Agents for treating or preventing ophthalmic conditions

Abstract

The present invention provides non-natural polypeptides for use in the treatment and / or prevention of ophthalmic conditions in a mammalian subject. The mammals well tolerate the administration of the polypeptide. The non-natural polypeptide is provided with high purity.

Description

[0001] Introduction Field of the Invention

[0002] The present invention provides a medicament for use in treating and preventing ophthalmic disorders, including but not limited to disorders involving damage and / or disorders of the optic nerve and retina. Background of the Invention

[0004] The optic nerve consists of a bundle of over one million nerve fibers that carry visual information. The optic nerve connects the retina to the brain. This connection involves retinal ganglion cells (RGCs) at the cellular level. Retinal ganglion cells (RGCs) are neurons located near the inner surface (ganglion cell layer) of the retina of the eye. RGCs vary in size, connectivity, and response to visual stimuli, but they all share a long axon that extends to the brain. These axons form the optic nerve.

[0005] Damage to the optic nerve can lead to vision loss. The type and severity of vision loss depend on the location where the damage occurs. It can affect one or both eyes.

[0006] There are many different types of optic nerve disorders, including:

[0007] - Glaucoma is a group of diseases that are a leading cause of blindness, especially in the United States. Glaucoma typically occurs when the fluid pressure inside the eye slowly increases and damages the optic nerve.

[0008] - Optic neuritis is an inflammation of the optic nerve. Causes include infections and immune-related diseases such as multiple sclerosis. Sometimes the cause is unknown.

[0009] - Optic nerve atrophy is damage to the optic nerve. Causes include poor blood flow to the eye, diseases, trauma, or exposure to toxic substances or drugs [ethambutol, isoniazid, digitalis, antibiotics (chloramphenicol, sulfonamides), and amiodarone].

[0010] - Optic nerve drusen are pockets of protein and calcium salts that accumulate in the optic nerve over time.

[0011] All of the above-reported diseases can cause damage to the RGC axons in the optic nerve and may ultimately lead to the death of these cells. For most optic nerve diseases, there are no available treatments, or the treatments can only prevent further vision loss. Therefore, enhancing the viability or function of RGCs remains a major goal in basic and translational research.

[0012] So far, there is no truly effective treatment on the market to counteract the loss of RCGs in ophthalmic diseases. Therefore, ophthalmic diseases, especially those involving loss or damage of RCGs, have been difficult to treat to date. Thus, there remains a very high medical need today.

[0013] Mesentier-Louro et al. have described some effects of ocular administration in experimental models of optic nerve damage in Mol. Neurobiol., Vol. 56, 2019, pp. 1056-1069.

[0014] In the rat optic nerve injury model, a large number of RGC degeneration usually occurs within 2 weeks after optic nerve crush due to reduced reverse transport of growth factors including nerve growth factor (NGF). According to the prior art, according to the preventive experimental paradigm (Mesentier-Louro et al., "Molecular Neurobiology (Mol. Neurobiol.)" 2019 Vol. 56 pp. 1056-1069), intravitreal and eye drop administration of recombinant human NGF (rhNGF) can only offset optic nerve compression in adult rats when administered immediately after optic nerve injury. However, this is different from the clinical situation of human ophthalmic diseases after all. For the intended treatment of human ophthalmic diseases, therapeutic intervention can only be performed some time after the first injury to the optic nerve, and these experimental results in the rat optic nerve crush model cannot be translated into clinical applications in humans. In particular, as a result of ONC, further downstream processes may be triggered on the optic nerve, including potential further damage to RCG. It is still unknown whether rhNGF or other molecules have a therapeutic effect when administered in the post-ONC injury stage of RGC. Therefore, when agents are administered in situations involving or resembling human ophthalmic diseases, whether the therapy is effective also remains elusive.

[0015] Thus, there remains a need for effective treatments for ocular conditions, particularly those affecting the optic nerve, which are not adversely affected, such as intolerable or otherwise undesirable side effects, and for therapeutic agents suitable for such purposes for administration to mammalian subjects, including humans, and which are available to practitioners in a reliable and acceptable purity.

[0016] Problem to be solved

[0017] The main purpose of the present invention is to provide a treatment or prevention of an ophthalmic condition without undesirable or painful side effects such as pain. It is also desirable to provide a therapeutic agent that can be easily used and applied by a practitioner. It is also desirable to provide a therapeutically active agent with a satisfactory yield and purity so that such treatment can be performed. Therefore, a further object of the present invention includes eliminating the disadvantages associated with the prior art. In view of the advantages obtained over the prior art, these and other further objects of the present invention will become apparent from the following detailed description. Specific objects include providing a reliable method for treating a subject suffering from an ophthalmic condition without undesirable side effects. Summary of the invention

[0018] The present invention provides polypeptides for treating and / or preventing ophthalmic disorders in mammalian subjects, wherein the polypeptides are selected from the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4. These polypeptides are characterized by mutations in the human NGF amino acid sequence (SEQ ID NO: 2), wherein the mutations are associated with reduced nociceptive activity. In particular, arginine at position 100 of hNGF is replaced by glutamate.

[0019] A particularly preferred polypeptide is the polypeptide of SEQ ID NO: 4. The polypeptide is characterized by the absence of at least proline at position 61, and more preferably proline at position 61 is replaced by another amino acid. In SEQ ID NO: 4, proline at position 61 of SEQ ID NO: 3 is replaced by serine.

[0020] Preferably, the mammalian subject is a human.

[0021] Preferably, the administration of the polypeptide of the present invention does not cause any adverse effects in the mammalian subject. Particularly preferably, the treatment and / or prevention according to the present invention does not cause hyperalgesia in the mammalian subject.

[0022] Preferably, the ophthalmic disorder involves damage and / or disorder of the optic nerve.

[0023] Preferably, the ophthalmic disorder is characterized by a disorder of retinal ganglion cells.

[0024] Preferably, the polypeptide is administered after optic nerve injury.

[0025] Preferably, the ophthalmic disorder includes at least one selected from glaucoma, neurotrophic keratitis, optic neuritis, optic atrophy, optic disc drusen, and optic pathway glioma.

[0026] Preferably, the polypeptide is for administration to the eye. More preferably, the administration is selected from topical administration to the eye and intravitreal administration, with topical administration being most preferred.

[0027] Preferably, the polypeptide is administered for at least four days after inducing optic nerve injury.

[0028] In one embodiment, the polypeptide is administered repeatedly. In a particularly preferred embodiment, the polypeptide is repeatedly administered at least three times a day.

[0029] In one embodiment, the polypeptide is repeatedly administered before complete cure of the ophthalmic disorder is observed or at least before improvement of the symptoms of the disorder. Alternatively, the polypeptide is repeatedly administered for three to 30 days, preferably seven to 14 days. Optionally, administration is stopped after completion of the interval.

[0030] Preferably, the dose / per dose has an amount of 0.3 to 30 μg of polypeptide per eye, more preferably 1 to 10 μg of polypeptide per eye, and most preferably 5 μg of polypeptide per eye. More preferably, these dosages are specifically for topical administration to the eye.

[0031] In one embodiment, the polypeptide is contained in an aqueous medium, and the aqueous medium is administered to a mammalian subject. More preferably, the polypeptide is contained in a composition comprising:

[0032] a) 0.2 to 20 mg / ml of the polypeptide,

[0033] b) 5 to 100 mM sodium acetate buffer,

[0034] c) 5 to 100 mM methionine,

[0035] d) pH 5.0 to 6.0.

[0036] More preferably, the polypeptide is contained in a composition comprising:

[0037] a) 2 mg / ml of the polypeptide,

[0038] b) 20 mM sodium acetate buffer,

[0039] c) 20 mM methionine,

[0040] d) pH 5.5.

[0041] In one embodiment, the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 is obtained from a biological source. This may include purification, i.e., separation from other molecules, which other molecules include other proteins, such as host cell proteins. Optionally, the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 can be obtained in a method comprising (re)folding and / or chromatographic purification and / or protease digestion and optionally adjusting the final protein concentration and / or preparing the desired formulation. In one embodiment, the polypeptide can be obtained by recombinant expression and purification, wherein the purification includes purification on a mixed-mode stationary phase. Preferably, the polypeptide for use according to the present invention is substantially free of degradation products of the polypeptide, especially substantially free of the des-nona variant of the polypeptide.

[0042] Furthermore, the present invention also provides the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 from recombinant sources and purified as described herein, for use in a method of treating the human or animal body by therapy, as described herein.

[0043] The present invention further relates to a composition of polypeptides comprising a polypeptide selected from SEQ ID NO: 3 and a polypeptide of SEQ ID NO: 4, wherein the composition is characterized by a pH of 5.0 to 6.0 (preferably pH 5.5), and comprising the following:

[0044] a) 0.2 to 20 mg / ml of said polypeptide (preferably 2 mg / ml),

[0045] b) 5 to 100 mM sodium acetate buffer (preferably 20 mM),

[0046] c) 5 to 100 mM methionine (preferably 20 mM). DETAILED DESCRIPTION OF THE INVENTION

[0048] This specification, together with the claims and the drawings, discloses in its entirety specific and / or preferred embodiments and variations of the various features of the present invention. The present invention also contemplates those embodiments that are particularly preferred, which are produced by combining two or more specific and / or preferred embodiments and variations described herein for the present invention. Accordingly, this disclosure also includes all entities, compounds, features, steps, methods or compositions mentioned or indicated individually or jointly in this specification, and any and all combinations or any two or more of said entities, compounds, features, steps, methods or compositions. Accordingly, unless otherwise expressly stated herein or required by the context, a reference to a single entity, compound, feature, step, method or composition shall be deemed to cover one or more (i.e., more than one, such as two or more, three or more or all) of these entities, compounds, features, steps, methods or compositions. Unless otherwise expressly stated or required by the context, each embodiment, aspect and example disclosed herein shall be considered applicable to any other embodiment, aspect or example disclosed herein and may be combined with any other embodiment, aspect or example disclosed herein.

[0049] Those of ordinary skill in the art will understand that the present invention described herein is susceptible to variations and modifications other than those specifically described. Accordingly, the scope of this disclosure is not limited by the specific embodiments described herein, and the specific embodiments provided herein are for purposes of illustration and example. Functional or other equivalent entities, compounds, features, steps, methods or compositions are within the scope of this disclosure. It will be apparent to those of ordinary skill in the art that this disclosure includes all variations and modifications of the entities, compounds, features, steps, methods or compositions literally described herein.

[0050] Each reference cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, presentations, etc.), whether above or below, is hereby incorporated by reference in its entirety. Nothing in this text should be construed as an admission that the present invention is not entitled to antedate a particular teaching and / or an admission that a particular reference, other than common general knowledge, contains sufficient clear and complete information for a person skilled in the art to carry out the present invention.

[0051] Generally, unless otherwise clearly defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art (e.g., in the fields of medicine, ophthalmology, neurology, genetics, molecular biology, gene expression, cell biology, cell culture, immunology, neurobiology, chromatography, protein chemistry, and biochemistry). Textbooks and review articles published, for example, in English generally define the meanings commonly understood by a person of ordinary skill in the art.

[0052] The expression “and / or”, e.g., “X and / or Y”, shall be understood to mean “X and Y” or “X or Y” and shall be regarded as clearly disclosing both “and” and “or” and both meanings (the meaning of “and” or the meaning of “or”).

[0053] As used herein, unless otherwise indicated, the terms “about”, “approximately”, and “substantially” all mean about or close to, and in the context of the numerical values or ranges set forth herein, preferably mean + / - 10% near the recited or claimed numerical values or ranges, more preferably + / - 5%.

[0054] Unless otherwise clearly stated, the use of the word “comprising” or variants such as “comprises” or “including” in the context of this document means that other members may optionally be present in addition to the members of the list introduced by “comprising”. However, as a specific embodiment of the present invention, the term “including” includes the possibility of the absence of other members, i.e., for the purposes of this embodiment, “including” shall be understood to have the meaning of “consisting of”.

[0055] Unless otherwise clearly stated, all indications of relative amounts with respect to the present invention are based on weight / weight. Indications of the relative amounts of components characterized by a general term are intended to refer to the total amount of all specific variants or members covered by said general term. If a component defined by a general term is specified to be present in a certain relative amount and if that component is further characterized as a specific variant or member covered by the general term, this means that no other variants or members covered by the general term are present additionally such that the total relative amount of the components covered by the general term exceeds the specified relative amount; more preferably, no other variants or members covered by the general term are present at all.

[0056] Unless otherwise specified herein or unless the context clearly dictates otherwise, all methods and processes described herein can be performed in any suitable order.

[0057] Unless otherwise indicated, the term "agent" as used herein generally refers to a compound or composition, preferably a compound. An agent is capable of acting on a living organism and / or cells from a living organism or cells derived from a living organism, for example by acting on cells and / or body tissues, or in the environment. The physical state of the agent is not particularly limited and, unless otherwise indicated, can be gaseous, aqueous and / or solid. Unless otherwise indicated, the type of agent is not particularly limited, and thus, the agent can be a chemical and / or biological molecule, such as a protein or nucleic acid. Specific agents defined herein can be used in the present invention.

[0058] As used herein, an "adverse effect" is an undesirable harmful effect caused by administering an agent (drug) to a subject. Adverse effects include, but are not limited to, morbidity, mortality, hyperalgesia syndrome, pain, weight change, enzyme levels, loss of function, or any pathological changes detected at the microscopic, macroscopic, or physiological level. Adverse reactions may result in reversible or irreversible changes, including an increase or decrease in an individual's sensitivity to other chemicals, foods, or procedures (such as drug interactions).

[0059] As used herein, the terms "chromatography", "chromatographic", etc. generally refer to techniques suitable for separating mixtures, in which a mixture is added to a non-liquid material called the "stationary phase" with the aim of at least partially separating one or more components of the mixture. For this purpose, the stationary phase can be exposed to a fluid and / or the mixture can be dissolved in the fluid; the fluid in contact with the stationary phase can also be referred to as the "mobile phase". Generally, any step described herein as "performed by chromatography" can be synonymously referred to as a "chromatographic step".

[0060] As used herein, the term "mobile phase" has the meaning commonly used in the art and can refer to all fluids in contact with the stationary phase during chromatography, i.e., the washing fluid as well as the fluid (mixture) containing the target protein, such as one or more proteins described herein. In the present invention, as described herein, the mixture subjected to chromatography generally contains one or more proteins, such as in particular the proteins described herein, such as polypeptides of SEQ ID NO: 3 or 4, precursors of any of these, proteases, and / or host cell proteins (HCPs).

[0061] "Stationary phase" generally comprises a base matrix as normally included, which is a water-insoluble material, usually in the form of particles or gels, such as resins. In many cases, including the embodiments described herein, the stationary phase comprises a base matrix and a moiety capable of binding to at least one component contained in the mixture to be chromatographed. The base matrix is generally a water-insoluble material, usually in the form of particles or gels. Non-limiting examples of the base matrix are agarose gels and agarose, such as high-rigidity agarose.

[0062] As used herein, a "chromatography step" refers to the action of adding a liquid containing at least one compound to be analyzed and / or purified to a chromatography material (preferably a stationary phase), which is preferably a protein (and in the context of the present invention, the protein is most preferably a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4), optionally washing the chromatography material with one or more washing solutions, and eluting the at least one compound. In this context, for illustration, a method characterized by two chromatography steps is characterized by adding a liquid containing at least one such compound to be analyzed and / or purified to a first chromatography material as described above, and, after eluting therefrom, adding a liquid containing at least one such compound to a second chromatography material as described above, from which it is also eluted. The purpose of any "chromatography step" is to include, in the mixture applied to the stationary phase, preferably in chromatography, the binding of at least one component to the stationary phase. Such compounds can be one or more proteins as described herein. The compounds can be recovered from the stationary phase, for example, by changing the mobile phase and / or by continuous exposure to the mobile phase over time.

[0063] When used in connection with chromatography, the term "binding" describes, for example, the binding ability of the stationary phase, without particular limitation, but generally refers to non-covalent binding. Thus, at least one component normally contained in the mixture, such as at least one protein, binds non-covalently to the stationary phase. The chromatography step optionally but preferably includes washing the stationary phase bound to at least one component. The at least one component can be at least one protein, such as at least one protein as described herein.

[0064] As used herein, the term "heterologous" describes something composed of multiple different elements.

[0065] In the context of the present invention, the terms "disulfide" and "disulfide bond" are used in their commonly understood meanings in the art. Generally, a "disulfide bond" refers to a functional group having the structure R-S-S-R'. This bond is also referred to as an "SS-bond" and is typically formed by the coupling of two thiol groups. Disulfide bonds in proteins are formed between the thiol groups of cysteine residues through an oxidative folding process; this specific disulfide bond between the thiol groups of two cysteine residues can also be referred to as a "disulfide bond". Without wishing to be bound by a particular theory, it is generally understood in the art that in eukaryotic cells, disulfide bonds are formed in the lumen of the endoplasmic reticulum (and the mitochondrial intermembrane space), but not typically in the cytosol, and for prokaryotes, disulfide bonds are formed in the periplasm (of the respective organism, particularly Gram-negative bacteria); disulfide bonds can also be found in proteins in the extracellular environment of eukaryotic and prokaryotic cells.

[0066] As used herein, terms such as "expression" and "gene expression" relate to the use of information from a gene in the synthesis of a functional gene product. Gene expression includes at least transcription and optionally includes one or more additional features, optionally selected from an open list including translation and post-translational modification. In the context of recombinant protein expression in a host cell, the term generally means that the protein is produced by the host cell (in any compartment of the cell and / or secreted and / or incorporated into inclusion bodies), unless the context indicates otherwise.

[0067] The terms "eye disorder" and "ophthalmic disorder" are used interchangeably herein and include all disorders affecting the eye. Without limitation, all disorders involving damage and / or dysfunction of the optic nerve and / or retina are included within the meaning of these terms.

[0068] As used herein, the term "heterologous" describes something composed of multiple different elements or sources. For example, in a non-human host cell containing a human gene (or a gene encoding a non-native polypeptide such as the polypeptides of the present invention), the gene is "heterologous" to the cell, and the cell may be capable of "heterologous" expression of the corresponding gene. Heterologous gene expression can also be referred to as "recombinant".

[0069] The term "inclusion body" has the meaning commonly used in the art and refers to aggregates or particles found in the cytosol or periplasm of a host cell; inclusion bodies typically contain proteins, such as, in particular, proteins recombinantly expressed in a host cell. Without wishing to be bound by any particular theory, it is understood that in the field of recombinant expression, inclusion bodies typically contain recombinantly expressed proteins, but relatively few host cell proteins (HCPs), ribosomal components, or DNA / RNA fragments. Without wishing to be bound by any particular theory, it should be understood that inclusion bodies typically contain at least partially misfolded proteins (misfolded proteins), especially misfolded recombinantly expressed proteins. It should be understood that inclusion bodies typically contain proteins in a non-correctly folded form, i.e., in the context of the present invention, they typically contain non-correctly folded polypeptides according to the present invention and / or their precursors. The term "misfolding" generally describes biomolecules, such as nucleic acids or polypeptides, that are not in their native conformation, i.e., in a non-correctly folded form.

[0070] "Isolated" refers to a material that is substantially or largely free of the components that it is normally associated with in its natural state. For example, as used herein, an "isolated peptide" or an "isolated protein" refers, respectively, to a peptide or protein that has been purified from the cellular environment and the extracellular environment (e.g., tissue) in which it is naturally surrounded, e.g., from the cell (e.g., host cell) that expresses it. In an alternative description, as used herein, an "isolated peptide" or an "isolated protein" refers, respectively, to a peptide or protein that has been isolated and / or purified in vitro from its natural cellular environment and from its association with other components of the environment in which the peptide or protein is normally present. In another example, as used herein, an "isolated cell" refers to a cell that has been purified from a cell and an extracellular environment such as tissue or cell colonies, the environment that surrounds it in its natural state, e.g., a host cell that has been removed from the environment that is normally adjacent to the cell. According to the above definition of the term "isolated", as used herein, "isolate" is a verb that describes the activity of obtaining an "isolated" material (e.g., an isolated cell or an isolated peptide or protein).

[0071] As used herein, the terms "plurality" and "a plurality of" refer to a plurality, i.e., any number of two or more.

[0072] As used herein, the term "mutation" refers to a change in the nucleotide sequence of the genome of an organism, a virus, extrachromosomal DNA, or other genetic element. The term also extends to mutations in amino acid sequences, particularly the amino acid sequence of a gene carrying at least one (non-silent) mutation. Unless otherwise specified, mutations in nucleotide sequences are permanent changes. Mutations present in the germline are generally heritable. In general, mutations in nucleotide sequences result in many different types of sequence changes: a gene mutation either has no effect, changes the gene's product, or prevents the gene from functioning normally or fully. Mutations can also be present in non-gene regions. Unless otherwise specified, the wild-type sequence is used as the reference sequence for describing a mutation. Thus, for example, when it is said that a given mutant is characterized by a mutation at position 100 of the polypeptide sequence, this indicates that the mutant does not have the same amino acid residue at position 100 as the wild-type polypeptide. Specific types of mutations in nucleotide sequences and / or amino acid sequences include alterations such as deletions, substitutions, additions, insertions, and splice variants. A "deletion" in a nucleotide sequence refers to the absence of one or more nucleotides from the nucleotide sequence. A "deletion" in an amino acid sequence refers to the absence of one or more amino acid residues from the polypeptide. An "addition" in a nucleotide sequence refers to the presence of one or more additional nucleotides in the nucleotide sequence. An "addition" in an amino acid sequence refers to the presence of one or more additional amino acid residues in the relevant polypeptide. A "substitution" in a nucleotide sequence refers to the replacement of one or more nucleotides in the nucleotide sequence by (one) other nucleotide(s). A "substitution" in an amino acid sequence refers to the replacement of one or more amino acid residues in the polypeptide by other amino acid residues. Additions, deletions, and substitutions in a nucleotide sequence, such as an open reading frame, can be at the 5'-end, 3'-end, and / or internal. Additions, deletions, and substitutions in a polypeptide can be at the amino terminus, carboxyl terminus, and / or internal. An "insertion" in a nucleotide sequence and / or polypeptide sequence refers to the addition of one or more nucleotides or one or more amino acid residues, respectively, particularly at an internal position in the respective sequence. The term "splice variant" is used to describe RNA encoding a polypeptide sequence that is spliced differently from the respective wild-type RNA, usually as a result of a mutation at the nucleic acid level, and generally results in a polypeptide translation product different from the wild-type polypeptide. The term "splice variant" can be used not only for the corresponding RNA, but also for the corresponding template DNA sequence (usually genomic DNA) and the sequence of the polypeptide encoded by such RNA.

[0073] The term "mutant" generally refers to a nucleic acid sequence or an amino acid sequence that is different from the wild-type sequence. Thus, a mutant nucleic acid sequence or amino acid sequence has at least one mutation relative to its respective wild-type sequence. In cases where polymorphisms exist in the nucleic acid sequence but are not reflected at the level of the corresponding encoded polypeptide (silent mutations, degeneracy of the genetic code), the term "mutant" at the nucleic acid level only refers to those nucleic acid variants that encode a mutant polypeptide. Mutants can contain different combinations of mutations, alone or in combination, including more than one mutation and different types of mutations.

[0074] In accordance with its general meaning in the art, the term "nerve growth factor" (abbreviated as "NGF" or "β-NGF") represents a neurotrophic factor and neuropeptide that participates in regulating the growth, maintenance, proliferation, and survival of certain neurons and other cells (see, for example, Levi-Montalcini, 2004, Progress in Brain Research, Volume 146, pages 525-527). Unless otherwise specified in the context, the term nerve growth factor only represents wild-type NGF and does not include the polypeptides of SEQ ID NO: 3 or 4. Wild-type NGF is the 2.5S, 26-kDa β subunit, which can be obtained from the NGF precursor and has biological activity: wild-type NGF binds to at least two classes of receptors: tropomyosin receptor kinase A (TrkA) and low-affinity NGF receptor (LNGFR / p75NTR). Unless otherwise specified, the term "NGF" refers to NGF of any species, preferably mammalian species; however, human NGF is always preferred. As used herein, "hNGF" represents human NGF. Unless otherwise specified in the context, the terms "NGF" and "hNGF" refer to wild-type NGF, that is, hNGF represents wild-type NGF. The amino acid sequence of wild-type human NGF corresponds to positions 121-239 of SEQ ID NO: 1. The sequences of non-human NGFs can be obtained, for example, in the scientific literature by sequence retrieval (such as BLAST) using positions 121-239 of SEQ ID NO: 1 as a bait and in public protein databases such as Swissprot.

[0075] The terms "NGF mutant protein" and "mutant protein of NGF", or "its mutant protein" with respect to NGF, are used interchangeably herein and refer to a polypeptide characterized by having at least one mutation compared to wild-type NGF, as described in further detail herein. The polypeptides of SEQ ID NO: 3 and SEQ ID NO: 4 are mutant proteins of NGF. Preferably, the mutant protein of NGF has 80 to 99.5% sequence identity with NGF, especially human NGF, and more preferably, the mutant protein has 90 to 99% sequence identity with NGF, especially human NGF.

[0076] The terms "mature moiety", "mature portion" with respect to NGF are used interchangeably with the term "β-NGF" and refer to the polypeptide of NGF which is characterized in that it does not contain the propeptide of NGF (and thus, of course, not the prepropeptide). Similarly, the term "mature portion" is also used to refer to the polypeptides of SEQ ID NO: 3 or 4, since these polypeptides likewise do not contain the propeptide (and thus, of course, not the prepropeptide). Preferably, the mature portion also does not contain the C-terminal cleavable peptide encoded by the wild-type NGF open reading frame; in the case of human NGF, this C-terminal cleavable peptide consists of the two amino acid residues "RA" (240 and 241 in SEQ ID NO: 1). More specifically, the mature portion can be obtained by, but is not limited to, cleavage of pro-NGF with the protease furin (and other proteases capable of precisely directly cleaving the first amino acid residue at the N-terminus of the polypeptide of NGF or SEQ ID NO: 3 or 4, respectively. For example, the furin cleavage site of human NGF and many orthologs is well known to consist of the sequence R1S2K3R4 (single letter amino acid code, sequence numbered from the N- to the C-terminus)). In mature NGF, generally neither the furin cleavage site nor any amino acids N-terminal to the furin cleavage site are present. By way of illustration, the mature portion of human NGF consists of the polypeptide represented by amino acid positions 122 - 239 of SEQ ID NO: 1. The mature portion of non-human NGF can be identified, for example, by sequence retrieval and / or sequence analysis, wherein the mature portion of human NGF is used for sequence alignment.

[0077] The terms "peptide" and "polypeptide" are used interchangeably herein and refer to a chain of amino acids linked by peptide (amide) bonds, and these two terms include, but are not limited to, all polypeptides shown in Figure 2.

[0078] As used herein, the term "precursor" with respect to NGF refers to any peptide sequence from which NGF can be obtained by proteolytic cleavage. By way of illustration, pro-NGF and propro-NGF and their variants are all typical examples of NGF precursors. As used herein, the term "precursor" can refer to a precursor whose most C-terminal amino acid residue is the most C-terminal residue of NGF, as well as a precursor that extends beyond the most C-terminal residue of NGF at the C-terminus, provided that NGF can be obtained from it by proteolytic cleavage: although the native precursor of wild-type human pro-NGF (SEQ ID NO: 1) contains the C-terminal dipeptide (amino acid residues 240 and 241 in SEQ ID NO: 1, bolded in Figure 1), the precursors of the present invention preferably do not contain the C-terminal cleavable peptide encoded by the wild-type NGF open reading frame; in the case of human NGF, this C-terminal cleavable peptide consists of the two amino acid residues "RA" (240 and 241 in SEQ ID NO: 1).

[0079] As used herein, the terms "propeptide" or "prosequence" are generally used interchangeably to refer to a polypeptide sequence encoded by a portion of the NGF open reading frame, with its N-terminus directly adjacent to the propeptide. By way of example: the propeptide is NGF and consists of a sequence comprising the contiguous sequence from residue 1 of SEQ ID NO:1 to residue 18 of SEQ ID NO:1. The sequences of the individual propeptides of non-human NGF precursors can be obtained, for example, in the scientific literature by sequence retrieval (such as BLAST) using the sequence of positions 1-18 of SEQ ID NO:1 as a bait and in public protein databases such as Swissprot. A polypeptide or protein consisting of a propeptide and pro-NGF, wherein the C-terminus of the propeptide is directly adjacent to the N-terminus of pro-NGF, may be referred to herein as "propro-NGF".

[0080] As used herein, the terms "propeptide" or "prosequence" are generally used interchangeably to refer to a polypeptide sequence encoded by a portion of the NGF open reading frame in nature, with its N-terminus directly adjacent to mature NGF, but this polypeptide sequence does not include the propeptide. By way of example: the propeptide is included in the wild-type precursor of NGF. The propeptide of the NGF precursor consists of a sequence comprising the contiguous sequence from residue 19 of SEQ ID NO:1 to residue 121 of SEQ ID NO:1. The sequences of the individual propeptides of non-human pro-NGF can be obtained, for example, in the scientific literature by sequence retrieval (such as BLAST) using the sequence of positions 19-121 of SEQ ID NO:1 as a bait and in public protein databases such as Swissprot.

[0081] "Pro-NGF", as used herein, refers to a peptide sequence that contains both the mature portion of NGF and the corresponding propeptide but does not contain the corresponding propeptide. Human pro-NGF consists of a sequence comprising the contiguous sequence from the 19th residue of SEQ ID NO:1 to at least the 239th residue of SEQ ID NO:1. Although wild-type human pro-NGF contains a C-terminal dipeptide (amino acid residues 240 and 241 in SEQ ID NO:1), preferably, the pro-NGF obtained and used in the present invention does not contain a C-terminal cleavable peptide encoded by the wild-type NGF open reading frame; in the case of human NGF, such a C-terminal cleavable peptide consists of the two amino acid residues "RA" (240 and 241 in SEQ ID NO:1). The sequences of non-human pro-NGF can be obtained, for example, in the scientific literature by sequence retrieval (such as BLAST) using the sequence of positions 19-239 of SEQ ID NO:1 as a bait and in public protein databases such as Swissprot.

[0082] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to RNA and DNA, including cDNA, genomic DNA, synthetic DNA, and DNA / RNA equivalents that include nucleotide analogs, phosphate analogs, and / or sugar analogs. Nucleic acids can be double-stranded or single-stranded (i.e., sense or antisense strands). Non-limiting examples of polynucleotides include genes, open reading frames, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, siRNA, microRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated nucleic acids of any type and sequence, nucleic acid probes and primers, and nucleic acid analogs. Nucleic acids can have any type of three-dimensional structure.

[0083] The term "peptide" according to the present invention includes oligopeptides and polypeptides and refers to substances that contain two or more, preferably more than 3, preferably more than 4, preferably more than 6, preferably more than 8, preferably more than 10, preferably more than 13, preferably more than 16, preferably more than 21, preferably up to 8, 10, 20, 30, 40, or 50, especially 100 amino acids, which are covalently linked in a chain by peptide bonds.

[0084] The term "protein" preferably refers to large peptides, preferably peptides having more than 100 amino acid residues, but in general, the terms "peptide", "polypeptide", and "protein" are synonyms and are used interchangeably herein unless the context indicates otherwise. Thus, the terms "polypeptide of SEQ ID NO:4" and "protein of SEQ ID NO:4" have the same meaning.

[0085] The term "pharmaceutically acceptable" generally describes a substance that can be administered to a subject in the dosage used, optionally and preferably in combination with a medicament, without causing intolerable side effects.

[0086] The terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" are used to refer to any one or more of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc., which are physiologically compatible and suitable for administration to a subject as described herein or do not interfere with such administration. Examples of such pharmaceutically acceptable carriers include, but are not limited to, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, etc., and combinations thereof. Especially in the case of liquid pharmaceutical compositions, an isotonic agent, such as sugar, polyols such as mannitol, sorbitol, or sodium chloride, is preferably included in the composition. Pharmaceutically acceptable carriers can also contain auxiliary substances, such as wetting agents or emulsifying agents, preservatives or buffering agents, which can enhance the shelf life or effectiveness of the medicament. Pharmaceutically acceptable carriers are generally included in the compositions according to the present invention.

[0087] The term "pharmaceutically active agent" refers to an agent that can be used to administer to a subject, wherein the agent will be beneficial for, for example, improving the symptoms of a disease or disorder. In addition, when administered to a subject in a therapeutically effective amount, the "pharmaceutically active agent" can have a positive or beneficial effect on the subject's disorder or disease state. Preferably, the pharmaceutically active agent has therapeutic properties and can be administered to improve, alleviate, mitigate, reverse, delay the onset of, or reduce the severity of one or more symptoms of a disease or disorder. The pharmaceutically active agent can have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathological condition. For example, as claimed, the agent of the present invention is considered herein to be a pharmaceutically active ingredient for the treatment of cystic fibrosis. In another example, a pharmaceutically active protein can be used to treat cells or individuals that normally do not express a protein, or do not reach the desired level, or misexpress a protein. For example, the pharmaceutically active protein can compensate for a mutation or lack of high enough expression by providing the desired protein. The term "pharmaceutically active peptide or protein" includes intact proteins or polypeptides, and can also refer to pharmaceutically active fragments thereof. It can also include pharmaceutically active analogs of peptides or proteins.

[0088] An "open reading frame" or "ORF" is a continuous stretch of codons that begins with a start codon and ends with a stop codon.

[0089] As used herein, the terms "subject" and "patient" refer to mammals. For example, mammals in the context of the present invention are humans, non-human primates, domesticated animals (including but not limited to dogs, cats, sheep, cows, goats, pigs, horses, etc.), laboratory animals (including but not limited to mice, rats, rabbits, etc.), and captive animals such as zoo animals. As used herein, the terms "subject" and "patient" specifically include humans. A subject (human or animal) has two sets of chromosomes; that is, the subject is diploid. The term "patient" refers to a subject who has a disorder, is at risk of having a disorder, has had a disorder, or is expected to have a disorder, and can be treated, for example, by administering an agent. The patient's condition may be chronic and / or acute. Thus, a "patient" can also be described as a subject who is receiving treatment and / or in need of treatment.

[0090] The term "therapy" should be understood broadly and refers to treatment in a subject that is aimed at preventing or treating a disorder of the subject. In a preferred embodiment, the therapy specifically includes administering an agent to the subject.

[0091] As used herein, the term "trypsin" generally refers to a proteolytic enzyme classified as EC 3.4.21.4. Trypsin typically cleaves peptide chains mainly on the carboxyl side of lysine or arginine amino acids, unless any of them is followed by proline. Without wishing to be bound by theory, it should be understood that trypsin is a serine protease and that trypsin occurs naturally in the digestive systems of many vertebrates, where it hydrolyzes proteins. In the present invention, preference is given to trypsin from recombinant sources. Although in vivo, trypsin is formed together with a propeptide (referred to as "trypsinogen"), the term "trypsin" as used herein preferably refers to mature trypsin without any propeptide. Proteolytic cleavage using trypsin may also be referred to as "tryptic proteolysis" or "trypsinization", and the protein produced by cleavage with trypsin is referred to as "trypsinized".

[0092] A "variant" of the NGF precursor or a polypeptide of SEQ ID NO: 3 or 4 refers to a polypeptide or protein in which the amino acid sequence that is not part of mature NGF (β-NGF) or not part of SEQ ID NO: 3 or 4, respectively, is characterized by at least one mutation compared to the wild-type precursor of NGF, such as wild-type pro-NGF or wild-type pro-pro-NGF; said at least one mutation is preferably located at the N-terminus of the amino acid sequence of mature NGF (β-NGF). Thus, as used herein, a "variant" of the NGF precursor, etc., refers to a peptide or protein in which the propeptide and / or prepropeptide is characterized by at least one mutation in the amino acid sequence of the propeptide and / or prepropeptide, such as but not limited to those variants described in WO 2013 / 092776 A1 and US2018 / 0086805 A1. By way of illustration, WO 2013 / 092776 A1 describes "variants" of pro-NGF in which the (wild-type) furin cleavage site is absent due to one or more specific mutations.

[0093] The term "vector" or "cloning vector" generally refers to a nucleic acid that can be introduced into a host cell. Exemplary vectors include but are not limited to plasmids, phages, and all other types of nucleic acids that can be introduced into a host cell. The term "vector" should be understood broadly and will include vectors encoding a peptide or protein for heterologous expression (such vectors can be used as templates for generating transcripts), as well as those that do not encode. The first type of vector will contain an open reading frame encoding a protein or peptide that can be expressed when the vector is present in a host cell. Although the type of vector that a person skilled in the art will choose will depend on the type of host cell that the person skilled in the art will choose, in a particular case, cloning vectors for all common host cells, including Escherichia coli, are commercially available, and thus the person skilled in the art will choose a particular vector with due consideration of the selected host cell.

[0094] The term "wild-type" is used herein to refer to a gene or protein that is typically found in nature, preferably in a healthy subject. A gene or protein that is not "wild-type" is referred to herein as "mutant" or "mutated", etc. By way of example, SEQ ID NO: 1 shows the amino acid sequence of the wild-type human NGF precursor; SEQ ID NO: 2 shows the amino acid sequence of wild-type human NGF.

[0095] The present invention is based on a number of discoveries that are interrelated and thus together have led the inventors to the various aspects of the present invention, which will be described separately hereinafter.

[0096] An agent according to the present invention

[0097] The present invention provides an agent for treating and / or preventing ophthalmic disorders in a mammalian subject. An agent that can be used to administer to a subject, wherein the agent will be beneficial, for example, in improving the symptoms of a disease or disorder. In particular, the agent that can be used in the present invention is a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. Accordingly, the present invention particularly provides a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for use in therapy. Therapy generally involves administering the polypeptide to a human or animal body, as described hereinafter.

[0098] According to the present invention, there is provided a polypeptide of SEQ ID NO: 3 and SEQ ID NO: 4 as a pharmaceutically active agent. By the present invention, there is provided a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for medical use, particularly for treating and / or preventing ophthalmic disorders in a mammalian subject. Optionally, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is from a recombinant source. Accordingly, the present invention also provides a recombinant polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for medical use, as described herein.

[0099] The agent according to the present invention will now be described in more detail, also referred to herein as "the polypeptide of SEQ ID NO:3" or "the polypeptide of SEQ ID NO:4". The term "the polypeptide of SEQ ID NO:3" and similar terms herein refer to a polypeptide comprising the amino acid sequence defined by SEQ ID NO:3 and / or an agent having equivalent biological activity. The term "the polypeptide of SEQ ID NO:4" and similar terms herein refer to a polypeptide comprising the amino acid sequence defined by SEQ ID NO:4 and / or an agent having equivalent biological activity. Thus, functional equivalent portions or analogs of such polypeptides are also included in these terms. An example of a biologically equivalent portion of a polypeptide can be a domain or subsequence of the polypeptide of SEQ ID NO:3 or the polypeptide of SEQ ID NO:4, which includes a binding site enabling the domain or subsequence to exert substantially the same biological activity as the full-length polypeptide of SEQ ID NO:3 or the full-length polypeptide of SEQ ID NO:4 or the gene encoding such a polypeptide. The term "substantially the same biological activity" means an equivalent portion or analog polypeptide having at least 50%, preferably at least 60%, more preferably at least 70%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% and most preferably at least 97%, at least 98% or at least 99% of the activity of the polypeptide of SEQ ID NO:3 or the polypeptide of SEQ ID NO:3 in the assay described in Example 4. Examples of biologically equivalent analogs of a polypeptide can be fusion proteins, which include at least a portion of the amino acid sequence of the polypeptide of SEQ ID NO:3 or the polypeptide of SEQ ID NO:4, but it can also be a homologous analog of the polypeptide. In addition, a fully synthetic molecule that mimics the specific biological activity of the polypeptide of SEQ ID NO:3 or the polypeptide of SEQ ID NO:4 will constitute a "biologically equivalent analog".

[0100] More preferably, the term "the polypeptide of SEQ ID NO:3" and similar terms herein refer to a polypeptide comprising the amino acid sequence defined by SEQ ID NO:3; such an agent is optionally a fusion protein, which particularly comprises the amino acid sequence defined by SEQ ID NO:3. Most preferably, the term "the polypeptide of SEQ ID NO:3" and similar terms herein refer to a polypeptide consisting of the amino acid sequence defined by SEQ ID NO:3; in this embodiment, the agent consists of a polypeptide composed of 118 amino acid residues arranged in the order defined by SEQ ID NO:3. In this and other embodiments, the polypeptide optionally bears one or two or three internal cysteine bonds such that cysteine (Cys, C) residues are covalently linked to each other to form intramolecular disulfide bonds. The cysteine bonds are preferably equivalent to those in wild-type human NGF.

[0101] Also more preferably, the term "polypeptide of SEQ ID NO:4" and similar terms herein refer to a polypeptide comprising the amino acid sequence defined by SEQ ID NO:4; such agents are optionally fusion proteins, which particularly comprise the amino acid sequence defined by SEQ ID NO:4. Most preferably, the term "polypeptide of SEQ ID NO:4" and similar terms herein refer to a polypeptide consisting of the amino acid sequence defined by SEQ ID NO:4; in this embodiment, the agent consists of a polypeptide composed of 118 amino acid residues arranged in the order defined by SEQ ID NO:4. In this and other embodiments, the polypeptide optionally bears one or two or three internal cysteine bonds such that cysteine (Cys, C) residues are covalently linked to each other to form intramolecular disulfide bonds. The cysteine bonds are preferably equivalent to those in wild-type human NGF.

[0102] The polypeptides of the present invention may optionally be characterized by further post-translational modifications. Such post-translational modifications optionally include glycosylation and / or phosphorylation. However, preferably, the polypeptides according to the present invention are not glycosylated and / or phosphorylated. In fact, considering that the experimental examples herein demonstrate the beneficial effects on the cure of ocular diseases and the beneficial benefit-side effect ratio, the polypeptides used are obtained by cytoplasmic recombinant expression in bacteria, which generally does not result in glycosylation and / or phosphorylation, so it is reasonable that the beneficial effects of the present invention do not depend on this type of post-translational modification. Therefore, in a preferred embodiment, the polypeptides according to the present invention are not characterized by glycosylation and / or phosphorylation.

[0103] Generally, the polypeptides according to the present invention are non-natural polypeptides, which are not naturally produced by the subject to which the polypeptide is administered. This is not only related to the advantage of detectability in the subject after administration, but also demonstrates the need to administer to the subject (from an external source, such as a composition prepared according to the present disclosure) to succeed in the treatment or prevention of the disease.

[0104] Preferably, the polypeptide according to the present invention is an isolated polypeptide. More preferably, the polypeptide according to the present invention is substantially free of host cell proteins, degradation products (such as des-nona variants), and proteases (such as trypsin). When the polypeptide according to the present invention is substantially free of host cell proteins, degradation products (such as des-nona variants), and proteases (such as trypsin), it may also be referred to as a "pure polypeptide". Preferably, the polypeptide according to the present invention is administered as a pure polypeptide. More preferably, the weight percentage of the pure polypeptide composed of SEQ ID NO: 3 and / or the pure polypeptide composed of SEQ ID NO: 4 is 90% or more, preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, more preferably 99.2% or more, more preferably 99.4% or more, more preferably 99.6% or more, more preferably 99.8% or more, more preferably 99.9% or more, relative to the total protein in the composition. Such pure polypeptides are obtainable based on the disclosure herein, including Examples 1 and 2. Most preferably, the pure polypeptide according to the present invention has a purity grade compatible with Good Manufacturing Practice (GMP).

[0105] As demonstrated in the experiments herein, particularly in Example 4, the administration dose of the agent according to the present invention did not induce any hyperalgesia syndrome (pain) in a separate experiment. The absence of pain is particularly remarkable because the agent was topically and repeatedly administered to the damaged eye, also in a chronic setting (see Examples for details).

[0106] Optionally, according to the present invention, an effective amount of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is administered to a subject in need thereof. Details of administration, effective amount, and subject in need are described below.

[0107] The polypeptides composed of SEQ ID NO: 3 and SEQ ID NO: 4 differ from the amino acid sequence of human nerve growth factor (NGF, also referred to as wild-type human NGF or wild-type NGF, see SEQ ID NO: 2) at one or two positions. The differences between the polypeptides of the present invention and the SEQ ID NO: 2 polypeptide have a significant effect on the treatment or prevention of ocular disorders and have no side effects, as detailed herein and supported by the experimental examples herein.

[0108] Nerve growth factor (NGF) is a neurotrophic factor required for the development and survival of specific neuronal populations. NGF is a homodimeric peptide that can naturally trigger neuronal proliferation and homeostasis. In vivo, NGF binds to at least two types of receptors: tropomyosin receptor kinase A (TrkA) and the low-affinity NGF neurotrophin receptor p75 (LNGFR / p75NTR / p75). Both are associated with certain diseases in humans and animals, although their respective mechanisms of action may differ. Several therapeutic applications of NGF have been proposed, but few have reached the mature market.

[0109] However, many of the therapeutic uses of NGF envisioned in the past have not matured into marketed therapeutic NGF products. One reason can be seen that in addition to its expected effects on neuronal proliferation and homeostasis, NGF is also associated with pain: when administered locally or systemically, it causes hyperalgesia (Lewin et al., 1994, Eur. J. Neurosci., vol. 6, pp. 1903 - 1912; Della Seta et al., 1994, Pharmacol. Biochem. Behav., vol. 49, p. 701; Dyck et al., 1997, Neurology, vol. 48, pp. 501 - 505; McArthur, et al., 2000, Neurology, vol. 54, pp. 1080 - 1088; Svensson et al., 2003, Pain, vol. 104, pp. 241 - 247; Ruiz et al., 2004, Brain Res., vol. 1011, pp. 1 - 6). As a solution, mutant versions of NGF ("mutant proteins") have been developed that are associated with reduced nociceptive activity ("painless NGF"), characterized by at least one mutation in the NGF domain that interacts with the TrkA receptor (WO 2008 / 006893 A1, Malerba et al. PLOS One, 2015, vol, e0136425). However, to date, such polypeptides of pharmaceutically acceptable purity have not been available to the public, and perhaps also taking into account the bias and general negative experience in the research of growth factors in this therapeutic area, no therapeutic or prophylactic ophthalmic conditions for the eye have been proposed or developed.

[0110] It is known that (wild-type) NGF affects target cells by binding to two independent receptors, (a) the receptor tyrosine kinase A (TrkA), leading to neuronal survival, and (b) the p75 neurotrophin receptor, which is involved in regulating cell death (Mesentier-Louro et al., 2017, Int. J. Mol. Sci., vol. 18(98)). Therefore, polypeptide sequence elements with wild-type NGF may also have the effect of exacerbating retinal degeneration by stimulating apoptosis after optic nerve crush, see Mesentier-Louro et al., 2018, Mol. Neurobiol., vol. 56, pp. 1056-1069. It is also known that the residue R100 of wild-type human NGF is involved in the binding of NGF to p75, and mutations in this residue affect p75 binding, see for example WO 2008 / 006893A1. Compared with wild-type NGF, hNGF, the polypeptides of the present invention have a lower binding affinity for p75 (see for example Malerba et al., 2015, PlosOne, 10(9): e0136425). Human NGF P61SR100E corresponds to the polypeptide of SEQ ID NO: 4. Among several mutants of human NGF (hNGF mutants), the mutant human NGF P61SR100E is considered to be the most promising, and it is mentioned that this mutant is particularly suitable for ophthalmic diseases. However, those references do not teach the specific therapeutic and / or prophylactic uses according to the present invention, and those references also fail to obtain the corresponding polypeptides in a sufficiently high purity to enable their use in humans for medical purposes. Therefore, the polypeptides used according to the present invention, as described and provided herein, offer several unexpected advantages compared to wild-type human NGF. In particular, the experimental findings of the present invention (see Examples) cannot be explained solely by the fact that the agent according to the present invention is "painless" because its ability to induce pain has never been experimentally studied in the innervated area of the eye (i.e., the area characterized by exposed nociceptors). In addition, even if the administration of the agent according to the present invention causes neural potentiation (see for example Example 3), the administration is not related to pain.

[0111] According to the present invention, the stability and thus the long-term purity of the polypeptide of SEQ ID NO: 3 or its SEQ ID NO: 4 can be obtained and / or improved by the aspects and embodiments described herein. Therefore, the present disclosure not only provides a new treatment or prophylaxis for ophthalmic disorders, but also provides an agent suitable for such treatment or prophylaxis, the purity grade of which is suitable for therapeutic applications, including administration to mammals. The agent of the present invention has not been available to the public in such a favorable purity grade before.

[0112] The polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 does not exist in nature and can also be referred to as a non-natural polypeptide. Therefore, the agent according to the present invention is not wild-type NGF, especially not wild-type human NGF.

[0113] Preferably, the non-natural polypeptide according to the present invention is provided in high purity. Optionally, the polypeptide contains internal disulfide bonds. Optionally, the polypeptide is properly folded. Optionally, the polypeptide is soluble in an aqueous medium.

[0114] The present invention is partly based on experiments on animal models of optic nerve injury or disorders (see Example 4). Compared with animals treated with placebo, the polypeptide induced significant and dose-dependent improvement in the healing time of disorders involving optic nerve injury and / or disorders. This improvement was evident at doses without pain-related side effects, thus demonstrating potential benefits over the prior art.

[0115] In particular, data generated in in vivo models of disorders involving optic nerve injury and / or disorders have demonstrated that the polypeptide of the present invention is painless but still retains the activity of targeting the NGF receptor system and thereby provides a therapeutic means for treating or preventing ophthalmic disorders. In fact, the polypeptide of the present invention retains the trophic properties of wild-type NGF for angiogenesis and nerve reinnervation, which is beneficial for the healing of the optic nerve without exerting the nociceptive pre-effect of wild-type NGF at the application site and the systemic level.

[0116] The present invention provides a polypeptide for treating and / or preventing an ophthalmic disorder in a mammalian subject, wherein the polypeptide is selected from the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4. Therefore, the present invention also provides the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for use in a method of treating the human or animal body by therapy as described herein.

[0117] More specifically, the present invention relates to the specific therapeutic use of the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4, wherein the specific therapeutic use is treating and / or preventing an ophthalmic disorder in a mammalian subject. Therefore, the present invention also provides the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 for use in a method of treating the human or animal body by therapy, wherein the therapy comprises treating and / or preventing an ophthalmic disorder in a mammalian subject. A mammalian subject is generally a subject characterized by the need for such treatment.

[0118] The polypeptides of SEQ ID NO: 3 and the polypeptides of SEQ ID NO: 4 are characterized by mutations in the amino acid sequence of human NGF (hNGF, SEQ ID NO: 2), wherein said mutations are associated with a decrease in nociceptive activity. In particular, the arginine at position 100 of hNGF is replaced by glutamate. The present invention is based in part on the surprising finding that therapeutic effects can be achieved without the side effects known in the prior art.

[0119] Without wishing to be bound by a particular theory, preferably the polypeptides according to the invention comprise one or more disulfide bonds, most preferably three disulfide bonds. Mature and correctly folded human NGF is characterized by three disulfide bonds (linkage positions Position numbering refers to SEQ ID NO: 1; see Wiesmann et al., 1999, Nature, vol. 401, pages 184-188). Without wishing to be bound by a particular theory, preferably, the polypeptides according to the invention comprise equivalent disulfide bonds (the position numbering of which can be obtained by a person skilled in the art by aligning the polypeptides according to the invention with the polypeptides of SEQ ID NO: 1 and Wiesmann et al.

[0120] Description of adverse reactions

[0121] Preferably, the treatment and / or prevention does not cause side effects or adverse effects in the subject to which the polypeptide has been administered or is being administered. Thus preferably, the administration of the polypeptides of the present invention does not result in any adverse effects in mammalian subjects.

[0122] One side effect or adverse effect that is preferably absent in this case is hyperalgesia or pain. Particularly preferably, the treatment and / or prevention according to the invention does not cause hyperalgesia in mammalian subjects. Thus, preferably, the administration of the medicament according to the invention does not induce any hyperalgesia syndrome (pain).

[0123] It is important to note that compared to the administration of a reference compound associated with pain (such as wild-type NGF), the absence of pain not only results in a more pleasant (or less unpleasant) treatment, but is at least part of the reason for the successful treatment or prevention of ocular disorders: considering that the polypeptides according to the invention are preferably administered locally, more preferably locally to the eye, the absence of pain will enable the treated subject to receive the administration of the polypeptide without adverse reactions, such as scraping or washing it off or otherwise removing it to avoid pain, and thus, the polypeptide will exert a therapeutically beneficial effect, such as treating or preventing ocular disorders. Thus, the absence of pain associated with the polypeptides of the present invention will be suitable for overcoming the reluctance of consumers and the concerns of regulatory authorities. In other words, compared to drugs associated with pain, the absence of pain is associated with a significant increase in the benefit-risk ratio.

[0124] In particular, preferably, the treatment and / or prevention do not cause hyperalgesia in a mammalian subject. In one embodiment, the subject to which the polypeptide of the invention is administered does not suffer from mechanical allodynia. More precisely, mechanical allodynia is not induced in the subject to which the polypeptide of the invention is administered, and thus the subject to which the polypeptide is administered does not suffer from mechanical allodynia.

[0125] Another side effect or adverse effect that is preferably absent in this case is malignancy or cancer. In particular, the administration of the polypeptide of the invention to a subject is preferably not associated with abnormal cell growth, and even more preferably not associated with abnormal cell growth that may invade or spread to other parts of the body. Particularly preferably, the administration of the polypeptide of the invention to a subject is not associated with eye cancer.

[0126] Thus, in summary, preferably, the administration of the polypeptide of the invention to a subject is not associated with side effects such as malignancy and / or pain.

[0127] Generally, the administration of the medicament according to the invention is well tolerated by the subject. In particular, preferably, the administration of the polypeptide according to the invention is not associated with the formation of anti-drug antibodies in the subject. Indeed, since the amino acid sequence of the polypeptide according to the invention differs from wild-type human NGF only at one or two amino acid positions, it seems reasonable that immunotolerance in humans is particularly advantageous, and it seems reasonable that the administration of the polypeptide of the invention is not associated with the formation of anti-drug antibodies in the human body.

[0128] Preferably, the administration according to the invention has a positive effect on one or more of the following: inflammation, extracellular matrix deposition, innervation, and angiogenesis.

[0129] Detectability of the polypeptide

[0130] Preferably, the polypeptide used according to the invention can be selectively recognized by a specific reagent for endogenous (e.g., human) NGF. The terms "selectively recognized" and "detectable" are used interchangeably herein and generally refer to the specific identification of a protein in a biological sample, preferably by molecular means.

[0131] In this regard, the polypeptide according to the invention is preferably detectable by an antibody or other immunoreactive molecule.

[0132] A protein that can be detected by an antibody or other immunoreactive molecule can also be referred to as an antigen. In some embodiments, a biological sample can be characterized by the presence or absence of one or more specific antigens. In the context of the present invention, a polypeptide administered to a subject is preferably detectable in a biological sample obtained from the subject after polypeptide administration. A non-limiting way of showing the presence of a protein is by Western blotting, but other immunological methods are equally included in the context of the present invention. The antibody or other immunoreactive molecule itself is labeled (e.g., with a fluorophore), or is recognized by a labeled secondary antibody or other immunoreactive molecule added for this purpose. Thus, in some cases, a secondary molecule that aids in detection is also added, such as an optionally labeled secondary antibody to facilitate detection.

[0133] According to the present invention, an antigen is said to be present in a biological sample if the antigen level is above the detection limit and / or if the level is high enough to allow binding of an antigen-specific antibody added to the sample. According to the present invention, an antigen is said not to be expressed on a cell if the expression level is below the detection limit and / or if the expression level is too low to allow binding of an antigen-specific antibody added to the sample.

[0134] An antibody or other immunoreactive molecule can recognize an epitope on a cell. The term "epitope" refers to an antigenic determinant in a molecule such as an antigen, i.e., the part or fragment of the molecule that is recognized (i.e., bound) by the immune system, such as by an antibody or other immunoreactive molecule. Detection of an epitope specific for any particular antigen can generally lead to the conclusion that the particular antigen is present on the cell being analyzed.

[0135] In one embodiment, a sample obtained from a subject, particularly a subject that has been administered a polypeptide according to the present invention, can be characterized by immunophenotyping. "Immunophenotyping" generally refers to the characterization of a cell or sample by antigen-specific molecules (such as antibodies or other immunoreactive molecules) that are added to the sample to determine the presence of an antigen. Immunophenotyping includes methods of cell sorting using various techniques (including flow cytometry), as well as methods of analysis of lysed cells and lysed samples, such as Western blotting.

[0136] In the present invention, polypeptides that can be specifically detected even in the presence of wild-type NGF, such as wild-type human NGF, are particularly preferred. Although any mutation in the amino acid sequence, such as any point mutation, can render the polypeptide specifically detectable even in the presence of the corresponding non-mutated wild-type polypeptide, thus each of the polypeptides of SEQ ID NO:3 and SEQ ID NO:4 can be initially specifically detected even in the presence of wild-type human NGF, especially the polypeptide of SEQ ID NO:4, which can be distinguished from wild-type human NGF by an antibody specific for said polypeptide (WO 2008 / 006893 A1).

[0137] Thus, preferably, the polypeptide is characterized by the absence of at least proline at position 61 (which is present at position 61 of SEQ ID NO: 2 for reference), and more preferably, by the substitution of proline at position 61 with another amino acid. In a particularly preferred embodiment, the proline at position 61 is substituted with serine. In this preferred embodiment, the polypeptide used according to the present invention is the polypeptide of SEQ ID NO: 4. This polypeptide is characterized by the absence of at least proline at position 61, and more preferably, the proline at position 61 is substituted with another amino acid. In SEQ ID NO: 4, the proline at position 61 of SEQ ID NO: 3 is substituted with serine.

[0138] Ophthalmic disorders

[0139] According to the present invention, the polypeptides according to the present invention are used for the treatment and / or prevention of ophthalmic disorders. For such uses and all of their embodiments, the polypeptide of SEQ ID NO: 4 is particularly preferred.

[0140] The ophthalmic disorders can be genetic and / or acquired ophthalmic disorders.

[0141] In some embodiments, the ophthalmic disorder is or includes an eye injury. In one embodiment, the eye injury includes an injury and / or disorder of the optic nerve and / or retina.

[0142] Although terms such as "ophthalmic disorder", "injury", "disorder", "eye", "optic nerve", "retina" are used herein in the singular form, the present invention also applies to subjects suffering from multiple ophthalmic disorders, injuries, disorders, and is applicable to administration and treatment of all (both) eyes, all (both) optic nerves, and all (both) retinas of a subject.

[0143] The preferred disorders to be treated or prevented according to the present invention are diseases involving the visual pathway. Generally, the visual pathway includes the retina, optic nerve, optic chiasm, optic radiation, and occipital cortex. Thus, the preferred disorders to be treated or prevented according to the present invention are disorders affecting any one or more of the retina, optic nerve, optic chiasm, optic radiation, and occipital cortex. An injury along the visual pathway can result in various visual field defects. Such visual field defects can be treated or prevented according to the present invention. Thus, the present invention also contemplates the treatment and / or prevention of visual field defects, including partial and complete vision loss.

[0144] Ophthalmic conditions include, but are not limited to, eye fatigue, red eyes, night blindness, lazy eye, crossed eyes (strabismus), nystagmus, color blindness, uveitis, presbyopia, blurred vision, eye pain, light sensitivity, floaters, dry eyes, excessive tearing, cataracts, glaucoma, retinal diseases, conjunctivitis, corneal diseases, eye pain, eyelid problems, changes in vision, vision loss, problems associated with contact lenses, diseases associated with optic nerve damage and / or disease, and diseases associated with retinal ganglion cell damage and / or disease. The polypeptides according to the present invention can be administered to a subject suffering from any of these conditions to treat the corresponding condition. Alternatively or additionally, the polypeptides according to the present invention can be administered to a subject at risk of suffering from any of these conditions to prevent the corresponding condition. The present invention is also applicable to a list of diseases including, but not limited to, glaucoma, neurotrophic keratitis, optic neuritis, optic atrophy, drusen of the optic nerve head, and optic pathway glioma. The optic neuropathies to which the present invention is applicable include, but are not limited to, the following: glaucoma, optic pathway glioma, anterior ischemic optic neuropathy, traumatic optic neuropathy, post-hydrocephalus optic atrophy, trans-synaptic degeneration of optic nerve fibers, compression of the pre-chiasmatic pathway, autosomal dominant hereditary optic atrophy, Leber hereditary optic neuropathy, Wolfram syndrome optic neuropathy.

[0145] The present invention also includes the treatment and prevention of one or more conditions from the following list, including but not limited to: autosomal dominant hereditary optic atrophy, Leber hereditary optic neuropathy, anterior ischemic optic neuropathy, traumatic optic neuropathy, optic pathway glioma, neurotrophic keratitis, corneal ulcer, glaucoma, retinitis pigmentosa, macular degeneration, diabetic retinopathy, retinal ganglion cell dysfunction.

[0146] Particularly preferred is the use of the polypeptides according to the present invention for the treatment and / or prevention of ophthalmic conditions involving the optic nerve. In some embodiments, the optic nerve is damaged or affected. As shown, for example, in Example 4, when administered in one embodiment of the present invention, the polypeptides of the present invention have a direct therapeutic effect on such ophthalmic conditions. Thus, preferably, the ophthalmic conditions treated and / or prevented by using the polypeptides according to the present invention involve damage and / or disease of the optic nerve. Subjects having such optic nerve damage and / or disease are described herein, and the present invention and the following description of specific damage and / or disease involving the optic nerve apply to all such subjects, unless the context otherwise indicates. Conditions involving optic nerve damage and / or disease include, but are not limited to, glaucoma, neurotrophic keratitis, optic neuritis, optic atrophy, drusen of the optic nerve head, and optic pathway glioma. The present invention includes the treatment and / or prevention of all of these conditions and other conditions involving the optic nerve. In such embodiments, a medicament according to the present invention is provided for the treatment or prevention of a condition involving optic nerve damage and / or disease.

[0147] Particularly preferred is the use of the polypeptide according to the invention for the treatment and / or prevention of ophthalmic disorders involving the retina. In some embodiments, the retina is damaged or affected. The retina is the light-sensitive tissue layer in the innermost layer of the mammalian eye. The eye creates a focused two-dimensional image of the visual world optically on the retina, which converts the image into nerve impulses in the brain to produce visual perception. The neural retina consists of several layers of neurons interconnected by synapses and is supported by an outer layer of pigment epithelial cells (photoreceptor cells). Preferably, the ophthalmic disorder is characterized by a disorder of the retina. For example, the disorder may be caused by a disorder of the neural retina and / or the retinal epithelial cells. Preferred retinal diseases include macular degeneration (age-related or not), retinopathy (diabetic or not), retinitis pigmentosa, retinoblastoma, retinitis pigmentosa (CORD), and retinal detachment (retinal separation). Retinitis pigmentosa is usually hereditary and causes loss of night vision and peripheral vision. Macular degeneration is characterized by loss of central vision due to cell death or damage in the macula. In retinal detachment, the retina detaches from the back of the eyeball. Hypertensive retinopathy and diabetic retinopathy are both characterized by damage to the tiny blood vessels supplying the retina and are caused by diabetes in some embodiments. Retinoblastoma is a cancer of the retina. In some embodiments, the optic nerve is physically damaged or affected, such as optic nerve crush (ONC, Example 4) and optic pathway glioma. The present invention encompasses the treatment and / or prevention of all these and other retinal diseases. In such embodiments, a medicament according to the invention is provided for the treatment and prevention of disorders involving retinal damage and / or disorders.

[0148] In some embodiments, the polypeptide according to the invention is administered to a subject suffering from neuropathy. In a preferred embodiment, a medicament according to the invention is used to administer to a subject suffering from neuropathy, such as in particular neuropathy of the optic nerve. Such a subject can be a diabetic or non-diabetic subject. The neuropathy may be local or systemic. In some embodiments, the administration according to the invention can reduce the neuropathy of the subject. The reduction of neuropathy can be local and / or systemic. In one embodiment, reducing neuropathy includes reducing neuropathy in the eye to which the polypeptide of the invention is administered. Optic nerve neuropathies to which the present invention is applicable include, but are not limited to, the following: glaucoma, optic pathway glioma, anterior ischemic optic neuropathy, traumatic optic neuropathy, post-hydrocephalic optic atrophy, trans-synaptic degeneration of optic nerve fibers, compression of the pre-chiasmatic pathway, autosomal dominant hereditary optic atrophy, Leber hereditary optic neuropathy, Wolfram syndrome optic neuropathy. The present invention specifically contemplates the administration to children, particularly but not limited to the following cases: post-hydrocephalic optic pathway glioma and optic atrophy.

[0149] Particularly preferred is the use of the polypeptides according to the invention for the treatment and / or prevention of ophthalmic disorders involving retinal ganglion cells. In some embodiments, the retinal ganglion cells are damaged or affected. Such diseases have been described, for example, by Garcia et al., 2016, Cytokin Groth Fact. Rev., Vol. 34, pp. 1359 - 1601 and Levin et al., 2002, Progr. Retin. Eye Res., Vol. 21, pp. 465 - 484. Generally speaking, retinal ganglion cells (RGCs) are neurons located near the inner surface (ganglion cell layer) of the retina of the eye. However, all retinal ganglion cells also have a long axon that extends into the brain. These axons form the optic nerve, the optic chiasm, and the optic tract. Thus, many diseases involving the optic nerve are also diseases involving retinal ganglion cells, and vice versa. These terms are not mutually exclusive. Similarly, many diseases involving the retina are also diseases involving retinal ganglion cells, and vice versa. These terms are not mutually exclusive. In any case, in a preferred embodiment of the invention, the ophthalmic disorder to be treated and / or prevented is preferably characterized as a retinal ganglion cell disorder. Disorders involving retinal ganglion cell damage and / or disorders include, but are not limited to, glaucoma, optic pathway glioma, anterior ischemic optic neuropathy, traumatic optic neuropathy, post - hydrocephalus optic atrophy, trans - synaptic degeneration of optic nerve fibers, compression of the pre - chiasmatic pathway, autosomal dominant hereditary optic atrophy, Leber hereditary optic neuropathy, Wolfram syndrome optic neuropathy. The present invention specifically contemplates administration to children, particularly but not limited to the following cases: post - hydrocephalus optic pathway glioma and optic atrophy. In some embodiments, the RCGs are physically damaged or affected, such as optic nerve crush (ONC, Example 4) and optic pathway glioma. The present invention encompasses the treatment and / or prevention of all these and other disorders involving RGC damage and / or disorders. In such embodiments, a medicament according to the invention is provided for the treatment and prevention of disorders involving RGC damage and / or disorders.

[0150] The most preferred ophthalmic diseases to be treated and / or prevented according to the invention are selected from glaucoma, optic pathway glioma, anterior ischemic optic neuropathy, traumatic optic neuropathy, post - hydrocephalus optic atrophy, trans - synaptic degeneration of optic nerve fibers, compression of the pre - chiasmatic pathway, autosomal dominant hereditary optic atrophy, Leber hereditary optic neuropathy, Wolfram syndrome optic neuropathy. The present invention specifically contemplates administration to children, particularly but not limited to the following cases: post - hydrocephalus optic pathway glioma and optic atrophy.

[0151] Particularly suitable subjects for the medicament of the invention

[0152] According to the present invention, a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 can be administered to a subject in need thereof. A subject in need thereof can be a subject suffering from a disorder described herein, a subject at risk of developing such a disorder, or a subject otherwise afflicted with such a disorder. The agent is administered to the subject in a therapeutically effective amount. The therapeutically effective amount can be determined by a physician based on the disclosure herein.

[0153] Specifically, the polypeptide according to the present invention is administered to a mammalian subject. The subject can also be referred to as a "patient". Most preferably, the mammalian subject is a human.

[0154] The subject can be an adult subject or a non - adult subject, such as a child or an adolescent. Administration to children is expressly contemplated in the present invention.

[0155] The present invention also relates to a method of treating a patient suffering from an ophthalmic disorder, wherein the method comprises administering to the patient an effective amount of a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. The terms "patient" and "subject" are used interchangeably herein, particularly referring to a patient / subject characterized by an ophthalmic disorder described herein.

[0156] Preferably, the ophthalmic disorder is characterized by damage and / or disorder of the optic nerve and / or retina and / or retinal ganglion cells of the subject. Such damage and / or disorder includes dysfunction and reduced function of retinal ganglion cells. Diseases characterized by damage and / or disorder of the optic nerve and / or retina and / or retinal ganglion cells have been described above, and the following description of the subject applies to all such damage and / or disorder of such subjects, unless the context otherwise requires.

[0157] In the context of the present invention, the term "prevention" should be understood broadly and includes not only preventing the onset of a disorder, but also preventing the progression of a disorder. In particular, in the context of disorders involving optic nerve damage and / or disorder, the term "prevention" also includes preventing further progression of optic nerve damage.

[0158] In the context of the present invention, the term "treatment" should be understood broadly and includes, but is not limited to, improvement of the symptoms of a disorder. In fact, it is preferred and also demonstrated by the experimental examples herein that achieving improvement of an ophthalmic disorder, such as (partial) restoration of vision or other improvement or amelioration of the condition or disorder, is a preferred component of the present invention as claimed herein. In fact, obtaining the claimed therapeutic effect is a functional technical feature of the present invention. The examples herein justify that the claimed functional technical feature can be achieved as a direct result of administering the polypeptide of the present invention. In other words, the inventors have determined that the polypeptide of the present invention is the cause of the improvement in a subject suffering from an ophthalmic disorder. The ophthalmic disorder is preferably characterized by damage and / or disorder of the optic nerve.

[0159] The present invention is particularly applicable to a subgroup of subjects suffering from ophthalmic disorders. Such subgroups are described herein. Specific subjects may also belong to one or more of the subgroups described herein; the administration of a polypeptide according to the present invention to a subject falling into one of the subgroups described herein and the administration of a polypeptide according to the present invention to a subject falling into more than one of the subgroups described herein are equally encompassed by the present invention.

[0160] The present invention is not limited to a specific cause of optic nerve injury and / or disorder. Mechanical causes as well as non-mechanical causes of optic nerve injury and / or disorder are included in the present invention. Moreover, diabetic causes as well as non-diabetic causes of optic nerve injury and / or disorder are included in the present invention.

[0161] In some embodiments, the polypeptide according to the present invention is optionally used for administration to a subject who has undergone surgery. Thus, the polypeptide according to the present invention is suitable for treating or preventing one or more postoperative complications in the eye. Alternatively, the polypeptide according to the present invention is also suitable for preventing surgery in a subject. For example, in optic pathway glioma, the optic nerve can be strengthened by administering the polypeptide of the present invention, which can render surgery unnecessary or optional. Thus, the present invention also provides a non-invasive administration.

[0162] The present invention is applicable to treating ophthalmic disorders in diabetic and non-diabetic subjects, such as in particular injury and / or disorder of the optic nerve. Further details of optic nerve injury and / or disorder are described above.

[0163] Diabetic patients may develop ophthalmic disorders. Such ophthalmic disorders can be treated and / or prevented based on the present invention.

[0164] In some embodiments, the mammal to which the polypeptide of the present invention is administered is preferably a human being suffering from diabetes or having a tendency to develop diabetes; the corresponding subject is referred to herein as a "diabetic subject".

[0165] Diabetes is a common and debilitating disease that affects multiple organs. Methods for detecting diabetes are well known in the art. In one embodiment, methods for detecting diabetes are not part of the present invention, but they can facilitate the treatment or prevention of ophthalmic disorders according to the present invention.

[0166] In typical embodiments, diabetes is selected from type 1 diabetes and type 2 diabetes.

[0167] Optionally, the ophthalmic disorder includes a disorder caused by diabetes or otherwise involving diabetes. Thus, the present invention is applicable to subjects with diabetes and subjects without diabetes. In some embodiments, the agent according to the present invention is for administration to a diabetic patient. Thus, the polypeptide of the present invention can be administered to the eyes of a diabetic subject. Thus, in one embodiment, the use of the polypeptide according to the present invention includes administration to the eyes of a diabetic subject.

[0168] Thus, the present invention provides treatment and / or prevention of ophthalmic disorders in subjects affected by diabetes. In fact, according to the present invention, effective medical treatment can not only help patients recover from these ocular complications, but also enable them to achieve a better quality of life and reduce medical care and / or costs.

[0169] Thus, the present invention provides advantages over current treatment methods, which generally do not provide an effective way to treat ophthalmic disorders. The present invention provides treatment and / or prevention of such ophthalmic disorders.

[0170] Ocular administration

[0171] Treatment or prevention according to the present invention can be carried out by administering, preferably topically, the polypeptide of the present invention. According to the present invention, the eyes of the subject are the preferred site of administration of the polypeptide of the present invention. Generally, when it is mentioned herein that a polypeptide is administered to the eyes of a subject, such administration can be on the surface of the eye or within the eye, unless the context otherwise indicates.

[0172] Preferably, the polypeptide is for administration to the eyes. More preferably, the administration is selected from topical administration to the eyes and intravitreal administration, with topical administration to the eyes being most preferred.

[0173] In a preferred embodiment, the agent according to the present invention is for the treatment or prevention of disorders involving optic nerve injury and / or disorders, and for this purpose, it is administered to the eyes. According to the present invention, the polypeptide is suitable for the treatment or prevention of injury and / or disorders of the optic nerve, and for this purpose, it is administered to the eyes.

[0174] The present invention is not limited to subjects with ophthalmic disorders in only one eye, nor to subjects with ophthalmic disorders in both eyes. Thus, the terms "eye" and "optic nerve", independent of whether they are used in the singular or plural form in the present disclosure, clearly include all those singular and plural embodiments.

[0175] In some embodiments, the administration is carried out in a hospital. In some embodiments, the treatment is not carried out in a hospital. For example, administration of eye drops generally does not require the subject to be hospitalized.

[0176] Optional but not mutually exclusive ophthalmic diseases include at least one mechanical injury. Accordingly, the present invention also encompasses the treatment or prevention of mechanical injury, where treatment of such injury is actually more meaningful than prevention. Such conditions include those involving optic nerve crush (ONC) and other conditions affecting the optic nerve.

[0177] In summary, as detailed herein, according to the present invention, a polypeptide is administered to the eye of a subject having or at risk of having an eye disease.

[0178] In another embodiment, the medicament according to the present invention is used for the treatment or prevention of ocular cancer, such as but not limited to optic pathway glioma, and / or ocular conditions associated with such cancer.

[0179] In a further embodiment, the medicament according to the present invention is used for the treatment or prevention of ophthalmic conditions caused by or affected by a genetic disorder of the subject.

[0180] Route of administration

[0181] The present invention provides a heterologous polypeptide for administration to a subject.

[0182] Preferably, the polypeptide is for topical administration. Accordingly, preferably, the polypeptide of the present invention is administered to the eye. In some embodiments, the polypeptide is administered intravitreally. In some embodiments, the polypeptide is topically administered to the eye.

[0183] More preferably, the polypeptide is administered to the surface of the eye. In other words, the polypeptide according to the present invention is preferably topically administered, more preferably topically applied to the eye. Most preferably, the polypeptide is applied to the conjunctiva of the subject. Administration to the conjunctiva of the subject is also referred to as "conjunctival" administration. Conjunctival administration is most preferably carried out by eye drops.

[0184] Administration according to the present invention generally does not involve surgery on the subject. In one embodiment, the administration of the polypeptide of the present invention does not include or encompass invasive steps representing a substantial physical intervention on the body, which requires professional medical expertise and which, even when carried out with the required professional care and expertise, poses significant health risks. Instead, in more typical embodiments, the administration of the polypeptide of the present invention, particularly topical administration, is generally considered safe for the subject, such that the polypeptide can be self-administered by the subject, particularly in the case of human subjects.

[0185] Optionally, the eye is covered with a plaster and / or an eye dressing before and / or during and / or after administration. The various types of plasters and / or eye dressings available are not limited by the present invention. Thus, any plaster and / or eye dressing can be used unless it is clearly technically inappropriate. A plaster and / or eye dressing suitable for covering the eye is preferred. In some embodiments, the polypeptide of the present invention is administered concomitantly with the administration of a plaster or an eye dressing; optionally, the plaster or eye dressing contains the polypeptide of the present invention, optionally in the form of an aqueous medium, and is administered to the eye dressing before administration.

[0186] In an alternative and more preferred embodiment, the polypeptide is administered repeatedly. In a particularly preferred embodiment, the polypeptide is repeatedly administered at least one to five times per day. In one embodiment, the polypeptide is administered once a day. In one embodiment, the polypeptide is administered twice a day. In one embodiment, the polypeptide is administered three times a day (see also Example 4). In one embodiment, the polypeptide is administered four times a day. In one embodiment, the polypeptide is administered five times a day. It is particularly preferred to administer the polypeptide twice a day to a human subject. As disclosed herein, all of the above administrations are preferably repeated over the course of several days. For example, the polypeptide can be repeatedly administered for three to 30 days, preferably seven to 14 days, preferably one to five times per day for these days.

[0187] Preferably, when the agent according to the present invention is administered to the eye as described herein, it does not cause a hyperalgesia syndrome (pain). Thus, the agent according to the present invention can contact nociceptive fibers (nerves) including the optic nerve without causing a hyperalgesia syndrome (pain). For this and other reasons, the present invention provides major advantages, particularly for the treatment and / or prevention of diseases involving the optic nerve.

[0188] Administration time

[0189] Generally, the polypeptide according to the present invention is administered repeatedly or once.

[0190] In one embodiment, the polypeptide is administered in a single administration. In this embodiment, the polypeptide is administered in a single administration and the administration is stopped after this single administration.

[0191] In an alternative embodiment, the polypeptide is repeatedly administered for three to 30 days, preferably seven to 14 days. Optionally, the administration is stopped after the interval is completed.

[0192] In one embodiment, the polypeptide is repeatedly administered. In one embodiment, the polypeptide is repeatedly administered before, for example, a complete cure of an ophthalmic condition is observed or at least a symptom improvement of the condition is observed. Alternatively, the polypeptide is repeatedly administered for three to 30 days, preferably seven to 14 days. Optionally, administration is stopped after the interval is completed. In a particularly preferred embodiment, the polypeptide is repeatedly administered at least three times a day.

[0193] Preferably, the polypeptide is administered following optic nerve injury.

[0194] Preferably, the polypeptide is administered as soon as possible after the diagnosis of optic nerve damage to minimize the extent of RGC death. "As soon as possible" includes embodiments in which one day or less after the diagnosis of optic nerve damage. However, if administered a few days after the optic nerve damage occurs, the polypeptide still has a neuroprotective effect. Preferably, the polypeptide is administered at least three days after the induction of optic nerve damage. Preferably, the polypeptide is administered at least four days after the induction of optic nerve damage. Preferably, the polypeptide is administered at least five days after the induction of optic nerve damage. Preferably, the polypeptide is administered at least six days after the induction of optic nerve damage. Preferably, the polypeptide is administered at least seven days after the induction of optic nerve damage. Preferably, the polypeptide is administered at least eight days after the induction of optic nerve damage. Preferably, the polypeptide is administered at least nine days after the induction of optic nerve damage. Preferably, the polypeptide is administered at least ten days after the induction of optic nerve damage. Most preferably, however, the polypeptide is administered at least four days after the induction of optic nerve damage. In all of these aforementioned embodiments, "at least (several) days after the induction of damage" is intended to mean that, in the case of repeated administration, the first dose is administered after a specified number of days. Optionally, further doses may be administered later, on the same day, and / or on subsequent days in accordance with the disclosure herein.

[0195] dose

[0196] The agents and compositions described herein are administered in an effective amount. According to the present invention, an "effective amount" is an amount or dosage that achieves a desired response or desired effect alone or together with other doses. In the case of treating a specific condition, the desired response preferably involves inhibiting the progression of the disease. This includes slowing the progression of the condition and preferably interrupting or reversing the progression of the condition. The desired response to treat a disease or condition may also include delaying the onset of the disease or condition or preventing the onset of the disease or condition. In some embodiments, the desired response includes completely curing the symptoms of the condition locally and / or systemically.

[0197] The effective amount of the agent or composition described herein will depend on the condition or disorder to be treated, the severity of the disorder, the individual parameters of the subject to which the agent is administered, such as age, physiological condition, concomitant conditions (if any), size and weight, the duration of treatment, the type of concomitant treatment (if any), the specific route of administration, and other parameters. Accordingly, the dosage of the agent described herein can depend on various such parameters. In cases where the initial dose is not sufficient for the response of the patient, higher doses (or effectively higher doses achieved by different, more local routes of administration) can be used.

[0198] According to the present invention, a suitable and therapeutically effective dose of a therapeutic agent for administration to a human subject for treating and / or preventing an eye disorder can be determined based on an experimentally determined suitable and therapeutically effective dose of a therapeutic agent for administration to a rodent subject for treating and / or preventing an eye disorder.

[0199] Animal models (Example 4) are useful for establishing a pharmacological response and for assessing the potential toxicity of a therapeutic product. In some embodiments, the dose administered to a subject is the dose disclosed in Example 4 or Example 5 or Example 6.

[0200] Preferably, the dose of the polypeptide is determined at or before the start of treatment. In one embodiment, the dose is adjusted for subsequent administrations based on the progress of the treatment. In an alternative embodiment, the dose is not adjusted for subsequent administrations, and thus subsequent doses correspond to the first dose.

[0201] The polypeptide is active both by local (e.g., conjunctival) and by intravitreal administration. In particular, for local (most preferably conjunctival) administration (preferably eye drops), the preferred dose / dose has an amount of 0.3 to 30 μg of polypeptide per eye, more preferably 1 to 10 μg of polypeptide per eye, and most preferably 5 μg of polypeptide per eye. Most preferably, these indicated doses are specifically for human eye administration.

[0202] Method for obtaining the polypeptide

[0203] In one embodiment, the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 are obtained from a biological source. Optionally in one embodiment, the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 are obtained from recombinant expression. For this purpose, the open reading frames encoding the respective polypeptides are introduced into a recombinant protein source, such as a host cell or a cell-free system for protein expression. In fact, considering that human NGF is only produced in trace amounts in vivo, murine NGF is usually produced as a heterogeneous mixture of various proteins (see WO 2000 / 022119A1), and according to the equivalent suggestions for wild-type NGF in the prior art, the polypeptides of the present invention are non-natural and thus are not produced in vivo at all. The most meaningful possibility of producing the polypeptides of the present invention is through recombinant expression (WO 2000 / 022119 A1, WO 2008 / 006893 A1; Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, pp. 3296-3303, US2018 / 0086805 A1). However, obtaining such polypeptides at a purity level sufficient for administration to mammals has always been a challenge. As detailed herein, the present invention has overcome this challenge (see also Examples 1 and 2).

[0204] Preferably, the polypeptides according to the present invention can be obtained by recombinant expression in bacteria. More preferably, the polypeptides according to the present invention can be obtained by recombinant cytoplasmic expression in bacteria. Generally, bacterial cells, especially Escherichia coli, are capable of recombinantly producing large amounts of recombinant proteins. However, as in the case of many other recombinantly expressed genes, the production of recombinant NGF and similar polypeptides in bacteria results in biologically inactive translation products that then accumulate in the cell (cytosol) in the form of aggregates (so-called inclusion bodies (IB) (WO 2000 / 022119A1; US2018 / 0086805 A1). Compared with NGF, proNGF is known to be quite unstable and requires great effort to refold and purify with a low recovery rate, which makes the process of producing NGF from proNGF in bacteria relatively difficult and expensive. Therefore, the main difficulties associated with NGF produced in bacteria and similar polypeptides produced in bacteria (through their respective pro-forms) involve the folding, processing, and purification of the recombinant proteins. These difficulties have now been solved (see Examples 1 and 2). As a result, the polypeptides of SE ID NO: 3 and SEQ ID NO: 4 become available at a purity level suitable for administration to mammals, including humans.

[0205] Preferably, the polypeptides according to the invention are expressed together with a presequence. Without limitation, a suitable presequence is the presequence of wild-type human NGF (amino acid positions 18 to 121 of SEQ ID NO: 1), which is typically fused to the N-terminus of the polypeptide of SEQ ID NO: 3 or 4. For wild-type NGF, although not part of mature NGF and thus not essential for the biological function of NGF, the presence of the covalently linked presequence has been shown to facilitate the refolding of recombinant NGF from inclusion bodies while the mature portion forms disulfide bonds (β-NGF). Thus, compared to the in vitro refolding of mature NGF from inclusion bodies, the presence of the covalently linked presequence has a positive effect on the yield and rate of refolding (Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, pages 3296 - 3303). Without wishing to be bound by a particular theory, this is reasonable and hypothesized herein for the polypeptides of SEQ ID NO: 3 and 4.

[0206] Thus, when the polypeptides according to the invention have been produced in inclusion bodies, correct folding is required, which is usually achieved post-translationally, as is the cleavage of the covalently linked pre-sequence; elaborate methods for folding, cleavage and purification have been proposed in the past, especially for wild-type human NGF. Notably, most published studies on NGF have applied the general refolding mechanism previously established by Rattenholl et al. (2001, Eur. J. Biochem, vol. 268, pages 3296 - 3303). In this original study, several parameters of protein refolding (such as temperature, refolding time, pH value of the refolding reaction, arginine, glutathione and protein concentration) were studied in detail and their effects on refolding efficiency were evaluated. The protocol of Rattenholl et al. relies on the renaturation of the pro-form, which has very poor solubility and can be obtained from inclusion bodies after recombinant production in prokaryotes, thus dissolving pro-NGF in a denaturing agent solution at denaturing concentration and transferring it to a non-denaturing or weakly denaturing solution, so as to maintain solubility and the dissolved pro-NGF can adopt a bioactive conformation, including forming disulfide bonds in native NGF, and then purifying NGF and removing the pre-sequence by proteolysis (WO 2000 / 022119 A1; Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, pages 3296 - 3303). Notably, in this study, it was found that compared with higher protein concentrations, lower protein concentrations led to a higher specific yield of correctly folded products. Exemplarily, a protein concentration of about 50 mg per liter of the refolding reaction resulted in a specific yield of correctly folded pro-NGF of about 25%, while at a protein concentration of 500 mg / L, this fraction decreased to 10%. Based on this, Rattenholl et al. suggested that the protein concentration in the refolding solution must be very low: according to Rattenholl et al., it was expected that 15 - 20 mg of correctly folded protein could be produced per liter of the refolding reaction as the yield. However, this would require scale-up (e.g., beyond laboratory scale) to purify even a few hundred milligrams of recombinant protein.

[0207] While human pro-NGF contains a natural cleavage site for the protease furin (Arg 1 -Ser 2 -Lys 3 -Arg 4 ; R 1 S 2 K 3 R 4) and furin cleaves this site of proNGF in vivo, but furin cannot be obtained in a commercially relevant purity or quantity. According to the present invention, when expressed together with a pro-sequence, for example in Escherichia coli, the polypeptide of the present invention is preferably cleaved by the commercially available protease trypsin (EC 3.4.21.4). In fact, it has been reported that for wild-type NGF, trypsin produces a satisfactory bioactive mature NGF, which can ultimately be purified (Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, pages 3296 - 3303), and trypsin-based proteolysis of recombinantly expressed proNGF has also been employed by others (e.g., D'Onofrio et al., 2011, PLoS One, vol. 6, e20839). However, it was later found that there are several drawbacks to cleaving wild-type proNGF with trypsin to produce β-NGF, because a small amount of trypsin results in low cleavage efficiency, while a large amount of trypsin further reduces the cleavage selectivity, since trypsin is capable of cleaving the C-terminus of any arginine and lysine residues (R and K residues). Thus, by trypsin digestion of proNGF containing R 1 S 2 K 3 R 4 several alternative digestion products will be obtained; therefore, using trypsin as the cleavage enzyme will result in a very low yield of correctly cleaved NGF and cause purification and yield problems, because different cleavage products cannot be economically separated under standard conditions. As a solution, a variant of proNGF was proposed in which the protease cleavage site R 1 S 2 K 3 R 4 in the propeptide is replaced by another amino acid at least at positions corresponding to positions 101 and 103 of the human wild-type proNGF sequence (SEQ ID NO: 1) R 1 and K 3 (WO 2013 / 092776A1). In one embodiment, R 1 and K 3 are replaced by valine (V) and alanine (A), respectively, converting the original furin cleavage site R 1 S 2 K 3 R 4 to V 1 S 2 A 3 R 4 , where trypsin can only specifically cleave R 4C-terminus; The trypsin-mediated cleavage of the corresponding pro-NGF can also be referred to as the "VSAR method". Although WO 2013 / 092776A1 does not mention the polypeptides of SEQ ID NO:3 or 4 according to the present invention, the VSAR method was initially proposed to be applicable to certain variants of pro-NGF mutant proteins, although it was reported that care was needed in titrating the proteolytic conditions (US2018 / 0086805 A1). In the process of implementing the present invention, the inventors found that, contrary to earlier suggestions, the VSAR technique cannot satisfactorily solve the purity problems associated with the recombinant production of the polypeptides of the present invention in satisfactory purity. In fact, it remains a challenge to purify recombinant-expressed β-NGF or its mutant proteins not only from host cell proteins (HCP), but also from trypsin (or other proteases used for cleavage); needless to say, it is required that there is no protease (such as trypsin) in the final formulation of the pharmaceutical protein to avoid proteolysis during polypeptide storage. Therefore, according to the present invention, at the time point of administration to a subject, the polypeptide is substantially pure and undegraded. As described herein, the inventors have solved this challenge. Thus, the present invention makes the polypeptides according to SEQ ID NO:3 or 4 obtainable in high purity and thus substantially free of trypsin and / or degradation products of the polypeptide. Although certain methods for producing NGF (such as W02013092776A1) and the polypeptide of SEQ ID NO:4 (such as Malerba et al., 2015, PLOS One, Volume 10, e0136425) have been previously described, the present disclosure surprisingly shows that the previously disclosed methods are not sufficient to obtain the corresponding polypeptides in high purity. As a solution to these deficiencies, the present disclosure provides a new process and related aspects, as described in detail herein.

[0208] According to the present invention, a method for obtaining the polypeptide of SEQ ID NO:3 and the polypeptide of SEQ ID NO:4 from recombinant expression in a host cell, for example, may include purification. In the broadest sense, purification refers to the separation of the polypeptide of SEQ ID NO:3 or SEQ ID NO:4 from other molecules, including other proteins, such as host cell proteins. Thus, purification may include separation from one or more other molecules, including other proteins, such as host cell proteins, proteases (such as trypsin) and / or degradation products of the polypeptide according to the present invention.

[0209] The production method of the polypeptide of SEQ ID NO:3 and the polypeptide of SEQ ID NO:4 according to the present invention preferably includes the following steps:

[0210] (a) Obtaining a precursor of the polypeptide of SEQ ID NO:3 or SEQ ID NO:4,

[0211] (d) Purification,

[0212] And the purification in step (d) generally includes purification on a mixed-mode stationary phase. Thus, in one embodiment, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 can be obtained by recombinant expression and purification, wherein the purification includes purification on a mixed-mode stationary phase. The term "on a mixed-mode stationary phase" should be understood broadly to mean that a mixture containing the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 or a precursor of any of these polypeptides and other molecular species is exposed to a mixed-mode stationary phase, for example, by chromatography or other suitable process steps. In fact, it is preferred to chromatograph a mixture containing the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 or a precursor of any of these polypeptides and other molecular species, such that the purification in step (d) includes purification by mixed-mode chromatography. Preferably, the mixed-mode chromatography includes using a stationary phase having a charged group, preferably a negatively charged group, and an aromatic group and / or a hydrophobic group.

[0213] In the broadest sense, according to the present invention, purification means that the polypeptide of SEQ ID NO: 3 or its SEQ ID NO: 4 is at least partially separated from other molecular species, including other proteins, such as host cell proteins, precursors, and / or degradation products. As a result, at least partially purified polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 can be obtained. Although other molecular species may optionally be discarded or not discarded, it is preferred to obtain and retain the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 as a result of purification.

[0214] Preferably, the mixed-mode chromatography includes using a stationary phase having a charged group, preferably a negatively charged group, and an aromatic group and / or a hydrophobic group.

[0215] Each of these steps can itself include several actions, which for simplicity can also be referred to as steps. By way of illustration, and as detailed below, step (d) can include more than one purification step, for example, on more than one stationary phase.

[0216] Any letter or number used herein in connection with one or more process steps, such as (a), (b), (c), (d), (d1), (d2), should not be construed as limiting, but is for reference. It should not be understood that the order of events in the process or use according to the present invention may be limited to the alphabetical order of the letters or the numerical order of the numbers. Despite the foregoing, it is highly preferred that the order of events in the method or use according to the present invention is the order described herein.

[0217] Other aspects of mixed-mode chromatography, in particular suitable stationary phases, will be described in more detail below, but these aspects generally apply to the present invention. Thus, in particular, all those stationary phases (including all their embodiments, which are described below as being particularly suitable for mixed-mode chromatography in step (d2)) are generally used for the purification of the polypeptide of SEQ ID NO: 3 and / or the polypeptide of SEQ ID NO: 4 according to the present invention and can be used in all types of embodiments, for example, in combination with or without the step of (d1) capture chromatography. In fact, Example 2B describes that some advantages can be achieved by using mixed-mode chromatography in a variant of the protocol according to the prior art.

[0218] Optionally, the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 can be obtained in a method comprising (re)folding and / or chromatographic purification and / or protease digestion and optionally adjusting the final protein concentration and / or preparing the desired formulation.

[0219] Thus, as disclosed herein, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 can also be administered to a subject in need thereof with an industrially relevant purity and yield of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4, which is obtainable to the person skilled in the art based on the disclosure herein. Accordingly, the present disclosure also describes methods for producing the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4.

[0220] The method for producing the polypeptides of SEQ ID NO: 3 and SEQ ID NO: 4 according to the present invention preferably comprises the following steps:

[0221] (a) Obtaining a precursor of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4, for example, by recombinant expression,

[0222] (d) Purification, wherein the purification comprises purification on a mixed-mode stationary phase.

[0223] The present invention also preferably subjects the precursor of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 to the step

[0224] (c) Exposure to a protease.

[0225] Said exposure is generally carried out before step (d).

[0226] A preferred feature of the method of the present invention is also that no chromatographic purification is carried out before exposure to the protease. In fact, the present disclosure (Examples 1, 2) shows that protease digestion also proceeds well and effectively in the crude fraction obtained from the host cell, i.e., when no chromatographic purification has been carried out before exposure to the protease.

[0227] Preferably, the step of obtaining (a) comprises expressing a precursor of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4, preferably recombinant expression. More preferably, the recombinant expression is in a host cell. After culturing the host cell, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is obtained in a fraction of the cell culture. This fraction may consist of the host cell, i.e., in the case where the protein is substantially not secreted from the host cell. This is the case, for example, when the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is produced in inclusion bodies and / or in intracellular compartments including the cytosol. Suitable host cells can be selected from prokaryotic and eukaryotic host cells, although prokaryotic host cells are preferred in typical embodiments. Preferred prokaryotic host cells include Escherichia coli (E. coli), preferably E. coli Rosetta (DE3). In one embodiment, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is obtained in a conformation and / or aggregate different from the native conformation, most preferably in inclusion bodies. Then, preferably, the method of the present invention comprises step (b) of (re)folding the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. Preferably, step (c) is carried out after step (b).

[0228] Preferably, in step (c), the protease is a protease capable of cleaving the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 in a manner that releases the (mature) polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. In a specific embodiment, the protease is trypsin, preferably porcine trypsin, optionally recombinantly expressed.

[0229] Preferably, the purification step (d) comprises the following steps, preferably in sequence:

[0230] (d1) Capture,

[0231] (d2) Refinement.

[0232] Preferably, the capture step (d1) is carried out by chromatography, preferably column chromatography. More preferably, the capture step (d1) is carried out using a cation exchange chromatography stationary phase or a mixed mode chromatography stationary phase. Even more preferably, the capture step (d1) is carried out using a mixed mode chromatography stationary phase, which is preferably Capto MMC.

[0233] Preferably, the refinement step (d2) is carried out by chromatography, preferably column chromatography. More preferably, the refinement step is carried out using a cation exchange chromatography stationary phase. Even more preferably, the capture step (d1) is carried out using SP Sepharose gel, preferably SP Sepharose gel with a small particle size. SP is the abbreviation of sulfopropyl.

[0234] Optionally, the method according to the invention comprises additional steps of adjusting the final protein concentration and / or preparing the desired formulation. As a result, a composition according to the invention can be obtained.

[0235] In other words, the present invention provides a mixed-mode chromatography for preparing the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. The mixed-mode chromatography can be used to prepare the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. In a preferred embodiment, a precursor of the polypeptide of SEQ ID NO: 3 or a precursor of the polypeptide of SEQ ID NO: 4 is exposed to a protease for digestion, and the mixed-mode chromatography is used in the steps after the exposure to the protease. In a preferred embodiment, no chromatographic purification of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is carried out before the exposure to the protease.

[0236] Purity of the polypeptide

[0237] The polypeptide of the present invention is substantially or largely free of the components that are normally associated with it in its natural state. The polypeptide of the present invention is isolated before administration. In one embodiment, an "isolated polypeptide" refers to a polypeptide purified from the cellular environment and extracellular environment (such as tissue) that surrounds it in its natural state, for example, from the cells (such as host cells) that express it. In another embodiment, an "isolated polypeptide" refers to in vitro isolation and / or purification of the polypeptide from its natural cellular environment and from its association with other components of the environment in which the polypeptide normally exists.

[0238] Preferably, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 used according to the present invention is substantially free of impurities. Such advantageously pure polypeptides of SEQ ID NO: 3 or SEQ ID NO: 4 can be obtained as described herein.

[0239] The polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 described herein is considered a pharmaceutically active peptide or protein.

[0240] In a particularly advantageous embodiment of the present invention, the resulting polypeptide according to the present invention is substantially free of degradation products of the polypeptide. In particular, the present disclosure shows that, contrary to the reports in the prior art regarding wild-type human NGF, exposure of the precursor of SEQ ID NO: 4 to trypsin will result in an inherent partial cleavage of the C-terminus of the precursor at arginine (Arg, R) residue 9 of SEQ ID NO: 4 if trypsin (des-nona variant, data not shown) cannot be completely removed before or after purification. By the specific purification method provided by the present invention, a polypeptide of the present invention substantially free of trypsin and / or des-nona variant can be obtained.

[0241] Preferably, the polypeptides obtainable as described above are substantially free of degradation products of the polypeptide. In particular, the present disclosure makes the polypeptides of the invention available in a new, improved purity grade, and preferably administers the polypeptides at such a high purity. Preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade of at least 90%. More preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade of at least 91%. More preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade of at least 92%. More preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade of at least 93%. More preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade of at least 94%. More preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade of at least 95%. More preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade of at least 96%. More preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade of at least 97%. More preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade of at least 98%. Even more preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade of at least 99%.

[0242] Most preferably, the polypeptides of the invention used according to the invention are characterized by a purity grade greater than 99.0%, such as a purity grade greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%, greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, greater than 99.9%.

[0243] Herein, the "purity grade" generally refers to the percentage of the weight (w) of the polypeptide of the present invention relative to the weight (w) of biological materials other than the polypeptide of the present invention. For illustration, generally, in the case of a purity grade of 99.0%, the polypeptide of the present invention is present in a relative amount (weight) of 99.0 units (e.g., 1.0 mg), and the sum of the weights of all biological materials other than the polypeptide of the present invention is 1.0 unit (e.g., 1.0 mg). Such biological materials other than the polypeptide of the present invention include, but are not limited to, host cell proteins, nucleic acids, proteases, such as inactivated or unactivated trypsin, degradation products of the polypeptide of the present invention, such as and macromolecules from other biological sources. In a specific embodiment, the "purity" grade refers to the purity relative to polypeptides other than the polypeptide of the present invention. For illustration, in that embodiment, in the case of a purity grade of 99.0%, the polypeptide of the present invention is present in a relative amount (weight) of 99.0 units (e.g., 1.0 mg), and the total weight of all polypeptides different from the polypeptide of the present invention is 1.0 unit (e.g., 1.0 mg). To avoid doubt, degradation products of the polypeptide of the present invention are included in the "polypeptides different from the polypeptide of the present invention". A specific degradation product is the des-nona variant (see Examples 1 and 2).

[0244] Particularly substantially free of the des-nona variant of the polypeptide. The des-nona variant is a previously uncharacterized degradation product of the polypeptide of the present invention, which is associated with the production of certain variants of NGF, including the polypeptide of the present invention, unless the polypeptide is produced by the new method disclosed herein (see, for example, Examples 1 and 2). "Substantially free" in this context means that the polypeptide of the present invention used according to the present invention is characterized by a purity grade greater than 99.0% relative to the des-nona variant, e.g., a purity grade greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%, greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, greater than 99.9%, all of which are purity grades relative to the des-nona variant. In the most preferred embodiment, the des-nona variant is undetectable and / or absent.

[0245] The polypeptide according to the invention is also preferably substantially free of any proteases (such as trypsin). "Substantially free" means in this context that the polypeptide according to the invention used according to the invention is characterized by a purity grade greater than 99.0% relative to the sum of all proteases (including trypsin), for example a purity grade greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%, greater than 99.5%, greater than 99.6%, greater than 99.7%, greater than 99.8%, greater than 99.9%, all of which are purity grades relative to the sum of all proteases (including trypsin). In the most preferred embodiment, trypsin is undetectable and / or absent.

[0246] In the above embodiments, such a high purity grade is related to the increased acceptability by regulatory authorities and qualifies the polypeptide according to the invention as a drug for mammalian subjects, particularly humans. Thus, the purity grade according to the invention enables for the first time the polypeptide to be administered to the eye, including the human eye, in a safe and reliable manner. With respect to proteases (trypsin), the high purity grade particularly enables the polypeptide to be stored in a non-frozen form.

[0247] Composition

[0248] Compositions comprising the polypeptide according to the invention will be described below. Such compositions are part of the invention both per se and in the specific context of use according to the invention for the prevention and / or treatment of ophthalmic disorders. Thus, the invention is directed both to the medical use of such compositions and to such compositions per se.

[0249] In the composition according to the invention, the polypeptide comprising SEQ ID NO: 3 or SEQ ID NO: 4 is used as the active ingredient. Other ingredients may be included.

[0250] In one embodiment, the polypeptide is contained in an aqueous medium. The aqueous medium is for administration to a mammalian subject.

[0251] A specific aqueous composition used according to the invention is a liquid composition suitable for use as an eye drop. Generally, an eye drop is a liquid drop suitable for ocular administration. The term "eye drop" is not particularly limited and generally refers to a composition, usually an aqueous liquid composition, which can be administered to the eye without causing harm to the eye. Generally, the risk of side effects of eye drops is lower than, for example, oral drugs or intravitreal injection drugs. For these and other reasons, eye drops are particularly preferred.

[0252] Thus, in one embodiment, the polypeptide is contained in a composition suitable for use as an eye drop.

[0253] The eye drops are expected to be administered to a mammalian subject. The eye drops are expected to be used for the ocular administration route to administer the medicament of the present invention. Preferably, the eye drops are topically administered to the eye.

[0254] Methods for preparing eye drops are known in the art. By way of non-limiting illustration only, methods for preparing eye drops have been described in WO2016 / 162812A1.

[0255] In some embodiments, the polypeptide of SEQ ID NO:3 or SEQ ID NO:4 described herein is included in a composition, such as an eye drop composition, which further comprises one or more carriers and / or one or more excipients. As used herein, the term "carrier" refers to an organic or inorganic component, natural or synthetic, which is combined with the active ingredient to effect, enhance or facilitate the application of the active ingredient. As used herein, the term "excipient" is intended to denote all substances that may be present in the pharmaceutical compositions of the present invention and that are not active ingredients.

[0256] Preferably, the composition according to the present invention comprises at least water as an excipient. In some embodiments, the composition according to the present invention comprises an aqueous medium, and more preferably the composition according to the present invention is in the form of an aqueous solution. In one embodiment, the polypeptide is included in an aqueous medium, and the aqueous medium is administered to a mammalian subject. The aqueous medium may be, for example, an aqueous solution. In some embodiments, the aqueous solution and other corresponding compositions can be obtained directly from the purification of NGF in an aqueous medium. For example, when the medicament according to the present invention is purified from a biological source, the corresponding aqueous composition can be obtained directly from the final purification step, such as the elution and / or filtration from the final chromatographic column (usually the polishing step). Alternatively, the corresponding composition can be obtained by adjusting the final protein concentration and / or additional steps for preparing the desired formulation. Such additional steps may include, for example, clarification or filtration steps as described herein, and / or the addition of one or more excipients and / or one or more carriers. Exemplary compositions that can be used in the present invention are described herein, but are not limited thereto.

[0257] Thus, the polypeptide of SEQ ID NO:3 or SEQ ID NO:4 described herein can be present in a composition, such as a pharmaceutical composition. The composition described herein is preferably sterile and preferably comprises the polypeptide of SEQ ID NO:3 or SEQ ID NO:4 as a pharmaceutically active peptide or protein, and optionally comprises other medicaments mentioned or not mentioned herein. The composition can be in any state, such as liquid, frozen, lyophilized, etc.

[0258] The compositions described herein may contain salts, buffering substances, preservatives, carriers, diluents, and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" describes things that are non-toxic and / or do not interact with the action of the active ingredients of the pharmaceutical composition.

[0259] Suitable buffering substances for use in the present invention include acetic acid in salts, citric acid in salts, boric acid in salts, and phosphoric acid in salts. For example, preferably, due to various aspects of the present invention, the polypeptides of the present invention may be obtained in a buffer having a pH between 4.5 and 6.5, preferably between 5.0 and 6.0. In one embodiment, an acetate buffer is a suitable buffer for such purposes and is thus particularly preferred. Thus, in one embodiment, the polypeptides of the present invention are obtained in an acetate buffer having a pH between 4.5 and 6.5, preferably between 5.0 and 6.0. In particular, acetate is considered to be the best buffer for stabilizing NGF and its derivatives in the pH range of pH 5.0 to pH 5.8.

[0260] Since NGF, and possibly also the polypeptides according to the present invention, are sensitive to oxidation of methionine residues, it is preferred to include methionine in the formulation.

[0261] The present invention further relates to a composition of polypeptides comprising a polypeptide selected from SEQ ID NO: 3 and a polypeptide of SEQ ID NO: 4, wherein the composition is characterized by a pH of 5.0 to 6.0 (preferably pH 5.5), and comprising the following:

[0262] a) 0.2 to 20 mg / ml of said polypeptide (preferably 2 mg / ml),

[0263] b) 5 to 100 mM sodium acetate buffer (preferably 20 mM),

[0264] c) 5 to 100 mM methionine (preferably 20 mM).

[0265] Thus, preferably, the polypeptide is included in a composition comprising the following:

[0266] a) 0.2 to 20 mg / ml of the polypeptide of the present invention,

[0267] b) 5 to 100 mM sodium acetate buffer,

[0268] c) 5 to 100 mM methionine,

[0269] d) pH 5.0 to 6.0.

[0270] A more preferred pH range is 5.0 to 5.8.

[0271] More preferably, the polypeptide is included in a composition comprising:

[0272] a) 2 mg / ml of said polypeptide,

[0273] b) 20 mM sodium acetate buffer,

[0274] c) 20 mM methionine,

[0275] d) pH 5.5.

[0276] Another preferred formulation of the present invention is as follows:

[0277] 1 mg / ml of the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 (preferably)

[0278] 10 mM sodium acetate buffer

[0279] 10 mM methionine

[0280] 154 mM NaCl

[0281] 0.1 mg / mL polysorbate 80

[0282] pH 5.5

[0283] It is contemplated that these and other formulations are suitable as pharmaceutical products for ophthalmic use.

[0284] The above composition is preferably an eye drop. The above composition is preferably an aqueous composition.

[0285] According to one or more of the following embodiments, the composition (formulation) of the present invention can be preserved, but not limited to:

[0286] - First embodiment: The above formulation can be stored frozen at -70 °C (data not shown) and thawed before administration.

[0287] - Second embodiment: The above formulation can be stored in a refrigerator, preferably in the temperature range of +2 to +8 °C (data not shown).

[0288] - Third embodiment: The above formulation can be dried or lyophilized and then stored, for example, at room temperature (data not shown). It can be reconstituted before administration.

[0289] All of the above embodiments provide certain advantages of freezing at, for example, -20 °C, as this avoids the need for transporting with dry ice, thawing, and remixing after thawing, etc. In such embodiments, the formulation according to the present invention overcomes certain inconveniences of prior art drug NGF formulations such as, in particular, oxervate (cenegermin).

[0290] A particularly preferred composition for administration according to the present invention is illustrated in Example 3.

[0291] Suitable preservatives for the compositions according to the invention include those known in the art, by way of illustration and not limitation, benzyl alcohol, benzalkonium salts and their salts, m-cresol, phenol, chlorobutanol, parabens and thimerosal. These and other preservatives are optionally included in the compositions according to the invention.

[0292] Accordingly, the present invention provides the use of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for therapeutic use, i.e., for use in a method of treating the human or animal body by therapy. Therapy may include the prevention and / or treatment of diseases. Given the potential for therapeutic use, the polypeptide may also be referred to as a pharmaceutically active protein or peptide.

[0293] Optionally, administration according to the invention is accompanied by administration of at least one antimicrobial agent, such as an antibiotic. The antimicrobial agent may be part of a composition comprising the polypeptide according to the invention, or may be administered separately to the same or different sites of the subject by the same or different routes of administration.

[0294] Industrial Applicability

[0295] The polypeptides of SEQ ID NO: 3 or SEQ ID NO: 4 described herein are suitable for a variety of purposes, such as for the therapeutic applications described herein.

[0296] The following examples and drawings are intended to illustrate some preferred embodiments of the present invention and should not be construed as limiting the scope of the present invention as defined by the claims. Examples

[0297] Materials and methods common to multiple examples

[0298] Unless otherwise specified, the following experimental examples specifically relate to the polypeptide of SEQ ID NO: 4, which is characterized by the substitutions P61S R100E relative to wild-type human NGF (referred to as "NGF P61S R100E", Malerba et al., PLOS One, 2015, Volume 10, e0136425, SEQ ID NO: 4), and its precursors, etc. The polypeptide of SEQ ID NO: 4 may also be referred to as "NGF mutant protein", but it must be kept in mind that according to the present invention and as demonstrated in the experimental examples, especially Example 4, the specific therapeutic applicability of this protein is significantly different from that of wild-type human NGF. Similarly, as described in Example 2, the purification of the polypeptide of SEQ ID NO 4 is different from the published purification protocol of wild-type NGF, and the specific method for preparing the polypeptide according to the present invention is suitable for achieving high purity, especially the absence of des-nona variants and trypsin.

[0299] The polypeptide of SEQ ID NO: 4 was recombinantly expressed as a precursor. For this purpose, SEQ ID NO: 4 was fused with the propeptide of wild-type human NGF (positions 1 - 121 of SEQ ID NO: 1). In other words, the precursor of the polypeptide of SEQ ID NO: 4 consists of the precursor of human wild-type NGF (SEQ ID NO: 1), except for the substitutions P61S R100E in the mature part of human wild-type NGF (however, for clarity, lacking the 2 most C-terminal amino acids that do not form part of the polypeptide sequence of SEQ ID NO: 1 of human wild-type NGF). The expression was carried out in Escherichia coli Rosetta(DE3) (strain: E. coli Rosetta(DE3) / pET11a-hpro NGF P61S R100E) in the form of insoluble inclusion bodies.

[0300] Equipment

[0301]

[0302]

[0303] Table 1 List of equipment used.

[0304] Protein parameters of the proteins and peptides of the present text

[0305] The theoretical values of the protein parameters of the relevant proteins were calculated using the ProtParam-Tool of ExPASy, which is available at http: / / web.expasy.org / protparam. These are shown in Table 2 as follows:

[0306]

[0307] Table 2: Theoretical Derived Properties of Related Proteins / Polypeptides

[0308] Analysis method

[0309] SDS-PAGE and Western blotting

[0310] SDS-PAGE and Western blotting were performed using standard procedures. For SDS-PAGE, a 12% Bis-TRIS NuPAGE gel (product number NP0342BOX from Thermo Fisher) was operated in NuPAGE MES running buffer (product number NP0002 from ThermoFisher) under reducing conditions at a constant voltage (175V). The primary antibody for Western blotting was purchased from Santa Cruz Biotechnology (NGF (H-20) sc-548).

[0311] Analytical CEX-HPLC

[0312] CEX-HPLC was performed using a ProPac SCX-10 from Dionex. A 50 mM citrate buffer, pH 5.5, was used to operate the column at 1 mL / min. For elution, 1 M NaCl (B) was added and a linear gradient from 0 - 100% B was performed over 50 minutes.

[0313] SE-HPLC

[0314] SE-HPLC was performed using a Superdex 200 Increase 10 / 300GL from GE Healthcare. The column was operated in PBS. The product was detected at 280 nm.

[0315] Endotoxin, DNA and HCP

[0316] Endotoxin, DNA, and host cell protein (HCP) were determined according to standard protocols.

[0317] Example 1: Expression of the Polypeptide of SEQ ID NO: 4 as a Precursor Protein

[0318] Strain production

[0319] The gene encoding pre-NGF was cloned into the pET11a expression plasmid. The gene is derived from Homo sapiens (H. sapiens), and two point mutations (i.e., P61S and R100E) were introduced into the open reading frame. Subsequently, chemically competent Rosetta(DE3) cells were transformed with the expression plasmid and a single colony was selected (the resulting strain is designated as E5901 STRAIN (= E. coli Rosetta(DE3) / pET11a-pre-NGF P61S R100ENGF RCB C-151101)). Aliquots were stored at < -60 °C in 1.0 mL.

[0320] In Example 1, the initial fermentation development based on strain E5901 STRAIN is described.

[0321] Equipment

[0322]

[0323]

[0324] Growth medium

[0325] Complex medium for fermentation

[0326] The complex medium for fermentation consisted of: 49.3 g / L yeast extract, 0.61 g / L MgSO4·7H2O, 0.5 g / L NH4Cl, 14.2 g / L K2HPO4·3H2O and 10 g / L glucose. The feed for this fermentation consisted of 263 g / L yeast extract and 133 g / L glucose.

[0327] Minimal medium (MM) for fermentation

[0328]

[0329]

[0330] For the batch phase, 30 g / L glucose was added to both basal media. Unless otherwise stated, the feed had the same composition as the corresponding batch medium but contained 300 g / L of the corresponding carbon source.

[0331] LB agar plate containing ampicillin and chloramphenicol

[0332] LB-agar plates were freshly poured. The medium consisted of 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl and 15 g / L agar. After autoclaving, 100 μg / ml ampicillin and 30 μg / mL chloramphenicol were added to the medium.

[0333] Fermentation

[0334] Unless otherwise stated, fermentation, in Example 1, was carried out in a 1 L stirred glass bioreactor controlled by a Biostat B unit from Sartorius. Generally, pO2 was controlled at 30% using 2 M phosphoric acid and 25% ammonium hydroxide, the culture temperature was set at 37 °C, and the pH was controlled at 7. Unless otherwise stated, the batch phase was followed by exponential feeding with F0 = 6 g / L / h and μ = 0.25 / h. For practical reasons, all exponential feeds were approximated by two linear feeds. Generally, product expression was induced by adding 1 mM IPTG, and after induction, a constant feeding rate of 10 g / L / h was applied. Cell biomass was collected by centrifugation using a Sorvall Evolution RC from ThermoScientific. The centrifuge was equipped with an SLC-6000 rotor, and the culture was centrifuged at 8500 rpm and 4 °C for 30 minutes.

[0335] Relative quantification of products in biomass samples

[0336] At a given time point, culture samples were diluted to OD 600 of 10, and 100 μL aliquots of biomass from this dilution were pelleted. The pellets were resuspended in 150 μl (non-reducing) Laemmli buffer, and the samples were boiled at 95 °C for 5 minutes. 10 μL of each sample was analyzed on a 10% Bis-Tris gel from Novex. Electrophoretic separation was carried out at 125 V for 90 minutes, and the gel was stained with Coomassie. The de-stained gel was scanned and the abundance of the band corresponding to the polypeptide precursor of SEQ ID NO: 4 was quantified by densitometry. To further correct for inconsistencies in the biomass utilized, the intensity of the band corresponding to the polypeptide precursor of SEQ ID NO: 4 was normalized to the intensity of a housekeeping protein.

[0337] Relative product accumulation was calculated from the increase in the band corresponding to the polypeptide precursor of SEQ ID NO: 4 after induction compared to before induction. It should be noted that the measurements represent specific yields (i.e., normalized to OD 600 = 10). For the absolute yield of a given fermentation, the actual cell density must be considered (see below).

[0338] Absolute quantification of products in biomass samples

[0339] The standard of the polypeptide precursor of SEQ ID NO: 4 was obtained from the European Brain Research Institute (EBRI, Rome, Italy). The standard was diluted to a concentration of 65 μg / mL in Laemmli buffer. The protein concentration was defined by EBRI. A standard curve was prepared with 260, 520, 780, 1040, and 1300 ng of the standard of the polypeptide precursor of SEQ ID NO: 4. Samples were analyzed on the same gel as the standard curve, and the absolute yield of the product at a given time was calculated taking into account the dilution factor of the samples.

[0340] Summary and conclusion of Example 1

[0341] Based on the above, it was concluded that the production strain (E5901 STRAIN, see above) was successfully used for fermentation on a 1 L scale. Although the ability of different medium compositions to promote bacterial growth and product expression has been evaluated, it has been proven that minimal medium MM I supplemented with 5 g / L yeast extract is advantageous in terms of expression yield and the cell density obtainable. In terms of product formation, no significant differences were observed whether or not antibiotics (ampicillin and chloramphenicol, data not shown) were used during the main culture.

[0342] Example 1 can be scaled up for the production of polypeptides on an industrial scale.

[0343] Example 2: Laboratory-scale purification, establishment of Capto MMC

[0344] The precursor of SEQ ID NO: 4 used in this example was obtained in inclusion bodies as described in Example 1.

[0345] Optimization starting point in view of the prior art

[0346] At the beginning, it was speculated that, in the absence of contrary indications, the basic basis of NGF purification previously reported in the literature could be followed to improve the method for purifying the polypeptide of the present invention. However, it should also be borne in mind that, for large-scale and efficient production, adjustments suitable for subsequent scale-up should be considered. Therefore, it was speculated that, based on Rattenholl et al. (ibid.), WO2013092776 A1, and other publications, the polypeptide of SEQ ID NO: 4 could also be obtained in its precursor form using non-specific digestion with trypsin and subsequent purification, at least in a laboratory-scale process. It was speculated that the mature polypeptide of SEQ ID NO: 4 with high purity could be obtained thereby. However, it was only through the specific adjustments and modifications reported in this example that the mature polypeptide of SEQ ID NO: 4 with high purity could be obtained. Therefore, particularly in view of the high purity of the polypeptide of SEQ ID NO: 4 as described herein, the polypeptide of SEQ ID NO: 4 can be administered to a subject in need.

[0347] Equipment, SEQ ID Production of the polypeptide of SEQ ID NO: 4

[0348]

[0349]

[0350] List of equipment used in Example 2.

[0351] Details of the manufacturing process according to this example, including the improvements described in Example 2B, are given in the process overview of Figure 1.

[0352] Unless otherwise stated, the analytical methods are as described in the "Analytical methods" section above.

[0353] Example 2A: Purification is carried out according to the previously described protocol.

[0354] Escherichia coli cells producing the polypeptide precursor of SEQ ID NO: 4 ("biomass") are generated as described in Example 1 and the cells are lysed by adding lysozyme and subsequent sonication on ice. Inclusion bodies ("IB") are (1) extracted from the host cells and washed with 6% Triton X100 (in 1.5 M NaCl, 60 mM EDTA), then, (2) dissolved in 6 M guanidine hydrochloride ("gHCl"), 0.1 M Tris-HCl pH 8.0, 1 mM EDTA, 100 mM (fresh) DTT. The IB is dissolved for 2 hours at room temperature. Thereafter, the pH is adjusted to 3 - 4 by adding 37% HCl. The resulting solution of the dissolved precursor ("lysate") containing the precursor of the polypeptide of SEQ ID NO: 4 is dialyzed against 6 M gHCl (pH 3 - 4).

[0355] Refolding of the polypeptide precursor of SEQ ID NO: 4 is carried out in 0.1 M Tris-HCl, 1 M L-arginine, 5

[0356] mM EDTA, 0.61 g / L oxidized glutathione and 1.53 g / L reduced glutathione (pH 9.5 at +4 °C). Thus, 50 μg of protein is added per hour per mL of refolding buffer. After refolding, the reaction is dialyzed against 50 mM sodium phosphate pH 7.0. A significant precipitate appears upon changing the buffer.

[0357] The precursor of the polypeptide of SEQ ID NO: 4 is purified by cation exchange chromatography (SP Sepharose HP using 50

[0358] Purified on a continuous sequence of anion exchange chromatography (Mono Q, operating with 50 mM sodium phosphate, pH 7.0, and eluting with a NaCl gradient) and subsequent hydrophobic interaction chromatography (Phenyl Sepharose HP, operating with 50 mM sodium phosphate, 1 M ammonium sulfate, pH 7.0). Subsequently, another dialysis was used to exchange the buffer of the sample with 50 mM sodium phosphate, pH 7.0 (note that this second dialysis can be omitted during this process). Throughout the process of reducing the buffer conductivity, a large amount of the product precipitated again.

[0359] Limited proteolysis of the precursor of the polypeptide of SEQ ID NO: 4 thus prepared was carried out by adding 1 mg of trypsin per 250 mg of pro-NGF. The precursor of the polypeptide of SEQ ID NO: 4 was exposed to the protease at 2 - 8 °C for 14 hours.

[0360] The mature NGF was finally refined on a cation exchanger (SP Sepharose XL, operating with 50 mM sodium phosphate, pH 7.0, and eluting with a NaCl gradient). Finally, the product was concentrated to 0.5 - 1 mg / mL and frozen at < -65 °C.

[0361] Example 2B: Improvements

[0362] Below, several improvements tested and implemented in the process of achieving the present invention compared to Example 2A are described. Unless otherwise specified in the context, all details not explicitly stated are as described in Example 2A above.

[0363] Optimization of IB dissolution

[0364] Although it has been previously reported that a small amount of IB (such as the example obtained from shake flask cultures) is readily soluble in a solubilization buffer (6 M guHCl, 0.1 M Tris-HCL pH 8.0, 1 mM EDTA, 100 mM (fresh) DTT), the IB obtained from high cell density fermentation could not be completely solubilized. This was resolved by adding 2 M urea to the solubilization buffer, which was shown to significantly improve the solubilization yield (data not shown). For the avoidance of doubt: in addition to 6 M guHCl and the other components, 2 M urea was present.

[0365] Refolding optimization

[0366] Initially based on Rattenholl et al. (2001, Eur. J. Biochem, vol. 268, pages 3296 - 3303; Rattenholl, 2001, Doctoral thesis (Dr.rer.nat.), Martin-Luther- In Halle-Wittenberg (Germany), but importantly also taking into account future (upward) scalability, it was decided to refold with 200 to 500 mg of the precursor of the polypeptide of SEQ ID NO: 4 per liter of refolding reaction, preferably 200 to 300 mg of the precursor of the polypeptide of SEQ ID NO: 4 per liter of refolding reaction. This led to a relatively good yield of the soluble precursor of the polypeptide of SEQ ID NO: 4. In particular, it is important to consider the increased amount of NGF compared to the volume of the refolding reaction. The refolding reaction contains relatively expensive components (such as glutathione and arginine), and relatively more precursors of the polypeptide of SEQ ID NO: 4 can be refolded per volume of the refolding reaction, which should make refolding economically feasible and also feasible on a production scale.

[0367] SEQ ID Purification of the polypeptide precursor of SEQ ID NO: 4

[0368] The precursor of the polypeptide of SEQ ID NO: 4 was purified after refolding by a method that utilizes the relatively high isoelectric point of proNGF and uses a cation exchange stationary phase (i.e., SP Sepharose). For running this type of chromatography, for technical reasons, the refolding buffer had to be changed to a buffer with low conductivity. In doing so, a large amount of the precursor of the polypeptide of SEQ ID NO: 4 precipitated (data not shown). This observation can be attributed to the decrease in the concentration of arginine in the buffer.

[0369] Therefore, measures were taken to replace the capture column with different capture columns (columns with different selectivities) that could be more tolerant of the presence of arginine in the refolding reaction. In the first attempt to do this, the performance of several stationary phases was evaluated, but none of the methods produced promising results (see Table 6). Therefore, the stationary phase used for the capture column remained the same as defined in the previous process. However, due to the relatively high isoelectric point (pI) of the precursor of the polypeptide of SEQ ID NO: 4, it was possible to increase the conductivity of the running buffer (by adding 250 mM L-arginine) without affecting performance. In this way, the refolded precursor of the polypeptide of SEQ ID NO: 4 could be stabilized to some extent and the amount of precipitated precursor decreased (data not shown).

[0370]

[0371]

[0372] The table above: Alternative selectivities for capturing the precursor of the polypeptide of SEQ ID NO: 4 were tested and evaluated.

[0373] However, with respect to mixed-mode chromatography, it should be understood, without wishing to be bound by a particular theory, that the precursor of the polypeptide of SEQ ID NO: 4 cannot be effectively eluted from mixed-mode chromatography, while the mature polypeptide of SEQ ID NO: 4 can.

[0374] Protease digestion to produce mature NGF

[0375] For the production of the polypeptide of SEQ ID NO: 4, a protease (trypsin) is required. Thus, it is inferred that ideally, the specific trypsin selected should meet the following criteria:

[0376] 1. Derived from a recombinant source. Certification of raw materials free of animal components is crucial for subsequent GMP-compliant processes.

[0377] 2. Low side activity of trypsin. It should be noted that trypsin can autolyze. This process may produce so-called pseudotrypsin, which has a broadened substrate spectrum and chymotrypsin-like activity. Ca 2+ (e.g., 1 mM CaCl2) can be added to reduce autolysis. However, nowadays, "modified trypsin" is usually applied to each protocol, which requires strict sequence specificity (e.g., peptide fingerprint). This modified trypsin is usually obtained by acylation of the ε-amino group of trypsin-exposed lysine residues.

[0378] 3. Low batch-to-batch variability to achieve a reproducible production process. Alternatively, the selected enzyme should be accompanied by a certificate stating the specific activity of the corresponding batch. Then the required amount of enzyme can be based on activity rather than mass.

[0379] Despite a comprehensive search, no trypsin meeting criteria 1 and 2 was found on the commercial market. The reason is that criterion 1 is more important. To reduce autolysis, the addition of CaCl2 may be sufficient. Therefore, recombinant "GMP-grade" trypsin from Roche (Roche 06369880103, lot number: 11534700) was selected as the raw material for this process. This enzyme is expressed in Pichia pastoris and its sequence is derived from Sus scrofa. According to its certificate, the specific activity of the trypsin batch used is 4997 U / mg (determined according to USP).

[0380] Omit the second purification step before trypsin digestion

[0381] In the initial screening to find the optimal enzyme / substrate ratio for the intended trypsin digestion, the precursor of the polypeptide of SEQ ID NO: 4 obtained from the capture column (see above) was used. Compared to the previously established method (European Brain Research Institute (EBRI), details not published, based on Rattenholl et al., ibid.), it was decided not to use additional hydrophobic interaction chromatography before trypsin digestion. The decision to omit the second column purification step before trypsin digestion was mainly based on two lines of thought: on the one hand, the product obtained after the capture column was already almost pure according to SDS-PAGE. On the other hand, trypsinization itself might contribute to improving the impurity profile by digesting the remaining host cell proteins (HCP).

[0382] Table 7 summarizes the condition matrix of the first round of screening. The results of trypsin digestion were studied by 12% SDS-PAGE (data not shown). The results (data not shown) indicated that trypsin digestion reproducibly produced a stable polypeptide of SEQ ID NO: 4 within a rather wide range of enzyme / substrate ratios (i.e., 1 - 5 μg of trypsin per 375 μg of the precursor of the polypeptide of SEQ ID NO: 4). The digestion time was not very critical. Thus, the time required to stop the reaction and load the reaction onto the purification column was clearly not limiting. This finding was particularly important because the reaction could not be quenched appropriately or economically on a preparative scale.

[0383] Some additional experiments were carried out to improve the optimal enzyme / substrate ratio for the envisaged trypsinization, and it was found that at an enzyme / substrate ratio of 1 / 100 to 1 / 200 (protein weight / protein weight), on the one hand, a good yield of the polypeptide of SEQ ID NO: 4 could be reproducibly obtained, and on the other hand, a small amount of truncated products could be obtained. It must be noted that under the conditions used (i.e., incubation (exposure to protease) for 2 to 6 hours in phosphate / arginine buffer (pH 7.0) at 2 - 8 °C), the quality of the digestion was not highly dependent on the enzyme / substrate ratio. This finding was particularly important because potential enzymatic digestion was prone to minor changes in the experimental setup (e.g., due to batch-to-batch differences or changes in trypsin activity caused by storage of the enzyme; time and temperature of the incubation step (exposure to protease); errors in protein concentration determination). In addition, this was also why it seemed meaningless to scale up fine-tuning within a small scale to further reduce potential truncated products. If the "optimal" conditions were determined within a small scale, there would still be a good chance of producing a slightly altered product pattern when repeating almost the same digestion on a larger scale the next time.

[0384] Refining chromatography aimed at obtaining pure polypeptide after trypsin digestion

[0385] Compared with the previously established process (European Brain Research Institute (EBRI), details not published, based on Rattenholl et al., ibid.) of using SP Sepharose stationary phase to purify the mature polypeptide (note: SP sepharose is a cation exchange stationary phase), a more suitable stationary phase was sought here based on the following considerations: To effectively load onto the SP Sepharose column, it is necessary to reduce the conductivity of the solution containing the precursor of the polypeptide of SEQ ID NO: 4, for example by buffer exchange. However, it is known (e.g., Example 2A) that a decrease in the ionic strength of the solution does cause precipitation of the target molecule, so changing the buffer to a low-conductivity buffer should be avoided. In addition, cation exchange stationary phases have been used to capture the precursor of the polypeptide of SEQ ID NO: 4, and for better separation of the remaining contaminants, orthogonal selectivity is preferred. The third and final argument against using the SP stationary phase to purify the trypsinized reaction is that the potential remaining polypeptide precursor of the polypeptide of SEQ ID NO: 4 will bind to the column and can be separated from the mature polypeptide of SEQ ID NO: 4 only by elution selectivity rather than by binding selectivity.

[0386] To establish such an orthogonal purification column for purifying the mature polypeptide of SEQ ID NO: 4, a hydrophobic interaction (HIC) column was first intended to be used. The selection of this stationary phase not only has orthogonal selectivity but also because there is no need to change the buffer to a low-conductivity buffer. Although several HIC stationary phases and conditions were tested (e.g., phenyl agarose and butyl agarose operated with 1M (NH4)2SO4 and 0.5M (NH4)2SO4 respectively), a satisfactory purification step based on HIC could not be implemented (data not shown).

[0387] However, in a further experimental setup for the purification step, the mixed-mode stationary phase Capto MMC was tested and could be successfully implemented. It was found that under optimized conditions, the stationary phase reversibly binds to the polypeptide of SEQ ID NO: 4, and the product can be eluted by increasing the pH (data not shown). In contrast, the precursor of the polypeptide of SEQ ID NO: 4 irreversibly binds to the stationary phase and can only be eluted by using 1M NaOH as the mobile phase (data not shown). In addition, it could be demonstrated that trypsin does not bind to the column operated under the same conditions (data not shown). These results provide clear evidence that the Capto MMC stationary phase can effectively separate the mature polypeptide of SEQ ID NO: 4 from trypsin and the remaining precursor of the polypeptide of SEQ ID NO: 4.

[0388] Establish additional membrane chromatography

[0389] To further remove endotoxins and DNA, an additional anion exchange membrane was added to the process. Generally, and as is well known, membrane chromatography is characterized by passing or flowing a solution containing the component to be analyzed or purified (in this case the polypeptide of SEQ ID NO: 4) across or through a membrane that is typically charged. For this purpose, in this case, a STIC membrane (Sartorius, Göttingen, Germany) was added at the position shown in Figure 1. It could be shown that the polypeptide of SEQ ID NO: 4 does not bind to the membrane, thus providing proof of concept that membrane chromatography is suitable for purifying the polypeptide of SEQ ID NO: 4. To illustrate the binding throughout the process, including membrane chromatography, see Figure 1.

[0390] Reproducibility of the method according to Example 2

[0391] To probe the robustness of the process, the process was run five times and the yields and purities of the resulting fractions were analyzed. During these runs, the process details were steadily optimized and buffer compositions, gradients, etc. were employed until the final optimized process details (see Figure 1) were established. The results showed that, on a laboratory scale, approximately 50 to 100 mg of the polypeptide of SEQ ID NO: 4 could be produced from a single consistent production run. Notably, by SDS polyacrylamide gel electrophoresis followed by Coomassie staining or silver staining, the resulting product was consistently found to be relatively pure (less than 5% contaminating host cell proteins and only trace amounts of truncated NGF, data not shown).

[0392] For the precursor of the polypeptide of SEQ ID NO: 4, a meaningful SE-HPLC method could not be established. In contrast, SE-HPLC analysis of the mature polypeptide of SEQ ID NO: 4 was straightforward and yielded a homogeneous product peak of approximately 16 kDa, which corresponded to the monomeric state of the polypeptide of SEQ ID NO: 4n (data not shown).

[0393] Summary and conclusion

[0394] For this process, SP Sepharose FF (“FF” stands for Fast Flow, i.e., a stationary phase with relatively large particles) was used to capture the refolded precursor of the polypeptide of SEQ ID NO: 4, which was subsequently treated with trypsin to produce mature NGF. For this purpose, the arginine concentration of the refolding reaction was reduced from 1 M (as recommended by the prior art) to 350 mM.

[0395] Controlling the proteolytic cleavage of the polypeptide precursor of SEQ ID NO: 4 to produce the mature polypeptide of SEQ ID NO: 4 is considered the most critical factor in this process. Here, conditions were determined that both reproducibly promote efficient cleavage and prevent the formation of NGF degradation products. The experimental data herein show that a significantly robust production process can be established over a fairly wide range of enzyme / substrate ratios. For trypsinization, the step yields are apparently good and no significant losses are expected at this stage of the process. The product patterns obtained are apparently not strongly dependent on the reaction conditions used (in terms of enzyme / substrate ratio and incubation time (time of exposure to the protease)). Notably, even with good yields of enzyme purification expected, at least 2×x grams of the polypeptide of SEQ ID NO: 4 must be processed to deliver x grams of the mature polypeptide of SEQ ID NO: 4.

[0396] Purification according to this example is a lean process consisting of only two chromatographic purification steps. The existing purification process was further optimized and several aspects were scaled up (see Figure 1). The exemplary, previously used cell disruption method was replaced by high-pressure homogenization, and all dialysis steps could be replaced by tangential flow filtration. The method so established is capable of delivering the polypeptide of SEQ ID NO: 4 in high purity.

[0397] Despite the above challenges, the entire process is capable of delivering a product of acceptable quality. In particular, the polypeptide of SEQ ID NO: 4 produced by this method is in a state that is substantially free of polypeptide degradation products, especially substantially free of the des-nona variant of the polypeptide. Thus, this method produces a polypeptide of high purity.

[0398] Figure 1 schematically depicts the complete process incorporating the improvements according to Example 2, including membrane chromatography.

[0399] Example 2 can be scaled up for the production of the polypeptide on an industrial scale.

[0400] Example 3: Preparation of the formulation of the present invention

[0401] Taking into account the recommendations of Eng et al., 1997, Anal. Chem., volume 69, pages 4184 - 4190, a formulation containing the polypeptide of SEQ ID NO: 4 previously prepared according to Example 2 was prepared.

[0402] Specifically, the prepared formulation has the following composition:

[0403] 2 mg / mL of the polypeptide according to SEQ ID: 4 obtained as described in Example 2

[0404] 20 mM sodium acetate buffer

[0405] 20 mM methionine

[0406] pH 5.5

[0407] In particular, acetate is considered to be a suitable buffer for stabilizing NGF and its derivatives in the pH range from pH 5.0 to pH 5.8. Since NGF, and possibly also the polypeptides according to the invention, are sensitive to the oxidation of methionine residues, methionine is included in the formulation.

[0408] The following embodiments are particularly suitable for the preservation of the formulation:

[0409] - First embodiment: The above-mentioned formulation can be stored frozen at -70 °C (data not shown) and thawed before administration.

[0410] - Second embodiment: The above-mentioned formulation can be stored in a refrigerator, preferably in the temperature range from +2 to +8 °C (data not shown).

[0411] - Third embodiment: The above-mentioned formulation can be dried or lyophilized and then stored, for example, at room temperature (data not shown). It can be reconstituted before administration.

[0412] All of the above embodiments offer certain advantages when frozen, for example, at -20 °C, as this avoids the need for shipping with dry ice, thawing, and remixing after thawing, etc. In such embodiments, the formulation according to the invention overcomes certain inconveniences of prior art NGF formulations, such as oxervate (cenegermin) in particular.

[0413] Example 4: Proof of concept in non-human mammals

[0414] The aim of this example was to study the efficacy of the polypeptide of SEQ ID NO: 4 in the treatment and / or prevention of ophthalmic disorders in non-human animals.

[0415] This invention is based in part on experiments on animal models.

[0416] Studies on the administration of the polypeptide of SEQ ID NO: 4 to non-human animals are reported herein. The polypeptide of SEQ ID NO: 4 can be obtained in high purity by expression as described in Example 1 and purification as described in Example 2. It is formulated as described in Example 3.

[0417] Materials and methods

[0418] Optic nerve crush (ONC) model

[0419] Rats (Sprague Dawley, male, 180 - 200 g, Charles River, Italy) were housed in a temperature - and humidity - controlled chamber (12 - hour dark / light cycle, free access to food and water). Behavioral experiments were conducted between 9:00 am and 5:00 pm in a quiet temperature - controlled room (20 to 22 °C) with the operator blinded to the drug treatment status.

[0420] For optic nerve crush (ONC), rats were anesthetized with a mixture of ketamine and xylazine (90 mg / kg and 3 mg / kg, intraperitoneal, i.p.) and the optic nerve was accessed through an incision in the periorbital conjunctiva. The left optic nerve was crushed for 10 seconds at 1 mm from the optic disc using a cross - action forceps applying a constant and uniform force. All procedures were performed on the left eye under sterile conditions. ONC was induced in the left experimental eye while the right eye served as an internal control. Before and after surgery, the fundus was observed through a surgical microscope to evaluate the integrity of retinal blood flow.

[0421] In the first experimental setting, retinas were dissected and immunodetected for retinal ganglion cells at 4, 7, and 14 days after ONC to evaluate the time - course of retinal damage. The un - touched and untreated contralateral right eye was used as a control for ONC - induced retinal ganglion cell loss. Day 7 after ONC was selected as the optimal time - point for a preliminary study of the effect of the polypeptide of SEQ ID NO: 4 because approximately 50% of the retinal ganglion cell population remained.

[0422] Rats were treated with drugs according to a rescue treatment protocol after intravitreal injection and eye drops to study the effect of the polypeptide of SEQ ID NO: 4 in an experimental setting that could potentially translate to the clinic:

[0423] · Treated with vehicle or the polypeptide of SEQ ID NO: 4: 20, 2, 0.2 μg / ml solutions; intravitreal injections of 2 μl volume were performed starting from day 4 after injury until day 7.

[0424] · Treated with vehicle or the polypeptide of SEQ ID NO: 4: 200 μg / ml solution; eye drops of 25 μl volume were performed starting from day 4 after injury until day 7, once, twice, or three times a day.

[0425] After treatment, retinas were dissected and immunodetected for retinal ganglion cells to quantify retinal damage and the effects of different treatments with the polypeptide of SEQ ID NO: 4.

[0426] Immunohistochemistry

[0427] The dissected retinas were post-fixed in formalin for 24 h and paraffin-embedded. Some formalin-fixed paraffin-embedded sections (5 μm) were stained with hematoxylin and eosin (H&E) for histological examination. Other sections were deparaffinized and rehydrated by successive incubation in xylene and a descending ethanol series (100%, 90%, and 70%). Antigen retrieval was achieved by microwaving the sections in 10 mM citrate buffer (pH 6.0) for 5 min. The sections were incubated with primary antibodies (Brn3a, 1:500; and RBPMS 1:500) in a humid chamber for 1 h. The sections were then incubated with pre-diluted biotinylated anti-mouse / rabbit IgG secondary antibody or biotinylated anti-guinea pig secondary antibody (1:200, #BA-7000, Vector Laboratories, Burlingame, USA) at room temperature for 30 min and then incubated in avidin-biotin complex solution (#PK-7200, Vectastain Elite ABC-HRP kit, Vector Laboratories, Burlingame, USA) at room temperature for 30 min. The sections were then transferred to peroxidase substrate (#K3468, ImmPACT DAB, Vector Laboratories, Burlingame, USA) for 4-6 min for color development reaction and rinsed with distilled water before mounting. The nuclei were counterstained with Mayer's hematoxylin. The tissues were visualized and digital images were captured using an optical microscope Leica DM2500 (Leica Microsystems, Milan, Italy).

[0428] After low-power magnification selection of immunopositive cells, total cells, Brn3a+, and RBPMS+ cells were quantified at high power fields (HPF, X400, Leica Microsystem) in a box of 7×10 4 μm 2 . For each rat, 3 different sections of each eye were analyzed.

[0429] Data (mean ± SEM) were compared using one-way ANOVA and then post hoc comparisons were performed using Bonferroni correction. GraphPad Prism (version 5.00, La Jolla, USA) was used for all analyses and P < 0.05 was considered statistically significant.

[0430] Results

[0431] In rats that began receiving intravitreal vehicle treatment on day 4 after ONC, consistent with the data generated, a reduction of approximately 60% in the number of retinal ganglion cells was observed during model establishment. The polypeptide of SEQ ID NO: 4 administered intravitreally at 3 different dose levels (20, 2, 0.2 μg / ml solution; injection volume 2 μl) exerted a dose-dependent protective effect on retinal ganglion cell loss, which reached statistical significance in rats receiving intravitreal injection with the 20 μg / ml solution. In this group, the number of retinal ganglion cells in the eyes subjected to ONC was similar to that in the healthy contralateral eyes( Figure 3 ).

[0432] In addition, in the group of rats that suffered ONC and began receiving vehicle eye drops treatment on day 4 after injury, the loss of retinal ganglion cells was superimposed compared to the loss quantified during the model establishment phase and in the experiment evaluating the effect of the polypeptide of SEQ ID NO: 4 after intravitreal injection. The rescue treatment effect of the eye drops of the polypeptide of SEQ ID NO: 4 (200 μg / ml) was studied by applying 3 different treatment regimens: once, twice, or three times a day. The data generated showed that the bioactivity of the 200 μg / ml solution administered three times a day was significantly improved, and the efficacy trend was also obvious even after administration once and twice a day( Figure 4 ).

[0433] Conclusion: The rescue effect of the polypeptide of SEQ ID NO: 4 administered four days after optic nerve injury was completely unexpected and opens the possibility that this compound could be effective in restoring the function of patients suffering from optic nerve and other retinal diseases due to trauma, ischemia, inflammation, metabolic, or tumor causes whenever RGC loss is part of the pathological process.

[0434] Example 5: Comparison of the nociceptive behavior and facial allodynia induced by the polypeptide of SEQ ID NO: 2 and the polypeptide of SEQ ID NO: 4 after ocular administration in mice

[0435] The objective of this example was to study the efficacy of the polypeptide of SEQ ID NO: 4 in the treatment and / or prevention of ophthalmic disorders in non-human animals.

[0436] This invention is based in part on experiments on animal models.

[0437] Studies on the administration of the polypeptide of SEQ ID NO: 4 to non-human animals are reported herein. The polypeptide of SEQ ID NO: 4 can be obtained in high purity by expression as described in Example 1 and purification as described in Example 2. It is formulated as described in Example 3.

[0438] The objective of this example:

[0439] 1. This example aims to compare the pain-inducing activities of the polypeptide of SEQ ID NO: 4 after ocular instillation with wild-type human NGF (the polypeptide of SEQ ID NO: 2) and wild-type mouse NGF (mNGF) in a mouse model of constriction of the infraorbital nerve (CION).

[0440] 2. The effects of the NGF analogues (the polypeptide of SEQ ID NO: 4) will be evaluated by comparing their ability to induce acute nociceptive responses after ocular instillation with the polypeptide of SEQ ID NO: 2, mNGF, and the pungent reference compound capsaicin, which is known to induce nociceptive responses. The following doses will be tested: 0.5, 1, 5, and 10 μg, 5 μl / eye, diluted with isotonic saline (0.9% NaCl). The test concentration of capsaicin will be 0.001 - 0.5 nmol / 5 μl / eye.

[0441] 3. Then the ability of subthreshold doses of the different compounds to induce nociceptive responses after ocular instillation will be tested in a constriction of the infraorbital nerve (CION) model.

[0442] Materials and methods

[0443] Mouse NGF (mouse NGF, mNGF)

[0444] Mouse NGF is highly purified native mouse NGF 2.5S (>95%) and was obtained by extraction and purification from the submandibular glands of mice according to the method described by Boccini et al. 1969, Pro. Natl. Acad. Sci. U S A, volume 64, pages 787 - 794. mNGF consists of residues 129 - 241 of the UniProt P01139 polypeptide sequence.

[0445] In vivo model of nociception

[0446] Animal experiments will be conducted in accordance with the EU guidelines for animal care procedures and Italian legislation (DLgs 26 / 2014) of EU Directive 2010 / 63 / EU. The study will be carried out under the University of Florence research permit #194 / 2015 - PR. C57BL / 6 mice (male, 25 - 30 g, Envigo, Milan, Italy) will be used for nociceptive testing. The animals will be housed in a temperature - and humidity - controlled chamber (12 - hour dark / light cycle, free access to food and water). Behavioral experiments will be performed between 9 am and 5 pm in a quiet temperature - controlled room (20 to 22 °C) by an operator who is unaware of the drug treatment status.

[0447] Contraction of the infraorbital nerve (CION)

[0448] It is reported that CION will be performed in C57BL / 6 mice (Vos et al., 1994, J. Neurosci., Vol. 14, pp. 2708 - 2723; Luiz et al., 2010, Neuropeptides, Vol. 44, pp. 87 - 92). Briefly, mice will be anesthetized by intraperitoneal (ip) injection of a mixture of ketamine (90 mg / kg) and xylazine (3 mg / kg), and the skin on the nasal side of the left upper lip will be incised to expose the rostral end of the infraorbital nerve. Then, two loose constricting ligatures (#6 / 0 silk suture) will be placed around the infraorbital nerve at a distance of 2 mm. In the sham operation, the left infraorbital nerve will be exposed but not ligated. Neomycin sulfate and sulfathiazole (powder, 0.05 g and 9.95 g respectively; Boehringer Ingelheim Italia S.p.A, Italy) will be applied to the wound and the incision will be sutured. The mice will be monitored, adequately hydrated and kept at a controlled temperature (37 °C) until fully recovered from anesthesia. All experiments will be performed on the 10th postoperative day. At the end of the experiment, the animals will be euthanized with inhaled CO2 plus 10 - 50% O2.

[0449] Mouse eye rubbing test

[0450] As previously described, ocular instillation (5 μl) of the test compound, the polypeptide of SEQ ID NO: 4, the polypeptide of SEQ ID NO: 2, mNGF (all, 0.5, 1, 5 and 10 μg, 5 μl / eye) and capsaicin (0.5 nmol / 5 μl / eye) or their respective vehicles (isotonic saline, 0.9% NaCl and 1% dimethyl sulfoxide, DMSO) will be used to induce an acute nociceptive response (DePetrocellis et al., 2010, Pharmacol. Res., Vol. 63, pp. 294 - 299). Mice will be placed individually in a plexiglass chamber and allowed to acclimatize for 20 minutes before stimulation. The number of eye rubbing movements after drug instillation into the eye will be recorded for a 5 - minute period and considered as an irritancy index.

[0451] On the 10th day after CION or sham operation, mice will receive ocular instillation (5 μl / eye) of a subthreshold dose of the polypeptide of SEQ ID NO: 2, mNGF and the polypeptide of SEQ ID NO: 4 or capsaicin, and the nociceptive response will be measured.

[0452] Results

[0453] In the first part of the example, the ability of different doses (0.001 - 0.5 nmol) of capsaicin administered by ocular instillation (5 μl / eye drop) to induce an acute nociceptive response was tested, and the number of eye rubs was measured over a 5-minute period. Capsaicin induced a dose-dependent nociceptive response, as evidenced by an increase in the eye rub response measured after capsaicin ocular instillation (0.001 - 0.5 nmol / 5 μl / eye)( Figure 5 ).

[0454] Next, different doses (0.001 - 5 μg 5 μl / eye) of mouse NGF (mNGF), human NGF (polypeptide of SEQ ID NO: 2), and polypeptide of SEQ ID NO: 4 were evaluated. All compounds induced a dose-dependent increase in the nociceptive response, which was measured as the number of eye rubs after ocular application. Compared with mNGF, and more importantly compared with the mutant form of the polypeptide of SEQ ID NO: 2, the application of the polypeptide of SEQ ID NO: 2 showed a stronger potency to induce a nociceptive response( Figure 6 ).

[0455] Next, the effects of subthreshold doses of different compounds in the chronic constriction injury of the infraorbital nerve (CION) model were evaluated. The CION model induced sensitivity to further nociceptive stimuli (Trevisan et al., 2016, Brain, 139(Pt 5), pp. 1361 - 1377).

[0456] On the 10th day after CION or sham surgery, mice received ocular instillation of subthreshold doses of capsaicin (0.001 nmol / 5 μl / eye), mNGF (0.001 nmol / 5 μl / eye), polypeptide of SEQ ID NO: 2 (0.001 nmol / 5 μl / eye), and polypeptide of SEQ ID NO: 4 (0.001 nmol / 5 μl / eye), and the nociceptive response of eye rubs was measured.

[0457] Data showed that in the sensitized model (CION model), compared with sham-operated mice, the nociceptive response produced by a subthreshold dose of capsaicin elicited a stronger response in CION-operated mice( Figure 7 ). The same result was obtained when mNGF and the polypeptide of SEQ ID NO: 2 were instilled into the eyes of CION-operated mice. Ocular instillation of the polypeptide of SEQ ID NO: 4 failed to induce an increase in the nociceptive response.

[0458] Example 6: Use in treating or preventing human ophthalmic disorders

[0459] The purpose of this example is to further support the efficacy of the polypeptide of SEQ ID NO: 4 in treating and / or preventing human ophthalmic disorders.

[0460] Those skilled in the art can determine the appropriate dosage of the polypeptide according to the guidance given herein.

[0461] The inventors expect to use in humans the same concentration (for eye drops at 200 μg / mL three times a day) found to be effective in rats with optic nerve injury, particularly for topical administration. Most preferably, it is conjunctival administration. Brief Description of the Drawings

[0463] Figure 1: Overview of the method according to Example 2, including the improvements described in Example 2B.

[0464] Figure 2: Polypeptide sequence. Asterisk (*) = position 61 in mature human NGF; cross (+): position 100 in mature human NGF.

[0465] A: Preprohuman NGF sequence encoded by the respective human open reading frame of SEQ ID NO:1.

[0466] Prepropeptide: amino acids 1 - 18; propeptide: amino acids 19 - 121; mature NGF: amino acids 122 - 239; C-terminal dipeptide: amino acids 240 - 241.

[0467] Disulfide bonds (in the correctly folded mature portion): connecting amino acid positions

[0468] Furin cleavage site (RSKR): amino acids 118 - 121.

[0469] B: Schematic diagram of prepropeptide, propeptide and mature NGF.

[0470] C: Sequence of mature human NGF of SEQ ID NO:2.

[0471] D: SEQ ID NO:3

[0472] E: SEQ ID NO:4

[0473] Figure 3 : Effect of the polypeptide of SEQ ID NO:4 (“CHF 6467”) after intravitreal administration. Statistical analysis was performed by One-Way-Anova, followed by Bonferroni post hoc analysis. N = 6 / group.

[0474] Figure 4 : Effect of the polypeptide of SEQ ID NO:4 after treatment with eye drops. Statistical analysis was performed by one-way analysis of variance, followed by Bonferroni post hoc analysis. N = 3 / group (see Example 4 for details).

[0475] Figure 5 : In C57BL / 6 mice, a dose-dependent eye-rubbing response induced by ocular instillation (5 μl / eye drop) of capsaicin (0.001 - 0.5 nmol). Error bars represent mean ± SEM, 6 - 8 mice per group. Veh is the vehicle for CPS. *P < 0.05, ***P < 0.001 vs. vehicle. One-way ANOVA with Bonferroni post-correction.

[0476] Figure 6 : In C57BL / 6 mice, a dose-dependent eye-rubbing response induced by ocular instillation (5 μl / eye drop) of murine NGF (mNGF), the polypeptide of SEQ ID NO: 2 (“human NGF”), and the polypeptide of SEQ ID NO: 4 (“mutated NGF”) (0.001 - 0.5 nmol). Error bars represent mean ± SEM, 6 - 8 mice per group. Veh is the vehicle for mNGF, the polypeptide of SEQ ID NO: 2, and the polypeptide of SEQ ID NO: 4. *P < 0.05, ***P < 0.001 vs. vehicle. One-way ANOVA with Bonferroni post-correction.

[0477] Figure 7 : In C57BL / 6 mice, the nociceptive responses produced by sub-threshold doses of capsaicin (0.001 nmol / 5 μl / eye), mNGF (0.001 nmol / 5 μl / eye), the polypeptide of SEQ ID NO: 2 (0.001 nmol / 5 μl / eye), and the polypeptide of SEQ ID NO: 4 (0.001 nmol / 5 μl / eye). Error bars represent mean ± SEM. Veh is the vehicle for capsaicin, mNGF, the polypeptide of SEQ ID NO: 2, and the polypeptide of SEQ ID NO: 4. *P < 0.05, ***P < 0.001 vs. vehicle. One-way ANOVA with Bonferroni post-correction.

Claims

1. A polypeptide selected from the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4, for treating and / or preventing ophthalmic disorders in a mammalian subject.

2. The polypeptide according to claim 1, wherein the mammalian subject is a human.

3. The polypeptide according to any one of the preceding claims, wherein the polypeptide is the polypeptide of SEQ ID NO:

4.

4. The polypeptide according to any one of the preceding claims, which is for administration to the eye.

5. The polypeptide according to claim 4, wherein the administration is selected from topical administration to the eye and intravitreal administration, and topical administration is preferred.

6. The polypeptide according to any one of the preceding claims, wherein the polypeptide is administered repeatedly.

7. The polypeptide according to claim 6, wherein the polypeptide is administered repeatedly at least three times a day.

8. The polypeptide according to claim 6 or claim 7, wherein the polypeptide is administered repeatedly for three to 30 days, preferably seven to 14 days.

9. The polypeptide according to any one of the preceding claims, wherein the ophthalmic disorder involves damage and / or disorder of the optic nerve.

10. The polypeptide according to claim 9, wherein the ophthalmic disorder is characterized by a disorder of retinal ganglion cells.

Images