromycin lactobionate,
SAS:clf 8123-111807-02 04/15/25 06621 erythromycin propionate, erythromycin stearate, josamycin, leucomycins, midecamycins, miokamycin, oleandomycin, primycin, rokitamycin, rosaramicin, roxithromycin, spiramycin, troleandomycin), polypeptides (for example, amphomycin, bacitracin, capreomycin, colistin, enduracidin, enviomycin, fusafungine, gramicidin s, gramicidin(s), mikamycin, polymyxin, pristinamycin, ristocetin, teicoplanin, thiostrepton, tuberactinomycin, tyrocidine, tyrothricin, vancomycin, viomycin, virginiamycin, zinc bacitracin), tetracyclines (for example, apicycline, chlortetracycline, clomocycline, demeclocycline, doxycycline, guamecycline, lymecycline, meclocycline, methacycline, minocycline, oxytetracycline, penimepicycline, pipacycline, rolitetracycline, sancycline, tetracycline), and others (e.g., cycloserine, mupirocin, tuberin). Agents of use also include synthetic antibacterials, such as 2,4-Diaminopyrimidines (for example, brodimoprim, tetroxoprim, trimethoprim), nitrofurans (for example, furaltadone, furazolium chloride, nifuradene, nifuratel, nifurfoline, nifurpirinol, nifurprazine, nifurtoinol, nitrofurantoin), quinolones and analogs (for example, cinoxacin, ciprofloxacin, clinafloxacin, difloxacin, enoxacin, fleroxacin, flumequine, grepafloxacin, lomefloxacin, miloxacin, nadifloxacin, nalidixic acid, norfloxacin, ofloxacin, oxolinic acid, pazufloxacin, pefloxacin, pipemidic acid, piromidic acid, rosoxacin, rufloxacin, sparfloxacin, temafloxacin, tosufloxacin, trovafloxacin), sulfonamides (for example, acetyl sulfamethoxypyrazine, benzylsulfamide, chloramine-b, chloramine-t, dichloramine t, mafenide, 4’- (methylsulfamoyl)sulfanilanilide, noprylsulfamide, phthalylsulfacetamide, phthalylsulfathiazole, salazosulfadimidine, succinylsulfathiazole, sulfabenzamide, sulfacetamide, sulfachlorpyridazine, sulfachrysoidine, sulfacytine, sulfadiazine, sulfadicramide, sulfadimethoxine, sulfadoxine, sulfaethidole, sulfaguanidine, sulfaguanol, sulfalene, sulfaloxic acid, sulfamerazine, sulfameter, sulfamethazine, sulfamethizole, sulfamethomidine, sulfamethoxazole, sulfamethoxypyridazine, sulfametrole, sulfamidocchrysoidine, sulfamoxole, sulfanilamide, sulfanilylurea, n-sulfanilyl-3,4- xylamide, sulfanitran, sulfaperine, sulfaphenazole, sulfaproxyline, sulfapyrazine, sulfapyridine, sulfasomizole, sulfasymazine, sulfathiazole, sulfathiourea, sulfatolamide, sulfisomidine, sulfisoxazole) sulfones (for example, acedapsone, acediasulfone, acetosulfone sodium, dapsone, diathymosulfone, glucosulfone sodium, solasulfone, succisulfone, sulfanilic acid, p- sulfanilylbenzylamine, sulfoxone sodium, thiazolsulfone), and others (for example, clofoctol, hexedine, methenamine, methenamine anhydromethylene-citrate, methenamine hippurate, methenamine mandelate, methenamine sulfosalicylate, nitroxoline, taurolidine, xibornol). [0092] Additional agents of use include antifungal antibiotics such as polyenes (for example, amphotericin B, candicidin, dennostatin, filipin, fungichromin, hachimycin, hamycin, lucensomycin, mepartricin, natamycin, nystatin, pecilocin, perimycin), others (for example, azaserine, griseofulvin, oligomycins, neomycin undecylenate, pyrrolnitrin, siccanin, tubercidin, viridin) allylamines (for example, butenafine, naftifine, terbinafine), imidazoles (for example, bifonazole, butoconazole, chlordantoin, chlormiidazole, cloconazole, clotrimazole, econazole, enilconazole, fenticonazole, flutrimazole, isoconazole, ketoconazole, lanoconazole, miconazole, omoconazole, oxiconazole nitrate,
SAS:clf 8123-111807-02 04/15/25 06621 sertaconazole, sulconazole, tioconazole), thiocarbamates (for example, tolciclate, tolindate, tolnaftate), triazoles (for example, fluconazole, itraconazole, saperconazole, terconazole) others (for example, acrisorcin, amorolfine, biphenamine, bromosalicylchloranilide, buclosamide, calcium propionate, chlorphenesin, ciclopirox, cloxyquin, coparaffinate, diamthazole dihydrochloride, exalamide, flucytosine, halethazole, hexetidine, loflucarban, nifuratel, potassium iodide, propionic acid, pyrithione, salicylanilide, sodium propionate, sulbentine, tenonitrozole, triacetin, ujothion, undecylenic acid, zinc propionate). Antineoplastic agents can also be of use including (1) antibiotics and analogs (for example, aclacinomycins, actinomycin, anthramycin, azaserine, bleomycins, cactinomycin, carubicin, carzinophilin, chromomycins, dactinomycin, daunorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin, epirubicin, idarubicin, menogaril, mitomycins, mycophenolic acid, nogalamycin, olivomycines, peplomycin, pirarubicin, plicamycin, porfiromycin, puromycin, streptonigrin, streptozocin, tubercidin, zinostatin, zorubicin), (2) antimetabolites such as folic acid analogs (for example, denopterin, edatrexate, methotrexate, piritrexim, pteropterin, trimetrexate), (3) purine analogs (for example, cladribine, fludarabine, 6-mercaptopurine, thiamiprine, thioguanine), (4) pyrimidine analogs (for example, ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, doxifluridine, emitefur, enocitabine, floxuridine, fluorouracil, gemcitabine, tagafur). [0093] Steroidal anti-inflammatory agents can also be used such as 21-acetoxypregnenolone, alclometasone, algestone, amcinonide, beclomethasone, betamethasone, budesonide, chloroprednisone, clobetasol, clobetasone, clocortolone, cloprednol, corticosterone, cortisone, cortivazol, cyclosporine, deflazacort, desonide, desoximetasone, dexamethasone, diflorasone, diflucortolone, difluprednate, enoxolone, fluazacort, flucloronide, flumethasone, flunisolide, fluocinolone acetonide, fluocinonide, fluocortin butyl, fluocortolone, fluorometholone, fluperolone acetate, fluprednidene acetate, fluprednisolone, flurandrenolide, fluticasone propionate, formocortal, halcinonide, halobetasol propionate, halometasone, halopredone acetate, hydrocortamate, hydrocortisone, loteprednol etabonate, mazipredone, medrysone, meprednisone, methylprednisolone, mometasone furoate, paramethasone, prednicarbate, prednisolone, prednisolone 25-diethylamino- acetate, prednisolone sodium phosphate, prednisone, prednival, prednylidene, rimexolone, tixocortol, triamcinolone, triamcinolone acetonide, triamcinolone benetonide, and triamcinolone hexacetonide. [0094] In addition, non-steroidal anti-inflammatory agents can be used. These include aminoarylcarboxylic acid derivatives (for example, enfenamic acid, etofenamate, flufenamic acid, isonixin, meclofenamic acid, mefenamic acid, niflumic acid, talniflumate, terofenamate, tolfenamic acid), arylacetic acid derivatives (for example, aceclofenac, acemetacin, alclofenac, amfenac, amtolmetin guacil, bromfenac, bufexamac, cinmetacin, clopirac, diclofenac sodium, etodolac, felbinac, fenclozic acid, fentiazac, glucametacin, ibufenac, indomethacin, isofezolac, isoxepac, lonazolac, metiazinic acid, mofezolac, oxametacine, pirazolac, proglumetacin, sulindac, tiaramide, tolmetin, tropesin, zomepirac), arylbutyric acid derivatives (for example, bumadizon, butibufen, fenbufen, xenbucin), arylcarboxylic acids (for example, clidanac, ketorolac, tinoridine), arylpropionic
SAS:clf 8123-111807-02 04/15/25 06621 acid derivatives (for example, alminoprofen, benoxaprofen, bermoprofen, bucloxic acid, carprofen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, loxoprofen, naproxen, oxaprozin, piketoprolen, pirprofen, pranoprofen, protizinic acid, suprofen, tiaprofenic acid, ximoprofen, zaltoprofen), pyrazoles (for example, difenamizole, epirizole), pyrazolones (for example, apazone, benzpiperylon, feprazone, mofebutazone, morazone, oxyphenbutazone, phenylbutazone, pipebuzone, propyphenazone, ramifenazone, suxibuzone, thiazolinobutazone), salicylic acid derivatives (for example, acetaminosalol, aspirin, benorylate, bromosaligenin, calcium acetylsalicylate, diflunisal, etersalate, fendosal, gentisic acid, glycol salicylate, imidazole salicylate, lysine acetylsalicylate, mesalamine, morpholine salicylate, 1-naphthyl salicylate, olsalazine, parsalmide, phenyl acetylsalicylate, phenyl salicylate, salacetamide, salicylamide o-acetic acid, salicylsulfuric acid, salsalate, sulfasalazine), thiazinecarboxamides (for example, ampiroxicam, droxicam, isoxicam, lornoxicam, piroxicam, tenoxicam), .epsilon.-acetamidocaproic acid, s- adenosylmethionine, 3-amino-4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, α- bisabolol, bucolome, difenpiramide, ditazol, emorfazone, fepradinol, guaiazulene, nabumetone, imesulide, oxaceprol, paranyline, perisoxal, proquazone, superoxide dismutase, tenidap, and zileuton. [0095] Following administration, the subject can be evaluated for response using any methods known in the art. This includes, but is not limited to, slit lamp examination. [0096] Also provided are a pharmaceutical pack or kit comprising one or more containers filled with the composition comprising the GDF-15. Associated with such container(s) can be a written instructions for the use of the composition. In some aspects, the kit includes one or more containers, including, but not limited to a vial, tube, ampule, bottle and the like, for containing the composition. The one or more containers also can be carried within a suitable carrier, such as a box, carton, tube or the like. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments. [0097] In some aspects, the container can hold a composition that is by itself or when combined with another composition effective for inhibiting PCO and may have a sterile access port (for example the container may be an vial having a stopper pierceable by a hypodermic injection needle). Alternatively, or additionally, the article of manufacture may further include a second (or third) container including a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate- buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. The container can include a delivery apparatus, such as a dropper for administering eye drops. [0098] The presently disclosed kits or pharmaceutical systems also can include associated instructions for using the compounds for inhibiting PCO, including information on dosage and timing of administration. In some aspects, the instructions include one or more of the following: a description
SAS:clf 8123-111807-02 04/15/25 06621 of the active compound; a dosage schedule and administration; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and references. The instructions can be printed directly on a container (when present), as a label applied to the container, as a separate sheet, pamphlet, card, or folder supplied in or with the container. The container can also include vials for administration, such as, but not limited to, sterile containers for the administration of eye drops, or a tube for delivery of an ointment to the eye. Overview [0099] Clause 1: A method for inhibiting posterior capsular opacification in a subject, comprising: a) selecting a subject with or at risk of developing posterior capsular opacification, and b) locally delivering to the eye of the subject a composition comprising an effective amount of growth differentiation factor 15 (GDF-15) and a pharmaceutically acceptable carrier to a lens with posterior capsular opacification, or to a lens at risk of developing posterior capsular opacification resulting from the cataract surgery, thereby inhibiting posterior capsular opacification in the subject. [0100] Clause 2: The method of clause 1, wherein the composition is administered topically to the eye of the subject. [0101] Clause 3: The method of clause 1 or clause 2, wherein the composition is formulated as an eye drop, an ointment, or a contact lens, or for posterior chamber injection. [0102] Clause 4: The method of any one of clauses 1-3, wherein the effective amount of GDF-15 reduces epithelial-mesenchymal transformation of cell in the lens. [0103] Clause 5: The method of any one of clauses 1-4, wherein the subject at risk of developing posterior capsular opacification has undergone, or is having, a cataract surgery. [0104] Clause 6: The method of clause 5, wherein the subject is having the cataract surgery, and the composition is administered prior to the cataract surgery. [0105] Clause 7: The method of clause 6, wherein the composition is administered within an hour prior to the cataract surgery. [0106] Clause 8: The method of clause 6 or clause 7, wherein the composition is administered to the eye of the subject at the time of the cataract surgery. [0107] Clause 9: The method of clause 5, wherein the subject has undergone the cataract surgery, and the composition is administered following the cataract surgery. [0108] Clause 10: The method of clause 9, the composition is administered within an hour following the cataract surgery. [0109] Clause 11: The method of clause 9 or clause 10, wherein the composition is administered two times a day, daily, biweekly or weekly following the cataract surgery. [0110] Clause 12: The method of any one of clauses 1-5, wherein the subject has the posterior capsular opacification.
SAS:clf 8123-111807-02 04/15/25 06621 [0111] Clause 13: The method of clause 12, wherein the composition is administered two times a day, daily, biweekly or weekly. [0112] Clause 14: The method of any one of clauses 1-13, wherein the subject is a human. [0113] Clause 15: The method of any one of clauses 1-14, wherein the GDF-15 is human GDF-15. [0114] Clause 16: The method of any one of clauses 1-15, wherein the composition improves visual acuity of the subject. [0115] Clause 17: The method of any one of clauses 1-16, further comprising evaluating the vision of the subject. [0116] Clause 18: The method of any one of clauses 1-17, further comprising evaluating the corneal opacity of the subject. [0117] Clause 19: A composition comprising an effective amount of GDF-15 and a pharmaceutically acceptable carrier for use in the method of any one of clauses 1-18. [0118] Clause 20. A pharmaceutical composition comprising an effective amount of GDF-15 and a pharmaceutically acceptable carrier for use in inhibiting posterior capsular opacification in a subject, wherein the pharmaceutical composition formulated for local administration to the eye of the subject. [0119] Clause 21: The pharmaceutical composition of clause 20, wherein the composition is formulated for topical administration to the eye of the subject. [0120] Clause 22. The pharmaceutical composition of clause 20 or clause 21, wherein the composition is formulated as an eye drop, an ointment, or a contact lens, or for posterior chamber injection. [0121] Clause 23: The pharmaceutical composition of any one of clauses 20-23, wherein the subject at risk of developing posterior capsular opacification, and has undergone, or is having, the cataract surgery. [0122] Clause 24: The pharmaceutical composition of clause 23, wherein the subject is having the cataract surgery, and the pharmaceutical composition is administered to the subject prior to the cataract surgery. [0123] Clause 25: The pharmaceutical composition of clause 24, wherein the pharmaceutical composition is administered to the subject a) within an hour prior to the cataract surgery; and/or b) at the time of the cataract surgery. [0124] Clause 26: The pharmaceutical composition of clause 23, wherein the subject has undergone the cataract surgery, and the composition is administered following the cataract surgery.
SAS:clf 8123-111807-02 04/15/25 06621 [0125] Clause 27: The pharmaceutical composition of clause 26, wherein the composition is administered within an hour following the cataract surgery. [0126] Clause 28 The method of any one of clauses 20-22, wherein the subject has the posterior capsular opacification. [0127] Clause 29: The pharmaceutical composition of any one of claims 26-28, wherein the composition is administered two times a day, daily, biweekly or weekly following the cataract surgery. [0128] Clause 30: The pharmaceutical composition of any one of clauses 20-29, wherein a) the subject is a human; and/or b) the GDF-15 is human GDF-15. [0129] The disclosure is illustrated by the following non-limiting Examples. EXAMPLES [0130] No matter how skillfully performed, cataract surgery disrupts integrity of the eye and triggers a wound-healing response (Wormstone et al., Prog Retin Eye Res 82:100905, 2021; Wallentin et al., Investigative Ophthalmology & Visual Science 39:1410-1418, 1998) that elevates levels of transforming growth factor beta (TGFβ) in the eye (Chang et al., Chem Biol Interact 276:149-154, 2017; Sun et al., Life Sci 265:118741, 2021). TGFβ can induce an epithelial-mesenchymal transition (EMT) by modulating transcription factors (Hachana et al., Cells 11, 2022; Kubo et al., Int J Mol Sci 19, 2018) in residual lens epithelial cells, which then proliferate and migrate to the posterior capsule instead of differentiating into lens fiber cells (Gotoh et al., Investigative Ophthalmology & Visual Science 48:4679-4687, 2007; Li et al., Exp Eye Res 92:173-179, 2011; Nahomi et al., J Cell Biochem 119:6814-6827, 2018; Nam et al., Biochem J 478:2285-2296, 2021). This abnormal differentiation leads to re-formation of cataracts in PCO (Zhang et al., Sci Rep 7:1117, 2017). Example 1: Materials and Methods Materials and cell culture [0131] Recombinant human TGFβ2 protein was purchased from R&D (302-B2). GDF-11 (120-11) and GDF-15 (120-28C) were obtained from PeproTech. The GDF-15 amino acid sequence is: ARNGDHCPLG PGRCCRLHTV RASLEDLGWA DWVLSPREVQ VTMCIGACPS QFRAANMHAQ IKTSLHRLKP DTVPAPCCVP ASYNPMVLIQ KTDTGVSLQT YDDLLAKDCH CI (SEQ ID NO: 2) [0132] NPCs were differentiated from H9 embryonic stem cells, a gift from Don Zack’s laboratory at Johns Hopkins University, following a previous protocol (Zhang et al., Restor Neurol Neurosci 38:131-140, 2020), and the characterization of NPC was confirmed by immunofluorescence staining of NPC markers. Human adult retinal pigment epithelial cells (RPEs) (ARPE-19; ATCC, CRL-2302)
SAS:clf 8123-111807-02 04/15/25 06621 and fetal human lens epithelial cells (FHL-124) were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, 11885-084) supplemented with 10% and 5% fetal bovine serum, respectively, and 100 units/mL of penicillin/streptomycin (Gibco, 15140122). Cells were cultured in a humidified incubator with 5% CO2 at 37℃. Ex vivo murine lenses assay and expression levels of EMT markers [0133] Both male and female 8–12-week-old C57BL/6 wild-type mice were used (from Jackson Laboratory). The animals were kept in microisolator cages on a 12-hour day/night cycle. Mice were euthanized by isoflurane liquid inhalant (Medline, 66794-017-25) and cervical dislocation (IACUC #21018446). To compare lens opacity, mouse lenses were isolated and cultured in DMEM supplemented with 0.1% bovine serum albumin, and 100 units/mL of penicillin/streptomycin in a humidified incubator with 5% CO2 at 37℃. Phosphate buffered saline (PBS, Fisher Scientific, BP39950) only, TGFβ2 (1, 5, or 10 ng/mL) with PBS, and TGFβ2 (10 ng/mL) with GDF-15 (50 ng/mL) one-hour pretreatments were added to the medium respectively and treated for seven days. Lenses were settled down on a metal grid and lens transparency was accessed using a EVOS M5000 microscope. To determine the expression levels of EMT markers (α smooth muscle actin 2, fibronectin 1, vimentin), lenses were treated with PBS only, TGFβ2 (1 ng/mL) with PBS, and TGFβ2 (1 ng/mL) with GDF-15 (50 ng/mL) for one hour pretreatments for three days. Cell assays [0134] To investigate if GDF-11 regulated TGFβ2 signaling pathway, embryonic stem cell-derived NPCs were plated in 6-well plates with a Matrigel pre-coating (Sigma-Aldrich, CLS354277). At 70% confluence, cells were pretreated with DMSO (0.1%, Sigma-Aldrich, D8418) or Smad2 inhibitor SB431542 (10 µM, Selleckchem, S1067) for 1 hour before the addition of PBS or GDF-11 (50 ng/mL) treatment and then cultured for seven days. To investigate the effects of GDF-15, NPCs were plated in 6-well plates with Matrigel pre-coating. At 70% confluence, cells were pretreated with PBS or GDF-15 (50 ng/mL) for 1 hour before the addition of phosphate buffered saline (PBS) or GDF-11 (16.7 ng/mL) treatment and then cultured for seven days. GDF-15 was added again after three days, whereas SB431542 and GDF-11 were added once at the beginning. Expression of nucleus and fibronectin were detected by immunofluorescence staining. [0135] To determine the expression levels of Smad2 and p-Smad2, and fibronectin, ARPE-19 cells were plated in 6-well plates. At 70% confluence, cells were pretreated with PBS or GDF-15 (50 ng/mL) for 1 hour, and then treated with PBS or TGFβ2 (1 ng/mL) for one hour or two days, respectively. Expression of Smad2, p-Smad2 and fibronectin were detected by Western blot.
SAS:clf 8123-111807-02 04/15/25 06621 Immunofluorescence staining [0136] NPCs were fixed in 4% paraformaldehyde (Fisher Scientific, 50-980-488) for ten minutes, then washed with PBS for five minutes three times. NPCs were blocked for 1 hour using cell blocking buffer [5% normal goat serum (Invitrogen, 10000C) and 0.2% Triton X-100 (Sigma, X-100) in PBS]. NPCs were incubated with anti-fibronectin antibody (1:500, Abcam, ab2413) overnight at 4℃. After being washed with PBS for five minutes three times, the cells were incubated with goat anti-rabbit Alexa Fluor™ 488 secondary antibody (1:500, Invitrogen, A11034), and counterstained with DAPI (30 nM, Invitrogen, D1306) for one hour at room temperature. Fluorescence images were acquired using an Olympus IX83 microscope. Western blot [0137] Cells were lysed in RIPA buffer (Thermo Scientific, 89900) with HALT™ protease and phosphatase inhibitor cocktail (1:100, Thermo Scientific, 78440) with sonication, and centrifuged at 12,800 rpm for ten minutes in 4℃. Supernatant was collected and protein concentration was quantified using a BCA assay (Thermo Scientific, 23225). Protein lysates were separated on am SDS- PAGE gel (4–20% Mini-PROTEAN® TGX™ precast protein gels, Bio-Rad, 4561094), and transferred to PVDF membranes (Bio-Rad, 1704272) using a semi-dry Trans-Blot Turbo Transfer System (Bio-Rad). After blocking with blocking buffer (diluted 1:1 in H2O; LI-COR, 927-60001) for one hour, membranes were probed with primary antibodies against Smad2 (1:1000; Cell Signaling Technology, 3103S), p-Smad2 (1:1000; Cell Signaling Technology, 18338S), fibronectin (1:1000; Abcam, ab2413), and GADPH (1:1000; Cell Signaling Technology, 2118S) overnight at 4℃. Membranes were washed with TBS (Bio-Rad, 1706435) containing 1% Tween-20 (Sigma, P9416), and then probed with fluorescence-conjugated secondary antibodies (800CW goat anti-rabbit IgG, LI- COR, 926-32211; 680RD donkey anti-mouse IgG, LI-COR, 926-68072). Protein expression was visualized using chemiluminescence detection reagent and a luminescence imaging system (Odyssey M Imaging System; LI-COR). The results were analyzed using ImageJ software. Real-time qPCR (RT-qPCR) [0138] FHL-124 was pretreated with GDF-15 (50 ng/mL) before the addition of TGFβ2 (5 ng/mL) treatment for 24 hours. Lenses were pretreated with GDF-15 (50 ng/mL) before the addition of TGFβ2 (10 ng/mL) treatment for three days. After treatment, RNA was extracted using a RNeasy Plus Mini Kit (Qiagen, 74134) and converted into cDNA by ISCRIPTTM cDNA synthesis kit (Bio-Rad, 1708891). RT-qPCR was performed to determine gene expression using ITAQTM Universal SYBR green Supermix (Bio-Rad, 1725121) and primer sets (OriGene). Quantitative PCR reactions were performed using an ABI QUANTSTUDIO™ 3 machine (Thermo Fisher Scientific). The comparative cycle threshold (CT) method was used for data analysis and the relative fold change was compared to
SAS:clf 8123-111807-02 04/15/25 06621 PBS. GAPDH was selected as an internal control for quantitative real-time PCR analysis. The primer sequences used for RT-qPCR are listed below: mouse fibronectin 1 (Fn1) forward 5’- CCCTATCTCTGATACCGT TGTCC-3’ (SEQ ID NO: 3), reverse 5’- TGCCGCAACTACTGTGATTCGG-3’ (SEQ ID NO: 4); mouse alpha smooth muscle actin 2 (Acta2) forward 5’- TGCTGACAGAGGCACCACTGAA -3’(SEQ ID NO: 5), reverse 5’- CAGTTGTACGTCCAGAGGCATAG -3’(SEQ ID NO: 6); mouse vimentin (Vim) forward 5- CGGAAAGTGGAATCCTTGCAGG -3’(SEQ ID NO: 7), reverse 5’- AGCAGTGAGGTCAGGCTTGGAA -3’(SEQ ID NO: 8); mouse GAPDH forward 5’ - CATCACT GCCACCCAGAAGACTG-3’(SEQ ID NO: 9), reverse 5’- ATGCCAGTGAGCTTCCCGTTCAG - 3’(SEQ ID NO: 10), human fibronectin 1 (FN1) forward 5’- ACAACACCGAGGTGACTGAGAC - 3’(SEQ ID NO: 11), reverse 5’- GGACACAACGATGCTTCCTGAG -3’ (SEQ ID NO: 12); human alpha smooth muscle actin 2 (ACTA2) forward 5’ -CTATGCCTCTGGACGCACAACT -3’(SEQ ID NO: 13), reverse 5’- CAGATCCAGACGCATGATGGCA -3’(SEQ ID NO: 14); human vimentin (VIM) forward 5- AGGCAAAGCAGGAGTCCACTGA -3’(SEQ ID NO: 15), reverse 5’- ATCTGGCGTTCCAGGGACTCAT -3’(SEQ ID NO: 16); human GAPDH forward 5’- GTCTCCTCTGACTTCAACAGCG -3’(SEQ ID NO: 17), reverse 5’ - ACCACCCTGTTGCTGTAGCCAA -3’(SEQ ID NO: 18). Statistical analysis [0139] All experiments were independently conducted at least three times. Continuous variables are presented as the mean ± standard error of the mean. Prior to analyzing group differences, the F test for equal variance was conducted. Student’s t-test was used for comparing two groups, while one-way ANOVA was employed for multiple groups. Post hoc analysis involved the application of the Tukey multiple-comparison post hoc test to correct for multiple comparisons. Target protein expression measured by immunoblotting was determined via densitometry and is expressed relative to an internal control or as phosphorylated protein relative to total protein expression. Statistical significance was defined as a p-value < 0.05. All analyses were performed using GraphPad Prism (version 10, GraphPad Software Inc.). Example 2: GDF-15 inhibits GDF-11-induced EMT in NPCs and LECs [0140] To determine whether GDF-11 induces EMT in NPCs (FIG.1A-B) and LECs (FIG.1C-D), cells were pretreated with DMSO or Smad2 inhibitor SB431542 followed by treatment with PBS or GDF-11. Immunofluorescence showed that fibronectin protein was significantly elevated following GDF-11 treatment and was suppressed by SB431542 pretreatment (FIG.1A, FIG.1C), demonstrating that GDF-11 induces EMT via Smad2 activation. Next it was investigated whether GDF-15 could inhibit GDF-11-induced EMT in NPCs. Cells were pretreated with PBS or GDF-15 followed by
SAS:clf 8123-111807-02 04/15/25 06621 treatment with PBS or GDF-11. GDF-15 pretreatment suppressed the GDF-11-induced increase in fibronectin expression, indicating that GDF-15 alleviates GDF-11-induced EMT (FIG.1B, FIG.1D). Example 3: GDF-15 inhibits GDF-11-induced EMT in NPCs [0141] TGFβ2 also induces EMT in RPEs (Huang et al., Int J Ophthalmol 14:973-980, 2021). To determine whether GDF-15 can inhibit TGFβ2-induced EMT in human ARPE-19 cells, cells were pretreated with PBS or GDF-15, followed by treatment with TGFβ2. TGFβ2 treatment increased phosphorylated Smad2 (FIG.2A) and fibronectin (FIG.2B), and these effects were inhibited by GDF- 15 pretreatment. Expression of FN1 in FHL-124 showed similar effects to TGFβ2 and GDF-15 treatment (FIG.2C). These results indicate that GDF-15 is capable of suppressing TGFβ2-induced Smad2 phosphorylation and its downstream signaling, including the expression of fibronectin. Example 4: GDF-15 alleviates the effects of TGFβ2-induced EMT in mice lenses [0142] To determine if TGFβ2 could induce EMT in mice lenses, extracted lenses were cultured and treated with different concentrations of TGFβ2. As shown in the images, higher opacity (less light transmission) appears darker. After seven days of treatment, the opacity of the lens increases with the concentration of TGFβ2 (FIG.3A). [0143] To investigate whether GDF-15 pretreatment could alleviate the effects of TGFβ2 on EMT, extracted mouse lenses were pretreated with PBS or GDF-15 for 1 hour before the addition of 10 ng/mL TGFβ2 treatment and cultured for seven days. TGFβ2 treatment induced lens opacity, while GDF-15 pretreatment reduced this effect (FIG.3B). In addition, qPCR revealed elevated expression of Acta2, Fn1, and Vim (Kalluri et al., J Clin Invest 119:1420-1428, 2009; Dongre et al., Nature Reviews Molecular Cell Biology 20:69-84, 2019) in lenses treated with TGFβ2 compared to PBS controls, and GDF-15 pretreatment significantly inhibited this effect (Fig.3C). These results suggest that GDF-15 could alleviate the effects of TGFβ2-induced EMT and opacity in the lens. [0144] Currently, the main treatment for cataracts is surgical removal of the opaque lens and replacement with a synthetic lens (Thompson et al., Prim Care 42:409-423, 2015). However, complications may occur after surgery, most commonly PCO (Wang et al., Investigative Ophthalmology & Visual Science 54:323-332, 2013; Ma et al., Biochem Biophys Res Commun 447:689-695, 2014). The standard treatment for PCO is Nd:YAG laser capsulotomy (Karahan et al., Med Hypothesis Discov Innov Ophthalmol 3:45-50, 2014), a safe and effective treatment (Liu et al., J Cataract Refract Surg 48:238-244, 2022). Nonetheless, many complications can still occur after laser capsulotomy, such as retinal edema and retinal detachment (Keates et al., J Am Intraocul Implant Soc 10:164-168, 1984). Development of novel therapeutics to treat or prevent PCO could further avoid the complications of laser capsulotomy. It is documented herein that GDF-15 inhibits Smad2 phosphorylation induced by TGFβ2 and thus alleviates TGFβ2-induced EMT in LECs, potentially
SAS:clf 8123-111807-02 04/15/25 06621 slowing progression of PCO (FIG.4). Accurate differentiation and stable functionality of RPE and retinal progenitor cells (RPC) are crucial for retinal development and the treatment of ocular diseases (Stern et al., Cell Stem Cell 22:834-849, 2018). EMT plays a pivotal role in neural differentiation. Neural stem/progenitor cells undergo EMT, characterized by the loss of epithelial traits, cell instability, and increased migration and invasion. For instance, alterations in epithelial polarity by neural stem/progenitor cells can lead to impaired cell polarity, contributing to neurodevelopmental disorders (Arai et al., Cell Stem Cell 22:834-849, 2018). RPE dysfunction is associated with retinal diseases, presenting as a loss of RPE barrier function and disrupted RPE polarization. In several degenerative retinal diseases such as proliferative vitreoretinopathy (PVR) (Zhou et al., Front Cell Dev Biol 8:501, 2020), RPE cells cease differentiation, initiate EMT, and exhibit enhanced migratory abilities. Persistent inflammation and wound healing occur after retinal injury in PVR. RPE cells contribute to the healing process by undergoing EMT and migrating to the epiretinal area. The subsequent formation and contraction of PVR membranes resulted in retinal folds and vision loss. Thus, GDF-15 is involved in NPC differentiation and can be a therapeutic agent for PVR. However, it was not clear, prior to the present studies, that GDF-15 would be effective for achieving EMT in the lens or lens epithelial cells. [0145] GDF-15, also known as macrophage inhibitory cytokine-1 (MIC-1), is a member of the TGFβ superfamily and plays a role in development, differentiation, and repair in the human body (Xue et al., Brain Behav 12:e2502, 2022). Some studies show that GDF-15 is involved in development and progression of diseases such as diabetes, coronary heart disease, and cancer (Yao et al., Neurodegenerative Diseases 17:251-260, 2017). Other studies show that GDF-15 is widely distributed in the nervous system, can be positively associated with stress response, and increases neurogenesis (Xue et al., Brain Behav 12:e2502, 2022; Kim et al., Stem Cells Dev 24:2378-2390, 2015). GDF-15 also is reported to help promote retinal ganglion cell differentiation (Chang et al., Curr Biol 29:1963-1975 e1965, 2019). [0146] GDF-15 was previously shown to be involved in proliferative vitreoretinopathy, it was uncertain if it would have any effect on cataract development. Indeed, dexamethasone is effective to treating proliferative vitreoretinopathy (see Trenado-Luna et al., Cirugía y Cirujanos 2023;91(5): 664- 671) but exposure to dexamethasone increases the incidence of cataracts in these patients (Banerjee et al., Am J Hematol.2024;99: E12–E14). For at least this reason, the ability of GDF-15 to inhibit the formation of cataracts was unexpected. The studies presented herein show that GDF-15 can be a therapeutic agent for PCO. In addition, it was demonstrated that GDF-15 attenuated TGFβ2-induced EMT by blocking Smad2 phosphorylation and slowing progression of lens opacity. Topical administration of other agents has been demonstrated to reduce PCO in animals (Lee et al., Vet. Ophthalmol.2024;27:170–176). Thus, similar topical formulations could be used for the delivery of GDF-15.
SAS:clf 8123-111807-02 04/15/25 06621 [0147] In view of the many possible aspects to which the principles of the disclosed invention may be applied, it should be recognized that the illustrated aspects are only preferred examples of the invention and should not be taken as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.