Conjugates for neuroretinal drug delivery

Abstract

The present invention is in the field of drug delivery. The present invention relates to the use of monocarboxylic acids as ligands for receptors present, for example, in the retina. It has been found that these receptors are capable of mediating the uptake of these ligands and conjugates of these ligands. Such conjugates can be used in methods for treating or preventing neurodegenerative diseases, such as retinal degeneration.

Description

Detailed Description of the Invention

[0001] [Field of the Invention] The present invention is in the field of drug delivery. The present invention relates to the use of monocarboxylic acids as ligands for receptors present, for example, in the retina. It has been found that these receptors are capable of mediating the uptake of these ligands and conjugates of these ligands. Such conjugates can be used in methods for treating or preventing neurodegenerative diseases, such as retinal degeneration.

[0002] [Background of the invention] There are few drug treatments available for patients suffering from retinal diseases, and a key reason for this is the lack of suitable delivery systems that can achieve sufficiently high drug uptake in the retina and its photoreceptors.

[0003] Inherited retinal degeneration (IRD) refers to a group of diseases, such as retinitis pigmentosa and Leber's congenital amaurosis, characterized by the progressive loss of photoreceptors, ultimately leading to blindness. Generally, IRD-type diseases manifest as a primary loss of rod photoreceptors, which are responsible for vision under scotopic conditions. Therefore, early disease symptoms include night blindness. As rod cells are lost, cone photoreceptors, which mediate color and fine visual acuity under photopic conditions, also degenerate, ultimately leading to complete blindness. To date, IRD-type diseases remain essentially untreatable, and the development of new therapies is highly needed.

[0004] In many types of IRD, elevated levels of cyclic guanosine monophosphate (cGMP) are found in rod photoreceptors, caused by a decrease in phosphodiesterase 6 (PDE6). PDE6 regulates cGMP levels, and dysregulation can lead to pathological concentrations of cGMP, resulting in the overactivation of key cGMP-dependent proteins and ultimately cell death. To mitigate this effect, drug candidates that are inhibitory analogs of cGMP have been shown to promote photoreceptor survival in mouse models of IRD (Vighi, E., et al., PNAS, 2018, 115(13):E2997-E3006). Rescue of rod photoreceptors can provide functional protection for cone photoreceptors.

[0005] To ensure successful translation for clinical use, drugs such as cGMP analogs must be delivered to photoreceptors at concentrations high enough to provide protective effects. Therefore, an appropriate drug delivery system is needed. Glutathione-conjugated liposomes have previously been shown to enhance the therapeutic efficacy of CN03 in an IRD mouse model after systemic administration (Vighi et al., supra). These liposomes were designed to allow the drug to penetrate the blood-retinal barrier. However, to achieve high drug concentrations at the target and limit systemic exposure, intravitreal (IVT) administration is preferred. There is a need for an administration method that can directly target photoreceptors to achieve therapeutic effects.

[0006] Monocarboxylate transporters (MCTs) are membrane-bound transporters highly expressed in tissues such as the retina, which have significant energy demands and therefore high metabolic turnover rates (Halestrap, AP and MC Wilson, IUBMB Life, 2012. 64(2): p. 109-19). MCTs are sometimes highly expressed on photoreceptors due to the demand for lactate shuttle between photoreceptors and Müller glial cells. MCTs have a variety of substrates, including lactate, pyruvate, ketone bodies, and short-chain fatty acids. MCTs have been used as a means of targeting drug delivery to the brain (Venishetty et al., doi:10.1016 / j.nano.2012.08.004).

[0007] There is a need for delivery strategies that target the retina or photoreceptors.There is a need for agents and compositions that can be used for targeting the retina or photoreceptors.

[0008] [Summary of the Invention] We identified strong lactate transporter (also known as monocarboxylate transporter, MCT) expression on photoreceptors as a target for drug delivery vehicles. Nanoparticles were conjugated with different monocarboxylic acids as ligands, including lactate, pyruvate, and cysteine. For example, monocarboxylic acid-conjugated, dye-loaded liposomes were tested in both human-derived cell lines and mouse retinal explant cultures. Nanoparticles conjugated with appropriate ligands were found to consistently exhibit higher cellular uptake than unconjugated nanoparticles. Pharmacological inhibition of MCT1 and MCT2 reduced internalization, demonstrating an MCT-mediated uptake mechanism. In a particularly attractive embodiment, pyruvate-conjugated liposomes loaded with various retinal drug substances reduced photoreceptor cell death in mouse rd1 and rd10 retinal degeneration models. Notably, free drug solutions did not achieve the same therapeutic effect in these models.

[0009] The present invention provides a conjugate comprising a monocarboxylate transporter (MCT) ligand and a pharmaceutically acceptable nanoparticle. Preferably, the monocarboxylate transporter is at least monocarboxylate transporter 1 (MCT1), monocarboxylate transporter 2 (MCT2), or monocarboxylate transporter 4 (MCT4). Preferably, the ligand specifically binds to or is taken up by target cells at a rate that is at least 10% improved compared to a control condition selected from: a) uptake in cells lacking expression of a monocarboxylate transporter; b) uptake in cells pretreated with an MCT inhibitor; and c) uptake of a reference conjugate lacking the monocarboxylate transporter ligand, when measured at least two hours after contacting the ligand with retinal cells.

[0010] In some embodiments, the pharmaceutically acceptable nanoparticles are liposomes, solid lipid nanoparticles, micelles, carrier proteins, metal nanoparticles, polyplex systems, lipoplex systems, or polymeric nanoparticles. Preferably, the pharmaceutically acceptable nanoparticles comprise one or more phospholipids. Preferably, the pharmaceutically acceptable nanoparticles comprise one or more non-cationic lipids.

[0011] In some embodiments, the pharmaceutically acceptable nanoparticles further comprise a pharmaceutical agent, preferably a neuroprotective agent, such as a photoreceptor rescue drug, preferably a cyclic guanosine monophosphate (cGMP) analogue.

[0012] The ligand preferably comprises a free carboxylic acid moiety contained in a short chain fatty acid, an amino acid, or a keto acid. The pharmaceutically acceptable nanoparticle preferably comprises a water-soluble polymer on its surface. It is highly preferred that the ligand is conjugated to a water-soluble polymer. Preferably, the ligand has the general formula (I): [ka] wherein X is S, O, Se, or NH; c is —CH—, —CH(CH)—, —C(═O)—, —C(═S)—, —C(═NH)—, —CH(—OH)—, —CH(NH)—, —CH(halogen)—, or —C(halogen)—; n is 1, 2, or 3; and R is —H, —CH, ═O, ═S, ═NH, —OH, —NH, or halogen; and * is a conjugation site for a pharmaceutically acceptable nanoparticle) It is of the type.

[0013] General formula (A): [ka] wherein X is S, O, Se, or NH; c is —CH—, —CH(CH)—, —C(═O)—, —C(═S)—, —C(═NH)—, —CH(—OH)—, —CH(NH)—, —CH(halogen)—, or —C(halogen)—; n is 1, 2, or 3; R is —H, —CH, ═O, ═S, ═NH, —OH, —NH, or halogen; and Q is a conjugate of a lipid and a water-soluble polymer, wherein the lipid is preferably a phospholipid. Also provided is a compound of the formula:

[0014] Also provided is a conjugate as defined above for use as a medicament. Preferably, the medicament is for treating a neurodegenerative disease or a retinal disease, such as inherited retinal degeneration (IRD), glaucoma, age-related macular degeneration, Stargardt disease, Usher syndrome, geographic atrophy, diabetic retinopathy, retinitis pigmentosa, Leber's congenital amaurosis, blindness, loss of rod photoreceptors, night blindness, loss of cone photoreceptors, color blindness, loss of color vision, and loss of high-level visual acuity. Accordingly, the present invention also provides a method for treating, delaying, or preventing a neurodegenerative disease or a retinal disease, the method comprising administering to a subject a conjugate as defined above.

[0015] [Description of the Invention] Surprisingly, it was found that a strong lactate transporter (also known as monocarboxylic acid transporter, MCT) is expressed on photoreceptors, and the present invention provides conjugates that allow targeting of nanoparticles to MCT and thus to photoreceptors. i) a ligand for the monocarboxylate transporter (MCT); ii) pharmaceutically acceptable nanoparticles; The present invention provides a conjugate comprising:

[0016] Such conjugates are referred to herein as "conjugates" or "conjugates according to the invention", as will be clear from the context. The nanoparticles and the ligand form the conjugate. The MCT ligand is conjugated to a pharmaceutically acceptable nanoparticle, sometimes referred to herein for short as nanoparticles.

[0017] A "conjugate" is defined herein as consisting of two entities bound to each other. Preferably, the two entities are conjugated by a covalent, non-covalent, or coordinate chemical bond. Preferably, the two entities are conjugated by a covalent or non-covalent bond. An example of a non-covalent bond is the interaction of biotin with avidin or streptavidin or another analogue. The two entities can be linked directly or via a (non-)cleavable spacer, linker, or other component. In a preferred embodiment, the MCT ligand is covalently bound to a hydrophobic moiety contained in the nanoparticle, more preferably contained in the lipid phase of the lipid nanoparticle, e.g., the lipid bilayer of a liposome. This hydrophobic moiety can be a lipid, a sterol, e.g., cholesterol, a steroid, a vitamin or a derivative thereof, e.g., vitamin D or a derivative thereof, vitamin E or a vitamin E derivative (e.g., as described in EP 05292820), a C8-30 alkane, a C6-30 (poly)cyclic alkane, or a C6-30 aromatic moiety. Preferably, the hydrophobic moiety is a lipid or a sterol, more preferably a lipid. Throughout this application, the preferred lipid is a phospholipid. Throughout this application, the more preferred lipid is a natural phospholipid. In this regard, it will be recognized by those skilled in the art that if the hydrophobic moiety is a lipid nanoparticle, it will phase-separate into the lipid phase of the nanoparticle. For example, if the hydrophobic moiety is a phospholipid, in the case where the nanoparticle is a liposome, it will associate to form the lipid bilayer of the liposome. These techniques are widely known in the art, and those skilled in the art will be able to select an appropriate hydrophobic moiety.

[0018] In a preferred embodiment, the MCT ligand is linked to the hydrophobic moiety via a linker or spacer. In a preferred embodiment, the ligand is solvent accessible. A preferred linker is a water-soluble polymer. A water-soluble polymer, as defined below, is conjugated to the MCT ligand at one end and to the hydrophobic moiety at the other end. In a preferred embodiment, the MCT ligand is linked to a phospholipid PEG, as disclosed, for example, in U.S. Patent Application Publication No. 2015 / 0157733. In the most preferred embodiment, it is conjugated to 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000 (DSPE-mPEG2000) or, advantageously, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000]. Those skilled in the art can select which chemistry is suitable for forming the required bond.

[0019] A variety of methods for conjugating ligands to nanoparticles, lipids, or polymers are known in the art. Such methods are described, for example, in Hermanson (1996, Bioconjugate Techniques, Academic Press), U.S. Pat. No. 6,180,084, and U.S. Pat. No. 6,264,914, and include methods commonly used in immunological applications, such as those used to link haptens to carrier proteins (see Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Given the wide variety of conjugation methods available, those skilled in the art can find one that does not affect or has minimal impact on the functionality of the linked entity. Suitable methods for conjugating ligands to agents or carriers include, for example, carbodiimide conjugation (Bauminger and Wilchek, 1980, Meth. Enzymol. 70:151-159). Alternatively, the agent or carrier can be conjugated to the ligand as described in Nagy et al. Proc. Natl. Acad. Sci. USA 95:1794-1799 (1998). Other methods of conjugation that may be suitably used include, for example, sodium periodate oxidation followed by reductive alkylation of suitable reactants and glutaraldehyde crosslinking. The use of active esters or Michael addition is highly preferred.

[0020] [Ligand] The ligand of MCT is preferably a monocarboxylic acid. In a preferred embodiment, the ligand preferably comprises a free carboxylic acid moiety contained in a short-chain fatty acid, an amino acid, or a keto acid. As known to those skilled in the art, the carboxylic acid may be converted into its conjugated base -COO depending on its environment. -The -COOH moiety can be: Preferred ligands contain a single carboxylic acid moiety. The carboxylic acid moiety is free and therefore not available for conjugation, for example, as a donor acid via the formation of an ester or amide. The ligand is conjugated to the nanoparticle via another moiety other than the carboxylic acid. Because the ligand is conjugated, it can be considered as a radical of the actual ligand.

[0021] Short-chain fatty acids are fatty acids having fewer than six carbon atoms. Suitable short-chain fatty acids are methanoic acid, ethanoic acid, propanoic acid, butanoic acid, 2-methylpropanoic acid, pentanoic acid, 3-methylbutanoic acid, and 2-methylbutyric acid. Examples of suitable radicals for short chain fatty acids are -X-COOH, -X-CH2COOH, -X-CH2CH2COOH, -X-CH2(CH2)2COOH, -XC(CH3)2COOH, -X-CH2CH(CH3)COOH, -X-CH2(CH2)3COOH, -XC(CH3)2CH2COOH, -X-CH2CH(CH3)CH2COOH, -X-CH2CH2CH(CH3)COOH, more preferably -X-CH2COOH, -X-CH2CH2COOH, -X-CH2(CH2)2COOH, -XC(CH3)2COOH, -X-CH2CH(CH3)COOH, -X-CH2(CH2) 3COOH, -XC(CH3)2CH2COOH, -X-CH2CH(CH3)CH2COOH, -X-CH2CH2CH(CH3)COOH, and even more preferably -X-CH2CH2COOH, -X-CH2(CH2)2COOH, -XC(CH3)2COOH, -X-CH2CH(CH3)COOH, -X-CH2CH(CH3)COOH, -X-CH2(CH2)3COOH, -XC(CH3)2CH2COOH, -X-CH2CH(CH3)CH2COOH, and -X-CH2CH2CH(CH3)COOH, where X is absent or a heteroatom, preferably O, N(H), Se, or S, and most preferably S. In some embodiments, X is S. In some embodiments, X is absent. A preferred fatty acid in this regard is -X-CH2(CH2)2COOH.

[0022] Amino acids are widely known. Preferred amino acids are short-chain fatty acids containing -NH2 at the carbon atom directly adjacent to the carbonyl moiety. Examples of suitable radicals for the ligand are -X-CH(NH2)COOH, -X-CH2CH(NH2)COOH, -X-(CH2)2CH(NH2)COOH, -X-CH2C(NH2)(CH3)COOH, -XC(CH3)2CH(NH2)COOH, -X-CH2CH(CH3)CH(NH2)COOH, and -X-CH2CH2C(NH2)(CH3)COOH, where X is absent or a heteroatom, preferably O, N(H), Se, or S, most preferably S. In some embodiments, X is S. In some embodiments, X is not absent. The amino acid is preferably an L-amino acid. When X is S, -X-CH2CH(NH2)COOH can be considered as cysteine ​​and is a highly preferred amino acid in this regard.

[0023] A keto acid is a carboxylic acid that also contains an oxo moiety. Preferred keto acids are those in which the oxo moiety is on the carbon atom immediately adjacent to the carboxylic acid moiety. Examples of suitable radicals for keto acids are -XC(=O)COOH, -X-CHC(=O)COOH, -X-CHCHC(=O)COOH, -X-CH(CH)C(=O)COOH, -XC(CH)C(=O)COOH, and -X-CHCH(CH)C(=O)COOH, more preferably -X-CHC(=O)COOH, -X-CHCHC(=O)COOH, -X-CH(CH)C(=O)COOH, -XC(CH)C(=O)COOH, and -X-CHCH(CH)C(=O)COOH, where X is absent or a heteroatom, preferably O, N(H), Se, or S, most preferably S. In some embodiments, X is S. In some embodiments, X is not absent. A highly preferred keto acid in this context is -X-CH2C(=O)COOH.

[0024] Preferably, the ligand specifically binds to or is taken up by target cells at a rate that is at least 10% improved compared to a control condition selected from: a) uptake in cells lacking expression of a monocarboxylic acid transporter; b) uptake in cells pretreated with an MCT inhibitor; and c) uptake of a reference conjugate lacking a ligand for a monocarboxylic acid transporter, when measured at least 2 hours after contacting the ligand with the retinal cells.

[0025] The term "specific binding" as used herein means binding that is somewhat different from non-specific interactions. Specific binding can be measured, for example, by determining the binding of a molecule (ligand) compared to the binding of a control molecule (ligand), which is generally a molecule of similar structure that does not have (specific) binding activity, for example, a peptide of similar size that lacks a specific binding sequence. Specific binding exists when the ligand has a measurably higher affinity for the receptor than the control ligand. The specificity of binding can be determined, for example, by competition with a control ligand known to bind to the target. The term "specific binding" as used herein includes low-affinity and high-affinity specific binding. Specific binding can be measured, for example, by a specific binding activity of at least about 10 -4 A low affinity targeting agent having a Kd of at least about 10 M can be used. For example, if a receptor has multiple binding sites for a ligand, a ligand with low affinity can still be useful. Specific binding can be achieved by a high affinity ligand, for example, at least about 10 M. -7 M, at least about 10 -8 M, at least about 10 -9 M, at least about 10 -10 M or at least about 10 -11 M or 10 -12 It can also be exhibited by a ligand that can have a Kd of at or above M. Both low and high affinity targeting ligands are useful for incorporation in the conjugates of the invention.

[0026] Specific binding or uptake is preferably enhanced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400% or more, more preferably at least 100% or more, and most preferably at least 250% or more, where enhancement is measured 2, 3, 4, 5, 6, 7, 8 hours or more after contacting the ligand with the target cells.

[0027] The cells lacking expression of monocarboxylate transporters are, for example, pancreatic beta cells or cells treated to reduce or eliminate MCT expression, for example, by RNA interference technology. The cells pretreated with an MCT inhibitor are preferably pretreated with AZD3965 and AR-C155858 or other MCT inhibitors known in the art. The reference conjugate lacking a ligand for the monocarboxylate transporter is preferably a conjugate that differs only in the presence or absence of a ligand. An example is the use of untargeted liposomes (Lp-OMe) in Table S3 of the Examples.

[0028] In a preferred embodiment, the ligand has the general formula (I): [ka] (In the formula, X is S, O, Se, or NH; X is preferably S or Se, more preferably S; in some embodiments, X is S, O, or NH; in other embodiments, X is S or O; c is —CH—, —CH(CH)—, —C(═O)—, —C(═S)—, —C(═NH)—, —CH(—OH)—, —CH(NH)—, —CH(halogen)— or —C(halogen)—, preferably cis —CH—, —CH(CH)—, —C(═O)— or —CH(NH)—, c is most preferably —CH—; n is 1, 2 or 3, n is preferably 1 or 2, and n is most preferably 1; and R is -H, -CH3, =O, =S, =NH, -OH, -NH2 or halogen, R is preferably -H, =O, =S, =NH, -OH or -NH2, R is more preferably =O, =S, =NH, -OH or -NH2, even more preferably R is =O, =S, =NH or -NH2, even more preferably R is =O, =S or -NH2, most preferably =O or -NH2, and * is a conjugation site for a pharmaceutically acceptable nanoparticle) It is of the type.

[0029] In general formula (I), [ka] The bond is double or single depending on the nature of R and the associated requirements of valency. As used herein, halogen is preferably F, Cl, Br or I, more preferably F or Cl, and most preferably F. Preferably, when multiple instances of C are present, at least one is -CH2-. In some embodiments, R is =O, =S or =NH. In some embodiments, R is -OH or -NH2. In some embodiments, R is =O. In some embodiments, R is -NH2.

[0030] In a preferred embodiment, X is S or O, preferably S; c is —CH—, —CH(CH)—, —C(═O)— or —CH(NH)—, most preferably —CH—; n is 1 or 2, most preferably 1, and R is =O, =S or -NH2, most preferably =O or -NH2, and * is a conjugation site for a pharmaceutically acceptable nanoparticle.

[0031] The conjugation method is not critical, and those skilled in the art can carry out the conjugation appropriately. For example, the nanoparticles or their antibiotics may contain a ring that can be opened by a leaving group, a Michael acceptor, or a nucleophile of the ligand, or may contain a carboxylic acid, amine, thiol, or hydroxyl moiety that can react by forming an amide, ester, or dithiol bridge. For example, if X is S in the above-mentioned ligand, the free ligand may have an -SH moiety, which is highly versatile for conjugation. Similarly, if X is O, the free ligand may have the corresponding -OH, and if X is NH, the free ligand may have -NH. Those skilled in the art can select an appropriate conjugation method. Preferred examples are nanoparticles containing Michael acceptor, active ester, or α-halogenacetic acid moieties on their surface. Preferred Michael acceptors are maleimide, vinyl ketone, acrylic, or methacrylic moieties. Preferred active esters are esters of nitrophenol, pentafluorophenol, or N-hydroxysuccinimide (NHS). Preferred α-haloacetic acid moieties are esters or amides of iodoacetic acid or bromoacetic acid. In a preferred embodiment, the ligand is conjugated to a water-soluble polymer as described herein below.

[0032] [Monocarboxylate transporter (MCT)] MCTs are a family of proton-coupled plasma membrane transporters that transport molecules with one carboxylic acid group (monocarboxylic acids) across biological membranes. They belong to the group of solute carrier (SLC) membrane transport proteins. Molecules suitable for transport by MCTs include lactate, pyruvate, and ketones. MCTs are widely expressed. High-grade tumors rely heavily on anaerobic glycolysis (the metabolism of glucose to lactate even in the presence of oxygen; the Warburg effect) and therefore efflux lactate into the tumor microenvironment via MCTs to maintain a strong glycolytic flux and prevent harmful accumulation of lactate within the tumor. At least 14 MCTs corresponding to the 14 solute carrier 16A transporters are known, namely SLC16A1, SLC16A2, SLC16A3, SLC16A4, SLC16A5, SLC16A6, SLC16A7, SLC16A8, SLC16A9, SLC16A10, SLC16A11, SLC16A12, SLC16A13 and SLC16A14.

[0033] In some embodiments, the MCT is at least one of MCT1, MCT2, MCT3, MCT4, MCT5, MCT6, MCT7, MCT8, MCT9, MCT10, MCT11, MCT12, MCT13, and MCT14. In preferred embodiments, the monocarboxylic acid transporter is at least monocarboxylic acid transporter 1 (MCT1), monocarboxylic acid transporter 2 (MCT2), monocarboxylic acid transporter 3 (MCT3), or monocarboxylic acid transporter 4 (MCT4). In more preferred embodiments, the monocarboxylic acid transporter is at least monocarboxylic acid transporter 1 (MCT1), monocarboxylic acid transporter 2 (MCT2), or monocarboxylic acid transporter 4 (MCT4), more preferably each of MCT1, MCT2, and MCT4. In certain embodiments, the monocarboxylic acid transporter is at least monocarboxylic acid transporter 3 (MCT3). In certain embodiments, the monocarboxylic acid transporter is at least monocarboxylic acid transporter 4 (MCT4). In a highly preferred embodiment, the monocarboxylic acid transporter is at least monocarboxylic acid transporter 1 (MCT1) or monocarboxylic acid transporter 2 (MCT2), more preferably both MCT1 and MCT2.

[0034] [Pharmaceutically acceptable nanoparticles] The present invention resides in the surprising discovery that nanoparticles, such as nanocarriers, can effectively target themselves and their cargo to photoreceptors. From this insight, one skilled in the art can select an appropriate pharmaceutically acceptable nanoparticle. In preferred embodiments, the pharmaceutically acceptable nanoparticle is a liposome, a solid lipid nanoparticle, a micelle, a carrier protein, a metal nanoparticle, a polyplex system, a lipoplex system, or a polymeric nanoparticle. More preferably, it is a liposome, a solid lipid nanoparticle, a micelle, a carrier protein, or a polymeric nanoparticle. Even more preferably, it is a liposome, a solid lipid nanoparticle, a micelle, or a carrier protein, and even more preferably, it is a liposome, a solid lipid nanoparticle, or a micelle. In certain embodiments, the nanoparticle is a liposome, a micelle, a carrier protein, a polyplex system, a lipoplex system, or a polymeric nanoparticle.

[0035] Good results have been achieved with lipid nanoparticles. Therefore, preferred pharmaceutically acceptable nanoparticles are lipid nanoparticles. Lipid nanoparticles can be liposomes, solid lipid nanoparticles, and lipid micelles. Lipid nanoparticles can also be lipid-polymer hybrid nanoparticles. Preferred polymers are biodegradable polymers such as polylactic acid. In some embodiments, the lipid nanoparticles are liposomes or lipid micelles. In some embodiments, the lipid nanoparticles are lipid micelles or solid lipid nanoparticles. In some embodiments, the lipid nanoparticles are solid lipid nanoparticles or liposomes. Liposomes are most preferred.

[0036] Nanoparticles are herein understood to be small objects that behave as a single integral unit in terms of their transport and properties.Preferably, when the nanoparticles of the present invention are solid lipid nanoparticles, they do not contain stabilizing surfactants.The size of lipid nanoparticles is preferably 1-300 nm.

[0037] Pharmaceutically acceptable nanoparticles preferably contain lipids, preferably at least two types of lipids. In a preferred embodiment, the pharmaceutically acceptable nanoparticles contain one or more phospholipids. Phospholipids preferably contain a single organic molecule, such as a diglyceride, a phosphate group, and choline. In particular, "phospholipids" include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidic acid (PA), phosphatidylinositol (PI), phosphatidylserine (PS), sphingomyelin, plasmalogens, and phosphatidylcholine lipid derivatives in which the two hydrocarbon chains are typically about 14 to 22 carbon atoms in length and have various degrees of unsaturation.

[0038] Phospholipid can comprise net negative charge or net positive charge.However, in the preferred embodiment of the present invention, one or more phospholipids are natural phospholipids.More preferably, all phospholipids are natural phospholipids.Natural phospholipids are herein understood as phospholipids that do not have net charge.

[0039] In a preferred embodiment, the lipid is 1,2-dilauroyl-sn-glycero-3-phosphate (DLPA), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dimyristoyl-sn-glycero-3-phosphate (DMPA), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dimyristoyl-sn-glycero-3-phosphoserine (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phosphate (DPPA), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2 -comprises one or more natural phospholipids selected from the group consisting of dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1,2-distearoyl-sn-glycero-3-phosphate (DSPA), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS) and hydrogenated soy phosphatidylcholine (HSPC). Furthermore, the one or more natural phospholipids used in the present invention can be soybean phosphatidylcholine (SPC), or egg yolk phosphatidylcholine (EYPC), or 1-palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC). However, SPC and EYPC are less preferred as lipids for (solid lipid) nanoparticles because their melting points may be below room temperature, which may limit the stability of lipid nanoparticles. In some embodiments, the one or more phospholipids are selected from DSPC, DSPE, and POPC. In some embodiments, the nanoparticles comprise DSPE and POPC.In some embodiments, the nanoparticles comprise DSPE and DSPC. Nanoparticles such as liposomes containing DSPC may exhibit slower drug release and are expected to be more suitable for in vivo applications. DSPE is advantageously included in the nanoparticles as part of a conjugate of DSPE with a water-soluble polymer such as PEG.

[0040] Lipid nanoparticles may contain a single or various lipids. In particular, nanoparticles according to the present invention may contain at least 1, 2, 3, 4, or 5 different lipids. Alternatively, nanoparticles may contain up to 1, 2, 3, 4, or 5 different lipids. Preferably, lipid nanoparticles contain two or more lipids, such as three different lipids, particularly for liposomes. In some embodiments, only a single lipid is contained, which is particularly attractive for micelles. The lipid is preferably a biocompatible lipid.

[0041] In some embodiments, the pharmaceutically acceptable nanoparticles comprise one or more non-cationic lipids. Preferred non-cationic lipids are sterols, more preferably cholesterol or its derivatives, most preferably cholesterol. The total amount of lipid preferably comprises at least about 1, 5, 10, 20, 25, 30, 35, 40, or 45% (w / w) cholesterol (derivative). Preferably, the lipid comprises at least 25 or 30%, e.g., about 30-35%, cholesterol (derivative). Alternatively, the molar ratio of phospholipid to non-cationic lipid ranges from 0.2:1 to 5:1, preferably 1:1 to 4:1, more preferably 1.5:1 to 2.5:1, with a ratio of about 2:1 being particularly preferred for better results. Alternatively, in some embodiments, the lipid comprises less than 0.1, 0.5, 1, 5, 10, 20, 30, 35, 40, or 45% (w / w) cholesterol (derivative).

[0042] A (stabilizing) water-soluble polymer is herein understood to be a polymer that contributes to the stability of nanoparticles, for example, by increasing tissue penetration / diffusion depth, circulation time, and / or reducing bloodstream clearance (so-called "stealth" properties) in vivo compared to the same nanoparticles without a water-soluble polymer.The water-soluble polymer used in the present invention may alternatively or additionally have a lubricating effect.The water-soluble polymer may be a stabilizing and / or humidifying water-soluble polymer.

[0043] Liposomes are lipid nanoparticles comprising a lipid bilayer and an aqueous interior or lumen. Liposomes are attractive for delivering hydrophilic pharmaceutical agents because they can be encapsulated in the lumen. Liposomes can have multiple bilayers, forming multivesicles. The lumen can contain additional vesicles, forming multivesicular vesicles. Solid lipid nanoparticles are lipid nanoparticles lacking an aqueous interior. They can be multilamellar or unstructured. Solid lipid nanoparticles are attractive for delivering hydrophobic pharmaceutical agents. Micelles are preferably lipid micelles, which are attractive for delivering hydrophobic pharmaceutical agents. Micelles are conveniently formed from a single surfactant, such as a single phospholipid or a single conjugate of a lipid and a water-soluble polymer. Micelles can also be formed from a phospholipid and a polymer-lipid conjugate described herein. Micelles can also be formed from a non-cationic lipid and a polymer-lipid conjugate described herein. The carrier protein can be any protein suitable for conjugation to a ligand, preferably including a pharmaceutical agent (e.g., via conjugation, encapsulation, or hydrophobic interactions). The metal nanoparticles can be, for example, imaging agents, anticancer agents, or diagnostic agents, such as those described by Liang et al. (doi:10.2147 / IJN.S75174). The polyplexes can be complexes of nucleic acids with polymers such as polylysine or polyethyleneimine. The lipoplexes can be complexes of lipids with nucleic acids. The polymeric nanoparticles can be, for example, layer-by-layer particles prepared by suspension polymerization, polymersomes, or solid polymeric nanoparticles, which are preferably biodegradable polymeric nanoparticles based on, for example, polylactic acid. The polymeric nanoparticles can also be hybrid nanoparticles containing lipids.

[0044] In a preferred embodiment, the pharmaceutically acceptable nanoparticles comprise a water-soluble polymer on their surface. Thus, the nanoparticles preferably have a surface comprising a water-soluble polymer. Preferably, the surface of the nanoparticles is at least partially covered by the water-soluble polymer. More preferably, the water-soluble polymer covers at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 99, or 100% of the surface of the nanoparticles. In a further embodiment, the nanoparticles have a surface consisting of a water-soluble polymer.

[0045] In a further embodiment, the lipid nanoparticles used in accordance with the present invention comprise a water-soluble polymer, the water-soluble polymer being i) a polyalkyl ether, preferably a polyalkyl ether that is a linear polyethylene glycol (PEG), a star PEG, or a multi-arm branched PEG; ii) homopolymers that are PEG substitutes or PEG alternatives, preferably homopolymers selected from the group consisting of polymethylethylene glycol (PMEG), polyhydroxypropylene glycol (PHPG), polypropylene glycol (PPG), polyvinylpyrrolidone (PVP), polyglycerol (PG), polymethylpropylene glycol (PMPG), polyhydroxypropylene oxide (PHPO), poly-oxazoline (POZ) and hydroxyethyl starch (HES); iii) Heteropolymers of small alkoxy monomers, preferably homopolymers containing polyethylene / polypropylene glycol (PEG / PPG) At least one of the following is true.

[0046] PEG is most preferred, and depending on its molecular weight is also known as polyethylene oxide (PEO) or polyoxyethylene (POE), which names may be used interchangeably herein. Water-soluble polymers may impart stealth-like and / or humidifying and / or stabilizing properties to the nanoparticles.

[0047] The nanoparticles may contain a single water-soluble polymer or a variety of water-soluble polymers. In particular, the nanoparticles may contain at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different water-soluble polymers. Alternatively, the nanoparticles may contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 different water-soluble polymers. Furthermore, instead of or in addition to the water-soluble polymers defined herein, the water-soluble polymers may be derivatives of the polyalkyl ethers, homopolymers, and / or heteropolymers defined above. In particular, the water-soluble polymers may be derivatized to contain functional groups such as, for example, carboxylic acids, maleimides, or amides, for covalent attachment of ligands, for example.

[0048] In a preferred embodiment, the water-soluble polymer has a molecular weight of at least about 120 Daltons and up to 20,000 Da. Preferably, the water-soluble polymer has a molecular weight of 300 to 10,000 Da, more preferably 500 to 5,000 Da, most preferably 750 to 3,000 Da, or 1,000 to 3,400 Da, or 1,000 to 2,000 Da, for example about 2000 Da.

[0049] In another preferred embodiment, the water-soluble polymer has a molecular weight of less than 20,000, 15,000, 10,000, 5,000, 4,500, 4,000, 3,500, 3,400, 3,300, or 3,200 Da. In a further preferred embodiment, the water-soluble polymer has a degree of polymerization of at least about 4, 5, 6, 7, 8, 9, 10, 25, 50, 75, 100, 125, 150, 175, 200, 209, 210, 211, 250, 300, 400, or 500. In particular, the water-soluble polymer preferably has a degree of polymerization of at least 6 or between about 6 and 210. In a further embodiment, the water-soluble polymer is conjugated to one of the lipids or the lipid. Preferably, the water-soluble polymer is covalently attached. In a particularly preferred embodiment, the conjugate of a lipid and a water-soluble polymer is a conjugate of a phospholipid, as defined herein, and a polymer, as defined above.

[0050] In a preferred embodiment, lipids such as phospholipids can be conjugated or linked to a water-soluble polymer. Such polymer-phospholipid conjugates can have a stabilizing effect on nanoparticles and a moisturizing and / or lubricating effect. Highly suitable for inclusion in such polymer-lipid conjugates is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), most preferably N-linked. More preferably, the conjugate is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-polyethylene glycol (DSPE-PEG). Most preferably, the conjugate is 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-mPEG2000) or d-α-tocopherol-N-[methoxy(polyethylene glycol)-1000] (TPEG1000). In an alternative embodiment of the invention, the conjugate is hydrogenated soybean phosphatidylcholine-polyethylene glycol (HSPC-PEG), and most preferably, the HSPC-PEG is hydrogenated soybean phosphatidylcholine-[methoxy(polyethylene glycol)-2000] (HSPC-mPEG2000).

[0051] Such polymer-lipid conjugates are preferably present in the nanoparticles in an amount ranging from 0.5 to 15% based on the total weight of lipid. As used herein, the amount of polymer-lipid conjugate is considered to contribute in its entirety to the total lipid weight. More preferably, it is present in an amount ranging from 1 to 10% based on the total weight of lipid, even more preferably from 2 to 8%, more preferably from 3 to 7%, and most preferably from 4 to 6%, e.g., about 5%.

[0052] In lipid nanoparticles, particularly liposomes, the phospholipid, non-cationic lipid and polymer-lipid conjugate are preferably present in a weight ratio in the range of 1-36:1-18:0.1-3, preferably in the range of 6-24:3-12:0.5-2, more preferably in the range of 8-16:4-8:0.7-1.3, for example in the range of about 12:6:1, and most preferably it is about 63.3:31.7:5.

[0053] In one embodiment of the present invention, the size of the lipid nanoparticles is 5 to 1000 nm, preferably 15 to 500 nm, more preferably 20 to 250 nm, more preferably 30 to 200 nm, even more preferably 40 to 100 or 50 to 90 nm. PEGylated liposomes with a diameter of less than 100 nm are expected to reach photoreceptors. The size of the nanoparticles can be determined by any method known in the art. Preferably, the size of the nanoparticles is determined by dynamic light scattering (Zetasizer).

[0054] Preferably, for solid lipid nanoparticles or micelles, the size of the nanoparticles is at least about 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nm and not more than about 350, 300, 250, 200, 175, 150, 125, 100, 75, or 50 nm. More preferably, the size of the nanoparticles is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nm. Most preferably, the size of the nanoparticles is 5-300 nm, 10-150 nm, 15-100 nm, 20-100 nm, 15-80 nm, 20-80 nm, 15-60 nm, or 20-60 nm.

[0055] In a further preferred embodiment, the present invention relates to a composition comprising a conjugate of the present invention and further comprising a pharmaceutically acceptable excipient such as water. Preferably, in such compositions, the nanoparticles have an average size of at least about 3, 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 nm and not more than about 350, 300, 250, 200, 175, 150, 125, 100, 75, or 50 nm. More preferably, the average size of the nanoparticles is about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nm. The nanoparticles in the composition can deviate from the average size by at least 0.01, 0.05, 0.10, 0.15, 0.20, 0.25, 0.30, 0.35, 0.40, 0.45, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 2.0, or 5.0 nm. Most preferably, the nanoparticles in the composition are about 5-300 nm, 10-150 nm, 15-100 nm, 20-100 nm, 15-80 nm, 20-80 nm, 15-60 nm, 20-60 nm, 15-50 nm, 20-50 nm, 15-40 nm, 20-40 nm, 15-30 nm, or 20-30 nm.

[0056] The nanoparticles of the present invention can be obtained using any method known in the art. The mixture containing the nanoparticles can then be sterilized using any conventional method. For example, the mixture containing the conjugate of the present invention can be sterilized by passing the mixture through a (sterilizing) filter. Preferably, the filter has a pore size of about 0.15, 0.2, 0.25, 0.3, 0.4, 0.5, 0.5, or 1.0 microns.

[0057] The conjugates or nanoparticles disclosed herein can be prepared using any conventional method known in the art. As a non-limiting example, nanoparticles can be prepared by dissolving lipids, water-soluble polymers, and diagnostic, lubricant, or therapeutic agents in a suitable solvent. Preferred solvents are water-miscible and pharmaceutically acceptable. Particularly preferred solvents are ethanol, methanol, and isopropanol, more preferably ethanol and methanol. The most preferred solvent is ethanol. The solution can be heated. Preferably, the solution is heated to about 40, 55, 60, 65, or 70°C. The lipid solution can then be added to a suitable aqueous solution, preferably having approximately the same temperature as the lipid solution. The lipid solution is preferably added slowly, e.g., in stages. Alternatively, an aqueous solution can be added to the lipid solution. Preferably, the aqueous solution is added slowly, e.g., in stages. Suitable aqueous solutions include water, saline, phosphate-buffered saline, or any other aqueous solution commonly known in the art. A preferred aqueous solution is water. The solvent / aqueous solution percentage is preferably about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, or 60% (w / w). After the solution has cooled, residual solvent can optionally be removed from the particle solution. Removal of residual solvent can be carried out using any conventional method known in the art, including, but not limited to, dialysis, diafiltration, or liquid chromatography.

[0058] Alternatively, liposomes can be prepared using the thin film rehydration method. Here, a chloroform solution of a lipid such as POPC, cholesterol, and a phospholipid-PEG conjugate can be mixed in a molar ratio of, for example, 63.3:31.7:5 to produce untargeted liposomes with PEG chains on their surface. For encapsulation of pharmaceutical agents, the lipid DSPC can be used instead of POPC. The entire lipid solution can be dried using a rotary evaporator under reduced vacuum (e.g., 300 mbar). After about 1 hour, the dried lipid can be rehydrated in a rehydration buffer. For example, the agent can be added at a drug:lipid molar ratio of 1:3. After the lipids are dissolved in the rehydration medium, about five freeze-thaw cycles are performed in liquid nitrogen and a 37°C water bath. The liposome solution can be extruded, for example, about 11 times, through a PC membrane with pores of about 100 nm. For conjugation with MCT ligands, a suitable nucleophilic analog, such as a thiol, can be prepared in 10 mM of a reducing agent, such as tris(2-carboxyethyl)phosphine, in a buffer, such as 25 mM HEPES (pH 7.4), and added to Michael acceptor-containing nanoparticles, such as PEG-maleimide-containing liposomes, with the ligand at a 2x maleimide concentration, followed by incubation at room temperature for 2 hours. To remove unencapsulated compounds and unbound ligand, the formulation can be dialyzed, for example, against isotonic saline at 4°C. A 2-hour dialysis can be performed to remove the agent. Nanoparticles, such as liposomes, can be sterile filtered and stored, for example, at 2-8°C.

[0059] [cargo] In some embodiments, the present invention provides a conjugate according to the present invention, wherein the pharmaceutically acceptable nanoparticles further comprise a pharmaceutical agent, preferably a neuroprotective agent, such as a photoreceptor rescue drug, preferably a cyclic guanosine monophosphate (cGMP) analogue. Nanoparticles, such as lipid nanoparticles, comprising pharmaceutical agents are widely used. Examples of suitable pharmaceutical agents include: a. neuroprotective agents, b.Central nervous system depressants, c. central nervous system stimulants, d. Psychopharmacological agents, e. Respiratory medications, f. Peripheral nervous system drugs, g. Drugs that act at synapses or neuroeffector junctions; h. smooth muscle acting drugs or drugs acting on skeletal or cardiac muscles; i. histaminergic agents, j. antihistamines, K. Cardiovascular drugs, l. Blood or hematopoietic system drugs, m. Gastrointestinal drugs, n.Steroids, o. Cytostatic or antitumor agents, P.Antibiotics, Q.Antifungal agents, r.Antimalarials, s. antiprotozoal drugs, t.Antibacterial agents, u.Anti-inflammatory agents, v. immunosuppressants, w. Cytokines, x. enzymes, y. imino sugar, Z. Ceramide analogues, aa. brain-acting hormones or neurotransmitters, bb. peptides, such as neuropeptides or derivatives thereof; cc. neurotrophic factors, dd. antibodies or fragments thereof; ee. Alzheimer's disease drugs or compounds; ff. nucleic acids such as siRNA, mRNA or miRNA, gg. contrast agents, hh. (organophosphate) antidote, ii. Anticancer drugs is.

[0060] The above pharmaceutical agents are described in detail in paragraphs

[0018] to

[0050] of U.S. Patent Application Publication No. 2014 / 0227185. In some embodiments, the pharmaceutical agent is a neuroprotective agent, a peptide, or a nucleic acid. The pharmaceutical agent is preferably present at a total concentration of 0.5 to 50 μM, more preferably 1 to 10 μM, and even more preferably 2 to 5 μM. Examples of anticancer agents include DNA replication inhibitors such as non-cell-cycle specific antitumor agents (e.g., cisplatin or oxaliplatin), topoisomerase inhibitors such as anthracyclines (e.g., doxorubicin), mitotic inhibitors (e.g., paclitaxel), or combinations thereof.

[0061] More preferably, the pharmaceutical agent is a neuroprotective agent or photoreceptor rescue drug, such as palmitoylethanolamide (PEA), sunitinib, or mycophenolic acid (MPA), and a particularly preferred photoreceptor rescue drug is a cyclic guanosine monophosphate (cGMP) analog. Rescue drugs, also known as cGMP-derived PKG inhibitors, cGMP analogs (e.g., Rp-8-Br-cGMPS, etc.), are known to protect rd1 and rd2 photoreceptors in both in vitro and in vivo mouse retinitis pigmentosa models (Paquet-Durand et al., 2009; Vighi, E., et al., PNAS, 2018, 115(13):E2997-E3006). cGMP analogs themselves are known in the art. International Publication No. 2012130829 describes boranophosphate analogs of cyclic nucleotides. WO 2018 / 010965 describes a multimeric complex of cGMP analogs. Butt et al. (FEBS Letters, 1990, 263(1):48, DOI: 10.1016 / 0014-5793(90)80702-K) describes the inhibition of cGMP-dependent protein kinase by (Rp)-guanosine 3',5'-monophosphorothioate.

[0062] Most preferably, the pharmaceutical agent is a cGMP analog. Preferably, the cGMP analog has the general formula (cGMP): [ka] (In the formula, X 2 is p' of H, where p' is a hydroxyl protecting group, preferably methoxymethyl (MOM), tetrahydropyranyl (THP), t-butyl (tBu), allyl (all), benzyl (Bn), (tri)alkylsilyl (such as t-butyldimethylsilyl (TBDMS), triisopropylsilyl (TIPS) or t-butyldiphenylsilyl (TBDPS)), acyl (such as acetyl (Ac), pivaloyl (Pv) or benzoyl (Bz)); h is H, halogen or Q; R 1 and R 2 are each independently H, -(CH2) n -H, -(CH2) n -C 3~9 Heterocyclyl, -(CH2) n - selected from ar and ar, where each n is independently selected from 0, 1, 2, 3, or 4, or R 1 and R 2 are both CH=C(ar)- or -(CH2) 1~4 Forming C(=O)- ar is, independently at each occurrence, a 5- or 6-membered aromatic or heteroaromatic ring, preferably phenyl or 2-furanyl, and each instance of ar is independently and individually optionally substituted with halogen, —OH, —SH, —NH, —NO, —OCH, —CH, —CHCH, —CH(CH), or —CF, and is optionally fused to a second instance of ar, preferably to form a naphthyl moiety; Q is -(CH2) n -S-(CH2) n -H, -S-(CH2) n -OH, -S-(CH2) n -NH2, -(CH2) n -O-(CH2) n -H, -O-(CH2) n -OH, -O-(CH2) n-NH2, -OC(CH3)3, -O-CH(CH3)2, -(CH2) n -N(-[CH2] n H)2, -NH-(CH2) n NH2, -NH-(CH2) n -OH, -(CH2) n -Nc 1 c 2 and c 1 and c 2 form a 3- to 8-membered heterocyclic ring together with the N to which they are attached, or 1 is H, and c 2 is a 3- to 8-membered heterocycle, -(CH2) n -H, -N3, -CF3, -(CH2) n -ar, -O-(CH2) n -(ar), -NH-(CH2) n -(ar), -S-(CH2) n -(ar), -(CH2) n -amide-ar, -O-(CH2) n -amide-(ar), -NH-(CH2) n -Amido-(ar), -S-(CH2) n -amide-(ar) or linker moiety, wherein any -H may be optionally replaced by halogen, and each instance of n is independently selected from 0, 1, 2, 3, 4, 5, 6, 7, or 8; and o 3 is H, -SH or -SC 1~12 hydrocarbon, borane, methylborane, dimethylborane, or cyanoborane, and o3 is preferably -SH, borane, methylborane, dimethylborane, or cyanoborane, most preferably -SH. or a salt thereof.

[0063] Preferably, the cGMP analogue of general formula (cGMP) has the general formula (cGMP-Rp): [ka] (In the formula, o 3 are SH, borane, methylborane, dimethylborane, and cyanoborane. However, the boron analogues are called Sp analogues due to the lower priority of boron compared to oxygen within the Cahn-Ingold-Prelog precedence rules.

[0064] Further preferred examples of cGMP analogs are: 1. 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP) or its phosphorothioate (8-Br-cGMPS), 2. 8-(2,4-dihydroxyphenylthio)guanosine-3',5'-cyclic monophosphate (8-o,pDHPT-cGMP) or its phosphorothioate 8-o,pDHPT-cGMPS, 3. 8-(2-aminophenylthio)guanosine-3',5'-cyclic monophosphate (8-APT-cGMP) or its phosphorothioate 8-APT-cGMPS, 4. 8-(4-hydroxyphenylthio)guanosine-3',5'-cyclic monophosphate (8-pHPT-cGMP) or its phosphorothioate 8-pHPT-cGMPS, 5. 8-(4-aminophenylthio)guanosine-3',5'-cyclic monophosphate (8-pAPT-cGMP) or its phosphorothioate 8-pAPT-cGMPS, 6.8-(4-chlorophenylthio)-s-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphate (8-pCPT-PET-cGMP) or its phosphorothioate 8-pCPT-PET-cGMPS, 7. 8-(4-chlorophenylthio)guanosine-3',5'-cyclic monophosphate (8-pCPT-cGMP) or its phosphorothioate 8-pCPT-cGMPS, 8. 8-(2,4-dichlorophenylthio)guanosine-3',5'-cyclic monophosphate (8-o,pDClPT-cGMP) or its phosphorothioate 8-o,pDClPT-cGMPS, 9. 8-(4-methoxyphenylthio)guanosine-3',5'-cyclic monophosphate (8-pMeOPT-cGMP) or its phosphorothioate 8-pMeOPT-cGMPS, 10.8-Bromo-β-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphate (8-Br-PET-cGMP) or its phosphorothioate 8-Br-PET-cGMPS, 11. 8-Bromo-(2-naphthyl-1,N 2 -etheno)guanosine-3',5'-cyclic monophosphate (8-Br-(2-N)ET-cGMP) or its phosphorothioate 8-Br-(2-N)ET-cGMPS, 12. 8-(4-hydroxyphenylthio)-s-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphate (8-pHPT-PET-cGMP) or its phosphorothioate 8-pHPT-PET-cGMPS, 13. 8-(4-chlorophenylthio)-β-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphate (8-pCPT-PET-cGMP) or its phosphorothioate 8-pCPT-PET-cGMPS, 14.2-Naphthyl-1,N 2 ethenoguanosine-3',5'-cyclic monophosphate ((2-N)ET-cGMP) or its phosphorothioate (2-N)ET-cGMPS, 15.β-phenyl-1,N 2 ethenoguanosine-3',5'-cyclic monophosphate (PET-cGMP) or its phosphorothioate PET-cGMPS, 16.4-Methoxy-β-phenyl-1,N 2 - ethenoguanosine-3',5'-monophosphate (pMeO-PET-cGMP) or its phosphorothioate pMeO-PET-cGMPS, 17.β-1,N 2 -acetyl-8-bromoguanosine-3',5'-cyclic monophosphorothioate (β-1,N 2-Ac-8-Br-cGMPS) and its phosphate (β-1,N 2 -Ac-8-Br-cGMP), 18.8-Bromo-δ-1,N 2 -Butyrylguanosine-3',5'-cyclic monophosphorothioate (8-Br-δ-1,N 2 -But-cGMPS) and its phosphate (8-Br-δ-1,N 2 -But-cGMP), 19. 8-Bromo-(4-methyl-β-phenyl-1,N 2 -etheno)guanosine-3',5'-cyclic monophosphorothioate (8-Br-pMe-PET-cGMPS) and its phosphate (8-Br-pMe-PET-cGMP), 20. 8-Bromo-(3-thiophene-yl)-1,N 2 -etheno)guanosine-3',5'-cyclic monophosphorothioate (8-Br-(3-Tp)ET-cGMPS) and its phosphate (8-Br-(3-Tp)ET-cGMP), 21. 1-benzyl-8-bromoguanosine-3',5'-cyclic monophosphorothioate (1-Bn-8-Br-cGMPS) and its phosphate (1-Bn-8-Br-cGMP), 22. 8-thioguanosine-3',5'-cyclic monophosphorothioate (8-T-cGMPS) and its phosphate (8-T-cGMP), 23. 8-(4-isopropylphenylthio)guanosine-3',5'-cyclic monophosphorothioate (8-pIPrPT-cGMPS) and its phosphate (8-pIPrPT-cGMP), 24. 8-Phenylamidomethylthioguanosine-3',5'-cyclic monophosphorothioate (8-PAmdMT-cGMPS) and its phosphate (8-PAmdMT-cGMP), 25.β-Phenyl-1,N 2 -etheno-8-phenylamidomethylthioguanosine-3',5'-cyclic monophosphorothioate (PET-8-PAmdMT-cGMPS) & phosphate (PET-8-PAmdMT-cGMP), 26. 8-(4-Isopropylphenylthio)-β-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphorothioate (8-pIPrPT-PET-cGMPS) and its phosphate (8-pIPrPT-PET-cGMP), 27. 8-(2-aminophenylthio)-β-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphorothioate (8-oAPT-PET-cGMPS) and its phosphate (8-oAPT-PET-cGMP), 28.β-Phenyl-1,N 2 -etheno-8-thioguanosine-3',5'-cyclic monophosphorothioate (PET-8-T-cGMPS) and its phosphate (PET-8-T-cGMP), 29.8-Methylthio-β-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphorothioate (8-MeS-PET-cGMPS) and its phosphate (8-MeS-PET-cGMP), 30. 8-methylthio-guanosine-3',5'-cyclic monophosphorothioate (8-MeS-cGMPS), preferably the sodium salt, and its phosphate (8-MeS-cGMP), 31. 8-Phenylguanosine-3',5'-cyclic monophosphorothioate (8-Phe-cGMPS) and its phosphate (8-Phe-cGMP), 32. 8-(2-Furyl)guanosine-3',5'-cyclic monophosphorothioate (8-(2-Fur)-cGMPS) and its phosphate (8-(2-Fur)-cGMP), 33. 8-(4-chlorophenyl)guanosine-3',5'-cyclic monophosphorothioate (8-pCP-cGMPS) and its phosphate (8-pCP-cGMP), 34. 8-Phenyl-β-phenyl-1,N 2 - ethenoguanosine-3',5'-cyclic monophosphorothioate (8-Phe-PET-cGMPS) and its phosphate (8-Phe-PET-cGMP), 35. 8-(4-chlorophenyl)-β-phenyl-1,N 2 -ethenoguanosine-3',5'-cyclic monophosphorothioate (8-pCP-PET-cGMPS) and its phosphate (8-pCP-PET-cGMP), as well as pharmaceutically acceptable salts thereof. The most preferred cGMP analog is 8-Br-PET-cGMP or its phosphorothioate, preferably the phosphorothioate. Highly preferred cGMP analogs are 8-Br-PET-cGMPS and 8-pCPT-PET-cGMPS, particularly Rp-8-Br-PET-cGMPS and Rp-8-pCPT-PET-cGMPS, even more preferably their sodium salts, sometimes referred to as CN03 and CN04, respectively. [ka]

[0065] In some embodiments, the pharmaceutically acceptable nanoparticles further comprise two or more pharmaceutical agents. In some embodiments, the pharmaceutical agents are contained in the aqueous lumen of the pharmaceutically acceptable nanoparticles. In some embodiments, the pharmaceutical agents are contained in the solid phase of the pharmaceutically acceptable nanoparticles, for example, in the lipid phase or lipid bilayer. One skilled in the art can select an appropriate dose of the pharmaceutical agents. Preferred doses range from 0.01 to 10 mM, preferably 0.2 to 2 mM.

[0066] [Compound] The present invention relates to a compound of general formula (A): [ka] (In the formula, X is S, O, Se or NH; c is -CH2-, -CH(CH3)-, -C(=O)-, -C(=S)-, -C(=NH)-, -CH(-OH)-, -CH(NH2)-, -CH(halogen)- or -C(halogen)2-; n is 1, 2 or 3, and R is -H, -CH3, =O, =S, =NH, -OH, -NH2 or halogen, and Q is a conjugate of a lipid and a water-soluble polymer, the lipid preferably being a phospholipid. The definitions of X, c, n, and R are preferably as described above for the ligand herein. The general formula (A) without Q can be seen as the ligand. As described above, the means of conjugation is not essential.

[0067] Q is a conjugate of a lipid and a water-soluble polymer. Preferably, the water-soluble polymer is as described elsewhere herein. Preferably, the lipid is as described elsewhere herein. Examples of Q include 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-3000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000], 1-pal Mitoyl-2-oleyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-1000], 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[bromoacetamide(polyethylene glycol)-2000], and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[acrylamide(polyethylene glycol)-2000].

[0068] It is advantageous if the ligand is at one end of the water-soluble polymer and the lipid is at the other end of the water-soluble polymer. An example of a compound of general formula (A) is a conjugate of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] with mercaptopyruvate or cysteine, preferably L-cysteine, as illustrated in the Examples.

[0069] [Application] The present invention provides a conjugate according to the present invention for use as a drug. This includes methods for treating, preventing, or diagnosing a disease, comprising administering the conjugate of the present invention to a subject in need thereof. The disease may preferably be a disease associated with MCT-expressing cells, such as cancer, or a neurodegenerative disease, or a retinal disease. Preferably, the drug is for treating a neurodegenerative disease or a retinal disease, such as hereditary retinal degeneration (IRD), glaucoma, age-related macular degeneration (AMD), Stargardt disease, Usher syndrome, geographic atrophy, diabetic retinopathy, retinitis pigmentosa, Leber's congenital amaurosis, blindness, loss of rod photoreceptors, night blindness, loss of cone photoreceptors, achromatopsia, loss of color vision, and loss of high-level visual acuity. Preferably, the drug is for treating a neurodegenerative disease that is also a retinal disease. Preferred conditions are hereditary retinal degeneration and cone-specific diseases such as achromatopsia or age-related macular degeneration, more preferably hereditary retinal degeneration. In some embodiments, the condition is a cone-specific disease such as color blindness or age-related macular degeneration.

[0070] As is evident from the examples, the conjugates according to the present invention are well suited for delivering pharmaceutical agents to the retina. Higher total drug exposure is achieved, which allows for the use of lower doses and reduces side effects. This also allows for the effective administration of higher doses, which may improve therapeutic efficiency or require less frequent administration to achieve a similar pharmaceutical window or steady state of the drug in the subject.

[0071] In certain embodiments, delivery of a pharmaceutical agent is enhanced compared to delivery of the same pharmaceutical agent when it is not present in a conjugate of the invention, e.g., compared to delivery of a "naked" drug. The conjugates disclosed herein can be administered at least once, twice, three times, four times, five times, six times, or more times per day. The lipid nanoparticles or compositions disclosed herein can be administered once every two, three, four, or five days, or even less frequently. In a preferred embodiment, delivery of an anticancer drug is enhanced compared to delivery of the same anticancer drug when it is not present in a conjugate of the invention.

[0072] The conjugates of the present invention can be said to be photoreceptor-targeted. The usefulness of photoreceptor-targeted nanoparticles, such as liposomes, for clinical use can depend on the drug release rate. Rapid drug release can limit the shelf life of the formulation. For non-targeted nanoparticles, a low drug release rate can lead to inferior results, as the available drug concentration in the target tissue is potentially too low to achieve efficacy. Conversely, for targeted nanoparticles, a lower release rate has been found to be beneficial to the therapeutic efficacy of the drug, even if there is no difference in the efficacy of sustained-release and fast-release non-targeted liposomes. This may be because the nanoparticles, and therefore the drug, are internalized into the target cells and released entirely at the relevant location without being distributed throughout the body. Therefore, a sustained release rate can be attractive for photoreceptor-targeted nanoparticles.

[0073] Complete release of CN04 from the liposomes of the present invention was demonstrated in an in vitro setup within 48 hours. The release rates of CN03 and CN04 can be reduced by precipitation with specific salts within the liposome cavity, using a remote loading technique similar to that performed for liposomal doxorubicin formulations. CN03 has previously been remotely loaded into similar liposomes using calcium acetate salt (Vighi, E., et al., PNAS, 2018, 115(13)).

[0074] In some embodiments, the conjugate used is for systemic or intravitreal (IVT) administration. In some embodiments, the conjugate used is for systemic administration. In some embodiments, the conjugate used is for IVT administration. Common routes of drug administration for the treatment of ocular diseases are topical, systemic, periocular, and intravitreal administration. Topical administration, preferably to the eye, is preferred due to high patient compliance and its non-invasive nature. When administered topically, drug absorption occurs through either the corneal route (cornea, aqueous humor, intraocular tissues) or the non-corneal route (conjunctiva, sclera, choroid / retinal pigment epithelium (RPE)). With regard to naked drugs, only a small fraction of topically applied drug, generally less than 5%, reaches intraocular tissues (Mishra GP et al. J. of Drug Delivery (2011) 2011:863-734). Factors responsible for poor ocular bioavailability after topical instillation are precorneal drainage and the lipoid nature of the corneal epithelium. Furthermore, most of the drug reaches the systemic circulation through the conjunctival blood vessels and nasolacrimal duct, which may cause adverse effects. Therefore, the topical route is not preferred. Systemic administration requires high doses due to the blood-aqueous humor barrier and the blood-retinal barrier. Such high doses may cause side effects. For untargeted nanoparticles, intravitreal administration may require frequent administration, which may lead to vitreous hemorrhage, retinal detachment, and endophthalmitis. Targeted nanoparticles can improve this by effectively delivering higher doses of drug.

[0075] Lactic acid and MCTs, particularly MCT1 and MCT4, are important contributors to tumor aggressiveness (Payen et al., Mol Metab. 2020 Mar;33:48-66). In some embodiments, the drug is for cancer treatment. In these embodiments, the conjugate preferably further comprises an anticancer agent. Suitable cancers to be treated using the conjugates of the present invention include, for example, adrenocortical carcinoma, bladder cancer, brain tumors, breast cancer, cervical cancer, colorectal cancer, gastric cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, lymphoma, ovarian cancer, prostate cancer, skin cancer, and soft tissue cancer, and optionally hematological malignancies such as leukemia. Preferred cancers are cervical cancer, lung cancer (particularly NSCLC), lymphoma (such as B-cell lymphoma and Burkitt's lymphoma), and skin cancer (particularly squamous cell skin cancer).

[0076] [method] The present invention provides a method of treating, delaying or preventing a neurodegenerative or retinal disease, comprising administering to a subject a conjugate according to the invention, the characteristics and definitions preferably being as defined above.

[0077] The present invention provides an in vivo, in vitro or ex vivo method for improving the delivery or efficacy of a pharmaceutical agent, comprising: i) incorporating a pharmaceutical agent into nanoparticles according to the invention to obtain nanoparticles containing the pharmaceutical agent; ii) contacting the cells with nanoparticles comprising a pharmaceutical agent; The cells are preferably cells that express an MCT, more preferably photoreceptor cells.

[0078] [General definition] In this specification and the claims, the verb "comprise" and its conjugations are used in their open-ended sense to mean that the elements following the term are included, but elements not specifically recited are not excluded. Furthermore, the verb "consisting of" can be replaced by "consisting essentially of," which means that a combination or composition defined herein may contain additional elements other than those specifically identified, and that such additional elements do not alter the inherent characteristics of the invention. Furthermore, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that a plurality of elements is present, unless the context clearly requires that one and only one element be present. Thus, the indefinite article "a" or "an" typically means "at least one."

[0079] Where a structural formula or chemical name is understood by one of ordinary skill in the art to have chiral centers, no chirality is implied and all three are individually referred to for each chiral center: the racemic mixture, the pure R enantiomer, and the pure S enantiomer.

[0080] Whenever parameters of a substance are discussed in connection with the present invention, unless otherwise specified, the parameters are determined, measured, or determined under physiological conditions, which are known to those skilled in the art and include aqueous solvent systems, atmospheric pressure, pH values ​​of 6-8, temperatures ranging from room temperature to about 37°C (about 20-40°C), and appropriate concentrations of buffer salts or other components.

[0081] The use of a substance as a medicament described herein can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for therapy or as a medicament, it may also be used in the manufacture of a medicament for therapy. The products used as medicaments described herein may be used in a therapeutic method, such a therapeutic method comprising the administration of the product used.

[0082] In the context of the present invention, a decrease or increase in an evaluated parameter means a change of at least 5% in the value corresponding to that parameter. More preferably, a decrease or increase in value means a change of at least 10%, more preferably at least 20%, at least 30%, at least 40%, at least 50%, at least 70%, at least 90%, or 100%. In this latter case, it may be the case that there is no longer a detectable value associated with that parameter. The term "about" or "approximately" when used in conjunction with a numerical value (e.g., about 10) preferably means that the value may be more than or less than 1% of the given value (10).

[0083] The embodiments identified in this specification may be combined with each other unless otherwise specified. The present invention has been described above with respect to a number of embodiments. Obvious variations of some elements of the embodiments will be envisioned by those skilled in the art. These are encompassed within the scope of protection defined in the appended claims. All patents and references mentioned are incorporated herein by reference in their entirety. [Brief explanation of the drawings]

[0084] [Figure 1A] Figure 1 shows the cellular uptake of ligand-conjugated liposomes. Free calcein or liposomally loaded calcein was incubated with HEK293T cells. The amount of calcein internalized relative to the total added calcein is shown, as determined by fluorescence intensity measurements on a microplate reader. AZD3965 and AR-C155858 are inhibitors of various MCTs. Results are shown as mean ± SD, * = p ≤ 0.05, **** = p ≤ 0.0001. [Figure 1B] Figure 1 shows the cellular uptake of ligand-conjugated micelles. Parameters are the same as in Figure 1A, except that either DSPE-mPEG (control) or DSPE-PEG-maleimide-mercaptopyruvate micelles were used and calcein was replaced with DiO (CAS no. 34215-57-1). [Figure 2A]Distribution of liposome-delivered calcein in organotypic retinal explant cultures is shown. Ligand-conjugated liposomes containing calcein (Lp-Pyr and Lp-Cys) were added to retinal cultures at postnatal day 15 for 6 hours and compared with untargeted liposomes (Lp-OMe), optionally in conjunction with MCT1-2 and the inhibitor AR-C155858. Liposomes were added to the surface closest to the ganglion cell layer (GCL). Representative images demonstrating calcein distribution in the retina are shown. IPL = inner plexiform layer, INL = inner nuclear layer, OPL = outer plexiform layer, ONL = outer nuclear layer, Seg. = photoreceptor inner and outer segments, DAPI = nuclear counterstain. [Figure 2B] Calcein signals from each retinal layer are shown as in 2A. Results represent the mean ± SD for n = 5-6, * = p ≤ 0.05, ** = p ≤ 0.01. Statistical analysis: Two-way ANOVA with Tukey's multiple comparison test. Scale bar: 50 μm. [Figure 3A] Treatment of organotypic retinal explant cultures derived from the rd1 mouse model is shown. Retinas were cultured at P5 and treated with liposome-encapsulated or non-encapsulated (free) neuroprotective drugs (CN03 or CN04) from P7-P11. Assuming equal distribution throughout the culture medium, the final drug concentration for all treatments was 3.14 μM. The amount of dead photoreceptors in tissue sections from cultures under various treatment conditions was assessed using the TUNEL assay. DAPI was used to distinguish the outer nuclear layer (ONL). INL = inner nuclear layer. Scale bar 50 μm. [Figure 3B]3A shows the percentage of dead (TUNEL+) cells in the ONL. Under non-treated (NT) conditions, the number of TUNEL+ cells is high in the rd1 model. Administration of free CN03 or CN04 had no significant effect on cell death. The same was confirmed for untargeted control liposomes (Lp-OMe) and targeted but empty pyruvate-conjugated liposomes (Lp-Pyr). Importantly, when loaded with CN03 or CN04, Lp-Pyr produced significant photoreceptor protection. Data are presented as mean ± SD for n = 5-9 animals; *** = p ≤ 0.001, **** = p ≤ 0.0001. Statistical analysis: one-way ANOVA with Tukey's multiple comparisons. Scale bar 50 μm. [Figure 4A] Treatment of organotypic retinal explant cultures from the rd10 mouse model is shown. Retinas were cultured at P9 and treated with free neuroprotective CN03 or CN03 encapsulated in pyruvate-liposomes (Lp-Pyr / CN03) from P11 to either P17 or P24. Representative images of retinal explant culture sections at P17 and P24. DAPI used as a nuclear stain. ONL = outer nuclear layer, INL = inner nuclear layer. [Figure 4B] As for 4A, the number of remaining photoreceptor rows in the tissue sections is shown. NT and CN03 are substantially overlapping. Results represent the mean ± SD. n = 4-6 at P17; n = 4-5 at P24. ** = p ≤ 0.01 between Lp-Pyr / CN03 and CN03 or NT groups. Statistical analysis: two-way ANOVA with Tukey's multiple comparison test. Scale bar 50 μm.

[0085] Example 1 - Methods [1.1 Animals] C3H Pde6b rd1 / rd1 (rd1), congenic C3H Pde6 b+ / + Wild-type (WT) and C57BL / 6J Pde6b rd10 / rd10(rd10) mice were housed under standard lighting conditions, had free access to food and water, and were used regardless of sex. All procedures were performed in accordance with the ARVO Declaration for the Use of Animals in Vision and Ophthalmology Research and the Animal Protection Act (Tierschutzgesetz) issued by the German Federal Government and approved by the Animal Welfare Office of the University of Tübingen. Every effort was made to minimize the use and suffering of the animals. Animals were not assigned to experimental groups prior to their sacrifice.

[0086] [1.2 Materials] 1-Palmitoyl-2-oleyl-sn-glycero-3-phosphocholine (POPC), distearoylphosphatidylcholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (mPEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] (maleimide-PEG), cholesterol, chloroform (99%, with 0.5-1% ethanol), tris(2-carboxyethyl)phosphine (TCEP), thiolactic acid (95%), L-cysteine, sodium mercaptopyruvate dihydrate, free calcium carbonate Rusein, hydrogen chloride, sodium hydroxide, disodium hydrogen phosphate dihydrate, sodium dihydrogen phosphate monohydrate, paraformaldehyde, Triton-X (t-octylphenoxypolyethoxyethanol), goat serum, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium chloride, proteinase K, fetal bovine serum (FBS), bovine serum albumin (BSA), Corning™ Transwell polycarbonate membranes (0.4 μm, sterile), polycarbonate membranes (0.1 μm pore size), and filter supports were obtained from Sigma-Aldrich (Darmstadt, Germany). Dulbecco's modified Eagle's medium (DMEM), 1% penicillin / streptomycin, R16 medium, and dialysis cassettes (Slide-A-Lyzer, cellulose, 100K molecular weight cut-off) were obtained from Thermo Fisher Scientific (Waltham, MA, USA). Drug compounds CN03 and CN04 were provided by Biolog Life Science Institute (Bremen, Germany).

[0087] 1.3 Preparation of lipid nanoparticles Liposomes with or without conjugation of monocarboxylic acid-bearing molecules were prepared using the thin film rehydration method. Here, chloroform solutions of the lipids POPC, cholesterol, and mPEG were mixed in a molar ratio of 63.3:31.7:5 to generate untargeted liposomes with PEG chains terminated by methoxy groups (Lp-OMe). Conversely, the lipids POPC, cholesterol, and PEG-maleimide were mixed in the same ratio to generate liposomes with maleimide groups at the PEG chain ends for subsequent surface conjugation. For CN03 encapsulation, the lipid DSPC was used instead of POPC. All lipid solutions were dried in a rotary evaporator operating at 105 rpm at room temperature under reduced vacuum (300 mbar). After 1 h, the dried lipids were rehydrated in the rehydration buffers listed in Table S1 (all media adjusted to pH 7.4). For CN03 and CN04 encapsulation, the drug was added at a drug:lipid molar ratio of 1:3. After dissolving the lipids in the rehydration medium, five freeze-thaw cycles were performed in liquid nitrogen and a 37°C water bath. The liposome solution was extruded at least 11 times through a PC membrane with approximately 100 nm pores. For conjugation with monocarboxylic acid molecules, thiolactic acid (Lp-Lac for lactate-liposomes), sodium mercaptopyruvate (Lp-Pyr for pyruvate-coated liposomes), or L-cysteine ​​(Lp-Cys for cysteine-coated liposomes) was prepared in 10 mM reducing agent tris(2-carboxyethyl)phosphine in 25 mM HEPES (pH 7.4) and added to the PEG-maleimide-containing liposomes at a 2x maleimide concentration, followed by incubation at room temperature for 2 hours. To remove unencapsulated compounds and unbound monocarboxylic acids, the formulation was dialyzed against isotonic saline at 4°C. To remove the drug, a 2-hour dialysis was performed. A 6-hour dialysis period was used with saline changed every 2 hours to remove calcein. Liposomes can be sterile filtered and stored at 2-8°C until further use. Drug concentrations were measured by ultra-performance liquid chromatography (UPLC) before and after dialysis.The hydrodynamic diameter and zeta potential were determined by dynamic light scattering (DLS) (for details, see Urimi, D., et al., Int J Pharm, 2021.602:p.120640).

[0088] [Table 1]

[0089] 1.4 Nanoparticle uptake in cell culture Human-derived cell line HEK293T was seeded in 48-well plates at 50,000 cells / well in cell culture medium (DMEM + 20% FBS + 1% penicillin / streptomycin) for 24 h at 37 °C and 5% CO2. Dye-loaded nanoparticles (Lp-OMe, Lp-Lac, Lp-Pyr, or Lp-Cys) were adjusted to 90 μM dye and diluted 1:1 in culture medium to obtain a final concentration of 45 μM. The same concentration of free dye was added. For liposomes, calcein was used as the dye, and for micelles, DiO (Cas number: 34215-57-1) was used. To determine the role of MCT in uptake, a portion of the cells was pretreated for 24 h with either the MCT inhibitor AZD3965 or AR-C155858 at concentrations of 2.5 μM and 1 μM, respectively, before adding calcein-loaded Lp-Pyr. For controls, nothing was added to the cells. After 2 hours of incubation, the wells were washed three times with prewarmed PBS. Fluorescence intensity (FI) was measured using a microplate reader (Spark 10M, Tecan, Mannedorf, Switzerland) at an excitation wavelength (Ex.) / emission wavelength (Em.) of 485 / 530 nm to quantify the amount of intracellular dye. FI from cells without dye was used as the background signal. 90 μM of dye in PBS was measured to establish the total FI signal (100%) for quantification. To prepare cells for imaging under a fluorescence microscope, sterile circular glass inserts were added to the wells 24 hours before seeding the cells. After incubation with nanoparticles, the cells were fixed in 4% paraformaldehyde for 15 minutes. The glass inserts were transferred to microscope slides (Superfrost Plus™, R. Langenbrinck, Emmendingen, Germany) and a drop of mounting medium containing DAPI was added (Vectashield, Vector Laboratories, Burlingame, CA, USA). Cells were imaged using a fluorescence microscope (Axio Imager Z2, Zeiss, Oberkochen, Germany) equipped with an ApoTome function, a CCD camera, and a 20x objective.The green channel (Ex. / Em. 493 / 517 nm) was used to measure the dye signal, and the blue channel (Ex. / Em. 353 / 465) was used to measure the DAPI signal.

[0090] [1.5 Immunostaining of HEK293T cells and mouse retina] Before seeding HEK293T cells (100,000 cells / well), sterile round glass inserts were added to 24-well plates. After 24 hours, the cells were fixed with 4% paraformaldehyde and incubated with 0.3% Triton-X in PBS for 5 minutes at room temperature, followed by three PBS washes. Then, 5% goat serum was added for 1 hour. Primary antibodies against MCT1-4 (see Table S2) were diluted in 5% goat serum, added to the cells, and incubated overnight at 2–8°C, followed by three washes. Secondary antibodies (1:350) dissolved in 5% goat serum were added and incubated with the cells for 1 hour at room temperature, followed by three washes.

[0091] For immunostaining of mouse retinas, wild-type mice were used at postnatal day (P) 30. Mice were sacrificed by CO2 asphyxiation and spinal dislocation. Eyeballs were removed, and retinas were isolated and fixed in 4% paraformaldehyde. They were then cryoprotected in sucrose as previously described (Belhadj, S., et al., J. Vis. Exp., 2020). Retinas were submerged in embedding medium (Tissue-Tek OCT Compound, Sakura Finetek Europe, Alphen aan den Rijn, The Netherlands) and frozen in liquid nitrogen. 12-μm-thick sections were prepared on microscope slides using a cryostat (NX50, Thermo Fisher Scientific, Weltham, MA, USA). Slides were dried and hydrated in PBS for 10 minutes. Blocking solution (10% goat serum, 1% BSA, and 0.3% Triton-X in PBS) was added to the slides for 1 h. Primary antibodies against MCT1-4 and glutamine synthetase (GS) were dissolved in blocking solution and added to the slides, which were then incubated overnight at 2–8°C. They were then rinsed three times with PBS, incubated with secondary antibodies in the dark for 1 h, and washed again with PBS. Mounting medium with DAPI was applied, and the slides were imaged using a fluorescence microscope. To detect MCT1 and GS, the following channels were used: Ex. / Em.557 / 572. For MCT2 detection, the channels were Ex. / Em.577 / 603. For MCT3 and MCT4 detection, the channels were Ex. / Em.493 / 517.

[0092] [Table 2]

[0093] 1.6 Uptake of monocarboxylic acid nanoparticles in organotypic retinal explant cultures Following a previously established protocol (Belhadj et al., Long-Term, Serum-Free Cultivation of Organotypic Mouse Retina Explants with Intact Retinal Pigment Epithelium. J.Vis.Exp., 2020), retinas were isolated from wild-type mice at postnatal day (P) 13, cultured, and maintained in culture until P15. A 20 μL drop of 5 mg / mL calcein-loaded liposomes (Lp-OMe, Lp-Pyr, or Lp-Cys) was then added to the culture at the surface corresponding to the vitreoretinal interface. Alternatively, 1 μM AR-C155858 was added to the organotypic medium before the addition of Lp-OMe or Lp-Pyr. After 6 hours of incubation (37°C, 5% CO), the retinal explant cultures were fixed in 4% paraformaldehyde, cryoprotected in sucrose, and frozen in liquid nitrogen according to the previously described protocol. Using a cryostat, 14 μm sections of the retinal explant cultures were obtained from the center of the tissue. The sections were hydrated in PBS for 10 minutes, and a drop of DAPI-containing mounting medium was added. Images were then captured under a fluorescence microscope to record the DAPI signal (Ex. / Em. 353 / 465 nm) and calcein signal (Ex. / Em. 493 / 517 nm). Z-stacks were obtained by recording 11 images spaced 1 μm apart. The stacks were projected using the maximum intensity projection (MIP) function. From these images, fluorescence intensity was measured for each of the following layers using acquisition software (ZEN2.6, Zeiss, Oberkochen, Germany): ganglion cell layer, inner plexiform layer, inner nuclear layer, outer plexiform layer, outer nuclear layer, and inner and outer photoreceptor segments. Furthermore, immunostaining of cone photoreceptors (cone-arrestin) was performed on sections from cultures incubated with calcein-loaded Lp-Pyr according to the immunostaining procedure described above for the mouse retinal sections.

[0094] 1.7 Therapeutic Effects of Nanoparticle-Delivered Drugs Retinas from rd1 or rd10 mouse models were cultured according to the protocol described above and treated with the CN03 or CN04 drugs. Treatment was performed by applying 20 μL of a solution containing 160 μM drug (loaded in liposomes or free solution) on top of the culture. Assuming uniform distribution in the medium, the final drug concentration in the medium would have been 3.14 μM. After fixation with 4% paraformaldehyde, the histological workup described above was followed, and 14 μm-thick sections were prepared. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed on rd1-derived culture sections according to the published protocol detailed in Prajapati, M., et al., Molecules, 2021.26(5)). Mounting medium containing DAPI was applied, and the sections were imaged under a fluorescent microscope. For TUNEL detection, an Ex. / Em. 548 / 561 nm filter was used. Eleven Z-stacks were recorded at 1 μm intervals, and the number of TUNEL-positive cells in the outer nuclear layer was manually counted from the projection images. For rd10-derived cultures, the average number of photoreceptor columns belonging to the outer nuclear layer was counted from microscopic images using the same imaging method. The treatment paradigm for organotypic retinal explant cultures derived from the rd1 or rd10 mouse models was as follows: rd1 treatment was initiated on postnatal day 7 of culture and discontinued on day 11; rd10 treatment was initiated on day 11 of culture and discontinued on days 17 and 24. Cultures were treated every other day and discontinued by chemical fixation at the indicated time points. The same paradigm was followed for untreated cultures.

[0095] Example 2 - Results [2.1 MCT expression in the retina] We first examined the expression of MCTs in the retina, specifically on photoreceptors, to assess whether these transporters could be used for liposome targeting. Immunostaining for MCT isoforms 1–4 was performed on retinal tissue sections. Different MCT isoforms were expressed in different regions of the retina. MCT1 and MCT2 were found to be expressed on photoreceptors. MCT1 was localized primarily in the inner segments of photoreceptors. MCT2 was found on cell bodies in the outer nuclear layer. Because of its localization near the outer border of the outer nuclear layer, these may have been cone photoreceptors. The majority of cells in the inner nuclear layer also expressed MCT2. MCT3 was not detected in the neural retina. MCT4 was primarily localized at the vitreoretinal interface. Co-staining with the Müller glial cell marker glutamine synthetase revealed co-expression of MCT4 and Müller glial endfeet.

[0096] 2.2 Characterization of Monocarboxylic Acid Nanoparticles Because MCTs are expressed on retinal photoreceptors, we designed a nanoparticle system to target MCTs, such as a liposome system conjugated with MCT substrates. Lactate and pyruvate are known substrates of MCTs, and liposomes conjugated with these were prepared. Cysteine, although structurally similar to both molecules but not generally considered a substrate for MCTs, was also used. All molecules were conjugated to the terminus of a water-soluble polymer, here poly(ethylene glycol) (PEG), and linked to the nanoparticle surface. To confirm successful liposome formation, dynamic light scattering (DLS) was performed to measure the hydrodynamic diameter and zeta potential (Table S3—data represent mean ± SD for n=3). The size of the conjugated liposomes was similar to that of untargeted, control liposomes. Pyruvate and lactate liposomes exhibited a more negative surface potential than control liposomes, indicating successful conjugation. Since cysteine ​​is neutral at pH 7, the cysteine-liposomes exhibited the same zeta potential as the control.

[0097] [Table 3]

[0098] 2.3 Cellular uptake of monocarboxylic acid-coated nanoparticles The cellular uptake of monocarboxylic acid-coated nanoparticles was investigated using a human embryonic kidney cell line (HEK293T). Immunostaining demonstrated the expression of MCT isoforms 1–4 in these cells. Liposomes and micelles were examined. All liposome formulations (monocarboxylic acid-conjugated or untargeted) were loaded with calcein, a cell-impermeable green fluorescent dye, and incubated with cells for 2 hours. Micelles were loaded with DiO (CAS No. 34215-57-1). Fluorescence images revealed higher uptake of pyruvate- and cysteine-coated liposomes compared to other conditions. Calcein uptake in cells was significantly increased with pyruvate- and cysteine-coated liposomes (Figure 1A), with the former showing higher uptake than the latter. Cysteine-coated FePt nanoparticles have previously been shown to be taken up by HEK293 cells (Liang, S., et al., Int J Nanomedicine, 2015.10:pp.2325-33). Micelles exhibited comparable behavior (Figure 1B).

[0099] Interestingly, when inhibitors of MCTs (AZD3965 and AR-C155858) were added, less calcein was delivered to cells by pyruvate-liposomes, demonstrating that its uptake is at least partly mediated by MCTs. This difference was significant when the inhibitor AR-C155858 was used. AZD3965 inhibited MCT1 (K i =1.6 nM), while AR-C155858 was selective for MCT1 and MCT2 (K i= 2.3 and < 10 nM), suggesting that pyruvate-liposomes are more likely to cause MCT2-dependent uptake. When the AR-C155858 inhibitor was added, there was still a trend toward higher calcein uptake by Lp-Pyr compared to Lp-OMe. This suggests that MCT3 or MCT4 may also mediate uptake in HEK293 cells. Both of these transporters have been shown to uptake lactate in vitro.

[0100] 2.4 Retinal uptake of pyruvate-coated liposomes To determine the potential of pyruvate-liposomes to deliver drugs to photoreceptors, their uptake in organotypic retinal explant cultures derived from mice was analyzed. Because the targeting compound is a hydrophilic molecule, a similarly hydrophilic dye, calcein, was used to predict where the drug would accumulate in the tissue. MCT-targeted pyruvate-liposomes (Lp-Pyr), cysteine-liposomes (Lp-Cys), or untargeted control liposomes (Lp-OMe) were loaded with calcein. These were added to the vitreous side of isolated retinas in retinal explant cultures on postnatal day 15 to simulate IVT administration. After 6 hours of incubation, the cultures were fixed, frozen, and sectioned. The amount of calcein dye in the sections was analyzed from fluorescence microscopy images (Figure 2A) and signal measured from distinct retinal layers (Figure 2B). Lp-Pyr achieved higher calcein uptake in the inner plexiform layer (IPL) and outer retina compared with Lp-OMe, from the outer plexiform layer (OPL) to the photoreceptor segments. A higher calcein signal could be detected in the ONL for Lp-Pyr than for Lp-Cys. This comparison is particularly relevant because the two formulations share very similar structures and sizes (see Table S3). The zeta potential of Lp-Pyr is more negative than that of Lp-Cys, which would normally be expected to reduce cellular uptake. This further demonstrates the ability of pyruvate conjugation to drive cellular uptake.

[0101] To determine whether the higher uptake was mediated by MCTs, retinal cultures were treated with the MCT1-2 inhibitor, AR-C155858, during the incubation period with Lp-Pyrt. Addition of the inhibitor resulted in an overall decrease in calcein signal, suggesting that uptake was mediated by MCTs. Because AR-C155858 is not considered an inhibitor of MCT4, our results indicate that MCT1-2 is important for the uptake of conjugated particles.

[0102] Because MCT regulates the flow of metabolites inside and outside cells, inhibiting these important transporters may limit or slow down the cell's ability to take up liposomes due to low energy conditions or toxic effects.To control this, we used Lp-OMe in combination with MCT inhibitors.We found that, unlike Lp-Pyr uptake, the addition of AR-C155858 did not cause a decrease in Lp-OMe, suggesting that the transporter itself is responsible for Lp-Pyr uptake.

[0103] MCT immunostaining indicated that cones primarily expressed MCT2. Because MCT2 is thought to be more specific for pyruvate transport, we tested whether Lp-Pyr was delivered to cones or rods in the outer retina. Immunostaining for the cone-specific marker cone-arrestin was performed on sections from cultures to which Lp-Pyr had been applied, and the relative amounts of calcein-containing rods and cones were determined. We found a trend toward proportionally greater cone uptake than rod uptake. Although the difference was not significant (p = 0.067), this suggests that Lp-Pyr may be more selective for cone photoreceptors, which may be relevant in the context of cone-specific diseases such as achromatopsia or age-related macular degeneration (AMD).

[0104] 2.5 Therapeutic effects of drug-loaded pyruvate-liposomes Because pyruvate-liposomes showed significant photoreceptor uptake, we next tested whether these liposomes could enhance the delivery of specific drugs to photoreceptors. We used retinal explant cultures derived from the rd1 photoreceptor degeneration mouse model and treated them from P7 to P11, a time point just before the peak of degeneration. We evaluated the efficacy of the liposomal system using both free CN03 and CN04 as well as encapsulated compounds. We used the TUNEL assay to detect dying photoreceptors in tissue cross-sections as a readout of the effects of different treatments (Figure 3).

[0105] Application of CN03 or CN04 to rd1-derived cultures did not result in a significant reduction in dying photoreceptors, but there was a trend toward reduced cell death. While CN03 and CN04 were protective in this model, the amount used here (160 μM in 20 μL diluted in 1 ml medium = 3.14 μM) was too low to achieve a reduction in cell death when free drug solution was used alone. The Lp-OMe system proved insufficient to deliver CN03 or CN04 to photoreceptors. However, the Lp-Pyr system loaded with either CN03 or CN04 was able to significantly protect photoreceptors. Compared to untreated controls (NT), cell death was approximately 50% less. This protection was significant compared to the free drug solution and the Lp-OMe system, suggesting that Lp-Pyr actively assisted in the delivery of the drug to photoreceptors. To confirm that pyruvate-liposomes themselves were not responsible for protecting photoreceptors, empty Lp-Pyr without drug was tested and found not to reduce dying photoreceptors. The concentration was chosen to be 2 mg / mL because this was the approximate concentration in the Lp-Pyr / CN03 sample. For Lp-Pyr, the encapsulation efficiency was 24.7 ± 6.5% for CN03 and 80.0 ± 5.9% for CN04. Due to the low encapsulation efficiency of CN03, these samples had the highest liposome concentrations.

[0106] The rate of cell death in the rd1 model is very rapid, with almost complete rod loss by P18, which is atypical of most IRD patients. A better representation of the human disease state is provided by the slower degenerating rd10 mouse model, in which the peak of photoreceptor cell death occurs around P20. Therefore, we tested whether pyruvate-liposomes could achieve similar benefits in this model. We therefore tested CN03 encapsulated in DSPC-containing liposomes, a formulation that results in slower drug release and is expected to be better suited for in vivo use. In the rd10 model, we assessed long-term photoreceptor survival by quantifying the number of photoreceptor rows remaining in the tissue at P17 and P24 (i.e., before and after the peak of degeneration) (Figure 4). Protection of rd10 photoreceptors was confirmed when CN03 was encapsulated in pyruvate-liposomes, whereas the same concentration of free drug did not achieve a rescue effect.

[0107] [2.6 Discussion] 2.6.1 Nanoparticle uptake in the retina and photoreceptors Efficient retinal drug delivery is a key concern, particularly in the context of IRD. In this study, we developed a novel approach to deliver cargo to photoreceptors using a targeted nanoparticle drug delivery system. For example, we demonstrate that efficient photoreceptor protection can be achieved by pyruvate-conjugated liposomes loaded with neuroprotective drugs.

[0108] We found that when MCT-ligands were conjugated to liposome-grafted PEG chains, greater MCT-dependent cellular uptake was observed in HEK293T cells and photoreceptor uptake was observed in retinal explant cultures. Furthermore, this photoreceptor uptake enhanced the therapeutic efficacy of photoreceptor rescue drugs in vitro. We attribute this effect to at least the MCT1-2 transporter expressed on photoreceptors. Surprisingly, lactate nanoparticles did not achieve the same benefit, likely due to structural differences between lactate and the other ligands used. This demonstrates that minor differences between targeting ligands and natural substrates can affect transport recognition. This is further illustrated by the fact that our cysteine-liposomes achieved relatively low uptake in the retina, despite being structurally similar to pyruvate and exhibiting good uptake in other cell types.

[0109] Regarding retinal uptake, nanoparticles, such as liposomes, may be taken up by neuroglial cells due to the phagocytic properties of these cells. Retinal penetration of nanoparticles has been described in vivo in the mouse retina (Lee, J., et al., Mol. Pharm., 2017, 14(2): pp. 423-430), which may occur via transcytosis by Müller cells and subsequent release into the interphotoreceptor matrix. Several lines of evidence indicate that transporter targeting can improve transcellular transport across tissue barriers, such as the blood-brain barrier and intestinal epithelium. This suggests that pyruvate-liposomes, for example, may be able to penetrate the retina better than conventional liposomes, possibly via MCT4-mediated uptake into Müller cells. Like other cells, such as Muller cells and RPE in the retina, photoreceptors are not prone to endocytosis, although one study found endocytic activity in the inner segments of photoreceptors (Hollyfield, JG and MERayborn, Exp Eye Res, 1987. 45(5): pp. 703-19). There is little information available about the fate of transporters after liposome uptake. One study found that after initial liposome uptake, the transporters were recycled to the cell surface, meaning that liposomes did not result in cell-induced degradation of the transporters.

[0110] We used mouse explant retinal cultures, which have several advantages over in vivo models because they are faster and more reliable, and allow for direct investigation of retinal-specific effects. In the explant culture system, we did not preserve the vitreous after culture, and therefore did not test the IVT mobility of nanoparticles. Previously, we documented the biodistribution of nanoparticles and found that they could reach the retina after IVT injection into ex vivo pig eyes (Eriksen, AZ, et al., Int J Pharm, 2017. 522(1-2): pp. 90-97).

[0111] Overall, our studies demonstrate that the nanoparticles of the present invention are useful as drug delivery systems for the active targeting of photoreceptors.

Claims

1. i) Ligands of monocarboxylic acid transporters (MCTs) and ii) Pharmaceutically acceptable nanoparticles and Includes, The ligand is of general formula (I): 【Chemistry 1】 (In the formula, X is S, O, Se, or NH. c is -CH2-, -CH(CH3)-, -C(=O)-, -C(=S)-, -C(=NH)-, -CH(-OH)-, -CH(NH2)-, -CH(halogen)-, or -C(halogen)2-. n is 1, 2 or 3, and R is -H, -CH3, =O, =S, =NH, -OH, -NH2 or halogen, and * indicates the conjugation site with the pharmaceutically acceptable nanoparticles. A conjugated form of [something].

2. The conjugate according to claim 1, wherein the pharmaceutically acceptable nanoparticles are liposomes, solid lipid nanoparticles, micelles, carrier proteins, metal nanoparticles, polyplex systems, lipoplex systems, or polymer nanoparticles.

3. The pharmaceutically acceptable nanoparticles comprise one or more phospholipids in the conjugate according to claim 1.

4. The conjugate according to claim 1, wherein the pharmaceutically acceptable nanoparticles comprise one or more noncationic lipids.

5. The conjugate according to claim 1, wherein the pharmaceutically acceptable nanoparticles further comprise a pharmaceutically active substance.

6. The conjugate according to claim 5, wherein the pharmaceutically acceptable nanoparticles are a neuroprotective agent.

7. The conjugate according to claim 6, wherein the neuroprotective agent is a photoreceptor rescue agent.

8. The conjugate according to claim 7, wherein the photoreceptor rescue agent is a cyclic guanosine monophosphate (cGMP) analog.

9. The conjugate according to claim 1, wherein the pharmaceutically acceptable nanoparticles include a water-soluble polymer on their surface.

10. The conjugate according to claim 9, wherein the ligand is conjugated to the water-soluble polymer.

11. The conjugate according to claim 1, wherein X is S, O, or NH.

12. General formula (A): 【Chemistry 2】 (In the formula, X is S, O, Se, or NH. c is -CH ２ -, -CH(CH ３ )-, -C(=O)-, -C(=S)-, -C(=NH)-, -CH(-OH)-, -CH(NH ２ ) -, -CH (halogen) - or -C (halogen) ２ - and n is 1, 2 or 3, and R is -H, -CH ３ , =O, =S, =NH, -OH, -NH ２ or halogen, and Q is a conjugate of a lipid and a water-soluble polymer. A compound of [unclear].

13. The compound according to claim 12, wherein the lipid is a phospholipid.

14. A pharmaceutical composition comprising the conjugate described in any one of claims 1 to 11.

15. The pharmaceutical composition according to claim 14, for use in the treatment of neurodegenerative diseases or retinal diseases.

16. The pharmaceutical composition according to claim 15, wherein the disease is hereditary retinal degeneration (IRD), glaucoma, age-related macular degeneration, Stargardt disease, Usher syndrome, geographic atrophy, diabetic retinopathy, retinitis pigmentosa, Leber congenital amaurosis, blindness, loss of rod photoreceptors, night blindness, loss of cone photoreceptors, total color blindness, loss of color vision, or loss of high vision.

17. The pharmaceutical composition according to claim 14, for use in the treatment of retinal diseases.

18. The pharmaceutical composition according to claim 14, which is administered to a subject via local administration, periorbital administration, or intravitreous administration.

19. The pharmaceutical composition according to claim 18, which is administered to a subject via intravitreous administration.

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