Polysialic acid-polymer conjugates and nanoparticles

Abstract

Disclosed herein are polysialic acid (PSA)-polymer conjugate compounds represented by structural formula (I): (wherein P is poly(lactide-co-glycolide)-poly(ethylene glycol) copolymer (PLGA-PEG), and p is an integer between 4 and 200), or pharmaceutically acceptable salts thereof, nanoparticles containing the same, and methods for treating ophthalmic diseases using the same.

Description

[Technical Field]

[0001] Related applications This application claims the benefits of U.S. Provisional Patent Application No. 63 / 459,486 filed on 14 April 2023, U.S. Provisional Patent Application No. 63 / 529,059 filed on 26 July 2023, U.S. Provisional Patent Application No. 63 / 542,147 filed on 3 October 2023, and U.S. Provisional Patent Application No. 63 / 546,950 filed on 2 November 2023. The entire contents of each of the aforementioned applications are incorporated herein by reference. [Background technology]

[0002] The ability to recognize oneself is a mechanism that downregulates the body's immune system to prevent the destruction of its own healthy host cells. The composition of a particular cell's glycome (the carbohydrate portion that covers all cells) determines whether the cell is recognized as self-related, non-self, or damaged. In the processes of immune surveillance and inflammation, the immune system checks the glycome signature of the cells it encounters to determine whether the cell requires elimination through immune activation, or whether the cell constitutes a non-damaged host cell that should signal immune activation or suppression of inflammation. The receptors or binding regions present on inflammatory cells that are responsible for recognizing this glycome signature are considered self-related pattern recognition receptors.

[0003] The largest family of self-associated molecular pattern recognition receptors is called siglecs (sialic acid-binding immunoglobulin lectins). Currently, 16 siglecs have been described. Siglecs are present on the surface of inflammatory cells and have various siglec expression patterns found on different inflammatory cells. When a specific sialic acid ligand pattern is presented on the surface of healthy host cells, the stimulated inhibitory siglec receptor activates an immunoglobulin tyrosine kinase inhibitory motif (ITIM), which recruits src homology 2-domain-containing protein tyrosine phosphatases 1 and 2 (SHP-1 and 2). Both of these phosphatases dephosphorylate kinases that keep inflammatory cells activated. This inhibitory siglec regulatory mechanism can lead to the resolution of inflammation by potently shutting down activated inflammatory cells. Different siglecs have various sialic acid signatures that bind to and stimulate their receptors, resulting in potent inactivation of inflammatory cells. [Overview of the project] [Problems that the invention aims to solve]

[0004] Improved compositions and methods for modulating, for example, stimulating sialic acid-binding self-associated pattern recognition receptors are needed to treat diseases resulting from acute, chronic, or abnormal immune system activation. [Means for solving the problem]

[0005] In one embodiment, the present invention relates to structural formula (I): [ka] A polysialic acid (PSA)-polymer conjugate compound represented by formula (I) or a pharmaceutically acceptable salt thereof. In formula (I), P is poly(lactide-co-glycolide)-poly(ethylene glycol) copolymer (PLGA-PEG), and p is an integer between 4 and 200.

[0006] In another embodiment, the present invention relates to structural formula (IV): [Chemical formula] It is a compound represented by or a pharmaceutically acceptable salt thereof. In formula (IV), p is an integer from 17 to 200.

[0007] In another embodiment, the present invention is a method of treating a subject suffering from an ophthalmic disease, comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., a compound of formula I or formula II), a particle described herein, or a pharmaceutical composition described herein.

[0008] In another embodiment, the present invention is a method of increasing the best corrected visual acuity (BCVA) score in a subject that requires it, comprising administering to the subject a therapeutically effective amount of a compound described herein (e.g., a compound of formula I or formula II), a particle described herein, or a pharmaceutical composition described herein.

[0009] In another embodiment, the present invention relates to the use of a compound of formula I or formula II or a pharmaceutically acceptable salt thereof or a particle or pharmaceutical composition described herein for the manufacture of a medicament for treating an ophthalmic disease in a subject suffering therefrom.

[0010] In another embodiment, the present invention relates to a compound of formula I or formula II and their pharmaceutically acceptable salts or a particle or pharmaceutical composition described herein for use in a method of treating a subject suffering from an ophthalmic disease.

[0011] In another embodiment, the present invention is the following structural formula (I): [Chemical formula] A method for preparing a poly sialic acid (PSA)-polymer conjugate compound represented by or a pharmaceutically acceptable salt thereof, comprising structural formula (III) [Chemical formula] The polymer represented by structural formula (IV) [ka] The method involves reacting a PSA precursor represented by with conditions sufficient to form a compound represented by structural formula (I). P is poly(lactide-co-glycolide)-poly(ethylene glycol) block copolymer (PLGA-PEG), and p is an integer between 4 and 200.

[0012] The above will become clear from the following more specific description of embodiments of the present invention, as shown in the attached drawings, and similar reference symbols refer to the same elements throughout the different drawings. The drawings are not necessarily to scale and are instead focused on illustrating embodiments of the present invention. [Brief explanation of the drawing]

[0013] [Figure 1A] This is a schematic diagram of non-equivalent hydrogen present in PSA amine. [Figure 1B] Table 1 shows the 1H NMR (400 MHz, D2O) peak assignments for the structure shown in Figure 1A. [Figure 1C] This is a schematic diagram of non-equivalent carbons present in PSA amine. [Figure 1D] Table 2 shows the peak assignments for 13C NMR (400 MHz, D2O-d6). [Figure 1E] This plot shows the LCMS Q-TOF deconvolution mass of PSA-amine. [Figure 2A-2E] These are reproductions of Tables 3, 4, and 4A-4C, respectively, and show a list of PSA-amine batch analysis results. [Figure 3] Reproduce Table 5, which describes this product. [Figure 4A-4C] Tables 6-8, which show the effects of homogenization pressure, time, and storage temperature on particle size distribution, are reproduced. [Figure 5] Table 9 shows a summary of the sample batch of this product. [Figure 6] Table 11 summarizes the results of the temperature and photostability tests. [Figure 7] This shows typical IR spectra of pharmaceuticals. [Figure 8] A typical chromatogram used to measure the PSA-amine content in the formulation is shown. [Figure 9] A typical histogram of the product's particle size distribution is shown. [Figure 10] A typical histogram of the zeta potential distribution of the product is shown. [Figure 11A-11D] Tables 14A to 14D summarize the batch analysis results of this product for intravitreal injection. [Figure 12A-12C] Tables 15, 16, and 17 summarize the results of the long-term stability tests. [Figures 13A-13B] This plot shows the gene transcripts (A) and protein expression levels (B) of Siglec-7, -9, and -11 in human eyes with exudative and non-exudative age-related macular degeneration compared to a healthy eye. [Figure 14] This is a plot of optical concentration as a function of total solids concentration, showing the binding affinity of AVD-104 to Siglec 7, 9, and 11. [Figures 15A-15B] This is a bar graph showing the levels of identified cytokines in the supernatant. [Figure 16A-16C] This is a bar graph showing the levels of identified cytokines in the supernatant. [Figure 17] This bar graph, measured by the CH50 assay, shows that this product reduces complement activity in the classical pathway. [Figure 18] This bar graph, measured by the AH50 assay, shows that this product reduced the activity of the alternative pathway. [Figures 19A-19B] This bar graph shows the effect of this product on retinal degeneration, as evaluated by optical coherence tomography (OCT). [Figure 20] This bar graph shows the dose-dependent suppression of TNF-α in the RPE / choroid by this product. [Figure 21]This bar graph shows the size of lesions as evaluated by fluorescein angiography. [Figure 22] This bar graph shows lesion size as evaluated by immunohistochemical staining for isolectin-B4. [Figure 23] This is a plot of the mean PEG concentration-time profile in plasma and tissue. [Figure 24A] This bar graph shows the effect of this product on intact C3 protein. [Figure 24B] This bar graph shows the effects of this product on stabilizing factors (CFD and CFP) of the complement alternative pathway. [Figure 24C] This bar graph shows the effects of this product on inhibitors of alternative complement pathways. [Figure 25] The map of the HITSIGREC 11 targeting vector is shown. [Figure 26] This plot shows the transcriptome analysis results of changes in Siglec RNA expression in AMD eyes. [Figure 27] This bar graph shows the results of administering this product to IL-12 levels in the supernatant of human M1 macrophages. [Figures 28A-28C] This bar graph shows the effects of administering this product on the C3a, C5a, and sC5b-9 levels in the supernatant of human M1 macrophages. [Figures 29A-29B] This bar graph shows the effect of administering this product on the levels of Ba and Bb in the supernatant of human M1 macrophages. [Figure 30] This bar graph shows the results of an assay evaluating the inhibitory effect of AVD-104 described herein on the binding of Siglec-7Fc and Siglec-9Fc proteins to sialic acid-expressing Panc-1 cells. [Figure 31A] This plot shows the cytotoxic effect of AVD-104 on macrophages activated by either LPS or oxidized (Ox)LDL, using an MTT cell viability assay. [Figure 31B]This bar graph shows the dose-dependent inhibition of TNF-α production in macrophages treated with either OxLDL or LPS by AVD-104. [Figure 32] The test design for NCT05839041 is represented as a graph. [Figure 33] The table below summarizes the interim results of the clinical trial of AVD-104 in patients with geographic atrophy. [Figure 34] This plot shows the hyper-AF area at the anterior edge of the lesion over a one-month period in two GA patients treated with AVD-104 for one month. [Figures 35A-35B] The plots of hyper-AF area and BCVA score for representative patient case studies are shown. [Figures 36A-36D] For representative patient case studies, plots showing the levels of IL-6, complement C3a and C4a, and complement factor H (CFH) over time are presented. [Figure 37] This document presents the design of a Phase 2 / 3 Part 2 US clinical trial for GA secondary to AMD. [Figures 38A-38B] The percentage change in lesion area from baseline (A) and the mean change in lesion area from baseline (B) are shown. [Figure 39-40] The mean change in lesion area as a function of time for cohorts 3 and 4 is shown, and this is plotted against similar data from clinical trials, including SoC. [Figure 41] This plot shows the percentage change in hyperautofluorescence for all patients (pooled cohort). [Figure 42] This plot shows the CVA score (mean change from baseline) for the entire cohort. [Figure 43] This bar graph shows the percentage of patients (all cohorts) classified by a decrease or increase in their BCVA score. [Figure 44A-44B] This bar graph shows the percentage of patients (cohorts 3 and 4) classified by a decrease or increase in their BCVA score. [Figures 45A-45B]This plot shows the mean change in BCVA as a function of time in cohorts 3 and 4, compared to Syfovre PM and lampalizumab (in the Chroma&Spectri-4qw and Chroma&Spectri-6qw trials). [Figure 46] This bar graph shows the change in BCVA score at 3 months for each patient in each cohort. [Figure 47] This bar graph demonstrates that human macrophages treated with AVD-104 showed significant TNF-α suppression compared to PSA ligand alone. [Figure 48] This bar graph demonstrates that human macrophages treated with AVD-104 having 20 or more DPs showed significantly greater TNF-α inhibition than nanoparticle compositions with lower PSA DPs. [Figure 49] This is the dose-response curve for TNF-α suppression in PMBC-derived macrophages by AVD-104. [Modes for carrying out the invention]

[0014] Examples of embodiments of the present invention will be described below.

[0015] As used herein, “sialic acid” refers to a monosaccharide, and “polysialic acid (PSA)” refers to a polysaccharide derivative of sialic acid that is a cognate of at least one sialic acid receptor. Sialic acid refers to either naturally occurring or synthetically obtained neuraminic acid or any chemical modification of neuraminic acid. The structural formula of neuraminic acid is reproduced below. [ka]

[0016] An example of a sialic acid derivative is the following structural formula: [ka] N-acetylneuraminic acid (Neu5Ac), represented by the following structural formula: [ka] One example is N-glycolylneuraminic acid (Neu5Gc), represented by [the formula shown].

[0017] As used herein, a carbohydrate residue is a monosaccharide modified at one or more positions for covalent bonding.

[0018] As used herein, “infectious pathogen” refers to a viral, bacterial, or parasitic pathogen, and the receptor may be a capsid, membrane, or nuclear glycan-binding molecule / protein / enzyme (lectin) such as hemagglutinin esterase, coronavirus spike protein, or viral neuraminidase / sialidase.

[0019] As used herein, “average cross-sectional width” refers to the widest portion of a non-spherical nanoparticle averaged across the entire aggregate of particles.

[0020] As used herein, the term “particles” includes fine particles and nanoparticles as defined herein.

[0021] This disclosure provides therapeutic agents containing polysialic acid for use as immune system modulators, i.e., immune system suppressors or activators, inhibitors of viral / bacterial / parasitic infectivity, and demasking of damage-associated molecular patterns (DAMPs) to enhance immune surveillance. Target cell populations include those expressing Siglec receptors, CFH CCP4-6, 19-20, viral HE, viral N, viral SP, and CD147. In specific embodiments, the delivery vehicle comprises a polymer formulated as nanoparticles or microparticles and anchored (conjugated or linked) to ligands containing PSA and derivatives thereof for presentation on the surface of the nanoparticles. The anchored PSA functions as a ligand for targeted binding of the nanoparticles to receptors such as Siglec receptors expressed on the surface of target cells.

[0022] In one embodiment, the disclosure provides nanoparticles comprising a polymer that achieves anchoring via covalent chemical coupling to PSA or a derivative thereof for presentation on the surface of the nanoparticles. To modulate inflammatory processes, nanoparticles can be used to contact immune cells expressing sialic acid-binding immunoglobulin-like lectins (Sigrec). It has been determined that inflammatory responses in target cells and associated environments can be modulated using PSA that can target and bind to immune cells expressing sialic acid-binding immunoglobulin-like lectins (Sigrec). Siglecs are members of the self-associated pattern recognition family of receptors and include Siglec isotypes that exhibit selectivity on different cell populations. Therefore, the ability to design nanoparticles that selectively bind to specific Siglec receptors enables targeted binding to desired cell populations of interest. Such binding of Siglec receptors to nanoparticles can be used as a means to modulate the signaling activity of Siglec receptors within cells of interest, thereby resulting in a reduction of inflammatory responses or an enhancement of anti-inflammatory responses in therapeutic targets.

[0023] The presentation of PSA on the surface of nanoparticles means that the PSA can be bound by Siglec receptors of target cells or organisms. Preferably, the PSA can be provided to bind to the receptor and activate or inhibit it. While we do not wish to be bound by theory, in order to present PSA on nanoparticles to modulate inflammatory responses, enhance immune surveillance, or inhibit infectivity, the PSA needs to be presented at a specific concentration density.

[0024] Single nanoparticles can be modified with a multivalent complex of PSA, enabling multivalent binding of this single nanoparticle to different Siglec receptors, thereby regulating the inflammatory response. Nanoparticles modified with a unique type of PSA moiety can also be mixed with other ligand-modified nanoparticles that can target different Siglec receptors, which also allows for desired regulation of the inflammatory response. In some embodiments, presentation of PSA on the surface of nanoparticles or microparticles can result in an increase in cellular uptake of the particles by at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, or at least about 10-fold. In some embodiments, presentation of PSA on the surface of nanoparticles or microparticles can reduce the inflammatory response. In non-limiting embodiments, the reduction in the inflammatory response is more than about 2-fold, more than about 3-fold, more than about 4-fold, more than about 5-fold, more than about 10-fold, more than about 20-fold, more than about 50-fold, more than about 100-fold, or more than about 1000-fold.

[0025] Nanoparticles or microparticles can be used for systemic or local delivery to targeted affected tissues in subjects requiring treatment, thereby modulating the inflammatory response in said subjects to dissipate innate and adaptive inflammation, activate innate and / or adaptive immunity if enhanced immune surveillance is desired, or reduce the infectivity of infectious organisms. Targeted immune cells or viruses must possess a Siglec receptor or a viral sialic acid ligand binding domain, respectively. Innate immune system activity includes, for example, the cellular response of the innate immune system, the innate immune system, the complement system, the surrogate complement pathway, the amplification loop of the surrogate complement pathway, and / or the noncellular / humoral response of the amplification loop of the surrogate complement pathway activated by complement factor H. Adaptive immune system activity includes dendritic cell maturation and presentation to T cells, T cell activation, T cell regulation, T cell checkpoint inhibition or activation, neutrophil NETosis, and B cell activation. Reduction of infectivity includes reduced viral entry into host cells, reduced proliferation of viral particles, or reduced inflammatory response to viral infection.

[0026] As used herein, “subject” refers to a subject receiving treatment according to the treatment method provided. A subject may be a human, primate, dog, cat, cattle, horse, mouse, etc. A subject also refers to an animal used in laboratory tests.

[0027] As used herein, “nanoparticles” refers to particles composed of one or more polymers, whose size in nanometers (nm) includes a linear dimension range of 10 nanometers to 2000 nanometers. As used herein, “linear dimension” refers to the distance between any two points on the surface of a nanoparticle measured in a straight line. The nanoparticles of this disclosure may be irregular, oblong, spindle-shaped, rod-shaped, cylindrical, pancake-shaped, disc-shaped, spherical, biconcave, or erythrocyte-shaped. Linear dimensions can be measured using several methods, including, but are not limited to, transmission electron microscopy or adjustable resistance pulse sensing, which are part of the standard means of measuring nanoparticle size. One widely used technique for measuring nanoparticle size is dynamic light scattering (DLS), which can confer the diameter and polydispersity of nanoparticles. DLS assumes that nanoparticles are essentially spherical and that the size of the nanoparticle is the average diameter (or radius) of such assumed spheres. In such measurements, nanoparticles may be described as having a size range of 10 nm to 1000 nm or 1 nm to 500 nm.

[0028] As used herein, “fine particles” refers to minute particles composed of one or more polymers, with a size in micrometers (μm) of less than 1000 μm and a maximum cross-sectional width of 1 μm or more.

[0029] Several types and configurations of nanoparticles are included in this disclosure. For example, nanoparticles may consist of a variety of materials, including, but not limited to, biodegradable polymers, biocompatible polymers, bioabsorbable polymers, or combinations thereof. Biocompatibility refers to polymers that do not unnecessarily interfere with the biological function of tissues. The terms biodegradable, bioabsorbable, and biodegradable, as well as degradation, erosion, and absorption, are used interchangeably (unless the context indicates otherwise) and refer to polymers and metals that can be broken down or absorbed when exposed to bodily fluids such as blood and enzymes and their components, and that can be gradually reabsorbed, absorbed, and / or eliminated by the body.

[0030] The polymer backbone of the nanoparticles may consist of naturally occurring polymers such as carbohydrates or proteins, or synthetic polymers, when sialic acid ligands are linked to it. The polymer backbone has unique terminal functional groups that allow for the anchoring of sialic acid ligands to the surface of the nanoparticles. The polymer backbone may be initially linked to multiple sialic acid ligands before the nanoparticles are formed by chemical bonding, or the functional groups displayed on the surface of the nanoparticles may be initially linked to the sialic acid ligands by chemical bonding after the polymer backbone has been formed into the nanoparticles.

[0031] Suitable nanoparticles include polymer particles and hydrogel particles. As used herein, “polymer” refers to a molecule composed of multiple repeating structural units linked by covalent bonds. As used herein, “polymer particles” refers to solid or porous particles, in contrast to the shell-like structure of liposomes and polymerosomes and the relatively open structure of hydrogel particles. As used herein, “hydrogel particles” refers to a cross-linked network of polymer chains that is absorbent in an aqueous environment but is stable.

[0032] Polymers that may be used to prepare nanoparticles include, but are not limited to, poly(N-acetylglucosamine)(chitin), chitosan, poly(3-hydroxyvalerate), poly(D,L-lactide-co-glycolide), poly(1-lactide-co-glycolide), poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxybutyrate-co-3-hydroxyvalerate), polyorthoester, poly Poly(L-Lactide), poly(glycolic acid), poly(glycolide), poly(L-lactic acid), poly(L-lactide), poly(D,L-lactic acid), poly(D,L-lactide)-b-poly(ethylene glycol)-azide, poly(D,L-lactide)-b-poly(ethylene glycol)-methyltetrazine, poly(D,L-lactide), poly(L-lactide-co-D,L-lactide), poly(D,L-lactide)-b-poly(ethylene glycol)-b-poly(ethylene glycol) )-carboxylic acid, poly(ethylene glycol) methyl ether-block-poly(lactide-co-glycolide), poly(lactide-co-glycolide)-b-poly(ethylene glycol)-b-poly(lactide-co-glycolide), poly(lactide-co-glycolide)-b-poly(ethylene glycol)-azide, poly(lactide-co-glycolide)-b-poly(ethylene glycol)-alkyne, poly((D,L)lactic acid)-b-poly(ethylene glycol)- Azide, poly((D,L)lactic acid)-b-poly(ethylene glycol)-alkyne, poly(caprolactone), poly(caprolactone)-b-poly(ethylene glycol), polycaprolactone-b-poly(ethylene glycol), poly(lactide-co-caprolactone)-b-poly(ethylene glycol)-b-poly(lactide-co-caprolactone), poly(L-lactide-co-caprolactone), poly(L-lactide-co-caprolactone), poly(D,L-lactide-cocaprolactone), poly(glycolide-cocaprolactone), poly(DL-lactide)-b-poly(ethylene glycol)-b-poly(DL-lactide), poly(trimethylene carbonate), polyesteramide, poly(glycolic acid-co-trimethylene carbonate), acrylate-poly(caprolactone)-b-poly(ethylene glycol)-alkyne, co-poly(ether ester) (e.g., PEO / PLA), poly(N-isopropylacrylamide-co-acrylic acid), poly(N-isopropylacrylamide-co-methoxypoly(ethylene glycol) methacrylate), polyphosphazene, biomolecules (fibrin, fibrin glue, fibrinogen, cellulose, starch, collagen and hyaluronic acid, elastin and hyaluronic acid, etc.), polyurethane, silicone, polyester, polyolefin, polyisobutylene and ethylene-alphaolefin copolymer, acrylics other than polyacrylate Polypolymers and copolymers, vinyl halogenated polymers and copolymers (such as polyvinyl chloride), polyvinyl ethers (such as polyvinyl methyl ether), polyvinylidene halogenated polymers (such as polyvinylidene chloride), poly(vinylidene fluoride), poly(vinylidene fluoride-co-hexafluoropropylene), polyacrylonitrile, polyvinyl ketone, polyvinyl aromatic compounds (such as polystyrene), polyvinyl esters (such as polyvinyl acetate), acrylonitrile-styrene copolymer, ABS resin, polyamide (such as nylon 66 and polycaprolactam), polycarbonates such as tyrosine-based polycarbonates, polyoxymethylene, polyimide, polyether, polyurethane, rayon, rayon triacetate, cellulose, cellulose acetate, cellulose butyrate, cellulose acetate butyrate, cellophane, cellulose nitrate, cellulose propionate, cellulose ether, carboxymethylcellulose, and fullerene.

[0033] In one embodiment, the nanoparticles are formed from the biodegradable polymer polycaprolactone, and in other embodiments, they are formed from polymers including polyglycolic acid, poly(L-lactic acid), poly(lactic acid-coglycolic acid), polycaprolactone, and poly(3-hydroxybutyric acid). In some embodiments, the nanoparticles may be polymer particles, in particular, the particles may be formed from biodegradable polyesters such as poly(lactide) (PLA), poly(glycolide) (PGA), polylactic acid-10-glycolic acid (PLGA), poly(butylcyanoacrylate) (PBCA), or N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. In another embodiment, the nanoparticles are formed from polymers such as poly(ethylene glycol), polyethylene oxide, Pluronic F127, Pluronic F68, poloxamer, poly(hydroxymethyl methacrylate), polyvinyl alcohol, and poly(vinylpyrrolidone).

[0034] In certain embodiments, the nanoparticles are formed from poly(lactic acid-co-glycolide)-poly(ethylene glycol) copolymer (PLGA-PEG), for example, (lactide-co-glycolide)block-poly(ethylene glycol). In one embodiment, the polymer is PLGA(10k)-PEG(5k).

[0035] Methods for synthesizing nanoparticles are well known to those skilled in the art (see, for example, Spence et al., Science Translational Medicine, 2015, 7:303 303ra140 and the references cited therein). For example, methods for synthesizing nanoparticles with known degradation rates are known to those skilled in the art and are described in U.S. Patent No. 6,451,338 by Gregoriadis et al., U.S. Patent No. 6,168,804 by Samuel et al., and U.S. Patent No. 6,258,378 by Schneider et al., which are incorporated herein by reference in their entirety.

[0036] Formulated nanoparticles or microparticles anchored to PSA are provided for use in selective binding to receptors expressed on target cells of interest. The term sialic acid refers to any monosialic acid, and the term PSA refers to any oligomeric sialic acid or polymeric sialic acid or polysialic acid, including disialic acid capable of binding to Siglek receptors, particularly sialic acid having binding specificity to inhibitory Siglek receptors such as Siglek 7. In some embodiments, the PSA for use in the compositions or methods disclosed herein may be any group of aminocarbohydrates that are components of mucoproteins and glycoproteins in animal tissues and blood cells. In some embodiments, sialic acid (also known as nonulosonic acid) is a member of the family of amino-containing sugars containing nine or more carbon atoms, such as N-acetylneuraminic acid (also known as 5-(acetylamino)-3,5-dideoxy-D-glycero-DD-galactononulosonic acid, lactamic acid, and O-sialic acid).

[0037] In several embodiments, the PSA is typically considered to be linked in an α-configuration of 2→8 and / or 2→9 and / or 2→6 and / or 2→3. In several embodiments, the PSA is anchored to the surface of nanoparticles or fine particles. The PSA is a homopolymer consisting of multiple sialic acid units. The PSA has lengths of less than 5 sialic acid units, less than 4 sialic acid units, less than 3 sialic acid units, and 2 sialic acid units. In several embodiments, the degree of polymerization (DP) can be in the range of DP2 to greater than DP250, for example, DP2 to DP200. The DP can be DP2 to DP100, DP2 to 90, DP2 to DP80, DP2 to DP70, DP2 to DP60, DP2 to DP50, DP2 to DP40, DP2 to DP30, DP2 to DP30, DP2 to DP20, and DP2 to DP10. In certain non-limiting embodiments, the degree of polymerization is DP3 to DP100. In alternative embodiments, the PSA may contain five or more sialic acid units. For example, polysialic acid may contain at least six sialic acid units, at least seven sialic acid units, or at least eight sialic acids. In several embodiments, the degree of polymerization (DP) may be in the range of DP5 to DP1000. For example, the degree of polymerization may be DP5 to DP500, DP5 to DP100, DP5 to DP90, DP5 to DP80, DP5 to DP70, DP5 to DP60, DP5 to DP50, DP5 to DP40, DP5 to DP30, DP5 to DP20, or DP15 to DP25. In certain non-limiting embodiments, the degree of polymerization is DP10 to DP400, DP20 to DP300, or DP30 to DP200. In certain embodiments, DP is DP5 to DP30, for example, DP5, DP10, DP15, DP20, DP25, DP30, DP35, DP40, DP45, or DP50. In certain embodiments, DP is DP5 to DP500. In example embodiments, DP is DP10 to DP30. In some embodiments, DP is DP20.

[0038] In certain embodiments, 1 The DP of PSA measured by 1H-NMR is approximately 17 to 60. For example, the DP is approximately 17 to 60, for instance, approximately 22 to 60. In some examples, the DP was approximately 22.

[0039] In certain embodiments, 1 The DP of PSA measured by 1H-NMR is approximately 17 to 60. For example, the DP can be approximately 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30.

[0040] In a particular embodiment, the DP of the PSA is selected from one of the following: 17-100, 17-60, 17-25, 20-100, 20-60, 20-30, 22-100, 22-60, 22-30, and 22-25.

[0041] In embodiments where sialic acid analogs are used, these analogs may have structural similarity to the sialic acid disclosed herein and possess binding affinity to specific Siglek receptors. Suitable analogs will likely be known in the art. The features that influence the binding of sialic acid ligands to Siglek receptors are thought to be the charge-distance-coordination relationships between the carboxylic acid functional groups of sialic acid.

[0042] In specific embodiments, PSA is NeuAcα2-3Galβ1-4Glc, NeuAcα2-3Galβ1-4GlcNAc, NeuAcα2-3Galβ1-3GlcNAc, NeuAcα2-3Galβ1-3GalNAc, NeuGcα2-3Galβ1-4GlcNAc, NeuGcα2-3Galβ1-3GlcNAc, NeuAcα2-6Galβ1-4Glc, NeuAcα2-6GalNAc, Galβ1-3(NeuAcα2-6)GalNAc, NeuGca2-6Galβ1-4Glc, NeuGcα2-6Galβ1-4GlcNAc, NeuG Examples include cα2-6GalNAc, NeuAcα2-8NeuAcα2-3Galβ1-4Glc, NeuAcα2-6Galβ1-4GlcNAc, NeuAcα2-3Galβ1-4[Fucα1-3]GlcNAc, NeuAcα2-6Galβ1-4GlcNAc6S, NeuAcα2-3Galβ1-4GalNAc, NeuAcα2-8NeuAc, NeuAcα2-3GalβSβ1-4GlcNAcα2-3Fuc, NeuAcα2-3Galβ1-4GlcNAc6Sα2-3Fuc, and NeuAcα2-8NeuAc or sialoside derivatives of such sialic acids, such as BPCNeuAc sialoside.

[0043] In certain embodiments, PSA may first be bonded to a polymer backbone by chemical bonding techniques, and then polymer-conjugated PSA constructs may be formed on the surface of nanoparticles. In other embodiments, the polymer may be formed on nanoparticles having functional groups exposed on the nanoparticle surface, thereby allowing these functional groups to be bonded to PSA.

[0044] PSAs, including oligomers and polymers, can be linked to each other by any combination of α2-3, α2-6, α2-8, or α2-9 glycosidic linkages. By adjusting the type of glycosidic linkage that links sialic acid or sialic acid analogs to the nanoparticle surface, binding affinity to the target Siglek receptor can be maximized, increasing specificity for a particular Siglek. Since different Sigleks are known to be expressed differentially by different cell types, the selection of a specific type of sialic acid linkage can be used to determine the type of cell that is contacted or targeted by the nanoparticles and sialic acid ligand. PSAs in oligomer and polymer forms can have linear or branched structures. Branched structures in oligomer or polymer forms can be formed by introducing different glycosidic links between adjacent glycosidic links. Oligomer and polymer forms can have a homogeneous composition composed of one type of sialic acid, or a heterogeneous composition composed of multiple sialic acids. The oligomer and polymer forms may also consist of other carbohydrate monomers such as galactose, N-acetylgalactoseamine, glucose, N-acetylglucoseamine, mannose, N-acetylmannosamine, fucose, or other sugars / carbohydrates, in addition to sialic acid and / or sialic acid analogs.

[0045] Sialic acid may be of natural origin (e.g., Neu5Ac, Neu5Gc, Neu5Ac9Ac, etc.) or may include sialic acid analogs prepared by any synthesis. Sialic acid analogs are known in the art. In some embodiments, such analogs may have a substitution at position C9. Analogs may also have substitutions at C1, C4, C5, C7, and C8. Analogs may include neuraminic acid derivatives, sialosides, and carbohydrate oligomers containing neuraminic acid molecules.

[0046] Sialic acid analogs can be prepared by mammalian or bacterial cell synthesis, recombinant methods, or genetic engineering methods, such as chemical synthesis, chemoenzyme synthesis (e.g., one-pot multiplex enzyme, OPME), or by cellular supply of precursor carbohydrates (e.g., mannose derivatives). PSA containing sialic acid analogs prepared for use as nanoparticle ligands can be prepared using one-pot synthesis or a microarray platform. Arrays of sialic acid analogs can be prepared in situ using the HTS method.

[0047] Chemical binding of sialic acid ligands to the surface of nanoparticles can be achieved through a variety of chemical reactions. In such reactions, a chemical reaction occurs between the terminal functional groups of the nanoparticle polymer and the terminal functional groups of PSA (referred herein to as "terminal functional group conjugate pairs"), resulting in the binding of the polymer to the PSA. The type of terminal functional groups present on the polymer surface and their binding partner ligands determine the type of chemical reaction used to chemically bind the PSA to the surface of the nanoparticles. Furthermore, by utilizing the selection of polymers with specific terminal functional groups, the type, density, and spatial arrangement of PSA conjugate partners to be presented on the surface of the nanoparticles can be controlled.

[0048] For example, the surface of the nanoparticles provides a position for the binding of PSA. In one example, the nanoparticles are formed from a PLGA-PEG polymer and PLGA-PEG-NHS (N-hydroxysuccinimide) having an ester (e.g., activated ester) moiety. In non-limiting embodiments, blends of various polymers having different terminal functional groups can be used. Such polymers include, for example, PLGA-PEG-alkynes, PLGA-PEG-esters, and PLGA-PEG-DBCO. In certain embodiments, a blend of PLGA-PEG-ester and PLGA-PEG-carboxylic acid can be prepared as nanoparticles. In another particular embodiment, PLGA-PEG-ester can be prepared as nanoparticles. In the examples of embodiments, the use of PLGA-PEG-NHS as the conjugation site to the PSA-amine is preferred over the use of other bindings (e.g., DBCO).

[0049] Preferably, the polymer or copolymer may be branched or linear and may have multiple terminal functional groups.

[0050] In other embodiments, the PSA includes terminal functional groups (i.e., conjugated sites) that achieve anchoring to the nanoparticle surface. Such terminal functional groups include azides, alkynes, aryl esters, amides, amines, arylamides, aldehydes, acetyls, substituted aryl esters, alkyl esters, alkyl ketones, aryl ketones, substituted aryl ketones, ketones, alkyl halides, amnioxys, alcohols, azailides, carboxylic acids, esters, amides, bicyclononones, dihydrazides, halocarbonyls, halosulfonyls, hydrazides, N-hydroxysuccinimide, norbornene, oxanorbornadiene, succinimidyl esters, and isothiocyanates. Examples include iodoacetamide, monofluorinated and difluorinated cyclooctin, maleimide, methylcyclopropene, isocyanopropanoate, hydrazine, nitrile, nitro, phosphine, phosphazide, tertadine, methyltetrazine, transcyclooctene, strain alkyne, dibenzocyclooctin, biarylazacyclooctinone, propargyl, isocyanide, azadibenzylcyclooctin, vinyl, sulfonyl ester, thioester, thiocarboxylate, thioester, halogenated sulfonyl, thiol, and thiolene.

[0051] Multiple PSAs and / or analogs, in the form of monomers, polymers, or oligomers and having adjacent glycans, can be anchored to the surface of nanoparticles by chemical bonding. Such chemical bonding may include, for example, click chemistry, carbodiimide chemistry, reductive amination, or chemiadsorption.

[0052] The functional groups of PSA can be located at different positions on the sialic acid unit, specifically at the C1, C2, C4, C5, C7, C8, or C9 positions. Therefore, binding of PSA to the surface of nanoparticles can occur via conjugation at the C1, C2, C4, C5, C7, C8, or C9 positions, which can result in different orientations of the ligand in three-dimensional space on the nanoparticle surface, affecting ligand presentation to target immune cells. Thus, ligand presentation can be controlled to induce a desired cellular response upon contact with immune cells via receptor binding.

[0053] PSA and its sialic acid analogs have ligands of known spacing and / or density, which are intended to be presented on the surface of nanoparticles as ligands for the Siglec receptor. By anchoring specific PSAs to the surface of nanoparticles, the nanoparticles can be brought into contact with a known set of immune cells expressing the Siglec receptor to induce specific biological responses and thereby modulate inflammation. The diversity of PSA composition, structure, density, and buildup presented on the surface of nanoparticles provides means for modulating the degree and direction of the modulation of immune cell responses.

[0054] In various embodiments, the average molecular weight of the polymer, such as PEG, PLGA, PEG-PLGA block copolymer, or polysialic acid, can be determined by any of the methods known in the art, including, in particular, anion exchange chromatography, gel permeation chromatography, and concentration measurement.

[0055] The anchoring of PSA or sialic acid and its analogs to the surface of nanoparticles is carried out in such a manner that it achieves the presentation of PSA for maximum binding affinity to Siglec receptors expressed on the surface of immune cells or sialic acid ligand receptors expressed on the surface of viral particles. The ligand density can be adjusted to provide desired multivalent or polyvalent ligand interactions with Siglec receptors upon contact with immune cells, because such interactions correlate with desired cellular immune responses. Multivalent or polyvalent sialic acid receptor interactions can be controlled based on the density of ligands brought to the nanoparticle surface, and this density can influence the response induced by immune cells upon contact.

[0056] Preferably, PSA can be immobilized on the surface of nanoparticles. PSA can be bound directly to the nanoparticles or via a linker. Nanoparticles can be derivatized or activated to enable PSA binding. Alternatively, nanoparticles can be derivatized or activated to enable linker binding to the nanoparticles, allowing the linker to bind to the PSA. By linking PSA to nanoparticles, the nanoparticles can be adapted to target cells containing Siglec receptors and induce Siglec receptor binding, resulting in inhibition of intracellular pro-inflammatory cytokine production or increased production of anti-inflammatory cytokines, thereby suppressing the pro-inflammatory immune response.

[0057] The density of different functional groups can be controlled by the ratio of different polymers, the polymer concentrations and types of conjugate pairs, the type of reaction, and the size and shape of the PSAs. The number of different ligands that can be presented on the surface can be determined by those skilled in the art. In a non-limiting embodiment, the number of different ligands present on the surface of the nanoparticles is in the range of 1 to 20. The number of different ligands is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another embodiment, the number of different ligands present on the surface of the nanoparticles is in the range of 2 to 20. The number of different ligands is, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In another embodiment, the nanoparticles will contain at least two different PSAs. In another embodiment, the nanoparticles will contain at least three different PSAs. In another embodiment, the nanoparticles may contain at least four different PSAs. In yet another embodiment, the nanoparticles may contain at least five different PSAs.

[0058] Generally, the density of functional groups on the surface of nanoparticles determines the maximum ligand density that can be anchored to the surface of the nanoparticles via covalent chemical bonds through click chemistry. Ligand density can be controlled and quantified with respect to the number of functional groups per square nanometer of surface area. The density achieved allows for the migration of ligands from mushroom confirmation to brush confirmation. Brush confirmation provides the maximum density packing of PSA. Adjusting the ligand density on the surface of nanoparticles provides a means to modulate the biological response of target immune cells contacted by the nanoparticles. The density of ligands presented on the surface of nanoparticles can be quantified as nmols of ligand per 1 mg of total nanoparticle solid. The density can range from 0.05 nmol / mg to 50 nmol / mg of nanoparticles. The particle size of the nanoparticles can range from 25 nm to 200 nm.

[0059] The ligand density on the surface of nanoparticles can be controlled by several methods, including chemical bonding techniques, ligand density on the polymer, ligand type, solvent, pH, and ionic strength. The ligand density on the surface of nanoparticles can be tuned to elicit immunomodulatory responses, including anti-inflammatory biological responses, upon contact between immune cells and such nanoparticles. Ligand density control can also be used to adjust the magnitude of the desired anti-inflammatory response.

[0060] In some embodiments, PSA can be presented on nanoparticles in groups of at least 2, at least 5, at least 10, at least 15, at least 20 or at least 25, at least 50, at least 100, at least 200 or at least 400. In some embodiments, the PSA can be spaced apart on the surface of the nanoparticles so that they or the nanoparticles can bind to two or more Siglec receptors. In some embodiments, the PSA can be spaced apart on the surface of the nanoparticles so that they or the nanoparticles can bind to multiple Siglec receptors presented to individual cell types, and the amount of Siglec receptors presented on their plasma membrane can vary.

[0061] In some embodiments, the nanoparticles may include polymers containing PSA at concentrations ranging from 0.05 nmol / mg of PSA to 250 nmol / mg of PSA per nanoparticle, preferably 0.5 nmol / mg to 25 nmol / mg, most preferably 0.5 to 15 nmol of PSA per mg of nanoparticles. In some embodiments, devices can be coated with such nanoparticles. In alternative embodiments, devices can be formed from polymers, for example, in which case the devices are fine particles or nanoparticles, and the PSA is provided in the polymer at concentrations ranging from 0.05 nmol / mg of PSA to 250 nmol / mg of PSA per nanoparticle, preferably 1 nmol / mg to 25 μg / mg, most preferably 2 to 15 nmol of PSA per mg of nanoparticles.

[0062] In some embodiments, the nanoparticles may have a maximum cross-sectional width or particle size of less than about 1000 nm, less than about 500 nm, less than about 250 nm, or less than about 200 nm. In some embodiments, the nanoparticles may have a width greater than about 1 nm, greater than about 10 nm, greater than about 50 nm, or greater than about 100 nm. In some embodiments, nanoparticles coated with sialic acid or a sialic acid analog may have a maximum cross-sectional width or diameter in the range of about 130 nm to about 170 nm, more preferably about 150 nm. In some embodiments, these size ranges may be the average width of the nanoparticles. In some embodiments, at least 80% of the nanoparticles are within the disclosed range.

[0063] Preferably, in some embodiments, at least 80%, more preferably at least 90%, of the particles have a maximum cross-sectional width of 130 nm to 170 nm. In some embodiments, the particles may have an average maximum cross-sectional width of 150 nm, and the particles do not have a width greater than or less than a value that is not within one standard deviation of 150 nm. In some embodiments, the nanoparticles may have a volume equivalent to a sphere having a diameter of 10 nm to 500 nm, preferably 50 nm to 250 nm, 100 nm to 200 nm, or 130 nm to 170 nm. In some embodiments, the average particle size is 80 nm to 120 nm, for example, 100 nm.

[0064] In non-limiting embodiments, the nanoparticles may have a volume equivalent to a sphere with a diameter of about 100 nm, for example, 70 nm to 130 nm. In some embodiments, the polydispersity index (PDI) of the nanoparticle size is less than 0.5, less than 0.4, less than 0.3, less than 0.2, less than 0.1, less than 0.05, less than 0.25, or less than 0.01. For example, the PDI may be less than 0.15.

[0065] In another aspect of the present invention, the linkage of nanoparticles to PSA provides a means for nanoparticles to evade opsonization and phagocytosis via the immune system, namely the reticuloendothelial system (RES). PEGylation of nanoparticles, i.e., coating of nanoparticles with polyethylene glycol, is known to provide a protective barrier against detection by immune cells. However, PEG has drawbacks such as toxicity, immunogenicity, reduced cell uptake, reduced binding, and non-biodegradability or bioabsorption properties. PSA coating of nanoparticles overcomes the drawbacks of PEG and provides a natural, non-immunogenic nanoparticle coating that can evade RES and immunodetection. Accordingly, the nanoparticles disclosed herein have the ability to evade immunodetection and mitigate immunogenic responses.

[0066] Nanoparticles or fine particles disclosed herein may further include bioactive agents encapsulated within the nanoparticles, attached to their surface, or incorporated into their structure. For example, nanoparticles may further include at least one of antibiotics, antivirals, anti-inflammatory agents, cytokines, cytokine inhibitors, immunomodulators, immunotoxins, anti-angiogenic agents, antihypertensive agents, anti-edema agents, radiosensitizers, oligonucleotides including DNA or RNA, peptides, or any combination thereof. Methods for preparing nanoparticles containing bioactive agents encapsulated within the nanoparticles, attached to their surface, or incorporated into their structure are known to those skilled in the art.

[0067] This disclosure further provides pharmaceutical or veterinary compositions comprising PSA-conjugated nanoparticles disclosed herein. Such pharmaceutical compositions are formulated to suit their intended route of administration. Examples of routes of administration include, for example, intravenous, intravitreous, oral, intraocular, subretinal, sub-Tenon, intrascleral, periorbital, intravenous, nasal and oral inhalation, and parenteral administration methods including intramuscular, intra-arterial, intraspinal, intrathecal, intracranial, intradermal, transdermal (topical), transmucosal, subcutaneous, lung lavage, gastric lavage, intrahepatic, subcutaneous and rectal administration.

[0068] Preferably, in some embodiments, nanoparticles can be administered parenterally. After parenteral administration, the nanoparticles can selectively accumulate in specific tissues or body locations. In some embodiments, the nanoparticles can deliver a therapeutic payload to cells or tissues. In some embodiments, the nanoparticles can enter affected tissue through enhanced permeability and retention effects.

[0069] Generally, pharmaceutical compositions are provided that contain an effective amount of nanoparticles together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants and / or carriers. Such compositions include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as detergents and solubilizers (e.g., Tween 80, polysorbate 80), antioxidants (e.g., ascorbic acid, sodium pyrosulfite), preservatives (e.g., thymelsol, benzyl alcohol), and fillers (e.g., lactose, mannitol). Such compositions may affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the nanoparticles. See, for example, Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042), pp. 1435-1712 (these are incorporated herein by reference). The composition may be prepared in liquid form or formulated as a dry powder, such as in a freeze-dried form.

[0070] This disclosure provides, but is not limited to, methods for treating immune and inflammatory-related diseases in affected subjects by administration of the above-mentioned pharmaceutical compositions, in any case including, but also including, dry and exudative macular degeneration, retinal vascular diseases, diabetic retinopathy, diabetic macular edema, cystoid macular edema, proliferative diabetic retinopathy, proliferative vitreoretinopathy, dry eye, allergic conjunctivitis, rheumatoid arthritis, inflammatory arthritis, lupus, nephritis, combined immune nephropathy, allergic esophagitis, allergic gastritis, hepatitis, hepatic fibrosis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, sepsis, bacterial and viral infections, influenza, SARS-CoV-1 and SARS-CoV-2, HIV / AIDS, group B streptococcal infection, and Neisseria infection. This disclosure provides a method for modulating an intracellular inflammatory response, the method comprising providing sialic acid or an analogue thereof to cells, the sialic acid or analogue thereof being presented on nanoparticles such that an intracellular pro-inflammatory response is suppressed or an intracellular anti-inflammatory response is increased. In several embodiments, the method provides suppression of a pro-inflammatory response. In alternative embodiments, the method provides enhancement of an anti-inflammatory response. In some embodiments, the method provides enhancement of a pro-inflammatory response in situations such as infection.

[0071] As used herein, the terms “treatment” or “to treat” characterize a method or process aimed at (1) delaying or preventing the onset of a disease, disorder, or condition; (2) slowing or halting the progression, worsening, or exacerbation of one or more symptoms of a disease, disorder, or condition; (3) improving the symptoms of a disease, disorder, or condition; (4) reducing the severity or incidence of a disease, disorder, or condition; or (5) curing a disease, disorder, or condition. Treatment may be administered before the onset of a disease, disorder, or condition for preventive or protective purposes. Alternatively, or in addition, treatment may be administered after the onset of a disease, disorder, or condition for therapeutic purposes.

[0072] Depending on the route of administration and the disease, the effective dose can be calculated based on the body weight, body surface area, size of the primary organ / tumor and / or number, size and / or type of metastases of the subject to be treated. Optimization of the appropriate dosage can be easily performed by those skilled in the art, taking into account pharmacokinetic data observed in human clinical trials. The final dosing plan will be determined by considering various factors that alter the action of the drug, such as the specific activity of the drug, the severity of the injury and the patient's responsiveness, the patient's age, condition, weight, sex and diet, the severity of any pre-existing infections, the timing of administration, the use (or non-use) of other treatments, and other clinical factors.

[0073] The compositions and nanoparticles described herein may be used to treat acute, life-threatening inflammation, including sepsis and cytokine storm sialic acid, but are not limited to these. Specific embodiments provide methods for treating multiple ocular inflammatory diseases, including macular degeneration, uveitis, optic neuritis, neuromyelitis, and inflammation resulting from eye infections, drug and toxin exposure, as well as common immunodisorders, including autoimmune diseases. Non-limiting embodiments provide methods useful for preventing, treating, or improving macular degeneration in patients, such as dry (atrophic) macular degeneration, exudative macular degeneration, geographic atrophy, geographic atrophy secondary to age-related macular degeneration (AMD), moderate macular degeneration, and age-related macular degeneration. Methods for treating, preventing, or improving ocular inflammation, including macular degeneration, include administering a PSA nanoparticle composition to a patient who has or is at risk of developing ocular inflammation, such as macular degeneration.

[0074] In certain non-limiting embodiments, this disclosure provides methods for treating immune and inflammation-related diseases, including, but not limited to, dry macular degeneration, exudative macular degeneration, retinal vascular diseases, diabetic retinopathy, diabetic macular edema, cystic macular edema, proliferative diabetic retinopathy, proliferative vitreoretinopathy, dry eye, and allergic conjunctivitis.

[0075] In non-limiting embodiments, methods are provided for preventing, treating, or improving macular degeneration in patients, such as dry macular degeneration, exudative macular degeneration, geographic atrophy, moderate macular degeneration, and age-related macular degeneration.

[0076] In some embodiments, the ophthalmic preparation is provided as eye drops, eye ointment, or ophthalmic injection. In the case of ophthalmic injection, intravitreous or subconjunctival injection may be used to administer the nanoparticles.

[0077] The concomitant administration of additional compounds having applications in methods for treating, preventing, or improving macular degeneration may be co-administered with nanoparticle-containing pharmaceutical compositions used to treat macular degeneration. For example, anti-angiogenic agents for treating exudative age-related macular degeneration, such as pegaptanib sodium, ranibizumab, bevacizumab, aflibrecept, and brolucizumab, can be used as concomitant agents. While specific embodiments of this disclosure are shown and described, it will be apparent to those skilled in the art that in its broader embodiments, variations and modifications can be made without departing from the disclosure. Accordingly, the appended claims shall encompass within their scope all variations and modifications that fall within the true spirit and scope of this disclosure.

[0078] Age-related macular degeneration (AMD) is the leading cause of moderate to severe vision impairment in adults over 60 worldwide, currently affecting approximately 11 million people in the United States. Because central vision is significantly and permanently impaired, patients may lose independence and experience limitations in many basic functions, including reading, driving, and facial recognition. Exudative, or neovascular, AMD develops due to the destruction of central retinal cells through abnormal vascular proliferation, bleeding, and scarring. Approved anti-VEGF therapies are accelerating the control and treatment of this type of disease. VEGF arises from chronic inflammation, particularly the polarization of macrophages into the M2d phenotype. Dry (atrophic), or non-neovascular, AMD is characterized in its early stages by drusen formation and changes in the retinal pigment epithelium (RPE), which can progress to geographic atrophy (GA), in which irreversible progressive destruction of central retinal cells and underlying blood vessels occurs due to chronic inflammation (with phagocytosis of retinal and RPE cells due to hyperactivated macrophage activity) and abnormal complement activation in the retinal photoreceptors, retinal pigment epithelium, and choroidal capillary network region of the fundus. Current therapies are under development to treat early and late atrophic AMD by reducing chronic inflammation and inhibiting elements of the complement cascade.

[0079] In the first embodiment, the present invention is a polysialic acid (PSA)-polymer conjugate compound. In the first aspect of the first embodiment, the conjugate compound has structural formula (I): [ka] It is represented by or a pharmaceutically acceptable salt thereof, where P is poly(lactide-co-glycolide)-poly(ethylene glycol) copolymer (PLGA-PEG), and p is an integer between 4 and 200.

[0080] In the second aspect of the first embodiment, P is PLGA(10k)-PEG(5k). The remaining values ​​and exemplary values ​​are as defined above with respect to the first aspect of the first embodiment.

[0081] In the third embodiment of the first example, the compound has the following structural formula (II): [ka] This is expressed as follows, where y is an integer between 1 and 1000, x is an integer between 1 and 1000, and m is an integer between 1 and 450. The remaining values ​​and exemplary values ​​are as defined above with respect to the first and second aspects of the first embodiment.

[0082] In the fourth aspect of the first embodiment, y is an integer between 1 and 500, x is an integer between 1 and 500, and m is an integer between 1 and 250. The remaining values ​​and exemplary values ​​are as defined above with respect to the first to third aspects of the first embodiment.

[0083] In the fifth aspect of the first embodiment, x is an integer between 90 and 140, y is an integer between 10 and 75, and m is an integer between 90 and 140. The remaining values ​​and exemplary values ​​are as defined above with respect to the first to fourth aspects of the first embodiment.

[0084] In the sixth aspect of the first embodiment, the value of p is selected from one of the following ranges: 10-20, 20-30, 30-40, 40-50, and 50-60. For example, p is 22. The remaining values ​​and exemplary values ​​are as defined above with respect to the first to fifth aspects of the first embodiment.

[0085] In the seventh aspect of the first embodiment, P is PLGA(10k)-PEG(5k), and p is 15-25. The remaining values ​​and exemplary values ​​are as defined above with respect to the first to sixth aspects of the first embodiment.

[0086] In the second embodiment, the present invention relates to particles comprising any compound of the first embodiment or a pharmaceutically acceptable salt thereof. For example, the particles may be nanoparticles.

[0087] In the first aspect of the third embodiment, the weight of PSA per unit weight of particles is 1 μg / mg to 1000 μg / mg.

[0088] In the second aspect of the third embodiment, the weight of PSA per unit weight of particles is 10 to 75 μg / mg.

[0089] In the third embodiment of the third example, the average particle size is 80 nm to 120 nm. The remaining features and exemplary features of the third embodiment are as defined above with respect to the first and second embodiments of the third embodiment.

[0090] In the third embodiment, the present invention is a pharmaceutical composition comprising particles described in any aspect of the second embodiment in a pharmaceutically acceptable carrier or diluent.

[0091] In one embodiment of the third embodiment, the composition is aqueous and further comprises sucrose. The remaining features and exemplary features of the third embodiment are as defined above with respect to any aspect of the second embodiment.

[0092] In the fourth embodiment, the present invention is a method for treating a subject suffering from an ophthalmic disease, comprising administering a therapeutically effective amount of a compound according to any aspect of the first embodiment, particles according to any aspect of the second embodiment, or a pharmaceutical composition according to any aspect of the third embodiment to the subject.

[0093] In the first aspect of the fourth embodiment, the ophthalmic disease is age-related macular degeneration (AMD). For example, the AMD may be atrophic AMD. In another example, the AMD is exudative AMD. The remaining features and exemplary features of the fourth embodiment are as defined above with respect to any aspect of the first, second, and third embodiments.

[0094] In the second aspect of the fourth embodiment, the ophthalmic disease is geographic atrophy secondary to AMD. The remaining features and exemplary features of the fourth embodiment are as defined above with respect to any aspect of the first, second, and third embodiments.

[0095] In the third aspect of the fourth embodiment, the ophthalmic disease is retinitis pigmentosa. The remaining features and exemplary features of the fourth embodiment are as defined above with respect to any aspect of the first, second, and third embodiments.

[0096] In the fourth aspect of the fourth embodiment, the ophthalmic disease is diabetic macular edema. The remaining features and exemplary features of the fourth embodiment are as defined above with respect to any aspect of the first, second, and third embodiments.

[0097] In the fifth aspect of the fourth embodiment, the administration is intravitreous. The remaining features and exemplary features of the fourth embodiment are as defined above with respect to any aspect of the first, second, and third embodiments.

[0098] As used herein, “retinitis pigmentosa (RP)” refers to one of a group of rare, hereditary ophthalmic disorders affecting the retina. The hereditary form of RP occurs in approximately 1 in 5,000 people worldwide and is a leading cause of irreversible blindness in young people. RP is associated with polymorphisms in genes that are typical of those involved in the visual cycle, genes that support retinal cell function, or genes that define the structure of retinal tissue. In some cases, RP is a complication of genetic syndromes such as Usher syndrome, Baldett-Beedl syndrome, Alport syndrome, Waardenberg syndrome, and Kearns-Sayre syndrome. Many of the genes that cause RP remain unidentified, and RP can also be caused by non-genetic factors such as toxic medications, infections, or eye injuries.

[0099] The clinical findings of photoreceptor degeneration and death, following the loss of retinal pigment epithelial cells, are characteristic of this series of retinal degenerative diseases. The complete loss of retinal cells, often seen in the late stages of these diseases, is mediated by phagocytosis of pre-apoptotic cells by microglia; inhibiting this process would preserve photoreceptors or other retinal cells. Because it takes decades to reach clinically complete blindness, therapeutic density ranges are provided to preserve lifelong vision. Due to this long progression period, except for a very small number of rare cases, genetic abnormalities are considered predisposing genes, and most cases of RP are not suitable for gene therapy. Repolarizing microglia into a neuroprotective homeostatic state would be a universally effective treatment for both hereditary and non-hereditary RP.

[0100] In the fifth embodiment, the present invention relates to structural formula (I): [ka] This is a method for preparing polysialic acid (PSA)-polymer conjugate compounds represented by or pharmaceutically acceptable salts thereof.

[0101] In the first aspect of the fifth embodiment example, this method is performed using structural formula (III) [ka] The polymer represented by structural formula (IV) [ka] The process involves reacting a PSA precursor represented by with conditions sufficient to form a compound represented by structural formula (I), where P is poly(lactide-co-glycolide)-poly(ethylene glycol) copolymer (PLGA-PEG) and p is an integer between 4 and 200.

[0102] In the second aspect of the fifth embodiment, P is PLGA(10k)-PEG(5k). The remaining values ​​and exemplary values ​​for the second aspect of the fifth embodiment are as defined above with respect to the first aspect of the fifth embodiment.

[0103] In the third aspect of the fifth embodiment, the compound represented by structural formula (I) is structural formula (II): [ka] (In the formula, y is an integer between 1 and 1000, x is an integer between 1 and 1000, and m is an integer between 1 and 450.) This is represented by [the given expression]. The remaining values ​​and illustrative values ​​for the third aspect of the fifth embodiment are as defined above with respect to the first and second aspects of the fifth embodiment.

[0104] In the fourth aspect of the fifth embodiment, y is an integer between 1 and 500, x is an integer between 1 and 500, and m is an integer between 1 and 250. The remaining values ​​and example values ​​for the fourth aspect of the fifth embodiment are as defined above with respect to the first to third aspects of the fifth embodiment.

[0105] In the fifth aspect of the fifth embodiment, x is an integer between 90 and 140, y is an integer between 10 and 75, and m is an integer between 90 and 140. The remaining values ​​and example values ​​for the fifth aspect of the fifth embodiment are as defined above with respect to the first to fourth aspects of the fifth embodiment.

[0106] In the sixth aspect of the fifth embodiment, the value of p is selected from one of the following ranges: 10-20, 20-30, 30-40, 40-50, and 50-60. The remaining values ​​and example values ​​for the sixth aspect of the fifth embodiment are as defined above with respect to the first to fifth aspects of the fifth embodiment.

[0107] In the seventh aspect of the fifth embodiment, the value of p is selected from one of the following ranges: 17-200, 17-100, 17-60, 17-25, 20-100, 20-60, 20-30, 22-100, 22-60, 22-30, and 22-25. The remaining values ​​and example values ​​for the seventh aspect of the fifth embodiment are as defined above with respect to the first to sixth aspects of the fifth embodiment.

[0108] In the eighth aspect of the fifth embodiment, P is PLGA(10k)-PEG(5k), and p is 15 to 25. The remaining and exemplary values ​​for the eighth aspect of the fifth embodiment are as defined above with respect to the first to sixth aspects of the fifth embodiment.

[0109] In the ninth aspect of the fifth embodiment example, the method is structural formula (V) [ka] The compound represented by structural formula (VI) [ka] The process further includes the step of preparing the compound represented by structural formula (IV) by reacting the compound represented by with under conditions sufficient to prepare the compound represented by structural formula (IV).

[0110] The remaining values ​​and exemplary values ​​for the ninth aspect of the fifth embodiment are as defined above with respect to the first to eighth aspects of the fifth embodiment.

[0111] In the sixth embodiment, the present invention provides a method for increasing the best corrected visual acuity (BCVA) score in a subject requiring such increase, comprising administering a therapeutically effective amount of a compound described in any embodiment of the first embodiment, particles described in any embodiment of the second embodiment, or a pharmaceutical composition described in any embodiment of the third embodiment to the subject.

[0112] In the first aspect of the sixth embodiment, the increase in the target BVCA score is 1 to 4 characters.

[0113] In the second aspect of the sixth embodiment, the increase in the target BVCA score is 5 to 9 characters.

[0114] In the third aspect of the sixth embodiment, the increase in the target BVCA score is 10 to 14 characters.

[0115] In the fourth aspect of the sixth embodiment, the increase in the target BVCA score is 15 characters or more.

[0116] In the seventh embodiment, the present invention relates to structural formula (IV): [ka] It is a compound represented by or a pharmaceutically acceptable salt thereof.

[0117] In the first aspect of the seventh embodiment, p is an integer between 17 and 200.

[0118] In the second embodiment of the seventh embodiment example, the value of p is selected from any one of the following ranges: 17-100, 17-60, 17-25, 20-100, 20-60, 20-30, 22-100, 22-60, 22-30, and 22-25.

[0119] In the third aspect of the seventh embodiment, p is 22. [Examples]

[0120] Examples I. Preparation and Characterization of PSA-Linker Conjugates Structural formula (IV): [ka] The PSA-linker conjugate represented by structural formula (IV) was prepared and characterized according to the following procedure. The compound represented by structural formula (IV) is also referred to herein as "PSA-amine".

[0121] The PSA-amine used in the experiments described below was found to have an average molecular weight of over 6,000 Da and a unit molecular weight of 309.27 g / mol, as determined by measurement.

[0122] PSA-amine was prepared according to Scheme 1. Scheme 1 [ka]

[0123] Briefly, commercially available sodium colomate was dissolved in sterile water for injection, and the solution was heated at approximately 85°C for 5 hours. After completion, the reaction mixture was cooled to room temperature (25°C). Next, sterile water for injection was added to each batch, and the batches were subjected to 30 kDa filtration four times by passing through a filter under pressure. This filtration was performed to remove unwanted short PSA fragments. The complete retention fraction was freeze-dried to produce free PSA.

[0124] The size of free PSA was controlled by mass measurement using liquid chromatography-quadrupole time-of-flight mass spectrometry (LC Q-TOF MS).

[0125] Lyophilized PSA was dissolved in 0.1 M sodium acetate buffer (pH 6), and 2-aminoethoxyamine dihydrochloride was added in an amount ranging from 1 to 20 equivalents of aminoethoxyamine dihydrochloride per equivalent of PSA. The recommended amount of aminoethoxyamine dihydrochloride was 5 equivalents. The pH was adjusted to 6.0-6.5 with 1 M NaOH. The reaction mixture was stirred at approximately 37°C for at least 10 hours. Water for injection was added to each batch, and the batch was subjected to 30 kDa filtration five times by passing through a filter under pressure. Water for injection was added to each batch, and the retained fraction was subjected to 10 kDa filtration three times by passing through a filter under pressure. This filtration was performed to remove trace amounts of excess buffer. The product-containing retained fraction was lyophilized to obtain PSA-amine, a white to off-white solid. The 10 kDa filtration residue was lyophilized until it became a solid with a loss of 5.0 w / w or less.

[0126] Using PSA-amine lot A022216873, 1 H and 13 Chemical structure evidence was obtained using 13C NMR. A 10 mg sample was dissolved in 0.6 mL of D2O (deuterium oxide, heavy water), transferred to an NMR tube, and the NMR spectrum was recorded.

[0127] NMR analysis confirmed that all protons and carbons were consistent with the expected structure of the product. Figure 1A is a schematic diagram of the non-equivalent hydrogens present in the PSA amine. Figure 1B is Table 1, showing the estimated 1H NMR (400 MHz, D2O) peak assignments. Figure 1C is a schematic diagram of the non-equivalent carbons present in the PSA amine. Figure 1D is Table 2, showing the estimated 13C NMR (400 MHz, D2O-d6) peak assignments. Based on LCMS Q-TOF analysis, the synthesized PSA amine has a mass of 6000 Da or greater. Figure 1E is a plot showing the LCMS Q-TOF deconvolution mass for PSA amine lot A022216873.

[0128] In addition to batch A022216873, five other batches were analyzed. The results are shown in Tables 3 and 4 to 4C, which are reproduced in Figures 2A to 2E.

[0129] In these tables, the following abbreviations are used: CAD = charged particle detector, BDL = below detection limit, EU = endotoxin units, GC = gas chromatography, GLP = good laboratory standards, HPLC = high-performance liquid chromatography, KF = Karl Fischer method, LAL = dynamic quantitative turbidimetry lysate endotoxin test, LC Q-TOF MS = liquid chromatography quadrupole time-of-flight mass spectrometer, NLT = above, NMR = nuclear magnetic resonance, ppm = parts per million, RI = refractive index, SEC = size exclusion chromatography, SEC-MALS = size exclusion chromatography using multi-angle light scattering, RRT = relative retention time. **Biosynth lot 0000044791 is the first batch of the manufactured active pharmaceutical ingredient. Therefore, the 1H-NMR spectrum was used as a reference for future lots of the active pharmaceutical ingredient. Because this lot was the first lot, no specifications or acceptance criteria were defined for this lot.

[0130] Measurement of the degree of polymerization (DP) of IA.PSA amines When used herein in relation to the structural formula (IV) above, p is, for example, 1 This is a DP value measured by any method known in the art, such as 1H-NMR. Those skilled in the art will understand that any given p value corresponds to the average value of the entire aggregate of molecules in a batch.

[0131] The degree of polymerization (DP) is a property that can be measured experimentally in an assembly of polymer molecules. Several analytical techniques exist to measure DP. For example, in the case of homopolymers such as PSA, the average molecular weight of the polymer can be measured by any known chromatographic technique, and the result is divided by the molecular weight of the repeating units.

[0132] In one embodiment, as a method for determining the DP of a polymer of formula (IV) 1¹H-NMR was used. In this method, the intensity of the integrated NMR signal generated by the sole equatorial hydrogen of the repeating unit was measured. The DP value was directly obtained from the ratio of this integrated intensity to the intensity of the reference signal generated by the hydrogen of the CH2-N moiety of the terminal unit (modified with an oxime). Thus, 1 The DP values ​​measured by 1H-NMR were averaged over the entire aggregate of polymer molecules in a given batch. Based on equation (IV), the value of DP was determined to be equal to the value of p averaged over the entire aggregate of molecules.

[0133] Furthermore, the LC-Q-TOF method can be used to measure the molecular weight of PSA. The DP can be estimated from the molecular weight. Similarly, the MALDI-TOF method, GPC method, or SEC-MALS method can be used. These methods can generate a molecular weight distribution profile. From this molecular weight, the P range of the PSA's D can be calculated along with the PDI value. For example, with a PDI value of 0.15, the average molecular weight of PSA may be 9000 daltons.

[0134] Overall, the PSA-amines generated during this study ranged in size from approximately 6,000 Da to 20,000 Da. The DP(p) values ​​of PSA were: 1 Measured by 1H-NMR, the DP ranged from approximately 17 to approximately 60, for example, approximately 20 to approximately 60, and even more specifically, approximately 22 to approximately 60. In some embodiments, the DP was approximately 22.

[0135] II. Preparation and Characterization of PSA-Polymer Conjugates Product Overview This product (also called the "pharmaceutical") is an intravitreous injection suitable for delivery to the posterior segment of the eye. This product contains polysialic acid amine (PSA-amine) as its active ingredient, which is coated onto biodegradable poly(lactide-co-glycolide)-poly(ethylene glycol)-(PLGA-PEG) nanoparticles. Sucrose and sterile water for injection are used as isotonic agents and vehicles, respectively. Sodium hydroxide can be added to adjust the pH of the formulation to 6.0-7.5. The final product is shown in Table 5, reproduced in Figure 3. As used herein, "AVD-104" refers to PSA-amine / PLGA-PEG nanoparticles.

[0136] Manufacturing process overview This product contains a PLGA-PEG-based nanoparticle core with a PSA (polysialic acid) amine conjugated to its surface. The nanoparticle core is composed of PLGA-PEG-NHS[poly(L-lactidecoglycolide)-polyethylene glycol-N-hydroxysuccinimide] polymer (lactide:glycolide (75:25), molecular weight approximately 10 kDa) and PEG blocks (molecular weight approximately 4.6 kDa, terminal group: NHS).

[0137] To compound the pharmaceutical, the following process was developed: An organic phase was prepared consisting of PLGA-PEG-NHS polymer dissolved in a mixture of N,N-dimethylformamide, benzyl alcohol, and ethyl acetate solvent. Separately, an aqueous phase was prepared by dissolving the active pharmaceutical ingredient (PSA) in water and N,N-dimethylformamide. The two phases were mixed, N,N-diisopropylethylamine was added to the mixture, and it was maintained overnight for conjugation of the active pharmaceutical ingredient and the polymer.

[0138] An additional aqueous phase was prepared by mixing 92% (v / v) water for injection (WFI) and 8% v / v ethyl acetate, and stored at 2–8°C. The organic phase was mixed with the aqueous phase under high-speed homogenization using a rotor-stator mixer to form a primary crude suspension. The suspension was further processed using a standard 3-stroke (3-pass) procedure with a microfluidizer under a pressure of 15,000 psi to reduce the final average particle size to approximately 100 nm. The resulting particles were purified by tangential flow filtration (TFF) and filtered through a 0.2 micron filter under sterile conditions.

[0139] Overview of intravitreal injection formulations For intravitreal injection, particle size definition is crucial to ensure target binding while avoiding endocytosis. The Siglec receptor is extracellular, and using particle sizes in the range of approximately 150 nm to 50 nm can facilitate the binding of nanoparticles to the receptor. Thus, particle size control was evaluated during process development.

[0140] To evaluate the effect of manufacturing process parameters on particle size, various parameters such as pressure, number of passes, time, and temperature effects were investigated using a drug-free formulation. After dissolving the polymer in a solvent, it was mixed with sterile water for injection to form a crude suspension. Next, this was processed using a microfluidizer. As is clear from Table 6 shown in Figure 4A, it was concluded that the desired particle size distribution was achieved by a three-pass process under a pressure of 15,000 psi.

[0141] In another study, formulations treated with different pass counts and fixed pressures showed no significant changes in particle size distribution up to 6 hours, as shown in Table 7 in Figure 4B. Similarly, formulations treated with fixed pressure and a fixed number of passes were stored under various temperature conditions, showing no significant changes in particle size, as shown in Table 8 in Figure 4C.

[0142] Table 9, shown in Figure 5, outlines the intravitreous injection development batches of this product prepared at Aviceda. These batches were prepared using PLGA-PEG-NHS polymer and PSA-amine active pharmaceutical ingredient. Batches of the active pharmaceutical ingredient with different degrees of polymerization of the sialic acid monomer were also evaluated. The manufacturing process showed similar particle size distribution profiles across all batches.

[0143] Product characteristics The product obtained by the procedure described above was confirmed to have the characteristics listed in Table 10.

[0144] [Table 1]

[0145] stability Based on stability tests, the intravitreal injection formulation of this product was confirmed to be stable for 3 months under long-term storage conditions (-20℃±5℃) and accelerated storage conditions (5℃±3℃). The results of the stability tests are shown in Table 11, which is shown in Figure 6.

[0146] Batch manufacturing Structural formula (I) [ka] (In the formula, P is poly(lactide-co-glycolide)-poly(ethylene glycol) block copolymer (PLGA-PEG), and p is an integer between 4 and 200.) Polysialic acid (PSA)-polymer conjugate compounds represented by were prepared by the following process.

[0147] PLGA-PEG-NHS was dissolved in N,N-dimethylformamide (DMF), benzyl alcohol, and ethyl acetate, and mixed until a clear to white translucent solution was obtained. PSA-amine was dissolved in sterile water for injection to obtain a clear, colorless solution. DMF was added to this solution and mixed to obtain a clear solution.

[0148] The polymer solution and the drug solution were mixed. Subsequently, N,N-diisopropylethylamine (DIPEA) was added to obtain a clear to cloudy solution. This solution was stirred at room temperature for 18 hours. An 8% v / v aqueous solution of ethyl acetate (water for injection) was prepared and maintained at 2-8°C for 18 hours.

[0149] The polymer solution was homogenized in an aqueous phase at approximately 9,500 RPM for approximately 2 minutes (mass ratio of aqueous phase to organic phase: 9:1) to obtain a turbid suspension. The crude suspension was processed in three passes using a microfluidizer operating at a pressure of approximately 15,000 psi to obtain the desired particle size distribution. The product temperature was maintained at 2–8°C during the process.

[0150] The homogenized product was quenched in sterile water for injection at 2-8°C for 60 minutes. The entire product was purified using tangential flow filtration (C / D / C) with a WFI that had already been cooled to 2-8°C. The product was concentrated to the target volume. 10% sucrose was added to the concentrated product volume and dissolved.

[0151] The product was filtered using sterile filtration following a pre-filtration step. The product was pre-filtered using a 0.8 μm filter, and then a 0.45 μm filter. After pre-filtration, the pharmaceutical product was filtered using sterile filtration with a sterile-grade 0.2 μm filter.

[0152] Next, the product was filled into 2 mL transparent USP Type I glass vials under sterile conditions.

[0153] The dose of this product administered to a subject requiring it may vary depending on the subject's condition, its severity, and their body weight. In certain embodiments, the dose is expressed as the weight of PSA delivered per eye. When calculated as the weight of PSA delivered per eye, the dose may range from 0.01 μg / eye to 1000 μg / eye. For example, the weight of PSA per eye may range from 0.01 to 0.1 μg / eye, 0.1 μg / eye to 1 μg / eye, 1 μg / eye to 10 μg / eye, 10 μg / eye to 100 μg / eye, or 100 μg / eye to 1000 μg / eye. For example, the weight of PSA per eye may be approximately 0.01 μg / eye, 0.1 μg / eye, 1 μg / eye, 10 μg / eye, 100 μg / eye, or 1000 μg / eye.

[0154] In certain embodiments, the dose of the product is expressed as the weight of "total solids," for example, as the weight of "total solids" per eye. This "total solids" is a measure of the weight of the product as expressed in a unit volume of aqueous suspension of the product. This can be calculated by direct measurement of concentration or by weighing lyophilized solids suspended in a known volume of sample. In several embodiments, the therapeutically effective dose of the product, expressed as "total solids," may range from 0.05 mg / eye to 2.0 mg / eye. For example, the dosages are 0.1 mg / eye, 0.15 mg / eye, 0.2 mg / eye, 0.25 mg / eye, 0.3 mg / eye, 0.35 mg / eye, 0.4 mg / eye, 0.45 mg / eye, 0.5 mg / eye, 0.55 mg / eye, 0.6 mg / eye, 0.65 mg / eye, 0.7 mg / eye, 0.75 mg / eye, 0.8 mg / eye, 0.85 mg / eye, 0.9 mg / eye, 0.95 mg / eye, 1 mg / eye, 1.1 mg / eye, 1.2 mg / eye, 1.3 mg / eye, 1.4 mg / eye, or 1.5 mg / eye.

[0155] In other embodiments, the therapeutically effective dose of this product, expressed as "total solids," may be 2 mg / eye to 5 mg / eye. For example, the doses are 2.1 mg / eye, 2.2 mg / eye, 2.3 mg / eye, 2.4 mg / eye, 2.5 mg / eye, 2.6 mg / eye, 2.7 mg / eye, 2.8 mg / eye, 2.9 mg / eye, 3.0 mg / eye, 3.1 mg / eye, 3.2 mg / eye, 3.3 mg / eye, 3.4 mg / eye, 3.5 mg / eye, 3.6 mg / eye, 3.7 mg / eye, 3.8 mg / eye, 3.8 mg / eye, 3.9 mg / eye, 4.0 mg / eye, 4.1 mg / eye, 4.2 mg / eye, 4.3 mg / eye, 4.4 mg / eye, 4.5 mg / eye, 4.6 mg / eye, 4.7 mg / eye, 4.8 mg / eye, 4.9 mg / eye, and 5.0 mg / eye. In one embodiment, the effective therapeutic dose is 3.0 mg / eye.

[0156] It is understood that the amount of PSA in a sample can be measured directly, for example, by UV spectroscopy or HPLC. For example, a nanoparticle sample can be subjected to chemical decomposition of PSA to release sialic acid monomers, which can then be detected. If both the weight of PSA and the weight of total solids in a sample of this product are known, the "weight of PSA per unit weight of total solids" can be calculated from the ratio of these two values. Knowing this value makes it possible to convert the dose expressed as the weight of "total solids" to the dose expressed as the weight of PSA, and furthermore, to directly compare two different samples.

[0157] In the specific tests described herein, the PSA concentration in the sample was measured by fluorescence detection of monomers after acid-catalyzed hydrolysis of PSA (4M acetic acid, 80°C for 3 hours, quenched by adding NaOH at room temperature). The Abcam sialic acid kit (Cat#ab83375) was used. Fluorescence was measured at excitation / emission of 535 / 587 nm.

[0158] In a specific test described in this specification, the PSA concentration in the sample was measured by colorimetric detection of the monomer after acid-catalyzed hydrolysis of PSA (4M acetic acid, 3 hours at 80 °C, quenching by adding NaOH at room temperature). The Abcam sialic acid kit (Cat# ab83375) was used. Absorbance was measured at 570 nm.

[0159] III. Analysis procedure Measurement of the molecular weight of PLG-PEG polymers by gel permeation chromatography The average molecular weight and polydispersity (PD) of PLGA10K-PEG5K-NHS are measured by gel permeation chromatography (GPC) using a refractive index detector. Approximately 50 mg of the sample is dissolved in 5 mL of diluent. The experimental parameters for this test are shown in Table 12 below.

[0160]

Table 2

[0161] It was confirmed that the number average molecular weight Mn was 15,000 Da ± 3000 Da and the PDI was 1.9 or less.

[0162] Identification of PSA-polymer conjugates by infrared spectroscopy The sample was thawed as follows. The sample vial stored at -20 °C was first thawed to a refrigerated state (2 - 8 °C) for 60 minutes or for an appropriate time until the frozen product was completely thawed. The vial was conditioned at room temperature for 60 minutes from refrigerated storage (2 - 8 °C). The thawed pharmaceutical sample was tested by the direct addition method. The identification of the pharmaceutical was carried out by confirming the amide bond between the PLGA-PEG polymer and the PSA-amine drug substance. As a criterion for confirmation, the presence of the amine carbonyl stretch on the IR spectrum at 1630 cm -1 ~1670 cm -1 was utilized. A representative IR spectrum of the pharmaceutical is shown in Figure 7.

[0163] Measurement of PSA amine content by HPLC The polysialic acid amine (PSA-amine) content is analyzed by high-performance liquid chromatography (HPLC) using UV / photodiode array (UV / PDA) detection technology. The PSA content is calculated based on the peak area of ​​the sample preparation relative to the peak area of ​​the standard.

[0164] The following conditions, materials, and equipment were used. Instrument: HPLC equipped with UV / PDA detection and quaternary pump. HPLC column: Waters XBridge Amide, 250 mm × 4.6 mm, 3.5 μm Column temperature: 40℃ (sample) Cooler temperature: 5℃ Flow rate: 0.8mL / min Injection volume: 20μL Wavelength: UV214nm Runtime: 45 minutes Erosion: Gradient Solvent: HPLC-grade water Needle washing: Mixture of water and acetonitrile (50:50% v / v) Mobile phase A: 1 mL of triethylamine was mixed with 1 liter of mill-Q water, the pH was adjusted to 6.5 ± 0.05 with diluted orthophosphate, and the mixture was filtered (0.2 μm). Mobile phase B: Acetonitrile (100%)

[0165] The gradient program is shown in Table 13.

[0166] [Table 3]

[0167] Samples were prepared as follows: Product samples were pooled from three vials (0.5 mL each), and a 1.0 mL sample was taken. This was diluted to 5 mL with a diluent and then tested (prepared twice). The standard solution was prepared as follows: 1000 μg / mL in the diluent (prepared twice). One standard bracket solution was injected after each of the six sample injections and at the end of the sequence. Multiple blanks were injected to obtain a stable baseline.

[0168] The samples were thawed using the following procedure: Sample vials stored at -20°C were first thawed at a refrigerated state (2-8°C) for 60 minutes, or over an appropriate period of time until the frozen product was completely thawed. The vials were then prepared at room temperature for 60 minutes after being stored at a refrigerated state (2-8°C).

[0169] PSA content (mg / mL) = (A SPL / A STD )×(W STD / D STD )×(D SPL / V SPL ) × (P / 100) The formula is as follows:

[0170] [Table 4]

[0171] Figure 8 shows a typical chromatogram used to measure PSA-amine content.

[0172] Measurement of particle size distribution using dynamic light scattering method The particle size distribution is measured by dynamic light scattering using a Zetasizer in accordance with USP 1430.3.

[0173] The following conditions, materials, and equipment were used. Instrument: Malvern Zetasizer Nano ZS Dispersant: Water (HPLC grade or Milli-Q water) Particle refractive index: 1.48 Particle absorption rate: 0.001 Dispersant refractive index: 1.330 Temperature: 25℃ Dispersant viscosity: 0.8872 cP (at 25°C) Equilibration time: 60 seconds Cell type: Disposable polystyrene sizing cuvettes Measurement angle: 173° backscattering (non-invasive backscattering default)

[0174] Sample thawing procedure: Sample vials stored at -20°C were thawed for 60 minutes until they first reached refrigerated conditions (2-8°C), or for an appropriate amount of time until the frozen product was completely thawed. The vials were then allowed to set at room temperature for 60 minutes after being stored refrigerated (2-8°C).

[0175] Sample preparation: Product samples were pooled from three vials (0.5 mL each), and a 1.0 mL sample was taken. This was diluted to 10 mL with a diluent before testing. Samples were prepared in singlets.

[0176] Calculation of particle size distribution parameters: The Z-mean and PDI values ​​were obtained using Zetasizer's dedicated software.

[0177] Reporting of results: The Z-mean value is reported in nanometers (nm), and the polydispersity index (PDI) value is reported as a numerical value.

[0178] Figure 9 shows a typical histogram of the particle size distribution.

[0179] Measurement of particle size distribution by electrophoretic light scattering method Zeta potential analysis is performed by electrophoretic light scattering using a Zetasizer in accordance with USP 1430.3.

[0180] The following conditions, materials, and equipment were used. Instrument: Malvern Zetasizer Nano ZS Dispersant: Water (HPLC grade or Milli-Q water) Particle refractive index: 1.59 Particle absorption rate: 0.010 Dispersant refractive index: 1.330 Temperature: 25℃ Dispersant viscosity: 0.8872 cP (at 25°C) Equilibration time: 60 seconds Cell type: Disposable foldable capillary cell Measurement angle: 173° backscattering (non-invasive backscattering default)

[0181] Sample thawing procedure: Sample vials stored at -20°C were thawed for 60 minutes until they first reached refrigerated conditions (2-8°C), or for an appropriate amount of time until the frozen product was completely thawed. The vials were then allowed to set at room temperature for 60 minutes after being stored refrigerated (2-8°C).

[0182] Sample preparation: Product samples were pooled from three vials (0.5 mL each), and a 1.0 mL sample was taken. This was diluted to 10 mL with a diluent and tested. Samples were prepared in singlets.

[0183] Calculation of particle size distribution parameters: Zeta potential values ​​were obtained using dedicated Zetasizer software.

[0184] Reporting of results: Zeta potential values ​​were reported in millivolts (mV).

[0185] Figure 10 shows a typical histogram of the zeta potential distribution.

[0186] Analysis of polymer-PSA conjugate concentration The analysis of total solids (product concentration) is performed using UV spectrophotometric techniques. The calculation is based on the UV absorbance of product nanoparticles in the sample relative to the UV absorbance of the PLGA-PEG-NHS polymer standard.

[0187] The following conditions, materials, and equipment were used. Instrument: UV spectrophotometer Wavelength: UV 230nm Diluent: Acetonitrile

[0188] Sample Preparation: Product samples from two vials (each 0.5 mL) were pooled, and a 0.5 mL sample was taken into a 10 mL volumetric flask. The sample was diluted to 10 mL with the diluent and dissolved. Next, the diluted sample was centrifuged, and the supernatant solution was used for the test. The sample was prepared as a singlet. Standard Preparation: Five standard preparations with concentrations ranging from 0.3125 mg / mL to 5 mg / mL were prepared using the PLGA-PEG-NHS polymer in the diluent, and a linearity curve was created using the UV absorbance values and their corresponding concentrations. For the test, a system suitability criterion with a regression coefficient of 0.990 or more was selected. Sample Thawing Procedure: The sample vial stored at -20 °C was thawed first for 60 minutes until it reached the refrigerated state (2 - 8 °C) or for an appropriate time until the frozen product was completely thawed. The vial was conditioned at room temperature for 60 minutes from refrigerated storage (2 - 8 °C).

[0189] Calculation of Results: The total solids were calculated using the following formula:

Number

[0190] Batch Analysis of the Product Figures 11A to 11D show Tables 14A to 14D, respectively, which summarize the batch analysis results of intravitreal injection products. The following abbreviations are used in these tables: BQL = less than limit of quantification, DIPEA = N,N-diisopropylethylamine, DMF = N,N-dimethylformamide, EU = endotoxin units, GC-HS = gas chromatography with headspace sampler, HPLC = high-performance liquid chromatography, IR = infrared, MTBE = methyl tert-butyl ether, N / A = not applicable, NHS = N-hydroxysuccinimide, NLT = greater than or equal to, NMT = less than or equal to, RI = refractive index, USP = United States Pharmacopeia, N / A = not applicable, PDI = polydispersity index, PSA = polysialic acid. 1 Preliminary tests for total solids content (AVD-104 concentration) were performed on batches SF22000208, SF22000414, SF22000417, and SF22000573 as in-process tests using freeze-drying during the manufacturing TFF step. Subsequently, UV spectrophotometry was developed as a final product inspection method and was used to measure technical batch FT22000012.

[0191] Long-term stability testing Specific batches of this product (intravitreal injection) were tested for long-term stability in reverse at -20°C ± 5°C. The results are shown in Tables 15 (Batch SF22000414), 16 (Batch SF22000417), and 17 (Batch SF22000573), which are reproduced in Figures 12A, 12B, and 12C, respectively.

[0192] Molecular weight of PSA polymer conjugates The molecular weight of the PSA-polymer conjugate was determined by adding the molecular weights of the PSA-amine and polymer, which were measured individually. In various embodiments, the molecular weight of the conjugate was found to be in the range of 10–50 kDa, 15–40 kDa, and 20–35 kDa. In various embodiments, the molecular weight of the conjugate was 15 kDa, 20 kDa, 25 kDa, or 30 kDa. In one example, the molecular weight was 20 kDa.

[0193] IV. Characterization of the biological activity of this product The term "AVD-104" shown in the diagram illustrating the test results described below refers to batch SF22000208, which is characterized in Tables 14A-14D, reproduced in Figures 11A-11D.

[0194] 1. Batch SF22000208 Batch SF22000208 is characterized in Tables 14A to 14D shown in Figures 11A to 11D.

[0195] 2. Formulation for clinical trials The final formulation of the product prepared for clinical trials is poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(sialic acid) and contains the following additives: poly(lactide-co-glycolide)-block-poly(ethylene glycol)-block-poly(lactide-co-glycolide)-succinimidyl ester, sucrose, and water. Sucrose was added for stability, and water for suspension.

[0196] 3. Protein and gene expression of Siglec 7, 9, and 11 In this study, protein expression levels were measured by Western blotting, and gene transcription levels were measured by real-time qRT-PCR. The results were normalized to those of healthy donor eyes.

[0197] This study demonstrated significant increases in the gene and protein expression of Siglec 7, 9, and 11 in retinal-RPE-choroidal complex tissue collected from three exudative AMD eyes and three non-exudative AMD eyes (from one female and two male donor eyes, respectively) aged 85 ± 10 years.

[0198] Exudative AMD donors showed increased Siglec 7, 9, and 11 gene expression by 90-fold, 64-fold, and 58-fold, respectively, compared to healthy donors (Figure 13A and table below, measured by RT-PCR). Similarly, non-exudative AMD donors showed increased Siglec 7, 9, and 11 gene expression by 77-fold, 32-fold, and 27-fold, respectively, compared to healthy donors.

[0199] [Table 5]

[0200] Figure 13B shows significant upregulation of Siglec 7, 9, and 11 in retinal / RPE / choroidal complex extracts from exudative and non-exudative AMD donors compared to healthy donors.

[0201] The data shown in Figures 13A and 13B represent the mean ± standard error in three exudative and three neAMD donor eyes (one female, two males, age 85 ± 10 years). (A) Proteins were extracted, and the expression of Siglec-7, -9, and -11 was measured by Western blot assay. After separating the proteins by gel electrophoresis, blotting was performed, and the eyes were stained with Siglec-7, -9, and -11, as well as β-actin as an internal control. The total band density was normalized with β-actin, and the magnification changes were compared with those of a normal donor eye. Tukey's multiple comparison test showed no significant difference between the exudative AMD group (light bars) and the neAMD group (dark bars). (B) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of Siglec-7, -9, and -11 mRNA expression in human exudative AMD and neAMD donors. Gene transcription levels were measured by real-time qRT-PCR. Data at the Sigrec-7, -9, and -11 gene transcript levels were normalized to qRT-PCR values ​​for glyceraldehyde-3-phosphate dehydrogenase (GAPDH, housekeeping gene). Tukey's multiple comparison test showed no significant difference between the exudative AMD group (light bars) and the neAMD group (dark bars).

[0202] These results suggest that in humans, macrophages bearing Siglec receptors 7, 9, and 11 are resident in the eyes of AMD patients. The higher expression of Siglec receptor proteins in the eyes of AMD patients compared to healthy controls further indicates that macrophages can be an active inflammatory source of the disease. It is suggested that these cells can be re-polarized to a resting state by glycomimetic therapy using AVD-104.

[0203] 4. Cell-free binding affinity of the product for Siglec 7, 9, and 11 In this study, the cell-free binding affinity of the product for Siglec-7, -9, and -11 was examined and confirmed.

[0204] The specific binding affinity of the product for Siglec-7, -9, and -11 was confirmed using a cell-free PEG-ELISA method. When batches AT-07-NP12 and SF22000208 were tested, both showed significant binding to Siglec-7, -9, and -11 (Figure 14), suggesting that the product interacts with macrophages through specific binding to these Siglecs. At a total solids content of 5 mg / mL, the binding affinity of the product for Siglec-7, -9, and -11 was higher than that of blank nanoparticles (a construct of PLGA and PEG without sialic acid). The relative binding affinity was Siglec-7 > Siglec-9 > Siglec-11. This enables the development of nanoparticles with multivalent affinity for all three Siglecs that target macrophages, monocytes, and microglia, which are the major cell types involved in non-resolving inflammation in GA.

[0205] Recombinant Fc proteins for Siglec-7, -9, and -11 (R&D Systems) were coated onto ELISA plates. Different concentrations of the product and blank control nanoparticles (0.1–5 mg / mL), measured as total solids, were added to the coated plates. Detection was performed using an anti-PEG biotin / horseradish peroxidase standard according to the manufacturer's ELISA protocol. Absorbance was measured at 490 nm. At a total solids concentration of 5 mg / mL, the binding affinity of the product to Siglec-7 (solid circle), Siglec-9 (solid triangle), and Siglec-11 (solid rhombus) was higher than that of the blank nanoparticles. A dose-dependent increase in binding of the product to all three Siglecs was observed.

[0206] 5. Cytokine analysis in THP-1-derived macrophages and normal human PBMC-derived M1 macrophages In this study, the modulation of TNF-α, IL-6, IL-10, and VEGF by this product was measured either in human macrophages derived from THP-1 or by polarization of M1 macrophages into M2c converging macrophages.

[0207] Polarization from M1 macrophages to M2c converging macrophages was demonstrated, as confirmed by the suppression of inflammatory mediators: TNF-α, IL-6, IL-1β, and VEGF, and the increase of the anti-inflammatory cytokine IL-10.

[0208] This product induced a 1.3-fold (p=0.087) and 1.35-fold (p<0.05) decrease in the release of inflammatory cytokines IL-6 and IL-1β in retinal macrophages compared to the control, and a 1.4-fold (p<0.05) increase in the production of the anti-inflammatory (converging) cytokine IL-10 in retinal macrophages compared to the control (Figures 15A and B, 16A, B and C).

[0209] THP-1 cells (ATCC TIB-202™, Gaythersburg, Maryland) were differentiated using 10 ng / mL of phorbol 12-myristart 13-acetate (PMA) and activated with 1 μg / mL of LPS. The cells were treated overnight with the product in a continuous dose range (batch SF22000208 (referred to as SF208 in Figures 15 and 16, 0.01-1 mg / mL)). LPS was used as a positive control. The supernatant was collected after treatment and assayed for (Figure 15A) TNF-α and (Figure 15B) IL-10 by ELISA (R&D Systems). In vitro, the product significantly downregulated TNF-α levels and significantly upregulated IL-10 levels.

[0210] PBMCs (Stem Cell Research, Cat No-70500.2) were activated for 48 hours with the M1 phenotype (interferon-γ: 50 ng / mL, LPS: 10 ng / mL). The cells were then treated overnight with the product (batch SF22000208, 0.01-1 mg / mL) in a continuous dose range. LPS was used as a positive control. The supernatant was collected and assayed for (16A)VEGF, (16B)IL-1β, and (16C)IL-6 by ELISA (R&D Systems). In vitro, AVD-104 significantly downregulated the levels of VEGF, IL-1β, and IL-6 in the supernatant derived from activated macrophages. *p<0.05 (Dunnett's multiple comparison test).

[0211] The dose-dependent response of PMBC-derived cells to AVD-104 and PSA ligand alone was also compared using a TNF-α ELISA assay. For this comparison, the AVD-104 batch used had more than 20 DPs (similar to the PSA control). The results are shown in Figure 47. Unlike the PSA ligand control, AVD-104 was found to induce a clear dose-dependent inhibition of TNF-α production.

[0212] Furthermore, using a TNF-α ELISA assay, the response of PMBC-derived cells to AVD-104 (a batch with DPs of 20 or higher PSA) was compared with that of DP8 and DP11 nanoparticles.

[0213] In this comparative study, each type of nanoparticle was synthesized according to the method described in Part II of the Examples section (conjugation of polymer and PSA-amine). The estimated molecular weight of the PSA portion was approximately 2500 Da for DP8 nanoparticles and approximately 3000 Da for DP11 nanoparticles. The total solid content was calculated to be 10 mg / ml per 10 ml of DP8 nanoparticles and 12.4 mg / ml per 10 ml of DP11 nanoparticles. The particle size was measured to be approximately 109 nm (PDI 0.23) for DP8 nanoparticles and approximately 61 nm (PDI 0.17) for DP11 nanoparticles.

[0214] The results are shown in Figure 48. Unlike DP8 and DP11 nanoparticles, AVD-104 (20 or more DPs) was found to induce a clear dose-dependent inhibition of TNF-α production.

[0215] The dose-dependent response of PMBC-derived cells to AVD-104 (a batch with DP of 20 or more PSAs) was measured using a TNF-α ELISA assay. Based on the dose-response curve shown in Figure 49, the IC of AVD-104-mediated inhibition of TNF-α production was measured. 50 The value was calculated. Specifically, the IC of "total solids". 50 This was calculated to be 191.0 μg / ml, which corresponds to the IC50 of 6.075 μg / ml of PSA on AVD-104 nanoparticles. 50 It corresponds to this.

[0216] 6. Recruitment of the receptor SHP-1 in macrophages (M1) derived from normal human peripheral blood mononuclear cells by this product. This study demonstrated the activation of ITIM and the recruitment of SHP-1, which exhibits agonist activity of Siglec.

[0217] SHP-1 consists of three domains: an N-terminal SH2 domain, a C-terminal SH2 domain, and a C-terminal catalytic protein tyrosine phosphatase (PTP) domain. The N-terminal SH2 domain is self-repressive, binding to the PTP domain until the C-terminal SH2 domain binds to a phosphorylated peptide ligand, thereby enabling a structural change and release of self-repression. This structural change reduces the molecular weight from 70 kDa to 60 kDa.

[0218] When THP-1 cells are activated by LPS, they polarize macrophages to the M1 state. Activation of Siglec releases the self-inhibited state of SHP-1, causing it to transition to the 60kDa state. Thus, the detection of a 70kDa signal indicates the self-inhibited state of SHP-1, while the detection of a 60kDa fragment by Western blotting indicates the released state of SHP-1.

[0219] The following assay was used to detect the state of SHP-1.

[0220] Immunoprecipitation (IP) was performed using anti-Siglec 7 and 9, followed by visualization of SHP-1 recruitment in THP-1 cells and M0 / M1 macrophage lysates by Western blotting. The 70kD band indicates a 25% increase in SHP-1 recruitment when THP-1 cells were incubated in the presence of sialic acid-coated NPs compared to sucrose control. A similar 10% increase was observed in total protein of M1 macrophages immunoprecipitated from THP-1 cell lysate (A) using anti-Siglec 7 and 9 (Cat#AF1138-SP and AF1139-SP, RD Systems, Minneapolis, Minnesota), and then attached to SHP-1 WB, which was revealed by electrochemiluminescence (ECL) (Thermofisher Scientific, Waltham, Massachusetts). The mean band intensity was quantified using ImageJ after applying background correction.

[0221] PMBC-derived M0 and M1 macrophage lysates were incubated in the presence or absence of LPS, sucrose control, and sialic acid-coated nanoparticles. After background removal, the mean band intensity was measured using ImageJ. The results described above were obtained by comparing the band intensities of the sucrose control and sialic acid-coated nanoparticles.

[0222] 7. This product modulates the classical and alternative complement pathways. In this study, in hemolysis assays (CH50 assay for total hemolytic complement and AH50 assay for alternative pathways (measuring 50% erythrocyte (RBC) lysis)), this product demonstrated the ability to attenuate both the alternative pathway and the classical complement pathway with efficacy equivalent to that of a neutralizing antibody against C3.

[0223] This product has been shown to regulate complement activation in vitro (Figures 17 and 18) and complement deposition in an in vivo retinal injury model (Karlstetter et al., 2017).

[0224] Referring to Figure 17, normal human serum (NHS, CompTech) was treated with either no treatment, sucrose vehicle control, the product (SF22000208) (0.3 mg / mL, 1 mg / mL), or C3 neutralizing antibody (C3nAb, Millipore, 0.1 mg / mL), and immediately used in the CH50 assay. Pre-activated normal human serum with cobra venom factor (NHS-CVF) was included as a negative control. Briefly, antibody-sensitized sheep erythrocytes (CompTech) were added to a dilution series of each serum sample in GVB++, incubated at 37°C for 60 minutes, and the remaining erythrocytes (RBCs) were centrifuged and pelletized. The supernatant (containing heme derived from lysed RBCs) was then transferred to a 96-well plate, and the absorbance at 560 nm was measured using a spectrophotometer. All data were normalized against a 100% positive control (buffer replaced with water or detergent) and a 0% negative control (no NHS). 50% hemolysis data were averaged across experiments. NHS exhibits robust classical pathway (CP) activity, NHS-CVF shows extremely low CP activity, and both C3nAb and this product inhibit CP activity.

[0225] As shown in Figure 18, normal human serum (NHS, CompTech) was treated with either no treatment, sucrose vehicle control, the product (SF22000208) (0.3 mg / mL, 1 mg / mL), or C3 neutralizing antibody (C3nAb, Millipore, 0.1 mg / mL), and immediately used in the AH50 assay. Normal human serum with C3 deletion (NHS-C3dpl, CompTech) was included as a negative control. Briefly, rabbit erythrocytes (CompTech) were added to a dilution series of each serum sample containing MgEGTA, incubated at 37°C for 30 minutes, and the remaining erythrocytes (RBCs) were centrifuged and pelletized. The supernatant (containing heme from lysed RBCs) was then transferred to a 96-well plate, and the absorbance at 560 nm was measured using a spectrophotometer. All data were normalized against a 100% positive control (buffer replaced with water or detergent) and a 0% negative control (no NHS or with EDTA). The data were graphed as 50% RBC lysis rate by serum dilution, and after determining the 50% hemolysis rate (AH50) for each group, the average of the two experiments was calculated. NHS has robust alternative pathway (AP) activity, NHS-C3dpl has low AP activity, and both C3nAb and this product inhibit AP activity.

[0226] 8. Efficacy of this product in a photo-induced retinal degeneration model in humanized Siglec 11 transgenic mice. This study was conducted using BALB / c and C57BL / 6 mice, as well as transgenic Siglec-11 humanized mice. A single dose of this product (SF22000208) resulted in a statistically significant reduction in retinal degeneration.

[0227] The mice were generated by knocking in the human Siglec 11 gene into the ROSA26 locus of C57BL / 6N mice using CRISPR / Cas genome engineering.

[0228] The mouse ROSA26 gene (NCBI reference sequence: NR_027008.1) is located on mouse chromosome 6. The human Siglec 11 gene (NCBI reference sequence: NM_052884.3) is located on human chromosome 19. In the knock-in (KI) model, the "CAG promoter-Kozak-Human Siglec 11 CDS-rBG pA" cassette was cloned in reverse orientation into intron 1 of ROSA26. To generate KI mice, Cas9 and gRNA were co-injected into fertilized eggs along with the targeting vector. Mouse genome fragments containing homology arms (HAs) were amplified from BAC clones using high-fidelity Taq DNA polymerase, and then sequentially assembled into the targeting vector along with the recombination site and selection marker.

[0229] Figure 25 shows the map of the Hitoshigrec 11 targeting vectors.

[0230] Referring to Figures 19A, 19B, and 20, ONL thickness was similar across the groups at baseline (Figure 19A), and similarly, total retinal thickness was similar across the groups (Figure 19B). On day 7, both ONL thickness and total retinal thickness decreased from baseline in all groups. However, in the vehicle groups, both measurements decreased more significantly than in the 3 mg / ml group (equivalent to a clinical dose of 0.35 mg / eye in humans) (ONL, p<0.01, total retinal thickness, p<0.0001) and the 19 mg / ml group (equivalent to a clinical dose of 2.3 mg / eye in humans) (both p<0.0001). Therefore, this product likely significantly reduced retinal damage in a dose-dependent manner. Furthermore, this product induced a dose-dependent decrease in TNF-α release in the RPE / choroid compared to the control (p<0.0001) (Figure 20), indicating that AVD-104 suppresses pro-inflammatory TNF-α.

[0231] Referring to Figures 19A and 19B, the eyes of the retinal degeneration model, Siglec-11 mice, were treated with a single IVT dose of this product (3 mg / ml or 19 mg / ml) or a 10% sucrose vehicle, and then exposed to 10,000 lux for 4 hours to induce BLD (blue light damage). (Figure 19A) Both ONL thickness and (Figure 19B) total retinal thickness showed significant thickness reduction between baseline and day 7, as evidenced by two-way ANOVA. This product showed a dose-dependent significant reduction in retinal degeneration compared to the vehicle (shown in the graph). At either dose, IVT administration of this product was not associated with ocular inflammation or toxicity.

[0232] Referring to Figure 20, ocular tissue samples collected on day 8 from the 3 mg / ml and 19 mg / ml groups of this product were homogenized, and the collected supernatant was analyzed for TNF-α by ELISA (R&D Systems). The data are representative of homogenized N-5 RPE / choroidal tissue. RPE = retinal pigment epithelium.

[0233] 9. Efficacy of this product in a laser-induced choroidal neovascularization (CNV) model using humanized Siglec 11 transgenic mice. This study was conducted using BALB / c and C57BL / 6 mice, as well as transgenic Siglec-11 humanized mice. A single dose of this product (SF22000208) resulted in clinically significant protection against lesion formation and lesion leakage, as well as inhibition of the C5b9 membrane attack complex.

[0234] In this in vivo study, laser trauma was used to induce CNV in Siglec-11 humanized mice, allowing for the evaluation of the product's effects on retinal damage (quantified in this study as lesion size and degree of vascular leakage). On day 1, animals received a 1 μL IVT injection of OU (both eyes). One group received a 10% sucrose vehicle (control group), one group received 3 mg / mL of AVD-104, and one group received 19 mg / mL of the product. Subsequently, four single laser-spot OUs were formed around the optic nerve using a 532 nm diode laser. On day 8, all animal OUs underwent fluorescence angiography. After euthanasia on day 8, 9-10 eyes were extracted from each group, the retinas were dissected, incubated with an antibody cocktail, and then flat-mounted.

[0235] On day 8, fluorescein angiography revealed that the control group had the largest mean ± standard deviation (SD) lesion area (5,269.8 ± 2,419.1 μm²). 2 ) has, and this product 3 mg / ml group (4,352.7 ± 2,014.7 μm 2 ) and the 19 mg / ml group of this product (3,496.0 ± 1,735.1 μm 2 (Figure 21 and data not included, see below) demonstrated that this was followed by the control group. Flat-mount immunohistochemistry (IHC) staining of isolectin B4 showed that the control group had the largest mean ± SD lesion area (25,026.8 ± 7,648.7 μm²). 2 ) has a product 3 mg / ml group (19,573.6 ± 10,369.3 μm 2 ), this product 19mg / ml group (19,818.9±4,915.3μm 2 (Figure 22 and data not included, described later) This was shown to be the case.

[0236] Analysis of representative fundus images and fluorescein angiography (not shown) images from day 8 revealed a dose-dependent decrease in lesion size and leakage in the CNV transgenic mouse model on day 8.

[0237] Referring to Figure 21, in the laser-induced CNV model, Siglec-11 mice, the eyes were treated with a single IVT dose of the product (3 mg / ml or 19 mg / ml) or a 10% sucrose vehicle. CNV was then induced by laser trauma using a 532 nm diode laser, which created four single laser spot OUs around the optic nerve. Fluorescein angiography was performed on all animal OUs on day 8. The product showed a clinically significant reduction in lesion area.

[0238] Referring to Figure 22, in the laser-induced CNV model, Siglec-11 mice, the eyeballs were treated with a single IVT dose of the product (3 mg / ml or 19 mg / ml) or a 10% sucrose vehicle. CNV was then induced by laser trauma using a 532 nm diode laser, which created four single laser spot OUs around the optic nerve. After euthanasia on day 8, 9-10 eyes were extracted from each group, the retinas were dissected, incubated with an antibody cocktail (anti-C5b-9 antibody and anti-Iba1 antibody), and then flat-mounted. For each treatment group, representative images of Iba1 expression within the lesion (isolectin B4, an endothelial cell vascular-specific antibody marker used to analyze the vascular structure of mouse eye tissue and quantify the area of ​​neovascularization) were evaluated (acquired using an Olympus Bx63 upright fluorescence microscope equipped with CellSens software).

[0239] After euthanasia on day 8, 9-10 eyes per group were treated for ocular flat-mount IHC to determine C5b-9 and Iba1 expression within the lesions (by incubating the flat mounts with the corresponding antibody cocktail). The data strongly suggested that a reduction in lesion size correlated with a reduction in C5b-9 membrane attack complexes. Treatment with this product also reduced infiltrating macrophage cells, as indicated by reduced staining of the Iba1 marker. Analysis of representative images of C5b-9 and Iba1 expression showed a reduction in both C5b-9 membrane attack complexes and Iba1 staining.

[0240] 10. Pharmacokinetics and Metabolism of Products in Animals Following two in vivo single-dose toxicity studies of this product in Dutch belted rabbits (N=6 eyes / group), all doses were well-tolerated, and no ocular or systemic safety concerns were observed in any of the animals. In the MTD study, three doses were administered: 0.05 mg / eye, 0.15 mg / eye, and 0.5 mg / eye. All of these doses were well-tolerated.

[0241] In another study conducted with the same rabbit breed, 2.0 mg / eye of this product resulted in moderate inflammation and vitreous opacity on day 4 (using the Hackett-McDonald method), with an average ocular inflammation score of 11 out of 60. By day 7, without any intervention, this inflammation had significantly decreased to an ocular score of 2, indicating mild inflammation. Therefore, the MTD (Mean Time Tolerance) was determined to be 1 mg / eye.

[0242] 11. Single-dose pharmacokinetic evaluation of AVD-104 in Dutch-belted rabbits This study demonstrated that PEG levels persisted in all ocular tissues until the final sample collection date. The order of exposure levels was RPE / choroid > retina > AH and VH. T for plasma and RPE / choroid max This was the final sample collection date.

[0243] In this study, all rabbits received bilateral in vitro injection of 0.5 μg / eye of the product (SF22000208) on day 0. Terminal blood (for plasma) and / or ocular tissue (vitreous humor, RPE / choroid, retina) were collected on days 1, 3, 7, 10, 14, 17, 21, 24, and 28.

[0244] Following injection, PEG became detectable in plasma on day 3 after administration, and this detection persisted until the final time point (day 24), with a steady increase in concentration throughout the entire study period (Figure 23).

[0245] Quantitative analysis of the product in the rabbit matrix was performed using the PEG ELISA kit (Cat. No. ab215546) from Abcam.

[0246] In short, the polyethylene glycol (PEG) RabMAb® ELISA kit operates based on competition between enzyme HRP-conjugated PEG and PEG-labeled molecules for a constant binding site on the surface of a 96-well plate coated with anti-PEG RabMAb® antibody. The degree of color development resulting from the interaction between HRP and substrate TMB is inversely proportional to the amount of PEGylated molecules in the sample. For example, a vivid blue color is produced when no PEGylated molecules are present in the sample, but the color is reduced or disappears when PEGylated molecules are present.

[0247] The PEG content in the batch used in this study was found to be 1.6 ng / mL.

[0248] Referring to Figure 23, the highest exposure levels to RPE / choroid (Cmax 22.6 ng / mL, AUClast 292 ng / mL) were observed after injection of the formulation. LLOQ was 0.1 ng / mL.

[0249] Similarly, PEG concentrations tended to increase in the RPE / choroid, with a higher concentration observed from day 7 to day 28. The highest detected PEG concentration was observed in the RPE / choroid. Retinal, aqueous humor, and vitreous humor levels remained relatively constant throughout the entire study period. The degree of exposure in ocular tissue was determined to be RPE / choroid > retina > AH and VH (Table 20).

[0250] [Table 6]

[0251] In most test tissues, PEG exposure persisted (AH, VH, retina) or increased (RPE / choroid, plasma) until the final time point. In VH, exposure peaked at a Cmax of 0.277 ng / mL on day 3.

[0252] The values ​​for the VH half-life were calculated based on data from days 24 and 28 after administration and are shown in Table 22. The measurement revealed that the half-life of VH is in the range of 25 to 40 hours.

[0253] [Table 7]

[0254] Due to a lack of elimination phase data, accurate prediction of PK parameters in other tissues is not possible. The average PEG residence time for RPE and AH was found to be 15.7–16.8 hours by measurement.

[0255] 12. Effects of this product on alternative complement activation The complement system is a central effector of the innate immune system and is activated in response to either a pathogenic threat or disease-related pathology. The complement system is an enzyme cascade that functions to recruit inflammatory cells (via anaphylatoxins C3a, Ba, C5a, C4a), opsonize cells / remote for phagocytosis (via opsonins C3b, C1q and receptor CR3), and further directly lyse cells (via C5b-9). There are three main pathways for complement activation: the classical pathway (C1q, C2, C2a, C4, C4a), the alternative pathway (CFB, Ba, CFD, CFH, CFP), and the lectin pathway (MBL, MASP). All pathways converge at C3 and share a single terminal pathway (C5, C5b-9). Complement proteins circulating in the serum are produced in the liver, but inflammatory cells (macrophages, neutrophils, microglia, and astrocytes) also produce and secrete complement proteins. The retina has multiple sources of complement proteins (serum due to BBB breakdown, focal cells, and infiltrating cells). Complement proteins and receptors are present in a normal retina and are increased in many clinical diseases and disease models. The complement system, particularly alternative pathways, is strongly involved in the onset and progression of AMD. Multiple GWAS and candidate gene studies have identified variants of C3, C2, CFB, CFH, CFI, and C9 as being associated with AMD. In studies measuring complement protein concentrations in human aqueous humor samples, levels of C3, C3a, CFB, Ba, CFI, and CFH were higher in AMD patients than in control non-AMD patients. Therefore, we investigated the in vitro effects of this product on macrophages and complement proteins related to AMD (i.e., central complement protein C3, alternative pathway convertase stabilizers CFD and CFP, and alternative pathway inhibitor CFH).

[0256] Treatment with this product (SF208) reduces the expression of complement proteins by macrophages in vitro (and increases the expression of complement inhibitors). THP1 cells (human macrophage cell line) were cultured in 96-well plates, immediately differentiated in PMA for 2 days, rested for 1 day, and then treated as follows: medium control, LPS control (1 μg / ml), sucrose vehicle control (1%), LPS + SF208 (0.1 mg / ml), LPS + SF208 (0.3 mg / ml), LPS + SF208 (1 mg / ml). SF208 is batch SF22000208 of AVD104. The supernatant was collected 1 day after treatment and stored at -20°C. The complement protein concentration of supernatant samples (1:2 dilution) was tested using Quidel's MicroVue Multiplex Complement ELISA kits (#A900, #A916) equipped with a Quansys Q-View imager LS (#104150GR).

[0257] The results shown in Figure 24A indicate that C3, the central protein of all complement pathways, is increased by LPS and decreased by this product. The results shown in Figure 24B indicate that complement factors D (CFD) and P (CFP), which are stabilizing factors of alternative pathway convertases, are decreased by this product. The results shown in Figure 24C indicate that complement factor H (CFH), a potent inhibitor of alternative pathways, may be slightly increased by this product.

[0258] 13. Data Overview and Future Research This product contains a uniquely sized sialic acid polymer designed to specifically interact with inhibitory Siglec receptors on immune cells, aiming to repolarize macrophages and microglia into a quiescent state. This product is a nanoparticle intended to address severe chronic "non-converging" inflammation in two ways: (1) by reprogramming inflammatory macrophages that modulate inflammatory pathobiology into a quiescent state, and (2) by downregulating the complement cascade, when administered via IVT injection to patients with AMD (particularly geographic atrophy secondary to AMD) and other ophthalmic diseases. In vitro and in vivo studies using this product have demonstrated the safety of this approach, and indications of efficacy have been shown in multiple preclinical models.

[0259] Based on non-clinical toxicity studies in monkeys, the no-observed-adverse-effect level (NOAEL) was determined to be 0.5 mg total solids / eye. The initial dose for human clinical trials will take into account all formulation release criteria from toxicological, PK, and pharmacodynamic studies in monkeys and rabbits, as well as in vivo and in vitro information. The initial trial will be a single-dose trial starting with a dose below therapeutic level and escalating to the pharmacological or predicted therapeutic range, as recommended in the M3(R2) Human Clinical Trial Implementation and Non-Clinical Safety Studies for Marketing Authorization Submissions (US FDA, Guidance for Industry, 2010). Dose escalation will be stopped when dose-limiting toxicity (DLT) is observed in a small participant group (n=6). Based on a 60 kg individual, this corresponds to a systemic exposure of 11 mg and 1.3 mg / eye. However, the planned initial dose in humans will be even lower, at 0.05 mg / eye.

[0260] The first-instance (FIH) human trial will be divided into two parts. Part 1 will be an open-label MTD trial, including a single intravenous dose administered to the test eye. Part 2 will consist of three randomized, blinded groups. The primary objective of Part 1 is to determine the safety and MTD of AVD-104 (product) after a single dose via IVT injection using a 3+3 design. Eligible participants will be those with evidence of GA secondary to AMD who meet the selection criteria.

[0261] In short, if three participants are enrolled in Cohort 1 and no DLTs are observed within the first 28 days, these three participants are enrolled in Cohort 2, which is the next higher dose. If DLTs are observed in the first three participants of either cohort, three additional participants are mobilized to that dose. If at least one of the six participants has a DLT, the dose can be escalated to the next dose level. If two or more participants have a DLT, the dose escalation is stopped, and the MTD is declared as the previous dose. If there are fewer than two DLTs at the highest dose, six additional participants are administered to both the intermediate and high doses, so that nine participants are administered to each dose level, providing more data and reliability at these doses. If two or more participants have a DLT in Cohort 1, the dose is reduced by 50% in the next cohort and used as the new lowest dose. After the initial 28-day DLT observation period, all participants are followed up for safety for three months.

[0262] Part 2 consists of three randomized, blinded groups: high-dose product, low-dose product, and sham injection. If supported by planned nonclinical multi-dose toxicity studies and Part 1 PK data, the dosing interval will be extended to every other month or longer for all groups.

[0263] The main selection criteria are the presence of GA secondary to AMD, age 55 years or older, and no prior treatment for neovascular AMD in the eyes being tested. The transparent media must be sufficiently clear, and pupillary dilation must be sufficient to enable high-quality fundus imaging. The area of ​​the GA should be 2.5 mm². 2 17.5mm 2 The following conditions apply, and at least one lesion ≥ 1.25 mm 2 Participants must have (0.5 times the optic nerve head area). In Part 1, participants may have a history of central GA (subfoveal) and CNV in the same eye (currently inactive). In Part 2, central GA and a history of CNV in either eye are excluded.

[0264] The primary exclusion criteria are the presence of a RPE tear or other macular lesions such as macular hole, epiretinal membrane, toxic maculopathy, or diabetic retinopathy. Additional exclusion criteria include aphakia, myopia >8 diopters, and a history of intraocular surgery (excluding cataract surgery) more than three months prior, corneal transplantation, uveitis, glaucoma, or herpes infection. The presence of any significant ophthalmic disease that may affect vision in the next two years is also an exclusion criterion.

[0265] Risks to participants in this study include standard risks associated with IVT injections (e.g., endophthalmitis, retinal tear or retinal detachment, or lens damage), all of which can cause vision loss or eye loss. Risks associated with the study drug include inflammation and loss of retinal function. Non-clinical studies in mice, rabbits, and NHP have shown a low risk of severe vision loss.

[0266] Benefits for participants in this study include more frequent evaluations, which increases the likelihood of detecting changes in eye condition. Early detection of significant visual changes in participants with AMD may promote better visual outcomes. Participants receiving this product may also experience an improvement in the clinical course of their underlying AMD condition.

[0267] V. Further characterization of the biological activity of this product 1. Transcriptome analysis of AMD ophthalmology Transcriptome analysis was performed to investigate changes in Siglec expression in eyes with AMD. The GSE135092 dataset, containing RNA-seq data from eyes clinically diagnosed with AMD using the AREDS classification and aged 59–98 years, was used for this analysis. This dataset is described below: Orozco, LD, et al., “Integration of eQTL and a Single-Cell Atlas in the Human Eye Identifies Causal Genes for Age-Related Macular Degeneration.” Cell Rep, 2020.30(4):p.246-1259.e6. This dataset consisted of bulk RNA-seq data obtained from retinal tissue in the macular and non-macular (peripheral) regions of 129 post-mortem donors (106 controls and 23 AMD patients). The analysis was performed using the R software described in Team, RC, “R: A Language and Environment for Statistical Computing.” 2021. Statistical analysis was performed using Student's t-test, or, in the case of non-parametric data, the Wilcoxon test.

[0268] The results are shown in Figure 26. The results clearly show an increase in Siglec 7 expression in AMD patients compared to healthy patients.

[0269] 2. Measurement of IL-12 production inhibition by this product Peripheral blood mononuclear cells (PBMCs, Stem Cell Research, Cat. No-70500.2) were activated to the M1 phenotype (interferon-γ 50 ng / mL, LPS 10 ng / mL) over 48 hours. Next, the cells were treated overnight with the product (batch SF22000208, 0.01-1 mg / mL) in a continuous concentration range. LPS was used as a positive control. The supernatant was collected and assayed for IL-12 by ELISA (R&D Systems, DY1270-05). In vitro AVD-104 significantly downregulated the amount of IL-12 in the supernatant of activated macrophages. ****p<0.0001 (Dunnett's multiple comparison test).

[0270] The results are shown in Figure 27.

[0271] 3. Measurement of complement inhibitory activity by this product in human macrophages Administration of this product reduces the expression of complement proteins by macrophages in vitro (and further increases the expression of complement inhibitors).

[0272] PBMCs (Stem Cell Research, Cat. No-70500.2) were activated to the M1 phenotype (interferon-γ 50 ng / mL, LPS 10 ng / mL) over 48 hours. Next, the cells were treated overnight with the product (batch FG2200012, 0.01-3 mg / mL) in a continuous concentration range. One day after treatment, the supernatant was collected and stored at -20°C. The supernatant samples (1:2 dilution) were tested for complement protein concentration using Quidel MicroVue Multiplex Complement ELISA kits (#A900, #A916) equipped with a Quansys Q-View imager LS (#104150GR).

[0273] The results are shown in Figures 28A-28C and 29A and 29B. As can be seen from the figures, this product reduces overall complement activity. Specifically, a decrease in the levels of C3a, C5a, and sC5b-9 (Figures 28A-28C) and a decrease in the levels of Ba and Bb (Figures 29A and 29B) were demonstrated.

[0274] VI. Further characterization of the biological activity of this product in non-human primates. 1. Summary of Results The purpose of this study is to determine the potential toxicity of AVD-104 when administered intravitreousally every two months or every month for 39 weeks to cynomolgus monkeys (also known as non-human primates or NHPs), and to evaluate the reversibility, persistence, or delayed onset of toxic effects after an 8-week recovery period.

[0275] For this one-month interim report, monkeys received a single intravitreal injection of 0 mg / eye of AVD-104 (vehicle, 5 / sex), 0.5 and 1.0 mg / eye of AVD-104 (3 / sex / group), and 1.5 mg / eye of AVD-104 (5 / sex) into their left eye. The right eye was left untreated in the vehicle group, or sham-injected in the AVD-104 group.

[0276] This one-month interim report evaluated the following parameters and endpoints: mortality, clinical observation, body weight, ophthalmology, intraocular pressure (IOP), and cardiovascular safety pharmacology (electrocardiogram [ECG], blood pressure, heart rate, and body temperature). Electroretinography (ERG), optical coherence tomography (OCT), clinicopathology, and toxicological results were not included.

[0277] No animals were found to be in a mortal state or confirmed to have died by the end of the one-month interim phase. No changes in body weight or cardiovascular safety pharmacological parameters related to the test substance were observed by the end of the one-month interim phase.

[0278] The main AVD-104-related findings in males and females administered 1.5 mg / eye were: acute, transient, mild, or severe aqueous humor flare (1+ or 4+, respectively) resolved by the end of the first week after treatment with TOBRADEX® and a mydriatic agent; secondary IOP decrease observed in females on day 7; mild to moderate cytoid opacities in the anterior chamber of the vitreous from day 6 to day 28 (1-2+) (severity reduced on day 28); and minor vitreous opacity (0.5+) on day 28. Cytoid vitreous opacities and vitreous opacities may be attributable to AVD-104 particles due to inflammatory cells and / or precipitation.

[0279] Ophthalmic findings at doses of ≤1.0 mg / eye were limited to dose-dependent vitreous cell-like opacities, which were mild at 0.5 mg / eye and mild to moderate on day 7 at 1.0 mg / eye, and partially resolved by day 28. All ophthalmic findings after a single intravitreal dose of ≤1.5 mg / eye were considered non-adverse events due to their transient onset or low severity.

[0280] 2. Conclusion In conclusion, administration of AVD-104 at doses of 0.5, 1.0, and 1.5 mg / eye via single unilateral intravitreal injection in conjunction with the 1-month interim evaluation did not cause any drug-related changes in body weight or cardiovascular safety pharmacological parameters. Non-adverse ophthalmic findings associated with AVD-104 at 1.5 mg / eye included acute, transient, mild, or severe aqueous humor flare that resolved by day 7, secondary IOP decrease on day 7, and mild vitreous opacity on day 28. At doses of 1.5 mg / eye or less, dose-related mild to moderate vitreous cell-like opacity was observed on day 7 and partially resolved by day 28.

[0281] At doses of 1.5 mg / eye or less, dose-related mild to moderate vitreous cell-like opacity was observed on day 7, and partially resolved by day 28.

[0282] Fundus photographs and optical coherence tomography (OCT) images were collected and analyzed after the 6th injection in the control group and the low-dose group (0.5 mg / eye), and after the 3rd injection in the intermediate-dose group (1 mg / eye) and the high-dose group (1.5 mg / eye). No abnormalities or adverse effects were observed.

[0283] Since no adverse effects were observed with 1.5 mg / eye NHP administration, the "clinical dose" in humans was set at 3 mg / eye.

[0284] VI. Further Characterization of AVD-104 1. AVD-104 inhibits the binding of Siglec-7 and Siglec-9 to human PANC-1 cells. The binding of AVD-104 to Siglec was evaluated by an ELISA-type binding assay that assessed the specific binding ability of AVD-104 to the extracellular domains of Siglec-7 and 9, which are conjugated to the C-terminal Fc tag.

[0285] Competitive binding assays revealed that AVD-104 inhibits the binding of Siglec-7 Fc and Siglec-9 Fc proteins to sialic acid-expressing Panc-1 cells (50% reduction in Siglec-9 binding at 0.5 mg / mL, and 28% reduction in Siglec-7 binding at 0.25 mg / mL (Figure 30, asterisked bars)). This result indicates that AVD-104 inhibits the binding of Siglec-7 / 9 Fc to Panc-1 cells, since AVD-104 binds to the latter.

[0286] 2. AVD-104 reduces oxLDL-mediated inflammation. The effect of AVD-104 on macrophages activated with LPS or oxidized (Ox)LDL was evaluated using an MTT cell viability assay. The results showed that no cytotoxic effect of AVD-104 was observed when used at concentrations of 1.0–0.01 mg / mL on OxLDL-treated macrophages (Figure 31A).

[0287] AVD-104 demonstrated dose-dependent inhibition of TNF-α production in both OxLDL and LPS-treated macrophages compared to a 10% sucrose vehicle (dashed line) (p<0.05 and p<0.001, one-way ANOVA) (Figure 31B).

[0288] VII. Clinical trials of AVD-104 in patients with geographic atrophy As demonstrated in in vitro and in vivo experiments, AVD-104 has the following dual mechanisms: In the cellular arm of the innate immune response, AVD-104 acts by binding to Siglec and repolarizing hyperactivated macrophages, and in the humoral arm, AVD-104 binds to complement factor H to downregulate complement overproduction.

[0289] Further investigations are being conducted on the effects of AVD-104 on patients with geographic atrophy (GA) (NCT05839041).

[0290] In short, an ongoing clinical trial validated AVD-104 as a treatment for GA secondary to age-related macular degeneration (AMD). Part 1 of the trial was a multicenter, open-label, single-dose safety study involving four cohorts to investigate safety, tolerability, and dose-limiting toxicity. The trial design is shown graphically in Figure 32.

[0291] The interim results of this study are shown in the table in Figure 33. In this study, the best corrected visual acuity (BCVA) was the number of characters on the Schellen visual acuity chart or an equivalent chart that the patient was able to read (also called the BCVA score). A baseline measurement was established. Increases or decreases were measured as the difference between the baseline BCVA score and the BCVA score at subsequent measurement points.

[0292] Initial data suggest that AVD-104 may have the potential to suppress the long-term progression of GA lesions from the anterior edge of the lesion. Data collected from patients 4 and 5 in Cohort 2 are shown in Figure 34, which demonstrate a significant reduction in hyper-AF area at the anterior edge of the lesion over a one-month period. The dose of AVD-104 is expressed in mg / eye.

[0293] The hyper-AF (HAF) area and BCVA score for patient 5 in Cohort 2 are also shown in Figures 35A and 35B (BCVA (OS test eye): 29 (screening), 28 (baseline), 33 (day 15), 33 (1 month later), 33 (2 months later), HAF area: 1.03 mm²). 2 (Baseline), 0.66mm 2 (One month later).

[0294] Representative aqueous humor biomarker analysis was also performed on patient 5 in Cohort 2. The measurement results for IL-6, complement C3a and C4a, and complement factor H (CFH) are shown in Figures 36A to 36D.

[0295] Figure 37 shows the design of the second part of the aforementioned AVD-104 clinical trial. This second part of the trial is a randomized, sham-controlled, active-controlled trial that evaluates the efficacy of reducing the proliferation rate of GA lesions. 290 patients are expected to be enrolled, of whom 250 will be randomized.

[0296] In summary, AVD-104 is a novel dual-mechanism therapy for GA. The mechanism of action of AVD-104 involves macrophage repolarization and complement inhibition to target the pathobiology of GA. While available complement inhibitors address only one arm of the innate immune response and can deplete complement factors, AVD-104 repolarizes hyperactivated macrophages and inhibits the hyperamplified complement system.

[0297] VIII. Additional results of the AVD-104 clinical trial in patients with geographic atrophy. Additional data on the efficacy of AVD-104 in patients with geographic atrophy (GA) were collected during the clinical trial NCT05839041 (see Part VII above).

[0298] The study design is shown graphically in Figure 32. Briefly, four cohorts were followed, each defined by AVD-104 dose (Cohort 1: 0.1 mg, Cohort 2: 0.5 mg, Cohort 3: 1 mg, Cohort 4: 3 mg / eye). Patients in each cohort suffered from bilateral GA. Each patient received treatment in one eye (test eye: SE) but not in the other eye (companion eye: FE). Furthermore, in certain cases, data collected during the study were categorized into "all lesions" and "excluding large lesions." Here, "large lesions" were defined as those with an area of ​​15.5 mm². 2 The above was considered to be the lesion.

[0299] Autofluorescence data (GA lesion area, mm²) obtained by combining the results of cohorts 3 and 4 2 The results are shown in Figure 38A (percentage change in lesion area from baseline) and Figure 38B (mean change in lesion area from baseline). These data indicate that AVD-104 slows the growth of GA lesions compared to the same eye, which also has GA.

[0300] The collected autofluorescence data enabled inter-study comparisons with standard of care (SoC) drugs such as Izervay and Syfovre. Specifically, for cohorts 3 and 4, the mean change in lesion area as a function of time was plotted against similar data from clinical trials using standard of care (SoC) drugs. The results excluding large lesions and out-of-window (late) measurements are shown in Figure 39 (Syfovre trial OAK and Derby) and Figure 40 (Iservay). Note that for the data shown in Figures 39 and 40, AVD-104 was administered by a single injection at baseline, Syfovre by three injections (monthly treatment), and Izervay by three injections (monthly treatment). The SoC data is 17.5 mm. 2Lesions exceeding this size are excluded. In this study, the size was 15.5 mm. 2 ~17.5mm 2 No patients had lesions. Since the lesions in this study were larger than the mean lesions in the Syfovre and Izervay public series, they should have shown a larger area increase than the active control group. Compared to the active control, which received injections three times a month, GA lesion growth in eyes treated with AVD-104 was smaller than that observed in eyes treated with either Izervay or Syfovre, even though AVD-104 was administered only as a single injection.

[0301] Figure 41 shows hyperautofluorescence data around the lesion (excluding pooled cohort and out-of-window evaluations). The periphery of the GA lesion, called the junction zone, is the site of most active inflammation, indicated by hyperautofluorescence (hyper-AF). This area of ​​hyperAF increased in the fellow eye and significantly decreased in the AVD-104 treated eye, demonstrating the positive effect of AVD-104 in reducing inflammation.

[0302] For each patient in the cohort, the Best Corrected Visual Acuity (BCVA) score was determined. The BCVA score is the number of characters on the ETDRS visual acuity chart that the patient was able to read. A baseline measurement was established. Increases or decreases were measured as the difference between the baseline BCVA score and the BCVA score at subsequent measurement points. The score was evaluated using the ETDRS visual acuity test.

[0303] Figure 42 shows the BCVA scores (mean change from baseline) for all cohorts. A clear dose-response relationship is observed in cohorts 3 and 4, where participants who received higher doses showed improved visual acuity during the study period. The natural course of GA is a decrease of 1-2 letters over 3 months.

[0304] Tables 18 (test eye) and 19 (fellow eye) show the increase or decrease in BCVA scores across the entire cohort. While most patients had stable or improved BCVA scores in the test eye, the majority of patients showed a decrease in BCVA scores in their fellow eye.

[0305] [Table 8]

[0306] [Table 9]

[0307] Figure 43 further shows an analysis of the number of patients whose letter count decreased or increased on the BCVA test for all cohorts. This figure shows that visual acuity improvement was not achieved by only one or two participants, but was maintained or improved by the majority of participants on the BCVA test.

[0308] An analysis of BCVA testing, similar to that shown in Figure 43, is presented in Figures 44A and 44B for the combined group of cohorts 3 and 4.

[0309] The collected BCVA data allowed for inter-trial comparisons with standard-of-care (SoC) drugs, SYFOVRE PM and lamparizumab (Chroma&Spectri trials at 4qw and 6qw). Specifically, the mean change in BCVA over time in cohorts 3 and 4 was plotted against similar data from clinical trials using SYFOVRE PM and lamparizumab. The results are shown in Figures 45A and 45B. Note that for the data shown in Figures 45A and 45B, AVD-104 was administered as a single injection at baseline, Syfovre as three injections (monthly treatment), and lamparizumab as either every 4 weeks or every 6 weeks. These graphs comprehensively show that patients treated with Syfovre or lamparizumab experienced a decline in visual acuity over time, while participants treated with AVD-104 actually recovered their visual acuity.

[0310] Figure 46 is a bar graph showing the change in BCVA score at 3 months for each patient in each cohort (each bar represents one patient). This waterfall plot reaffirms that the majority of participants in cohorts 3 and 4 showed improvement in visual acuity.

[0311] In this study, multifocal electroretinography (mfERG) was performed on specific patients using equipment and methods designed by Diagnosys LLC, in accordance with the manufacturer's protocol.

[0312] Multifocal electroretinography (mfERG) measures the electrical signals of the entire macula as an indicator of visual function. In this test, the macula is divided into multiple hexagonal regions. The stimulus is a pseudo-random signal sequence consisting of alternating black and white hexagons several times per second. The signals are recorded using a standard ERG electrode configuration, and multifocal waveforms are generated using mathematical extraction. A typical multifocal waveform consists of a peak (P1) with troughs (N1 and N2, respectively) before and after it. The MfERG response originates primarily from cone on and off bipolar cells, with additional contributions from cone photoreceptors. The main cellular components of the mfERG response are on and off bipolar cells, with less contribution from cone photoreceptors. Therefore, mfERG can be readily used to distinguish between macular and generalized retinal dystrophy, and to locate retinal defects.

[0313] The data obtained in this clinical trial demonstrated excellent inter-trial reliability. In the Phase I trial, 3 out of 6 patients showed an increase in P1 voltage above normal range (≥20%) at 2 months, and in 5 out of 6 patients, the fellow eye remained within normal range (≤20%) at 3 months. At 3 months, the mean BCVA change from baseline in the eye treated with AVD-104 reached +6.0 letters. All subjects showed extremely poor response in the area of ​​severe RPE loss (GA area during FAF imaging). Three subjects (50% of those examined) showed significant improvement in the waveform around the lesion (waveforms in the atrophy area continued to show severe decline). This improvement in the waveform around the lesion generally lasted for 2 months, returning to the poor baseline response at 3 months.

[0314] Evaluation of eyes treated with AVD-104 showed that the majority of the improved mfERG response occurred in areas adjacent to the GA lesion. These data, based on completely objective evidence, demonstrate that AVD-104 has the ability to improve the function of residual photoreceptors in areas where GA is incomplete.

[0315] IX. Phase II trial of intravitreous AVD-104 in diabetic macular edema Patients with diabetic macular edema will be examined during the clinical trial NCT06181227.

[0316] This is a Phase II trial to determine the safety and preliminary efficacy of intravitreal injection of AVD-104 in reducing macular edema associated with diabetic retinopathy. The primary objective is to evaluate the tolerability and therapeutic effect of intravitreal injection (IVT) of AVD-104 in participants with diabetic macular edema (DME). Participants will receive either three intravitreal injections of low-dose AVD-104 (1.0 mg) at 28-day intervals or two intravitreal injections of high-dose AVD-104 (2.0 mg) at 56-day intervals. Continuous optical coherence tomography (OCT), ultra-wide-field fluorescein angiography, and OCT-A angiography will be performed to evaluate the therapeutic effect on foveal retinal thickness (CST) and non-perfusion areas. All participants will be followed up for safety until day 84. Planned enrollment will be limited to a maximum of 30 participants.

[0317] Participation criteria: • Diagnosis of diabetes (type 1 or type 2) as defined by the World Health Organization and / or the American Diabetes Association • Visual acuity loss due to DME (VA), BCVA character score based on ETDRS-like chart: 75-20 characters (equivalent to 20 / 32-20 / 320 Snellen) • DME:CST ≥ 325 μm, as indicated by macular thickening based on SD-OCT, including the central macula.

[0318] Exclusion criteria: • Any IVT anti-vascular endothelial growth factor (VEGF) treatment within 3 months prior to randomization • Any history of panretinal photocoagulation (PRP) treatment • Any use of Iluvien® (Alimera Sciences, Inc., Alpharetta, GA) within the past 3 years or Ozurdex® (Abbvie, Chicago, Illinois) or Xipere (Bausch & Lomb, Vaughan, Ontario, Canada) within the past 6 months • History of macular laser photocoagulation treatment • Any signs of high-risk proliferative diabetic retinopathy (PDR)

[0319] The arms and intervention items are listed in Table 21 below.

[0320] [Table 10]

[0321] Although the present invention has been specifically illustrated and described with reference to examples of its embodiments, those skilled in the art will understand that various modifications to the form and details can be made without departing from the scope of the invention as encompassed in the appended claims.

Claims

1. Structural formula (I): 【Chemistry 1】 (In the formula, P is poly(lactide-co-glycolide)-poly(ethylene glycol) copolymer (PLGA-PEG), and p is an integer between 4 and 200. A polysialic acid (PSA)-polymer conjugate compound represented by or a pharmaceutically acceptable salt thereof.

2. The compound according to claim 1, wherein P is PLGA(10k)-PEG(5k).

3. The following structural formula (II): 【Chemistry 2】 (In the formula, (where y is an integer between 1 and 1000, x is an integer between 1 and 1000, and m is an integer between 1 and 450) The compound according to claim 1, represented by [the given expression].

4. The compound according to claim 3, wherein y is an integer from 1 to 500, x is an integer from 1 to 500, and m is an integer from 1 to 250.

5. x is an integer between 90 and 140. y is an integer between 10 and 75, and The compound according to claim 4, wherein m is an integer between 90 and 140.

6. The compound according to any one of claims 1 to 5, wherein the value of p is selected from any one of the following ranges: 10 to 20, 20 to 30, 30 to 40, 40 to 50, and 50 to 60.

7. The compound according to any one of claims 1 to 5, wherein the value of p is selected from any one of the following ranges: 17-200, 17-100, 17-60, 17-25, 20-100, 20-60, 20-30, 22-100, 22-60, 22-30, and 22-25.

8. The compound according to any one of claims 1 to 7, wherein the value of p is 22.

9. The compound according to any one of claims 1 to 7, wherein P is PLGA(10k)-PEG(5k) and p is 15 to 25.

10. Particles comprising the compound described in any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof.

11. The particles according to claim 10, which are nanoparticles.

12. The particle according to claim 10 or 11, wherein the weight of the PSA per unit weight of the particle is 1 μg / mg to 1000 μg / mg.

13. The particle according to any one of claims 10 to 12, wherein the weight of PSA per unit weight of P is 10 to 75 μg / mg.

14. The particles according to any one of claims 10 to 13, wherein the average particle size is 80 nm to 120 nm.

15. A pharmaceutical composition comprising particles according to any one of claims 10 to 14 in a pharmaceutically acceptable carrier or diluent.

16. The pharmaceutical composition according to claim 15, which is aqueous and further comprises sucrose.

17. A method for treating a subject suffering from an ophthalmic disease, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 9, a particle according to any one of claims 10 to 14, or a pharmaceutical composition according to claim 15 or 16.

18. The method according to claim 17, wherein the ophthalmic disease is age-related macular degeneration (AMD).

19. The method according to claim 18, wherein the age-related macular degeneration is atrophic age-related macular degeneration.

20. The method according to claim 18, wherein the age-related macular degeneration is exudative age-related macular degeneration.

21. The method according to claim 17, wherein the ophthalmic disease is geographic atrophy secondary to AMD.

22. The method according to claim 17, wherein the ophthalmic disease is retinitis pigmentosa.

23. The method according to claim 17, wherein the ophthalmic disease is diabetic macular edema.

24. The method according to any one of claims 17 to 23, wherein the administration is performed intravitreously.

25. Structural formula (I): 【Transformation 3】 A method for preparing a polysialic acid (PSA)-polymer conjugate compound represented by or a pharmaceutically acceptable salt thereof, Structural formula (III) 【Chemistry 4】 The polymer represented by structural formula (IV) 【Transformation 5】 The PSA precursor represented by is reacted with the compound represented by structural formula (I) under conditions sufficient to form it. Including, in the formula, P is poly(lactide-co-glycolide)-poly(ethylene glycol) copolymer (PLGA-PEG), and p is an integer between 4 and 200.

26. The method according to claim 25, wherein P is PLGA(10k)-PEG(5k).

27. The compound represented by the above structural formula (I) is structural formula (II): 【Transformation 6】 (In the formula, (where y is an integer between 1 and 1000, x is an integer between 1 and 1000, and m is an integer between 1 and 450) The method according to claim 25, as represented by [the specified method].

28. The method according to claim 27, wherein y is an integer from 1 to 500, x is an integer from 1 to 500, and m is an integer from 1 to 250.

29. x is an integer between 90 and 140. y is an integer between 10 and 75, and The method according to claim 28, wherein m is an integer between 90 and 140.

30. The method according to any one of claims 25 to 29, wherein the value of p is selected from one of the following ranges: 10 to 20, 20 to 30, 30 to 40, 40 to 50, and 50 to 60.

31. The method according to any one of claims 25 to 29, wherein the value of p is selected from any one of the following ranges: 17 to 200, 17 to 100, 17 to 60, 17 to 25, 20 to 100, 20 to 60, 20 to 30, 22 to 100, 22 to 60, 22 to 30, and 22 to 25.

32. The method according to any one of claims 25 to 30, wherein P is PLGA(10k)-PEG(5k) and p is 15 to 25.

33. The process further includes the step of preparing the compound represented by the structural formula (IV), wherein the structural formula (V) 【Transformation 7】 The compound represented by the structural formula (VI) 【Transformation 8】 The method according to any one of claims 25 to 32, comprising reacting a compound represented by with conditions sufficient to prepare the compound represented by the structural formula (IV).

34. A method for increasing the best corrected visual acuity (BCVA) score in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a compound according to any one of claims 1 to 9, a particle according to any one of claims 10 to 14, or a pharmaceutical composition according to claim 15 or 16.

35. The method according to claim 34, wherein the increase in the BCVA score of the target is 1 to 4 characters.

36. The method according to claim 34, wherein the increase in the BCVA score of the target is 5 to 9 characters.

37. The method according to claim 34, wherein the increase in the BCVA score of the target is 10 to 14 characters.

38. The method according to claim 34, wherein the increase in the BCVA score of the target is 15 characters or more.

39. Structural formula (IV): 【Chemistry 9】 (In the formula, p is an integer between 17 and 200.) A compound represented by or a pharmaceutically acceptable salt thereof.

40. The compound according to claim 39, wherein the value of p is selected from any one of the following ranges: 17-100, 17-60, 17-25, 20-100, 20-60, 20-30, 22-100, 22-60, 22-30, and 22-25.

41. The compound according to claim 39, wherein p is 22.

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