Ophthalmic pharmaceutical compositions and uses thereof

Abstract

Provided are stable and well-tolerated ophthalmic pharmaceutical compositions for use in the treatment of retinal neurodegenerative diseases, more particularly for use in the treatment of diabetic retinopathy, age-related macular degeneration, glaucoma, and retinitis pigmentosa. [Solution] In a first aspect, the present invention provides an ophthalmic pharmaceutical composition comprising a peptide having a sequence length of 13 to 50 amino acids or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients or carriers, wherein the N-terminal region of the peptide consists of a specific sequence, and the pH value of the composition is 4.0 to 4.8, and the osmolality is in the range of 0.5 to 200 mOsm / kg.

Description

[Technical Field]

[0001] This application claims the benefit of European Patent Application EP20382101.2, filed February 13, 2020. The present invention relates to the field of pharmaceutical compositions for ocular diseases, particularly retinal neurodegenerative diseases. The present invention provides pharmaceutical compositions for topical application to the eye, comprising peptides and methods for their preparation. The present invention further relates to ophthalmic pharmaceutical compositions for use in the topical ocular treatment and / or prevention of retinal neurodegenerative diseases. [Background technology]

[0002] Retinal neurodegenerative diseases refer to retinal conditions characterized by progressive neuronal loss. Diabetic retinopathy, age-related macular degeneration, glaucoma, and retinitis pigmentosa are considered retinal diseases in which neurodegeneration plays an important role.

[0003] A detailed analysis of these diseases, their critical locations, and possible methods of protection and recovery can be extracted from Schmidt et al., "Neurodegenerative Diseases of the Retina and Potential for the Protection and Recovery", Current Neuropharmacology - 2008, Vol. No. 6, pp.:164-178.

[0004] Diabetic retinopathy (DR) is the most common complication of diabetes and remains the leading cause of blindness in working-age individuals in developed countries. Current treatments for DR, such as laser photocoagulation and intravitreal injections of corticosteroids or anti-VEGF agents, are indicated too early in the disease's progression and are associated with significant side effects. Furthermore, all of these treatments are very expensive, have a reduced benefit / risk ratio, require vitreoretinal specialists, and most of them are invasive. Consequently, new therapies are urgently needed to treat the early stages of the disease.

[0005] Diabetic retinopathy (DR) has classically been considered a microcirculatory disorder of the retina. However, in recent years, increasing evidence has clearly suggested that retinal neurodegeneration is an early event in the pathogenesis of DR and is involved in the microcirculatory abnormalities that occur in DR, as can be inferred from Simo et al. ("Neurodegeneration is an early event in diabetic retinopathy: therapeutic implications", Br. J. Ophthalmol., 2012, vol. 96, pp. 1285-1290), on behalf of the European Consortium for the Early Treatment of Diabetic Retinopathy (EUROCONDOR).

[0006] In DR, neurodegeneration (loss of effective neurons) begins early in the disease, leading to functional abnormalities such as loss of both color discrimination and contrast sensitivity. These changes can be detected by specific electrophysiological studies in diabetic patients even before the duration of diabetes reaches two years, i.e., before microvascular lesions can be detected by ophthalmologic examination. Furthermore, delayed multifocal electroretinogram (ERG) latency (mfERG-IT) can detect early microvascular abnormalities. Furthermore, neuroretinal degeneration initiates and / or activates several metabolic and signaling pathways involved in the microangiopathy process and the breakdown of the blood-retinal barrier (a key factor in the pathogenesis of DR).

[0007] Although there is currently no cure for the early stages of retinal neurodegenerative disease or neurodegeneration associated with these conditions, there are treatments that would prevent advanced pathologies, such as microcirculatory problems that lead to retinal neovascularization. Therefore, in the early stages, especially in DR, no treatment is applied, and patients are followed up with standard follow-up.

[0008] Diabetes mellitus is a group of chronic diseases characterized by hyperglycemia. To prevent diabetic complications, reducing hyperglycemia using systemic hypoglycemic agents is essential. Therefore, glucose-lowering drugs could theoretically be beneficial for preventing or preventing diabetic complications, including DR. However, information on the direct effects of antidiabetic drugs on DR, independent of their blood glucose-lowering effects, is lacking. For example, glucagon-like peptide 1 agonists known as exenatide (Byetta, Amylin Pharmaceuticals) and liraglutide (ictoza, Novo Nordisk) are used to treat type 2 diabetes by promoting blood glucose reduction. Furthermore, these agonists are known to improve metabolic syndrome-related conditions, such as obesity and hypertension. Patent application WO200706434 also discloses an intranasally administered pharmaceutical composition delivering the same glucagon-like peptide 1 (GLP-1) to treat metabolic syndrome and diabetic complications, including DR.

[0009] Therefore, administration of such glucagon-like peptide 1 agonists is known to improve or alleviate DR symptoms. This is because the primary cause or origin of the disease, particularly high blood glucose levels, is ultimately corrected. Nevertheless, these treatments do not eliminate systemic adverse effects. Furthermore, if these substances are to reach the retina in therapeutic concentrations and cross the so-called blood-retinal barrier, high doses are required, increasing adverse effects.

[0010] In developed countries, diabetic patients are included in well-defined treatment protocols derived from widely adopted guidelines, but despite such systemic and effective glucose-lowering treatment, more than 30% of the diabetic population develop DR, suggesting that standard systemic control is not sufficient to efficiently prevent the development of DR.

[0011] Currently, there are no specific treatments for retinal neurodegenerative diseases. In the specific case of DR, this means there are no specific treatments to protect the neural retina from damage (leading to neuronal loss), especially in cases of background retinopathy or non-proliferative DR. Therefore, new pharmacological treatments for this disease are needed, especially in the early stages when neurodegeneration appears to have begun. Early treatment of DR is effective in preventing progression to an advanced stage, which requires aggressive treatments such as laser photocoagulation or intravitreal injections.

[0012] WO2014131815 discloses a peptide having a sequence length of 13 to 50 amino acids, and the N-terminal region of the peptide has the sequence HXaa 1 EGTFTSDXaa 2 SXaa 3 Xaa 4 (SEQ ID NO: 1), wherein Xaa 1 is an amino acid selected from alanine and glycine; Xaa 2 is an amino acid selected from valine and leucine; 3 is an amino acid selected from serine and lysine; 4 is an amino acid selected from tyrosine and glutamine; histidine is the N-terminal residue; and is for use in the topical treatment and / or prevention of retinal neurodegenerative diseases, particularly diabetic retinopathy. It has been disclosed that the glucagon-like peptide 1 receptor (GLP-1R) is present in the human retina, and contrary to all previous assumptions, peptidic substances with molecular weights ranging from 3.35 kDa to 4.18 kDa reach the retina when applied topically to the eye (i.e., to the cornea). Therefore, the topical use (topical ocular use) of peptides containing 13 to 50 amino acids, including SEQ ID NO: 1, which are thought to be involved in GLP-1R activation and are also present in mammalian GLP-1, has been proposed.

[0013] However, to date, no practical ophthalmic pharmaceutical compositions have been developed that are safe, highly bioavailable, and stable enough for frequent drug delivery, and that can be used to administer peptides containing 13 to 50 amino acids, such as GLP-1(7-36)NH2 (SEQ ID NO: 1), and other peptide incretins. One problem with this approach is that, among the various known biologically active polypeptides, certain peptides, including GLP-1(7-36)NH2, which have an isoelectric point (pI) in the acidic or neutral pH range, tend to be unstable in acidic or neutral solutions.

[0014] For example, our observations have revealed that some solutions prepared with GLP-1(7-36)NH2 render the peptide insoluble when the solution is stored for several days. Therefore, there is a need to develop a stable solution containing solubilized peptide suitable for topical ocular application.

[0015] Therefore, it is practically difficult to find a stable liquid composition for administering these peptides in the form of a solution formulation. The goal of an ophthalmic drug delivery system is to achieve a therapeutic concentration of the active drug in the target tissue for an appropriate period of time.

[0016] It is therefore an object of the present invention to provide a stable and well-tolerated ophthalmic pharmaceutical composition for use in the treatment of retinal neurodegenerative diseases, more particularly for use in the treatment of diabetic retinopathy, age-related macular degeneration, glaucoma, and retinitis pigmentosa.

[0017] A challenge remains in the art to provide topical ophthalmic preparations that contain low concentrations of GLP-1 but that retain stability and efficacy over a period that translates into an acceptable shelf life for the composition. The present invention provides topical ophthalmic compositions containing peptides that, when applied topically to the eye (i.e., to the cornea or conjunctival hole), are capable of reaching the retina despite their high molecular weight and achieving effective concentrations to inhibit the progression of retinal neurodegenerative diseases. Summary of the Invention

[0018] The present inventors have discovered topical ophthalmic preparations containing low concentrations of peptides that exhibit pharmaceutical stability, which translates into acceptable shelf life for the compositions. These topical ophthalmic formulations allow for more convenient topical administration of peptides containing 13-50 amino acids, including SEQ ID NO: 1, which are believed to be involved in activation of the GLP-1R and are also present in mammalian GLP-1.

[0019] Thus, in a first aspect, the present invention relates to an ophthalmic pharmaceutical composition comprising a peptide having a sequence length of 13 to 50 amino acids or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients or carriers, wherein the N-terminal region of said peptide has the sequence: HXaa 1 EGTFTSDXaa 2 SXaa 3 Xaa 4 (SEQ ID NO: 1), wherein: Xaa 1 is an amino acid selected from alanine and glycine; Xaa 2 is an amino acid selected from valine and leucine; Xaa 3 is an amino acid selected from serine and lysine; Xaa 4 is an amino acid selected from tyrosine and glutamine; Histidine is the N-terminal residue; The pH value of the composition is 4.0 to 4.8, and the osmolality is in the range of 0.5 to 200 mOsm / kg.

[0020] GLP-1 (glucagon-like peptide-1) is an endogenous insulinotropic peptide secreted by L-cells in the gastrointestinal tract in response to food (the "incretin response"). Acting through its receptor (GLP-1R), GLP-1 exerts potent effects on glucose-dependent insulin secretion, insulin gene expression, pancreatic islet beta cell neogenesis, gastrointestinal motility, energy homeostasis, and food intake. The GLP-1 receptor (GLP-1R) is a member of the peptide hormone binding class B1 (secretin-like receptor) family of seven-transmembrane heterotrimeric G protein-coupled receptors (GPCRs). GLP-1R is widely distributed and found in the pancreas, adipose tissue, muscle, heart, gastrointestinal tract, and liver. Furthermore, GLP-1R is found throughout the central nervous system (i.e., hypothalamus, striatum, brainstem, substantia nigra, subventricular zone, and even retina), and there is some evidence that GLP-1R stimulation by GLP-1 exerts neuroprotective effects in both the central and peripheral nervous systems.

[0021] Human GLP-1 is a 30- or 31-amino acid residue peptide derived from preproglucagon, which is produced and secreted by enteroendocrine L-cells in the distal ileum, pancreas, and brain. Human preproglucagon is identified by UniProt database accession number P01275, version 3, February 6, 2007. Processing of preproglucagon to produce GLP-1(7-36)amide, GLP-1(7-37), and GLP-2 occurs primarily in L-cells. A simple system is used to describe fragments and analogs of this peptide. Thus, for example, Gly 8 -GLP-1(7-37) denotes a fragment (analog) of GLP-1 formally derived from GLP-1 by deleting amino acid residues 1 to 6 and substituting the naturally occurring amino acid residue (Ala) at position 8 with Gly. Similarly, Lys 34 (N ε-tetradecanoyl)-GLP-1(7-37) refers to GLP-1(7-37) in which the ε-amino group of the Lys residue at position 34 is tetradecanoylated.

[0022] The designation GLP-1(1-36) indicates that the peptide fragment in question comprises amino acid residues numbered 1 (inclusive) through 36 (inclusive), when counted from the N-terminus of the parent peptide, GLP-1. Similarly, the designation GLP-1(7-37) specifies that the fragment in question comprises amino acid residues numbered 7 (inclusive) through 37 (inclusive), when counted from the N-terminus of the parent peptide, GLP-1. The amino acid sequence of GLP-1(7-36)amide (SEQ ID NO:2) corresponds to: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg, wherein the C-terminus is -CONH2, while the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 3) corresponds to the following: His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly.

[0023] These peptides have been reported to reach the retina and exert neuroprotective effects therein, offering the therapeutic advantage of providing local action in the eye and minimizing associated systemic side effects.

[0024] As shown below, a combination of an acidic pH in the range of 4-4.8 and an osmolality in the range of 0.5-200 mOsm / kg provides long-term stability (up to 12 months) of peptides formulated into ophthalmic compositions.

[0025] The topical treatment and / or prevention is a topical ocular treatment and / or prevention, and thus the peptide is on the surface of the eye (i.e., the cornea or conjunctival hole) so that it can reach the retina when applied topically to the eye. This applies to any of the embodiments and combinations of embodiments disclosed in the present invention.

[0026] In a second aspect, the present invention relates to a lyophilisate comprising a peptide as defined in the first aspect of the invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable amount of a stabilizer or buffer, said lyophilisate being suitable, upon reconstitution, for preparing an ophthalmic pharmaceutical composition according to any of the preceding claims.

[0027] Alternatively, this second aspect may include: a) a pharmaceutically effective amount of a peptide as defined in the first aspect and / or a pharmaceutically acceptable salt thereof; b) a pharmaceutically acceptable amount of a stabilizer or buffer; and c) water, and The lyophilisate is suitable for preparing, upon reconstitution, an ophthalmic pharmaceutical composition according to any of the preceding claims.

[0028] In a third aspect, the present invention relates to a process for preparing an ophthalmic pharmaceutical composition of the first aspect, comprising the step of reconstituting a lyophilisate as defined in the second aspect of the invention with an aqueous vehicle composition comprising one or more pharmaceutically acceptable carriers or excipients, in particular an aqueous vehicle composition comprising at least one viscosity increasing agent and optionally at least one preservative.

[0029] The present invention also provides a process for preparing the ophthalmic pharmaceutical composition of the first aspect, comprising: a) providing a lyophilisate comprising a pharmaceutically effective amount of a peptide as defined in the first aspect and / or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable amount of a stabiliser or buffer; b) providing a vehicle composition comprising at least one thickening agent and optionally at least one preservative; c) reconstituting the lyophilized product of step a) with the vehicle composition of step b) to form the ophthalmic pharmaceutical composition.

[0030] In a fourth aspect, the present invention relates to an ophthalmic pharmaceutical composition obtainable by the process of the third aspect.

[0031] In a fifth aspect, the present invention relates to a kit comprising a lyophilisate as defined in the second aspect of the invention and a physiologically acceptable vehicle composition comprising one or more pharmaceutically acceptable excipients or carriers for reconstituting the peptide.

[0032] The components of the formulation can be included in the kit in the form of a mixed powder or a liquid. In the kit of the present invention, all components can be included in a mixed solution, or some can be included in a mixed solution and some can be in the form of a powder. In one embodiment, the physiologically acceptable vehicle includes at least one thickening agent and optionally at least one preservative.

[0033] In a sixth aspect, the present invention provides a kit comprising the ophthalmic pharmaceutical composition of the first and fourth aspects, a container for holding the pharmaceutical composition, and a drop dispenser adapted to administer the composition in a volume, for example, about 10 to 100 μl per drop, preferably about 10 to 50 μl, more preferably about 20 to 40 μl.

[0034] In a final aspect, the present invention relates to an ophthalmic pharmaceutical composition of the first or fourth aspect of the present invention for use in the topical ocular treatment and / or prevention of a retinal neurodegenerative disease. Alternatively, this aspect can be formulated as use of the ophthalmic pharmaceutical composition of the first or fourth aspect of the present invention in the manufacture of a medicament for the treatment and / or prevention of a retinal neurodegenerative disease. Alternatively, this aspect can be formulated as a method for the treatment and / or prevention of a retinal neurodegenerative disease, the method comprising administering a therapeutically effective amount of the ophthalmic pharmaceutical composition of the first or fourth aspect of the present invention to a subject in need thereof. DETAILED DESCRIPTION OF THE INVENTION

[0035] For the sake of understanding, the following definitions are included:

[0036] The expression "neuroprotection in the early stages of diabetic retinopathy" relates to any therapeutic or preventive method implemented before the advanced stage of DR (proliferative DR (PDR)) is established. The "early stage of diabetic retinopathy" should be understood as the time when functional and microvascular abnormalities of the eye (i.e., abnormalities in color discrimination, contrast sensitivity, and electroretinogram) due to the presence of diabetes can be detected, but the characteristic neovascularization of PDR has not yet been fully established.

[0037] The terms "human glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide)" and "human glucagon-like peptide-1(7-37) (GLP-1(7-37))" refer to fragments derived from human proglucagon and comprising amino acids 7 to 36 or amino acids 7 to 37, respectively, of the amino acid sequence of human proglucagon described above.

[0038] By "analog of human GLP-1(7-37)" is to be understood a peptide in which one or more amino acid residues of GLP-1(7-37) have been replaced by another amino acid residue and / or one or more amino acid residues of GLP-1(7-37) have been deleted and / or one or more amino acid residues have been added to GLP-1(7-37).

[0039] As used herein, the phrase "therapeutically effective amount" refers to that amount of a compound (i.e., peptide) that, when administered, is sufficient to prevent progression of the disease being addressed or to alleviate to some extent one or more of its symptoms. The particular dose of a compound administered in accordance with the present invention will, of course, be determined by the particular circumstances surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and similar considerations.

[0040] As used herein, the term "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of a subject (e.g., a human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Each carrier, excipient, etc. must also be "acceptable" in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Suitable carriers, excipients, etc. can be found in standard pharmaceutical textbooks; examples include preservatives, flocculants, humectants, emollients, and antioxidants.

[0041] As used herein, the term "pharmaceutically acceptable salts" refers to pharmaceutically acceptable salts derived from a variety of organic and inorganic counterions well known in the art, including, by way of example only, sodium, potassium, calcium, magnesium, ammonium, and tetraalkylammonium, and when the molecule contains a basic functional group, salts of organic or inorganic acids such as hydrochloride, hydrobromide, tartrate, mesylate, phosphate, acetate, trifluoroacetate, citrate, tosylate, maleate, and oxalate.

[0042] The phrase "excipient and / or carrier" refers to an acceptable material, composition, or vehicle. Each component must also be pharmaceutically acceptable in the sense of being compatible with the other components of the composition. It must also be suitable for use in contact with the tissues or organs of humans and non-human animals without undue toxicity, irritation, allergic response, immunogenicity, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Examples of suitable acceptable excipients include solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, solid binders, lubricants, and the like. Except insofar as any conventional excipient medium is incompatible with the substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated within the scope of the present invention.

[0043] Those skilled in the art will appreciate that pharmaceutically acceptable salts of compounds can be prepared either in situ during the final isolation and purification of the compounds, or by separately reacting the purified compounds in their free acid or free base form with a suitable base or acid, respectively.

[0044] The compounds of the present invention may be in the form of a salt, eg a pharmaceutically acceptable salt, or a solvate, eg a hydrate.

[0045] A "solvate" or "solvates" of a compound refers to those compounds, as defined above, bound to a stoichiometric or non-stoichiometric amount of solvent. In certain embodiments, the solvent is volatile, non-toxic, and / or acceptable for administration to humans in trace amounts and / or water.

[0046] The "N-terminal region" or "N-terminus" (also known as the amino terminus, NH2 terminus, N-terminus, or amine terminus, all of which are used interchangeably herein) refers to the beginning of a protein or polypeptide, ending with an amino acid bearing a free amine group (-NH2). The convention for writing a peptide sequence is to place the N-terminus on the left and write the sequence from N-terminus to C-terminus. When a protein is translated from messenger RNA, it is created from N-terminus to C-terminus. With respect to the "N-terminal residue," it should be understood that a residue in a peptide bearing an amino group that is free or at least not acylated by another amino acid residue (e.g., which may be acylated or formylated) is referred to as the N-terminus and is at the N-terminus. A residue that has a free carboxyl group or at least not acylated by another amino acid residue (e.g., which may be acylated with ammonia to give -NH-CHR-CO-NH2) is referred to as the C-terminus.

[0047] For the purposes of the present invention, a pharmaceutical composition containing an active agent is considered to be "stable" if said component degrades substantially slower than itself and / or known pharmaceutical compositions.

[0048] As mentioned above, the present inventors propose for the first time an ophthalmic pharmaceutical composition of GLP-1 and analogues for retinal neurodegenerative diseases (retinal diseases in which neurodegeneration plays an important role), which, in addition to being non-aggressive, is useful in the treatment of the early stages of these diseases, in particular in the treatment of DR.

[0049] Ocular administration of drugs is primarily associated with the need to treat ophthalmic diseases. The ocular surface is the most easily accessible site for topical administration of drugs. Ophthalmic formulations are sterile products, appropriately formulated and packaged for instillation into the eye. They are easily administered by nurses or patients themselves, and they have rapid absorption and effectiveness, fewer visual and systemic side effects, a longer shelf life, and better patient compliance.

[0050] In a particular embodiment of the present invention, the ophthalmic pharmaceutical composition according to the first aspect has a sequence length of up to 30 to 50 amino acids.

[0051] For purposes of the present invention, any range given includes both the lower and upper endpoints of the range.

[0052] Unless otherwise specified, one or more of the amino acids forming the peptides of the present invention can have the L- or D-configuration.

[0053] Another particular embodiment is an ophthalmic pharmaceutical composition comprising a peptide having a sequence length of 30 to 40 amino acids or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients or carriers, wherein the N-terminal region of the peptide has the sequence: HXaa 1 EGTFTSDXaa 2 SXaa 3 Xaa 4 (SEQ ID NO: 1), wherein: Xaa 1 is an amino acid selected from alanine and glycine; Xaa 2 is an amino acid selected from valine and leucine; Xaa 3 is an amino acid selected from serine and lysine; Xaa 4 is an amino acid selected from tyrosine and glutamine; Histidine is the N-terminal residue; The pH value of the composition is 4.0 to 4.8, and the osmolality is in the range of 0.5 to 200 mOsm / kg.

[0054] In another particular embodiment, the ophthalmic pharmaceutical composition according to the first aspect further has a sequence length of 13 to 40 amino acids.

[0055] In a preferred embodiment of the pharmaceutical composition disclosed herein, the pharmaceutically acceptable salt of the peptide is selected from acetate, hemitartrate, and hydrochloride. Preferably, the pharmaceutically acceptable salt of the peptide is acetate.

[0056] The compounds of the invention refer to the therapeutically active compounds as well as any prodrugs thereof, and pharmaceutically acceptable salts, hydrates and solvates of the compounds and prodrugs.

[0057] In another embodiment of the invention, the peptide has at the N-terminal region of the amino acid sequence Xaa 1 is alanine, Xaa 2 Valine, Xaa 3 is serine, and Xaa 4 These peptides include those consisting of SEQ ID NO: 1, where SEQ ID NO: 1 is tyrosine. That is, they contain the amino acid sequence of SEQ ID NO: 4 (HAEGTFTSDVSSY). These peptides are particularly intended for the topical treatment and / or prevention of DR.

[0058] In another embodiment, the peptide according to the present invention is a mammalian glucagon-like peptide-1, which contains at its N-terminus (N-terminal region) the sequence identified as SEQ ID NO: 4, which is conserved in most mammals, including humans, pigs, and monkeys, and is the sequence most commonly recognized by GLP-1R.

[0059] Thus, in a preferred embodiment, a peptide according to the invention consists of human glucagon-like peptide-1 of amino acid sequence SEQ ID NO: 2, corresponding to HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR-NH2, and variations of this human peptide. This peptide according to the invention can also be referred to as native glucagon-like peptide-1(7-36)amide. In some embodiments, this peptide according to the invention can also be referred to as native glucagon-like peptide-1(7-36)amide, which is available as the acetate salt.

[0060] In another preferred embodiment, the peptide according to the invention consists of human glucagon-like peptide-1 of amino acid sequence SEQ ID NO: 3, corresponding to HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRG, and variations of this human peptide, which can also be referred to as human glucagon-like peptide-1(7-37).

[0061] Although the variations relate to variations between individuals, these variations do not affect the interaction with GLP-1R and do not deprive the peptide from acting through this receptor (in particular as an agonist or activator of subsequent signaling pathways that result in neuroprotection or lowering of blood glucose levels). By "variation" is meant any deletion of one or two amino acids, as well as any substitution or addition of conservative amino acids.

[0062] Thus, the present invention also encompasses a mammalian glucagon-like peptide-1(7-37) or an analogue thereof for use in the topical (ocular) treatment of retinal neurodegenerative diseases, particularly DR, wherein the analogue of glucagon-like peptide-1(7-37) is a) deletion of at least one amino acid residue of glucagon-like peptide-1(7-37); b) substitution of at least one amino acid residue of glucagon-like peptide-1(7-37) with another amino acid residue; c) the addition of at least one amino acid residue to the C-terminus of glucagon-like peptide-1(7-37), while they contain in the N-terminal region the amino acid sequence of SEQ ID NO: 1. The above analogs are further peptide agonists of the human glucagon-like peptide-1 receptor and are capable of stimulating the formation of cAMP when tested in the presence of the receptor.

[0063] Alternatively, in another embodiment, the peptide is (a) a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof; or, alternatively, (b) a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof; or, alternatively, (c) a peptide having an amino acid sequence with a degree of identity of at least 85% to SEQ ID NO: 2, 3, or a pharmaceutically acceptable salt thereof, wherein the N-terminal region is as defined in the first aspect of the invention; or alternatively, (d) a peptide having a sequence length of up to 50 amino acids comprising an amino acid sequence having a degree of identity of at least 85% with SEQ ID NO: 2, 3, or a pharmaceutically acceptable salt thereof, wherein the N-terminal region is as defined in the first aspect of the invention; or alternatively, (e) a fragment of a peptide having a degree of identity of at least 85% to SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein the fragment has 14 to 29 amino acids and includes the N-terminal region as defined in the first aspect of the invention; or, alternatively, (f) A fragment of a peptide having a degree of identity of at least 85% with SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof, wherein the fragment has 14 to 30 amino acids and comprises the N-terminal region as defined in the first aspect of the present invention.

[0064] In another embodiment of the first aspect of the invention, the peptide or salt thereof is a peptide having 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 2 or SEQ ID NO: 3. In another embodiment of the first aspect of the invention, the peptide or salt thereof is a peptide having 100% identity to SEQ ID NO: 2. In another embodiment of the first aspect of the invention, the peptide or salt thereof is a peptide having 100% identity to SEQ ID NO: 3. In another embodiment of the first aspect of the invention, the peptide is a pharmaceutically acceptable salt of the sequence of SEQ ID NO: 2, in particular the acetate salt of the sequence of SEQ ID NO: 2. Alternatively, in another embodiment of the first aspect of the invention, the peptide is the sequence of SEQ ID NO: 2.

[0065] In the present invention, the term "identity" refers to the percentage of residues that are identical in two sequences when the sequences are optimally aligned. In optimal alignment, if a position in a first sequence is occupied by the same amino acid residue as the corresponding position in a second sequence, the sequences exhibit identity at that position. The level of identity (or "percent sequence identity") between two sequences is measured as the ratio of the number of identical positions shared by the sequences to the size of the sequences (i.e., percent sequence identity = (number of identical positions / total number of positions) × 100). In the context of the present invention, a peptide having a degree of at least 85% amino acid sequence identity to SEQ ID NO: 2 or 3 will retain the N-terminal region defined in the first aspect of the present invention and any of the above embodiments.

[0066] Several mathematical algorithms for rapidly obtaining optimal alignments and calculating the identity between two or more sequences are known and are incorporated into several available software programs. Examples of such programs include, among others, the MATCH-BOX, MULTAIN, GCG, FASTA, and ROBUST programs for amino acid sequence analysis. Preferred software analysis programs include ALIGN, CLUSTAL W, and BLAST programs (e.g., BLAST 2.1, BL2SEQ, and later versions thereof).

[0067] For amino acid sequence analysis, a weight matrix such as a BLOSUM matrix (e.g., BLOSUM45, BLOSUM50, BLOSUM62, and BLOSUM80 matrices), a Gonnet matrix, or a PAM matrix (e.g., PAM30, PAM70, PAM120, PAM160, PAM250, and PAM350 matrices) is used to determine identity.

[0068] The BLAST program aligns a selected sequence against multiple sequences in a database (e.g., GenSeq) or, using BL2SEQ, provides an analysis of at least two amino acid sequences between two selected sequences. The BLAST program is preferably modified by a low-complexity filtering program, such as the DUST or SEG programs, which are preferably integrated into the BLAST program operation. When using a gap existence cost (or gap score), the gap existence cost is preferably set between about -5 and -15. Similar gap parameters can be used in other programs as needed. The BLAST program and its underlying principles are further described, for example, in Altschul et al., "Basic local alignment search tool," 1990, J. Mol. Biol., vol. 215, pages 403-410.

[0069] For multiple sequence analysis, the CLUSTALW program can be used. The CLUSTAL W program is preferably run using the "dynamic" (rather than "fast") setting. Amino acid sequences are evaluated using a variable set of BLOSUM matrices depending on the level of identity between the sequences. The CLUSTAL W program and its basic operating principles are further described, for example, in Higgins et al., "CLUSTAL V: improved software for multiple sequence alignment," 1992, CABIOS, 8(2), pages 189-191.

[0070] In particular, mammalian glucagon-like peptide-1(7-37) or analogs thereof can be used to treat and / or prevent retinal neurodegenerative diseases, particularly DR. When applied topically to the eye, the peptide acts as a neuroprotectant (avoiding neurodegeneration in the case of prophylactic treatment).

[0071] In another embodiment of the present invention, the ophthalmic pharmaceutical composition of the first aspect has a pH value of 4.1 to 4.8, preferably 4.2 to 4.7.

[0072] The inventors have found that pH can affect the chemical stability, potency, and efficacy of the peptides of the present invention. An optimal pH avoids adverse effects, ensures that the drug provides optimal therapeutic benefit, and ensures that all components are optimized. Buffers are used in ophthalmic compositions when pH is important and must be within a specific range. As used herein, the term "buffer" refers to a mixture of an acid (usually a weak acid, e.g., acetic acid or citric acid) and its conjugate base (e.g., acetic acid or a citrate salt, e.g., sodium acetate or sodium citrate) in a ratio that resists pH changes when dissolved in aqueous solution. Ideally, the pH of an eye drop should be equivalent to the pH of tears, which is 7.4. However, the decision to add a buffer should be based on stability considerations. The selected pH must be optimal for both the stability and physiological tolerance of the active pharmaceutical ingredient.

[0073] In another embodiment of the present invention, the osmolality of the ophthalmic pharmaceutical composition of the first aspect is in the range of 1 to 150 mOsm / kg. In a preferred embodiment, the osmolality is in the range of 1 to 90 mOsm / kg, preferably 1 to 80 mOsm / kg, more preferably 1 to 70 mOsm / kg, and even more preferably 1 to 50 mOsm / kg.

[0074] In a preferred embodiment, the osmolality is in the range of 1 to 10 mOsm / kg, hi another preferred embodiment, the osmolality is in the range of 85 to 150 mOsm / kg.

[0075] Tonicity refers to the osmotic pressure exerted by salt in an aqueous solution. An eye drop solution is isotonic with another solution if the solutions have the same magnitude of colligative capacity. An eye drop solution is considered isotonic if its tonicity is equal to that of a 0.9% sodium chloride solution (290 mOsm / kg). Because human tears are isotonic and very similar to a 0.9% sodium chloride solution, tonicity was thought to be important for ophthalmic formulations.

[0076] In the present invention, the inventors have surprisingly found that the ophthalmic compositions of the present invention are well tolerated, stable and effective even when they are not isotonic and have a pH below 7.4, which is contrary to the majority of prior art ophthalmic compositions.

[0077] The osmolality of a solution therefore ranges from 0.5 to 200 mOsm / kg. The osmolality of a real solution corresponds to the molarity of an ideal solution containing undissociated solutes and is expressed in osmoles or milliosmoles per kilogram of solvent (Osmol per kg or mOsmol per kg, respectively), which are units similar to the molarity of a solution. Osmolality is therefore a measure of the osmotic pressure exerted by a real solution across a semipermeable membrane. The osmolality of a solution is usually determined by measuring the freezing point depression of the solution.

[0078] This apparatus, an osmometer for measuring freezing point depression, consists of: a means for cooling the vessel used for the measurement; a temperature-sensitive resistor (thermistor) equipped with a suitable amperometric or potentiometric device that can be stepped with temperature or osmolality; and a means for mixing the sample. Osmolality is determined by the method described in Pharmacopeial Forum: Volume No. 34(1) Page 157, chapter <785> Osmolarity and Osmolarity were measured using methods in accordance with the FDA.

[0079] In another embodiment of the present invention, the ophthalmic pharmaceutical composition of the first aspect further comprises at least one compound selected from the group consisting of stabilizers, thickeners, buffers, and mixtures thereof.

[0080] The stabilizer is introduced in small amounts to maintain both the pH and osmolality low, particularly lower than those typically achieved by the state of the art. In particular, in another embodiment of the present invention, the stabilizer is aspartic acid or glutamic acid. In a preferred embodiment, the weight ratio of stabilizer to peptide in the composition is in the range of 1:5 to 1:50, preferably 1:8 to 1:30, and more preferably 1:10 to 1:20.

[0081] As used herein, "stabilizer" refers to a component that helps maintain the structural integrity of a biologic drug, particularly during storage (especially when exposed to stress) and in solution. This stabilizing effect can occur for a variety of reasons, but typically, such stabilizers function as osmolytes to reduce protein denaturation or aggregation. Typical stabilizers include amino acids (i.e., free amino acids that are not part of a peptide or protein—e.g., glycine, arginine, histidine, aspartic acid, lysine), and sugar stabilizers such as sugar polyols (e.g., mannitol, sorbitol), and / or disaccharides (e.g., trehalose, sucrose, maltose, lactose).

[0082] In another embodiment of the present invention, the buffer is acetic acid / acetate or citric acid / citrate. In a preferred embodiment, the total amount of buffer in the composition is 0.05% to 5.0% w / w, more preferably 0.08% to 2.0% w / w, more preferably 0.1% to 1.5%. In another preferred embodiment, the buffer strength is between 20 mM and 100 mM, more preferably between 30 mM and 70 mM.

[0083] As used herein, "strength" refers to ionic strength, which is a measure of the concentration of ions in a solution. It is based on the dissociation that salts, acids, and bases undergo when in aqueous solution. It is expressed in concentration units, such as molar concentration.

[0084] In another embodiment of the invention, the thickening agent is selected from hydroxyethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, sodium hyaluronate, carbopol, polyacrylamide, sodium chondroitin sulfate, and mixtures thereof, hi a preferred embodiment, the thickening agent is hydroxypropyl methyl cellulose or polyvinylpyrrolidone.

[0085] As used herein, the term "thickener" refers to a component that thickens an ophthalmic liquid vehicle, particularly an aqueous solution, to increase the contact time of the drug with the eye and minimize drainage into the nasolacrimal system. Viscosity enhances drug absorption and therapeutic efficacy. The compositions of the present invention may also include a thickener.

[0086] In another preferred embodiment of the present invention, the total amount of thickener in the composition is 0.1% to 5% w / w, preferably 0.3% to 4% w / w, more preferably 0.5% to 3% w / w.

[0087] In another embodiment of the invention, the viscosity is in the range of 1 to 50 cSt, preferably in the range of 1 to 20 cSt, more preferably in the range of 1 to 10 cSt at room temperature and pressure. Viscosity was measured using the capillary viscometer method described in European Pharmacopeia 7.0 2.2.9.

[0088] In another embodiment of the present invention, the ophthalmic pharmaceutical composition of the first aspect further comprises an effective amount of a preservative. Commonly known preservatives are contemplated herein, including detergent preservatives, oxidizing preservatives, and ionic buffer preservatives. In a preferred embodiment, the preservative is selected from the group consisting of sodium edetate, benzalkonium chloride, centrimonium chloride, sodium perborate, stabilized oxychloro complexes, sorbic acid, thimerosal, polyquaternium-1, polyhexamethylene biguanide, chlorobutanol, phenylethyl alcohol, methylparaben, propylparaben, boric acid, sorbic acid, and propylene glycol, and mixtures thereof. More preferably, the preservative is benzalkonium chloride.

[0089] In another preferred embodiment, the total amount of preservatives in the composition is 0.005% to 0.5% w / w, preferably 0.008% to 0.3% w / w, more preferably 0.01% to 0.1% w / w.

[0090] As used herein, "preservative" refers to a substance that prevents or inhibits the growth of microorganisms and extends the shelf life of a pharmaceutical product, such as in an eye drop solution. The use of preservatives in topical ophthalmic treatments is ubiquitous in any product intended for more than one patient use, to prevent microorganisms that may be introduced into the product after the first use from multiplying and infecting the patient during subsequent uses of the product.

[0091] Antimicrobial preservatives are not included in single-use vials of eye drops because they are aseptically manufactured or sterilized, the product is used once, and the dispenser is discarded.

[0092] For ophthalmic formulations requiring sterilization, an appropriate, validated sterilization method should be determined based on the specific product and container characteristics. Filtration of the formulation through a 0.22 μm filter into a sterile final container is a commonly used method.

[0093] In another embodiment of the present invention, the concentration of the peptide in the composition is in the range of 1 to 50 mg / mL, preferably in the range of 1 to 25 mg / mL, more preferably in the range of 1 to 10 mg / mL, and even more preferably in the range of 1 to 5 mg / mL.

[0094] In another embodiment of the present invention, the ophthalmic pharmaceutical composition of the first aspect is in the form of a solution such as eye drops. The administration of peptides in the form of eye drops has the great advantage that they are easy to use and not uncomfortable for the subject in need thereof.

[0095] In another embodiment of the present invention, the ophthalmic pharmaceutical composition is selected from creams, lotions, ointments, emulsions, aerosol and non-aerosol sprays, gels, ointments, and suspensions.

[0096] Additionally, the compositions of the present invention may contain other ingredients such as fragrances, colorants, and other ingredients known in the state of the art for use in topical formulations.

[0097] The topical compositions of the present invention can be prepared according to methods well known in the state of the art. Suitable excipients and / or carriers, as well as their amounts, can be easily determined by those skilled in the art depending on the type of formulation to be prepared.

[0098] In a second aspect, the present invention relates to a lyophilisate.

[0099] In one embodiment of the second aspect, the lyophilisate is in the form of a lyophilised cake or powder.

[0100] In another embodiment of the second aspect, the water content of the lyophilisate is less than 5.0% by weight of the total amount of the lyophilisate, preferably less than 3.0% by weight of the total amount of the lyophilisate, more preferably less than 2.0% by weight of the total amount of the lyophilisate.

[0101] In a third aspect, the present invention relates to a process for preparing the ophthalmic pharmaceutical composition of the first aspect.

[0102] In one embodiment of the third aspect, the process comprises in step a) freeze-drying the solution comprising freezing the solution, primary drying and secondary drying, wherein the freeze-drying takes less than 40 hours, preferably 10 to 35 hours, more preferably 15 to 30 hours, from the initial step of freezing the solution to the end of secondary drying.

[0103] In another embodiment of the third aspect, the process above results in step a) in a lyophilisate of the second aspect.

[0104] In a fourth aspect, the present invention relates to an ophthalmic pharmaceutical composition obtainable by the process of the third aspect.

[0105] In a fifth aspect, the present invention relates to a kit comprising a lyophilisate and a physiologically acceptable vehicle composition comprising one or more pharmaceutically acceptable excipients or carriers for reconstituting the peptide.

[0106] The kit optionally further comprises instructions for carrying out the reconstitution of the lyophilisate to obtain the composition of the invention.

[0107] The lyophilisate and vehicle can be contained in separate containers (vials), or alternatively in a two-compartment container (vial) in which one compartment contains the lyophilisate and the other compartment contains the vehicle.

[0108] In one embodiment of the fifth aspect, the physiologically acceptable vehicle comprises at least one viscosity increasing agent and optionally at least one preservative.

[0109] In a sixth aspect, the present invention relates to a kit comprising the ophthalmic pharmaceutical composition of the first and fourth aspects, a container for holding the pharmaceutical composition, and a drop dispenser adapted to administer the composition in a volume of about 10 to 100 μl per drop, preferably about 10 to 50 μl, more preferably about 20 to 40 μl.

[0110] In another embodiment of the fifth and sixth aspects, the container and / or drop dispenser are made from a thermoplastic material or glass, preferably the thermoplastic material is selected from polyethylene or polypropylene. In a preferred embodiment of the fifth aspect, the container is made from polypropylene and the drop dispenser is made from polyethylene selected from low density or high density polyethylene. In another preferred embodiment of the fifth aspect, the container and drop dispenser are made from glass.

[0111] The final container must be appropriate for the ophthalmic product and its intended use and must not interfere with the stability and efficacy of the formulation.

[0112] Protection from retinal neurodegeneration detected by several ophthalmic examination methods represents an excellent approach to treating DR. Neurodegeneration is present in the early stages of DR (which can be detected by loss of both color discrimination and contrast sensitivity, glial activation, and neuronal apoptosis). The ophthalmic pharmaceutical composition of the present invention is useful in retinal degenerative diseases, particularly DR, especially in the early stages when treatment is not indicated and only follow-up is recommended until more advanced stages of DR (clinically significant diabetic macular edema and / or proliferative diabetic retinopathy) are established.

[0113] Treatment of DR at an early stage has the real advantage of avoiding further complications, namely microaneurysms, microhemorrhages, hard exudates, neovascularization, capillary occlusion, and breakdown of the blood-retinal barrier (BRB).

[0114] In another embodiment of the invention, the ophthalmic pharmaceutical composition of the first or fourth aspect, or the kit of the fifth aspect, is for use in the topical ocular treatment and / or prevention of a retinal neurodegenerative disease.

[0115] In a preferred embodiment, the retinal neurodegenerative disease is selected from the group consisting of diabetic retinopathy (DR), age-related macular degeneration, glaucoma, and retinitis pigmentosa, hi a more preferred embodiment, the retinal neurodegenerative disease is diabetic retinopathy.

[0116] In another more preferred embodiment, the ophthalmic pharmaceutical composition of the first or fourth aspect, or the kit of the fifth aspect, is for use in the topical treatment of early stages of diabetic retinopathy.

[0117] In another more preferred embodiment, the composition is administered one to four times per day, preferably once per day, preferably twice per day, preferably three times per day, preferably four times per day.

[0118] The present invention will now be described in more detail with reference to the following examples, which should not be construed as limiting the scope of the invention in any way.

[0119] Further aspects and embodiments of the present invention are described in the following clauses.

[0120] Clause 1. An ophthalmic pharmaceutical composition comprising: A peptide having a sequence length of 13 to 50 amino acids and one or more pharmaceutically acceptable excipients or carriers, wherein the N-terminal region of the peptide has the sequence: HXaa 1 EGTFTSDXaa 2 SXaa 3 Xaa 4 (SEQ ID NO: 1), wherein: Xaa 1 is an amino acid selected from alanine and glycine; Xaa 2 is an amino acid selected from valine and leucine; Xaa 3 is an amino acid selected from serine and lysine; Xaa 4 is an amino acid selected from tyrosine and glutamine; histidine is the N-terminal residue; The ophthalmic pharmaceutical composition, wherein the solution has a pH value of 4.0 to 4.8 and an osmolality in the range of 0.5 to 200 mOsm / kg.

[0121] Clause 2: The ophthalmic pharmaceutical composition of the preceding clause, wherein the sequence length is 30 to 40 amino acids.

[0122] Article 3 Xaa 1 is alanine, and Xaa 2 is valine, and Xaa 3 is serine, and Xaa 4 10. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein is tyrosine.

[0123] Clause 4. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein said peptide is mammalian glucagon-like peptide-1, or a pharmaceutically acceptable salt thereof.

[0124] Clause 5: The peptide (a) a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof; or, alternatively, (b) a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof; or, alternatively, (c) a peptide having an amino acid sequence having a degree of identity of at least 85% with SEQ ID NO: 2, 3, or a pharmaceutically acceptable salt thereof, wherein said N-terminal region is as defined in the first aspect of the invention; or alternatively, (d) a peptide having a sequence length of up to 50 amino acids comprising an amino acid sequence having a degree of identity of at least 85% with SEQ ID NO: 2, 3, or a pharmaceutically acceptable salt thereof, wherein said N-terminal region is as defined in the first aspect of the invention; or alternatively, (e) a fragment of a peptide having a degree of identity of at least 85% to SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein said fragment has 14 to 29 amino acids and comprises the N-terminal region defined in the first aspect of the invention; or, alternatively, (f) An ophthalmic pharmaceutical composition according to any one of the preceding clauses, wherein the fragment has a degree of identity of at least 85% with SEQ ID NO: 3, or a pharmaceutically acceptable salt thereof, wherein the fragment has 14 to 30 amino acids and comprises the N-terminal region as defined in the first aspect of the present invention.

[0125] Clause 6. An ophthalmic pharmaceutical composition according to any one of clauses 1 to 4, wherein the peptide is a pharmaceutically acceptable salt of the sequence of SEQ ID NO: 2, in particular an acetate salt of the sequence of SEQ ID NO: 2, or alternatively the peptide is the sequence of SEQ ID NO: 2.

[0126] Clause 7. The ophthalmic pharmaceutical composition according to any one of the preceding clauses, wherein the pH value is 4.1 to 4.8, preferably the pH value is 4.2 to 4.7.

[0127] Clause 8. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein the osmolality is in the range of 1 to 150 mOsm / kg.

[0128] Clause 9. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein the osmolality is in the range of 1 to 90 mOsm / kg.

[0129] Clause 10. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein the osmolality is in the range of 1 to 80 mOsm / kg.

[0130] Clause 11. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein the osmolality is in the range of 1 to 70 mOsm / kg.

[0131] Clause 12. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein the osmolality is in the range of 1 to 50 mOsm / kg.

[0132] Clause 13. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein the osmolality is in the range of 1 to 10 mOsm / kg.

[0133] Clause 14: The ophthalmic pharmaceutical composition according to any one of Clauses 1 to 9, wherein the osmolality is in the range of 85 to 150 mOsm / kg.

[0134] Clause 15. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein at least one of the one or more pharmaceutically acceptable excipients or carriers is selected from the group consisting of stabilizers, thickeners, buffers, and mixtures thereof.

[0135] Clause 16. The ophthalmic pharmaceutical composition of the preceding clause, wherein the stabilizer is aspartic acid or glutamic acid.

[0136] Clause 17. An ophthalmic pharmaceutical composition according to any one of the two preceding clauses, wherein the weight ratio of the stabilizer to the peptide in the composition is in the range of 1:5 to 1:50, preferably 1:8 to 1:30, more preferably 1:10 to 1:20.

[0137] Clause 18. The ophthalmic pharmaceutical composition of any one of the three preceding clauses, wherein said buffering agent is acetic acid / acetate or citric acid / citrate.

[0138] Clause 19. The ophthalmic pharmaceutical composition of any one of the four preceding clauses, wherein the total amount of buffer in the composition is 0.05% to 5.0% w / w, preferably 0.08% to 2.0% w / w, more preferably 0.1% to 1.5%.

[0139] Clause 20. The ophthalmic pharmaceutical composition of any one of the five preceding clauses, wherein the buffer strength ranges from 20 mM to 100 mM, preferably from 30 mM to 70 mM.

[0140] Clause 21. The ophthalmic pharmaceutical composition of any one of the six preceding clauses, wherein the viscosity increasing agent is selected from hydroxyethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyvinylpyrrolidone, sodium hyaluronate, carbopol, polyacrylamide, sodium chondroitin sulfate, and mixtures thereof.

[0141] Clause 22. The ophthalmic pharmaceutical composition of the preceding clause, wherein the viscosity increasing agent is hydroxypropyl methylcellulose or polyvinylpyrrolidone.

[0142] Clause 23. An ophthalmic pharmaceutical composition according to any one of the two preceding clauses or clause 11, wherein the total amount of thickening agent in the composition is from 0.1% to 5% w / w, preferably from 0.3% to 4% w / w, more preferably from 0.5% to 3% w / w.

[0143] Clause 24. The ophthalmic pharmaceutical composition of any one of the preceding clauses, having a viscosity in the range of 1 to 50 cSt, preferably in the range of 1 to 20 cSt, more preferably in the range of 1 to 10 cSt at room temperature and pressure.

[0144] Clause 25. The ophthalmic pharmaceutical composition of any one of the preceding clauses, further comprising an effective amount of a preservative.

[0145] Clause 26. The ophthalmic pharmaceutical composition of the preceding clause, wherein the preservative is selected from sodium edetate, benzalkonium chloride, centrimonium chloride, sodium perborate, stabilized oxychloro complexes, sorbic acid, thimerosal, polyquaternium-1, polyhexamethylene biguanide, chlorobutanol, phenylethyl alcohol, methylparaben, propylparaben, boric acid, sorbic acid, and propylene glycol, and mixtures thereof.

[0146] Clause 27. The ophthalmic pharmaceutical composition of any one of the two preceding clauses, wherein the preservative is benzalkonium chloride.

[0147] Clause 28. The ophthalmic pharmaceutical composition of any one of the three preceding clauses, wherein the total amount of preservatives in the composition is 0.005% to 0.5% w / w, preferably 0.008% to 0.3% w / w, more preferably 0.01% to 0.1% w / w.

[0148] Clause 29. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein the concentration of the peptide in the composition is in the range of 1 to 50 mg / mL, preferably in the range of 1 to 25 mg / mL, more preferably in the range of 1 to 10 mg / mL, and even more preferably in the range of 1 to 5 mg / mL.

[0149] Clause 30. The ophthalmic pharmaceutical composition of any one of the preceding clauses, wherein said composition is in the form of a solution.

[0150] Article 31 a) a pharmaceutically effective amount of a peptide as defined in any one of clauses 1 to 6 and / or a pharmaceutically acceptable salt thereof; b) a pharmaceutically acceptable amount of a stabilizer or buffer; and c) water, and a lyophilisate obtained by lyophilisation of a solution comprising The lyophilisate, which upon reconstitution is suitable for preparing an ophthalmic pharmaceutical composition according to any one of the preceding clauses.

[0151] Clause 32. A lyophilisate comprising a peptide as defined in any one of clauses 1 to 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable amount of a stabilizer or buffer, said lyophilisate being suitable for preparing, upon reconstitution, an ophthalmic pharmaceutical composition according to any one of the preceding clauses.

[0152] Clause 33. A lyophilisate according to any one of the two preceding clauses, wherein said lyophilisate is in the form of a lyophilised cake or a powder.

[0153] Clause 34. A lyophilisate according to any one of the three preceding clauses, wherein the water content of the lyophilisate is less than 5.0% by weight of the total amount of the lyophilisate, preferably less than 3.0% by weight of the total amount of the lyophilisate, and more preferably less than 2.0% by weight of the total amount of the lyophilisate.

[0154] Clause 35. A process for preparing an ophthalmic pharmaceutical composition according to any one of clauses 1 to 30, comprising the step of reconstituting a lyophilisate as defined in any one of clauses 31 to 34 with an aqueous vehicle composition comprising one or more pharmaceutically acceptable carriers or excipients, in particular an aqueous vehicle composition comprising at least one viscosity increasing agent and optionally at least one preservative.

[0155] Clause 36. A process for preparing an ophthalmic pharmaceutical composition according to any one of clauses 1 to 30, comprising: a) providing a lyophilisate comprising a pharmaceutically effective amount of a peptide as defined in any one of clauses 1 to 6 and / or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable amount of a stabilizer or buffer; b) providing a vehicle composition comprising one or more pharmaceutically acceptable excipients or carriers, such as at least one viscosity enhancing agent and optionally at least one preservative; c) reconstituting the lyophilized product of step a) with the vehicle composition of step b) to form an ophthalmic pharmaceutical composition.

[0156] Clause 37. The process according to the preceding clause, wherein the process comprises in step a) freeze-drying the solution comprising the steps of freezing the solution, primary drying and secondary drying, and wherein the freeze-drying takes less than 40 hours, preferably between 10 and 35 hours, more preferably between 15 and 30 hours, from the initial step of freezing the solution to the end of the secondary drying.

[0157] Clause 38. An ophthalmic pharmaceutical composition obtainable by a process as defined in any one of clauses 35 to 37.

[0158] Clause 39. A kit comprising a lyophilisate as defined in any one of clauses 31 to 34 and a physiologically acceptable vehicle composition comprising one or more pharmaceutically acceptable excipients or carriers for reconstituting said peptide.

[0159] Clause 40. The kit of the preceding clause, wherein said physiologically acceptable vehicle composition comprises at least one viscosity increasing agent and optionally at least one preservative.

[0160] Clause 41. A kit comprising an ophthalmic pharmaceutical composition as defined in clauses 1 to 30 or 38, a container for holding said pharmaceutical composition, and a drop dispenser adapted to administer the composition in a volume, for example about 10 to 100 μl per drop, preferably about 10 to 50 μl, more preferably about 20 to 40 μl.

[0161] Clause 42. A kit according to the preceding clause, wherein the container and / or drop dispenser are manufactured from a thermoplastic material or glass, preferably the thermoplastic material is selected from polyethylene or polypropylene.

[0162] Clause 43. A kit according to any one of the two preceding clauses, wherein the container is made from polypropylene and the drop dispenser is made from polyethylene selected from low density or high density polyethylene.

[0163] Clause 44. An ophthalmic pharmaceutical composition according to any one of clauses 1 to 30 or 38, or a kit according to any one of clauses 39 to 42, for use in the topical ocular treatment and / or prevention of a retinal neurodegenerative disease.

[0164] Clause 45. An ophthalmic pharmaceutical composition or kit for use according to the preceding clause, wherein said retinal neurodegenerative disease is selected from the group consisting of diabetic retinopathy (DR), age-related macular degeneration, glaucoma, and retinitis pigmentosa.

[0165] Clause 46. An ophthalmic pharmaceutical composition or kit for use according to any one of the two preceding clauses, wherein said retinal neurodegenerative disease is diabetic retinopathy.

[0166] Clause 47 An ophthalmic pharmaceutical composition or kit for use according to any one of the three preceding clauses for use in the topical treatment of non-proliferative diabetic retinopathy.

[0167] Clause 48. The ophthalmic pharmaceutical composition or kit for use according to any one of the four preceding clauses, wherein the composition is administered from once to four times a day, preferably once a day, preferably twice a day, preferably three times a day, preferably four times a day. [Example]

[0168] Example 1 Preparation of a ready-to-use eye drop solution of GLP-1(7-36) amide at a concentration of 2 mg / mL in acetic acid / acetate buffer at pH 4.4 A 2 mg / ml aqueous ophthalmic composition of synthetic human glucagon-like peptide (7-36) amide was prepared on a 60 mL scale. [Table 1]

[0169] 136 mg of sodium acetate and 77 mg of ammonium acetate were weighed and adjusted to a volume of 100 mL with irrigation water in a ready-to-use 250 mL sterile container. The mixture was stirred until completely dissolved. 0.25 mL of acetic acid was then added to the solution. The pH of the acetic acid / acetic acid solution was 4.4.

[0170] 99 g of the previous solution was sampled in a new reactor equipped with a magnetic stirrer. 1 g of polyvinylpyrrolidone K90 was weighed and added stepwise to the reactor as a thickener, followed by stirring for 30 minutes to ensure good dissolution.

[0171] 59.87 g of the previous solution was weighed and added to a reactor equipped with a magnetic stirrer. 133.5 mg of GLP-1(7-36) amide (acetate salt, batch: 020217, pure peptide content #90.78%) was weighed and added with stirring until completely dissolved. The pH of the final solution was 4.5.

[0172] The final solution was filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) filter membrane. 2 mL of the pre-filtered solution was placed in a Type I glass vial (2 mL). The vial was closed using 13 mm bromobutyl rubber and sealed with a 13 mm aluminum cap. 28 vials were obtained, with a final yield of approximately 93% w / w.

[0173] The resulting ophthalmic solution was then characterized. The solution had a clear appearance. Furthermore, the pH (Metrohm 780), osmolality (Osmomat 030-D), and GLP-1(7-36) amide content and purity by RP-HPLC were measured over a period of up to 12 months at two storage conditions: 5°C and 25°C / 60% RH. The RP-HPLC method used in all examples is described below. [Table 2]

[0174] The table below shows the visual appearance, pH, osmolality, GLP-1(7-36) amide content and purity at two storage conditions: 5°C and 25°C / 60% RH for up to 12 months. [Table 3]

[0175] Example 2 Preparation of a ready-to-use eye drop solution of aspartic acid containing GLP-1(7-36) amide at a concentration of 2 mg / mL A 2 mg / ml aqueous ophthalmic composition of synthetic human glucagon-like peptide (7-36) amide was prepared on a 60 mL scale. [Table 4]

[0176] 16.1 mg of aspartic acid was weighed out and then adjusted to a volume of 100 mL with water for irrigation in a ready-to-use 250 mL sterile container. The mixture was stirred until completely dissolved.

[0177] 99 g of the previous solution was sampled in a new reactor equipped with a magnetic stirrer. 1 g of polyvinylpyrrolidone K90 was weighed and added stepwise to the reactor as a thickener, followed by stirring for 30 minutes to ensure good dissolution.

[0178] 59.87 g of the previous solution was weighed and added to a reactor equipped with a magnetic stirrer. 133.5 mg of GLP-1(7-36) amide (batch: 020217, pure peptide content #90.78%) as acetate salt was weighed and added to the previously weighed solution and stirred until completely dissolved. The pH of the final solution was 4.5.

[0179] The final solution was filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) filter membrane. 2 mL of the pre-filtered solution was placed in a Type I glass vial (2 mL). The vial was closed using 13 mm bromobutyl rubber and sealed with a 13 mm aluminum cap. 29 vials were obtained, with a final yield of approximately 97% w / w.

[0180] The resulting ophthalmic solution was then characterized. The solution had a clear appearance. Furthermore, the pH, osmolality, and GLP-1(7-36) amide content and purity by RP-HPLC were measured at two storage conditions: 5°C and 25°C / 60% RH for up to 12 months. The RP-HPLC method used is described in Example 1.

[0181] The table below shows the visual appearance, pH, osmolality, GLP-1(7-36) amide content and purity at two storage conditions: 5°C and 25°C / 60% RH for up to 12 months. [Table 5]

[0182] Example 3 Preparation of a ready-to-use aqueous solution of aspartic acid containing GLP-1(7-36) amide at a concentration of 2 mg / mL and benzalkonium chloride as a preservative A 2 mg / ml aqueous ophthalmic composition of synthetic human glucagon-like peptide (7-36) amide was prepared on a 50 mL scale. [Table 6]

[0183] 16.1 mg of aspartic acid was weighed out and adjusted to a volume of 100 mL with water for irrigation in a ready-to-use 250 mL sterile container. The mixture was stirred until completely dissolved.

[0184] 99 g of the previous solution was sampled in a new reactor equipped with a magnetic stirrer. 1 g of polyvinylpyrrolidone K90 was weighed and gradually added to the reactor, stirring for 30 minutes to ensure complete dissolution. 20 mg of benzalkonium chloride was then added and stirred until completely dissolved.

[0185] 49.90 g of the previous solution was weighed and added to a reactor equipped with a magnetic stirrer. 111.3 mg of GLP-1(7-36) amide (batch: 020217, pure peptide content #90.78%) as acetate salt was weighed and added to the previously weighed solution and stirred until completely dissolved. The pH of the final solution was 4.4.

[0186] The final solution was filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) filter membrane. 2 mL of the pre-filtered solution was placed in a Type I glass vial (2 mL). The vial was closed using 13 mm bromobutyl rubber and sealed with a 13 mm aluminum cap. 24 vials were obtained, with a final yield of approximately 96% w / w.

[0187] The resulting ophthalmic solution was then characterized. The solution had a clear appearance. Furthermore, the pH, osmolality, and GLP-1(7-36) amide content and purity by RP-HPLC were measured at two storage conditions: 5°C and 25°C / 60% RH for up to 6 weeks. The RP-HPLC method used is described in Example 1.

[0188] The table below shows the visual appearance, pH, GLP-1(7-36) amide content and purity for up to 6 weeks at two storage conditions: 5°C and 25°C / 60% RH. [Table 7]

[0189] Example 4 Preparation of a ready-to-use aqueous solution of aspartic acid containing GLP-1(7-36) amide at a concentration of 24 mg / mL and benzalkonium chloride as a preservative A 24 mg / ml aqueous ophthalmic composition of synthetic human glucagon-like peptide (7-36) amide was prepared on a 5 mL scale. [Table 8]

[0190] 193.6 mg of aspartic acid was weighed out and adjusted to a volume of 100 mL with water for irrigation in a ready-to-use 250 mL sterile container. The mixture was stirred until completely dissolved.

[0191] 99 g of the previous solution was sampled in a new reactor equipped with a magnetic stirrer. 1 g of polyvinylpyrrolidone K90 was weighed and gradually added to the reactor, stirring for 30 minutes to ensure complete dissolution. 20 mg of benzalkonium chloride was then added and stirred until completely dissolved.

[0192] 4.87 g of the previous solution was weighed and added to a reactor equipped with a magnetic stirrer. 134.5 mg of GLP-1(7-36) amide (batch: 1065094, pure peptide content #89.3%) as acetate salt was weighed and added to the previously weighed solution and stirred until completely dissolved. The pH of the final solution was 4.5.

[0193] The final solution was filtered through a syringe filter with a 0.2 μm pore size polytetrafluoroethylene (PTFE) membrane. 2 mL of the pre-filtered solution was placed in a Type I glass vial (2 mL). The vial was closed using 13 mm bromobutyl rubber and sealed with a 13 mm aluminum cap.

[0194] The resulting ophthalmic solution was then characterized. The solution had a clear appearance. Furthermore, the pH, osmolality, and GLP-1(7-36) amide content and purity by RP-HPLC were measured at two storage conditions: 5°C and 25°C / 60% RH for up to 6 weeks. The RP-HPLC method used is described in Example 1.

[0195] The table below shows the visual appearance, pH, osmolality, GLP-1(7-36) amide content and purity at 5°C and 25°C, 60% HR for up to 12 months. [Table 9]

[0196] Example 5 Preparation of an aspartic acid ophthalmic solution containing GLP-1(7-36) amide at a concentration of 2 mg / mL, pH 4.5 Step 1: Preparation of a lyophilized product containing GLP-1(7-36) amide and aspartic acid at a dose of 4 mg / vial [Table 10]

[0197] 40.25 mg of aspartic acid was weighed into a ready-to-use 250 mL sterile container and adjusted to a volume of 100 mL with water for irrigation. The mixture was stirred until completely dissolved.

[0198] 59.67 g of the previous solution was weighed and added to a reactor equipped with a magnetic stirrer. 334 mg of GLP-1(7-36) amide (acetate salt, batch: 020217, pure peptide content #90.78%) was weighed and added with stirring until completely dissolved. The pH of the final solution was 4.4.

[0199] The final solution, consisting of GLP-1(7-36) at a concentration of 5 mg / mL, was filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) filter membrane.

[0200] Prior to freeze-drying, 0.8 mL of the solution was filled into 2 mL type I glass vials intended for freeze-drying, resulting in a freeze-dried product containing a dose of 4 mg / vial of GLP-1(7-36) amide after freeze-drying.

[0201] The lyophilized vials were stoppered inside the lyophilizer under 500 mbar N2 using 13 mm bromobutyl rubber and sealed with 13 mm aluminum caps. 67 lyophilized vials were obtained, resulting in a final yield of approximately 89%.

[0202] The appearance of the lyophilized vials and the content and purity of GLP-1(7-36) amide were then characterized by RP-HPLC under three storage conditions: 5°C, 25°C / 60% RH, and 40°C / 75% RH for up to 12 months. The RP-HPLC method used is described in Example 1.

[0203] The resulting cakes showed good, intact appearance. The table below shows the GLP-1(7-36) amide content and purity for up to 12 months at three storage conditions: 5°C, 25°C / 60% RH, and 40°C / 75% RH. [Table 11]

[0204] Step 2: Preparation of an aqueous reconstitution vehicle consisting of an aqueous solution containing 1% polyvinylpyrrolidone K90 as a thickening agent [Table 12]

[0205] 2 g of polyvinylpyrrolidone K90 was weighed as a thickener and gradually added to the reactor using 198 g of pre-weighed water for irrigation under stirring. The mixture was stirred for 30 minutes to ensure good dissolution. The solution was filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) filter membrane.

[0206] The reconstituted vehicle solution was then characterized by means of its visual appearance, which indicated the appearance of a clear solution.

[0207] Step 3. Preparation of reconstituted product at a concentration of 2 mg / mL of GLP-1(7-36) amide with aspartic acid A total of 27 vials from Example 5 in Step 1 were reconstituted with 2 mL from the reconstitution vehicle from Example 5 in Step 2 to provide an ophthalmic solution at a GLP-1(7-36) amide concentration of 2 mg / mL with the composition described below. [Table 13]

[0208] The resulting ophthalmic solution was then characterized. The solution had a clear appearance. Furthermore, the pH (Metrohm 780) and the GLP-1(7-36) amide content and purity by RP-HPLC were measured at two storage conditions: 5°C and 25°C / 60% RH for up to 6 weeks. The RP-HPLC method used is described in Example 1.

[0209] The table below shows the visual appearance and pH for up to 6 weeks at two storage conditions: 5°C and 25°C / 60% RH. [Table 14] The table below shows the GLP-1(7-36) amide content and purity for up to 6 weeks at three storage conditions: 5°C and 25°C / 60% RH. [Table 15]

[0210] Example 6 Preparation of an ophthalmic solution of acetic acid / acetate buffer at pH 4.5 containing GLP-1(7-36) amide at a concentration of 2 mg / mL Step 1: Preparation of a lyophilized product containing GLP-1(7-36) amide and mannitol at a dose of 4 mg / vial [Table 16]

[0211] 445 mg of GLP-1(7-36) amide acetate (batch: 020217, pure peptide content #90.78%), 800 mg of mannitol, and 78.8 g of water for irrigation were weighed into a ready-to-use 250 mL sterile container. The mixture was stirred until completely dissolved. The pH of the final solution was 4.9.

[0212] The final solution, consisting of GLP-1(7-36) at a concentration of 5 mg / mL, was filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) filter membrane.

[0213] Prior to freeze-drying, 0.8 mL of the solution was filled into 2 mL type I glass vials intended for freeze-drying, resulting in a freeze-dried product containing a dose of 4 mg / vial of GLP-1(7-36) amide after freeze-drying.

[0214] The lyophilized vials were stoppered inside the lyophilizer under 500 mbar N2 using 13 mm bromobutyl rubber and sealed with 13 mm aluminum caps. 92 lyophilized vials were obtained, with a final yield of approximately 92%.

[0215] The appearance of the lyophilized vials and the content and purity of GLP-1(7-36) amide were then characterized by RP-HPLC under three storage conditions: 5°C, 25°C / 60% RH, and 40°C / 75% RH for up to 6 weeks. The RP-HPLC method used is described in Example 1.

[0216] The resulting cake had a good appearance. The table below shows the GLP-1(7-36) amide content and purity for up to 6 weeks under three storage conditions: 5°C, 25°C / 60% RH, and 40°C / 75% RH. [Table 17]

[0217] Step 2: Preparation of an aqueous reconstitution vehicle consisting of acetic acid / acetate buffer solution (pH 4.4) containing 1% polyvinylpyrrolidone K90 as a viscosity enhancer [Table 18]

[0218] 272 mg of sodium acetate and 154 mg of ammonium acetate were weighed into a ready-to-use 250 mL sterile container and adjusted to a volume of 200 mL with water for irrigation. The mixture was stirred until completely dissolved. 0.50 mL of acetic acid was then added to the solution. The pH of the acetic acid / acetic acid solution is 4.4.

[0219] 198 g of the previous solution was sampled in a new reactor equipped with a magnetic stirrer. 2 g of polyvinylpyrrolidone K90 was weighed and gradually added to the reactor under stirring for 30 minutes to ensure complete dissolution. The solution was then filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) filter membrane.

[0220] The reconstituted vehicle solution was then characterized by means of its visual appearance, which indicated the appearance of a clear solution.

[0221] Step 3: Preparation of reconstituted product at a concentration of 2 mg / mL of GLP-1(7-36) amide with acetic acid / acetate buffer A total of 26 vials of Example 6 in Step 1 were reconstituted with 2 mL from the reconstituted vehicle form of Example 6 in Step 2 to obtain an ophthalmic solution with a GLP-1(7-36) amide concentration of 2 mg / mL, with the composition described below.

[0222] [Table 19]

[0223] The resulting ophthalmic solution was then characterized. The solution had a clear appearance. Furthermore, the pH and GLP-1(7-36) amide content and purity by RP-HPLC were measured over a period of up to 6 weeks at two storage conditions: 5°C and 25°C / 60% RH. The RP-HPLC method used is described in Example 1.

[0224] The table below shows the visual appearance and pH for up to 6 weeks at two storage conditions: 5°C and 25°C / 60% RH. [Table 20]

[0225] The table below shows the GLP-1(7-36) amide content and purity for up to six weeks at three storage conditions: 5°C, 25°C / 60% RH, and 40°C / 75% RH. [Table 21]

[0226] Example 7 Preparation of an aspartic acid ophthalmic solution containing GLP-1(7-36) amide at a concentration of 2 mg / mL, pH 4.4 Step 1 from Example 6: Preparation of a lyophilized product containing GLP-1(7-36) amide and mannitol at a dose of 4 mg / vial Step 2: Preparation of an aqueous reconstitution vehicle consisting of an aqueous solution of aspartic acid containing 1% polyvinylpyrrolidone K90 as a thickening agent [Table 22]

[0227] 32.2 mg of aspartic acid was weighed into a 250 mL sterile ready-to-use container and then poured into 200 mL of irrigation water. The mixture was stirred until completely dissolved.

[0228] 198 g of the previous solution was sampled in a new reactor equipped with a magnetic stirrer. 2 g of polyvinylpyrrolidone K90 was weighed and gradually added to the reactor under stirring for 30 minutes to ensure complete dissolution. The solution was then filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) filter membrane.

[0229] The reconstituted vehicle solution was then characterized by means of its visual appearance, which indicated the appearance of a clear solution.

[0230] Step 3: Preparation of reconstituted product at a concentration of 2 mg / mL of GLP-1(7-36) amide with mannitol and aspartic acid A total of 25 vials of the formulation from Example 6 in Step 1 were reconstituted with 2 mL of the reconstituted vehicle formulation from Example 7 in Step 2 to provide an ophthalmic solution at a GLP-1(7-36) amide concentration of 2 mg / mL, with the composition described below. [Table 23]

[0231] The resulting ophthalmic solution was then characterized. The solution had a clear appearance. Furthermore, the pH and GLP-1(7-36) amide content and purity by RP-HPLC were measured over a period of up to 6 weeks at two storage conditions: 5°C and 25°C / 60% RH. The RP-HPLC method used is described in Example 1.

[0232] The table below shows the visual appearance and pH for up to 6 weeks at two storage conditions: 5°C and 25°C / 60% RH. [Table 24]

[0233] The table below shows the GLP-1(7-36) amide content and purity for up to 6 weeks at two storage conditions: 5°C and 25°C / 60% RH. [Table 25]

[0234] Example 8 Preparation of an aspartic acid ophthalmic solution containing GLP-1(7-36) amide at a concentration of 2 mg / mL, pH 4.5 Step 1: Preparation of a lyophilized product containing GLP-1(7-36) amide and aspartic acid at a dose of 10 mg / vial [Table 26]

[0235] 480.0 mg of aspartic acid was weighed and poured into a 1 L reactor equipped with a magnetic stirrer containing 500 mL of irrigating water. The mixture was stirred until completely dissolved.

[0236] 6.72 g of GLP-1(7-36) amide (89.3% pure peptide content) as acetate salt was weighed and added with stirring until completely dissolved. The pH of the final solution was 4.5. The solution was finally brought to a final volume of 900 mL with irrigation water.

[0237] The final solution, consisting of GLP-1(7-36) at a concentration of 6.67 mg / mL, was filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) filter membrane.

[0238] Prior to freeze-drying, 1.5 mL of the solution was filled into 6 mL type I glass vials intended for freeze-drying, resulting in a freeze-dried product containing a dose of 10 mg / vial of GLP-1(7-36) amide after freeze-drying.

[0239] The lyophilized vials were stoppered inside the lyophilizer under 500 mbar N2 using 18 mm bromobutyl rubber and sealed with 18 mm aluminum caps. 495 lyophilized vials were obtained, with a final yield of approximately 93.4%.

[0240] The appearance of the lyophilized vials and the content and purity of GLP-1(7-36) amide were then characterized by RP-HPLC at two storage conditions: 5°C and 25°C / 60% RH for up to 3 months. The RP-HPLC method used is described in Example 1.

[0241] The resulting cake had a good, intact appearance. The table below shows the purity for up to 18 months at two storage conditions: 5°C and 25°C / 60%RH. [Table 27]

[0242] Step 2: Preparation of an aqueous reconstitution vehicle consisting of an aqueous solution containing 1% polyvinylpyrrolidone K90 as a thickening agent [Table 28]

[0243] 50 g of polyvinylpyrrolidone K90 was weighed as a thickener and gradually added to the reactor using 3 L of pre-weighed water for irrigation under stirring. The mixture was stirred for 1 hour to ensure good dissolution.

[0244] 1.25 g of benzalkonium chloride was weighed and added to the mixture until it was completely dissolved. The solution was brought to a final volume of 5 L and then filtered through a 0.22 μm pore size polyvinylidene fluoride (PVDF) filter membrane.

[0245] The reconstituted vehicle solution was then characterized by means of its visual appearance, which indicated the appearance of a clear solution.

[0246] Step 3. Preparation of reconstituted product at a concentration of 2 mg / mL of GLP-1(7-36) amide with aspartic acid A total of 152 vials from Example 8 in Step 1 were reconstituted with 5 mL from the reconstituted vehicle from Example 8 in Step 2 to provide an ophthalmic solution at a GLP-1(7-36) amide concentration of 2 mg / mL with the composition described below. [Table 29]

[0247] The resulting ophthalmic solution was then characterized. The solution appeared as a clear solution with a viscosity of 3.8 cSt at 20°C. Additionally, the pH and purity by RP-HPLC were measured at two storage conditions, 5°C and 25°C / 60% RH, for up to 3 months. The RP-HPLC method used is described in Example 1.

[0248] The table below shows the visual appearance and pH for up to 6 months at two storage conditions: 5°C and 25°C / 60%RH. [Table 30]

[0249] The table below shows purity for up to 3 months at two storage conditions: 5°C and 25°C / 60%RH. [Table 31]

[0250] Example 9 Preparation of a ready-to-use glutamic acid ophthalmic solution containing GLP-1(7-36) amide at a concentration of 2 mg / mL [Table 32]

[0251] 1.80 mg of glutamic acid was weighed and then poured into 10 mL of irrigation water in a ready-to-use 15 L sterile container. The mixture was stirred until completely dissolved.

[0252] 11.45 mg of GLP-1(7-36) amide in acetate (pure peptide content #90.78%) was weighed into a 6 mL vial.

[0253] Five mL of the previous glutamic acid solution was added to the 6 mL vial containing GLP-1(7-36) amide in acetate and vortexed to completely dissolve. The pH of the final solution was 4.6.

[0254] Example 10 Preparation of a ready-to-use glutamic acid ophthalmic solution containing GLP-1(7-36) amide at a concentration of 2 mg / mL [Table 33]

[0255] 1.80 mg of glutamic acid was weighed and then poured into 10 mL of irrigation water in a 15 mL sterile ready-to-use container. The mixture was stirred until completely dissolved.

[0256] 9.9 g of the previous solution was sampled in a new reactor equipped with a magnetic stirrer. 0.1 g of polyvinylpyrrolidone K90 as a thickener was weighed and added stepwise to the reactor, followed by 9.9 g of the previously prepared solution, stirring for 30 minutes to ensure good dissolution.

[0257] 10.0 g of the previous solution was weighed and added to a reactor equipped with a magnetic stirrer. 22.3 mg of GLP-1(7-36) amide (acetate salt, batch: 020217, pure peptide content #90.78%) was weighed and added to the previous solution and stirred until completely dissolved. The pH of the final solution was 4.6.

[0258] References cited in this application - Schmidt et al., “Neurodegenerative Diseases of the Retina and Potential for the Protection and Recovery”, Current Neuropharmacology, 2008, Vol. No. 6, pp. 164-178. - Simo et al., “Neurodegeneration is an early event in diabetic retinopathy:therapeutic implications”, Br. J. Ophthalmol., 2012, vol. 96, pp. 1285-1290 - W02007062434 - Altschul et al., “Basic local alignment search tool”, 1990, J.Mol.Biol, v.215, pages 403-410 [Sequence List Free Text]

[0259] SEQ ID NO: 1 <221> VARIANT <222> (2)..(2) <223> X is an amino acid selected from alanine and glycine. <221> VARIANT <222> (10)..(10) <223> X is an amino acid selected from valine and leucine. <221> VARIANT <222> (12)..(12) <223> X is an amino acid selected from serine and lysine. <221> mutation <222> (13)..(13) <223> X is an amino acid selected from tyrosine and glutamine. SEQ ID NO: 2 <221> MOD_RES <222> (30)..(30) <223> AMIDATION

[0260] JPEG2026041726000034.jpg216149 JPEG2026041726000035.jpg228111 JPEG2026041726000036.jpg216111

Claims

1. An ophthalmic pharmaceutical composition comprising: A peptide having a sequence length of 13 to 50 amino acids or a pharmaceutically acceptable salt or solvate thereof, and one or more pharmaceutically acceptable excipients or carriers, wherein the N-terminal region of the peptide has the sequence: HXaa １ EGTFTSDXaa ２ SXaa ３ Xaa ４ (SEQ ID NO: 1), wherein: Xaa １ is an amino acid selected from alanine and glycine; Xaa ２ is an amino acid selected from valine and leucine; Xaa ３ is an amino acid selected from serine and lysine; Xaa ４ is an amino acid selected from tyrosine and glutamine; histidine is the N-terminal residue; The ophthalmic pharmaceutical composition, wherein the pH value of the composition is 4.0 to 4.8 and the osmolality is in the range of 0.5 to 200 mOsm / kg.

2. The ophthalmic pharmaceutical composition according to the preceding claims, wherein the sequence length is between 30 and 40 amino acids.

3. Xaa １ is alanine, and Xaa ２ is valine, and Xaa ３ is serine, and Xaa ４ 10. The ophthalmic pharmaceutical composition of any one of the preceding claims, wherein is tyrosine.

4. 10. The ophthalmic pharmaceutical composition of any one of the preceding claims, wherein the peptide is mammalian glucagon-like peptide-1, or a pharmaceutically acceptable salt thereof.

5. The peptide is (a) a peptide comprising or consisting of the amino acid sequence of SEQ ID NO: 2 or a pharmaceutically acceptable salt thereof; or, alternatively, (b) a peptide having an amino acid sequence having a degree of identity of at least 85% with SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein the N-terminal region is as defined in claim 1; or, alternatively, (c) a peptide having a sequence length of up to 50 amino acids comprising an amino acid sequence having a degree of identity of at least 85% with SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein the N-terminal region is as defined in claim 1; or alternatively, (d) an ophthalmic pharmaceutical composition according to any one of the preceding claims, wherein the fragment has a degree of identity of at least 85% with SEQ ID NO: 2, or a pharmaceutically acceptable salt thereof, wherein the fragment has an amino acid length of 14 to 49 amino acids and comprises the N-terminal region as defined in claim 1.

6. 6. The ophthalmic pharmaceutical composition according to any one of claims 1 to 5, wherein the peptide is a pharmaceutically acceptable salt of the sequence SEQ ID NO: 2, in particular an acetate salt of the sequence SEQ ID NO: 2, or alternatively the peptide is the sequence SEQ ID NO:

2.

7. 10. The ophthalmic pharmaceutical composition according to any one of the preceding claims, wherein the pH value is between 4.1 and 4.

8.

8. 10. The ophthalmic pharmaceutical composition according to any one of the preceding claims, wherein the osmolality is in the range of 1 to 150 mOsm / kg.

9. 10. The ophthalmic pharmaceutical composition of any one of the preceding claims, wherein at least one of the one or more pharmaceutically acceptable excipients or carriers is selected from the group consisting of stabilizers, thickeners, buffers, and mixtures thereof.

10. 10. The ophthalmic pharmaceutical composition of claim 9, wherein the stabilizer is aspartic acid or glutamic acid.

11. 10. An ophthalmic pharmaceutical composition according to any one of the preceding claims, further comprising an effective amount of a preservative.

12. 10. An ophthalmic pharmaceutical composition according to the preceding claim, wherein the preservative is selected from sodium edetate, benzalkonium chloride, centrimonium chloride, sodium perborate, stabilized oxychloro complexes, sorbic acid, thimerosal, polyquaternium-1, polyhexamethylene biguanide, chlorobutanol, phenylethyl alcohol, methylparaben, propylparaben, a combination of boric acid, sorbic acid and propylene glycol, and mixtures thereof, preferably the preservative is benzalkonium chloride.

13. 10. A lyophilisate comprising a peptide as defined in any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable amount of a stabilizer and / or buffer, said lyophilisate being suitable for preparing, upon reconstitution, an ophthalmic pharmaceutical composition as defined in any one of the preceding claims.

14. A process for preparing an ophthalmic pharmaceutical composition according to any one of claims 1 to 12, comprising the step of reconstituting a lyophilisate as defined in claim 13 with an aqueous vehicle composition comprising one or more pharmaceutically acceptable carriers or excipients, in particular an aqueous vehicle composition comprising at least one viscosity increasing agent and optionally at least one preservative.

15. An ophthalmic pharmaceutical composition according to any one of claims 1 to 12 for use in the local ocular treatment and / or prevention of retinal neurodegenerative diseases.

16. A kit comprising a lyophilisate as defined in claim 13 and a physiologically acceptable vehicle composition comprising one or more pharmaceutically acceptable excipients or carriers for reconstituting the peptide.

17. 13. A kit comprising an ophthalmic pharmaceutical composition as defined in any one of claims 1 to 12, a container for holding said pharmaceutical composition, and a drop dispenser adapted to administer a quantity of said composition.

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