Peptide compositions and uses thereof

Abstract

Disclosed herein are compositions / pharmaceutical compositions and kits comprising a peptide and a stabilizing agent; wherein the peptide comprises an ester, and is between 2 and 10 amino acids in length; and wherein the stabilizing agent is selected from the group consisting of a diol, a polyol, a monosaccharide, a disaccharide, an oligosaccharide, and a polysaccharide, including any combination thereof.

Description

[0001] Cross-references to related applications This application claims priority to IL Patent Application No. 304603, filed July 19, 2023. The entire contents of the above application are incorporated herein by reference as if they were fully set forth herein. Technical Field

[0002] This invention generally relates to the field of peptide-based formulations, and further to solid or aqueous compositions comprising peptides and stabilizers. Background Technology

[0003] The use of peptides as therapeutic active ingredients in drug therapy has emerged because they can promote targeted intracellular protein interactions, which is almost impossible with small molecule drugs.

[0004] However, the solution stability of peptide solutions is limited, especially solutions containing peptides with limited water solubility. In most cases, peptide solutions are stable for no more than 24 hours at room temperature.

[0005] Therefore, the need to improve the stability of aqueous solutions containing poorly water-soluble peptides has not yet been met. Summary of the Invention

[0006] In one aspect of the invention, a composition is provided comprising: (i) a peptide, including any enantiomer thereof, its salt, its hydrate or solvate, or any combination thereof; and (ii) a stabilizer; wherein: the peptide has a length between 2 and 10 amino acids; the peptide comprises an ester; and wherein the stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, including any combination thereof.

[0007] In some embodiments, the ester is a carboxyl-terminated ester.

[0008] In some embodiments, the composition is an aqueous solution.

[0009] In some embodiments, the aqueous solution includes citric acid and / or citrate.

[0010] In some embodiments, the concentration of the peptide in the aqueous solution is at most 40% by weight.

[0011] In some embodiments, the composition is stable at a temperature of 2 to 40°C for at least 72 hours.

[0012] In some embodiments, the peptide and stabilizer are present in the composition in a molar ratio of 1:1 to 10:1.

[0013] In some embodiments, the peptide comprises that of Formula 1: The structure is represented by R, where R is a C1-C5 alkyl group.

[0014] In some embodiments, the peptide is an L-leucyl-L-leucine ester, its salt, its hydrate or solvate, or any combination thereof.

[0015] In some implementations, R is a methyl group.

[0016] In some embodiments, the composition is in powder form, characterized by a water content of less than 5% w / w.

[0017] In another aspect, a pharmaceutical composition is provided comprising: (i) a peptide, including any enantiomer thereof, its salt, its hydrate or solvate, or any combination thereof; (ii) a stabilizer; and (iii) a pharmaceutically acceptable carrier; wherein the peptide has a length between 2 and 10 amino acids, and the peptide comprises an ester; and wherein the stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, including any combination thereof.

[0018] In some implementations, the pharmaceutically acceptable carrier is an aqueous solution.

[0019] In another aspect, a kit is provided comprising: (i) a peptide, including any enantiomer thereof, its salt, its hydrate or solvate, or any combination thereof; and (ii) a stabilizer; wherein the peptide has a length between 2 and 10 amino acids, and the peptide comprises an ester; and wherein the stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, and any combination thereof.

[0020] In some implementations, the peptides and stabilizers are in the form of a mixture.

[0021] In some implementations, the peptides and stabilizers are stored in separate containers.

[0022] In some implementations, the kit also includes a citrate-water buffer.

[0023] In some implementations, the peptide and stabilizer are present in the kit in a molar ratio of 1:1 to 10:1.

[0024] In some implementations, the peptide and citrate aqueous buffer are present in the kit at a weight ratio of 1:500 to 1:10000.

[0025] In some embodiments, the kit includes instructions for mixing peptides, stabilizers, and citrate-water buffer to obtain the compositions of the present invention. Detailed Implementation

[0026] In the following detailed description, numerous specific details are set forth in order to provide a thorough understanding of the invention. However, those skilled in the art will understand that the invention can be practiced without these specific details. In other instances, well-known methods, procedures, and components have not been described in detail so as not to obscure the invention.

[0027] According to one aspect of the invention, a composition is provided comprising a peptide and a stabilizer; wherein the peptide is characterized by having a water solubility of less than 200 mg / ml at a temperature of 20 to 30°C and a pH of 5 to 8; and wherein the stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, including any combination thereof.

[0028] According to another aspect of the invention, a composition is provided comprising a peptide and a stabilizer; wherein the peptide is a low-soluble peptide, characterized in that its water solubility is less than 200 mg / ml at a temperature of 20 to 30°C and a pH of 5 to 8; and wherein the stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, including any combination thereof.

[0029] In some embodiments, the low-soluble peptide is characterized by a water solubility of up to 200 mg / ml, up to 150 mg / ml, up to 100 mg / ml, up to 40 mg / ml, up to 35 mg / ml, up to 30 mg / ml, up to 20 mg / ml, up to 25 mg / ml, up to 15 mg / ml, and up to 10 mg / ml, encompassing any range between these values, at temperatures of 20°C to 30°C and pH values ​​of 5 to 8, 5 to 6, or 6 to 7.5. In some embodiments, the low-soluble peptide is characterized by the water solubility as described above, and further characterized by the stability of the aqueous peptide solution for less than 72 hours, less than 48 hours, or less than 24 hours when stored at 20-30°C; and wherein said aqueous peptide solution consists substantially of the peptide and water or an aqueous buffer solution, and is free of the stabilizers disclosed herein.

[0030] In some embodiments, the peptide is characterized by its water solubility at temperatures of 20°C to 30°C and pH values ​​of 5 to 8, 5 to 6, or 6 to 7.5 in the range of about 1 mg / ml to about 200 mg / ml, about 1 mg / ml to about 150 mg / ml, about 1 mg / ml to about 130 mg / ml, about 1 mg / ml to about 100 mg / ml, about 1 mg / ml to about 40 mg / ml, about 1 mg / ml to about 30 mg / ml, about 1 mg / ml to about 25 mg / ml, about 5 mg / ml to about 40 mg / ml, about 5 mg / ml to about 30 mg / ml, or about 5 mg / ml to about 25 mg / ml, including any range in between.

[0031] The term "water solubility" refers to the ability of a compound to completely dissolve in an aqueous solution (such as water or an aqueous buffer such as citrate buffer), thereby forming a solution substantially free of particulate matter (i.e., undissolved aggregates or solid particles of the compound); and wherein said aqueous solution is substantially free of organic solvents (e.g., DMSO, ethanol, etc.). In some embodiments, the average particle size of the particulate matter is greater than 100 nm or greater than 200 nm. In some embodiments, the particulate matter in the solution is tested immediately after dissolution or within a time period of 1 to 48 hours, 1 to 24 hours, or 1 to 72 hours (inclusive) after dissolution.

[0032] The presence of particulate matter in a solution can be determined by various methods, such as DLS, optical microscopy, or visual inspection. Furthermore, the substantial absence of particulate matter can be assessed by filtering the solution through a 0.22-micron filter and calculating the content (e.g., w / w concentration) of the target compound in the filtered solution compared to the unfiltered solution (e.g., by HPLC). A solution is considered substantially free of particulate matter when the content in the filtered solution is greater than 90% or greater than 95% relative to the content in the unfiltered solution.

[0033] In some embodiments, the substantially organic solvent-free aqueous solution has a v / v concentration of organic solvent of up to 1%, up to 0.5%, up to 0.1%, up to 100 ppm, up to 10 ppm, or up to 1 ppm, including any range therebetween. In some embodiments, the aqueous solution is completely free of organic solvent (i.e., contains an amount of organic solvent below the detection limit of the GC instrument). In some embodiments, the water solubility of the peptide refers to the solubility of the peptide in its protonated amine form (i.e., at least one amino group of the peptide is in the protonated form -NH3). + (e.g., HCl salts of peptides).

[0034] In some embodiments, the peptide is 2 to 10 amino acids, 2 to 8 amino acids, 2 to 6 amino acids, 2 to 3 amino acids, or 2 to 4 amino acids, or 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, including any range or value therein. In some embodiments, the peptide is a therapeutically active peptide. In some embodiments, the peptide is the sole pharmaceutically active agent in the composition of the present invention.

[0035] As used herein, the term “peptide,” including any enantiomer thereof, any salt thereof, any hydrate thereof, or any solvate thereof, or any combination thereof, refers to a polymer or oligomer of amino acid residues linked together by peptide bonds.

[0036] As used herein, the term "peptide" includes natural peptides, linear or cyclic peptides, peptide derivatives such as β-peptides, peptide mimics (which typically include non-peptide bonds or other synthetic modifications), and peptide analogs such as peptoids and semi-peptoids, or any combination thereof. In another embodiment, the term "peptide" applies to amino acid polymers in which at least one amino acid residue is an artificial chemical derivative or analog of the corresponding natural amino acid.

[0037] The term "derivative" or "chemical derivative" includes any chemical derivative of a polypeptide having one or more residues chemically derived via a reaction on a side chain or any functional group within the peptide. Such derivatized molecules include, for example, peptides with one or more protecting groups (e.g., side chain protecting groups and / or N-terminal protecting groups), and / or peptides in which the free amino group has been derivatized to form an amine hydrochloride, p-toluenesulfonyl, benzyloxycarbonyl, tert-butoxycarbonyl, acetyl, or formyl group. Free carboxyl groups can be derivatized to form their amides, salts, alkyl esters such as methyl and ethyl esters, or other types of esters or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine. Also included as chemical derivatives are those peptides containing one or more naturally occurring amino acid derivatives of twenty standard amino acid residues. For example, 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and Dab, Daa, and / or ornithine (O) can replace lysine. The term "derivative" or "chemical derivative" also includes alkyl esters of peptides, such as carboxyl-terminated alkyl esters.

[0038] Furthermore, peptide derivatives can differ from the natural sequence of the peptides of the present invention by means of chemical modification, including but not limited to terminal -NH2 acylation, acetylation, or mercaptoacetate amidation, and amidation of terminal and / or side chain carboxyl groups, for example with ammonia, methylamine, etc. The peptides can be linear, cyclic, or branched, etc., having any conformation, which can be achieved using methods known in the art.

[0039] As used herein, the term "amino acid" includes amino acid residues linked to each other by at least one peptide bond. This term includes naturally occurring amino acids (i.e., val, ala, arg, asn, asp, cys, glu, gln, gly, his, met, ile, leu, lys, phe, pro, ser, thr, trp, and tyr), protected amino acids (e.g., those containing one or more protecting groups on their carboxyl, amine, and / or amino acid side chains), unusual non-naturally occurring amino groups (such as D-amino acids), and amino acids known to exist in free or combined forms but not typically present in proteins. The term also includes modified and unusual amino acids, such as those disclosed in Roberts and Vellaccio (1983) The Peptides. 5: 342-429. Modified, unusual, or non-naturally occurring amino acids include, but are not limited to, D-amino acids, hydroxylysine, 4-hydroxyproline, N-Cbz-protected aminovaleric acid (Nva), ornithine (O), aminooctanoic acid (Aoc), 2,4-diaminobutyric acid (Abu), homoarginine, norleucine (Nle), N-methylaminobutyric acid (MeB), 2-naphthylalanine (2Np), aminoheptanoic acid (Ahp), phenylglycine, β-phenylproline, tert-leucine, 4-aminocyclohexylalanine (Cha), N-methyl-nonoleucine, 3,4-dehydroproline, N,N-dimethylaminoglycine, N-methylaminoglycine, 4-aminopiperidin-4-carboxylic acid, 6-aminohexanoic acid, and trans-4-(amino (Methyl)-cyclohexanecarboxylic acid, 2-,3- and 4-(aminomethyl)-benzoic acid, 1-aminocyclopentanecarboxylic acid, 1-aminocyclopropanecarboxylic acid, cyanopropionic acid, 2-benzyl-5-aminovaleric acid, n-valine (Nva), 4-O-methylthreonine (TMe), 5-O-methylhomoserine (hSM), tert-butylalanine (tBu), cyclopentylalanine (Cpa), 2-aminoisobutyric acid (Aib), N-methylglycine (MeG), N-methylalanine (MeA), N-methylphenylalanine (MeF), 2-thienylalanine (2Th), 3-thienylalanine (3Th), O-methyltyrosine (YMe), 3-benzothienylalanine (Bzt), and D-alanine (DAl).

[0040] In some implementations, the term "peptide bond" refers to the bond formed by formula A: The secondary amide represented, or by formula B: The term "tertiary amide" refers to an amino acid side chain, a C1-C5 alkyl group, or a proline residue. In some embodiments, the term "peptide bond" includes formulas A and / or B, where R is a native amino acid side chain.

[0041] In some embodiments, the number of amino acid residues in the peptide is between 1 and 9, 1 and 7, 1 and 5, 1 and 3, or 2, including any range therebetween. In some embodiments, the peptide is a dipeptide, tripeptide, or tetrapeptide. In some embodiments, the peptide is a linear or cyclic dipeptide or a linear or cyclic tripeptide. In some embodiments, the peptide comprises a hydrophobic amino acid selected from gly, ala, leu, iso, val, pro, met, phe, tyr, and trp. In some embodiments, the peptide does not contain polar and / or charged amino acids such as ser, thr, asn, gln, asp, glu, arg, his, and lys.

[0042] In some embodiments, the peptide further includes an ester. In some embodiments, the ester is a carboxyl-terminated ester. In some embodiments, the C-terminus of the peptide, at least one carboxyl group of the peptide's side chain, or both are esterified. In some embodiments, the carboxyl-terminal amino acid is an esterified amino acid. In some embodiments, the ester refers to a C1-C... 10 Alkyl esters, or C1-C 10 The substituted alkyl ester comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, wherein each carbon atom is optionally substituted with a substituent. In some embodiments, the alkyl ester comprises up to 5 carbon atoms (e.g., 1, 2, 3, 4, or 5 carbon atoms). In some embodiments, the ester is a methyl ester.

[0043] The term "substituent" is as disclosed herein.

[0044] In some embodiments, a composition is provided comprising a peptide, including any enantiomer thereof, its salt, its hydrate or solvate, or any combination thereof; and a stabilizer; wherein the peptide is of length between 2 and 10 amino acids, and the peptide further comprises an ester (i.e., esterified amino acid); and wherein the stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, including any combination thereof.

[0045] In some embodiments, the ester is a carboxyl-terminated ester. In some embodiments, the C-terminus of the peptide, at least one carboxyl group of the peptide's side chain, or both are esterified. In some embodiments, the carboxyl-terminal amino acid is an esterified amino acid. In some embodiments, the ester refers to a C1-C... 10 Alkyl esters, or C1-C 10 Substituted alkyl esters contain 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 carbon atoms, wherein each carbon atom is optionally substituted with a substituent. In some embodiments, the alkyl ester contains up to 5 carbon atoms (e.g., 1, 2, 3, 4, or 5 carbon atoms).

[0046] In some embodiments, the peptide is a poorly soluble peptide, as described above.

[0047] In some embodiments, the peptide is represented by Formula 1: This includes any salt thereof, any tautomer thereof, any stereoisomer thereof, any hydrate thereof, or any combination thereof, wherein R is a C1-C5 alkyl group.

[0048] As used herein, the term "C1-C5 alkyl" refers to any straight-chain or branched alkyl chain comprising 1 to 5, 1 to 3, 1 to 4, or 1, 2, 3, 4, or 5 carbon atoms, including any range therein. In some embodiments, C1-C5 alkyl comprises any one or any combination of methyl, ethyl, propyl, isopropyl, butyl, pentyl, isopentyl, tert-butyl. In some embodiments, C1-C5 alkyl is a substituted C1-C5 alkyl. In some embodiments, C1-C5 alkyl as described herein also includes an unsaturated bond, wherein the unsaturated bond is located at the 1st, 2nd, 3rd, 4th, or 5th position of the C1-C5 alkyl.

[0049] In some embodiments, the peptide comprises a mixture of diastereomers. In some embodiments, the peptide comprises a single diastereomer. In some embodiments, the peptide comprises a mixture of enantiomers (e.g., a racemic mixture). In some embodiments, the peptide is rich in the enantiomer of interest. In some embodiments, the peptide is a single enantiomer. In some embodiments, the peptide is a single diastereomer.

[0050] In some implementations, the peptide is represented by Formula 2: This includes any salt thereof, any tautomer thereof, any hydrate or solvate thereof, or any combination thereof, wherein R is as described above. In some embodiments, the salt is a pharmaceutically acceptable salt.

[0051] In some implementations, R is a methyl group.

[0052] In some embodiments, the composition comprises multiple peptides, each of which is as described herein. In some embodiments, each of the multiple peptides differs in its chemical structure and / or chirality. In some embodiments, the multiple peptides consist of chemically distinct peptides. In some embodiments, the multiple peptides consist of peptides having the same chemical composition and / or the same chemical structure or chirality. In some embodiments, each peptide in the composition has the same amino acid sequence. In some embodiments, the peptides in the composition have at least two different amino acid sequences. In some embodiments, the peptides in the composition are in the form of a racemic mixture. In some embodiments, the peptides in the composition are in the form of a single enantiomer. In some embodiments, the peptides in the composition are characterized by an enantiomer purity of 70% to 100%, 90% to 100%, 80% to 99%, or 90% to 95%, including any range therebetween.

[0053] In some embodiments, the stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, or polysaccharides, including any combination thereof.

[0054] In some embodiments, non-limiting examples of stabilizers are sucrose, lactose, maltose, trehalose, cellobiose, chitobiose, kojibiose, nigerose, isomaltose, β,β-trehalose, α,β-trehalose, sophorose, laminaribiose, gentiobiose, turanose, trehalulose, maltulose, and Leuconostoc disodium disodium (Leuconostoc disodium) The stabilizer comprises ucrose, isomaltulose, 6-o-β-d-glucopyranosyl-d-fructose, mannobiose, melibiose, isolaxose, melibiulose, lactulose, rutinulose, xylobiose, mannitol, sorbitol, β-cyclodextrin, raffinose, aspergillus triose, maltotriulose, pinotriulose, maltotriulose, sucrose triose, ethylene glycol, glycerol, polyethylene glycol, propylene glycol, or any combination thereof. In some embodiments, the stabilizer does not contain polymer stabilizers. In some embodiments, the stabilizer is a small molecule with a molecular weight (MW) of less than 1000 Da or less than 500 Da.

[0055] In some embodiments, the stabilizer includes disaccharides (e.g., lactose, trehalose, etc.). In some embodiments, the stabilizer is trehalose.

[0056] In some embodiments, the stabilizer is a trisaccharide (e.g., fructose, raffinose, etc.). In some embodiments, the stabilizer is raffinose.

[0057] In some embodiments, the stabilizer can increase the water solubility of the insoluble peptide compared to the same original peptide without the stabilizer. In some embodiments, the stabilizer can form stable hydrogen bonds with the insoluble peptide in aqueous solution, thereby improving or enhancing the water solubility of the insoluble peptide.

[0058] In some embodiments, the weight ratio between the peptide and the stabilizer in the composition of the present invention is 1:1 to 10:1, 1:1 to 8:1, 1:1 to 6:1, 1:1 to 4:1, 1:1 to 1:2, 1:5 to 1:10 or 1:6 to 1:9, including any range therebetween.

[0059] In some embodiments, the compositions of the present invention are capable of increasing the water solubility of low-soluble peptides compared to peptides. In some embodiments, the water solubility of low-soluble peptides in the compositions of the present invention is increased by at least 20%, at least 50%, at least 2 times, at least 5 times, at least 10 times, at least 15 times, or at least 20 times, including any range therein, compared to the same original peptide.

[0060] In some embodiments, the low-soluble peptides in the compositions of the present invention are characterized by a water solubility of up to 600 g / L, up to 500 g / L, up to 400 g / L, up to 300 g / L, 40 g / L to 400 g / L, 50 g / L to 400 g / L, 50 g / L to 300 g / L, 50 g / L to 450 g / L, 50 g / L to 500 g / L, including any range therebetween, wherein the water solubility is measured at a pH of about 5.5 and a temperature of 20 to 25°C.

[0061] In some embodiments, the compositions of the present invention are free of surfactants and / or dispersants. In some embodiments, the compositions of the present invention are free of lipids. In some embodiments, the compositions of the present invention are free of additional stabilizers that are not stabilizers of the present invention. In some embodiments, the compositions of the present invention are free of additional peptides that are not the low-soluble peptides disclosed herein.

[0062] In some embodiments, the compositions of the present invention are used to treat a disease in a subject of need. In some embodiments, the disease is an ocular or retinal degenerative disease. In some embodiments, the compositions of the present invention are formulations for ocular application, such as in the form of eye drops or via intravitreal injection.

[0063] solution In some embodiments, the compositions of the present invention are in the form of a stable solution (also referred to herein as a "solution"). In some embodiments, the solution is a clear solution. In some embodiments, the solution is colorless (i.e., color is visible). In some embodiments, the solution of the present invention is a homogeneous solution. In some embodiments, clarity is determined by visual observation.

[0064] In some embodiments, as described above, the stable solution is substantially free of particulate matter, such as aggregates, precipitates, crystals, or undissolved substances. Various methods for detecting aggregates / particles in the solution are described above. In some embodiments, solution stability is determined by visual inspection. In some embodiments, the solution is free of solid particles with an average particle size greater than 10 nm, greater than 20 nm, greater than 30 nm, greater than 50 nm, greater than 100 nm, or greater than 200 nm, including any range therebetween.

[0065] In some embodiments, the solutions of the present invention are stable at temperatures ranging from 2°C to 50°C, 2°C to 40°C, 2°C to 25°C, 10°C to 30°C, 5°C to 40°C, 20°C to 30°C, 20°C to 27°C, or 5°C to 35°C, including any range or value therebetween.

[0066] In some embodiments, the solution of the present invention is stable over a period of at least 72 hours, at least 48 hours, or at least 26 hours, or 10 to 90 hours, 20 to 80 hours, 20 to 75 hours, or 20 to 72 hours, including any range therebetween.

[0067] In some embodiments, the solutions of the present invention are stabilized at temperatures of 2°C to 40°C, 2°C to 25°C, 10°C to 30°C, 5°C to 40°C, 20°C to 30°C, 20°C to 27°C, or 5°C to 35°C for the time periods described above, including any range or value therebetween.

[0068] In some embodiments, the solutions of the present invention are characterized by extended stability compared to a control (i.e., the same solution without the stabilizer of the present invention). In some embodiments, the extended stability is an extension of at least 20%, at least 30%, at least 50%, at least 70%, at least 100%, at least 150%, at least 200%, at least 300%, at least 500%, and any range therein.

[0069] In some embodiments, the solution is an aqueous solution. In some embodiments, the aqueous solution is substantially free of organic solvents, as described above. In some embodiments, the aqueous solution is completely free of organic solvents.

[0070] In some embodiments, the solution may be diluted up to 1000 times, wherein the diluted solution is substantially free of particulate matter, characterized in that it has at least the same solution stability as the solution of the present invention.

[0071] In some embodiments, the solution is characterized by a pH value between 5 and 8, 5 and 7, 5 and 6.5, 5.1 and 6.3, 5 and 6, 5.2 and 6, 5 and 5.8, and 5.3 and 5.7, including any range therebetween. In some embodiments, the solution is a buffered aqueous solution. In some embodiments, the buffered aqueous solution is characterized by a pH value between 5 and 8, 5 and 7, 5 and 6.5, 5.1 and 6.3, 5 and 6, 5.2 and 6, 5 and 5.8, and 5.3 and 5.7, including any range therebetween.

[0072] Examples of aqueous solutions include, but are not limited to, buffer solutions (e.g., phosphate buffer, bicarbonate buffer, etc.), salt solutions (e.g., inorganic salts), or aqueous salt solutions.

[0073] In some embodiments, the aqueous solution is a buffer solution. In some embodiments, the aqueous solution includes a citrate buffer solution. In some embodiments, the aqueous solution is a citrate buffer solution.

[0074] In some embodiments, the solutions of the present invention do not contain liquid particles (e.g., micelles, liposomes, nanodroplets, etc.). In some embodiments, the peptides are not encapsulated in liquid particles. In some embodiments, the solutions of the present invention do not contain emulsions, suspensions, or dispersions.

[0075] In some embodiments, the water content of the solution is at least 60%, at least 70%, at least 80%, at least 90%, at least 96%, at least 97%, or at least 98% by weight of the aqueous solution, including any range or value between these values. In some embodiments, the solution contains water as the sole solvent.

[0076] In some embodiments, the peptide content in the solution of the present invention is up to 40 wt%, up to 35 wt%, up to 30 wt%, or up to 25 wt%, including any range therebetween. In some embodiments, the weight percentage of peptides in the solution is 0.5 to 40 wt%, 1 to 40 wt%, 5 to 15 wt%, 1 to 10 wt%, 1 to 20 wt%, 5 to 25 wt%, 15 to 25 wt%, 25 to 35 wt%, 20 to 40 wt%, or 5 to 20 wt%, including any range therebetween.

[0077] In some embodiments, the weight percentage of the stabilizer in the solution is at least 1 wt%, at least 3 wt%, or at least 5 wt%, including any range or value therebetween. In some embodiments, the weight percentage of the stabilizer in the composition is 1 to 40 wt%, 1 to 50 wt%, 1 to 35 wt%, 1 to 33 wt%, 3 to 7 wt%, or 3 to 15 wt%, 1 to 10 wt%, or 3 to 38 wt%, including any range therebetween.

[0078] In some embodiments, the weight ratio between the peptide and the stabilizer in the solution is between 1:1 and 10:1, between 1:1 and 8:1, between 1:1 and 6:1, between 1:1 and 4:1, between 1:1 and 1:2, between 1:5 and 1:10, or between 1:6 and 1:9, including any range therebetween.

[0079] In some embodiments, the solution of the present invention comprises primarily peptides, stabilizers, and an aqueous buffer solution, wherein the peptides and stabilizers are as disclosed herein. In some embodiments, the solution of the present invention comprises at least 93%, at least 95%, at least 97%, at least 99%, or 90 to 99%, 95 to 99%, 95% to 100% by weight of peptides, stabilizers, and an aqueous buffer solution, wherein the peptides and stabilizers are as disclosed herein. In some embodiments, the solution of the present invention is a citrate buffer solution with a pH between 5 and 7, or between 5 and 6.

[0080] powder In some embodiments, the compositions of the present invention are solid compositions. In some embodiments, the compositions are solid at temperatures below 100°C. In some embodiments, the compositions of the present invention are powdered compositions. In some embodiments, the compositions of the present invention are powdered bulk materials.

[0081] In some embodiments, the powder is a homogeneous powder. In some embodiments, the powder is a homogeneous mixture of all the components of the powdered composition. In some embodiments, a homogeneous powder refers to a material that cannot be easily separated into individual components (e.g., peptides and stabilizers). In some embodiments, the entire bulk volume of the homogeneous powder has substantially the same concentration (with a deviation of up to 10%) of all the components of the powdered composition.

[0082] The terms “powderous composition” and “powder” are used interchangeably in this document.

[0083] In some embodiments, the powder is flowable. In some embodiments, the powder is a free-flowing powder.

[0084] In some embodiments, the powder is a white powder. In some embodiments, the powder is a pale yellow (light yellow) powder.

[0085] In some embodiments, the powder is a dry powder, characterized by a water content of less than 5 wt%, less than 4 wt%, or less than 3 wt%, including any range or value therebetween. In some embodiments, the dry powder is lyophilized. In some embodiments, the water content of the dry powder is between 0.001 and 5 wt%, 0.001 and 0.01 wt%, 0.001 and 0.1 wt%, 0.1 and 5 wt%, 0.1 and 4 wt%, 0.1 and 4 wt%, 0.1 and 3 wt%, 0.1 and 2 wt%, 0.1 and 1 wt%, 1 and 5 wt%, 2 and 5 wt%, including any range or value therebetween.

[0086] In some embodiments, the weight ratio between the peptide and the stabilizer in the powder is between 1:1 and 10:1, between 1:1 and 8:1, between 1:1 and 6:1, between 1:1 and 4:1, or between 1:1 and 1:2, including any range therebetween.

[0087] In some embodiments, the peptides in the powder of the present invention are 10 to 90 wt%, 45 to 65 wt%, 45 to 85 wt%, 45 to 55 wt%, 50 to 55 wt%, or 50 to 60 wt%, 45 to 75 wt%, including any range therebetween.

[0088] In some embodiments, the weight percentage of stabilizer in the powder of the present invention is 10 to 55 wt%, 10 to 90 wt%, 45 to 60 wt%, 45 to 55 wt%, 50 to 55 wt%, or 50 to 60 wt%, including any range therebetween.

[0089] In some embodiments, the powder of the present invention further includes a buffer. In some embodiments, the weight ratio of the buffer to the peptide in the powder of the present invention is 1:200 to 1:10000, 1:200 to 1:800, 1:200 to 1:600, 1:200 to 1:400, 1:400 to 1:600, or 1:500 to 1:600, including any range therebetween. In some embodiments, the weight ratio of the buffer to the peptide in the powder of the present invention is 100:1 to 1:100, 50:1 to 1:50, 20:1 to 1:20, 20:10000 to 20:1000, 20:500 to 10:500, including any range therebetween. In some embodiments, the powder of the present invention contains a sufficient amount of buffer to obtain a buffer solution upon dilution of the powder of the present invention, wherein the dilution is for obtaining a therapeutically effective amount of peptide in the resulting buffer solution.

[0090] In some embodiments, the buffer is a salt of a weak acid. In some embodiments, the weak acid is a weak organic acid. In some embodiments, the weak acid includes any one of citric acid, formic acid, acetic acid, lactic acid, trichloroacetic acid, benzoic acid, and dihydrogen phosphate. In some embodiments, the weak acid salt is a pharmaceutically acceptable salt of a weak acid.

[0091] In some implementations, the buffer is or includes citrate (e.g., sodium citrate).

[0092] In some embodiments, the powder is primarily composed of peptides, stabilizers, and buffers (e.g., weak acid salts); wherein the weight ratio of peptides to stabilizers within the powder is as described above, and wherein the weight ratio of buffers to peptides within the powder is approximately 100:1 to 1:100. In some embodiments, on a dry weight basis, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 90 to 99.9% of the powder is composed of peptides, stabilizers, and buffers. In some embodiments, on a dry weight basis, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 90 to 99.9% of the powder is composed of peptides, trehalose, and buffers. In some embodiments, on a dry weight basis, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 90 to 99.9% of the powder is composed of peptides, trehalose, and weak acid salts (e.g., citrate).

[0093] In some embodiments, the powder is a free-flowing powder. Methods for measuring powder flowability are well known to those skilled in the art, such as powder rheometers.

[0094] In some embodiments, the powder is stable. In some embodiments, the powder of the present invention is stable at temperatures of up to 70°C, up to 60°C, up to 50°C, and up to 40°C, including any range or value therebetween. In some embodiments, the powder of the present invention is stable over time periods of up to 5 years, up to 3 years, up to 1 year, and up to 6 minutes, including any range therebetween. The powder is considered stable when it maintains at least 90% by weight, at least 95% by weight, or at least 97% by weight of the initial concentration of the pharmaceutical active agent.

[0095] In some embodiments, the powder of the present invention is water-soluble. In some embodiments, the powder of the present invention is characterized by a water solubility of at least 1 g / L, at least 10 g / L, at least 50 g / L, at least 100 g / L, at least 200 g / L, at least 300 g / L, or at most 900 g / L, at most 800 g / L, at most 500 g / L, or at most 750 g / L, including any range therebetween, wherein the water solubility is determined at the temperature and pH values ​​disclosed above.

[0096] In some embodiments, the powder of the present invention is characterized in that its water solubility is between 10 g / L and 800 g / L, 100 g / L and 400 g / L, 10 g / L and 200 g / L, 50 g / L and 150 g / L, 50 g / L and 250 g / L, 150 g / L and 250 g / L, 200 g / L and 400 g / L, or 50 and 400 g / L, including any range therebetween.

[0097] In some embodiments, the powder of the present invention is primarily composed of peptides, stabilizers, and buffers. In some embodiments, on a dry weight basis, at least 93%, at least 95%, at least 97%, at least 99%, or 90 to 99%, 95 to 99%, or 95 to 100% of the powder of the present invention is composed of peptides, stabilizers, and buffers. In some embodiments, on a dry weight basis, 93% to 100%, 93% to 95%, 93% to 97%, or 95% to 99% of the powder of the present invention is composed of peptides, stabilizers, and optionally buffers.

[0098] In some embodiments, the powder of the present invention is completely soluble in water. In some embodiments, the resulting aqueous solution after reconstitution of the powder of the present invention is characterized by a peptide recovery rate of at least 90%, at least 95%, at least 97%, or 90 to 97%, including any range therebetween.

[0099] As used herein, the term "peptide recovery" refers to the recovery relative to the theoretical concentration of the peptide (based on the amount of dry powder and the amount of aqueous solution used for reconstitution). Recovery can be determined by HPLC.

[0100] Pharmaceutical Composition According to another aspect, a pharmaceutical composition is provided comprising: (i) a peptide; (ii) a stabilizer; and (iii) a pharmaceutically acceptable carrier; wherein the peptide and stabilizer are as disclosed herein.

[0101] As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic, inert solid or semi-solid liquid filler, diluent, encapsulating material, any type of pharmaceutical adjuvant, or a simple sterile aqueous medium such as saline. Some examples of materials that can be used as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragali powder; malt, gelatin, and talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers; alginic acid; pyrogen-free water; isotonic saline, Ringer's solution; ethanol and phosphate buffer solutions; wetting agents and lubricants such as sodium dodecyl sulfate; and colorants, flavoring agents, excipients, stabilizers, antioxidants, preservatives, and other non-toxic and compatible substances used in pharmaceutical formulations. Any non-toxic, inert, and effective carrier may be used to formulate the compositions considered herein. In this regard, suitable pharmaceutically acceptable carriers, excipients, and diluents are well known to those skilled in the art, such as those described below: The Merck Index, 13th edition, edited by Budavari et al., Merck, Inc., Ravi, NJ (2001); the International Cosmetic Ingredient Dictionary and Handbook, 10th edition (2004), CTFA (Cosmetic, Toiletries and Fragrance Association); and the Inactive Ingredient Guide, Office of Management, Center for Drug Evaluation and Research (CDER), U.S. Food and Drug Administration (FDA), all of which are incorporated herein by reference in their entirety.

[0102] In some embodiments, the compositions described herein may also be incorporated into artificially created structures such as liposomes, ISCOMS, sustained-release particles, and other carriers that increase the half-life of peptides or polypeptides in serum. Liposomes used with the peptides described herein are formed from standard vesicle-forming lipids, which typically comprise neutral and negatively charged phospholipids and sterols such as cholesterol. The choice of lipids generally depends on factors such as liposome size and stability in the blood. Various methods are available for the preparation of liposomes, as reviewed in Current Protocols in Protein Science, by Coligan, JE, et al., 1999, John Wiley & Sons, Inc., New York, and also see U.S. Patent Nos. 4,235,871, 4,501,728, 4,837,028, and 5,019,369.

[0103] In some embodiments, the pharmaceutically acceptable carrier is a liquid carrier. In some embodiments, the liquid carrier is an aqueous solution. In some embodiments, the liquid carrier is a buffer solution. In some embodiments, the liquid carrier is characterized by a pH value between 5 and 8, 6 and 7.5, 5 and 6.5, 5.1 and 6.3, 5 and 6, 5.2 and 6, 5 and 5.8, and 5.3 and 5.7, or any range therein. In some embodiments, the liquid carrier is an aqueous saline solution. In some embodiments, the liquid carrier is a buffered aqueous saline solution. In some embodiments, the liquid carrier is a citrate buffer solution.

[0104] In some embodiments, the carrier comprises, by weight, 0.1 to 99.99%, 0.1 to 50%, 0.2 to 30%, 0.1 to 70%, 30 to 99.99%, 50 to 99.99%, and any range therein, of the pharmaceutical composition described herein.

[0105] In some embodiments, the peptides have pharmaceutical-grade purity. In some embodiments, all components of the compositions of the present invention and pharmaceutical compositions have pharmaceutical-grade purity, characterized by a chemical purity of at least about 90%, at least about 95%, greater than 95%, or greater than 99%, and any range between 90 and 99.999%, 90 and 95%, 90 and 97%, and 95 and 99%. The chemical purity of the compounds / compositions can be determined by HPLC and / or LC / MS.

[0106] In some embodiments, the pharmaceutical composition consists essentially of the composition of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition consists primarily of the peptide of the present invention as the therapeutically active ingredient. In some embodiments, the peptide of the present invention is the sole active ingredient in the pharmaceutical composition. In some embodiments, the pharmaceutical composition does not contain any additional therapeutically active ingredients.

[0107] In some embodiments, the pharmaceutical composition comprises a therapeutically effective amount of peptide. In some embodiments, the pharmaceutical composition is a solution, wherein the therapeutically effective amount of peptide in the solution is between about 1% and about 5%, including any range therebetween.

[0108] In some embodiments, the pharmaceutical composition is an ophthalmic composition. In some embodiments, the pharmaceutical composition is formulated for ocular application. In some embodiments, the ophthalmic composition comprises additives. In some embodiments, the additives include, but are not limited to, tension modulators, preservatives, electrolytes, and / or any mixtures thereof. In some embodiments, the w / w concentration of the additives in the ophthalmic composition is between 0.01 and 20%, including any range therebetween.

[0109] In some embodiments, the ophthalmic composition contains an effective amount of a preservative. In some embodiments, the effective amount is an effective amount for preservative purposes. In some embodiments, the effective amount is an effective amount for antibacterial purposes. In some embodiments, according to USP... <51> Or any alternative to PET testing, based on which the effective amount of preservative is determined.

[0110] In some embodiments, the preservative is or includes a quaternary ammonium cation, such as benzalkonium (BAK), including any salt thereof, such as benzalkonium chloride. In some embodiments, the quaternary ammonium cation is a pharmaceutically acceptable compound. In some embodiments, the quaternary ammonium cation includes a pharmaceutically acceptable counter anion. Other preservatives (i.e., preservatives suitable for ophthalmic compositions) are known in the art.

[0111] In some embodiments, when measured at 25°C, the liquid ophthalmic composition is characterized by a viscosity between 1 and 150 cps.

[0112] In some embodiments, the ophthalmic composition is characterized by a tension between 200 and 600 mOsmols / kg, 200 and 500 mOsmols / kg, 200 and 300 mOsmols / kg, 300 and 350 mOsmols / kg, 350 and 400 mOsmols / kg, 400 and 450 mOsmols / kg, 450 and 500 mOsmols / kg, 500 and 550 mOsmols / kg, and 550 and 600 mOsmols / kg, including any range or value therebetween. In some embodiments, the ophthalmic composition is characterized by a tension between 250 and 450 mOsmols / kg.

[0113] In some embodiments, the pharmaceutical composition consists essentially of pharmaceutical-grade ingredients. In some embodiments, all components of the pharmaceutical composition are pharmaceutical-grade compounds. In some embodiments, the pharmaceutical composition is sterile. In some embodiments, the effective amount of preservative in the pharmaceutical composition of the present invention is sufficient to maintain its sterility when the pharmaceutical composition is exposed to non-sterile conditions (e.g., an environmental atmosphere containing one or more microorganisms), wherein the maintenance time ranges from 1 day to 2 months, 1 day to 1 month, 1 to 20 days, 1 to 60 days, 1 to 50 days, 10 to 60 days, 1 to 10 days, 10 to 50 days, 10 to 40 days, 10 to 30 days, and any range in between. The term "sterility" as used herein refers to the sterility requirements, microbial load, etc., of the composition (e.g., the ophthalmic composition of the present invention) or article as recorded in the United States Pharmacopeia or the European Pharmacopoeia, respectively.

[0114] In some embodiments, the pharmaceutical composition is packaged in unit dosage form. In some embodiments, the pharmaceutical composition is prepared by any method known in the pharmaceutical field. In some embodiments, the unit dosage form is in the form of tablets, capsules, lozenges, wafers, patches, ampoules, vials, or pre-filled syringes.

[0115] In some embodiments, each unit dose comprises an effective dose of the peptide of the present invention. The term "effective dose" refers to a daily dose of the composition / peptide of the present invention applied to a subject that is sufficient to induce the desired biological response (e.g., relief of symptoms, treatment of disease, etc.) in the subject.

[0116] In some embodiments, the pharmaceutical composition is used to treat or prevent a disease or disorder in a subject in need.

[0117] In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a laboratory animal. Examples of laboratory animals include, but are not limited to, mice, rats, rabbits, hamsters, dogs, cats, and monkeys. In some embodiments, the subject requires the composition of the present invention. In some embodiments, the subject requires treatment.

[0118] In some embodiments, the disease is an ocular disease. In some embodiments, the disease is an ocular or retinal degenerative disease. In some embodiments, the composition of the invention is administered via ocular or intraocular application. In some embodiments, ocular application is via intravitreal injection.

[0119] In some embodiments, the pharmaceutical compositions disclosed herein comprise a therapeutically effective amount of the composition of the invention. In some embodiments, a therapeutically effective amount refers to an amount that is effective in treating a disease or disorder in a mammal. The term "therapeuticly effective amount" means an effective amount that, within the necessary dosage and time period, achieves the desired therapeutic or preventative effect in a subject. The exact dosage form and regimen will be determined by a physician based on the patient's condition.

[0120] Reagent test kit According to another aspect, a kit is provided that comprises peptides, stabilizers, and optional buffers, as disclosed herein.

[0121] In some embodiments, the reagent kit components are stored in separate compartments. In some embodiments, peptides and stabilizers are stored in the same compartment. In some embodiments, peptides, stabilizers, and buffers are stored in separate compartments. In some embodiments, peptides, stabilizers, and buffers are stored in separate compartments.

[0122] In some embodiments, the peptide is in powder form. In some embodiments, the stabilizer is in solution or powder form. In some embodiments, the buffer is in powder or solution form.

[0123] In some embodiments, the kit also includes a diluent. In some embodiments, the diluent is an aqueous solution. In some embodiments, the diluent is a pharmaceutically acceptable carrier. In some embodiments, the diluent is an aqueous solution.

[0124] In some embodiments, the weight ratio of peptide to stabilizer in the kit is 1:1 to 10:1, 1:1 to 8:1, 1:1 to 6:1, 1:1 to 4:1 or 1:1 to 2:1, including any range therebetween.

[0125] In some embodiments, the peptide and citrate buffer are present in the kit at weight ratios of 1:500 to 1:10,000, 1:500 to 1:5,000, 1:500 to 1:1,000, 1:100 to 1:10,000, 1:5000 to 1:10,000, or 1:500 to 1:7,500, including any range therebetween.

[0126] In some embodiments, the kit also includes instructions for mixing kit components (dry or liquid components) in a predetermined ratio to obtain the composition of the invention.

[0127] In some embodiments, the kit also includes instructions for mixing or contacting the kit components with a predetermined amount of diluent to obtain the solution of the present invention. In some embodiments, mixing or contacting is performed at a temperature of 10 to 50°C, with sufficient mixing time to obtain a clear solution.

[0128] In some implementations, the kit is used in conjunction with the method of the present invention.

[0129] According to one embodiment of the present invention, the pharmaceutical composition and / or the pharmaceutical composition described above are packaged in packaging material, and a label for treating the disease or disorder as described herein is printed inside or on the packaging material.

[0130] According to another embodiment of the invention, the pharmaceutical composition / reagent kit is packaged in packaging material and has printed markings inside or on the packaging material for monitoring diseases or disorders as described herein.

[0131] If desired, the product of this invention can be packaged in a packaging or dispenser device, such as a U.S. Food and Drug Administration (FDA) approved kit, which may contain one or more unit dosage forms containing the disclosed composition. For example, the packaging may include metal or plastic foil, such as blister packs. The packaging or dispenser device may be accompanied by instructions for use. The packaging or dispenser may also be accompanied by a notification related to the container, in the form prescribed by the government agency regulating the manufacture, use, or sale of the drug, reflecting that agency's approval of the composition form or human or veterinary administration. For example, such a notification might be an FDA-approved prescription drug label or an approved product label.

[0132] method According to another aspect, a method is provided for treating a disease, disorder, or condition in a subject in need, comprising administering a therapeutically effective amount of the pharmaceutical composition of the present invention to the subject, thereby treating the disease, disorder, or condition.

[0133] In some embodiments, administration includes systemic administration. In some embodiments, administration includes local administration. In some embodiments, administration includes intravenous administration. In some embodiments, administration includes ocular administration. In some embodiments, administration includes intraocular administration. In some embodiments, administration includes intravitreal injection.

[0134] In some implementations, the method is used to reduce at least one symptom associated with a disease or condition in the subject.

[0135] In some embodiments, the disease is an eye disease. In some embodiments, the disease is a degenerative disease. Non-limiting examples of degenerative diseases include, but are not limited to, retinal diseases, familial drusen of cell senescence, glaucoma, Stargardt disease, and Best disease.

[0136] In some implementations, the subject has a retinal degenerative disease. Non-limiting examples of retinal degenerative diseases include, but are not limited to, age-related macular degeneration (AMD), wet AMD, diabetic retinopathy, or retinitis pigmentosa.

[0137] As used herein, the terms “administering,” “administration,” and similar terms refer to any method, in sound medical practice, of delivering a composition containing an active agent to a subject in a manner that provides a therapeutic effect.

[0138] The application steps can be repeated if necessary. The method of application of this composition depends on its form. For example, if the composition is a solution, it can be applied to the eye by drops, such as from a conventional eye dropper, or it can be injected into the eye.

[0139] In some embodiments, the amount (dosage) of the composition to be applied depends, of course, on the subject being treated, the condition being treated, the severity of the pain, the method of application, the prescribing physician's judgment, etc.

[0140] In some embodiments, the daily dose administered to each eye (i.e., the amount of peptide disclosed herein per eye per day) is in the range of 25 to 15,000 µg, 50 to 10,000 µg, 100 to 10,000 µg, 200 to 2000 µg, 50 to 100 µg, 100 to 200 µg, 200 to 300 µg, 300 to 400 µg, 400 to 500 µg, 500 to 600 µg, 600 to 700 µg, 700 to 800 µg, 800 to 900 µg, 900 to 1000 µg, 1000 to 1100 µg, 1100 to 1300 µg, 1300 to 1500 µg, 1500 to 1800 µg, 1000 to 15,000 µg, 1800 to 2000 µg. Between µg, including any range or value in between.

[0141] In some embodiments, the composition is applied 1, 2, 3, 4, 5, 6, 8, 9, 10 times daily, or any range thereof.

[0142] definition As used herein, the term "stable" refers to a clear solution that is free of precipitates, crystals, and / or particles.

[0143] As used herein, the term "mainly composed of" means that a composition may include other ingredients, provided that such other ingredients do not materially alter the fundamental and novel characteristics of the claimed composition. As used herein, the terms "treatment" or "treating" a disease, disorder, or condition include relieving at least one symptom, reducing its severity, or inhibiting its progression. Treatment does not necessarily mean a complete cure of the disease, disorder, or condition. For a composition to be an effective treatment, it is sufficient to reduce the severity of the disease, disorder, or condition, decrease the severity of associated symptoms, or improve the quality of life of the patient or subject.

[0144] As used herein, the term “prevention” of a disease, disorder, or condition includes delaying, preventing, suppressing, or containing the onset of a disease, disorder, or condition. As used in the context of the subject matter currently described, the term “prevention” relates to a preventative process in which a subject is exposed to the active ingredient currently described prior to the induction or onset of a disease / disorder process. This can be done in cases where an individual has a genetic lineage indicating a susceptibility to the occurrence of the disease / disorder to be prevented. For example, this may be true for individuals whose ancestors showed a predisposition to certain types of inflammatory disorders.

[0145] The term "suppression" is used to describe a situation where a disease / disorder has begun but no obvious symptoms have yet appeared. Therefore, an individual's cells may have a disease / disorder, but no external signs of the disease / disorder have yet been clinically detected. In either case, the term prophylaxis can be used to encompass both prevention and suppression.

[0146] Conversely, the term "treatment" refers to the clinical application of active agents to combat an existing disease whose clinical manifestations have already been achieved in a patient.

[0147] As used herein, the term “substituted” or the term “substituent” refers to one or more (e.g., 2, 3, 4, 5, or 6) substituents, wherein the substituents are as described herein.

[0148] As used herein, the term "substituent" includes hydrogen, halogen, -NO2, -CN, -OH, -CONH2, -CONR'2, -CNNR'2, -CSNR'2, -CONH-OH, -CONH-NH2, -NHCOR, -NHCSR, -NHCNR, -NC(=O)OR, -NC(=O)NR', ​​-NC(=S)OR', -NC(=S)NR', ​​-SO2R', -SOR', -SR', -SO2OR', -SO2N(R')2, -NHNR'2, -NNR', C1-C6 haloalkyl, optionally substituted C1-C6 alkyl, -NH2, -NH(C1-C6 alkyl), -N(C1-C6 alkyl)2, C1-C6 alkoxy, C1-C6 haloalkoxy , hydroxy (C1-C6 alkyl), hydroxy (C1-C6 alkoxy), alkoxy (C1-C6 alkyl), alkoxy (C1-C6 alkoxy), C1-C6 alkyl -NR'2, C1-C6 alkyl -SR', -CONH (C1-C6 alkyl), -CON (C1-C6 alkyl)2, -CO2H, -CO2R', -OCOR, -OCOR', -OC(=O)OR', -OC(=O)NR', ​​-OC(=S)OR', -OC(=S)NR', ​​or combinations thereof, wherein each R' independently represents hydrogen, or is selected from optionally substituted C1-C 10 Alkyl, optionally substituted C3-C 10 cycloalkyl, optionally substituted C3-C 10 Heterocyclic groups, optionally substituted heteroaryl groups, optionally substituted aryl groups, or combinations thereof.

[0149] As used herein, the term “substantially” means at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 60 to 99.9%, 70 to 80%, 70 to 90%, 80 to 90%, 90 to 95%, 95 to 99.9%, or any range or value in between.

[0150] In this discussion, unless otherwise stated, adjectives such as “substantially” and “approximately” that modify one or more features of an embodiment of the invention should be understood as meaning that the condition or feature is defined within an operationally acceptable tolerance range for the intended application of that embodiment. Unless otherwise stated, the word “or” in the specification and claims is considered inclusive rather than exclusive and indicates at least one or any combination of the items it connects.

[0151] It should be understood that the terms “a” and “an” as used above and elsewhere herein refer to “one or more” of the listed components. Those skilled in the art will appreciate that, unless otherwise specified, the use of the singular includes the plural. Therefore, the terms “a”, “an”, and “at least one” are used interchangeably in this application.

[0152] To better understand this teaching, and without limiting its scope in any way, all figures and other numerical values ​​representing quantities, percentages, or proportions used in the specification and claims, unless otherwise stated, should in all cases be understood to be modified by the term "about". Therefore, unless otherwise indicated, the numerical parameters described in the following specification and appended claims are approximate values ​​that may vary depending on the desired characteristics sought. At a minimum, each numerical parameter should be interpreted according to the number of significant figures reported and using conventional rounding techniques.

[0153] In the specification and claims of this application, each verb “comprise,” “include,” and “have,” and its related words, are used to indicate that one or more objects of the verb are not necessarily a complete enumeration of the components, elements, or parts of one or more subjects of the verb.

[0154] Other terms used herein are meant to be defined by their well-known meaning in the field.

[0155] Unless otherwise specified or obvious from the context, the term “or” as used herein should be understood to be inclusive.

[0156] In this specification and claims, the word “comprise” or variations such as “comprises” or “comprising” means that any integer or group of integers included in any statement is included, but does not exclude any other integer or group of integers.

[0157] As used herein, the terms “comprises,” “comprising,” “containing,” “having,” etc., can mean “includes,” “including,” etc.; “consisting essentially of” or “consistsessentially” also have the meanings provided for in U.S. patent law, and the term is open-ended, allowing for more than said content, as long as the essential or novel features of said content are not altered by the presence of more than said content, excluding prior art embodiments. In one embodiment, the terms “comprises,” “comprising,” and “having” can be used interchangeably with “consisting.”

[0158] Although the invention has been described in conjunction with specific embodiments thereof, it will be apparent to those skilled in the art that many alternatives, modifications, and variations will be apparent. Therefore, the invention is intended to cover all such alternatives, modifications, and variations falling within the spirit and broad scope of the appended claims.

[0159] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, to the same extent that each individual publication, patent, or patent application is specifically and individually indicated to be incorporated herein by reference. Furthermore, any reference or identification of any reference in this application should not be construed as an admission that such reference is prior art to the invention. The use of section headings should not be construed as an inherent limitation.

[0160] Example Materials and Methods Material L-Leucine, L-Leucyl-methyl ester monohydrochloride (CAS#6491-83-4) was purchased from Angene. Polysorbate 80 (Tween 80) was purchased from Croda. Polysorbate 20 (Tween 20), trehalose dihydrate, citrate monohydrate, and sodium hydroxide were purchased from Merck. Potassium dihydrogen phosphate was purchased from Fisher Chemical, deionized (DI) water was purchased from Formulex, and trisodium citrate dihydrate was purchased from Sigma Aldrich.

[0161] Citrate buffer (0.1M, pH 5.5) Mix 23.3 mL of citric acid solution (21.01 g citric acid in 1 L DI water) with 76.7 mL of sodium citrate solution (29.0 g sodium citrate in 1 L DI water) to obtain 100 mL of citrate buffer.

[0162] Phosphate buffer (0.1M, pH 7.4) Mix 50.0 mL of KH2PO4 solution (27.22 g KH2PO4 in 1 L DI water) with 39.1 mL of sodium hydroxide solution (8.0 g sodium hydroxide in 1 L DI water), and add 110.9 mL of DI water to prepare 200 mL of phosphate buffer.

[0163] Example 1 - Solubility of an exemplary dipeptide of the present invention First, as described below, an exemplary low-soluble peptide (L-leucyl-L-leucine methyl ester hydrochloride) and its free carboxyl counterpart (L-Leu-L-Leu-OH) were tested. The water solubility of HCl is relatively high.

[0164] Weigh 100.6 mg of L-Leu-L-Leu OH and place it in a clear glass test tube. Add approximately 800 μL of 1M HCl (approximately twice the excess) using a micropipette and mix thoroughly. Add approximately 4200 μL of acetonitrile using a micropipette to form an azeotrope and mix thoroughly. Heat the mixture to 80°C in a water bath to evaporate the liquid. Weigh the contents of the test tube containing dried L-Leu-L-Leu OH. A test tube containing HCl. At room temperature, add L-Leu-L-Leu-OH... Add water dropwise to the HCl tube. When a nearly clear solution is observed (visual inspection), stop adding water. Mix the solution overnight until completely clear. Weigh the resulting solution. Calculate 115.3 mg L-Leu-L-Leu-OH. Solubility of HCl in the resulting volume (mL = 1 mg) of water. L-Leu-L-Leu-OH The calculated water solubility of HCl is 35.7 mg / mL.

[0165] Weigh 100.3 mg of L-leucyl-L-leucine methyl ester hydrochloride (LLME) Add HCl to a transparent glass test tube. Weigh the contents of a test tube containing dry LLME. A test tube containing HCl. At room temperature, add LLME... Add water dropwise to the HCl tube. When a nearly clear solution is observed (visual inspection), stop adding water. Mix the solution overnight until completely clear. Weigh the resulting solution. Calculate the LLME. Solubility of HCl in the resulting volume (mL = 1 mg) of water. LLME The calculated water solubility of HCl is 19.9 mg / mL.

[0166] Subsequently, the solubility and solution stability of LLME-HCl in 1–10% phosphate buffer (pH 7.4) and 1–10% citrate buffer were examined in the presence of trehalose, Tween 20, and Tween 80 as potential stabilizers. All compositions were visually evaluated to determine their clarity and absence of precipitation over two weeks. Exemplary compositions were prepared by mixing LLME-HCl and trehalose at a temperature of 40°C to 50°C, according to Table 1.

[0167] Table 1: Formulation variables and observed visual stability. Citrate buffer pH 5.5; phosphate buffer pH 7.4.

[0168] Citrate buffer increases peptide stability. Furthermore, the presence of trehalose in the formulation stabilizes the peptide solution for 2 to 14 days at concentrations up to 30% w / w, depending on the trehalose-to-peptide ratio and peptide concentration.

[0169] Furthermore, no significant differences were observed when the preparation was filtered using a 0.22 μm nylon filter, indicating that sterilization was feasible.

[0170] The inventors have demonstrated that aqueous solutions containing the compositions of the present invention significantly improve the solution stability of LLME-HCl compared to similar solutions without stabilizers.

[0171] Surprisingly, the inventors found that the presence of surfactants significantly reduced the stability of the exemplary solutions of the present invention.

[0172] Example 2 - Enhancing stability with various stabilizers Exemplary stabilizers and their effects on the stability of the peptide (LLME-HCl) were tested. Briefly, 0.98 g of LLME-HCl and 0.81 g of various stabilizers were added to 8.21 g or 18.21 g of citrate aqueous buffer (pH 5.5) to obtain different solutions with final w / w concentrations of LLME-HCl of 9.8% or 4.9%, respectively. Stability was examined by visually observing crystal formation in the solution. The stabilizers tested are listed in Table 2 below. Surprisingly, all the stabilizers tested improved the stability of LLME-HCl solution by at least two times compared to the same solution without stabilizer. All tested solutions remained stable for at least four days. This demonstrates that trehalose and raffinose exhibit excellent stabilizing activity.

[0173] Example 3 Reconstitution of Exemplary Powdered Compositions Powder preparation The exemplary powder of this invention was obtained by freeze-drying (at -50°C, 10 Pa, and a drying time of 20 to 24 hours) in a 10 L citrate buffer solution containing 1.18 g LLME-HCl and 1 g trehalose at pH 5.5 using a Company Freeze Dryer. The raw material was divided into 10 vials for replication. The freeze-dried powder content is shown in Table 3.

[0174] Table 3: Lyophilized content of the tested powdered composition The inventors were able to demonstrate that the amount of LLME-HCl in the powder corresponds to the theoretical amount of peptide.

[0175] Reconstitute A high-performance liquid chromatography (HPLC) method was developed for the determination of LLME-HCl, and the amount of LLME-HCl in the composition reconstituted from lyophilized powder was examined. Results showed that at least 95% peptide recovery was obtained after reconstitution. Table 4 summarizes the results of the reconstitution test.

[0176] Table 4: Determination of LLME-HCl content in the reconstituted composition. The reconstitution of the exemplary powder at the final peptide concentration was tested. Surprisingly, even at a final LLME concentration of 30%, sufficient peptide recovery was observed without compromising the stability of the formulation.

[0177] In another experiment, a composition containing raffinose as a stabilizer (0.98 g LLME-HCl, 0.81 g raffinose, and citrate buffer at pH 5.5) was reconstituted. In this experiment, the peptide recovery rate reached over 90%.

[0178] Example 4 The inventors compared the water solubility of methyl esters of additional dipeptides and tripeptides in the exemplary compositions of the present invention or in citrate buffers (i.e., without trehalose).

[0179] General procedure for determining the solubility of dipeptides and tripeptides in 0.1 M citrate buffer at pH 5.5. Weigh 100 mg of di-tripeptide methyl ester sample and place it in a transparent glass test tube or beaker.

[0180] Weigh the test tube containing the dried di-tripeptide methyl ester sample.

[0181] Add 0.1 M citrate buffer solution dropwise to a test tube containing a sample of dipeptide methyl ester at room temperature. Vortex the mixture periodically.

[0182] When a clear solution is observed (visual inspection), stop adding 0.1M citrate buffer solution.

[0183] Weigh the resulting solution.

[0184] Calculate the solubility of 100 mg dipeptide methyl ester sample in the volume (mL) of 0.1 M citrate buffer.

[0185] The initial solubility and solution stability (whether precipitation occurred) were measured after 2, 4, 6, and 72 hours.

[0186] General procedure for determining the solubility of dipeptides and tripeptides in 10% trehalose-0.1M citrate buffer at pH 5.5. Weigh 100 mg of di-tripeptide methyl ester sample and place it in a transparent glass test tube or beaker.

[0187] Weigh the test tube containing the dried di-tripeptide methyl ester sample.

[0188] At room temperature, add a solution of 10% trehalose in 0.1M citrate buffer (pH 5.5) dropwise to a test tube containing the dipeptide methyl ester sample. Vortex the mixture periodically.

[0189] When a clear solution is observed (visual inspection), stop adding 10% trehalose to the solution in 0.1M citrate buffer at pH 5.5.

[0190] Weigh the resulting solution.

[0191] Calculate the solubility (mL) of 100 mg dipeptide methyl ester sample in a solution of 10% trehalose in 0.1 M citrate buffer at pH 5.5.

[0192] The initial solubility and solution stability (whether precipitation occurred) were measured after 2, 4, 6, and 72 hours.

[0193] Table 5 summarizes the solubility results of dipeptides and tripeptides in 0.1 M citrate buffer at pH 5.5.

[0194] Table 5: Solubility of dipeptides and tripeptides in 0.1 M citrate buffer at pH 5.5 Table 6 summarizes the solubility results of dipeptides and tripeptides in 10% trehalose-0.1M citrate buffer at pH 5.5.

[0195] Table 6. Solubility of dipeptides and tripeptides in 10% trehalose-0.1M citrate buffer at pH 5.5 Table 7 shows the sum of the solubility differences (%) between the solubility in citrate buffer at pH 5.5 and the solubility of 10% trehalose in citrate buffer.

[0196] Table 7. Summary of solubility differences (%) between trehalose in citrate buffer at pH 5.5 and 10% trehalose in citrate buffer. As shown in Table 7, the dipeptides and tripeptides have at least 20% higher solubility in trehalose / citrate buffer compared to citrate buffer alone.

[0197] To test the stabilizing effect of 10% trehalose in citric acid buffer solution, the concentrations of dipeptides and tripeptides were made equal in both media by adding a certain amount of trehalose solution to each dipeptide or tripeptide solution. The solution concentration was then increased by slowly evaporating water until peptide precipitation was observed. Table 8 summarizes the observations.

[0198] Table 8. Observed stability of dipeptides and tripeptides in citrate buffer at pH 5.5 and in a solution of 10% trehalose in citrate buffer. Therefore, compared with a citrate buffer solution without a stabilizer, the solubility of peptides dissolved in the compositions of the present invention (e.g., an aqueous solution containing trehalose as a stabilizer and a citrate buffer solution) is improved, as is the stability of the aqueous solution.

[0199] Although the invention has been described in conjunction with specific embodiments, it will be apparent to those skilled in the art that many alternatives, modifications, and variations exist. Therefore, it is intended to cover all such alternatives, modifications, and variations falling within the spirit and broad scope of the appended claims.

[0200] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety, as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated herein by reference. Furthermore, any reference or designation of any reference in this application should not be construed as an admission that such reference is prior art to the invention. The use of section headings should not be construed as necessarily limiting.

Claims

1. A composition comprising: (i) a peptide, including any enantiomer thereof, its salt, its hydrate or solvate, or any combination thereof; and (ii) a stabilizer; wherein: The peptide is between 2 and 10 amino acids in length; The peptide includes esters; and The stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, including any combination thereof.

2. The composition according to claim 1, wherein the ester is a carboxyl-terminated ester.

3. The composition according to claim 1 or 2, wherein the composition is an aqueous solution.

4. The composition according to claim 3, wherein the aqueous solution is a citrate buffer solution.

5. The composition according to claim 3 or 4, wherein the concentration of the peptide in the aqueous solution is at most 40% by weight; and wherein the length of the peptide is between 2 and 5 amino acids.

6. The composition according to any one of claims 3 to 5, wherein the composition is stable at a temperature of 2 to 40°C for at least 72 hours.

7. The composition according to any one of claims 1 to 6, wherein the peptide and the stabilizer are present in the composition in a molar ratio of 1:1 to 10:

1.

8. The composition according to any one of claims 1 to 7, wherein the peptide comprises a structure represented by Formula 1: , where R is a C1-C5 alkyl group.

9. The composition according to claim 8, wherein the peptide is an L-leucyl-L-leucine ester, its salt, its hydrate or solvate, or any combination thereof.

10. The composition according to claim 9, wherein R is methyl.

11. The composition according to claim 1 or 2, wherein the composition is in powder form, and is characterized in that... Water content is less than 5% w / w.

12. The composition according to claim 11, further comprising a buffer.

13. The composition of claim 12, wherein the buffer comprises citrate.

14. The composition according to any one of claims 1 to 13, wherein the stabilizer comprises trehalose.

15. A pharmaceutical composition comprising: (i) a peptide, including any enantiomer thereof, its salt, its hydrate or solvate, or any combination thereof; (ii) a stabilizer; and (iii) a pharmaceutically acceptable carrier; wherein the peptide has a length between 2 and 10 amino acids, and the peptide comprises an ester; and The stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, including any combination thereof.

16. The pharmaceutical composition of claim 15, wherein the pharmaceutically acceptable carrier is an aqueous solution.

17. The pharmaceutical composition of claim 16, wherein the aqueous solution is a buffer solution containing a pharmaceutically acceptable buffer.

18. The pharmaceutical composition of claim 17, wherein the buffer solution is characterized by a pH value between 5 and 8.

19. The pharmaceutical composition according to claim 17 or 18, wherein the pharmaceutically acceptable buffer comprises a pharmaceutically acceptable citrate.

20. The composition according to any one of claims 15 to 19, wherein the stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, including any combination thereof.

21. The composition of claim 20, wherein the disaccharide comprises trehalose.

22. A kit comprising: (i) a peptide, including any enantiomer thereof, its salt, its hydrate or solvate, or any combination thereof; and (ii) a stabilizer; wherein the peptide has a length between 2 and 10 amino acids, and the peptide comprises an ester; and The stabilizer is selected from diols, polyols, monosaccharides, disaccharides, oligosaccharides, and polysaccharides, and any combination thereof.

23. The kit according to claim 22, wherein the peptide and the stabilizer are in the form of a mixture.

24. The kit of claim 22, wherein the peptide and the stabilizer are stored in separate containers.

25. The kit according to any one of claims 22 to 24, wherein the kit further comprises citrate or citrate aqueous buffer.

26. The kit of claim 25, wherein the peptide and the citrate aqueous buffer are present in the kit at a weight ratio of 1:500 to 1:10000.

27. The kit according to claim 25 or 26, comprising instructions for mixing the peptide, the stabilizer, and optionally the citrate aqueous buffer to obtain the composition of any one of claims 3 to 10.

28. The kit according to any one of claims 22 to 27, wherein the peptide and the stabilizer are present in the kit in a molar ratio of 1:1 to 10:1.

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