Agent for treatment of dermatological disorders

Abstract

To provide an agent suitable for the treatment of subjects having dermatological disorders, without accompanying undesirable side effects.SOLUTION: The present invention provides a non-natural polypeptide having a specific sequence for treatment or prevention of skin disorders in a mammal. Administration of the polypeptide is well tolerated by the mammal. The non-natural polypeptide is provided at high purity.SELECTED DRAWING: Figure 26

Description

[Technical Field]

[0001] Introduction

[0002] FIELD OF THE INVENTION

[0003] The present invention provides medicaments suitable for the treatment and prevention of dermatological disorders, including but not limited to skin ulcers.

[0004] Background of the Invention

[0005] Skin disorders, including chronic wounds (e.g., ulcers), involve pain in the skin or mucous membranes and are often accompanied by tissue breakdown. Typically, such skin disorders can result in loss of the epidermis, and often a portion of the dermis, and even subcutaneous fat. Such skin disorders can be caused or initiated by a variety of factors, including, but not limited to, impaired blood circulation. Skin ulcers are common in humans, including subjects with diabetes (Ndip et al., 2012, Int. J. Gen. Med., vol. 5, pp. 129-134). Such skin disorders represent serious medical and social problems.

[0006] Currently, no suitable curative treatment is available for this type of dermatological disorder. According to current estimates, patients must be treated for months and in some cases for years, which incurs considerable costs and a high burden on patients and society (Buchberger et al., 2010, GMS Health Technol. Assess., vol. 1(6), doc. 12). In some cases, alternative measures, such as pressure relief through the use of cast removal devices, are the primary option for managing such disorders (Ndip et al., 2012, Int. J. Gen. Med., vol. 5, pp. 129-134), but such alternative measures do not provide any curative treatment.

[0007] Skin disorders frequently occur in diabetic patients. Diabetes is common in modern society, and it is estimated that one in four patients with diabetes will develop skin disorders, especially foot ulcers, during their lifetime (Ndip et al., 2012, Int. J. Gen. Med., vol. 5, pp. 129-134). This disorder often requires long-term hospitalization, rehabilitation, home care, and social services. Thus, ulcers as a result of diabetes are a serious problem with a significant impact on the global disease burden in light of the increasing prevalence of diabetes, and the current lack of curative treatment options is detrimental to the subjects involved as well as to society.

[0008] In the search for curative treatments, some human growth factors have been proposed in the past for the treatment and potential healing of skin disorders, but it is now accepted that there is limited evidence supporting the use of human growth factors in the treatment of skin ulcers (Ndip et al., 2012, Int. J. Gen. Med., vol. 5, pp. 129-134). One example of a growth factor previously considered for treating skin disorders is platelet-derived growth factor (becaplermin, trade name Regranex), but its administration has been found to be associated with severe side effects, including malignancies (Buchberger et al., 2010, GMS Health Technol. Assess., vol. 1(6), doc. 12; https: / / www.rxlist.com / regranex-side-effects-drug-center.htm#professional). Another example that has been tested is granulocyte colony-stimulating factor (G-CSF), but it was found that G-CSF did not significantly affect the resolution of infection or the likelihood of wound healing (Cruciani et al., 2005, Diabetes Care, vol. 28, pp. 454-460). A further example that has been proposed in the literature is epidermal growth factor (EGF), which was initially proposed to have unexpected positive healing effects (e.g., WO / 2003 / 075949 A1), but EGF-based treatments are not actually commercially available, suggesting that the initial hopes for this agent have not found support or confirmation.Alternatively, human nerve growth factor (hNGF), which has been proposed to have pro-angiogenic properties and promote wound repair (Graiani et al., 2004, Diabetologia, vol. 47, pp. 1047-1054), has been proposed for the treatment of certain neuropathic clinical conditions, but clinical testing has been disappointing (Apfel et al., 2000, J. Amer. Med. Assoc., vol. 284, pp. 2215-2221), and as a result, no NGF-based pharmaceuticals have been developed (see, e.g., https: / / www.gene.com / media / press-releases / 4875 / 1999-04-08 / phase-iii-trial-with-nerve-growth-factor). For example, Graiani et al. did not specifically comment on the allogenic effects of NGF, but it is generally known that human NGF induces pain (Dyck et al. 1997, Neurology, vol. 48, pp. 501-505; Svensson et al., 2003, Pain, vol. 104, pp. 241-247). As a result, human NGF has not been established as a suitable therapeutic agent for the treatment of skin ulcers, particularly due to its pain-inducing activity and / or lack of efficacy at tolerated doses. Consequently, given that growth factor-based treatments have had limited success or proven effective, particularly due to undesirable side effects, the medical community has investigated other potential drugs. Based on the above, interleukins and other non-growth factor molecules have more recently been proposed for the treatment of certain skin ulcers. For example, it has been proposed by Genentech that derivatives of interleukins, in particular interleukin 22 (which has a proposed role in regulating the immune system), may be suitable for treating or preventing skin ulcers (including diabetic skin ulcers) (see, e.g., https: / / www.gene.com / stories / mechanisms-of-healing), however, no such medicines are available to patients and it is now certain that this may eventually change.

[0009] Thus, there remains a need for effective treatment of skin conditions that do not suffer from adverse effects (such as intolerable or otherwise undesirable side effects), and for therapeutic agents suitable for such purposes that are available to practitioners in reliable and acceptable purity for administration to mammalian subjects (including humans).

[0010] A problem to be solved.

[0011] A primary object of the present invention is to provide a treatment or prevention for dermatological disorders (including ulcers) in diabetic and non-diabetic subjects that is not associated with unwanted or painful side effects. It is also desirable to provide therapeutically active agents in sufficient yield and purity to enable such treatment. Thus, an object of the present invention includes eliminating the disadvantages associated with the state of the art. A particular object includes providing a reliable method for treating subjects with dermatological disorders without unwanted side effects.

[0012] Summary of the Invention The present invention provides polypeptides for use in the treatment and / or prevention of dermatological disorders in mammalian subjects, wherein the polypeptide is selected between the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4. These polypeptides are characterized by a mutation in the amino acid sequence of human NGF (SEQ ID NO: 2), wherein said mutation is associated with reduced nociceptive activity. In particular, the arginine at position 100 of hNGF is replaced by glutamic acid.

[0013] A particularly preferred polypeptide is the polypeptide of SEQ ID NO: 4. Said polypeptide is characterized by the absence of proline at least at position 61, more preferably by the substitution of proline at position 61 with another amino acid. In SEQ ID NO: 4, the proline at position 61 of SEQ ID NO: 3 is substituted with serine.

[0014] Preferably, the mammalian subject is a human.

[0015] Preferably, the dermatological disorder is characterized by a wound surface on at least a portion of the subject's body. Preferably, the dermatological disorder is characterized by a wound surface. More preferably, the dermatological disorder is a skin lesion, preferably characterized by at least partial excision of the dermis, and optionally excision of the dermis.

[0016] Preferably, the dermatological disorder comprises at least one ulcer, preferably selected from the group consisting of a diabetic ulcer, a traumatic ulcer, a surgical ulcer, a pressure ulcer, a chronic ulcer, and any combination of these ulcers. In alternative, but not mutually exclusive, embodiments, the dermatological disorder comprises a burn or a mechanical injury.

[0017] Preferably, the mammal, preferably a human, suffers from or is predisposed to suffer from diabetes mellitus. In an exemplary embodiment, the diabetes mellitus is selected between type 1 diabetes mellitus and type 2 diabetes mellitus.

[0018] In one embodiment, the polypeptide is administered in a single dose.

[0019] In an alternative more preferred embodiment, the polypeptide is administered repeatedly. In a particularly preferred embodiment, the polypeptide is administered repeatedly 1 to 5 times per day, preferably 2 times per day.

[0020] In one embodiment, the polypeptide is repeatedly administered until the wound surface is closed. Alternatively, the polypeptide is repeatedly administered over a period of 3 to 30 days, preferably 7 to 14 days. Optionally, administration is discontinued after the completion of the interval.

[0021] In one embodiment, the polypeptide is administered to a subject with a diabetic neuropathic foot ulcer (DFU), preferably to the subject's foot below the ankle.

[0022] Preferably, the polypeptide is for topical administration. More preferably, the polypeptide is administered to a wound on the body surface.

[0023] Preferably, the dose of polypeptide to be administered is determined based on the area of ​​the wound body surface to be treated. Preferably, this determination is made at the beginning of treatment. In one embodiment, the dosage is adjusted for subsequent administrations depending on the area of ​​the wound body surface at the time of such subsequent administration. In an alternative embodiment, the dosage is not adjusted for subsequent administrations, so that the dosage of administration depends only on the area of ​​the wound body surface to be treated at the start of administration (first administration), and subsequent administrations correspond to the initial dose.

[0024] In one embodiment, the dose / each dose is calculated based on the mm of wound body surface to be treated. 2 Amount of polypeptide of 0.3 to 6 μg per unit volume (0.3 to 6 μg / mm 2 )

[0025] In one embodiment, the polypeptide is contained in an aqueous medium, and the aqueous medium is administered to a mammalian subject.

[0026] Preferably, the treatment and / or prevention does not result in hyperalgesia in the mammalian subject.

[0027] In one embodiment, the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 is obtainable from a biological source. This may involve purification, i.e., separation from other molecules, including other proteins (e.g., host cell proteins). Optionally, the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 is obtainable in a process that includes (re)folding and / or chromatographic purification and / or protease digestion, and optionally adjustment to a final protein concentration and preparation of a desired formulation. In one embodiment, the polypeptide is obtainable by recombinant expression and purification, wherein purification includes mixed-mode stationary phase purification.

[0028] Thus, 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 methods for therapeutic treatment of the human or animal body as described herein.

[0029] Detailed Disclosure of the Invention

[0030] The specification in its entirety, together with the claims and drawings, discloses particular and / or preferred embodiments and variations of individual features of the invention. The present invention also contemplates, as particularly preferred embodiments, those embodiments produced by combining two or more of the particular and / or preferred embodiments and variations described herein for the invention. As such, the present disclosure also includes all of the entities, compounds, features, steps, methods, or compositions referred to or shown herein, and any and all combinations of said entities, compounds, features, steps, methods, or compositions, or any two or more of said entities, compounds, features, steps, methods, or compositions, individually or collectively. Thus, unless specifically stated otherwise herein or unless the context requires otherwise, reference to a single entity, compound, feature, step, method, or composition shall be interpreted to encompass one and more (i.e., more than one, e.g., two or more, three or more, or all, etc.) of that entity, compound, feature, step, method, or composition. Unless specifically stated otherwise or unless the context requires otherwise, each embodiment, aspect, and example disclosed herein shall be construed as being applicable to and combinable with any other embodiment, aspect, or example disclosed herein.

[0031] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. Thus, the disclosure is not limited in scope by the specific embodiments described herein, which are provided herein for purposes of illustration and illustration. Functionally or otherwise equivalent entities, compounds, features, steps, methods, or compositions are within the scope of the disclosure. It will be apparent to those skilled in the art that the disclosure includes all variations and modifications of the entities, compounds, features, steps, methods, or compositions literally described herein.

[0032] Each of the references cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, presentations, etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein should be construed as an admission that the invention is not entitled to antedate any particular teaching and / or that any particular reference, other than common general knowledge, contains information sufficiently clear and complete for it to be practiced by one of ordinary skill in the art.

[0033] Generally, unless specifically defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., medicine, dermatology, neurology, genetics, molecular biology, gene expression, cell biology, cell culture, immunology, neurobiology, chromatography, protein chemistry, and biochemistry). Published textbooks and review articles (e.g., in English) typically define the meanings as commonly understood by one of ordinary skill in the art.

[0034] The expression "and / or", e.g., "X and / or Y", shall be understood to mean either "X and Y" or "X or Y", and shall be interpreted as providing an explicit disclosure of "and", "or", and both meanings ("and" or "or").

[0035] As used herein, unless otherwise specified, the terms "about," "approximately," and "substantially" all mean approximately or nearly, and in the context of any numerical value or range set forth herein, preferably specify + / - 10%, more preferably + / - 5%, around the recited or claimed numerical value or range.

[0036] Unless expressly specified otherwise, the word "comprise", or variations such as "comprises" or "comprising", are used in the context of this document to indicate that further members may optionally be present in addition to the members of the list introduced by "comprising". It is however contemplated that, as a specific embodiment of the present invention, the term "comprising" encompasses the possibility that no further members are present, i.e., for the purposes of this embodiment, "comprising" should be understood as having the meaning of "consisting of".

[0037] Unless otherwise expressly specified, all indications of relative amounts in the present invention are made on a weight / weight basis. An indication of a relative amount of a component characterized by a general term is meant to refer to the total amount of all specific variants or members encompassed by said general term. When a specific component defined by a general term is specified to be present in a specific relative amount, and this component is further characterized as a specific variant or member encompassed by the general term, no other variants or members encompassed by the general term are additionally present, and the total relative amount of the components encompassed by the general term exceeds the specified relative amount; more preferably, no other variants or members encompassed by the general term are present at all.

[0038] All AI methods and processes described herein can be performed in any suitable order unless otherwise indicated herein or unless the context clearly dictates otherwise.

[0039] As used herein, the term "drug" generally refers to a compound or composition, preferably a compound, unless otherwise specified. A drug can produce an effect by acting on a living organism and / or on cells from or derived from a living organism, e.g., on cells and / or bodily tissues, or in the environment. The physical state of the drug is not particularly limited, and unless otherwise specified, the drug can be in air, water, and / or a solid state. The type of drug is not particularly limited, and thus, unless otherwise specified, the drug can be a chemical substance and / or a biological molecule (e.g., a protein or nucleic acid, etc.). Specific drugs as defined herein are useful in the present invention.

[0040] "Adverse effects," as used herein, are unwanted, harmful effects resulting from the administration of a pharmaceutical agent (drug) to a subject. Adverse effects include, but are not limited to, morbidity, mortality, hyperalgesia, pain, changes in body weight, enzyme levels, loss of function, or any pathological change detected at a microscopic, macroscopic, or physiological level. Adverse effects can result in reversible or irreversible changes (including increases or decreases in an individual's sensitivity to other chemicals, foods, or procedures, such as drug interactions).

[0041] As used herein, the terms "chromatography," "chromatographic," and the like generally refer to techniques suitable for the separation of mixtures, in which the mixture is applied to a non-liquid material called a "stationary phase," with the goal of at least partially separating one or more components of the mixture. To that end, the stationary phase may be exposed to a liquid, and / or the mixture may be dissolved in a liquid; the fluid in contact with the stationary phase may also be referred to as a "mobile phase." In general, any process "performed by chromatography," as described herein, may be synonymously referred to as a "chromatographic process."

[0042] The term "mobile phase," as used herein, has the meaning typically used in the art and can refer to all liquids that come into contact with the stationary phase during chromatography, i.e., wash liquids, as well as the liquid (mixture) containing the protein of interest, such as one or more of the proteins described herein. In the present invention, the mixture subjected to chromatography typically contains one or more proteins, such as, in particular, the proteins described herein, such as the polypeptides of SEQ ID NO: 3 or 4, precursors of any of these, proteases, and / or host cell proteins (HCPs), as specified herein.

[0043] A "stationary phase" typically comprises a base matrix, which is a water-insoluble material, usually in particulate or gel form (e.g., a resin). In many cases (including the embodiments described herein), the stationary phase comprises a base matrix and a moiety that can bind to at least one component contained in a mixture to be chromatographed. The base matrix is ​​usually a water-insoluble material, usually in particulate or gel form. Non-limiting examples of base matrices are sepharose and agarose, e.g., highly rigid agarose.

[0044] As used herein, a "chromatographic step" refers to the act of adding to a chromatographic material (preferably a stationary phase) a liquid comprising at least one compound to be analyzed and / or purified, preferably a protein (and in the context of the present invention, said protein is most preferably the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4), optionally washing the chromatographic material with one or more wash solutions, and eluting said at least one compound. In that context, a process characterized by two chromatographic steps for illustration purposes is characterized in that a liquid comprising at least one such compound to be analyzed and / or purified is added to a first chromatographic material, as described above, and after elution therefrom, a liquid comprising at least one such compound is added to a second chromatographic material, from which it is also eluted, as described above. The aim of any "chromatographic step" is the binding of at least one component to the stationary phase, preferably comprised in the mixture applied to chromatography. Such a compound may be one or more proteins as described herein. The compound may be recovered from the stationary phase, for example, by exchanging the mobile phase and / or by continued exposure to the mobile phase over time.

[0045] The term "binding," when used in reference to chromatography (e.g., to describe the binding capacity of a stationary phase), is not particularly limited, but typically refers to a non-covalent bond. Thus, typically, at least one component (e.g., at least one protein) contained in the mixture is non-covalently bound to the stationary phase. The chromatography step optionally, but preferably, includes washing the stationary phase to which the at least one component is bound. The at least one component can be at least one protein, such as at least one protein described herein.

[0046] The term "heterologous" as used herein describes something that is made up of multiple different elements.

[0047] The terms "disulfide" and "disulfide bond" are used within the meaning commonly used in the art in the context of the present invention. Generally, "disulfide" refers to a functional group with the structure RSS-R'. This linkage, also known as an "SS bond," is usually generated by the coupling of two thiol groups. Disulfide bonds in proteins are formed between the thiol groups of cysteine ​​residues by the process of oxidative folding; such a specific disulfide bond between the thiol groups of two cysteine ​​residues may also be referred to as a "disulfide bridge." Without wishing to be bound by any particular theory, it is generally understood in the art that in eukaryotic cells, disulfide bridges are formed in the lumen of the endoplasmic reticulum (and the mitochondrial intermembrane space), but generally not in the cytosol, and that for prokaryotes, disulfide bridges are formed in the periplasm (of the respective organisms, particularly gram-negative bacteria); disulfide bridges are also found in proteins in the extracellular environment of both eukaryotic and prokaryotic cells.

[0048] The terms "express," "expressed," and "expression," "gene expression," and the like, as used herein, 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 of many additional features, optionally selected from an open list, including translation and post-translational modifications. In the context of recombinant expression of a protein in a host cell, the term usually 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 dictates otherwise.

[0049] As used herein, the term "heterologous" describes something that is composed of multiple different elements or origins. For example, in a non-human host cell that contains a human gene (or a gene encoding a non-native polypeptide, such as a polypeptide of the invention), the gene is "heterologous" to the cell, and the cell may be capable of "heterologous" expression of the respective gene. Heterologous gene expression may also be referred to as "recombinant."

[0050] The term "inclusion body" has the meaning typically used in the art and is meant to refer to aggregates or particles found in the cytosol or periplasm of a host cell; inclusion bodies typically contain proteins, such as proteins that are 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 is understood that inclusion bodies typically contain, at least in part, improperly folded proteins (misfolded proteins), particularly misfolded recombinantly expressed proteins. It is understood that inclusion bodies typically contain proteins in an improperly folded form, i.e., in the context of the present invention, they typically contain the polypeptide according to the present invention and / or its precursor in an improperly folded form. The term "misfolded" generally describes a biological molecule (such as a nucleic acid or polypeptide) that is not in its native conformation, i.e., is in an improperly folded form.

[0051] By "isolated" is meant material that is substantially or essentially free from components that normally accompany it in its native state. For example, an "isolated peptide" or an "isolated protein," as used herein, refers to a peptide or protein that has been purified from the cellular and extracellular environment (e.g., tissue, etc.) that surrounds it in its naturally occurring state, e.g., from the cell (e.g., host cell, etc.) in which it was expressed. In alternative descriptions, an "isolated peptide" or an "isolated protein," etc., as used herein, refers to the in vitro isolation and / or purification of a peptide or protein from its native cellular environment and from association with other components of the environment in which the peptide or protein normally resides, respectively. In another example, an "isolated cell," as used herein, refers to a cell that has been purified from the cellular and extracellular environment (e.g., tissue or cell colony, etc.) that surrounds it in its naturally occurring state, e.g., a host cell that has been removed from the environment normally adjacent to the cell. In accordance with the above definition of the word "isolated," "to isolate," as used herein, is a verb describing an activity to obtain "isolated" material, such as, for example, an isolated cell or an isolated peptide or protein.

[0052] As used herein, the terms "plurality" and "plurality" mean a large number, i.e., any number of two or more.

[0053] The term "mutation," as used herein, refers to a change in the nucleotide sequence of the genome of an organism, virus, or extrachromosomal DNA or other genetic element. This term also encompasses mutations in amino acid sequences, particularly those of genes with at least one (non-silent) mutation. Unless otherwise specified, a mutation in a nucleotide sequence is a permanent change. Mutations present in the germline are usually inherited. Generally, a mutation in a nucleotide sequence can result in many different types of changes in the sequence: a mutation in a gene can have no effect, alter the gene product, or prevent the gene from functioning properly or fully. Mutations can also occur in nongenic regions. Unless otherwise specified, the wild-type sequence is used as the reference sequence to describe a mutation. Thus, for example, if a given variant is said to be characterized by a mutation at position 100 of a polypeptide sequence, this indicates that the variant does not have the same amino acid residue at position 100 as the wild-type polypeptide. Specific types of mutations in nucleotide and / or amino acid sequences include alterations such as deletions, substitutions, additions, insertions, and splice variants. A "deletion" with respect to a nucleotide sequence refers to the absence of one or more nucleotides in a nucleotide sequence. A "deletion" with respect to an amino acid sequence refers to the absence of one or more amino acid residues in a polypeptide. An "addition" with respect to a nucleotide sequence refers to the presence of one or more additional nucleotides in a nucleotide sequence. An "addition" with respect to an amino acid sequence refers to the presence of one or more additional amino acid residues in a related polypeptide. A "substitution" with respect to a nucleotide sequence refers to the replacement of one or more nucleotides with other nucleotides in a nucleotide sequence. A "substitution" with respect to an amino acid sequence refers to the replacement of one or more amino acid residues with other amino acid residues in a polypeptide. Additions, deletions, and substitutions to a nucleotide sequence, such as in an open reading frame, can be at the 5'-terminus, 3'-terminus, and / or internally. Additions, deletions, and substitutions to a polypeptide can be at the amino-terminus, carboxy-terminus, and / or internally.An "insertion" in reference to a nucleotide sequence and / or polypeptide sequence is the addition of one or more nucleotides or one or more amino acid residues, respectively, especially at an internal position in the respective sequence. The term "splice variant" is used to describe that an RNA encoding a polypeptide sequence is spliced ​​differently from the respective wild-type RNA, typically as a result of a mutation at the nucleic acid level, usually resulting in a polypeptide translation product that differs from the wild-type polypeptide. The term "splice variant" can be used not only in reference to the respective RNA, but also in reference to the respective template DNA sequence (typically genomic DNA) and in reference to the sequence of the polypeptide encoded by such RNA.

[0054] The term "mutant" is generally intended to refer to a nucleic acid sequence or amino acid sequence that differs from the wild-type sequence. A mutant nucleic acid sequence or amino acid sequence thus has at least one mutation with respect to the respective wild-type sequence. In cases where polymorphisms in the nucleic acid sequence exist but are not reflected at the level of the respective encoded polypeptide (silent mutations, degeneracy of the genetic code), the term "mutant" at the nucleic acid level specifically refers only to those nucleic acid variants that encode the mutant polypeptide. Mutants can contain different combinations of mutations (including more than one mutation and different types of mutations) alone or in combination.

[0055] The term "nerve growth factor," abbreviated as "NGF" or "beta-NGF," refers, according to its common meaning in the art, to a neurotrophic factor and neuropeptide involved in regulating the growth, maintenance, proliferation, and survival of certain neurons and other cells (see, e.g., Levi-Montalcini, 2004, Progress in Brain Research, vol. 146, pp. 525-527). Unless the context dictates otherwise, the term "nerve growth factor" refers only to wild-type NGF and does not include the polypeptide of SEQ ID NO: 3 or 4. Wild-type NGF is the 2.5S, 26 kDa beta subunit obtainable from the NGF precursor, which is biologically active: wild-type NGF binds to at least two classes of receptors: tropomyosin receptor kinase A (TrkA) and low-affinity NGF receptor (LNGFR / p75NTR). The term "NGF," unless otherwise specified, refers to NGF of any species, preferably a mammalian species; however, human NGF is always preferred. "hNGF," as used herein, refers to human NGF. Unless the context dictates otherwise, the terms "NGF" and "hNGF" refer to wild-type NGF, i.e., hNGF refers to wild-type NGF. The amino acid sequence of wild-type human NGF corresponds to positions 121-239 of SEQ ID NO: 1 (gray in Figure 24). Sequences of non-human NGFs are available, for example, in the scientific literature, through sequence searches (e.g., BLAST, etc.) using positions 121-239 of SEQ ID NO: 1 as bait, and in public protein databases (e.g., Swissprot, etc.).

[0056] The terms "NGF mutein" and "mutein of NGF" (or with reference to "NGF mutein thereof") are used interchangeably herein and refer to a polypeptide characterized by at least one mutation compared to wild-type NGF, as described in more detail herein. The polypeptides of SEQ ID NO: 3 and SEQ ID NO: 4 are muteins of NGF. Preferably, the mutein of NGF has 80 to 99.5% sequence identity with NGF, particularly human NGF, and more preferably, the mutein has 90 to 99% sequence identity with NGF, particularly human NGF.

[0057] The term "mature portion" refers to NGF and is used interchangeably with the term "beta-NGF" to refer to a polypeptide of NGF characterized by not including the propeptide (and therefore, of course, not including the prepropeptide) of NGF. Similarly, the term "mature portion" is also used to refer to the polypeptide of SEQ ID NO: 3 or 4, because these polypeptides similarly do not include the propeptide (and therefore, of course, not including the prepropeptide). Preferably, the mature portion also does not include the 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 two amino acid residues "RA" (240 and 241 in SEQ ID NO: 1). In particular, the mature portion can be obtained by cleavage of proNGF or of the polypeptide of SEQ ID NO: 3 or 4, respectively, with, but not limited to, the protease furin and other proteases capable of cleaving precisely and directly N-terminal to the first amino acid residue of NGF. For example, the furin cleavage site of human NGF and of many orthologs is the sequence R 1 S 2 K 3 R 4 (single-letter amino acid code, sequence numbered from N-terminus to C-terminus; boxed in Figure 25). In mature NGF, the furin cleavage site or any amino acids N-terminal to the furin cleavage site are usually absent. For illustrative purposes, the mature portion of human NGF consists of the polypeptide represented by amino acid positions 122 to 239 of SEQ ID NO: 1. The mature portion of non-human NGF can be identified, for example, by sequence search and / or sequence analysis, in which the mature portion of human NGF is used for sequence alignment.

[0058] The term "precursor," as used herein with reference to NGF, refers to any peptide sequence from which NGF can be obtained by proteolytic cleavage. For illustrative purposes, both proNGF and preproNGF, as well as their variants, are typical examples of precursors of NGF. The term "precursor," as used herein, can refer to precursors whose most C-terminal amino acid residue is the most C-terminal residue of NGF, and also to precursors that extend C-terminally beyond the most C-terminal residue of NGF (as long as NGF can be obtained therefrom by proteolytic cleavage): While the naturally occurring precursor of wild-type human proNGF (SEQ ID NO: 1) contains a C-terminal dipeptide (amino acid residues 240 and 241 in SEQ ID NO: 1, bold in FIG. 1), it is preferred in the present invention that the precursor does not contain the 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 two amino acid residues "RA" (240 and 241 in SEQ ID NO: 1).

[0059] The terms "prepeptide" or "presequence," as used herein, generally refer interchangeably to a polypeptide sequence encoded by a portion of the NGF open reading frame immediately N-terminally adjacent to the propeptide. For illustrative purposes, a prepeptide is an NGF consisting of a sequence comprising the contiguous sequence ranging from residue 1 of SEQ ID NO:1 to residue 18 of SEQ ID NO:1. The sequences of the prepeptides of each of the precursors of non-human NGF are available, for example, in the scientific literature through sequence searches (e.g., BLAST) using positions 1-18 of SEQ ID NO:1 as bait, and in public protein databases (e.g., Swissprot). A polypeptide or protein consisting of a prepeptide and proNGF, in which the C-terminus of the prepeptide is immediately adjacent to the N-terminus of proNGF, may be referred to herein as "prepro-NGF."

[0060] The terms "propeptide" or "prosequence," as used herein, generally refer interchangeably to a polypeptide sequence naturally encoded by a portion of the NGF open reading frame immediately N-terminally adjacent to mature NGF, but where the polypeptide sequence does not include the prepeptide. For illustrative purposes, the propeptide is contained in the wild-type precursor of NGF. The propeptide of the precursor of NGF consists of a sequence comprising the contiguous sequence ranging from residue 19 of SEQ ID NO:1 to residue 121 of SEQ ID NO:1. The sequences of the respective propeptides of non-human proNGFs are available, for example, in the scientific literature, through sequence searches (e.g., BLAST, etc.) using positions 19-121 of SEQ ID NO:1 as bait, and in public protein databases (e.g., Swissprot, etc.).

[0061] As used herein, "proNGF" refers to a peptide sequence that includes both the mature portion of NGF and the respective propeptides, but not the respective prepeptides. Human proNGF consists of a sequence that includes the contiguous sequence ranging from residue 19 of SEQ ID NO:1 to at least residue 239 of SEQ ID NO:1. While wild-type human proNGF includes a C-terminal dipeptide (amino acid residues 240 and 241 in SEQ ID NO:1), it is preferred that the proNGF obtained and used in the present invention does not include the C-terminal cleavable peptide encoded by the wild-type NGF open reading frame; such a C-terminal cleavable peptide, in the case of human NGF, consists of the two amino acid residues "RA" (240 and 241 in SEQ ID NO:1). Sequences of non-human proNGFs are available, for example, in the scientific literature through sequence searches (e.g., BLAST) using positions 19-239 of SEQ ID NO:1 as bait, and in public protein databases (e.g., Swissprot).

[0062] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein and refer to both RNA and DNA (including cDNA, genomic DNA, synthetic DNA, and DNA / RNA equivalents containing nucleotide analogs, phosphate analogs, and / or sugar analogs). Nucleic acids can be double-stranded or single-stranded (i.e., sense or antisense). 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, any type of isolated nucleic acid and sequenced nucleic acid probe, and primers, as well as nucleic acid analogs. Nucleic acids can have any type of three-dimensional structure.

[0063] The term "peptide" according to the present invention includes oligopeptides and polypeptides and refers to a substance comprising two or more, preferably three or more, preferably four or more, preferably six or more, preferably eight or more, preferably ten or more, preferably thirteen or more, preferably sixteen or more, preferably twenty-one or more, and preferably 8, 10, 20, 30, 40, or 50, especially 100, amino acids covalently linked in a chain by peptide bonds.

[0064] The term "protein" preferably refers to large peptides, preferably peptides with more than 100 amino acid residues, but in general the terms "peptide," "polypeptide," and "protein" are synonymous and are used interchangeably herein unless the context dictates otherwise. Thus, the terms "polypeptide of SEQ ID NO: 4" and "protein of SEQ ID NO: 4" have the same meaning.

[0065] The term "pharmaceutically acceptable" generally describes that a particular substance can be administered to a subject, optionally, and preferably in combination with a drug, at the dosages used without the drug causing intolerable adverse effects.

[0066] The terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" refer to any one or more of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonicity and absorption delaying agents, etc. that are physiologically compatible and suitable for administration to a subject as described herein, or that do not otherwise 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. In the case of liquid pharmaceutical compositions in particular, it may be preferable to include an isotonicity agent, such as a sugar, a polyalcohol (e.g., mannitol, sorbitol, etc.), or sodium chloride in the composition. A pharmaceutically acceptable carrier may further contain auxiliary substances (e.g., wetting or emulsifying agents, preservatives, or buffers, etc.), which enhance the shelf life or effectiveness of the agent. A pharmaceutically acceptable carrier is typically included in the compositions of the present invention.

[0067] The term "pharmaceutically active agent" refers to an agent that can be used for administration to a subject, where the agent can be beneficial, for example, in ameliorating the symptoms of a disease or disorder. Also, a "pharmaceutically active agent" can have a positive or beneficial effect on the subject's condition or pathology when administered to a subject in a therapeutically effective amount. Preferably, a pharmaceutically active agent has curative properties and can be administered to ameliorate, alleviate, relieve, reverse, delay the onset of, or reduce the severity of, one or more symptoms of a disease or disorder. A pharmaceutically active agent can also have prophylactic properties and can be used to delay the onset of a disease or reduce the severity of such a disease or pathology. For example, the agents of the present invention, as claimed, are contemplated herein as pharmaceutically active ingredients for the treatment of cystic fibrosis. In another example, a pharmaceutically active protein can be used to treat cells or individuals that do not normally express a protein, or that do not express the protein at the desired level, or that incorrectly express the protein; for example, the pharmaceutically active protein can compensate for a mutation or lack of sufficiently high expression by providing the desired protein. The term "pharmaceutically active peptide or protein" includes the entire protein or polypeptide and can also refer to pharmaceutically active fragments thereof. It can also include pharmaceutically active analogs of the peptide or protein.

[0068] An "open reading frame" or "ORF" is a contiguous stretch of codons beginning with a start codon and ending with a stop codon.

[0069] The terms "subject" and "patient," as used herein, 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 (e.g., zoo animals, etc.). As used herein, the terms "subject" and "patient" particularly 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 is suffering from, at risk of suffering from, has suffered from, or is predicted to suffer from a condition, and who may be subjected to treatment, for example, by administration of a drug. A patient's condition may be chronic and / or acute. Thus, a "patient" may also be described as a subject who is subjected to and / or in need of treatment.

[0070] The term "treatment" should be understood broadly and refers to the treatment of a subject with the goal of preventing or treating a condition in the subject. In preferred embodiments, treatment specifically involves the administration of a drug to the subject.

[0071] The term "trypsin," as used herein, generally refers to a proteolytic enzyme classified as EC 3.4.21.4. Trypsin cleaves peptide chains primarily at the carboxyl side of the amino acids lysine or arginine, unless either is usually followed by proline. Without wishing to be bound by theory, it is understood that trypsin is a serine protease and is naturally found in the digestive systems of many vertebrates, where it hydrolyzes proteins. Preferred in the present invention is trypsin from a recombinant source. Although in vivo, trypsin is formed with a propeptide (called "trypsinogen"), the term "trypsin," as used herein, preferably refers to mature trypsin lacking any propeptide. The use of trypsin for proteolytic cleavage is also referred to as "trypsin proteolysis" or "trypsinization," and proteins resulting from cleavage with trypsin are said to be "trypsinized."

[0072] A "variant" of a precursor of NGF or of the polypeptide of SEQ ID NO: 3 or 4 refers to a polypeptide or protein in which the amino acid sequence, which is not part of mature NGF (beta-NGF) or is not part of SEQ ID NO: 3 or 4, respectively, is characterized by at least one mutation (preferably found at the N-terminus of the amino acid sequence of mature NGF (beta-NGF)) in comparison with a wild-type precursor of NGF, such as wild-type pro-NGF or wild-type pre-pro-NGF. Thus, as used herein, a "variant" of a precursor, such as NGF, refers to a peptide or protein in which the pre- and / or propeptide is characterized by at least one mutation, such as, but not limited to, the mutations described in WO 2013 / 092776 A1 and US 2018 / 0086805 A1, relative to the amino acid sequence of the pre- and / or propeptide. By way of example, WO 2013 / 092776 A1 describes "mutants" of proNGF in which the (wild-type) furin cleavage site is absent due to one or more specific mutations.

[0073] The term "vector" or "cloning vector" generally refers to a nucleic acid that can be introduced into a host cell. Examples of 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 includes vectors that encode peptides or proteins for heterologous expression (such vectors can serve as templates for the generation of transcripts), as well as non-encoding vectors. The first type of vector will contain an open reading frame encoding a protein or peptide, which can be expressed when the vector is present in a host cell. The type of vector that a skilled artisan can select will depend on the type of host cell that the skilled artisan can select; however, in certain cases, cloning vectors for all common host cells (including E. coli) are commercially available, and the skilled artisan will thus select a particular vector with due consideration of the host cell of choice.

[0074] 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 a "mutant" or "variant," etc. For illustrative purposes, SEQ ID NO: 1 shows the amino acid sequence of the precursor of wild-type human NGF; SEQ ID NO: 2 shows the amino acid sequence of wild-type human NGF.

[0075] The present invention is based on several discoveries, which are interrelated and which thus led the inventors to arrive at the various aspects of the invention, all of which are described individually below.

[0076] Medicaments according to the present invention

[0077] The present invention provides agents for the treatment and / or prevention of dermatological disorders in mammalian subjects. The agents can be used for administration to subjects where the agents will be beneficial, for example, in ameliorating the symptoms of a disease or disorder. In particular, agents useful in the present invention are polypeptides of SEQ ID NO: 3 or SEQ ID NO: 4. Thus, the present invention particularly provides polypeptides of SEQ ID NO: 3 or SEQ ID NO: 4 for use in therapy. Treatment typically involves administration of the polypeptide to the human or animal body, as described herein below.

[0078] According to the present invention, polypeptides of SEQ ID NO:3 and SEQ ID NO:4 are provided as pharmaceutically active agents. According to the present invention, polypeptides of SEQ ID NO:3 or SEQ ID NO:4 are provided for medical use, particularly for the treatment and / or prevention of dermatological disorders in mammalian subjects. Optionally, the polypeptides of SEQ ID NO:3 or SEQ ID NO:4 are from recombinant sources. Thus, the present invention also provides recombinant polypeptides of SEQ ID NO:3 or SEQ ID NO:4 for medical use, as described herein.

[0079] Agents according to the present invention are also referred to herein as "polypeptides of SEQ ID NO:3" or "polypeptides of SEQ ID NO:4," and are described in more detail herein. The term "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 with equivalent biological activity. 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 and / or an agent with equivalent biological activity. Thus, functionally equivalent portions or analogs of such polypeptides are also included within these terms. An example of a biologically equivalent portion of a polypeptide would 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 that enables 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 alternatively, a gene encoding such a polypeptide. The term "substantially the same biological activity" refers to 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:4 in the assays described in Examples 3 and 4. An example of a biologically equivalent analog of a polypeptide would be a fusion protein containing 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 could also be a homologous analog of the polypeptide. Also, a completely synthetic molecule that mimics a specific biological activity of the polypeptide of SEQ ID NO:3 or the polypeptide of SEQ ID NO:4 would constitute a "biologically equivalent analog."

[0080] More preferably, the term "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 comprising, inter alia, the amino acid sequence defined by SEQ ID NO:3. Most preferably, the term "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 consisting of 118 amino acid residues in the consecutive order defined by SEQ ID NO:3. In this and other embodiments, the polypeptide optionally has one, two, or three internal cysteine ​​bonds, and the cysteine ​​(Cys, C) residues are covalently linked to each other and form intramolecular disulfide bridges. The cysteine ​​bonds are preferably equivalent to those in wild-type human NGF.

[0081] Equally 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 an agent is optionally a fusion protein comprising, inter alia, 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 consisting of 118 amino acid residues in the consecutive order defined by SEQ ID NO:4. In this and other embodiments, the polypeptide optionally has one, two, or three internal cysteine ​​bonds, and the cysteine ​​(Cys, C) residues are covalently linked to each other to form intramolecular disulfide bridges. The cysteine ​​bonds are preferably equivalent to those in wild-type human NGF.

[0082] The polypeptides of the present invention may optionally be characterized by further post-translational modifications. Such post-translational modifications may optionally include glycosylation and / or phosphorylation. Preferably, however, the polypeptides according to the present invention do not contain glycosylation and / or phosphorylation. Indeed, considering that the experimental examples herein demonstrate beneficial effects and a beneficial benefit-to-harmful effect ratio on the healing of skin disorders, the polypeptides used are obtained by cytosolic recombinant expression in bacteria, which typically does not result in glycosylation and / or phosphorylation, and it is therefore plausible that the beneficial effects of the present invention are not dependent on such types of post-translational modifications. Therefore, in a preferred embodiment, the polypeptides according to the present invention are not characterized by glycosylation and / or phosphorylation.

[0083] Typically, the polypeptides according to the present invention are non-naturally occurring polypeptides that are not naturally produced by the subject to which the polypeptide is administered, which has the associated advantage not only of detectability in the subject after administration, but also of evidence that administration (from an external source, such as, for example, a composition prepared according to the present disclosure) needs to be administered to the subject to achieve success in treating or preventing the disorder.

[0084] Preferably, the polypeptides according to the present invention are isolated polypeptides. More preferably, the polypeptides according to the present invention are essentially free from host cell proteins, degradation products (e.g., des-nona variants), and proteases (e.g., trypsin). When the polypeptides according to the present invention are essentially free from host cell proteins, degradation products (e.g., des-nona variants), and proteases (e.g., trypsin), they can also be referred to as "pure polypeptides." Preferably, the polypeptides according to the present invention are administered as pure polypeptides. More preferably, the pure polypeptide consisting of SEQ ID NO: 3 and / or the pure polypeptide consisting of SEQ ID NO: 4 has a weight percentage of 90% or more, preferably 92% or more, more preferably 93% or 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, and more preferably 99.9% or more of the total protein in the composition. Such pure polypeptides are obtainable based on the disclosure herein, including Examples 1 and 2. Most preferably, pure polypeptides according to the present invention have a purity grade that meets Good Manufacturing Practice (GMP).

[0085] As demonstrated in the experiments herein, particularly in Examples 3 and 4, administration of the agent according to the present invention did not induce any hyperalgesic syndrome (pain), despite the fact that the agent directly contacted fully exposed nociceptive fibers (nerves); these were considered fully exposed due to the lack of skin, and they were considered overactivated as a result of the skin lesion. The absence of pain in this extreme setting is particularly remarkable, since the agent was administered topically and repeatedly to damaged skin, even in a chronic setting (see Examples for details). This is also particularly remarkable given the discouraging early studies using human NGF exposed to innervated areas, i.e., areas characterized by exposed nociceptors (Svensson et al., 2003, Pain, Vol. 104, pp. 241-247). This surprising finding cannot be explained solely by the fact that the agent according to the present invention has previously been described as "analgesic." This is because its ability to induce pain has never been experimentally studied in innervated areas, i.e., areas characterized by exposed nociceptors, not to mention overactivated nerves, as in the case of skin lesions. Moreover, even if administration of the agent according to the present invention is responsible for tissue reinnervation (see, e.g., Example 3), administration is not associated with pain. In addition, the positive effect of the agent according to the present invention on angiogenesis (see Experimental Examples) is surprising and not predictable based on the state of the art. Angiogenesis is understood to be particularly important for tissue formation and wound closure. In summary, the combination of these beneficial effects is very surprising in light of the state of the art.

[0086] Optionally, in accordance with the present invention, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is administered in an effective amount to a subject in need thereof. Details of administration, effective amounts, and subjects in need thereof are provided herein below.

[0087] The polypeptides consisting of SEQ ID NO: 3 and SEQ ID NO: 4, respectively, differ at one or two positions 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). The differences of the polypeptides according to the present invention relative to the polypeptide of SEQ ID NO: 2 have significant effects on the treatment or prevention of skin disorders and the absence of side effects, as disclosed in detail herein and supported by the experimental examples herein.

[0088] Nerve growth factor (NGF) is a neurotrophin required for the development and survival of specific neuronal populations. NGF is a homodimeric peptide that naturally induces neuronal proliferation and homeostasis. In the body, NGF binds to at least two types of receptors: tropomyosin receptor kinase A (TrkA) and low-affinity NGF neurotrophin receptor p75 (LNGFR / p75NTR / p75). Both are associated with specific disorders in humans and animals, although their mechanisms of action are likely distinct. Several therapeutic applications for NGF have been proposed, but few have matured for commercial use.

[0089] However, many of the therapeutic uses of NGF that have been envisioned in the past have not matured into commercially available therapeutic NGF products, and one reason for this, besides its desirable effects on neuronal proliferation and homeostasis, is that NGF is associated with pain: it can cause hyperalgesia when administered locally or systemically (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. 1080-1088). (pp. 241-247; Ruiz et al., 2004, Brain Res., vol. 1011, pp. 1-6). As a solution, mutant versions of NGF ("muteins") have been developed, which are associated with reduced nociceptive activity ("painless NG") and are characterized by at least one mutation in the domain of NGF that interacts with the TrkA receptor (WO 2008 / 006893 A1; Malerba et al. PLOS One, 2015, vol. 10, e0136425). However, such polypeptides have not yet been publicly available in pharmaceutically acceptable purity and, perhaps also due to the prejudice and generally negative experiences associated with research on growth factors in this therapeutic area, have not been proposed or developed for the treatment or prevention of dermatological disorders of the skin.

[0090] According to the present invention, the stability and, thus, long-term purity of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 can be obtained and / or improved by the aspects and embodiments described herein. Thus, the present disclosure not only makes available new treatments or preventions for dermatological disorders, but also provides agents suitable for such treatments or preventions in purity grades suitable for therapeutic applications (including administration to mammals). The agents of the present invention have not previously been publicly available in such advantageous purity grades.

[0091] The polypeptides of SEQ ID NO: 3 or SEQ ID NO: 4 are not found in nature and may also be referred to as non-naturally occurring polypeptides. Thus, the agents according to the invention are not wild-type NGF, and in particular are not wild-type human NGF.

[0092] Preferably, the non-natural polypeptides according to the present invention are provided in high purity. Optionally, the polypeptides contain internal disulfide bridges. Optionally, the polypeptides are properly folded. Optionally, the polypeptides are soluble in aqueous media.

[0093] The present invention is based in part on the experiment of two animal models of skin ulcer.In these models, skin ulcer is induced in diabetic mice by circular biopsy punch or by cycle of pressure loading, and the polypeptide of the present invention is applied topically.Compared with animals treated with placebo, the polypeptide induces significant and dose-dependent improvement in the healing time of ulcer.This improvement is evident at a dose that does not cause pain-related side effects, thus demonstrating the potential advantage over the state of the art.

[0094] In particular, data-generating in vivo models of diabetic skin ulcers have demonstrated that the polypeptides of the invention are painless yet retain activity targeting the NGF receptor system, thereby providing a therapeutic tool for the treatment or prevention of dermatological disorders. Indeed, the polypeptides of the invention retain the trophic properties of wild-type NGF for angiogenesis and reinnervation, which favor ulcer healing, without exerting the pro-nociceptive effects of wild-type NGF at the site of topical application and at the systemic level.

[0095] The present invention provides polypeptides for use in the treatment and / or prevention of dermatological disorders in mammalian subjects, wherein the polypeptide is selected from the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4. Thus, the present invention also provides the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for use in a method for use in the treatment of the human or animal body by therapy, as described herein.

[0096] In particular, the present invention relates to particular therapeutic uses of the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4, wherein the particular therapeutic use is the treatment and / or prevention of dermatological disorders in a mammalian subject. Thus, the present invention also provides the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 for use in methods for use in the treatment of the human or animal body by therapy, wherein the treatment includes the treatment and / or prevention of dermatological disorders in a mammalian subject. The mammalian subject is typically a subject characterized by a need for such treatment.

[0097] The polypeptide of SEQ ID NO: 3 as well as the polypeptide 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 reduced nociceptive activity. In particular, the arginine at position 100 of hNGF is replaced by glutamic acid. The present invention is based in part on the surprising finding that a therapeutic effect can be achieved without the side effects known from the prior art.

[0098] Without wishing to be bound by any particular theory, it is preferred that the polypeptides according to the present invention comprise one or more disulfide bridges, and most preferably three disulfide bridges. Mature and properly folded mature human NGF is characterized by three disulfide bridges (linkage positions 136←→201, 179←→229, 189←→231, position numbers refer to SEQ ID NO: 1; see Wiesmann et al., 1999, Nature, vol. 401, pp. 184-188). Without wishing to be bound by any particular theory, it is preferred that the polypeptides according to the present invention comprise the equivalent disulfide bridges (the position numbers are available to those skilled in the art by aligning the polypeptides according to the present invention with SEQ ID NO: 1 and the polypeptides of Wiesmann et al., supra).

[0099] A statement of the presence or absence of adverse effects

[0100] Preferably, the treatment and / or prophylaxis does not cause side or adverse effects in the subject to whom or to whom the polypeptide is administered. One side or adverse effect that is preferably not present in this context is hyperalgesia or pain. Thus, preferably, administration of an agent according to the present invention does not induce any hyperalgesia syndrome (pain).

[0101] It is important to note that the absence of pain not only makes treatment more comfortable (or less unpleasant) than administration of a reference compound associated with pain (e.g., wild-type NGF), but also accounts, at least in part, for the successful treatment or prevention of skin disorders themselves: considering that the polypeptides according to the present invention are preferably administered topically, more preferably locally onto the site of a skin disorder (e.g., an ulcer), the absence of pain allows the treated subject to accept administration of the polypeptide onto the body surface for use in pain without adverse reactions (e.g., scraping, washing, or otherwise removing it), so that the polypeptide remains exposed to the injured body surface and exerts its therapeutically beneficial effect (e.g., treatment or prevention of skin disorders). Thus, the absence of pain associated with the polypeptides of the present invention may be suitable for overcoming consumer reluctance and regulatory concerns. In other words, the absence of pain is associated with a significant increase in the benefit-to-risk ratio compared to drugs associated with pain.

[0102] In particular, preferably, the treatment and / or prevention does not cause hyperalgesia in the 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 the subject to which the polypeptide is administered does not suffer from mechanical allodynia.

[0103] In one embodiment, a subject to whom a polypeptide of the invention is administered does not suffer from thermal allodynia, more precisely, thermal allodynia is not induced in a subject to whom a polypeptide of the invention is administered, and a subject to whom a polypeptide is administered does not suffer from thermal allodynia.

[0104] In this context, a further side effect or adverse effect that is preferably absent is malignancy or cancer. In particular, administration of the polypeptide of the present invention to a subject is preferably not associated with abnormal cell growth, and even more preferably not associated with abnormal cell growth with the potential to invade or spread to other parts of the body. It is particularly preferred that administration of the polypeptide of the present invention to a subject is preferably not associated with cancer of the skin, particularly of the dermis or epidermis. In this regard, treatment or administration according to the present invention is associated with significant advantages, for example, compared to the state of the art, such as commercial treatments using platelet-derived growth factor (becaplermin, trade name Regranex). Thus, the absence of malignancy associated with the polypeptide of the present invention is expected to be suitable for overcoming consumer reluctance and regulatory concerns. In other words, the absence of malignancy is associated with a significantly increased benefit-to-risk ratio compared to drugs associated with malignancy.

[0105] Thus, in summary, preferably, administration of a polypeptide of the present invention to a subject is not associated with adverse effects (such as malignancies and / or pain).

[0106] Typically, the administration of the agent according to the present invention is well tolerated by the subject. In particular, preferably, the administration of the polypeptide according to the present 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 present invention differs from that of wild-type human NGF at only one or two amino acid positions, it is likely that the immunological tolerance in humans is particularly advantageous, and it is likely that the administration of the polypeptide according to the present invention is not associated with the formation of anti-drug antibodies in humans.

[0107] Preferably, administration according to the present invention positively affects one or more of the following: inflammation, extracellular matrix deposition, innervation, and angiogenesis.

[0108] Detectability of polypeptides

[0109] Preferably, polypeptides for use according to the invention can be selectively recognized by a specific reagent with respect to 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.

[0110] In that regard, the polypeptides according to the present invention are preferably detectable by antibodies or other immunoreactive molecules.

[0111] A protein detectable by an antibody or other immunoreactive molecule may also be referred to as an antigen. In some embodiments, a biological sample may be characterized by displaying or not displaying 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 administration of the polypeptide. One non-limiting method for demonstrating the presence of a protein is by Western blot, but other immunological methods are equally encompassed within the context of the present invention. The antibody or other immunoreactive molecule may itself be labeled (e.g., fluorophore-labeled) or may be recognized by a labeled secondary antibody or other immunoreactive molecule added for that purpose. Thus, in some cases, a secondary molecule (e.g., an optionally labeled secondary antibody, etc.) that aids in detection may also be added to facilitate detection.

[0112] According to the present invention, an antigen is said to be present in a biological sample if the level is above the detection limit and / or if the level is high enough to allow binding by an antigen-specific antibody added to the sample. According to the present invention, an antigen is said to not be expressed on a cell if the level of expression is below the detection limit and / or if the level of expression is too low to allow binding by an antigen-specific antibody added to the sample.

[0113] An antibody or other immunoreactive molecule may recognize an epitope on a cell. The term "epitope" refers to an antigenic determinant in a molecule (such as an antigen), i.e., a portion or fragment of a molecule that is recognized (i.e., bound) by the immune system, e.g., recognized by an antibody or other immunoreactive molecule. Detection of an epitope specific for any particular antigen usually allows one to conclude that that particular antigen is present on the cell being analyzed.

[0114] In one embodiment, samples obtained from subjects, particularly subjects to which a polypeptide according to the present invention has been administered, can be characterized by immunophenotyping. "Immunophenotyping" generally means that cells or samples can be characterized by antigen-specific molecules (such as antibodies or other immunoreactive molecules) added to the sample to determine whether the antigen is present. Immunophenotyping includes cell sorting using a variety of methods (including flow cytometry) and analytical methods on lysed cells and lysed samples (such as Western blotting).

[0115] Particularly preferred in the present invention are polypeptides that can be specifically detected even in the presence of wild-type NGF (such as wild-type human NGF). Any mutation in the amino acid sequence (such as any point mutation) will make the polypeptide specifically detectable, for example, even in the presence of the respective non-mutated wild-type polypeptide, and thus each of the polypeptides of SEQ ID NO: 3, but the polypeptide of SEQ ID NO: 4 can at least be specifically detected even in the presence of wild-type human NGF, particularly the polypeptide of SEQ ID NO: 4 for which an antibody is available that can distinguish said polypeptide from wild-type human NGF (WO 2008 / 006893A1).

[0116] Thus, preferably, the polypeptide is characterized by the absence of proline at least at position 61 (which, for reference, is present at position 61 in SEQ ID NO: 2), more preferably by substitution of the proline at position 61 by another amino acid. In a particularly preferred embodiment, the proline at position 61 is substituted by a serine. In this preferred embodiment, the polypeptide for use according to the invention is the polypeptide of SEQ ID NO: 4. This polypeptide is characterized by the absence of proline at least at position 61, more preferably by substitution of the proline at position 61 by another amino acid. In SEQ ID NO: 4, the proline at position 61 of SEQ ID NO: 3 is substituted by a serine.

[0117] wound body surface

[0118] According to the present invention, the wound body surface is subjected to administration of a polypeptide of the present invention.

[0119] Injured body surfaces include, but are not limited to, ulcers (venous ulcers, arterial ulcers, pressure ulcers, diabetic ulcers), post-surgical wounds, bedsores, burns, lacerations, incisions, bruises, abrasions, puncture wounds), etc. Subjects having such wounded body surfaces are further described below, and the description of wounded body surfaces below is applicable to all such subjects unless the context dictates otherwise.

[0120] In one embodiment, a medicament according to the invention is provided herein for the prophylactic treatment of a skin disorder, wherein the skin disorder is selected from ulcers, post-operative wounds, bedsores, burns, lacerations, incisions, bruises, abrasions, and puncture wounds.

[0121] In one embodiment, a medicament according to the invention is provided herein for the treatment of prevention of ulcers, wherein the ulcers are selected from venous ulcers, arterial ulcers, pressure ulcers, and diabetic ulcers.

[0122] In some embodiments, the wound body surface has a diameter of 1 mm or more. Generally, when referring to the "diameter" of a wound body surface herein, for non-circular wound body surfaces, the term "diameter" refers to the maximum diameter of the wound body surface measured from one boundary of the wound body surface across the wound body surface to the opposite boundary of the wound body surface. For circular wound body surfaces, the diameter is of course the same in any direction of measurement from one boundary of the wound body surface across the wound body surface to the opposite boundary of the wound body surface. The diameter can be determined on the outer surface of the wound body surface using a ruler or other suitable means.

[0123] In some embodiments, the wound body surface has a diameter of 1 mm to 50 cm. In some embodiments, the wound body surface has a diameter of 2 mm to 20 cm. Wound body surfaces with a diameter of 0.5 cm or more, preferably 1 cm or more, may also be referred to herein as "large" wound body surfaces. The present invention is also suitable for the treatment of large wound body surfaces (such as large ulcers) (see, e.g., Example 4). In some embodiments, the wound body surface has a diameter of 3 mm to 10 cm. In some embodiments, the wound body surface has a diameter of 4 mm to 5 cm. In some embodiments, the wound body surface has a diameter of 5 mm to 4 cm. In some embodiments, the wound body surface has a diameter of 6 mm to 3 cm. In some embodiments, the wound body surface has a diameter of 7 mm to 1 cm. In some embodiments, the wound body surface has a diameter of 8 mm to 1 cm. In some embodiments, the wound body surface has a diameter of about 6 mm. In some embodiments, the wound body surface has a diameter of about 12 mm.

[0124] Subjects for whom the medicament according to the invention is particularly suitable

[0125] According to the present invention, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 can be administered to a subject in need of such administration. The subject in need of such administration can be a subject suffering from a disorder described herein, a subject at risk of suffering from 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 in light of the disclosure herein.

[0126] In particular, the polypeptides according to the present invention are administered to a mammalian subject, which may also be referred to as a "patient." Most preferably, the mammalian subject is a human.

[0127] The present invention also relates to a method of treating a patient suffering from a dermatological disorder, wherein the method comprises administering to the patient an effective amount of the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4. The terms "patient" and "subject" are used interchangeably herein and particularly refer to patients / subjects characterized by a dermatological disorder as described herein.

[0128] Preferably, the dermatological disorder is characterized by a wound surface on at least a portion of the subject's body. Preferably, the dermatological disorder is characterized by a wound surface (wounded body surface). Wounded body surfaces, for example, have been described above, and the description of the subject below is applicable to all such wounded body surfaces in such subjects, unless the context dictates otherwise.

[0129] More preferably, the dermatological disorder is or comprises a skin lesion, preferably a skin lesion characterized by at least partial ablation of the dermis, and optionally ablation of the dermis. In one embodiment, the wound body surface is or comprises a lesion, particularly a skin lesion.

[0130] Although terms such as "dermatological disorder," "wound," "wound body surface," "chronic wound," "ulcer," and other terms are used in the singular herein, the present invention is also applicable to subjects having multiple dermatological disorders, wounds, wound body surfaces, chronic wounds, ulcers, and other such disorders.

[0131] Preferably, the wounded body surface comprises at least one chronic wound. Thus, the administration of the agent according to the present invention is suitable for the treatment or prevention of at least one chronic wound. In the context of the present invention, the term "chronic wound" should be broadly understood and includes, but is not limited to, all types of ulcers, bed sores, burns, and mechanical skin excisions, regardless of whether they are explicitly mentioned in the present disclosure. In particular, wounds that do not heal within the typical time frame for healing in healthy subjects of each species are included within this term. In addition, all wounds on the body surface of a subject that have not healed and / or closed for 7 days or more, for example, 14 days or more, 21 days or more, 1 month or more, or 1 year or more, are included within the term "chronic wound". The agent according to the present invention may be administered to all such types of chronic wounds to treat or prevent such chronic wounds.

[0132] In the context of the present invention, the term "preventing" should be understood broadly and includes not only the prevention of the onset of a disorder, but also the prevention of the progression of a disorder. In particular, in the context of a wound body surface (such as a chronic wound, e.g., an ulcer), the term "preventing" also includes the prevention of further progression of the expansion of the wound body surface, such as further deepening of the wound body surface and / or an increase in the diameter of the wound body surface.

[0133] In the context of the present invention, the term "treat" should be understood broadly and includes, but is not limited to, the alleviation of symptoms of a disorder. Indeed, the experimental examples herein demonstrate that achieving the alleviation of a dermatological disorder, such as (partial) closure of a wound, is a preferred and essential part of the invention claimed herein. In fact, achieving the claimed therapeutic effect is a functional technical feature of the invention. The examples herein make it plausible that the functional technical feature can be achieved as a direct result of administering the polypeptide of the present invention. In other words, the inventors demonstrate that the polypeptide of the present invention is responsible for achieving alleviation in a subject suffering from a dermatological disorder. The dermatological disorder is preferably characterized by a wound on the body surface.

[0134] The present invention is particularly suitable for subgroups of subjects suffering from dermatological disorders. Such subgroups are described herein. A particular subject may be divided into one or more of the subgroups described herein; administration of a polypeptide according to the present invention to a subject who is divided into one of the subgroups described herein is equally encompassed by the present invention as administration of a polypeptide according to the present invention to a subject who is divided into two or more of the subgroups described herein.

[0135] The present invention is not limited to a particular cause of the wound body surface, for example, diabetic causes are included in the present invention as well as non-diabetic causes.

[0136] The wounded body surface can be in any one or more parts of the body. Preferred are wounded body surfaces of the extremities, such as the arms (including hands) and legs (including feet), but wounded body surfaces of the trunk or head or other parts of the body can similarly be subjected to administration of the polypeptides of the present invention. In some embodiments, the wounded body surface is the leg or foot, and more preferably the foot. Such embodiments are frequent in diabetic subjects, but administration to such specific wounded body surfaces is not limited to diabetic subjects.

[0137] In some embodiments, the polypeptides according to the invention are for administration to a subject who has undergone surgery. Thus, the polypeptides according to the invention are suitable for treating or preventing one or more post-operative complications (such as bedsores) and / or for treating surgical wounds.

[0138] Preferably, the wound body surface comprises at least one ulcer. According to the present invention, the polypeptide may be administered to at least a portion of the wound body surface. "At least a portion of," as used herein, includes any proportion between 0% and 100%, such as between 10% and 90%, between 20% and 80%, between 30% and 70%, between 40% and 60%, and about 50%; thus, the polypeptide may be administered to the entire wound body surface or any portion thereof. Optionally, administration also includes the skin area adjacent to the wound body surface.

[0139] Preferably, the dermatological disorder comprises at least one ulcer. According to the present invention, the polypeptide may be administered to at least a portion of an ulcer. "At least a portion of," as used herein, includes any proportion between 0% and 100%, such as between 10% and 90%, between 20% and 80%, between 30% and 70%, between 40% and 60%, and about 50%; thus, the polypeptide may be administered to the entire surface of the ulcer or any portion thereof. Optionally, administration also includes the skin area adjacent to the ulcer.

[0140] Diabetes mellitus is a common, debilitating disease affecting various organs, including the skin. It is currently estimated that 30-70% of patients with diabetes mellitus, both type 1 and type 2, will develop skin complications of diabetes mellitus at some point during their lifetime. Regardless of such theoretical considerations (which do not limit the present invention in any way), methods for detecting diabetes are well known in the art. While methods for detecting diabetes are not part of the present invention in one embodiment, they are useful in determining subgroups of subjects at risk for suffering from dermatological disorders (such as those described herein) and who may benefit from treatment or prevention of such dermatological disorders according to the present invention. In some embodiments, the medicaments according to the present invention are for administration to diabetic subjects suffering from neuropathy, particularly peripheral neuropathy. Methods for detecting neuropathy and predicting the development of foot ulcers in humans with conditions such as diabetes mellitus are known (e.g., but not limited to, WO / 2010 / 128519 A1).

[0141] The polypeptide according to the present invention may be administered to a skin lesion in a diabetic subject, preferably a skin lesion characterized by at least partial excision of the dermis, and optionally excision of the dermis, in such a subject. In one embodiment, the wound body surface is or comprises a lesion, particularly a lesion of the skin of such a subject. Preferably, administration comprises administration to an ulcer, particularly a foot ulcer, in a diabetic subject.

[0142] Diabetic ulcers, particularly diabetic foot ulcers, are a major complication of diabetes mellitus. Within the context of the present invention, the term "diabetic ulcer" is not particularly limiting, apart from the precise definition that the ulcer is an ulcer in a diabetic subject. According to some estimates, diabetic subjects may have a 5-15 times higher risk of non-traumatic amputation compared with non-diabetic subjects (e.g., WO / 2003 / 075949 A1). If left untreated or unsuccessfully treated, diabetic foot ulcers may be difficult to heal in some subjects and may even require amputation, especially if accompanied by other complications or disorders (e.g., infection). Indeed, diabetes mellitus can affect multiple organ systems. The dermatological manifestations of diabetes mellitus have a variety of health implications, ranging from cosmetic concerns to even life-threatening if left untreated. The dermatological implications of diabetes mellitus are described, for example, by Rosen et al., 2000, Endotext, De Groot et al., Eds., South Dartmouth (MA, USA), MDText.com, Inc. The present invention provides treatment and / or prevention of such dermatological implications of diabetes.

[0143] In some embodiments, the polypeptides according to the invention are for administration to a diabetic subject who has undergone surgery. Thus, the polypeptides according to the invention are suitable for treating or preventing one or more post-operative complications (such as bedsores) and / or for treating surgical wounds in diabetic subjects.

[0144] In general, diabetic ulcers, particularly diabetic foot ulcers, as well as bedsores and large / deep surgical wounds, can be difficult to heal even under medication, possibly due to the large size of the area involved. If these wounds are not treated in time, they can worsen and subsequently become incurable and life-threatening. The present invention provides treatment and / or prevention for such dermatological consequences of diabetes. Indeed, according to the present invention, effective medical treatment not only helps patients recover from these skin complications, but may also lead to a better quality of life, reduced medical care or costs, or even an extended lifespan.

[0145] The present invention is also suitable for treating large-sized wounds on the body surface, particularly ulcers, in diabetic and non-diabetic subjects. In some embodiments, the present invention is suitable for treating large wounds on the body surface, such as those with a diameter of 5 mm or more, e.g., 1 cm or more. Further details of wound body surfaces, including specific embodiments of the diameter of the wound body surface, are described herein above.

[0146] Thus, the present invention provides advantages over current treatment methods, which often fail to provide an effective method for treating large wounds. The present invention provides treatment and / or prevention for such dermatological conditions, including large wounds, in diabetic and non-diabetic subjects.

[0147] According to the present invention, the polypeptides are suitable for the treatment or prevention of pressure injuries, including chronic pressure injuries, which in particular include pressure ulcers, pressure sores, decubitus ulcers, and bed sores.

[0148] In a preferred embodiment, the medicament according to the present invention is for use in the treatment or prevention of ulcers, and for that purpose is administered to an ulcer. According to the present invention, the ulcer to which the polypeptide is administered is preferably selected from the group consisting of diabetic ulcers, traumatic ulcers, surgical ulcers, pressure ulcers, chronic ulcers, and any combination of these ulcers. In certain embodiments, the ulcer is selected from diabetic trauma ulcers, diabetic surgical ulcers, diabetic pressure ulcers, diabetic chronic ulcers, traumatic diabetic ulcers, traumatic surgical ulcers, traumatic pressure ulcers, traumatic chronic ulcers, chronic surgical ulcers, chronic pressure ulcers, and other ulcers. In some embodiments, the ulcer is selected from traumatic ulcers, surgical ulcers, pressure ulcers, and chronic ulcers in diabetic subjects. In some embodiments, the ulcer is selected from traumatic ulcers, surgical ulcers, pressure ulcers, and chronic ulcers in non-diabetic subjects.

[0149] The present invention is not limited to subjects with one ulcer, nor to subjects with multiple ulcers. Among subjects with multiple ulcers, the present invention is not limited to treatment of only one of the ulcers, or to treatment of a particular number of the ulcers, or to treatment of all of the ulcers. Thus, the terms "ulcer" and "ulcers" are independent of their use in the singular or plural forms in this disclosure and are expressly inclusive of all such embodiments, and are not limited to any particular number of ulcers in a subject or any particular number of ulcers being treated.

[0150] It is well established that diabetic foot ulcers are associated with either neuropathy (neuropathic ulcers), peripheral vascular disease (ischemic ulcers), or both (neuroischemic ulcers), although the final pathogenic pathway may involve a combination of these major risk factors and other causative factors (e.g., trauma, etc.). Thus, in one embodiment, the polypeptides of the present invention are for the prevention and / or treatment of ischemic ulcers (including ischemic foot ulcers). In an alternative embodiment, the polypeptides of the present invention may be for the prevention and / or treatment of neuropathic ulcers (including neuropathic foot ulcers). Finally, the polypeptides of the present invention may be for the prevention and / or treatment of neuroischemic ulcers (including neurooischemic foot ulcers). All of the aforementioned ulcers are optionally diabetic ulcers, although this is not a requirement.

[0151] In some embodiments, a polypeptide according to the present invention is administered to a subject suffering from ischemia. The ischemia can be local or systemic. In some embodiments, administration according to the present invention can reduce ischemia in the subject. The reduction in ischemia can be local or systemic.

[0152] In some embodiments, the polypeptides according to the present invention are administered to a subject suffering from neuropathy. In a preferred embodiment, the medicament according to the present invention is for administration to a subject suffering from neuropathy (especially peripheral neuropathy). Such subjects may be diabetic or non-diabetic. In fact, it has been reported that the majority of diabetic ulcer patients have underlying neuropathy (Ndip et al., 2012, Int. J. Gen. Med., vol. 5, pp. 129-134). Thus, in a preferred embodiment, the polypeptides according to the present invention are administered to a diabetic subject suffering from neuropathy. The neuropathy may be local or systemic. In some embodiments, administration according to the present invention can reduce neuropathy in a subject. The reduction in neuropathy may be local and / or systemic. In one embodiment, the reduction in neuropathy includes a reduction in neuropathy in the area to which the polypeptide of the present invention is administered. In one embodiment, the reduction in neuropathy includes a reduction in neuropathy in the organ to which the polypeptide of the present invention is administered.

[0153] Treatment or prevention according to the present invention may be achieved by administration, preferably local administration, of a polypeptide of the present invention. In some embodiments, administration occurs in a hospital. In some embodiments, treatment does not occur in a hospital.

[0154] Optionally, but not mutually exclusive, the dermatological disorder includes at least one burn or mechanical injury. Thus, the present invention also includes the treatment or prevention of burns and mechanical injuries, whereby the treatment of such injuries is more practically meaningful than prevention.

[0155] Preferably, the mammal, preferably a human, to whom the polypeptide of the invention is administered is suffering from or predisposed to suffering from diabetes mellitus; each subject is referred to herein as a "diabetic subject." In exemplary embodiments, the diabetes mellitus is selected between type 1 diabetes mellitus and type 2 diabetes mellitus.

[0156] Foot ulcers and other dermatological disorders are common in diabetic subjects. Such other dermatological disorders can be treated and / or prevented according to the present invention. One of the main causes of foot ulcers in diabetic subjects is neuropathy (nerve damage), which makes it difficult to identify injuries to the foot (such as cuts, bruises, and pressure).

[0157] Thus, in one embodiment, the polypeptide is administered to a subject with a foot ulcer. In one embodiment, the polypeptide is administered to a subject with a diabetic foot ulcer (DFU). In fact, foot ulcers and their associated complications are frequent in subjects with diabetes, the majority of whom have underlying neuropathy (Ndip et al., 2012, Int. J. Gen. Med., vol. 5, pp. 129-134). Such ulcers are also referred to as diabetic neuropathic foot ulcers. Thus, preferably, the polypeptide is administered to a subject with a diabetic neuropathic foot ulcer.

[0158] In some cases, administration of a polypeptide according to the present invention also includes aspects that improve the aesthetic appearance of a subject, particularly the body surface of a subject. In some embodiments, wound closure is achieved as a result of administration according to the present invention. In some embodiments, scar formation is minimal. Thus, administration according to the present invention also provides cosmetic benefits to a treated subject compared to an untreated subject. Thus, the present invention also relates to a method of cosmetically treating a subject, wherein the method comprises administering a polypeptide according to SEQ ID NO: 3 or 4.

[0159] In another embodiment, the medicament according to the present invention is for the treatment or prevention of cancers on the skin (such as, but not limited to, hemangiomas) and / or skin disorders associated with such cancers.

[0160] In a further embodiment, the medicament according to the invention is for the treatment or prevention of a dermatological disorder caused by or affected by a genetic disorder in a subject.

[0161] Administration

[0162] The present invention provides heterologous polypeptides for administration to a subject.

[0163] Preferably, the polypeptide is for topical administration. Thus, preferably, the polypeptide of the present invention is administered to the skin, or to the surface of the body where the skin would be found if the skin is injured or absent, or where the skin would be found if the skin is not injured or absent. In some embodiments, the polypeptide is administered onto the epidermis. In some embodiments, the polypeptide is administered onto the dermis. In some embodiments, the polypeptide is administered to tissues normally found below the epidermis (such as, but not limited to, the subcutaneous region).

[0164] More preferably, the polypeptide is administered onto the surface of a wound. In other words, the polypeptide according to the present invention is preferably administered topically, more preferably onto the site of a skin disorder (e.g., an ulcer).

[0165] Administration in accordance with the present invention typically does not involve surgery on the subject. In one embodiment, administration of a polypeptide of the present invention does not involve or involve an invasive step that requires specialized medical expertise and represents a substantial physical intervention on the body, which, even when performed with the required professional care and expertise, carries substantial health risks. In contrast, in more typical embodiments, administration, particularly topical administration, of a polypeptide of the present invention is generally considered safe for the subject, and thus the polypeptide may be administered by the subject themselves, particularly in the case of human subjects.

[0166] Optionally, the ulcer is covered with a wound dressing before and / or during and / or after administration. The vast variety of types of wound dressings available is not limited by the present invention. Thus, any wound dressing may be used unless technically clearly inappropriate. In some embodiments, the polypeptide according to the present invention is administered simultaneously with the application of the wound dressing; optionally, the wound dressing comprises the polypeptide of the present invention in the form of an aqueous medium, which is applied to the wound dressing before administration.

[0167] Preferably, the polypeptide is administered to a subject with a foot ulcer on said subject's foot below the ankle.

[0168] In one embodiment, the polypeptide is administered in a single dose.

[0169] In an alternative and more preferred embodiment, the polypeptide is administered repeatedly. In a particularly preferred embodiment, the polypeptide is administered repeatedly 1 to 5 times per day. In one embodiment, the polypeptide is administered once per day (see also Example 3). In one embodiment, the polypeptide is administered twice per day (see also Example 5). In one embodiment, the polypeptide is administered three times per day. In one embodiment, the polypeptide is administered four times per day. In one embodiment, the polypeptide is administered five times per day. It is particularly preferred that the polypeptide is administered twice per day to a human subject. All the above administrations, as disclosed herein, are preferably repeated over the course of several days. For example, the polypeptide may be administered repeatedly over a period of 3 to 30 days, preferably 7 to 14 days, and preferably 1 to 5 times per each of these days.

[0170] In one embodiment, the polypeptide is repeatedly administered until the wound surface is closed. Alternatively, the polypeptide is administered in a single dose, and administration is stopped after the single dose. Alternatively, the polypeptide is repeatedly administered over a period of 3 to 30 days, preferably 7 to 14 days. Optionally, administration is stopped after the completion of the interval.

[0171] In some embodiments, the agent is administered in such a manner that it comes into direct contact with nociceptive fibers (nerves). In some embodiments, the agent is administered in such a manner that it comes into direct contact with fully exposed nociceptive fibers (nerves); nociceptive fibers (nerves) are considered fully exposed in the absence of skin, as is typical in the case of a wounded body surface. In some embodiments, the agent is administered in such a manner that it comes into direct contact with overactivated nociceptive fibers (nerves); nociceptive fibers (nerves) are considered overactivated as a result of a skin lesion. In some embodiments, the agent is administered in such a manner that it comes into direct contact with overactivated nociceptive fibers (nerves). Preferably, particularly in such embodiments, the agent does not cause hyperalgesia (pain). Agents with such properties have not previously been available to the medical community. For this and other reasons, the present invention provides a major advantage.

[0172] dose

[0173] The agents and compositions described herein are administered in an effective amount. According to the present invention, an "effective amount" is an amount or dose that alone, or together with further doses, achieves the desired response or desired effect. In the case of the treatment of a particular disorder, the desired response preferably relates to the inhibition of the course of the disease. This includes slowing the progression of the disease, and preferably preventing or reversing the progression of the disease. The desired response in the treatment of a disease or condition can also include delaying or preventing the onset of the disease or condition. In some embodiments, the desired response includes complete cure of the symptoms of the disorder, locally and / or systemically.

[0174] The effective amount of the agents or compositions described herein will depend on the condition or disorder being treated, the severity of the disorder, the individual parameters of the subject to whom the agent is administered (e.g., age, physiological condition, concomitant conditions (if any), etc.), size and weight, duration of treatment, type of concomitant therapy (if any), the particular route of administration, and other parameters. Thus, the dose administered of the agents described herein may depend on various such parameters. In cases where the patient's response is inadequate using the initial dose, a higher dose (or an effectively higher dose achieved by a different, more localized route of administration) may be used.

[0175] In accordance with the present invention, suitable therapeutically effective dosages for administration of therapeutic agents for administration to human subjects for the treatment and / or prevention of skin disorders (e.g., chronic skin ulcers and burns) can be determined based on experimentally determined suitable therapeutically effective dosages for administration of therapeutic agents for administration to rodent subjects, particularly mice, for the treatment and / or prevention of skin disorders (e.g., chronic skin ulcers and burns). Guidance is available in "Guidance for Industry Chronic Cutaneous Ulcer and Burn Wounds - Developing Products for Treatment," published by the U.S. Department of Health and Human Services, Food and Drug Administration, in 2006.

[0176] Animal wound models (Examples 3 and 4) are useful in establishing pharmacological responses and assessing potential toxicity of wound treatment products. In some embodiments, the dose administered to a subject is the dose disclosed in Example 3 or Example 4 or Example 5.

[0177] Preferably, the dose of polypeptide to be administered is determined based on the area of ​​the wound body surface to be treated. Preferably, this determination is made at the beginning of treatment. In one embodiment, the dosage is adjusted for subsequent administrations depending on the area of ​​the wound body surface at the time of such subsequent administration. In an alternative embodiment, the dosage is not adjusted for subsequent administrations, so that the dosage of administration depends only on the area of ​​the wound body surface to be treated at the beginning of administration (first administration), and subsequent administrations correspond to the first dose.

[0178] In one embodiment, the dose / each dose is calculated based on the mm of wound body surface to be treated. 2 0.3-6μg per (0.3-6μg / mm 2 ) the amount of polypeptide.

[0179] Optionally, in all embodiments according to the present invention, the dose is calculated based on the actual size of the wound body surface (e.g., ulcer) at the time of treatment. In other words, the dose can be calculated (recalculated) at every time of administration based on the actual size of the wound body surface (e.g., ulcer) at that time.

[0180] Process for Obtaining Polypeptides

[0181] In one embodiment, the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 are obtainable from a biological source. Optionally, the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 are obtainable by recombinant expression. To that end, an open reading frame encoding the respective polypeptide is introduced into a source of recombinant protein, such as a host cell or a cell-free system for protein expression. In fact, considering that human NGF is produced in vivo only in trace amounts, mouse NGF is usually produced as a heterogeneous mixture of various proteins (see WO 2000 / 022119 A1), and the polypeptides of the present invention are non-natural and thus not produced in vivo at all, the most meaningful possibility for producing the polypeptides of the present invention is by recombinant expression according to the state-of-the-art proposals for wild-type NGF (WO 2000 / 022119 A1, WO 2008 / 006893 A1; Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, pp. 3296-3303, US 2018 / 0086805 A1). However, obtaining such polypeptides in a purity grade sufficient for mammalian administration has been a persistent challenge. This challenge has been overcome by the present inventors, as disclosed in detail herein (see also Examples 1 and 2).

[0182] Preferably, the polypeptides according to the invention are obtainable by recombinant expression in bacteria, more preferably by cytosolic recombinant expression in bacteria. Generally, bacterial cells, especially E. coli, allow for the recombinant production of large amounts of recombinant proteins. However, as is the case for many other recombinantly expressed genes, the production of recombinant NGF and similar polypeptides in bacteria results in a biologically inactive translation product, which then accumulates in the cell (cytosol) in the form of aggregates (so-called inclusion bodies (IBs) (WO 2000 / 022119 A1; US ​​2018 / 0086805 A1). In contrast to NGF, proNGF is known to be quite unstable and requires high efforts for refolding and purification with low recovery rates, making the process of NGF production via proNGF in bacteria relatively difficult and expensive. Thus, the main difficulties associated with bacterially produced NGF and similar bacterially produced polypeptides relate to the folding, processing, and purification of the recombinant proteins via their respective proforms. These problems have now been resolved (see Examples 1 and 2). As a result, the polypeptides of SEQ ID NO: 3 and SEQ ID NO: 4 are available in purity grades suitable for administration to mammals (including humans).

[0183] Preferably, the polypeptides of the present invention are expressed with a prosequence. A suitable prosequence is, but is not limited to, the prosequence of wild-type human NGF (amino acid positions 18 to 121 of SEQ ID NO: 1), typically fused to the N-terminus of the polypeptide of SEQ ID NO: 3 or 4. For wild-type NGF, the presence of a covalently attached prosequence, which is not part of mature NGF and therefore not required for the biological function of NGF, has been shown to promote the refolding of recombinant NGF from inclusion bodies with concomitant disulfide bond formation of the mature portion (beta-NGF). Thus, the presence of a covalently attached prosequence positively affects the refolding yield and rate when compared with the in vitro refolding of mature NGF from inclusion bodies (Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, pp. 3296-3303). Without wishing to be bound by any particular theory, the same is plausibly assumed herein for the polypeptides of SEQ ID NOs: 3 and 4.

[0184] Thus, when the polypeptides according to the present invention are produced in inclusion bodies, correct folding is required, and this is usually achieved post-translationally, such as by cleavage from a covalently attached pro-sequence; sophisticated methods for folding, cleavage, and purification have been proposed in the past, particularly for wild-type human NGF. Notably, the majority of published studies on NGF have applied the general refolding regime previously established by Rattenholl et al. (2001, Eur. J. Biochem, vol. 268, pp. 3296-3303). Within this initial study, several parameters of protein refolding (e.g., temperature, refolding time, pH of the refolding reaction, arginine, glutathione, and protein concentration) were studied in detail, and their effects on refolding efficiency were assessed. The protocol by Rattenholl et al. relies on the re-naturalization of the pro-form, which has very poor solubility and is available from inclusion bodies after recombinant production in prokaryotes. Thereby, proNGF is solubilized in a solution of a denaturing agent at a denaturing concentration, transferred into a non-denaturing or weakly denaturing solution, maintaining solubility, and the dissolved denatured proNGF assumes a biologically active conformation (including the formation of disulfide bonds as in native NGF), and then NGF is purified and the prosequence is proteolytically removed (WO 2000 / 022119 A1; Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, pp. 3296-3303). Notably, in this study, it was found that a low protein concentration leads to a higher specific yield of correctly folded product compared to a higher protein concentration. An exemplary protein concentration of about 50 mg / liter in the refolding reaction resulted in a specific yield of correctly folded proNGF of about 25%, but this fraction decreased to 10% at a protein concentration of 500 mg / liter.Based on this, Rattenholl et al. suggest that the protein concentration in the refolding solution must be very low: they expect a yield of 15-20 mg of correctly folded protein per liter of refolding reaction. However, this may require scale-up (e.g., beyond laboratory scale) for the purification of several hundred mg of recombinant protein.

[0185] Human proNGF is synthesized by the protease furin (Arg1-Ser2-Lys3-Arg4; R 1 S 2 K 3 R 4 Although proNGF contains a natural cleavage site for furin, and furin cleaves proNGF at that site in vivo, furin is not commercially available in relevant purity or quantity. According to the present invention, when expressed together with the pro-sequence, for example in E. coli, the polypeptide according to the present invention is preferably cleaved by the commercially available protease trypsin (EC 3.4.21.4). Indeed, for wild-type NGF, trypsin has been reported to yield satisfactory biologically active, mature NGF, which can ultimately be purified (Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, pp. 3296-3303), and trypsin-based proteolysis of recombinantly expressed proNGF has meanwhile been employed by others (e.g., D'Onofrio et al., 2011, PLoS One, vol. 6, e20839). However, it was later shown that cleavage of wild-type proNGF with trypsin to produce beta-NGF is associated with several drawbacks, since a small amount of trypsin can lead to inefficient cleavage, whereas a large amount of trypsin can further reduce the selectivity of cleavage, since trypsin is capable of cleaving any arginine and lysine residues (R and K residues) at the C-terminus, and the R residues by trypsin are 1 S 2 K 3 R 4Digestion of proNGF containing trypsin can result in several alternative digestion products; therefore, the use of trypsin as a cleavage enzyme can lead to very low yields of correctly cleaved NGF, as well as purification and yield problems (because the different cleavage products cannot be economically separated under standard conditions). As a solution, it has been proposed to express a mutant of proNGF, in which the protease cleavage site R1S2K3R4 of the propeptide is replaced by at least positions R1S2K3R4, which correspond to positions 101 and 103 of the human wild-type proNGF sequence (SEQ ID NO: 1). 1 and K. 3 In one example, R1 and K3 are replaced by valine (V) and alanine (A), respectively, to close the original furin cleavage site R 1 S 2 K 3 R 4 V 1 S 2 A 3 R 4where trypsin is capable of specifically cleaving only the C-terminus of R4; trypsin-mediated cleavage of each proNGF can be referred to as the "VSAR method." WO 2013 / 092776 A1 was initially proposed to be applicable to certain variants of proNGF muteins, although it is silent for the polypeptides of SEQ ID NO: 3 or 4 according to the present invention, although it was reported that proteolysis conditions must be carefully titrated (US2018 / 0086805A1). In the course of arriving at the present invention, the inventors found that, contrary to earlier proposals, the VSAR technique does not satisfactorily resolve the purity issues associated with recombinant production of the polypeptides of the present invention with satisfactory purity. Indeed, the purification of recombinantly expressed beta-NGF or its muteins not only from host cell proteins (HCPs), but also from trypsin (or other proteases used for cleavage) remains a challenge; although, of course, proteolytic enzymes (such as trypsin) may be required to be absent from the final preparation of the pharmaceutical protein to avoid proteolysis during storage of the polypeptide, the polypeptide, according to the present invention, is substantially pure and not degraded at the time of its administration to a subject. The inventors have solved this problem as described herein. Thus, the present invention makes available a polypeptide according to SEQ ID NO: 3 or 4 in high purity, thus essentially free from trypsin and / or polypeptide degradation products. While certain methods for the production of NGF (e.g., WO2013092776 A1) and the polypeptide of SEQ ID NO: 4 (e.g., Malerba et al., 2015, PLOS One, vol. 10, e0136425) have previously been described, the present inventors surprisingly discovered that the previously published processes were insufficient for obtaining the respective polypeptides in high purity. As a solution to these inadequacies, the present inventors arrived at a new process and related aspects, as described in detail herein.

[0186] According to the present invention, the process for obtaining the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4, for example, from recombinant expression in a host cell, can include purification. Purification, in its broadest sense, means that the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is separated from other molecules, including other proteins (e.g., host cell proteins). Thus, purification can include separation from one or more other molecules (including other proteins, e.g., host cell proteins, proteases (e.g., trypsin), and / or degradation products of the polypeptide according to the present invention).

[0187] The process for the production of the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 according to the present invention preferably comprises the following steps: (a) obtaining a precursor of the polypeptide of SEQ ID NO:3 or SEQ ID NO:4; (d) Purification; and the purification in step (d) typically comprises purification on a mixed-mode stationary phase. Thus, in one embodiment, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is obtainable by recombinant expression and purification, wherein the purification comprises purification on a mixed-mode stationary phase. The term "on a mixed-mode stationary phase" should be understood broadly and means that a mixture containing the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 or a precursor of either of these, together with other molecular species, is exposed to a mixed-mode stationary phase, for example, by chromatography or other suitable process step. Indeed, preferably, a mixture containing the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 or a precursor of either of these, together with other molecular species, is subjected to chromatography, and the purification in step (d) comprises purification by mixed-mode chromatography. Preferably, the mixed-mode chromatography comprises the use of a stationary phase having charged groups, preferably negatively charged groups, as well as aromatic and / or hydrophobic groups.

[0188] Purification in the broadest sense according to the present invention means that the SEQ ID NO:3 or SEQ ID NO:4 polypeptide has been at least partially separated from other molecular species, including other proteins (e.g., host cell proteins, precursors, and / or degradation products). As a result, an at least partially purified SEQ ID NO:3 or SEQ ID NO:4 polypeptide is obtained. Other molecular species may or may not, as the case may be, be discarded, but the SEQ ID NO:3 or SEQ ID NO:4 polypeptide is preferably obtained and retained as a result of the purification.

[0189] Preferably, mixed mode chromatography involves the use of a stationary phase having charged groups, preferably negatively charged groups, as well as aromatic and / or hydrophobic groups.

[0190] Each of these steps may itself involve several operations, but for simplicity they may also be referred to as steps. By way of illustration, and as detailed below, step (d) may involve more than one purification step, e.g., on more than one stationary phase.

[0191] Any letter or number designation used herein with respect to one or more process steps, e.g., (a), (b), (c), (d), (d1), (d2), etc., should not be understood as limiting, but rather as a reference. The order of events in a process or use according to the invention should not be understood to be limited by alphabetical letter or numerical number designation. Notwithstanding the above, it is strongly preferred that the order of events in a process or use according to the invention is the order set forth herein.

[0192] Additional aspects of mixed-mode chromatography, particularly suitable stationary phases, are described in some more detail below, but these aspects are generally applicable to the present invention. Thus, all those stationary phases (including all their embodiments) described below as being particularly useful for mixed-mode chromatography in step (d2) are generally useful for purifying 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 (e.g., in combination with or without the step of (d1) capture chromatography). Indeed, Example 2B describes that some advantages can be achieved by using mixed-mode chromatography in variations of state-of-the-art protocols.

[0193] Optionally, the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 is obtained in a process that includes (re)folding and / or chromatographic purification and / or protease digestion, and optionally adjustment to a final protein concentration and / or preparation of a desired formulation.

[0194] Thus, administration of the polypeptide of SEQ ID NO:3 or SEQ ID NO:4 to a subject in need thereof, as disclosed herein, is also made possible through industrially relevant purity and yield of the polypeptide of SEQ ID NO:3 or SEQ ID NO:4, which is available to one of skill in the art based on the disclosure herein. Thus, the present disclosure also describes processes for the production of the polypeptide of SEQ ID NO:3 or SEQ ID NO:4.

[0195] The process for the production of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 according to the present invention preferably comprises the following steps: (a) obtaining a precursor of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4, for example by recombinant expression; (d) Purification, wherein purification includes mixed-mode stationary phase purification.

[0196] It is also preferred in accordance with the present invention that the precursor of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is subjected to the following steps: (c) Exposure to proteases.

[0197] The exposing typically occurs before step (d).

[0198] The process of the invention is also preferably characterized in that no chromatographic purification is carried out before exposure to the protease. Indeed, the inventors have surprisingly found that digestion with a protease also works well and efficiently in crude fractions obtained from host cells, i.e., when no chromatographic purification is carried out before exposure to the protease.

[0199] Preferably, the step of obtaining (a) comprises expression, preferably recombinant expression, of a precursor of the polypeptide of SEQ ID NO:3 or SEQ ID NO:4. 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. The fraction may consist of the host cell (i.e., in cases where the protein is not substantially 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 other intracellular compartments (including the cytosol). Suitable host cells can be selected from prokaryotic and eukaryotic host cells, although prokaryotic host cells are preferred in exemplary embodiments. Preferred prokaryotic host cells include Escherichia coli (E. coli), preferably E. coli Rosetat (DE3). In one embodiment, the polypeptide of SEQ ID NO:3 or SEQ ID NO:4 is obtained in a conformation other than its native conformation and / or in aggregates, most preferably in inclusion bodies. Preferably, the process of the invention then comprises a 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).

[0200] 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 such a manner as to release the (mature) polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. In a particular embodiment, said protease is trypsin, preferably porcine trypsin, optionally recombinantly expressed.

[0201] Preferably, step (d) of purification comprises the following steps, preferably in sequential order: (d1) capture; (d2) Polishing.

[0202] Preferably, the step (d1) of capture is carried out by chromatography, preferably by column chromatography. More preferably, said step (d1) of capture is carried out using a cation exchange chromatography stationary phase or a mixed mode chromatography stationary phase. Even more preferably, said step (d1) of capture is carried out using a mixed mode chromatography stationary phase, which is preferably Capto MMC.

[0203] Preferably, the polishing step (d2) is carried out by chromatography, preferably column chromatography. More preferably, said polishing step is carried out using a cation exchange chromatography stationary phase. Even more preferably, said capture step (d1) is carried out using SP Sepharose, preferably SP Sepharose with a small particle size. SP is an abbreviation for sulfopropyl.

[0204] Optionally, the process according to the invention comprises additional steps of adjusting the final protein concentration and / or preparing the desired formulation, so that the composition according to the invention is obtained.

[0205] In other words, the present invention provides mixed-mode chromatography for the preparation of a polypeptide of SEQ ID NO:3 or SEQ ID NO:4. Mixed-mode chromatography is useful in the preparation of a 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 SEQ ID NO:4 is exposed to a protease for digestion purposes, and mixed-mode chromatography is used in a step following exposure to the protease. In a preferred embodiment, chromatographic purification of the polypeptide of SEQ ID NO:3 or SEQ ID NO:4 is not performed prior to said exposure to the protease.

[0206] Polypeptide Purity

[0207] Polypeptides of the present invention are substantially or essentially free from components that normally accompany them in their native state. Polypeptides of the present invention are isolated prior to administration. In one embodiment, an "isolated polypeptide" refers to a polypeptide that has been purified from its cellular and extracellular environment, such as the tissue that surrounds it in its naturally occurring state, e.g., from the cell (e.g., host cell) in which it is expressed. In another embodiment, an "isolated polypeptide" refers to the in vitro isolation and / or purification of a polypeptide from its native cellular environment and from association with other components of the environment in which the polypeptide normally resides, respectively.

[0208] Preferably, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for use according to the 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.

[0209] The polypeptides of SEQ ID NO: 3 or SEQ ID NO: 4 described herein are considered to be pharmaceutically active peptides or proteins.

[0210] In a particularly advantageous embodiment of the present invention, the polypeptides according to the invention are obtained essentially free of degradation products of said polypeptides. In particular, the inventors have observed that, contrary to what has been reported for wild-type human NGF in the state of the art, exposure of the precursor of SEQ ID NO: 4 to trypsin inherently partially cleaves said precursor at the C-terminus of arginine (Arg, R) residue 9 of SEQ ID NO: 4, either before or after purification, unless trypsin is completely removed by purification (des-nona mutants, data not shown). The specific purification methods provided in the present invention allow the polypeptides according to the invention to be obtained essentially free of trypsin and / or des-nona mutants.

[0211] Preferably, the polypeptides obtainable as described above are essentially free of polypeptide degradation products. In particular, the present disclosure makes the polypeptides of the present invention available in new and improved purity grades, and it is preferred that the polypeptides be administered at such high purity. Preferably, the polypeptides of the present invention for use in accordance with the present invention are characterized by a purity grade of at least 90%. More preferably, the polypeptides of the present invention for use in accordance with the present invention are characterized by a purity grade of at least 91%. More preferably, the polypeptides of the present invention for use in accordance with the present invention are characterized by a purity grade of at least 92%. More preferably, the polypeptides of the present invention for use in accordance with the present invention are characterized by a purity grade of at least 93%. More preferably, the polypeptides of the present invention for use in accordance with the present invention are characterized by a purity grade of at least 94%. More preferably, the polypeptides of the present invention for use in accordance with the present invention are characterized by a purity grade of at least 95%. More preferably, the polypeptides of the present invention for use in accordance with the present invention are characterized by a purity grade of at least 96%. More preferably, the polypeptides of the present invention for use in accordance with the present invention are characterized by a purity grade of at least 97%. More preferably, the polypeptides of the invention for use according to the invention are characterized by a purity grade of at least 98%. Even more preferably, the polypeptides of the invention for use according to the invention are characterized by a purity grade of at least 99%.

[0212] Most preferably, the polypeptides of the invention for use according to the invention are characterized by a purity grade of greater than 99.0%, such as 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%, etc.

[0213] As used herein, "purity grade" generally refers to the percentage by weight (w) of a polypeptide according to the present invention relative to the weight (w) of biological material other than the polypeptide of the present invention. For illustrative purposes, in a 99.0% purity grade, 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 material other than the polypeptide of the present invention is 1.0 unit (e.g., 1.0 mg). Such biological material other than the polypeptide of the present invention includes, but is not limited to, host cell proteins, nucleic acids, proteases (e.g., trypsin, etc.) (inactivated or not), degradation products of the polypeptide of the present invention, and other macromolecules of biological origin. In certain embodiments, the "purity" grade refers to purity relative to polypeptides other than the polypeptide of the present invention. For illustrative purposes, in that embodiment, 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 polypeptides below that are not identical to the polypeptide of the present invention is 1.0 unit (e.g., 1.0 mg). Degradation products of the polypeptides of the invention are included, for the avoidance of doubt, in "polypeptides that are not identical to the polypeptides of the invention." Particular degradation products are des-nona variants (see Examples 1 and 2).

[0214] In particular, the polypeptides are essentially free of des-nona variants of the polypeptides. Des-nona variants are previously uncharacterized degradation products of the polypeptides of the invention that are associated with the production of specific variants of NGF, including the polypeptides of the invention, when the polypeptides are not produced by the novel methods disclosed herein (see, e.g., Examples 1 and 2). "Essentially free" in this context is intended to mean that the polypeptides of the invention for use according to the invention are characterized by a purity grade with respect to des-nona variants of greater than 99.0%, e.g., 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 with respect to des-nona variants). In the most preferred embodiment, des-nona variants are undetectable and / or absent.

[0215] It is also preferred that the polypeptides according to the invention are essentially free of any proteases (such as trypsin). "Essentially free" in this context is intended to mean that the polypeptides of the invention for use according to the invention are characterized by a purity grade of greater than 99.0% relative to the sum of all proteases (including trypsin), such as 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 relative to the sum of all proteases (including trypsin)). In a most preferred embodiment, trypsin is undetectable and / or absent.

[0216] Such a high purity grade, in the above embodiment, is associated with improved acceptability by regulatory authorities, qualifying the polypeptide of the present invention as a medicine for use in mammalian subjects, including humans in particular. Thus, the purity grade according to the present invention allows for the first time the use of the polypeptide for administration to wound body surfaces, including human wound body surfaces, and in particular ulcers, in a safe and reliable manner. The high purity grade, particularly with respect to the protease (trypsin), allows for the preservation of the polypeptide even in a non-frozen form.

[0217] composition

[0218] In some embodiments, the polypeptides of SEQ ID NO: 3 or SEQ ID NO: 4 described herein are included in a composition that additionally comprises one or more carriers and / or one or more excipients. As used herein, the term "carrier" refers to an organic or inorganic component, of natural or synthetic nature, with which an active ingredient is combined to enable, enhance, or facilitate application of the active ingredient. As used herein, the term "excipient" is intended to refer to any substance that may be present in the pharmaceutical composition of the present invention and that is not an active ingredient.

[0219] Preferably, compositions according to the present invention comprise at least water as an excipient. In some embodiments, compositions according to the present invention comprise an aqueous medium, and more preferably, compositions according to the present invention are in the form of an aqueous solution. In one embodiment, the polypeptide is contained in an aqueous medium, and the aqueous medium is administered to a mammalian subject. The aqueous medium can be, for example, an aqueous solution. The aqueous solution and each other composition can, in some embodiments, be obtained directly from the purification of NGF in the aqueous medium. For example, if a pharmaceutical agent according to the present invention is obtained by purification from a biological source, each aqueous composition can be obtained directly from the final purification step, such as elution and / or filtration from the final chromatography column (usually a polishing step). Alternatively, each composition can be obtained through additional steps of adjusting the final protein concentration and / or preparing the desired formulation. Such additional steps can include, for example, a clarification or filtration step as described herein, and / or the addition of one or more excipients and / or one or more carriers. Exemplary compositions useful in the present invention are described herein, but are not limited thereto.

[0220] Thus, the polypeptides of SEQ ID NO:3 or SEQ ID NO:4 described herein can be present in a composition, for example, a pharmaceutical composition. The compositions described herein are preferably sterile and preferably comprise the polypeptide of SEQ ID NO:3 or SEQ ID NO:4 as a pharmaceutically active peptide or protein, and optionally additional agents mentioned or not mentioned herein. The composition can be in any state (e.g., liquid, frozen, lyophilized, etc.).

[0221] The compositions described herein may include salts, buffers, preservatives, carriers, diluents, and / or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" describes something that is not toxic and / or does not interact with the action of the active ingredients of the pharmaceutical composition.

[0222] Suitable buffer substances for use in the present invention include acetic acid in a salt, citric acid in a salt, boric acid in a salt, and phosphoric acid in a salt. For example, the polypeptides of the present invention, as a result of various aspects of the present invention, are preferably available in a buffer having a pH between 4.5 and 6.5, preferably between 5.0 and 6.0. In one embodiment, acetate buffers are suitable buffers for such purposes and are therefore particularly preferred. Thus, in one embodiment, the polypeptides of the present invention are available in acetate buffers having a pH between 4.5 and 6.5, preferably between 5.0 and 6.0.

[0223] Suitable preservatives for use in compositions according to the present invention include those known in the art, including, by way of example but not limitation, benzyl alcohol, benzalkonium and its salts, M-cresol, phenol, chlorobutanol, parabens, and thimerosal.

[0224] Thus, the present invention provides polypeptides of SEQ ID NO: 3 or SEQ ID NO: 4 for therapeutic use, i.e., for use in methods of treatment of the human or animal body by therapy. Treatment may include prevention and / or treatment of a condition. In view of their potential therapeutic use, the polypeptides may also be referred to as pharmaceutically active proteins or peptides.

[0225] Optionally, administration according to the present invention involves administration of at least one antimicrobial agent (such as an antibiotic), which may be part of a composition comprising a polypeptide according to the present invention, or may be administered separately to the subject at the same or a different site, by the same or a different route of administration.

[0226] Industrial Applicability.

[0227] The polypeptides of SEQ ID NO: 3 or SEQ ID NO: 4 described herein are suitable for a variety of purposes, for example for the therapeutic applications described herein.

[0228] The following examples and figures are intended to illustrate some preferred embodiments of the invention and should not be construed as limiting the scope of the invention, as defined by the claims.

[0229] Example.

[0230] Materials and methods common to more than one example.

[0231] Unless otherwise specified, the following experimental examples specifically relate to the polypeptide of SEQ ID NO: 4, characterized by the substitution P61S R100E (referred to as "NGFP61S R100E"; Malerba et al., PLOS One, 2015, vol. 10, e0136425, SEQ ID NO: 4), as well as its proform, relative to wild-type human NGF. The polypeptide of SEQ ID NO: 4 may also be referred to as an "NGF mutein," but it should be noted that the specific therapeutic relevance of this protein is significantly different from wild-type human NGF, according to the present invention and as demonstrated in the experimental examples, particularly in Examples 3 and 4. Similarly, as described in Example 2, the purification of the polypeptide of SEQ ID NO: 4 differs from published purification protocols for wild-type NGF, and the specific process for preparing the polypeptide according to the present invention is suitable for achieving high purity, particularly the absence of des-nona variants and trypsin.

[0232] The polypeptide of SEQ ID NO: 4 was recombinantly expressed as a precursor. To this end, SEQ ID NO: 4 was fused to 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 consisted of the precursor of human wild-type NGF (SEQ ID NO: 1) except for the substitutions P61S R100E in the mature portion of human wild-type NGF (SEQ ID NO: 1) (but, for clarity, lacked the two most C-terminal amino acids of SEQ ID NO: 1, which do not form part of the polypeptide sequence of human wild-type NGF). Expression was carried out in the form of insoluble inclusion bodies in E. coli Rosetta (DE3) (strain: E. coli Rosetta (DE3) / pET11a-hpro NGF P61S R100E).

[0233] Equipment. [Table 1]

[0234] Protein parameters for the proteins and peptides described herein.

[0235] Theoretical protein parameters of the relevant proteins were calculated using the ProtParam-Tool of ExPASy, available at http: / / web.expasy.org / protparam, and are shown in Table 2 as follows: [Table 2]

[0236] Analysis method

[0237] SDS-PAGE and Western blot

[0238] SDS-PAGE and Western blotting were performed using standard procedures. For SDS-PAGE, 12% Bis-TRIS NuPAGE gels (product number NP0342BOX from Thermo Fisher Scientific) were run under reducing conditions at constant voltage (175V) in NuPAGE MES running buffer (product number NP0002 from Thermo Fisher Scientific). The primary antibody for Western blotting was purchased from Santa Cruz Biotechnology (NGF(H-20)sc-548). Example results are shown, for example, in Figures 9A and 10.

[0239] Analytical CEX-HPLC

[0240] CEX-HPLC was performed using a ProPacSCX-10 from Dionex. The column was operated at 1 mL / min with 50 mM citrate buffer (pH 5.5). For elution, 1 M NaCl (B) was added and a linear gradient from 0 to 100% B over 50 min was performed. An example of the results is shown in Figure 9B.

[0241] SE-HPLC

[0242] SE-HPLC was performed using a Superdex200 Gain 10 / 300GL (GE Healthcare). The column was operated in PBS. Products were detected at 280 nm.

[0243] Endotoxin, DNA, and HCP

[0244] Endotoxin, DNA, and host cell proteins (HCP) were determined by standard protocols.

[0245] Example 1: Expression of the polypeptide of SEQ ID NO: 4 as a precursor protein.

[0246] producing strain

[0247] The gene encoding proNGF was cloned into the pET11a expression plasmid. This gene was derived from H. sapiens, and two point mutations (i.e., P61S and R100E) were introduced into the open reading frame. Chemically competent Rosetta (DE3) cells were then transformed with the expression plasmid, and single colonies were selected (the resulting strain was designated E5901 STRAIN (= E. coli Rosetta (DE3) / pET11a-proNGF P61S R100E This was designated NGF RCB C-151101). Aliquots were stored in 1.0 mL volumes at <-60°C.

[0248] Example 1 describes the initial fermentation development based on strain E5901 STRAIN.

[0249] Equipment. [Table 3]

[0250] Growth medium

[0251] Complex media for fermentation

[0252] The complex medium used for the 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 used for this fermentation consisted of 263 g / L yeast extract and 133 g / L glucose.

[0253] Minimal medium (MM) for fermentation [Table 4]

[0254] In the batch phase, both basal media were supplemented with 30 g / L of glucose. Unless otherwise stated, the feeds had the same composition as the respective batch media but contained 300 g / L of the respective carbon source.

[0255] LB agar plates containing ampicillin and chloramphenicol

[0256] Freshly poured LB agar plates were prepared. The medium consisted of 10 g / L peptone, 5 g / L yeast extract, 5 g / L NaCl, and 15 g / L agar. After autoclaving, the medium was supplemented with 100 μg / mL ampicillin and 30 μg / mL chloramphenicol.

[0257] fermentation

[0258] Unless otherwise stated, fermentations were carried out in 1 L stirred glass bioreactors controlled by a Biostat B unit from Sartorius in this Example 1. Typically, pO2 was controlled at 30%, the culture temperature was set at 37°C, and the pH was controlled at 7 using 2 M phosphoric acid and 25% ammonium hydroxide. Unless otherwise stated, the batch phase was followed by an exponential feed of F = 6 g / L / h and μ = 0.25 / h. For practical reasons, all exponential feeds were approximated by two linear feeds. Typically, induction of product expression was performed by adding 1 mM IPTG, and a constant feed rate of 10 g / L / h was applied after induction. Cell biomass was collected by centrifugation using a Sorvall Evolution RC from Thermo Scientific. The centrifuge was equipped with an SLC-6000 rotor, and the culture was centrifuged at 8500 rpm and 4°C for 30 minutes.

[0259] Relative quantification of products in biomass samples.

[0260] At a given time point, culture samples were analyzed for OD 600The biomass was diluted to 100 μL, and a 100 μL aliquot of this dilution was pelleted. The pellet was resuspended in 150 μL (non-reducing) Laemmli buffer, and the sample was boiled at 95°C for 5 minutes. 10 μL of each sample was analyzed on a 10% Bis-Tris gel (Novex). Electrophoretic separation was performed at 125 V for 90 minutes, and the gel was stained with Coomassie. The destained gel was scanned, and the abundance of the band corresponding to the precursor of the polypeptide of SEQ ID NO: 4 was quantified by densitometry. To further correct for variability in the biomass utilized, the intensity of the band corresponding to the precursor of the polypeptide of SEQ ID NO: 4 was normalized to the intensity of a housekeeping protein.

[0261] The relative product accumulation was calculated from the increase in the band corresponding to the precursor of the polypeptide of SEQ ID NO: 4 after induction and before induction. It is worth noting that the measurements were taken as a function of the specific yield (i.e., OD 600 = 10). For absolute yields of a given fermentation, the actual cell density must be taken into consideration (see below).

[0262] Absolute quantification of products in biomass samples

[0263] Standards for the precursor of the polypeptide of SEQ ID NO: 4 were obtained from the European Brain Research Institute (EBRI, Rome, Italy). The standards were diluted in Laemmli buffer to a concentration of 65 μg / mL. The stated protein concentrations were defined by EBRI. A standard curve was prepared using 260, 520, 780, 1040, and 1300 ng of standards for the precursor of the polypeptide of SEQ ID NO: 4. Samples were analyzed on the same gel as the calibration curve, and the dilution factor of the samples was taken into account to calculate the absolute product yield at a given time.

[0264] Summary and Conclusions of Example 1

[0265] Based on the above, it was concluded that the production strain (E5901 STRAIN, see above) was successfully used for fermentation in 1 L scale. Different medium compositions have been assessed for their ability to promote bacterial growth and product expression, and minimal medium MMI supplemented with 5 g / L yeast extract proved advantageous in terms of expression yield and obtainable cell density. No significant differences in terms of product formation were observed when the main culture was performed with or without antibiotics (ampicillin and chloramphenicol, data not shown).

[0266] Example 1 can be scaled up to produce polypeptides on an industrial scale.

[0267] Example 2: Lab-scale purification, establishment of Capto MMC.

[0268] The precursor of SEQ ID NO: 4, as used in this example, was obtained in inclusion bodies as described in Example 1.

[0269] A starting point for state-of-the-art optimization.

[0270] At the outset, the inventors reasoned that process development could follow the basic foundations of NGF purification previously reported in the literature, in the absence of any indications to the contrary. However, they also kept in mind that for efficient production on a large scale, suitable adaptations for subsequent scale-up should be considered. Thus, based on Rattenholl et al. (supra), WO2013092776 A1, and other publications, it was reasoned that the polypeptide of SEQ ID NO: 4 could similarly be obtained, at least in a laboratory-scale process, via its proform using nonspecific digestion with trypsin and subsequent purification. It was reasoned that a highly pure mature polypeptide of SEQ ID NO: 4 could thereby be obtained. However, it was only through the specific adaptations and modifications reported in this example that a highly pure mature polypeptide of SEQ ID NO: 4 was obtained. Thus, administration of the polypeptide of SEQ ID NO: 4 to a subject in need thereof becomes possible, particularly in view of the high purity of the polypeptide of SEQ ID NO: 4 described herein.

[0271] Equipment, production of polypeptide of SEQ ID NO:4. [Table 5]

[0272] List of equipment used in Example 2.

[0273] Details of the manufacturing process according to this example (including the improvements described in Example 2B) are provided in the process overview in FIG.

[0274] Unless otherwise specified, analytical methods are as described above in the section "Analytical Methods."

[0275] Example 2A: Purification according to previously described protocol.

[0276] E. coli cells expressing the precursor of the polypeptide of SEQ ID NO:4 ("biomass") were produced as described in Example 1, and the cells were lysed by the addition of lysozyme followed by sonication on ice. Inclusion bodies ("IBs") were (1) extracted from the host cells and washed with 6% Triton X100 (in 1.5 M NaCl, 60 mM EDTA), and (2) solubilized in 6 M guanidinium HCl ("gHCl"), 0.1 M Tris-HCl pH 8.0, 1 mM EDTA, 100 mM (fresh) DTT. The IBs were solubilized for 2 hours at room temperature. The pH was then lowered to 3-4 by the addition of 37% HCl. The resulting solution, containing the solubilized precursor of the polypeptide of SEQ ID NO:4 ("solubilate"), was dialyzed against 6 M gHCl (pH 3-4).

[0277] Refolding of the precursor of the polypeptide of SEQ ID NO: 4 was carried out at +4°C in 0.1 M Tris-HCl, 1 M L-arginine, 5 mM EDTA, 0.61 g / L oxidized glutathione, and 1.53 g / L reduced glutathione (pH 9.5). Therefore, 50 μg of protein was added per mL of refolding buffer every hour. After refolding, the reaction was dialyzed against 50 mM sodium phosphate (pH 7.0). Significant precipitation occurred during buffer exchange.

[0278] The precursor of the polypeptide of SEQ ID NO: 4 was purified over a sequential sequence of cation exchange chromatography (SP Sepharose HP operated in 50 mM sodium phosphate, pH 7.0, and eluted with a NaCl gradient) followed by hydrophobic interaction chromatography (Phenyl Sepharose HP operated in 50 mM sodium phosphate, 1 M ammonium sulfate, pH 7.0). The sample was then buffer exchanged against 50 mM sodium phosphate, pH 7.0 using another dialysis (note, however, that such a second dialysis can be omitted in the process of Example 5). Again, a significant amount of product precipitated throughout the process of decreasing the buffer conductivity.

[0279] The precursor of the polypeptide of SEQ ID NO: 4 thus prepared was subjected to limited proteolysis by adding 1 mg of trypsin per 250 mg of proNGF. The precursor of the polypeptide of SEQ ID NO: 4 was exposed to the protease for 14 hours at 2-8°C.

[0280] The mature NGF was finally polished on a cation exchanger (SP Sepharose XL run in 50 mM sodium phosphate, pH 7.0, and eluted with a NaCl gradient). Finally, the product was concentrated to 0.5–1 mg / mL and frozen at <−65°C.

[0281] Example 2B: Improvement.

[0282] Below, some improvements (compared to Example 2A) are described as they were tested and implemented by the inventors in the course of arriving at the present invention. Unless the context dictates otherwise, all details not explicitly stated were as described above for Example 2A.

[0283] Optimization of IB solubilization.

[0284] It has previously been reported that small amounts of IB (exemplary as received from shake flask cultures) readily dissolve in solubilization buffer (6 M GHCL, 0.1 M TRIS-HCl pH 8.0, 1 mM EDTA, 100 mM (fresh) DTT), whereas IB obtained from high cell density fermentations could not be completely degraded. This could be resolved by the addition of 2 M urea to the solubilization buffer, which we found to significantly improve solubilization yields (data not shown). For the avoidance of doubt: 2 M urea was present in addition to 6 M GHCL and other components.

[0285] Refolding optimization.

[0286] Initially based on Rattenholl et al. (2001, Eur. J. Biochem, vol. 268, pp. 3296-3303; Rattenholl, 2001, Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.), Martin-Luther-Universitat Halle-Wittenberg, Germany), but importantly taking into account later scalability, the refolding is carried out using 200 to 500 mg of precursor of the polypeptide of SEQ ID NO: 4 per liter of refolding reaction, preferably 200 to 300 mg of precursor of the polypeptide of SEQ ID NO: 4 per liter of refolding reaction. This leads to a relatively good yield of solubilized precursor of the polypeptide of SEQ ID NO: 4. It is important to consider that with such an increased amount of NGF, especially compared to the volume of the refolding reaction, and considering that the refolding reaction involves relatively expensive components (e.g., glutathione and arginine), relatively more precursor of the polypeptide of SEQ ID NO: 4 can be refolded per volume of the refolding reaction, which should make refolding economically feasible even at production scale.

[0287] Purification of the precursor of the polypeptide of SEQ ID NO:4.

[0288] The purification of the precursor of the polypeptide of SEQ ID NO: 4 was carried out by an approach that takes advantage of the fairly high isoelectric point of proNGF after refolding and uses a cation exchange stationary phase (i.e., SP Sepharose) for purification. To perform this type of chromatography, for technical reasons, the refolding buffer must be exchanged for a buffer with a low conductivity. During this process, a significant amount of the precursor of the polypeptide of SEQ ID NO: 4 precipitated (data not shown). This observation may be due to the reduced arginine concentration in the buffer.

[0289] Therefore, some efforts were made to replace the capture column with another column (one with different selectivity) that would be more tolerant of the presence of arginine in the refolding reaction. Initial attempts to do this assessed the performance of several stationary phases, but none of the approaches yielded promising results (see Table 6). Therefore, the stationary phase used for the capture column was maintained as defined by the previous process. However, due to the high isoelectric point (pI) of the precursor of the polypeptide of SEQ ID NO:4, an increase in the conductivity of the running buffer (by adding 250 mM L-arginine) was possible without affecting performance. This allowed the refolded precursor of the polypeptide of SEQ ID NO:4 to be stabilized to some extent and reduced the amount of precipitated precursor (data not shown). [Table 6]

[0290] With respect to mixed-mode chromatography, but without wishing to be bound by any particular theory, it is understood by the inventors that the precursor of the polypeptide of SEQ ID NO: 4 does not elute efficiently from mixed-mode chromatography, whereas the mature polypeptide of SEQ ID NO: 4 does elute.

[0291] Protease digestion to yield mature NGF.

[0292] For the production of the polypeptide of SEQ ID NO: 4, it was reasoned that a protease (trypsin) is essential and, therefore, ideally, the particular trypsin chosen should fulfill the following criteria: 1. Derived from recombinant sources. Certification of animal-free raw materials is crucial for GMP compliance of the subsequent processes required. 2. Low side activity of trypsin. Notably, trypsin can undergo autolysis. This process can result in so-called pseudotrypsins, which have a broadened substrate spectrum and chymotrypsin-like activity. 2+ Autolysis may be reduced by adding HCl (e.g., 1 mM CaCl2). However, nowadays, "modified trypsin" is typically applied for all protocols that require strict sequence specificity (e.g., for peptide fingerprinting). This modified trypsin is typically obtained by acylation of the exposed ε-amino groups of lysine residues of trypsin. 3. Low batch-to-batch variability to allow for a reproducible production process. Alternatively, the enzyme of choice should be delivered with a certificate stating the specific activity of each batch. The amount of enzyme required can then be based on activity rather than mass.

[0293] Despite a comprehensive search, no trypsin was identified on the commercial market that fulfilled both criteria 1 and 2. We reasoned that criterion 1 was more important. To reduce autolysis, the addition of CaCl2 may be sufficient. As a result, recombinant "GMP-grade" trypsin from Roche (Roche 06369880103, lot: 11534700) was chosen as the raw material for the process. The sequence of this enzyme, expressed in Pichia pastoris, is derived from wild boar. According to its certificate, the utilized trypsin batch has a specific activity of 4997 U / mg (as determined by USP).

[0294] Omission of the second purification step before trypsinization.

[0295] In an initial screening search for the optimal enzyme / substrate ratio for the intended trypsinization, we used the precursor of the polypeptide of SEQ ID NO: 4 obtained from the capture column (see above). In contrast to a previously established process (European Brain Research Institute (EBRI), details not published, based on Rattenholl et al., supra), we decided not to use an additional hydrophobic interaction chromatography step before trypsinization. The decision to omit such a second column purification step before trypsinization was based mainly on two considerations: on the one hand, the product obtained after the capture column was already substantially pure by SDS-PAGE. On the other hand, the trypsinization itself could help improve the impurity profile by digesting remaining host cell proteins (HCPs).

[0296] Table 7 summarizes the matrix of conditions screened within the first round. The results of trypsinization were studied by 12% SDS-PAGE (data not shown). The results (data not shown) indicate that trypsinization reproducibly yields a stable polypeptide of SEQ ID NO: 4 over a fairly wide range of enzyme / substrate ratios (i.e., 1-5 μg of trypsin per 375 μg of precursor polypeptide of SEQ ID NO: 4). The timing of digestion is not highly critical; therefore, the time required to stop the reaction and load it onto the polishing column is not clearly limiting. This finding is particularly important because the reaction cannot be quenched adequately or economically on a preparative scale.

[0297] Several additional experiments were performed to refine the optimal enzyme / substrate ratio for the expected trypsin digestion. Enzyme / substrate ratios ranging from 1 / 100 to 1 / 200 (protein weight / protein weight) reproducibly yielded good yields of the polypeptide of SEQ ID NO:4, on the one hand, and small amounts of cleavage products, on the other. It should be noted that under the conditions used (i.e., in phosphate / arginine buffer (pH 7.0) at 2-8°C and incubation (protease exposure) for 2-6 hours), the quality of the digestion was not highly dependent on the enzyme / substrate ratio. This finding is particularly important because the underlying enzymatic digestion is prone to small variations in experimental setup (e.g., changes in trypsin activity due to batch-to-batch variability or enzyme storage; timing and temperature of the incubation step (protease exposure); errors in determining protein concentration). Furthermore, this also explains why extended fine-tuning on a small scale to further reduce potential cleavage products seems pointless. If "optimal" conditions can be identified on a small scale, then there is still a good chance that when substantially the same digest is repeated on a larger scale, it will produce a slightly altered product pattern.

[0298] Trypsinization followed by polishing chromatography with the aim of obtaining pure polypeptides.

[0299] In contrast to a previously established process using an SP Sepharose stationary phase for polishing the mature polypeptide (European Brain Research Institute (EBRI), details not published, based on Rattenholl et al., supra) (note: SP Sepharose is a cation exchange stationary phase), a search for a more suitable stationary phase was conducted herein based on the following considerations: To efficiently load the SP Sepharose column, a decrease in the conductivity of the solution containing the precursor of the polypeptide of SEQ ID NO: 4 is required, e.g., by buffer exchange. However, a decrease in the ionic strength of the solution is known to result in precipitation of the target molecule (e.g., Example 2A), and therefore, buffer exchange to a low-conductivity buffer should be avoided. Furthermore, although a cation exchange stationary phase has already been used for capture of the precursor of the polypeptide of SEQ ID NO: 4, orthogonal selectivity is preferred to achieve better separation of the remaining contaminants. The third and final argument against using an SP stationary phase for purification of the trypsinization reaction is that potentially remaining precursors of the precursor polypeptide of the polypeptide of SEQ ID NO: 4 may bind to this column and be separated from the mature polypeptide of SEQ ID NO: 4 solely by elution selectivity, not by binding selectivity.

[0300] To establish such an orthogonal polishing column for the purification of the mature polypeptide of SEQ ID NO: 4, we intended to use a hydrophobic interaction (HIC) column in the first example. This stationary phase was chosen not only for its orthogonal selectivity, but also because it does not require buffer exchange to a low-conductivity buffer. Despite testing several HIC stationary phases and conditions (e.g., phenyl-Sepharose and butyl-Sepharose operated with 1 M (NH4)2SO4 and 0.5 M (NH4)2SO4, respectively), we were unable to perform a satisfactory polishing step based on HIC (data not shown).

[0301] However, in a further experimental setup for the polishing step, the mixed-mode stationary phase Capto MMC was successfully tested. Using optimized conditions, it was found that the stationary phase reversibly bound the polypeptide of SEQ ID NO:4, and the product could be eluted by increasing the pH (data not shown). In contrast, the precursor of the polypeptide of SEQ ID NO:4 bound irreversibly to the stationary phase and could only be eluted by using 1 M NaOH as the mobile phase (data not shown). Furthermore, it was shown that trypsin did not bind at all to the column operated under the same conditions (data not shown). These results provide clear evidence that the Capto MMC stationary phase is capable of efficiently separating the mature polypeptide of SEQ ID NO:4 from trypsin and from the remaining precursor of the polypeptide of SEQ ID NO:4.

[0302] Establishment of additional membrane chromatography.

[0303] To further deplete endotoxins and DNA, an additional anion exchange membrane was included in the process. Generally, and as is commonly known, membrane chromatography is characterized in that a solution containing the component to be analyzed or purified (in this case, the polypeptide of SEQ ID NO: 4) is passed over or through a membrane (usually charged). For this purpose, in this case, a STIC membrane (Sartorius, Göttingen, Germany) was incorporated at the location shown in Figure 1. It could be shown that the polypeptide of SEQ ID NO: 4 does not bind to the membrane, and thus provided proof of concept that membrane chromatography is suitable for the purification of the polypeptide of SEQ ID NO: 4. For an illustration of the incorporation in the entire process (including membrane chromatography), see Figure 1.

[0304] Reproducibility of the process according to Example 2.

[0305] To explore the robustness of the process, the process was run five times, and the resulting fractions were analyzed for yield and purity. Through these runs, steady optimization of process details was pursued, and buffer compositions, gradients, etc. were adapted until the final optimized process details (see Figure 1) were established. Results indicate that laboratory-scale production of approximately 50-100 mg of SEQ ID NO:4 polypeptide can be obtained from a single, consistent production run. Notably, the resulting product was consistently found to be relatively pure (less than 5 percent of contaminating host cell proteins and only traces of cleaved NGF, data not shown) by SDS-polyacrylamide gel electrophoresis followed by Coomassie or silver staining.

[0306] No meaningful method for SE-HPLC could be established for the precursor of the polypeptide of SEQ ID NO: 4. 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, consistent with the monomeric state of the polypeptide of SEQ ID NO: 4 (data not shown).

[0307] Summary and conclusions.

[0308] For this process, the refolded precursor of the polypeptide of SEQ ID NO: 4 was captured using SP Sepharose FF ("FF" stands for Fast Flow, i.e., a stationary phase with relatively large particles) and then treated with trypsin to yield mature NGF. To that end, the arginine concentration of the refolding reaction was reduced from 1 M (as recommended by the prior art) to 350 mM.

[0309] Control of the proteolytic cleavage of the precursor of the polypeptide of SEQ ID NO:4 to yield the mature polypeptide of SEQ ID NO:4 is considered the most critical factor for the process. Herein, conditions were identified that reproducibly promote high-efficiency cleavage and prevent the formation of NGF degradation products. The experimental data herein demonstrate that a robust production process can be established over a fairly wide range of enzyme / substrate ratios. For trypsin treatment, the process yields are clearly good, and no significant losses are expected at this stage of the process. The resulting product pattern does not clearly depend strongly on the reaction conditions used (in terms of enzyme / substrate ratio and incubation time (time of exposure to protease)). Notably, even when a good yield is expected from enzyme polishing, at least 2*× grams of the polypeptide of SEQ ID NO:4 must be processed to deliver × grams of the mature polypeptide of SEQ ID NO:4.

[0310] The purification according to this example is a lean process consisting of only two chromatographic purification steps. The existing purification process was further optimized, and some aspects were adopted for scale-up (see Figure 1). Exemplary, the previously used cell disruption method was replaced by high-pressure homogenization, and all dialysis steps were replaced by tangential flow filtration. The thus established process is capable of delivering the polypeptide of SEQ ID NO: 4 with high purity.

[0311] Despite the named challenges, the overall process appears to be capable of delivering a product that appears to be of acceptable quality.

[0312] The complete process (including membrane chromatography) incorporating the improvements according to Example 2 is depicted schematically in FIG.

[0313] Example 2 can be scaled up to produce polypeptides on an industrial scale.

[0314] Example 3: Proof of concept in vitro and in non-human mammals.

[0315] The present invention is based, in part, on experiments using two animal models of skin ulcers, in which skin ulcers are induced in diabetic mice by circular biopsy punch or by cycles of pressure loading, and a polypeptide of the invention is applied topically.

[0316] Reported herein is a study of the administration to non-human animals of the polypeptide of SEQ ID NO: 4. 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.

[0317] The purpose of this example is to study the effectiveness of topical application of the polypeptide of SEQ ID NO: 4 on wound healing, associated histopathology, pain threshold, and plasma human NGF (hNGF) levels in diabetic mice. Reference compounds (human NGF (SEQ ID NO: 2) and mouse NGF, amino acid sequences available in public sources) are also included in the study.

[0318] The animal to which the polypeptide of SEQ ID NO: 4 is administered is characterized by a skin disorder as described herein, and the animal represents an animal model of a human suffering from or predisposed to suffering from diabetes mellitus, e.g., type 1 diabetes mellitus or type 2 diabetes mellitus.

[0319] The polypeptide of SEQ ID NO: 4 may be administered in a single dose or in multiple doses. The polypeptide of SEQ ID NO: 4 may be administered to subjects with diabetic ulcers, animal models for diabetic neuropathic foot ulcers (DFUs).

[0320] This embodiment includes the following sections:

[0321] Example 3A: In vitro PC12 neurite outgrowth test. The purpose of this section was to establish the effectiveness of the polypeptide of SEQ ID NO: 4. For this purpose, a conventional in vitro neurite outgrowth test in NGF-sensitive cells (PC12) was used.

[0322] Example 3B: In vivo efficacy studies The purpose of this section was to determine whether topical application of the polypeptide of SEQ ID NO: 4 improved wound healing (surgical lesions) in diabetic mice.

[0323] The following groups were included: - db / db, intact N=8, each time point - db / db, wound + vehicle N=8, each time point - db / db, wound + polypeptide of SEQ ID NO: 4, 1 μg / day; N=8, each time point - db / db, wound + polypeptide of SEQ ID NO: 4, 10 μg / day; N=8, each time point - db / db, wound + polypeptide of SEQ ID NO: 4, 30 μg / day; N=8, each time point - db / db, wound + hNGF, 10 μg / day N=8, each time point - db / db, wound + mNGF, 10 μg / day N=8, each time point (Dose (μg) refers to the respective dose administered per wound (each animal with one wound)

[0324] In this section of the study, animals were sacrificed 7 and 30 days after wound induction and the following endpoints were used: time to closure; lesion histology (N=4) and immunohistochemistry (N=4).

[0325] Example 3C: Mechanism: Exploratory Studies. The purpose of this section was to explore the molecular mechanisms underlying the positive effects of the polypeptide of SEQ ID NO: 4 on wound healing in diabetic mice, focusing on inflammation, extracellular matrix deposition, innervation, and angiogenesis.

[0326] The following groups were included: - db / db, intact N=6 - db / db, wound + excipient N=6 - db / db, wound + polypeptide of SEQ ID NO: 4, 1 μg / day N=6 - db / db, wound + polypeptide of SEQ ID NO: 4, 10 μg / day N=6 - db / db, wound + polypeptide of SEQ ID NO: 4, 30 μg / day N=6 - db / db, wound+hNGF, 10μg / day N=6 - db / db, wound+mNGF, 10μg / day N=6 (Dose (μg) refers to the respective dose administered per wound (each animal with one wound)

[0327] In this section of the study, animals were sacrificed 14 days after wound induction (corresponding to 50% wound healing) and the following endpoints were used: exploration of possible mechanisms involved in the therapeutic effect of the polypeptide of SEQ ID NO: 4 on the expression and regulation of mRNA (N=252) encoding proteins involved in extracellular matrix biology (N=84), angiogenesis (N=84), growth factor and neurotrophin biology (N=84).

[0328] Materials and Methods in this Example.

[0329] Animals and monitoring.

[0330] Mice homozygous for the diabetic spontaneous mutation (Leprdb) (genetic background C57BL / 6J) and their respective heterozygous controls from the same colony were used at 8–12 weeks of age (Charles River Laboratories - Calco - Lecco, T / BKS.CG-M+ / +LEPR DB / J and S / BKS.CG-M DB / +). See the introduction for group composition and animal sacrifice.

[0331] Animals were housed in single cages with free access to food pellets and water and a 12-hour light-dark cycle. All animal protocols described herein were performed in accordance with the European Communities Council Directive (2010 / 63 / EU), approved by the Ministry of Health (n°350 / 2015-PB), and conformed to the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals.

[0332] Blood glucose levels were measured (Contour XT, Bayer, Basel, Switzerland) before treatment, the day after the last treatment, and before sacrifice.

[0333] Experimental schedule for the 8-day cohort: [Table 7]

[0334] Experimental schedule for the 30-day cohort: First week: For the 8-day cohort Next: Photos twice a week until slaughter Glycemia on day 28 Sacrificed on day 29

[0335] In this example, the experiments are presented as "8-day" and "30-day" cohorts, while the days of testing or sacrifice are shown as reported in the experimental schedule.

[0336] Lesions, medications, and monitoring.

[0337] A 6 mm diameter circular, full-thickness wound was created by skin punch biopsy on the central back of each mouse. Briefly, animals were deeply anesthetized with isoflurane (+2 l / min O2). The skin on the back was shaved using wax makeup and disinfected with chlorhexidine 4% ("Clorexyderm" ICF srl Industria Chimica Fine - CR - Italy) or povidone-iodine 10% ("Poviderm" Nuova Farmec srl - VR - Italy). A sterile 6 mm diameter punch biopsy tool was used to create a full-thickness open wound on the animal's back. The wound area was immediately covered with semi-occlusive Tegaderm medication (Tegaderm Roll - 3M Health Care, St. Paul, MN, USA), and a 1.5 cm thick band was created around the chest to prevent the mouse from biting the dressing. Using a 26-gauge needle, 50 μl of medication was injected through the Tegaderm into the wound bed on post-wounding days 0 to 6. Notably, the Tegaderm dressing completely prevented leakage of solution from the lesion.

[0338] A circular full-thickness wound with a diameter of 6 mm is 28.26 mm 2 The surface of the Based on this, the doses administered to the animals were as follows: 1μg dose: 0.0035μg / mm 2 10μg dose: 0.35μg / mm 2 30μg dose: 1.05μg / mm 2 Animals were monitored daily for dressing integrity and absence of infection. Tegaderm was changed weekly in all animals until complete wound healing.

[0339] Photographs of the wounds containing a ruler were then taken and the lesion areas were measured by computer image analysis (NIS Elements, Nikon) three times during the first week and then twice weekly until the end of the experiment.

[0340] Administration of the polypeptide of SEQ ID NO:4.

[0341] The polypeptide of SEQ ID NO: 4 was diluted in phosphate buffered saline (PBS) and divided into daily aliquots. All procedures were performed on ice and final aliquots were stored at -80°C.

[0342] Human NGF (hNGF, recombinant, Escherichia coli, Cat. No.: N-245, Alomone, Jerusalem, Israel) and recombinant mouse NGF (mNGF, Cat. No.: 1156-NG, R&D System) were used as control NGF.

[0343] Test compound was administered daily for 7 days starting from the day of wound induction. 50 μl of test compound solution at each concentration was injected into the wound area under the Tegaderm band using a 26-gauge needle. The elasticity of Tegaderm allowed the needle to close after retraction, and no leakage of liquid solution was observed.

[0344] Pain threshold monitoring.

[0345] Thermal hyperalgesia was assessed in freely moving animals by the method of Hargreave using a thermal plantar test apparatus (Ugo Basile-Comerio, Varese). Animals were allowed to acclimate for 15 minutes in the Plexiglas box of the apparatus. A constant intensity radiant heat source (beam diameter 0.5 cm and intensity 25 I.R.) was placed under the hind paw, and the withdrawal latency (seconds) was recorded as the time from the start of radiant heat application to paw withdrawal. The average of four measurements was used for statistical analysis. Animals were tested using the plantar test on day -3 (before surgery) and day 7 (24 hours after the last application of test compound).

[0346] Tissue collection and processing.

[0347] On the day of sacrifice, mice were deeply anesthetized (isoflurane + 2 l / min O2), and skin samples (1 cm x 1 cm) were taken from the wound area. For Test B, in each group, four samples were collected for immunohistochemistry and four samples were collected for histology. For Test C, a 6 mm skin area was removed using an excision punch (wound area), and an 8 mm ring around this (periwound area) and a 6 mm area from the intact skin were collected.

[0348] Samples collected for histology were embedded in paraffin, sectioned, and stained using hematoxylin and eosin (H&E); samples collected for immunohistochemistry were post-fixed, washed in sucrose PBS, cryosectioned, and processed for indirect immunofluorescence.

[0349] Immunohistochemistry and quantitative analysis.

[0350] The skin was immersed in 4% (w / v) paraformaldehyde and saturated aqueous picric acid in 0.1M Sorensen buffer (pH 7) for 24 hours, then washed in 5% sucrose in 0.1M phosphate buffer for at least 48 hours. After freezing in CO2, sections (14 μm thick) were cut using a cryostat (HM550 Micron, Bio-Optica). The sections were first collected on gelatin-coated slides incubated in 0.1M PBS for 20 minutes at room temperature, followed by overnight incubation at 4°C in a humid atmosphere with primary antibodies diluted in 0.3% PBS-Triton X-100 (v / v). The following antisera were used in this study: laminin (rabbit, Sigma, 1:1000); protein gene product 9.5 (PGP-9.5) (rabbit, Boheringer, 1:2000). After rinsing in PBS for 20 min (2 × 10 min), sections were incubated for 30 min in a humid atmosphere at 37° C. with secondary antiserum conjugated with Rhodamine Red™-X conjugated affinity-pure donkey anti-rabbit IgG (Jackson Immunoresearch) diluted in PBS Triton 0.3%. Sections were then rinsed in PBS (as above) and mounted in glycerol containing 1,4-phenylenediamine (0.1 g / l).

[0351] Immunohistological images were captured using a Nikon Eclipse E600 microscope equipped with a digital CCD camera, Q Imaging Retiga-2000RV (Q Imaging, Surrey, British Columbia, Canada). Analysis was performed using Nis-Elements AR3.2 software. The laminin and PGP9.5 immunoreactive areas were calculated as a percentage of the epidermal layer at 7 and 30 days after the induction of skin lesions. The germination index was estimated by observing the number of sections in which PGP9, 5IR approached the border of the ulcer. For all morphological analyses, five images were analyzed for each animal and two levels / animal. All analyses were performed in a blinded manner. The mean values / animal were used for statistical analysis.

[0352] hNGF quantification.

[0353] Blood was drawn into EDTA-K2 Vacuum Tubes and centrifuged within 30 minutes at 3000×g for 10 minutes at 4° C. Plasma was collected, aliquoted into polypropylene tubes, and stored at −80° C. until use.

[0354] The Human Adipokine Magnetic Bead Panel 2 kit (HADK2MAG-61K, EMD Millipore Corporation, Billerica, Massachusetts, USA) was used to quantify hNGF in plasma samples using xMAP technology and the MAGPIX Luminex platform. This technology is based on the use of different populations of color-coded beads conjugated to monoclonal antibodies specific to specific proteins, thus enabling simultaneous capture and detection of specific analytes with high sensitivity from small sample volumes. We used a simple version of the kit containing only one population of beads conjugated to human NGF-β monoclonal antibodies. The assay was performed according to the manufacturer's specifications with minor modifications.

[0355] Briefly, after overnight incubation of a specific human NGF-β monoclonal antibody-conjugated bead population with plasma samples (25 μl) at RT, the beads were washed and incubated first with the detection antibody solution for 1 hour at RT, and then with the streptavidin-phycoerythrin conjugate solution for 30 minutes at RT. After washing, the beads were resuspended in 100 μl of drive solution and read on the MAGPIX instrument. Data were analyzed using xPONENT 4.2® software, and results were expressed as pg / mL. We obtained values ​​within the dynamic range of the standard curve (10,000–0.128 pg / mL) for all samples. The standard curve had a correlation coefficient (R2) value >0.98. The accuracy of the results was further verified through the values ​​obtained for the quality control solutions (QC1 and QC2) included in the kit, which were within the range specified by the kit manufacturer. The detection limit of human NGF-β was 0.3-0.7 pg / mL.

[0356] This assay was chosen due to its high sensitivity and specificity for hNGF compared to other ELISA methods.

[0357] PC12 culture and treatment.

[0358] The cells were cultured in a T25cm 2 Cells were maintained under standard culture conditions in medium (DMEM, 10% horse serum, 5% FBS, and 1x penicillin / streptomycin) in flasks (NUNC). After at least two passages, cells were seeded (1,000 cells / well) into cell culture-treated 96-well flat-bottom cell plates (NUNC). After 1 day in vitro (DIV), the culture medium was removed, and cells were maintained in depletion medium (DMEM, 1% horse serum, 0.5% FBS, and 1x penicillin / streptomycin). 24 hours after serum depletion, cells were treated with three different concentrations of all test compounds (50, 100, and 200 nM). After 2 DIV, the medium was refreshed, and on DIV 7, cells were fixed and stained for beta-III-tubulin antigen using indirect immunofluorescence (Figure 1).

[0359] Immunocytochemistry.

[0360] At 7 DIV, cells were fixed with 4% cold paraformaldehyde for 20 minutes. After treatment with blocking solution (PBS, 0.3% Triton X-100, 1% BSA, and 1% normal donkey serum) for 1 hour, cells were incubated with primary antiserum (mouse anti-beta III tubulin, 1:1000; R&D) overnight at 4°C. Cells were then incubated with secondary anti-mouse antibody (donkey anti-mouse Alexa-488 conjugate, 1:500; Jackson) for 30 minutes at 37°C. Finally, cells were incubated with the nuclear dye Hoechst 33258 for 20 minutes at RT.

[0361] Cell-based high-content screening assay.

[0362] Neurite outgrowth analysis was performed using the CellInsight™ CX5 High Content Screening (HCS; Thermo Scientific) with the Neuronal Profiling BioApplication. The software is able to recognize all cells in each well by the presence of nuclear dye fluorescence. Each nucleus is identified as a target, and every target corresponds to a single cell. The system recognizes green fluorescence (beta-III-tubulin immunoreactivity) around the nucleus, which identifies the cell body. The Neuronal Profiling tool is able to recognize and track all neurites emerging from each cell body, allowing all neurites from every cell to be counted and measured. Cell aggregates were not recognized as single-cell dimensional objects and were excluded from the analysis.

[0363] 2000-4000 single cells / well and 6 wells / treatment were analyzed.

[0364] RT-PCR.

[0365] Exploratory studies on the possible mechanisms underlying the positive effect of the polypeptide of SEQ ID NO: 4 on wound healing in diabetic mice were carried out using an exploratory strategy (pathway-focused gene expression analysis using RT2 Profiler PCR arrays) and focusing on the main molecular pathways involved in wound healing (e.g., angiogenesis, extracellular matrix and adhesion proteins, growth factors) in 50% of the repair process. Samples were collected from the center of the lesion (6 mm diameter), and RNA was extracted from all animals (6 animals / group), quantified (Nanodrop 2000 spectrophotometer), and pooled (100 ng / animal). Thus, 600 ng of RNA / group was used for reverse transcription.

[0366] A single PCR array was performed for each group using a CFX96 real-time PCR instrument (BioRad). The same threshold was used for all plates, and relative gene expression was calculated using the 2-ΔΔCq comparative method. Mouse angiogenesis, extracellular matrix and adhesion protein (ECM), and growth factor (GF) Rt2Profiler™ arrays (QIAGEN) were used to profile the expression of 250 key genes (84 genes each) involved in angiogenesis, ECM, and GF, using cDNA synthesized using the RT2First Strand kit (QIAGEN) according to the manufacturer's instructions.

[0367] result. The results are presented in the following order: -PC12 in vitro assay - Effectiveness: 8 days - Effectiveness: 30 days - Organization, 14th

[0368] Example 3A: In vitro PC12 neurite outgrowth assay.

[0369] The efficacy of the polypeptide of SEQ ID NO: 4 was tested in vitro in PC12 cells (see FIG. 1 for experimental design) and neurite outgrowth was measured using a cell-based high-content screen using the following parameters: - Mean neurite average length: represents the average neurite length / cell; -Average neurite length: represents the total neurite length / cell; - Maximum high neurite length (%): represents the percentage of cells showing the longest neurite equal to the length of the cell body.

[0370] First, all test compound doses were analyzed and compared with the vehicle group (data not shown). This report presents only the results for the more effective doses of each test compound (Figure 1). Cells exposed to effective doses of all test compounds showed increases in mean neurite mean length (A; mNGF, P ​​= 0.0394; hNGF, P ​​= 0.0196; polypeptide of SEQ ID NO: 4, P = 0.0033) and mean total neurite length (B; mNGF, P ​​= 0.0338; hNGF, P ​​= 0.0006; polypeptide of SEQ ID NO: 4, P = 0.0211; Aloe, P < 0.0001) compared with vehicle-treated cells. Only the polypeptide of SEQ ID NO: 4 (P = 0.0367) was able to increase the percentage of cells showing long neurites.

[0371] Example 3B: Efficacy Study, 8-day Cohort.

[0372] Animal monitoring.

[0373] Animals were monitored for glucose blood levels before wounding. The results are reported in Figure 3. Glycemia, as measured in pilot studies, was higher in diabetic mice than in control animals. No differences were observed between animals assigned to different experimental groups.

[0374] Weight gain between days 0 and 7 post-lesion is reported in Figure 4. No differences were observed with either time (day 0 vs. day 7) or treatment.

[0375] Hyperalgesia.

[0376] Thermal hyperalgesia was assessed in freely moving animals by the Hargreave method using a thermal plantar test device on day -3 (before skin lesion) and the day after the last test compound application (day 7). The results are illustrated in Figure 5. No intergroup differences were observed on day 0. By comparing the mean paw withdrawal latency on day 0 and day 7 in the same treatment group, a significant decrease was observed in hNGF-treated animals on day 7. No hyperalgesia to thermal stimuli was observed in other groups. Notably, a higher threshold was observed in the polypeptide of SEQ ID NO: 4-treated mice on day 7, indicating a higher pain threshold in this group.

[0377] Time until closure.

[0378] Wound healing was assessed by macroscopic observation of photographs taken every 2 days during the first week, starting on day 0 (the day of surgery). "Time to closure" was measured on these wound images using an NIS element (Nikon). Results are reported in Figure 6, where both the external and internal areas are plotted. A two-way ANOVA showed a time effect (F(4,203) = 41.25, p < 0.0001) and a group effect (F(5,203) = 2.565, P = 0.0282).

[0379] The time to closure at day 7 is reported in Figure 7. Despite the fact that a dose-dependent trend in promoting wound healing is observed in the polypeptide of SEQ ID NO: 4 treatment group, no differences are observed between the groups.

[0380] NGF plasma levels.

[0381] Blood was collected at the time of sacrifice, thus 48 hours after the last application of test compound. NGF plasma levels were determined by an antibody-based assay using antibodies capable of detecting human NGF, mouse NGF, and also the polypeptide according to SEQ ID NO: 4. Herein, the NGF plasma levels thus determined are referred to as "total NGF plasma levels." The results are reported in Figure 8. A dose-dependent increase in total NGF plasma levels was observed in treated mice, reaching values ​​greater than 350 pg / ml. Furthermore, an increase was also observed in the mNGF-treated group. This is not surprising, since the recombinant mouse NGF-β used for treatment is a homodimer of two amino acid polypeptides that share approximately 90% identity with human NGF at the amino acid level and are recognized by the same antibody.

[0382] Example 3C: Efficacy Study, 30-Day Cohort.

[0383] Animal monitoring.

[0384] Animals were monitored for glucose blood levels before wounding, after the end of test compound administration, and at the time of sacrifice. The results are reported in Figure 9. We observed a gradual increase in glucose blood levels in all experimental groups over the 28-day observation period. No differences between treatment groups were observed at different times.

[0385] Weight gain from day 0 to day 28 post-lesion is reported in Figure 10. No differences were observed with either time or treatment.

[0386] Hyperalgesia.

[0387] Thermal hyperalgesia was assessed in freely moving animals by the Hargreave method using a thermal plantar test apparatus on day -3 (before skin lesion) and day 7, 24 hours after the last NGF application. The results are illustrated in Figure 11. No differences between groups were observed on day 0. By comparing latencies on days 0 and 7 in the same treatment group, a non-significant trend toward a decrease in paw withdrawal latency was observed on day 7 in hNGF-treated animals. However, when data from the 8-day and 30-day cohorts were pooled, a significant decrease in pain threshold was observed in hNGF-treated animals, thus suggesting that this hNGF formulation induces hyperalgesia. No differences were observed in the other groups. The results are shown in Figure 12.

[0388] Time until closure.

[0389] Wound healing was assessed by macroscopic observation with photographs taken every 2 days for the first week starting on day -3 (the day of surgery), then twice weekly until sacrifice. "Time to closure" was measured on these wound images using an NIS element (Nikon).

[0390] Individual data for "time to closure" are presented in tabular form (Figure 13). Serial external wound area measurements are plotted against time in Figure 14. A two-way ANOVA showed a time effect (F(50,434) = 417.3, p < 0.0001), a treatment effect (F(5,434) = 21.45, p < 0.0001), and an interaction between treatment and time (F(50,434) = 1.638, p = 0.0055).

[0391] In mice treated with the polypeptide of SEQ ID NO: 4, the rate of wound healing was significantly accelerated in a dose-dependent manner compared to vehicle-treated animals.

[0392] The time to closure at day 8 is reported in Figure 15, where data from both the 8-day and 30-day cohorts are pooled. Already at this time point, a significant decrease in wound healing is observed in the group treated with the polypeptide of SEQ ID NO: 4 at a dose of 30 μg / day compared to the vehicle-treated group.

[0393] NGF plasma levels.

[0394] Blood was collected at the time of sacrifice. NGF plasma levels were determined by an antibody-based assay using antibodies capable of detecting human NGF, mouse NGF, and also the polypeptide according to SEQ ID NO: 4. The NGF plasma levels so determined are referred to herein as "total NGF plasma levels." The results are reported in Figure 16. Total NGF plasma levels were very low (compared to Figure 8), below 10 pg / ml, and similar in all groups.

[0395] Histology (8 days and 30 days) (see also pilot study report).

[0396] Skin samples containing the ulcerated area, including a 5 mm border of intact skin, were excised, embedded in paraffin, and serially sectioned according to the schematic diagram presented in Figure 17B. The sections were then stained (H&E), and representative low-magnification images at different levels of the wound are reported in Figure 17A. High-magnification photomicrographs illustrate the re-epithelialization process at the wound border (Figure 17D), where the epidermal migratory tongue (MET) is evident, and extensive granulation tissue in the dermis below the epidermal layer, characterized by inflammation, cell proliferation, matrix deposition (Figure 17E), and angiogenesis (Figure 17F). Re-epithelialization was assessed by measuring the thickness of the epidermal layer. Representative images from intact animals, mice treated with vehicle, the polypeptide of SEQ ID NO: 4 (1 μg / day), and the polypeptide of SEQ ID NO: 4 (30 μg / day) are presented in Figure 18. The polypeptide of SEQ ID NO: 4 induces a dose-dependent thickening of the epidermal layer, which appears to be much higher than in intact skin. The basal layer of the epidermis is characterized by cellular hyperplasia in the basal and spinous layers, likely reflecting increased cell proliferation. Furthermore, the dermis is thicker and more intensely stained, suggesting higher extracellular matrix deposition, and is enriched with dermal appendages (glands and hair follicles) according to the dose. The thickness of the epidermis in all groups is presented in the graph. The polypeptide of SEQ ID NO: 4 induces a dose-dependent thickening of the epidermal layer, which grows much thicker than the vehicle group. Furthermore, mNGF and hNGF induce the same effect, comparable to the dose-matched groups treated with the polypeptide of SEQ ID NO: 4.

[0397] Immunohistochemistry (days 8 and 30).

[0398] Skin reinnervation was analyzed by immunostaining for the protein PGP9.5, a highly sensitive neuroectodermal marker widely used to visualize skin innervation. The anatomy of skin innervation is presented in Figure 19, where PGP9.5-IR fibers are visualized in intact mouse skin. In particular, the subcutaneous, deep cutaneous, and subepidermal plexuses are visualized. The subepidermal plexus provides intraepidermal free nerve endings to the epidermis.

[0399] The effect of topical application of the polypeptide of SEQ ID NO: 4 on nerve regrowth in repaired skin was analyzed using the "eruption index" at the lesion border 8 days after the lesion, and PGP9.5-IR in the epidermis and dermis of the repaired area. Representative images are reported in Figure 20. Panels A, B, and C illustrate PGP9.5-IR at 30 days in intact animals, mice treated with vehicle, and mice treated with the polypeptide of SEQ ID NO: 4 (30 μg / day), respectively. The results of morphometric analysis are presented in Figure 21. Application of the polypeptide of SEQ ID NO: 4 at 30 μg / day induces a significant increase in sprouting at 8 days (e.g., hNGF). At 30 days, innervation has not yet been restored in vehicle-treated animals, but no difference between intact and NGF-treated animals was observed when the polypeptide of SEQ ID NO: 4 (all doses) and mNGF were administered. Conversely, hyperinnervation was observed when hNGF was used.

[0400] The effect of topical application of the polypeptide of SEQ ID NO: 4 on angiogenesis was estimated using laminin as a marker. Laminin is a basement membrane marker and thus labels several structures in the skin, including endothelial cells. Other endothelial markers, such as PECAM (also known as CD31), von Willebrand factor, and collagen, provided staining that was not suitable for quantification under the fixation conditions used in this study. Representative images are reported in Figure 22. Panel A illustrates the epidermal layers visualized by conventional histology (H&E). The arrow indicates the basal layer. Panel B illustrates the basement membrane underlying the epidermis (arrow); panel C illustrates the sensory innervation of the epidermis derived from the subependimal nerve plexus; panel D illustrates the ulcer border and associated innervation on day 8 (E) and after skin repair (F). Panels GI illustrate angiogenesis at day 8 (G, EE; H, laminin-IR) and day 30 (I).

[0401] The results from the morphometric analysis are presented in Figure 23. Application of the polypeptide of SEQ ID NO: 4 (30 μg / day) induced a significant increase in laminin-IR (e.g., hNGF) at day 8, which was still present at day 30, likely reflecting undergoing angiogenesis.

[0402] Mechanism: Exploratory study.

[0403] Regulation of gene expression.

[0404] The objective of this study was to explore the molecular mechanisms underlying the positive effects of the polypeptide of SEQ ID NO: 4 on wound healing in diabetic mice, focusing on inflammation, extracellular matrix deposition, innervation, and angiogenesis. An exploratory strategy (pathway-focused gene expression analysis using RT2 Profiler PCR Arrays) was used to identify key molecular pathways in wounds at 50% of the repair process. Mouse angiogenesis, extracellular matrix and adhesion proteins (ECM), and growth factors (GFs) were profiled using the RT2 Profiler™ to profile the expression of 252 key genes (84 genes each) involved in angiogenesis, ECM, and GFs.

[0405] Expression analysis for each panel (angiogenesis, ECM, growth factors) is presented below: - Gene list; - Heatmap providing a graphical representation of fold-adjusted expression data between two groups overlaid on a PCR array plate layout; - A scatter plot comparing the normalized expression of all genes on the array between the two groups by plotting them against each other, for quick visualization of large gene expression changes, and a list of genes whose expression changes are greater than a selected boundary (≥ 3).

[0406] Scatter plots illustrate group comparisons as follows: -db / db vs. WT mice (WT as control group) - db / db vehicle vs. db / db intact (db / db intact as control group) -db / db NGF (polypeptide of sequence number; mNGF, hNGF) vs. db / db vehicle (db / db vehicle as control group) -db / db NGF (polypeptides of sequence numbers; mNGF, hNGF) vs. db / db intact (db / db intact as control group)

[0407] The results are evaluated as extracellular matrix and adhesion molecules and growth factors and neurotrophins (figure not shown).

[0408] The main findings and conclusions from this analysis are as follows: Genotype Effects: Comparison of ‐db / db intact with WT intact showed that numerous angiogenic and ECM genes were differentially regulated according to genotype, and most GF genes were not differentially expressed, thus suggesting that ECM and angiogenesis are processes primarily affected by the diabetic state for wound repair. - Lesion effect in db / db: -The lesion induced downregulation of numerous angiogenic and ECM genes and upregulation of a few GF genes, thus suggesting that ECM and angiogenesis are the main processes driving wound repair even in diabetic mice. -Effect of polypeptide of SEQ ID NO: 4 in db / db (vs. vehicle): - The polypeptide of SEQ ID NO: 4 downregulates several genes, including: angiogenesis: akt, Ccl2 (chemokine (CC motif) ligand 2), Ctgf (connective tissue growth factor), Hif1a, MMP14, thbs2 (thrombospondin 2); - The polypeptide of SEQ ID NO: 4 upregulates several genes, only some of which are in turn also regulated by hNGF and mNGF. - The polypeptide of SEQ ID NO: 4 does not regulate GF genes, some of which are regulated by hNGF and mNGF.

[0409] STRING analysis was also performed (data not shown). STRING is a biological database and web resource of known and predicted protein-protein interactions, which has been widely used to search for interaction relationships among differentially expressed genes. The "clustering" analysis by STRING software is based on all genes regulated in the different arrays.

[0410] Conclusion.

[0411] The main conclusions from this example are: 1. PC12 cells coupled to HCS as an analytical approach is a suitable approach to assess the in vitro efficacy of the polypeptide of SEQ ID NO: 4; 2. The polypeptide of SEQ ID NO: 4 improves wound healing in a dose-dependent manner. 3. The polypeptide of SEQ ID NO: 4 strongly increases the repair of the epidermal layer and induces a strong increase in thickness; the polypeptide of SEQ ID NO: 4 also positively affects reinnervation and angiogenesis (as assessed by laminin-IR). All these parameters in mice treated with the polypeptide of SEQ ID NO: 4 were higher than in control uninjured mice, suggesting that the remodeling stage of wound healing should be evaluated in further studies. 4. Exploratory studies suggest that the AKT-mTOR pathway may be involved in the effects of the polypeptide of SEQ ID NO: 4. Akt and mTOR are considered to be promoters of survival and cell growth, and it has been suggested that transient pharmacological activation of the PI3K-Akt-mTOR signaling axis may represent a novel clinical intervention strategy for accelerating healing. Notably, impaired AKT-mTOR pathway has been implicated as a possible cause of impaired wound healing in diabetic mice, and the AKT-mTOR pathway has been demonstrated to be involved in improved wound healing in diabetic mice by several molecules, such as notoginsenoside Ft1; acemannan; SR-0379; and the microRNA-99 family.

[0412] Thus, the polypeptide of SEQ ID NO: 4 is a recombinant protein with a polypeptide sequence similar to human nerve growth factor, but with at least one mutation that renders it painless (hNGFp) and therapeutically effective. [Table 8]

[0413] Example 4: Proof of concept: Pressure ulcer model in mice.

[0414] Reported herein is a study of the administration to non-human animals of the polypeptide of SEQ ID NO: 4. 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.

[0415] method.

[0416] Control (C57BL6, albino) and genetically diabetic C57BL / KsJ-m+ / + Lepr db (db / db) male mice (Jackson Laboratories, 8-10 weeks old) were included in the experiment. Under gas anesthesia, the animals' backs were shaved, and the shaved area was thoroughly cleaned to prevent skin irritation. The skin fold was elevated, and two magnetic ceramic disks (average weight 2.4 g, magnetic force 1000 G) measuring 12 mm in diameter and 5.0 mm thick (Magnetic Fountain, Castle Rock, Colorado) were applied to the skin, leaving a skin "bridge" of approximately 5.0 mm between the two magnets. This process creates a compressive pressure of 50 mmHg between the two plates, as documented to be necessary to induce local tissue ischemia (Peirce et al., 2000, Wound Repair Regen., Vol. 8, pp. 68-76). Three cycles of ischemia-reperfusion (I / R) were administered to induce the formation of two ulcers of uniform severity. A single I / R cycle consisted of a 12-hour period for magnet application starting at 8:00 AM, followed by a 12-hour rest period without the magnet. Treatment with vehicle and test compounds began 3 days after the end of the I / R cycle to allow surgical debridement of the ulcers, consisting of removal of fibrin exudate and necrotic tissue.

[0417] Some mice were subjected to a study treatment with the polypeptide of SEQ ID NO: 4, which may be referred to as painless recombinant human mutant nerve growth factor (hNGFp). In particular, the following experimental groups were studied: -db / db, excipient -db / db, polypeptide of SEQ ID NO: 4, 1 μg / cm 2 / day -db / db, polypeptide of SEQ ID NO: 4, 10 μg / cm 2 / day -db / db, polypeptide of SEQ ID NO: 4, 100 μg / cm 2 / day.

[0418] Treatment was continued daily for 14 consecutive days, and then twice weekly at the same dosage (dosage calculated according to the size of the ulcer at the time of treatment) until closure. The ulcer was monitored by visual inspection to determine the date of closure. In addition, photographs of the wound with a ruler were taken, and the lesion area was measured by computerized image analysis when the ulcer was medicated twice weekly. Pressure ulcer assessment was performed by measuring the wound area according to a standardized scale and by computerized image analysis.

[0419] The effect of the compounds on pain threshold was assessed in freely moving animals at the site of injury using an electronic Von Frey from Bioseb, an electronic device that allows the determination of mechanical pain sensitivity threshold in rodents.

[0420] The histology, innervation, and vascularization of the lesions are analyzed by histology and immunohistochemistry and computerized image analysis.

[0421] result.

[0422] By placing a magnet on the skin folds on the backs of diabetic mice, irreversible damage was induced, including the entire dermal-epidermal tissue below the site of compression. Both visual inspection (presence of necrotic and hemorrhagic areas) and histological analysis confirmed that three I / R cycles were able to induce lesions reminiscent of pressure ulcers.

[0423] In all groups treated with the polypeptide of SEQ ID NO: 4, the rate of wound healing was accelerated compared to animals treated with vehicle. Ulcer closure in animals treated with the polypeptide of SEQ ID NO: 4 was evident starting from days 17 and 21, whereas animals treated with vehicle underwent healing starting from day 23.

[0424] On day 28 (the last day of observation), skin ulcers were completely closed in more than 80% of animals treated with the polypeptide of SEQ ID NO: 4; as shown in Figure 25; compared to animals treated with vehicle, the effect was statistically significant at all doses of the polypeptide of SEQ ID NO: 4, with the probability of healing in the former group being less than 60%. These data are consistent with literature evidence showing that wound healing rates are impaired in animal models of diabetes, and together with data generated in a surgical ulcer model (Example 3), they suggest that the polypeptide of SEQ ID NO: 4 can normalize the delayed healing process in diabetic mice.

[0425] In addition to the positive effect on wound healing, the histological and immunohistochemical data provide further evidence that the polypeptide of SEQ ID NO: 4 has the biological ability to improve the degree of wound healing parameters in diabetic mice with impaired healing. At the histological level, complete re-epithelialization and restoration of normal skin anatomy in the repair area were observed after treatment with the polypeptide of SEQ ID NO: 4. Immunohistochemistry showed that the polypeptide of SEQ ID NO: 4 also positively affected innervation (measured by PGP9.5 immunoreactivity in the epidermis) and angiogenesis (measured by PECAM immunoreactivity in the skin) (Figures 26A and B).

[0426] Because wild-type NGF has been reported to increase pain sensitivity at the site of administration, the mechanical pain threshold was assessed after 14 consecutive days of treatment with the polypeptide of SEQ ID NO: 4 by applying mechanical stimulation to the ulcer border. No alteration in the mechanical pain threshold was observed compared to diabetic mice treated with vehicle, suggesting that the polypeptide of SEQ ID NO: 4 after chronic local treatment can exert its positive trophic effect on the skin over a long administration interval without causing nociceptor sensitization (Figure 27).

[0427] Example 5: A randomized, double-blind, placebo-controlled study to study the safety, tolerability, pharmacokinetic and pharmacodynamic profile of the polypeptide of SEQ ID NO: 4 after single and repeated ascending doses in subjects with diabetic neuropathic foot ulcers (DFUs).

[0428] Reported herein is a study of the administration of single and repeated ascending doses of the polypeptide of SEQ ID NO: 4 in participants with diabetic neuropathic foot ulcers (DFUs). The participants are human subjects.

[0429] The clinical trial described in this example has received ethical approval from the respective authorities.

[0430] The polypeptide of SEQ ID NO: 4 may also be referred to as painless recombinant human mutant nerve growth factor (hNGFp). The polypeptide of SEQ ID NO: 4 may alternatively be referred to as "recombinant human nerve growth factor (RHNGF)" or "SUB75752" and has the full molecular formula C580H895N163O176S8. The polypeptide of SEQ ID NO: 4 may be obtained from biological / biotechnological sources (other than the Advanced Therapy IMP (ATIMP)). It is a recombinant pharmaceutical product (see also Example 1). In particular, the polypeptide of SEQ ID NO: 4 may be obtained by expression as described in Example 1 and purification as described in Example 2. In particular, the high purity obtainable as described in Example 2, preferably under GMP standards, allows the polypeptide of SEQ ID NO: 4 to be used as a pharmaceutical.

[0431] As used in this example, the polypeptide of SEQ ID NO: 4 is an investigational medicinal product (IMP). According to Directive 2001 / 20 / EC, "IMP" is "a pharmaceutical form of an active substance or placebo that is being tested or used as a reference in clinical trials, including products already with a marketing authorization but that are used or assembled (formulated or packaged) in a way different from the approved form, or that are used for an unapproved indication, or that are used to obtain further information about the approved form." Herein, the polypeptide of SEQ ID NO: 4 is the IMP used in the first-in-human clinical trial. Thus, this example describes the first-in-human clinical trial. No risk factors have been identified according to the first-in-human guidance.

[0432] The IMP used in this example is provided as a clear, colorless solution of hNGFp prepared with 1 mg / ml of the polypeptide of SEQ ID NO: 4. The polypeptide of SEQ ID NO: 4 is adjusted to the desired concentration and filled into a glass vial. The concentration of the solution is 1 mg / ml.

[0433] The polypeptide of SEQ ID NO: 4 is administered to a human subject in need thereof. The polypeptide of SEQ ID NO: 4 is administered as a skin solution. This is not a specific pediatric formulation.

[0434] The human subject in need of administration of the polypeptide of SEQ ID NO: 4 is a subject with a diabetic neuropathic foot ulcer (DFU). No risk factors have been identified by initial guidance in humans.

[0435] The polypeptide of SEQ ID NO: 4 is administered topically. Thus, the polypeptide of SEQ ID NO: 4 is for topical use (non-flowing).

[0436] The polypeptide of SEQ ID NO: 4 is 0.3 to 6 μg / mm 2 Administer a total dose of 1000 mm 2 " refers to the area of ​​the ulcer. The amount (μg) indicated refers to the amount of polypeptide administered per day.

[0437] The polypeptide of SEQ ID NO: 4 is administered twice daily for 14 consecutive days, after which administration is discontinued.

[0438] In this study, there is a placebo. The placebo is referred to as PL1. The placebo is a skin solution. The placebo is for topical use (non-flowing). The placebo is a placebo for the polypeptide of SEQ ID NO: 4 (PR1). The placebo is otherwise identical to IMP (PR1). The placebo is administered identically to the polypeptide of SEQ ID NO: 4.

[0439] Both PR1 and Placebo 1 are prepared for the trial by and at Klifo A / S, Smedeland 36, 2600 (Glostrup, Denmark).

[0440] There are no other (comparison) medicinal products in this study.

[0441] The subject of this test is suffering from, suffering from, or predisposed to a skin and connective tissue disease. In particular, the subject of this test is a subject with diabetic neuropathic foot ulcer (DFU). Diabetic foot ulcer is a major complication of dermal diabetes, and is a non-healing or poorly healing full-thickness wound that penetrates through the dermis below the ankle in individuals with diabetes. [Table 9]

[0442] The trial will have an independent data monitoring committee.

[0443] The initial estimate for the duration of the trial is 2 years and 1 month.

[0444] The planned number of subjects is 92 (60 of whom are in the age range 18-64 years; 32 of whom are in the age range 65 years or older). The study subject population consists of patients and does not include healthy volunteers. Certain vulnerable populations are included.

[0445] Treatment or care after a subject completes his / her participation in a clinical trial is the standard of care.

[0446] Ethical approval from the authorities has been obtained. A favorable opinion was given.

[0447] Clinical trial objectives:

[0448] Primary Objective: To assess the safety and tolerability of single and multi-day topical dosing with the polypeptide of SEQ ID NO: 4 in subjects with DFU.

[0449] Secondary Objectives: (a) To assess the pharmacokinetic profile of systemically available drug following single-day and multi-day topical dosing with the polypeptide of SEQ ID NO: 4 in subjects with DFU. (b) To assess the pharmacodynamic effects of multi-day topical administration of the polypeptide of SEQ ID NO: 4 on the healing of DFUs over a 12-week period.

[0450] There are no sub-exams.

[0451] Key inclusion criteria:

[0452] Part 1 SD and Part 2 MD:

[0453] Subjects must meet all of the following criteria to be eligible for enrollment in the study: 1. Subject's written informed consent obtained prior to any study-related procedures; 2. Male or female subjects aged 18-80 years (including extremes) diagnosed with type I or type II diabetes mellitus with glycated hemoglobin (HbA1c) ≤ 10%. 3. Female subjects of non-childbearing potential (WONCBP): - They must report surgical sterilization (performed at least 6 months prior to screening) or menopause (no regular menstrual bleeding for at least 1 year prior to screening, age ≥ 45 years, and FSH ≥ 40 mIU / ml at screening). 4. Female subjects of childbearing potential (WOCBP): They must be using one or more of the following reliable methods of contraception during the study period and for at least 90 days after the last study drug dose: a) Placement of an intrauterine device (IUD) or intrauterine system (IUS). b) Hormonal contraception (implant, patch, oral). c) Barrier method of contraception: condoms or occlusive caps (diaphragm or cervical vault / cap) with spermicidal foam / gel / film / cream / suppository. d) Sterilization of the male partner (with appropriate post-vasectomy documentation of the absence of sperm in the ejaculate). 5. Male subjects; they must be using two effective methods of contraception during the entire study period and must not have donated sperm within 90 days after the last study drug dose. 6. Presence of at least one diabetic foot ulcer meeting the following criteria: a) Diagnosed as a full-thickness neuropathic DFU located at or distal to the ankle (excluding interdigital ulcers but including heel ulcers). b) SD: present for 6 weeks to 12 months and 3-5 cm in the area after sharp debridement 2 , confirmed at screening. MD: 3-5 cm in areas present for 6 weeks to 12 months and after sharp debridement 2 , checked after a two-week break-in period. c) A minimum 2 cm margin between the eligible study ulcer and any other identified ulcer on the foot. d) After initial sharp debridement, depth ≥ 5 mm and grade 1A according to "The University of Texas Staging System for Diabetic Foot Ulcers" (22), with no exposure of the bursa, tendon, or bone and no tunnels, erosions, or sinus tracts. 7. The subject must be able to hold the target ulcer in a position and orientation that allows the investigational medication to be applied without significant loss of material through runoff until the dressing is applied. 8. Adequate vascular perfusion of the affected limb demonstrated within 30 days prior to screening, as defined by at least one of the following: a) Ankle-brachial index (ABI) ≥ 0.9 and ≤ 1.2 confirmed by transcutaneous oxygen tension (TcPO2) > 50 mmHg. b) Toe pressure (plethysmography) >50mmHg c) Doppler ultrasound (biphasic or triphasic waveform) of at least two vessels at the ankle consistent with adequate blood flow to the affected limb as determined by SoC.

[0454] Key exclusion criteria: Part 1 SD and Part 2 MD: To be eligible for enrollment in the study, a subject must not meet any of the following criteria: 1. For females only: Pregnant or lactating female subjects, confirmed by a positive serum pregnancy test at screening and a urine test on Day 1. 2. Subject with: a) Infected cellulitis, osteomyelitis, or ulcers with clinical signs or symptoms of infection according to the Infectious Diseases Society of America (IDSA) guidelines (19). b) Gangrene or necrosis in any part of the affected limb. c) Active or chronic Charcot foot in the test limb. d) Planned vascular surgery, angioplasty, or thrombolytic therapy or revascularization performed within 1 month prior to enrollment. e) Ulcers involving exposure of tendons, bones, or joint capsules (it is acceptable for the ulcer to extend through the dermis into the subcutaneous tissue with the presence of granulation tissue). f) Ulcers of non-diabetic etiology. g) Previous major amputation in the same target foot. h) Current or recent (3 weeks) antibiotic treatment for any reason. i) Bedridden subjects or subjects with a life expectancy of less than 1 year. 3. Use of any other growth factor therapy in the 6 months prior to screening. 4. A history of malignancy or a strong family history of cancer (e.g., familial cancer disorder) in the 5 years prior to screening, excluding definitively treated squamous cell or basal cell carcinoma of the skin. 5. Clinically significant cardiovascular, pulmonary, renal, endocrine, hepatic, neurological, psychiatric, immunological, gastrointestinal, hematological, or metabolic disease that, in the opinion of the investigator, has not been stabilized or may otherwise affect the subject's safety or study results (in cases of doubt, the sponsor's clinical research physician should be consulted). 6. Subjects undergoing hemodialysis or peritoneal dialysis or with chronic renal failure (plasma creatinine > 2 mg / dl). 7. Subjects with significantly abnormal primary laboratory parameters that interfere with patient safety, as judged by the PI.

[0455] Scope of the trial / parts of the trial: The trial has two parts: - Part 1 SD - Single Ascending Dose - Part 2MD - Multiple Ascending Dose Dosage: SD: 0.3, 1, 3, and 6 μg / mm 2 MD: 1 and 3 μg / mm 2

[0456] Evaluation items:

[0457] Primary endpoint: Part 1 SD: Safety: Adverse Events (AEs) and Adverse Drug Reactions (ADRs) Vital signs: Systolic (SBP) and diastolic (DBP) blood pressure 12-lead ECG parameters extracted from Holter (HR, PR, QRS, QTcF, QT) · Clinical laboratory evaluation (chemistry, hematology, and urinalysis). Part 2MD: Safety: ·AEs and ADRs; Vital signs: SBP, DBP; temperature; ·Triple 12-lead electrocardiogram; (If any ECG / cardiovascular findings emerge from Part 1 of the study, the SAC may also perform Holter monitoring for part or all of Part 2, as indicated). · 12-lead ECG parameters extracted from Holter (HR, PR, QRS, QTcF, QT). Abnormal 24-hour Holter ECG findings (total pauses >2.5 seconds, atrial fibrillation and atrial flutter, premature atrial contractions, premature ventricular contractions (PAC) load, premature ventricular contractions (PVC) load, abnormal morphology); heart rate (from 24-hour Holter ECG) and hourly average HR from 0 to 24 hours. · Clinical laboratory evaluation (chemistry, hematology, and urinalysis).

[0458] Time points for assessment of this endpoint: As shown above.

[0459] Secondary endpoints:

[0460] Part 1 SD:

[0461] Pharmacokinetic variables:

[0462] The following PK parameters are derived from the serum concentrations of the polypeptide of SEQ ID NO:4. ·AUC0-12h, AUC00-24h, AUC0-t, AUC0-∞, Cmax, tmax, t 1 / 2, CL / F, Vd / F; ·AUC0-12h DN, AUC00-24h DN, AUC0-t DN, AUC0-∞DN, CmaxDN.

[0463] Immunogenicity variables: ADA Ct

[0464] Part 2MD:

[0465] Pharmacokinetic variables:

[0466] The following PK parameters are derived from the serum concentrations of the polypeptide of SEQ ID NO:4. Day 1: AUC 0-12h, Cmax, and tmax; Days 2 to 13: Ctrough On the last day of drug administration (Day 14): AUC 0-12h, AUC0-t, AUC0-∞, Ctrough, Cmax, Cmin, tmax, tmin, Cav, and Rac, t 1 / 2, CL / F, and Vd / F.

[0467] Immunogenicity variables: Serum concentrations of anti-drug antibodies (ADA) will be assessed on Day 1 before the first dose, Day 15 before discharge, Day 24 (Week 4), Day 52 (Week 8), and Day 80 (Week 12). ·Ct Pharmacodynamic / Efficacy Variables: · Mean reduction in target ulcer area and volume from baseline to D14, D21, D28, D56, and D84. Time to healing of the target ulcer area and volume. Healing is defined as "complete recovery". Different "definitions of healing" also apply (partial reduction: 50%, 66%, 75%).

[0468] Time points for assessment of this endpoint: As shown above.

[0469] Single ascending doses (SAD) will be administered at the following doses: Cohort A: 0.3 μg / mm 2 Cohort B: 1 μg / mm 2 Cohort CA: 3 μg / mm 2Cohort D: 6 μg / mm 2 Each cohort will be composed of SEQ ID NO: 4 native subjects to avoid potential carryover effects between cohorts. This is particularly important with regard to hyperalgesia, which is known for wild-type human NGF. Dose levels will be adjusted if necessary, and washout periods may be included, if necessary, to meet study objectives.

[0470] In the single ascending dose (SAD), standard of care (SOC) was given at screening and at each successive visit until the end of the follow-up period, and continued for two consecutive visits if complete re-epithelialization / healing did not occur. In this case, SOC could be discontinued and the subject's foot managed according to the investigator's assessment / decision. SOC consisted of the following procedures: Debridement of the target ulcer (any possible bleeding caused by debridement was controlled by leg compression and elevation only), Dressing of the lesion with paraffin gauze and covering with a protective bandage made of sterile gauze. Use of a removable walker (which cannot be removed during the observation period) for unloading after bandaging.

[0471] In case of infection of the lesions during the study, the lesions were sampled for microbial culture, and the subject had to be prescribed systemic empirical antibiotic treatment as determined by the investigator, who had to adjust the treatment according to the culture results. All infections had to be evaluated and assessed for their severity, especially if severe in intensity.

[0472] Multiple ascending doses (MAD) were administered in two cohorts, each with a sequential naive subject. The target daily dose level was 1 μg / mm 2 with the polypeptide of SEQ ID NO: 4 (20) or placebo (10). 2 and 3 μg / mm 2After screening, eligible subjects are treated according to the standard of care (SOC; run-in period); enrollment is confirmed after measuring ulcer size. If the ulcer area decreases by 50% or more during this run-in period, the subject will not be enrolled.

[0473] Scope of the trial:

[0474] Determining the safety, pharmacokinetics, pharmacodynamics, and other (tolerability) properties of IMP in humans.

[0475] result.

[0476] Single ascending dose (SOC).

[0477] Single ascending doses (SAD) were administered in four consecutive cohorts at the following doses: Cohort A: 0.3 g / mm 2 Cohort B: 1 g / mm 2 ; Cohort CA: 3□g / mm 2 Cohort D: 6 g / mm 2 (In addition to standard treatment). In all of these cohorts, quantifiable levels of the polypeptide of SEQ ID NO: 4 were detectable in the systemic blood circulation of each human subject. Thus, the administered polypeptide is present in the body of the subject after administration.

[0478] Following the administration of a single dose, subjects are monitored for adverse events for 28 days.There is no adverse event associated with the administration of the polypeptide of SEQ ID NO: 4.Thus, the administration of the polypeptide of SEQ ID NO: 4 is not associated with any observable adverse drug reaction.As a result of the absence of adverse drug reaction, it is concluded that the polypeptide of SEQ ID NO: 4 is safe and well tolerated by human subjects.

[0479] opinion.

[0480] The biological activity of the polypeptide of SEQ ID NO: 4 derives from its ability to promote the growth, maintenance, proliferation and survival of cells, particularly neuronal cells.

[0481] As a result of this example, the safety, tolerability, pharmacokinetic and pharmacodynamic profile of the polypeptide of SEQ ID NO: 4 after single and multiple ascending doses in humans is studied and confirmed. [Brief explanation of the drawings]

[0482] [Figure 1] FIG. 1: Overview of the process according to Example 2, including the improvements described in Example 2B. [Figure 2] Figure 2: In vitro PC12 neurite outgrowth assay. Representation of the mean neurite length (A; mean length per well), total neurite length (B; mean total length per cell), and percentage of cells showing neurites longer than the cell body length (C). Bars represent mean ± SEM. Statistical analysis: One-way ANOVA followed by Tukey's post hoc vs. vehicle-treated group (*P<0.05; **P<0.01; ***P<0.001). CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 3] Figure 3. Efficacy study, glucose blood levels in db / db mice measured at the time of skin biopsy of the 8-day cohort. No differences were observed between animals assigned to different treatment groups. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 4] Figure 4. Efficacy study, 8-day cohort. Weight gain over the study time period (days 0 and 7 after skin lesion). CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 5] Figure 5: Efficacy study, 8-day cohort. Thermal threshold. Latency to paw withdrawal in the plantar test performed on days -3 and 7 after skin biopsy and NGF administration. Data are expressed as mean + SEM. Statistical analysis performed by Student's t-test, *p<0.05. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 6]Figure 6. Efficacy study, 8-day cohort. "Time to closure" measured as the external and internal area of ​​the ulcer from skin biopsy to day 8 (see report from pilot study for details). Results are expressed as % of lesion area at day 0; data are expressed as mean + SEM. See text for statistical analysis by two-way ANOVA. CHF6467 = Polypeptide of SEQ ID NO: 4 [Figure 7] Figure 7.B. Efficacy study, day 8. Time to closure of the ulcer (external area) on day 8 expressed as a percentage of ulcer closure compared to day 0. Data are expressed as mean + SEM. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 8] Figure 8: B. Efficacy study, 8-day cohort. NGF plasma levels at sacrifice. Data are mean + SEM; statistical analysis: one-way ANOVA followed by post-hoc Dunnett's test. *p<0.05, **p<0.01. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 9] Figure 9. Efficacy study, 30-day cohort. Glucose blood levels were measured at skin biopsy (d-1), after the end of treatment with the polypeptide of SEQ ID NO: 4 (d-8), and at sacrifice (d28). See text for details. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 10] Figure 10. Efficacy study, 30-day cohort. Weight gain over the study period. No differences were observed between the study groups. CHF6467 = Polypeptide of SEQ ID NO: 4 [Figure 11] Figure 11: Efficacy study, 30-day cohort. Results from skin biopsies and plantar examinations on days 0 and 7 after administration of the polypeptide of SEQ ID NO: 4 (N=8 in each group). Data obtained in the 30-day cohort are presented as mean + SEM; statistical analysis performed by Student's t-test. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 12]Figure 12: Efficacy study, 30-day cohort. Thermal hyperalgesia measured on days 0 and 7 after punch biopsy. In this graph, data from both the 8-day and 30-day experiments are pooled (N=16 in each group); data are mean + SEM. Statistical analysis: Student's t-test, *p<0.05. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 13] Figure 13: Efficacy study, 30-day cohort. This table lists the days of closure over the observation period, derived from clinical observations. "No" means that no closure was observed. CHF6467 = Polypeptide of SEQ ID NO: 4 [Figure 14] Figure 14: Efficacy study, 30-day cohort. Time course of the healing process measured as the external area of ​​the ulcer from skin biopsy to day 29. Results are expressed as % of the lesion area on day 0; data are mean + SEM. For statistical analysis, see text. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 15] Figure 15: Efficacy study, 8+30 day cohort. Time to closure of ulcer (external area) on day 8. Data depicted in the graph are obtained by pooling values ​​from both the 8 day and 30 day experiments; N=16. Data are expressed as mean + SEM. Statistical analysis: One-way ANOVA followed by post-hoc Dunnett's multiple comparison test: F(5,77)=3.007, P=0.0156. CHF6467=Polypeptide of SEQ ID NO: 4 [Figure 16] Figure 16: Efficacy study, 30-day cohort. NGF plasma levels measured on day 30. Data are expressed as mean + SEM; statistical analysis: one-way ANOVA and post-hoc Dunnett's test. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 17] Figure 17: Efficacy study, 30-day cohort. Photomicrographs illustrating wound histology (H&E staining) on ​​day 7, sampled according to the schematic presented in A, B. Photomicrographs of wound borders illustrating DF.MET (epidermal migratory tongue, D, E) and angiogenesis (F). [Figure 18]Figure 18: Efficacy study, 30-day cohort. Representative photomicrographs of the re-epithelialization process taken from mice treated with vehicle, SEQ ID NO: 4 (1, 10, and 30 μg / day). For comparison purposes, photomicrographs of intact skin are also reported. The thickness of the epidermal layer in the different groups is reported in the graph. Data are reported as mean + SEM; statistical analysis: one-way ANOVA followed by post-hoc Tukey's test, **p<0.01; ****p<0.0001. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 19] Figure 19: Efficacy study, 30-day cohort. Anatomy of innervation in the mouse dorsal skin visualized by PGP-9.5 immunostaining. [Figure 20] Figure 20: Efficacy study, 8+30 day cohort. PGP9.5-IR in the skin of intact animals (A), vehicle (B), and mice treated with SEQ ID NO: 4 (30 μg / day) (C) (30 days after lesion induction). D, E: ROI (region of interest, D) and threshold settings (E) for the computerized image analysis procedure used to assess cutaneous innervation at 30 days. [Figure 21] Figure 21: Efficacy study, 8+30 day cohort. Morphometric analysis of PGP9.5-IR in skin 8 days (A) and 30 days (B) after wound induction. Data are expressed as mean + SEM; statistical analysis: one-way ANOVA followed by Dunnett's post-hoc test. *p<0.05; **p<0.01. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 22] Figure 22: Efficacy study, 8+30 day cohort. Histological (H&E staining) and immunohistochemical analysis of repaired skin in the group treated with SEQ ID NO: 4 (30 μg / day). AB: Repaired skin layer above the basement membrane as shown by laminin staining (B, arrow); reinnervation comes from the subepidermal plexus and protrudes through the basement membrane (C). The MET (D) is highly innervated both 8 (E) and 30 (F) days post-lesion; angiogenesis is observed in the MET as visualized by H&E staining (G), laminin-IR at low (H) and high (I) magnification. Abbreviations: ep, epidermal layer; MET, epidermal migratory tongue [Figure 23]Figure 23: Efficacy study, 8+30 day cohort. Morphometric analysis of laminin-IR in skin 8 (A) and 30 (B) days after wound induction. Gray horizontal bars in graph B represent laminin-IR in intact skin. Data are expressed as mean + SEM. Statistical analysis: One-way ANOVA followed by Dunnett's post-hoc test. *p<0.05; ***p<0.001; ****p<0.0001. [Figure 24] Figure 24: Polypeptide sequences. Asterisk (*) = position 61 in mature human NGF; cross (+): position 100 in mature human NGF. A: SEQ ID NO: 1: Sequence of preprohuman NGF encoded by the respective human open reading frame. Prepeptide: amino acid positions 1-18; propeptide: amino acid positions 19-121; mature NGF: amino acid positions 122-239; C-terminal dipeptide: amino acid positions 240-241. Disulfide bond (correctly folded mature portion): amino acid positions 136←→201, 179←→229, 189←→231. Furin cleavage site (RSKR): amino acid positions 118-121. B: Schematic diagram of prepeptide, propeptide, and mature NGF. C: SEQ ID NO: 2: Sequence of mature human NGF. D: SEQ ID NO: 3E: SEQ ID NO: 4 [Figure 25] Figure 25: Rate of wound healing in diabetic mice after treatment with vehicle or polypeptide of SEQ ID NO: 4 (1-10-100 μg / cm2 / day), expressed as "percentage of non-healed mice" over the course of the study. Statistical analysis: Kaplan-Meier survival curve estimation. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 26] Figure 26: Morphometric analysis of PGP9.5-IR (A) and PECAM1-IR (A) in repaired skin 28 days after wound induction. Data are expressed as mean ± SEM (n = 15-18). Statistical analysis: One-way ANOVA followed by Dunnett's post-hoc test; *p<0.05; **p<0.01. CHF6467 = polypeptide of SEQ ID NO: 4 [Figure 27]Figure 27: The effect of the polypeptide of SEQ ID NO: 4 on the mechanical pain threshold in the area surrounding the ulcer border was assessed after 14 days of chronic topical treatment using an electronic Von Frey device from Bioseb. Data are expressed as mean ± SEM (n = 15-18). Statistical analysis: One-way ANOVA.

Claims

1. 10. A polypeptide selected between the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 for use in the treatment and / or prevention of a dermatological disorder in a mammalian subject.

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

3. 3. A polypeptide for use according to claim 1 or 2, wherein the polypeptide is the polypeptide of SEQ ID NO:

4.

4. The polypeptide for use according to any one of claims 1 to 3, wherein the dermatological disorder is characterized by a wound surface on at least a part of the body of the subject.

5. 5. The polypeptide for use according to claim 4, wherein the dermatological disorder characterized by a wound surface is preferably a skin lesion characterized by at least partial excision of the dermis and optionally by excision of the dermis.

6. 6. The polypeptide for use according to any one of claims 1 to 5, wherein the dermatological disorder comprises at least one ulcer, preferably selected from the group consisting of diabetic ulcers, traumatic ulcers, surgical ulcers, pressure ulcers, chronic ulcers, and any combination of these ulcers, or the dermatological disorder comprises a burn or a mechanical injury.

7. Polypeptide for use according to any one of claims 1 to 6, wherein the mammal, preferably a human, is suffering from or predisposed to suffering from diabetes mellitus.

8. 8. The polypeptide for use according to claim 7, wherein the diabetes is selected between type 1 diabetes mellitus and type 2 diabetes mellitus.

9. The polypeptide for use according to any one of claims 1 to 8, wherein the polypeptide is administered in a single dose.

10. The polypeptide for use according to any one of claims 1 to 8, wherein the polypeptide is administered repeatedly.

11. The polypeptide for use according to claim 10, wherein the polypeptide is administered repeatedly 1 to 5 times per day, preferably about 2 times per day.

12. 12. The polypeptide for use according to claim 10 or 11, wherein the polypeptide is administered repeatedly over a period of 3 to 30 days, preferably 7 to 14 days, or until closure of the wound body surface.

13. 13. The polypeptide for use according to any one of claims 1 to 12, wherein the polypeptide is administered to a subject with a diabetic foot ulcer (DFU), preferably a subject with a diabetic neuropathic foot ulcer (DFU).

14. A polypeptide for use according to any one of claims 1 to 13 for topical administration.

15. The polypeptide for use according to claim 14, wherein the polypeptide is administered to a wound body surface.

16. Dose / Each dose is measured in mm of the wound body surface being treated. 2 0.3 to 6 μg per (0.3 to 6 μg / mm 2 16. The polypeptide for use according to claim 15, wherein the amount of the polypeptide is 0.01 to 0.1%.

17. The polypeptide for use according to any one of claims 1 to 16, wherein the treatment and / or prevention does not cause hyperalgesia in a mammalian subject.

18. The polypeptide for use according to any one of claims 1 to 17, wherein the polypeptide is contained in an aqueous medium, and the aqueous medium is administered to a mammalian subject.

19. 19. The polypeptide for use according to any one of claims 1 to 18, wherein the polypeptide is obtainable by recombinant expression and purification, the purification comprising purification on a mixed-mode stationary phase.

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