RU2799211C2 - Agent for the treatment of dermatological diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method of treating skin ulcers in a mammalian subject. A method of treating skin ulcers in a mammalian subject comprising administering a polypeptide of SEQ ID NO: 4, where the polypeptide is administered topically and applied to the surface of the damaged body part and, in this case, the dose/each dose has an amount of 0.3–6 μg of the polypeptide per mm² of the treated damaged body surface, which is 0.3–6 μg/mm².

EFFECT: above invention makes it possible to treat skin ulcers in mammals.]

12 cl, 27 dwg, 9 tbl, 5 ex

Description

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

The present invention provides an agent suitable for the treatment and prevention of dermatological disorders, including, without limitation, skin ulcers.

BACKGROUND OF THE INVENTION

Skin diseases, including chronic wounds such as ulcers, include ulcers on the surface of the skin or mucous membrane, often accompanied by tissue breakdown. As a rule, such skin diseases can lead to the loss of the epidermis, and often parts of the dermis and even subcutaneous fat. Such skin diseases can be caused by a variety of factors, such as, but not limited to, circulatory disorders. Skin ulcers are common in humans, including diabetic patients (Ndip et al., 2012, Int. J. Gen. Med., Vol. 5, pp. 129-134). Such skin diseases represent a serious medical and social problem.

There is currently no suitable curative treatment for this type of dermatological disease. According to current data, patients have to be treated for months and sometimes even years, which leads to significant economic 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 reduction through the use of mechanical relief devices, are the main treatment option for such diseases (Ndip et al., 2012, Int. J. Gen. Med., Vol. 5, p. 129-134) , but such alternative measures provide no cure at all.

Skin diseases often occur in diabetic patients: Diabetes is common in today's society, and there are statistics that one in four diabetic patients will develop a skin disease, in particular a foot ulcer, during their lifetime (Ndip et al., 2012, Int. J Gen. Med., vol. 5, pp. 129-134). This disorder often requires long hospital stays, rehabilitation, home care, and social services. Thus, diabetes-related ulcers are a major problem with a huge impact on the overall global burden of disease due to the increasing prevalence of diabetes and the lack of treatment options currently sufficient for both individuals and society.

In the search for therapeutic treatment, the use of some human growth factors for the therapy and potential treatment of skin diseases has been proposed in the past, but it is now generally accepted that there is only limited evidence supporting the effectiveness of 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 that has been proposed in the past for the treatment of skin diseases is a platelet-derived growth factor (beclapermin, brand name Regranex), but its administration has been found to be associated with serious 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 variant tested is granulocyte colony stimulating factor (G-CSF), but G-CSF was not found to significantly affect the likelihood of infection resolution or wound healing (Cruciani et al., 2005, Diabetes Care, vol. 28, p. 454- 460). Another example proposed in the course of research is the epidermal growth factor (EGF), which is supposed to initially have an unexpected positive therapeutic effect (for example, WO/2003/075949 A1), but the EGF-based treatment has in fact turned out to be commercially unavailable, which suggests that the initial hopes placed on this agent did not materialize. Alternatively, human nerve growth factor (hNGF), which has been proposed to have pro-angiogenic properties and facilitate wound healing (Graiani et al., 2004, Diabetologia, vol. 47, p. 1047-1054), has been proposed for the treatment of certain neuropathic clinical conditions, but clinical trials have yielded inconsistent results (Apfel et al., 2000, J. Amer. Med. Assoc., vol. 284, p. 2215-2221), and as a result, an NGF-based drug has not been developed (see, for example, https://www.gene.com/media/press-releases/4875/1999-04-08/phase-iii-trial-with-nerve-growth-factor). For example, although Graiani et al. does not specifically discuss the allogenic effect of NGF, human NGF is well known to cause pain (Dyck et al. 1997, Neurology, vol. 48, p. 501-505; Svensson et al., 2003, Pain, vol. 104, p. 241 -247). As a result, human NGF has not been approved as a suitable therapeutic agent for skin ulcers; inter alia, due to its pain-producing activity and/or lack of efficacy at tolerated doses. Therefore, given that growth factor-based treatments have had limited success, there is insufficient evidence for a cure, and due to undesirable side effects, among other things, the medical community has continued to search for other potential cures. Based on the foregoing, interleukins and other non-growth factor molecules have recently been proposed for the treatment of certain skin ulcers. For example, Genentech has proposed derivatives of interleukins, in particular r-interleukin 22, which is believed to play a role in modulating the immune system, and may be useful in the treatment or prevention of skin ulcers, including diabetic skin ulcers (see e.g. https ://www.gene.com/stories/mechanisms-of-healing), but such a drug is not available to patients and it is currently unknown if this may change over time.

Thus, there is still a need for an effective treatment of skin diseases, in the absence of side effects, for example, completely intolerable or otherwise undesirable side effects caused by a therapeutic agent suitable for this purpose and available to practitioners, having a reliable and acceptable purity for administration. mammals, including humans.

Problem to be solved

The main object of the invention is to provide treatment or prevention of dermatological disorders, including ulcers, without causing unwanted or painful side effects in diabetic and non-diabetic subjects. It is also desirable to provide a therapeutically active agent in sufficient yield and purity to enable such treatment. Thus, the aim of the present invention is to overcome the problems identified in the prior art. Specific objectives include providing a reliable method of treating a subject with a dermatological disease without unwanted side effects.

SUMMARY OF THE INVENTION

The present invention relates to a polypeptide for use in the treatment and/or prevention of a dermatological disease in a mammal, where the polypeptide is selected from 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 ), while this mutation is associated with reduced nociceptive activity. In particular, the arginine at position 100 of hNGF is replaced by glutamic acid.

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

Preferably, the mammalian subject is a human.

Preferably, the dermatological disease is characterized by a damaged surface of at least a portion of the subject's body. Preferably, the dermatological disease is characterized by a damaged surface. More preferably, the dermatological disease is a skin lesion, preferably characterized by at least partial destruction of the dermis, although this is not necessary.

Preferably, the dermatological disease consists of at least one ulcer, preferably selected from the group consisting of diabetic ulcers, traumatic ulcers, surgical ulcers, decubitus ulcers, chronic ulcers, and combinations of any of these ulcers. In alternative but not mutually exclusive embodiments, the dermatological disease also includes a burn or mechanical injury.

Preferably the mammal, preferably the human, is diabetic or has a predisposition to diabetes. In exemplary embodiments, diabetes mellitus is selected from type 1 diabetes and type 2 diabetes.

In one embodiment, the polypeptide is administered once.

In an alternative and more preferred embodiment, the polypeptide is administered multiple times. In a particularly preferred embodiment, the polypeptide is administered repeatedly from one to five times a day, preferably twice a day.

In one embodiment, the polypeptide is administered repeatedly until the damaged body surface has healed. Alternatively, the polypeptide is administered repeatedly over a period of three to 30 days, preferably seven to 14 days. Optionally, the introduction is stopped after the specified interval.

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

Preferably, the polypeptide is for topical use. More preferably, the polypeptide is applied to the surface of the injured part of the body.

Preferably, the dose of the administered polypeptide is determined based on the area of the damaged body surface to be treated. Preferably, such a determination is carried out at the beginning of treatment. In one embodiment, the dosage is adjusted for later administration depending on the area of the injured body surface at the time of such later administration. In an alternative embodiment, the dosage is not adjusted for later administration so that the dosage of administration depends solely on the area of the injured body surface to be treated at the start of treatment (first dosage) and subsequent dosages correspond to the first dose.

In one embodiment, dose/each dose has an amount of 0.3 to 6 μg of polypeptide per mm ² of the injured body surface treated (0.3 to 6 μg/mm ² ).

In one embodiment, the polypeptide is contained in an aqueous medium, and the aqueous medium is administered to a mammal.

Preferably, the treatment and/or prophylaxis does not cause hyperalgesia in the mammal.

In one embodiment, the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 can be obtained from a biological source. This may include purification, ie separating it from other molecules, including other proteins, such as those of the host cell. Optionally, the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 can be obtained in a process that includes (re)folding and/or chromatographic purification, and/or cleavage with a protease, possibly bringing to a final protein concentration and/preparation of the desired drug. In one embodiment, the polypeptide can be obtained by recombinant expression and purification, where the purification includes purification in a mixed stationary phase.

Thus, the present invention also provides a polypeptide of SEQ ID NO: 3 and a polypeptide of SEQ ID NO: 4 from a recombinant source: purified as described herein for use in a method of treating human or animal body surfaces with therapy as described herein.

DETAILED DESCRIPTION OF THE INVENTION

This description as a whole, together with the claims and drawings, discloses specific and/or preferred embodiments and variations of individual features of the invention. The present invention also considers as particularly preferred embodiments those embodiments that are created by combining two or more specific and/or preferred embodiments and variants described herein for the present invention. Thus, the present disclosure also includes all of the items, compounds, features, steps, methods, or compositions mentioned or referred to in this specification, alone or in combination, and any or all combinations thereof, or any two or more of these items, compounds, features, steps, methods or compositions. Thus, unless otherwise specifically noted herein or the context otherwise requires, reference to a single entity, compound, feature, step, method, or composition should encompass both one and multiple (i.e., more than one, e.g., two or more , three or more, or all) of these objects, compounds, features, steps, methods, or compositions. Unless otherwise specifically noted or the context requires otherwise, each embodiment, aspect, and example disclosed herein is to be considered as applicable to, or in combination with, any other embodiment, aspect, or example disclosed herein.

Those skilled in the art will appreciate that the invention described herein is susceptible to changes and modifications other than those specifically described. Thus, the present disclosure is not limited in scope to the specific embodiments described herein as well as those presented herein for purposes of illustration and explanation. Functionally or otherwise, all equivalent entities, compounds, features, steps, methods, or compositions are included within the scope of this disclosure. It will be obvious to a person skilled in the art that the present disclosure includes all variations and modifications of the entities, compounds, features, steps, methods, or compositions described herein.

Each reference herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, presentations, etc., cited in the text) is hereby incorporated by reference in its entirety. Nothing in this document is to be construed as an admission that the present invention has no right to predate a particular doctrine and/or as an admission that a particular reference, other than those generally known, does not contain information clear and complete enough to be beyond the ability of a person skilled in the art to use. in this field of technology.

Generally, unless otherwise specifically defined, all technical and scientific terms used in this document have the same meaning as commonly understood by one of skill in the art (e.g., medicine, dermatology, neurology, genetics, molecular biology, gene expression, cell biology, cell culture, immunology, neuroscience, chromatography, protein chemistry and biochemistry). Published textbooks and review articles, for example, in English, usually define the meaning generally understood by a person skilled in the art.

The expression "and/or", for example, "X and/or Y" should be understood to mean "X and Y" or "X or Y" and should be considered to provide explicit disclosure of "and", "or", as well as both meanings simultaneously (and "and" and "or").

In the context of the present description, unless otherwise indicated, the terms "about", "approximately". and "substantially" all mean "approximately" or "almost", and in the context of the numerical value or range set forth herein, preferably means +/- 10%, more preferably +/- 5% of the numerical value or range specified or declared.

Unless expressly stated otherwise, the word "comprise", or variants thereof such as "comprises" or "comprising", are used in the context of this document to indicate that additional elements may optionally be present in addition to the list elements introduced by the word "comprising" . However, as a specific embodiment of the present invention, the term "comprising" is intended to cover the possibility of the absence of additional elements, i.e. for the purposes of a particular embodiment, the term "comprising" should be understood to mean "consisting of".

Unless specifically stated otherwise, all indications of relative amounts in relation to the present invention are made on a weight/weight basis. Indications of the relative amounts of a component characterized by a general term are intended to indicate the total amount of all specific variants or elements covered by the specified general term. If a certain component, defined by a general term, is indicated as being present in a certain relative amount, and if this component is further characterized as a specific variant or element covered by the general term, then this means that no other variants or elements covered by the general term are additionally present. so that the total relative amount of the components covered by the general term exceeds the indicated relative amount; more preferably, other variants or elements covered by the general term are not present at all.

All methods and processes described herein may be performed in any suitable order, unless otherwise indicated herein or unless the context clearly dictates otherwise.

The term "agent" as used herein, unless otherwise indicated, generally refers to a compound or composition, preferably a compound. The agent is capable of exerting an effect on a living organism and/or a cell of a living organism, or originating from a living organism, such as acting on a cell and/or tissue of the body, or on the environment. The physical state of the agent is not specifically limited and, unless otherwise indicated, it may be in a gaseous, liquid and/or solid state. The type of the agent is not particularly limited unless otherwise indicated, and thus the agent may be a chemical and/or a biomolecule such as a protein or a nucleic acid. The agents defined here are used in the present invention.

"Adverse effect", as used here, is an undesirable negative effect resulting from the introduction of an agent (drug) to the subject. Side effects include, without limitation, increased lesion, mortality, hyperalgesic syndrome, pain, changes in body weight, enzyme levels, loss of function, or any pathological change detectable at the microscopic, macroscopic, or physiological level. Adverse effects may cause reversible or irreversible changes, including an increase or decrease in the individual's susceptibility to other chemicals, foods, or processes such as drug interactions.

The terms "chromatography", "chromatographic" and the like are used herein. generally refers to a technique suitable for separating a mixture in which the mixture is added to a non-liquid material, referred to as a "stationary phase", in order to separate, at least in part, one or more components of the mixture. To this end, the stationary phase may be exposed to the liquid and/or the mixture may be dissolved in the liquid; said liquid in contact with the stationary (stationary) phase may also be referred to as the "mobile phase". In general, any step that is "performed by chromatography" as described herein can be synonymously referred to as a "chromatographic step".

As used herein, the term "mobile phase" has the meaning commonly used in the art and can refer to all liquids brought into contact with the stationary (stationary) phase during chromatography, i.e. washing liquids, as well as liquids (mixtures) containing proteins of interest, such as one or more of the proteins described herein. In the present invention, the mixture subjected to chromatography as defined herein typically includes 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 them, proteases and/or host cell proteins (HCPs).

The "stationary phase" typically includes a base matrix, which is a water-insoluble material, usually in the form of particles or a gel, such as a resin. In many cases, including the embodiments described herein, the stationary phase includes a host matrix and moieties that can bind to at least one component of the mixture to be chromatographed. The base matrix is usually a water-insoluble material, usually in the form of particles or a gel. Non-limiting examples of base matrices are sepharose and agarose, eg very hard agarose.

"Chromatographic step" in the context of the present description refers to the act of adding to the chromatographic material (preferably stationary (stationary) phase) a liquid containing at least one compound to be analyzed and/or purified, which is preferably a protein (and in the context of the present invention specified the protein is most preferably a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4); possibly washing the chromatographic material with one or more wash solutions and eluting at least one of said compounds. In this context, the two chromatographic step process provided for illustration is characterized in that a liquid containing at least one such compound to be analyzed and/or purified is added to the first chromatographic material as described above and, after elution, a liquid containing at least one such compound is added to the second chromatographic material, from which it is also eluted, as described above. The purpose of any "chromatographic step" is that at least one component contained in the mixture applied to the stationary (stationary) phase, preferably used in chromatography, is associated with the stationary (stationary) phase. Such a connection may be one or more of the proteins described here. The compound can be removed from the stationary phase, for example by changing the mobile phase and/or continuing exposure to the mobile phase over time.

The term "binds" when used in relation to chromatography, for example to describe the binding capacity of a stationary (stationary) phase, is not particularly limited, but generally refers to non-covalent binding. Thus, typically at least one component contained in the mixture, such as at least one protein, is non-covalently bound to the stationary phase. The chromatographic step optionally, but preferably, includes washing the stationary phase to which at least one component is associated. At least one component may be at least one protein, such as one described here.

The term "heterologous" as used here describes something that is made up of many different elements.

The terms "disulfide" and "disulfide bond" are used in the context of the present invention in the meanings commonly used in the art. In general, "disulfide" refers to a functional group with the structure R-S-S-R'. The bond is also referred to as an "SS bond" and is usually formed by combining two thiol groups. Disulfide bonds in proteins are formed between the thiol groups of cysteine residues during 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 relying on any particular theory, it is commonly understood in the art that in eukaryotic cells, disulfide bridges form in the lumen of the endoplasmic reticulum (and mitochondrial intermembrane space) but not in the cytosol, and in the case of prokaryotes, disulfide bridges form in the periplasm (of the relevant organisms). , in particular gram-negative bacteria); disulfide bridges can also be found in proteins of the extracellular environment of both eukaryotic and prokaryotic cells.

The terms "express", "expression", "expressed", "gene expression" and the like, as used herein, refer to the conversion of gene information into a functional gene product during synthesis. Gene expression includes at least transcription and optionally includes one or more additional functions, optionally selected from a list including, but not limited to, translation and post-translational modification. In the context of recombinant expression of a protein in a host cell, this term generally means that the protein is produced by the host cell (in any cell compartment and/or secreted and/or incorporated into inclusion bodies) unless the context dictates otherwise.

As used here, the term "heterologous" describes something that is made up of many different elements or derived from other sources. For example, in a non-human host cell containing a human gene (or a gene encoding a non-natural polypeptide, such as a polypeptide of the invention), said gene is "heterologous" to the cell, and the cell may be capable of "heterologous" expression of the corresponding gene. Heterologous expression of a gene may also be referred to as "recombinant".

The term "inclusion bodies" has the meaning commonly used in the art and is intended to refer to aggregates or particles found in the cytosol or periplasm of the host cell; inclusion bodies typically contain a protein, such as, in particular, a protein recombinantly expressed in the host cell. Without wishing to be bound by any particular theory, it is to be understood that in the region of recombinant expression, inclusion bodies typically contain the recombinantly expressed protein as well as relatively small amounts of host cell proteins (HCPs), ribosomal components, or DNA/RNA fragments. Without wishing to be bound by any particular theory, it is to be understood that inclusion bodies typically contain, at least in part, a misfolded protein (misfolded protein), in particular a misfolded, recombinantly expressed protein. It is understood that inclusion bodies typically contain the protein in a misfolded form, ie, in the context of the present invention, they typically include a polypeptide of the present invention and/or its precursor in a misfolded form. The term "misfolded" generally describes a biomolecule, such as a nucleic acid or polypeptide, that is not in its native conformation, ie is in a misfolded form.

By "isolated" is meant a material that is substantially or substantially free of the components normally associated with it in its native state. For example, "isolated peptide" or "isolated protein" in the context of the present description refers, respectively, to a peptide or protein purified from the cellular and extracellular environment, such as tissue, surrounding it in the native environment, i. to a peptide or protein isolated, for example, from the cell in which it was expressed, for example, from the host cell. Alternatively, "isolated peptide" or "isolated protein" and the like. in the context of the present description, refer to the in vitro isolation and/or purification of a peptide or protein, respectively, from its native cellular environment and isolated from association with other components of the environment in which the peptide or protein is usually found. In another example, "isolated cell" in the context of the present description refers to a cell that has been cleared of cellular and extracellular environment, such as tissue or cell colonies surrounding it in its natural state, for example, a host cell removed from the environment, usually adjacent to the cell. In accordance with the above definition of the term "isolated", "isolate", as used herein, is a term describing the activity of obtaining an "isolated" material, such as, for example, an isolated cell or an isolated peptide or protein.

The terms "multi" and "multiple" in the context of the present description means many, that is, any number consisting of two or more.

As used herein, the term "mutation" refers to a change in the nucleotide sequence of the genome of an organism, virus, extrachromosomal DNA, or other genetic elements. The term also covers mutations in the amino acid sequence, in particular the amino acid sequence of a gene carrying at least one non-silence mutation. Unless otherwise indicated, mutation of a nucleotide sequence is a permanent change. Mutations present in the germline are usually inherited. In general, mutation of a nucleotide sequence can result in many different types of sequence changes: mutations in genes can either have no effect, change the gene product, or prevent the gene from functioning correctly or completely. Mutations may also be present in non-gene regions. Unless otherwise indicated, the wild-type sequence is used as a reference sequence for describing a mutation. Thus, for example, if a given mutant is said to have a mutation at position 100 of the polypeptide sequence, this indicates that the mutant at position 100 does not have the same amino acid residue as the wild-type polypeptide. Specific types of nucleotide sequence and/or amino acid sequence mutations include changes such as deletions, substitutions, additions, insertions, and splicing variants. "Deletion" in relation to a nucleotide sequence refers to the absence of one or more nucleotides in a nucleotide sequence. "Deletion" in relation to an amino acid sequence refers to the absence of one or more amino acid residues in a polypeptide. "Addition" in relation to a nucleotide sequence refers to the presence of one or more additional nucleotides in a nucleotide sequence. "Addition" in relation to the amino acid sequence refers to the presence of one or more additional amino acid residues in the corresponding polypeptide. "Substitution" in relation to a nucleotide sequence refers to the replacement of one or more nucleotides with another(s) nucleotide(s) in a nucleotide sequence. "Substitution" in relation to the amino acid sequence refers to the replacement of one or more amino acid residues with other amino acid residues in a polypeptide. Nucleotide sequence additions, deletions and substitutions, for example in the open reading frame, may be at the 5' end, 3' end and/or within the gene. Additions, deletions and substitutions of the polypeptide may be at the amino terminus, carboxy terminus and/or within the polypeptide. An "insertion" with respect to a nucleotide sequence and/or a polypeptide sequence is the addition of one or more nucleotides or one or more amino acid residues, respectively, precisely within the corresponding sequence. The term "splicing variant" is used to describe a variant in which the RNA encoding a polypeptide sequence is spliced differently than the corresponding wild-type RNA, usually 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 "splicing variant" can be used not only in relation to the corresponding RNA, but also in relation to the corresponding template DNA sequence (usually genomic DNA) and in relation to the sequence of the polypeptide encoded by such RNA. The term "mutant" is generally intended to mean a nucleic acid sequence or amino acid sequence that differs from the wild-type sequence. Thus, a mutant nucleic acid sequence or a mutant amino acid sequence has at least one mutation with respect to the corresponding wild-type sequence. In cases where there are polymorphisms in the nucleic acid sequence, which, however, are not reflected at the level of the corresponding encoded polypeptide (silent (silence) mutations, degeneracy of the genetic code), the term "mutant" at the nucleic acid level specifically refers only to those variants nucleic acids that encode the mutated polypeptide. The mutants may contain various combinations of mutations, either alone or in a combination of more than one mutation and/or different types of mutations.

The term "nerve growth factor", abbreviated "NGF" or "beta-NGF", refers to a neurotrophic factor and neuropeptide involved in the regulation of growth, maintenance, proliferation and survival of certain neurons and other cells, in accordance with the generally accepted meanings of the terms in the art (see ., for example, 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 polypeptides of SEQ ID NO: 3 or 4 Wild-type NGF is a 2.5S, 26-kDa beta subunit derived from the NGF precursor, which is biologically active: wild type NGF binds to at least two classes of receptors: the tropomyosin receptor kinase A (TrkA) and the low affinity NGF receptor (LNGFR/p75NTR). The term "NGF", unless otherwise indicated, refers to NGF of any species, preferably mammalian species; however, human NGF is most preferred. "hNGF" as used herein means human NGF. Unless the context dictates otherwise, the terms "NGF" and "hNGF" refer to wild-type NGF, ie. hNGF means wild-type NGF. The amino acid sequence of wild-type human NGF corresponds to positions 121-239 of SEQ ID NO: 1 (gray in Fig. 24). Non-human NGF sequences are available, for example, in the scientific literature using a sequence search algorithm such as BLAST using positions 121-239 of SEQ ID NO: 1 as the entry point, as well as in public protein databases such as Swissprot.

The terms "NGF mutein" and "NGF mutein", or, with reference to NGF, "mutein such", are used interchangeably herein to refer to a polypeptide that has at least one mutation compared to wild-type NGF, as further detailed herein. The polypeptides of SEQ ID NO: 3 and SEQ ID NO: 4 are NGF muteins. Preferably, the NGF mutein has 80 to 99.5% sequence identity with NGF, especially human NGF, more preferably, the mutein has 90 to 99% sequence identity with NGF, especially human NGF.

The terms "mature portion", "mature fragment" in relation to NGF are used interchangeably with the term "beta-NGF" and refer to an NGF polypeptide that is characterized in that it does not contain the propeptide (and therefore does not contain the pre-propeptide) NGF. By analogy, the term "mature portion" is also used to refer to polypeptides of SEQ ID NO: 3 or 4, since these polypeptides also do not contain a propeptide (and therefore do not contain a pre-propeptide). Preferably, the mature portion also does not contain a C-terminally 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 two amino acid residues "RA" (240 and 241 in SEQ ID NO: 1). More specifically, the mature portion can be obtained by cleavage of pro-NGF with the furin protease Furin (and other proteases capable of accurately cleaving directly the N-terminus of the first amino acid residue of NGF or polypeptides of SEQ ID NO: 3 or 4, respectively, without being limited to such methods. For example, well the furin cleavage site of human NGF and many orthologues is known to consist of the sequence R ¹ S ² K ³ R ⁴ (single letter amino acid code; sequences are numbered N to C-terminus; and are boxed in Fig. 25)). In mature NGF, neither the furin cleavage site nor any amino acid at the N-terminus of the furin cleavage site is usually present. By way of illustration, the mature portion of human NGF consists of the polypeptide represented by amino acid positions 122-239 in SEQ ID NO: 1. sequence alignment.

The term "precursor" as used herein in relation to NGF refers to any peptide sequence from which NGF can be obtained by proteolytic cleavage. Presented for illustrative purposes, both pro-NGF and pre-pro-NGF, as well as variants thereof, are typical examples of NGF precursors. The term "precursor" as used herein may refer to precursors whose C-terminal amino acid residue is the C-terminal residue of NGF, as well as precursors whose C-terminus extends beyond the C-terminal residue of NGF. , provided that NGF can be obtained from it by proteolytic cleavage: although the naturally occurring wild-type human pro-NGF precursor (SEQ ID NO: 1) contains a C-terminal dipeptide (amino acid residues 240 and 241 in SEQ ID NO: 1 are isolated in bold in Fig. 1), preferably, in the present invention, the precursor does not contain a C-terminally cleavable peptide encoded by the wild-type NGF open reading frame; such a C-terminal cleavage peptide in the case of human NGF consists of two amino acid residues "RA" (240 and 241 in SEQ ID NO: 1).

As used herein, the terms "prepeptide" or "presequence" generally refer interchangeably to the polypeptide sequence encoded by the portion of the NGF open reading frame immediately adjacent to the propeptide at its N-terminus. For illustrative purposes, a prepeptide is an NGF consisting of a sequence containing a contiguous sequence ranging from residue 1 of SEQ ID NO: 1 to residue 18 of SEQ ID NO: 1. Sequences of the corresponding non-human NGF precursor prepeptides are available, for example, in the scientific literature, using sequence search algorithm such as BLAST using positions 1-18 of SEQ ID NO: 1 as input, as well as in public protein databases such as Swissprot. A polypeptide or protein consisting of a prepeptide and pro-NGF, where the C-terminus of the pre-peptide is immediately adjacent to the N-terminus of pro-NGF, may be referred to here as "pre-pro-NGF".

The terms "propeptide" or "prosequence" as used herein generally refer interchangeably to a polypeptide sequence naturally encoded by the portion of the NGF open reading frame N-terminally immediately adjacent to a mature NGF sequence whose polypeptide sequence does not include a prepeptide. Illustratively, the propeptide is contained in the wild-type NGF precursor. The NGF precursor propeptide consists of a sequence containing a contiguous sequence from residue 19 of SEQ ID NO: 1 to residue 121 of SEQ ID NO: 1. Sequences of the corresponding propeptides of non-human origin, or pro-NGF, are available, for example, in the scientific literature using a sequence search algorithm , such as BLAST, using positions 19-121 of SEQ ID NO: 1 as input, as well as in public protein databases such as Swissprot.

"Pro-NGF" in the context of the present description refers to a peptide sequence containing both the mature part of NGF and the corresponding pro-peptide, but not including the corresponding pre-peptide. Human pro-NGF consists of a sequence containing a contiguous sequence ranging from residue 19 of SEQ ID NO: 1 to at least residue 239 of SEQ ID NO: 1. Although wild-type human pro-NGF includes a C-terminal dipeptide (amino acid residues 240 and 241 in SEQ ID NO: 1 in bold in Figure 25), it is preferable that the pro-NGF produced and used in the present invention does not contain a C-terminal cleavage peptide encoded by the wild-type NGF open reading frame, such as a C-terminal cleavage peptide, in the case of human NGF, consisting of two amino acid residues "RA" (240 and 241 in SEQ ID NO: 1). Non-human pro-NGF sequences are available, for example, in the scientific literature using a sequence search algorithm such as BLAST using positions 19-121 of SEQ ID NO: 1 as input, and also in public protein databases such as Swissprot.

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. The nucleic acid may be double-stranded or single-stranded (ie, sense strand or antisense strand). Non-limiting examples of polynucleotides include genes, open reading frames, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, miRNA, miRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated nucleic acids of any type, nucleic acid probe sequences and primers, and nucleic acid analogs. Nucleic acids can have any type of three-dimensional structure.

The term "peptide" according to the invention includes oligo- and polypeptides and refers to substances comprising two or more, preferably 3 or more, preferably 4 or more, preferably 6 or more, preferably 8 or more, preferably 10 or more, preferably 13 or more , preferably more than 16, preferably 21 or more and preferably up to 8, 10, 20, 30, 40 or 50, in particular up to 100 amino acids covalently linked in a chain by peptide bonds.

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 here, unless the context dictates otherwise. Thus, the terms "polypeptide SEQ ID NO: 4" and "protein SEQ ID NO: 4" have the same meaning.

The term "pharmaceutically acceptable" generally describes that a particular substance can be administered to a subject, optionally and preferably in combination with an agent, without an agent causing intolerable side effects, at the dosage used.

The terms "pharmaceutically acceptable carrier" and "pharmaceutically acceptable excipient" are used to refer to one or more of solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic agents and absorption delaying agents, etc., which are physiologically compatible and suitable for administration to a subject as described herein, or otherwise interfere with such administration. Examples of such pharmaceutically acceptable carriers include, without limitation, one or more of the list consisting of water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, as well as combinations thereof. In particular, in the case of liquid pharmaceutical compositions, it may be advantageous to include isotonic agents, for example sugars, polyhydric alcohols such as mannitol, sorbitol or sodium chloride, in the composition. Pharmaceutically acceptable carriers may further include adjuvants such as wetting or emulsifying agents, preservatives or buffers to increase the shelf life or effectiveness of the agent. A pharmaceutically acceptable carrier is usually included in the composition according to the present invention.

The term "pharmaceutically active agent" refers to an agent that can be used when administered to a subject in such a way that the agent is able to provide a benefit, such as ameliorating the symptoms of a disease or disorder. In addition, a "pharmaceutically active agent" may have a beneficial or beneficial effect on a subject's condition or course of disease when administered to the subject in a therapeutically effective amount. Preferably, the pharmaceutically active agent has medicinal properties and may be administered to alleviate, reduce, arrest, regress, delay the onset of a disease, or reduce the severity of one or more symptoms of a disease or disorder. The pharmaceutically active agent may have prophylactic properties and be used to delay the onset of a disease or to lessen the severity of such a disease or condition. For example, the claimed agent of the invention is considered here as a pharmaceutically active ingredient for the treatment of cystic fibrosis. In another example, a pharmaceutically active protein can be used to treat a cell or individual that does not normally express the protein at all or at desired levels, or that does not express the protein correctly, e.g., the pharmaceutically active protein can compensate for mutations resulting in a lack of high enough expression, by supplying the desired protein. The term "pharmaceutically active peptide or protein" includes whole proteins or polypeptides, and may also refer to their pharmaceutically active fragments. It may also include pharmaceutically active analogs of the peptide or protein.

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

The terms "subject" and "patient" in the context of the present description refer to a mammal. For example, mammals in the context of the present invention are humans, primates, domesticated animals, including, but not limited to, dogs, cats, sheep, cattle, goats, pigs, horses, etc., laboratory animals, as well as mice, rats, rabbits, etc., as well as animals in captivity, such as animals kept in zoos. The terms "subject" and "patient" as used herein specifically include a human. The subject (human or animal) has two sets of chromosomes; that is, it is diploid. The term "patient" refers to a subject who suffers from a condition, is at risk of and is susceptible to a disease state, suffers from a condition, or is predicted to suffer from a condition, and who can be treated, for example, by administering an agent. The patient's condition may be chronic and/or acute. Thus, a "patient" can also be described as a subject undergoing and/or in need of therapy.

The term "therapy" should be understood in a broad sense, such as the application to a subject to prevent or treat a condition in the subject. In preferred embodiments, the implementation of therapy, in particular, includes the introduction of an agent to the subject.

As used herein, the term "trypsin" generally refers to a proteolytic enzyme classified as EC 3.4.21.4). Trypsin cleaves peptide chains mainly at the carboxyl end at the amino acids lysine or arginine, usually except when they are followed by proline. Without being limited by theory, it should be understood that trypsin is a serine protease and that trypsin is found naturally in the digestive system of many vertebrates, where it hydrolyzes proteins. In the present invention, trypsin from recombinant sources is preferred. Although trypsin is formed in vivo with a propeptide (referred to as "trypsinogen"), the term "trypsin" as used herein preferably refers to mature trypsin lacking any propeptide. The use of trypsin for proteolytic cleavage may also be referred to as "trypsin proteolysis" or "trypsinization", and proteins resulting from trypsin cleavage are referred to as "trypsinized".

A "variant" NGF precursor or polypeptide of SEQ ID NO: 3 or 4 refers to a polypeptide or protein whose amino acid sequence does not correspond to part of mature NGF (beta-NGF) or does not correspond to part of SEQ ID NO: 3 or 4, respectively; characterized by at least one mutation compared to a wild-type NGF precursor, such as wild-type pro-NGF or wild-type pre-pro-NGF; where the specified at least one mutation is preferably found at the N-terminus of the amino acid sequence of Mature NGF (beta-NGF). Thus, in the context of the present description, a "variant" of an NGF precursor or similar protein refers to a peptide or protein in which the prepeptide and/or propeptide is characterized by at least one mutation with respect to the amino acid sequence of the prepeptide and/or propeptide, without limitation. , for example, are those described in WO 2013/092776 A1 and US 2018/0086805 A1. For illustrative purposes, WO 2013/092776 A1 describes pro-NGF "variants" in which a furin cleavage site (wild type) is missing due to one or more specific mutations.

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 to include vectors encoding a peptide or protein for heterologous expression (such vectors may serve as templates for generating transcripts) and vectors that do not. Vectors of the first type will contain an open reading frame encoding a protein or peptide that will be expressed if the vector is present in the host cell. Although the type of vector chosen by one of skill in the art will depend on the type of host cell chosen by one of skill in the art, in a particular case, cloning vectors for all common host cells, including E. coli, are commercially available, and thus one of skill in of the art may choose a particular vector with full regard for the characteristics of the selected host cell.

The term "wild type" is used here to refer to a gene or protein that is usually found in nature, preferably in a healthy subject. A gene or protein that is not "wild type" is referred to here as "mutant" or "mutated" or the like. For illustrative purposes, SEQ ID NO: 1 shows the amino acid sequence of the wild-type human NGF precursor; SEQ ID NO: 2 shows the amino acid sequence of wild-type human NGF.

The present invention is based on several related discoveries which, taken together, led the inventors to various aspects of the invention, described individually below.

Agent of the present invention

The present invention relates to an agent for the treatment and/or prevention of a dermatological disease in a mammal. The agent may be used to administer to a subject as needed, for example, to alleviate the symptoms of a disease or disorder. In particular, the agent of the present invention is a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. Thus, the present invention specifically provides a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for use in therapy. Therapy usually includes the introduction of the specified polypeptide in the human or animal body, as described below.

According to the present invention, the polypeptides of SEQ ID NO: 3 and SEQ ID NO: 4 are presented as pharmaceutically active agents. In accordance with the present invention, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is intended for medical use, in particular for the treatment and/or prevention of a dermatological disease in a mammal. Optionally, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is from a recombinant source. Thus, the present invention also provides a recombinant polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for medical use as described herein.

The agent of the present invention, also referred to here as "polypeptide of SEQ ID NO: 3" or "polypeptide of SEQ ID NO: 4", will now be described in more detail. The term "polypeptide of SEQ ID NO: 3" and similar terms refer here to a polypeptide containing the amino acid sequence indicated in SEQ ID NO: 3 and/or an agent with equivalent biological activity. The term "polypeptide of SEQ ID NO: 4" and similar terms refer here to a polypeptide containing the amino acid sequence indicated in SEQ ID NO: 4 and/or an agent with equivalent biological activity. Thus, these terms also include functionally equivalent parts or analogs of such polypeptides. One example of a biologically equivalent portion of a polypeptide may be a domain or subsequence of a polypeptide of SEQ ID NO: 3 or a polypeptide of SEQ ID NO: 4 that includes a binding site allowing the domain or subsequence to exhibit substantially the same biological activity as the full-length polypeptide. SEQ ID NO: 3 or the full length polypeptide of SEQ ID NO: 4, or alternatively the gene encoding such a polypeptide. The term "substantially the same biological activity" refers to an equivalent portion or analogs of a 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 polypeptide analog would be a fusion protein that includes at least a portion of the amino acid sequence of a polypeptide of SEQ ID NO: 3 or a polypeptide of SEQ ID NO: 4, as well as a homologous polypeptide analog. In addition, fully synthetic molecules that mimic the specific biological activity of the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 will also be "biologically equivalent analogues".

More preferably, the term "polypeptide of SEQ ID NO: 3" and similar terms here refer to a polypeptide containing the amino acid sequence specified in SEQ ID NO: 3; such agents are optionally fusion proteins comprising, inter alia, the amino acid sequence set forth in 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 set forth in SEQ ID NO: 3. SEQ ID NO: 3; in this embodiment, the agent consists of a polypeptide consisting of 118 amino acid residues in sequential order as set forth in SEQ ID NO: 3. In this and other embodiments, the polypeptide optionally bears one, two, or three internal cysteine bonds such that the residues cysteine (Cys, C) covalently linked to each other with the formation of intramolecular disulfide bridges. The cysteine bonds are preferably equivalent to those in wild-type human NGF.

Equally more preferably, the term "polypeptide of SEQ ID NO: 4" and similar terms here refer to a polypeptide containing the amino acid sequence specified in SEQ ID NO: 4; such agents are optionally fusion proteins that comprise, inter alia, the amino acid sequence set forth in SEQ ID NO: 4. in SEQ ID NO: 4; in this embodiment, the agent consists of a polypeptide consisting of 118 amino acid residues in the sequential order as indicated in SEQ ID NO: 4. In this and other embodiments, the polypeptide optionally bears one, two, or three internal cysteine bonds, such that the cysteine residues (Cys, C) are covalently linked to each other to form intramolecular disulfide bridges. The cysteine bonds of the polypeptide are preferably equivalent to those in wild-type human NGF.

The polypeptide of the present invention may optionally be characterized by additional post-translational modifications. Such post-translational modifications optionally include glycosylation and/or phosphorylation. However, preferably, the polypeptide of the present invention is not glycosylated and/or phosphorylated. Indeed, in view of the fact that the experimental examples presented here demonstrate a positive effect on the healing of skin diseases and an acceptable ratio of positive and negative effects, and with the understanding that the polypeptide used was obtained by cytosolic recombinant expression in bacteria, usually not leading to glycosylation and/or phosphorylation, there is a possibility that the beneficial effect of the product of the present invention is independent of this type of post-translational modification. Therefore, in preferred embodiments, the implementation of the polypeptide according to the present invention is not subject to glycosylation and/or phosphorylation.

Typically, a polypeptide of the present invention is a non-natural polypeptide that is not normally produced by the subject to which the polypeptide is administered. This not only provides an advantage in detecting the agent in the subject after administration, but also suggests that administration to the subject (from an external source, such as, for example, compositions prepared in accordance with the present description) may be necessary to achieve success in treatment or disorder prevention.

Preferably, the polypeptide of the present invention is a single (isolated) polypeptide. More preferably, the polypeptide of the present invention is substantially free of host cell proteins, degradation products (such as the des-nona variant, for example) and proteases (eg, trypsin). If a polypeptide of the present invention is substantially free of host cell proteins, degradation products (such as des-nona variant, for example), and proteases (eg, trypsin), then such a polypeptide may also be referred to as a "pure polypeptide". Preferably, the polypeptide of the present invention is administered in the form of a pure polypeptide. 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 percent of 90% or more, preferably 92% or more, more preferably 93% or more, more preferably 94% or more, more preferably 96% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more, more preferably 99.2% or more, more preferably 99.4% or more, more preferably 99.6% or more, more preferably 99.8% or more, more preferably 99.9% or more, based on the total protein in the composition. Such pure polypeptide is available in accordance with the disclosure in this document, including Examples 1 and 2. Most preferably, the pure polypeptide according to the present invention has a degree of purity that is compatible with good manufacturing practices (GMP, Good manufacturing practices).

As demonstrated in the experiments in this document, in particular in Examples 3 and 4, the administration of the agent of the present invention does not cause a hyperalgesic syndrome (pain), despite the fact that the agent was in direct contact with fully exposed nociceptive fibers (nerves); these are considered fully open due to lack of skin and hyperactivated as a result of skin lesions. The absence of pain under these extreme conditions is particularly remarkable as the agent has been applied topically and repeatedly to damaged skin, including in chronic conditions (details are shown in the examples). This is particularly noteworthy given the discouraging data from earlier studies with human NGF placed in contact with an innervated area, ie, an area characterized by exposed nociceptors (Svensson et al., 2003, Pain, vol. 104, p. 241-247). The above unexpected results cannot be explained solely by the fact that the agent of the present invention has previously been described as "painless", since its ability to cause pain in the innervated area, i. in the area of exposed nociceptors, not to mention hyperactivated nerves, as in the case of skin lesions, has never been experimentally investigated. In addition, even though the administration of the agent of the present invention causes tissue reinnervation (see, for example, Example 3), the administration itself is not associated with pain. In addition, the positive effect of the agent of the present invention on angiogenesis (see Experimental Examples) is unexpected and was not predicted based on prior art data. It is understood that angiogenesis is of particular importance for tissue formation and wound closure. Thus, the combination of these beneficial effects is highly unexpected in light of the current state of the art.

Optionally, according to 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. The details of the administration, the effective amount and the quality of the subject in need thereof are described below.

The polypeptides consisting of SEQ ID NO: 3 and SEQ ID NO: 4, respectively, differ in one or two positions from the amino acid sequence of human nerve growth factor (NGF, also referred to as wild-type NGF, see SEQ ID NO: 2). It is the difference between the polypeptide of the present invention and the polypeptide of SEQ ID NO: 2 that causes a significant effect on the treatment or prevention of skin diseases and the absence of side effects, as detailed herein and confirmed by experimental data in the examples.

Nerve growth factor (NGF) is a neurotrophin required for the development and survival of certain populations of neurons. NGF is a homodimeric peptide that naturally triggers neuronal proliferation and homeostasis. In the body, NGF binds to at least two types of receptors: the tropomyosin receptor kinase A (TrkA) and the low affinity neurotrophin p75 receptor for NGF (LNGFR/p75NTR/p75). Both are associated with specific disorders in humans and animals, although the respective mechanisms of action are likely to be different. Several therapeutic uses for NGF have been proposed, but few have been commercialized.

However, although many therapeutic applications of NGF have been proposed in the past, they have not been developed into commercially sold NGF therapeutic products, and the reason for this may be that NGF, in addition to its desired effect on neuronal proliferation and homeostasis, is associated with pain: it is able to cause hyperalgesia with local or systemic administration. (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, 501-505; McArthur, et al., 2000, Neurology, vol. 54, pp. 1080-1088; Svensson et al., 2003, Pain, vol. 104, pp. 241- 247; Ruiz et al., 2004, Brain Res., Vol. 1011, pp. 1-6). As a solution, mutant versions of NGF ("muteins") have been developed which are associated with reduced nociceptive activity ("painless NG") and which are characterized by at least one mutation in the NGF domain interacting with the TrkA receptor (WO 2008/006893 A1, Malerba et al PLOS One, 2015, vol 10, e0136425). However, such polypeptides are not yet available for sale to the public in pharmaceutically acceptable purity and have not been proposed or developed for the treatment or prevention of dermatological skin conditions, perhaps due to bias and the general negative experience of growth factor research in this therapeutic field in general.

According to the present invention, stability and thus long-term purity of the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 can be achieved and/or improved based on the aspects and embodiments described herein. Thus, the present invention not only makes available a new treatment or prevention of dermatological disorders, but also provides an agent suitable for such treatment or prevention in a purity suitable for therapeutic use, including administration to a mammal. The agent of the present invention has not previously been available to the general public with such an effective degree of purity.

The polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 does not occur in nature and may also be referred to as a non-natural polypeptide. Thus, the agent of the present invention is not a wild-type NGF, and in particular is not a human wild-type NGF.

Preferably, the non-natural polypeptide of the present invention is of high purity. The polypeptide optionally contains internal disulfide bridges. The polypeptide is optionally correctly folded. The polypeptide is optionally soluble in an aqueous medium.

The present invention is based in part on experiments with two animal models of skin ulcers. In these models, skin ulcers are induced in diabetic mice by circular biopsy or pressure cycles and the polypeptide of the present invention is applied topically. The polypeptide produced a significant and dose-dependent improvement in ulcer healing time compared to placebo-treated animals. This improvement was evident at doses devoid of side effects associated with pain, and thus demonstrates a potential advantage over the current state of the art.

In particular, data from in vivo models of diabetic skin ulcers have demonstrated that the polypeptide of the present invention is painless but retains activity directed at the NGF receptor system and thus is a therapeutic agent for the treatment or prevention of dermatological diseases. Indeed, the polypeptide of the invention retains the angiogenesis and reinnervation trophic properties of wild-type NGF, which promotes ulcer healing without inducing the pro-nociceptive effects of wild-type NGF at the topical and systemic level.

The present invention provides a polypeptide for use in the treatment and/or prevention of dermatological diseases in a mammal, the polypeptide being selected from the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4. Thus, the present invention also relates to the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for use in a method of treating a human or animal through therapy as described herein.

More specifically, the present invention relates to a specific therapeutic use of the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4, where the specific therapeutic use is the treatment and/or prevention of a dermatological disease in mammalian subjects. Thus, the present invention also provides a polypeptide of SEQ ID NO: 3 and a polypeptide of SEQ ID NO: 4 for use in a method of treating a human or animal through therapy, wherein the therapy comprises treating and/or preventing a dermatological disorder in a mammal. A mammalian subject is typically one in need of such treatment.

The polypeptide of SEQ ID NO: 3, as well as the polypeptide of SEQ ID NO: 4, are characterized by a mutation in the amino acid sequence of human NGF (hNGF, SEQ ID NO: 2), while this mutation is associated with a decrease in the intensity of nociceptive action. In particular, the arginine at position 100 of hNGF is replaced by glutamic acid. The present invention is based in part on the unexpected discovery that a therapeutic effect can be achieved without the side effects known in the prior art.

Without wishing to be bound by any particular theory, it is preferred that the polypeptide of the present invention contains one or more disulfide bridges, most preferably three disulfide bridges. Mature and properly folded human NGF is characterized by three disulfide bridges (when bound at positions 136 ↔ 201, 179 ↔ 229, 189 ↔ 231, where position numbering refers 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 polypeptide of the present invention comprise equivalent disulfide bridges (wherein the linking position numbers are clear to the skilled artisan when aligning the polypeptide of the present invention with the polypeptide of SEQ ID NO: 1 and Wiesmann et al., see . higher.)

Description of the presence and absence of side effects

Preferably, the treatment and/or prophylaxis does not cause side or undesirable effects in the subject to which the polypeptide is or has been administered. One side effect or side effect that is preferably not present in this context is hyperalgesia or pain. Thus, preferably, the administration of the agent of the present invention does not cause a hyperalgesic syndrome (pain).

It is important to note that the absence of pain does not merely provide a more pleasant (or less unpleasant) treatment than administration of a reference pain-associated compound (e.g., wild-type NGF), but is at least partly responsible for the success of treating or preventing skin diseases as such: considering that the polypeptide of the present invention is preferably administered topically, more preferably administered topically to a site affected by a skin disease (e.g., ulcers), the absence of pain allows the subject to be treated to accept the administration of the polypeptide to the surface of the body without adverse reactions such as scraping, rinsing or otherwise removing it to relieve pain, whereby the polypeptide will continue to act on the affected surface of the body, providing a therapeutically beneficial effect, for example, in the treatment or prevention of a skin disease. Thus, the absence of pain associated with the polypeptide of the present invention would be an appropriate condition to overcome consumer resistance and regulatory concerns. In other words, the absence of pain is associated with a significant increase in the benefit/risk ratio compared to agents associated with pain.

In particular, it is preferred that the treatment and/or prophylaxis does not cause hyperalgesia in the mammal. 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 a subject to which a polypeptide of the invention is administered, so that a subject to which a polypeptide is administered does not suffer from mechanical allodynia.

In one embodiment, the subject to which the polypeptide of the invention is administered does not suffer from thermal allodynia. More precisely, thermal allodynia is not induced in a subject to which a polypeptide of the invention is administered, so that a subject to which a polypeptide is administered does not suffer from thermal allodynia.

Another side effect or undesirable effect, which is preferably not present in this context, is a malignant neoplasm or cancer. In particular, the 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 not only with abnormal cell growth, but also with the possibility of penetration or spread of cells to other parts of the body. It is especially preferred that the administration of the polypeptide of the present invention to a subject is preferably not associated with skin cancer, in particular of the dermis or epidermis. In this regard, the treatment or administration according to the present invention is associated with significant advantages over the prior art, for example over commercial treatment with platelet-derived growth factor (beclapermin, brand name Regranex). Thus, the absence of malignancies associated with the polypeptide of the present invention is expected to help overcome consumer resistance and regulatory concerns. In other words, the absence of malignancy is associated with a significant increase in the benefit/risk ratio compared to that of agents associated with the risk of malignancy.

Thus, it is preferred that administration of a polypeptide of the present invention to a subject is not accompanied by side effects such as cancer and/or pain.

Generally, administration of an agent of the present invention is well tolerated by the subject. Particularly preferably, the administration of the polypeptide of the present invention does not result in the formation of anti-drug antibodies in the subject. Indeed, since the amino acid sequence of the polypeptide of the present invention differs in only one or two amino acid positions from wild-type human NGF, it is likely that immunological tolerance in humans is a particularly attractive factor, and it is likely that administration of the polypeptide of the present invention does not induce the formation of antibodies against drugs in humans.

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

Polypeptide detectability

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

In this regard, the polypeptide according to the present invention is preferably detected by an antibody or other immunoreactive molecule.

A protein detected by an antibody or other immunoreactive molecule may also be referred to as an antigen. In some embodiments, the implementation of the biological sample may be characterized by the presentation - or lack of presentation of one or more specific antigens. In the context of the present invention, the polypeptide administered to a subject is preferably found in a biological sample obtained from the subject after administration of the polypeptide. One non-limiting way to demonstrate the presence of a protein is Western blot, but other immunological methods are equally included in the context of the present invention. The antibody or other immunoreactive molecule is either itself labeled (eg, with a fluorophore) or is recognized by a labeled secondary antibody or other immunoreactive molecule that is added for that purpose. Thus, in some cases, a secondary detection aiding molecule, for example optionally a labeled secondary antibody, is also added to facilitate detection.

According to the invention, an antigen is considered present in a biological sample if its level is above the detection limit and/or if the level is high enough to allow binding by antigen-specific antibodies added to the sample. According to the invention, an antigen is considered unexpressed in a cell if the expression level is below the detection limit and/or if the expression level is too low to allow binding by the antigen-specific antibodies added to the sample.

An antibody or other immunoreactive molecule can recognize an epitope on a cell surface. The term "epitope" refers to an antigenic determinant of a molecule, such as an antigen, i. to a portion or fragment of a molecule that is recognized by, ie bound to, the immune system, for example, that is recognized by an antibody or other immunoreactive molecule. The detection of an epitope specific for any particular antigen will usually lead to the conclusion that that particular antigen is present in the analyzed cell.

In one embodiment, a sample obtained from a subject, in particular a subject to whom a polypeptide of the present invention has been administered, can be characterized by immunophenotyping. "Immunophenotyping" generally means that a cell or sample can be characterized by antigen-specific molecules, such as antibodies or other immunoreactive molecules, which are added to the sample to determine the presence of an antigen. Immunophenotyping involves sorting cells using a variety of techniques, including flow cytometry, as well as methods for analyzing lysed cells and lysed samples, such as Western blotting.

Particularly preferred in the present invention is a polypeptide that can be specifically detected even in the presence of wild-type NGF, eg, wild-type human NGF. While any amino acid sequence mutation, such as, for example, any point mutation, can make a polypeptide specifically detectable even in the presence of the corresponding wild-type wild-type polypeptide, therefore, each polypeptide of SEQ ID NO: 3 and each polypeptide of SEQ ID NO: 4 can be , prima facie are specifically detected even in the presence of wild-type human NGF, in particular the polypeptide of SEQ ID NO: 4, for which an antibody is already available capable of distinguishing said polypeptide from wild-type human NGF (WO 2008/006893 A1).

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

Affected body surface

In accordance with the present invention, the polypeptide of the invention is administered to the affected body surface.

Body surfaces affected include, but are not limited to, ulcers (venous ulcers, arterial ulcers, bedsores, diabetic ulcers), postoperative wounds, bedsores, burns, tears, incisions, bruises, abrasions, puncture wounds), and the like. Subjects having such ​body surface lesions will be described below, and the following description of the affected body surface applies to all such subjects unless the context clearly dictates otherwise.

In one embodiment, an agent of the present invention is provided for the treatment and prevention of a skin disease, the skin disease being selected from ulcers, postoperative wounds, bedsores, burns, lacerations, incisions, bruises, abrasions, and puncture wounds.

In one embodiment, an agent of the present invention is provided herein for the treatment and prevention of ulcers, wherein the ulcer is selected from venous ulcers, arterial ulcers, decubitus ulcers, and diabetic ulcers.

In some embodiments, the implementation of the surface of the affected body has a diameter of 1 mm or more. In general, when reference is made herein to "diameter" of the affected body surface, for non-circular affected body surfaces, the term "diameter" refers to the largest diameter of the affected body surface measured from one border of the affected body surface across the affected surface to the opposite border of the affected body surface. . For round affected body surfaces, the diameter is, of course, the same in any direction of measurement from one border of the affected surface through the affected surface to the opposite border of the affected body surface. The diameter can be determined with a ruler or other suitable instrument on the outer surface of the affected body surface.

In some embodiments, the affected body surface has a diameter of 1 mm to 50 cm. In some embodiments, the affected body surface has a diameter of 2 mm to 20 cm. The affected body surfaces are 0.5 cm or more in diameter, preferably 1 cm or more, also may be referred to here as "large" affected body surfaces. The present invention is also suitable for the treatment of large diseased body surfaces such as large ulcers (see, for example, Example 4). In some embodiments, the affected body surface has a diameter of 3 mm to 10 cm. In some embodiments, the affected body surface has a diameter of 4 mm to 5 cm. In some embodiments, the affected body surface has a diameter of 5 mm to 4 cm. In some embodiments, the affected body surface has a diameter of 5 mm to 4 cm. embodiments, the affected body surface has a diameter of 6 mm to 3 cm. In some embodiments, the affected body surface has a diameter of 7 mm to 1 cm. In some embodiments, the affected body surface has a diameter of 8 mm to 1 cm. In some embodiments, the implementation of the affected body surface has a diameter of 8 mm to 1 cm. the affected surface of the body has a diameter of about 6 mm. In some embodiments, the affected body surface is about 12 mm in diameter.

Subjects for which the agent of the present invention is particularly suitable

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 an administration. A subject in need of such an administration may be a subject diagnosed with a disorder as described herein, a subject at risk for such a disorder, or otherwise suffering from such a disorder. The agent is administered to the subject in a therapeutically effective amount. Therapeutically effective amount can be determined by the physician in view of the present description.

In particular, the polypeptide according to the invention is administered to a mammal. A subject may also be referred to as a "patient". Most preferably, the mammalian subject is a human.

The present invention also relates to a method of treating a patient suffering from a dermatological disease, wherein the method comprises administering to the patient an effective amount of a polypeptide of SEQ ID NO: 3 or a polypeptide of SEQ ID NO: 4. The terms "patient" and "subject" are used interchangeably herein, in particular in relation to to a patient/subject characterized by a dermatological disease as described herein.

Preferably, the dermatological disorder is characterized by a damaged surface on at least a portion of the subject's body. Preferably, the dermatological disease is characterized by the affected surface (affected body surface). Affected body surfaces have been described, for example, above, and the following description applies to all such body surface lesions of subjects unless the context dictates otherwise.

More preferably, the dermatological disorder is or includes a skin lesion, preferably a skin lesion characterized by at least partial ablation of the dermis and optionally the dermis. In one embodiment, the affected body surface is or contains a lesion, in particular a skin lesion.

Although terms such as "dermatological disease", "wound", "damaged body surface", "chronic lesion", "ulcer" and other terms are used here in the singular, the present invention is also applicable to subjects suffering from multiple dermatological disorders, wounds , or having affected body surfaces, chronic wounds, ulcers and other similar diseases.

Preferably, the affected body surface contains at least one chronic lesion. Thus, the administration of an agent of the present invention is suitable for the treatment or prevention of at least one chronic lesion. In the context of the present invention, the term "chronic lesion" should be understood in a broad sense and includes, without limitation, ulcers of all types, whether they are explicitly mentioned in this disclosure or not, bedsores, burns, and mechanical ablations of the skin. In particular, this term includes wounds that do not heal within the time periods typical for healing in healthy subjects of the corresponding species. In addition, all wounds on the subject's body surface that have not healed and/or closed for seven days or more, such as 14 days or more, 21 days or more, 1 month or more, or one year or more, classified as "chronic". The agent of the present invention can be administered for all such types of chronic lesions to treat or prevent such chronic lesions.

In the context of the present invention, the term "prevent" should be understood in a broad sense, where it includes not only preventing the onset of the disorder, but also preventing the progression of the disorder. In particular, in the context of an affected body surface, such as a chronic wound, such as an ulcer, the term "prevent" also includes preventing a further increase in the area of the affected body surface, for example, further deepening the wound surface of the body and/or increasing the diameter of the affected body surface.

In the context of the present invention, the term "treat" should be understood broadly. The term includes, without limitation, reduction of the symptoms of a disorder. Indeed, it is preferable that the achievement of an improvement in a dermatological disorder, such as, for example, in the case of (partial) wound closure, as demonstrated by experimental examples, is a preferred integral part of the invention claimed here. Indeed, the achievement of the claimed therapeutic effect is a functional technical feature of the present invention. The examples given here demonstrate that the specified functional performance is achievable as a direct result of the introduction of the polypeptide of the present invention. In other words, the authors of the present invention have found that the polypeptide of the present invention is the cause of improvement in a subject suffering from a dermatological disease. The dermatological disease is preferably characterized by a damaged body surface.

The present invention is particularly suitable for subgroups of subjects suffering from a dermatological disease. Such subgroups are described here. It is also possible that a particular subject falls into one or more of the subgroups described herein; and administering a polypeptide of the present invention to subjects falling within one of the subgroups described herein is equally within the scope of the present invention as "administering a polypeptide of the present invention to subjects falling within more than one of the subgroups described herein."

The invention is not limited to specific causes of damage to the surface of the body. For example, diabetic causes as well as non-diabetic causes are included in the invention.

The surface of the lesion may be on one or more parts of the body. Affected body surfaces on extremities such as hands (including fingers) and legs (including feet) are preferred, but affected body surfaces on the trunk or head or other parts of the body may also be treated with a polypeptide of the invention. In some embodiments, the affected body surface is on the leg or foot, and more preferably on the foot. In such embodiments, diabetic patients are frequently encountered, but administration to such a particular affected body surface is not limited to diabetic patients.

In some embodiments, the implementation of the polypeptide according to the present invention is intended for administration to a subject who has undergone surgery. Accordingly, the polypeptide of the present invention is suitable for the treatment or prevention of one or more postoperative complications such as pressure sores and/or for the treatment of surgical wounds.

Preferably, the affected body surface contains at least one ulcer. According to the present invention, the polypeptide can be administered (applied) to at least a portion of the affected body surface. The term "at least one part" as used herein includes any ratio from 0 to 100%, such as 10 to 90%, 20 to 80%, 30 to 70%, 40 to 60%, and about 50%; thus, the polypeptide can be administered to the entire surface of the lesion or to any part of it. Optionally, the administration also includes an area of skin adjacent to the affected body surface.

Preferably, the dermatological disease includes at least one ulcer. According to the present invention, the polypeptide can be administered to at least a portion of one ulcer. "At least one part" as used herein includes any ratio from 0 to 100%, such as 10 to 90%, 20 to 80%, 30 to 70%, 40 to 60%, and about 50 %; thus, the polypeptide can be administered to the entire surface of the ulcer or to any part of it. Optionally, the administration also includes an area of skin adjacent to the ulcer.

Diabetes mellitus is a common and debilitating disease that affects many organs, including the skin. It is currently estimated that thirty to seventy percent of patients with both type 1 and type 2 diabetes will have skin complications of diabetes at some point in their lives. Regardless of such theoretical considerations, which in no way limit the present invention, methods for detecting diabetes are well known in the art. Methods for detecting diabetes, in one embodiment, are not part of the present invention, but they are helpful in identifying a subset of subjects at risk for a dermatological disorder such as those described herein and may be helpful in the treatment or prevention of such a dermatological disorder in accordance with the present invention. invention. In some embodiments, the agent of the present invention is for administration to diabetic patients suffering from neuropathy, such as, in particular, peripheral neuropathy. Methods for detecting neuropathy and predicting the development of foot ulcers in people with diseases such as diabetes mellitus are known (for example, described in WO/2010/128519 A1, without limitation).

The polypeptide of the invention can be administered to the affected skin of a diabetic subject, preferably to skin lesions characterized by at least partial ablation of the dermis and optionally (only) the dermis of such a subject. In one embodiment, the affected body surface is or includes a lesion, in particular a skin lesion, of such a subject. Preferably, administration comprises administering intravenously to an ulcer, in particular a foot ulcer, in a diabetic patient.

Diabetic ulcers, in particular diabetic foot ulcers, are a serious complication of diabetes mellitus. In the context of the present invention, the term "diabetic ulcer" is not specifically limited, except that an ulcer is an ulcer in a diabetic subject. According to some estimates, diabetic patients may have a high risk of non-traumatic amputation compared to non-diabetics, five to fifteen times higher (eg, WO/2003/075949 A1). Left untreated or without successful treatment, diabetic foot ulcers can be difficult to treat in some subjects and may even require amputation, especially if they are accompanied by other complications or disorders such as infection. Indeed, diabetes can affect several organ systems. The dermatological manifestations of diabetes mellitus have a variety of health consequences, ranging from those related to aesthetics to those that, if left untreated, can become life-threatening. The dermatological consequences of diabetes mellitus are described, for example, in 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 consequences of diabetes.

In some embodiments, the polypeptide of the present invention is for administration to a diabetic patient undergoing surgery. Accordingly, the polypeptide of the present invention is suitable for the treatment or prevention of one or more postoperative complications such as pressure sores in a diabetic patient and/or for the treatment of surgical wounds.

In general, wounds other than diabetic ulcers, especially diabetic foot ulcers, as well as decubitus ulcers and extensive/deep surgical wounds, can be difficult to heal even with medication, probably due to the large size of the lesions. If such wounds are not treated in time, they will worsen and subsequently become incurable and life-threatening. The present invention provides treatment and/or prevention of such dermatological manifestations of diabetes. Indeed, according to the present invention, effective medical treatment can not only help patients get rid of these skin complications, but can also provide them with a better quality of life, reduced medical costs, or even increased life expectancy.

The present invention is also suitable for the treatment of affected body surfaces, in particular large ulcers, in diabetics and non-diabetics. In some embodiments, the implementation of the present invention is suitable for the treatment of large lesions of the body with a diameter of 5 mm or more, such as 1 cm or more. Additional details of the affected body surface, including certain variants of the diameter of the affected body surface, are described above.

Thus, the present invention provides an advantage over current treatments, which often cannot provide an effective way to treat large wounds. The present invention provides treatment and/or prevention of such dermatological manifestations, including large wounds, in diabetic and non-diabetic subjects.

According to the present invention, the polypeptide is suitable for the treatment or prevention of pressure ulcers, including chronic pressure ulcers. Specifically, pressure sores (pressure injuries) include pressure sores in various stages and pressure sores of various types.

In preferred embodiments, the agent of the present invention is for the treatment or prevention of an ulcer and is administered to an ulcer for this purpose. According to the present invention, the ulcer into which the polypeptide is administered is preferably selected from the group consisting of diabetic ulcers, traumatic ulcers, surgical ulcers, decubitus ulcers, chronic ulcers, and combinations of any of the above. In particular embodiments, the ulcers are selected from diabetic trauma ulcers, diabetic surgical ulcers, diabetic decubitus ulcers, diabetic chronic ulcers, traumatic diabetic ulcers, traumatic surgical ulcers, traumatic decubitus ulcers, traumatic chronic ulcers, chronic surgical ulcers, chronic decubitus ulcers, and other ulcers. In some embodiments, the ulcers are selected from traumatic ulcers, surgical ulcers, decubitus ulcers, and chronic ulcers of subjects with diabetes. In some embodiments, ulcers are selected from traumatic ulcers, surgical ulcers, decubitus ulcers, and chronic ulcers in non-diabetic subjects.

The present invention is not limited to the treatment of subjects with a single ulcer or subjects with multiple ulcers. Among subjects suffering from multiple ulcers, the present invention is not limited to treating only one of these ulcers, nor to treating a certain number of these ulcers, nor to treating all of these ulcers. Thus, the terms "ulcer" and "ulcers", whether used in the singular or plural in this disclosure, are expressly inclusive of all of these embodiments and are not limited to any particular number of ulcers in a subject, or any particular number of ulcers, to be treated.

It has been found that diabetic foot ulcer may be associated with either neuropathy (neuropathic ulcer) or peripheral vascular disease (ischemic ulcer) or both (neuroischemic ulcer), although the ultimate etiopathogenetic pathway may involve a combination of these underlying risk factors and others. causative factors such as trauma. Thus, in one embodiment, the polypeptide of the invention is for the prevention and/or treatment of ischemic ulcers, including ischemic foot ulcers. In an alternative embodiment, the polypeptide of the invention is for the prevention and/or treatment of neuropathic ulcers, including neuropathic foot ulcers. Finally, the polypeptide of the invention can be used for the prevention and/or treatment of neuroischemic ulcers, including neuroischemic foot ulcers. All of the above ulcers are not necessarily diabetic ulcers, although this is not a requirement.

In some embodiments, a polypeptide of the present invention is administered to a subject suffering from ischemia. Ischemia can be local or systemic. In some embodiments, administration according to the present invention may reduce ischemia in a subject. Reduction of ischemia may be local or systemic.

In some embodiments, a polypeptide of the present invention is administered to a subject suffering from neuropathy. In preferred embodiments, the agent of the present invention is for administration to a subject suffering from neuropathy, such as, in particular, peripheral neuropathy. Such subjects may be diabetic or non-diabetic. Indeed, the majority of diabetic ulcer patients have been reported to have underlying neuropathy (Ndip et al., 2012, Int. J. Gen. Med., Vol. 5, p. 129-134). Thus, in a preferred embodiment, the polypeptide of the present invention is administered to a diabetic subject suffering from neuropathy. Neuropathy can be local or systemic. In some embodiments, the implementation of the introduction according to the present invention can reduce neuropathy in the subject. The reduction in neuropathy may be local and/or systemic. In one embodiment, the reduction of neuropathy includes the reduction of neuropathy in the area in which the polypeptide of the present invention is introduced. In one embodiment, the reduction of neuropathy includes the reduction of neuropathy in the organ into which the polypeptide of the invention is administered.

Treatment or prophylaxis in accordance with the present invention may be carried out by administration, preferably topical administration, of the polypeptide of the invention. In some embodiments, the implementation of the introduction is carried out in a hospital (hospital). In some embodiments, treatment is not carried out in a hospital.

Optionally, but without mutual exclusion, a dermatological disease includes at least a burn or mechanical injury. Thus, the present invention also includes the treatment or prevention of burns and mechanical injuries, and the treatment of such injuries is practically more important than prevention.

Preferably, the mammal to which the polypeptide of the invention is administered, preferably a human, is diabetic or predisposed to diabetes mellitus; the corresponding subject is referred to here as a "diabetic subject". In exemplary embodiments, diabetes mellitus is selected from type 1 diabetes and type 2 diabetes.

Foot ulcers and other dermatological conditions are common in diabetic patients. Other dermatological disorders can also be treated and/or prevented based on the present invention. One of the main causes of foot ulcers in patients with diabetes is neuropathy (nerve damage), which makes it difficult for a person to identify foot injuries such as cuts, bruises, and pressure.

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). Indeed, foot ulcers and their associated complications are common in diabetic patients, most of whom have underlying neuropathy (Ndip et al., 2012, Int. J. Gen. Med., Vol. 5, p. 129-134). These ulcers are also called diabetic neuropathic foot ulcers. Thus, preferably, the polypeptide is administered to a subject with a diabetic neuropathic foot ulcer.

Optionally, the administration of the polypeptide of the present invention also includes the aspect of improving the aesthetic appearance of the subject, in particular the surface of the subject's body. In some embodiments, wound closure is achieved by administration in accordance with the present invention. In some embodiments, scarring is minimal. Thus, administration in accordance with the present invention also provides a cosmetic benefit to a treated subject compared to untreated subjects. Therefore, the present invention also provides a method for cosmetically treating a subject, wherein the method comprises administering a polypeptide of SEQ ID NO: 3 or 4.

In another embodiment, the agent according to the present invention is for the treatment or prevention of skin cancer, for example, hemangioma, and/or skin diseases associated with such cancers, without limitation.

In a further embodiment, the agent of the present invention is for the treatment or prevention of a dermatological disorder that results from or is affected by a genetic disorder in a subject.

Agent Introduction

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

Preferably, the polypeptide is for topical use. Thus, preferably, the polypeptide of the invention is administered to the skin or, if the skin is damaged or absent, to the surface of the body at a location where the skin would be present if it were not damaged or absent. In some embodiments, the polypeptide is administered to the epidermis. In some embodiments, the polypeptide is administered to the dermis. In some embodiments, the implementation of the polypeptide is introduced into tissue, which is usually located under the epidermis, for example, in the subcutaneous region, without limitation.

More preferably, the polypeptide is administered to the affected body surface. In other words, the polypeptide of the present invention is preferably administered topically, more preferably administered topically at the site of a skin disease (eg, ulcer).

The introduction according to the present invention is usually not associated with surgery. In one embodiment, the administration of a polypeptide of the invention is not a surgical intervention and does not include an invasive step that is a significant physical intervention in the body that requires professional medical knowledge and that entails a significant risk to health, even when carried out with the required professionalism and experience. . In contrast, in more typical embodiments, administration of a polypeptide of the invention, in particular topical administration, is generally considered safe for the subject, and therefore the polypeptide can be administered by the subject himself, especially in the case of a human subject.

Optionally, the ulcer is closed with a bandage before and/or during and/or after administration. The vast variety of types of dressings available is not limited to the present invention. Thus, any dressing can be used, as long as it is not clearly technically inappropriate. In some embodiments, the implementation of the polypeptide according to the invention is administered simultaneously with the imposition of a dressing on the wound; optionally, the wound dressing contains a polypeptide of the invention, optionally in the form of an aqueous medium applied to the dressing prior to administration.

Preferably, the polypeptide is administered (applied) to a subject with a foot ulcer on said subject's foot below the ankle.

In one embodiment, the polypeptide is administered once.

In an alternative and more preferred embodiment, the polypeptide is administered repeatedly. In a particularly preferred embodiment, the polypeptide is administered repeatedly from one to five times a day. In one embodiment, the polypeptide is administered once a day (see also Example 3). In one embodiment, the polypeptide is administered twice daily (see also Example 5). In one embodiment, the polypeptide is administered three times a day. In one embodiment, the polypeptide is administered four times a day. In one embodiment, the polypeptide is administered five times a day. It is particularly preferred that the polypeptide be administered to a human twice daily. All of the above introductions are preferably repeated within a few days, as described in this document. For example, the polypeptide can be administered repeatedly over a period of three to 30 days, preferably seven to 14 days, and preferably one to five times on each of those days.

In one embodiment, the polypeptide is administered repeatedly until the affected body surface is covered. Alternatively, the polypeptide is administered once and the administration is stopped after that single administration. Alternatively, the polypeptide is administered repeatedly over a period of three to 30 days, preferably seven to 14 days. Optionally, the introduction is stopped after the specified interval.

In some embodiments, the agent is administered such that it comes into direct contact with the nociceptive fibers (nerves). In some embodiments, the agent is administered such that it is in direct contact with fully exposed nociceptive fibers (nerves); nociceptive fibers (nerves) are considered to be completely exposed in the absence of skin, which is typical of the affected body surface. In some embodiments, the agent is administered such that it comes into direct contact with hyperactivated nociceptive fibers (nerves); nociceptive fibers (nerves) are thought to be hyperactivated as a result of the skin lesion. In some embodiments, the agent is administered such that it comes into direct contact with hyperactivated nociceptive fibers (nerves). Preferably, in particular, in such embodiments, the agent does not cause a hyperalgesic syndrome (pain). Previously, an agent with such properties has not been made available to the medical community. For this and other reasons, the present invention has a significant advantage.

Dose

The agents and compositions described herein are administered in effective amounts. In accordance with the present invention, an "effective amount" is that amount or dose that achieves the desired response or desired effect, either after a single dose or in combination with additional doses. In the case of treating a particular disorder, the desired response is preferably related to the inhibition of the course of the disease. This includes slowing down the progress of the disease and preferably interrupting or regressing the disease. The desired response in the treatment of a disease or condition may also include delaying the onset of the disease or preventing said disease or said condition. In some embodiments, the desired response includes complete healing of the symptoms of the disorder locally and/or systemically.

The effective amount of an agent or composition 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, such as age, physiological state, comorbid condition (if any), height and weight, duration of treatment, type of concomitant therapy (if any), specific route of administration, and other parameters. Accordingly, the administered doses of the agents described herein may depend on various such parameters. In the event that the patient's response to the initial dose is insufficient, higher doses (or, in fact, higher doses achieved with a different, more localized route of administration) can be used.

According to the present invention, suitable and practically effective dosages for administering a therapeutic agent to a human subject for the treatment and/or prevention of a skin disease such as chronic skin ulcers and burn wounds can be determined based on experimentally determined suitable and therapeutically effective doses for administering a therapeutic agent to a subject. in a rodent, in particular a mouse, for the treatment and/or prevention of a skin disease such as chronic skin ulcers and burn wounds. Guidance is available in the "Guidance for Industry Chronic Cutaneous Ulcer and Burn Wounds - Developing Products for Treatment" published by the U.S. Food and Drug Administration, 2006 (Guidance for Industry Chronic Cutaneous Ulcer and Burn Wounds - Developing Products for Treatment”, published by U.S. Department of Health and Human Services Food and Drug Administration, 2006).

Animal models of skin lesions (wounds) (Examples 3 and 4) will help to establish pharmacological reactions, as well as to evaluate the potential toxicity of products in the treatment of wounds. In some embodiments, the dose to be administered to the subject is the dose described in Example 3, or in Example 4, or in Example 5.

Preferably, the dose of the administered polypeptide is determined based on the area of the affected body surface to be treated. Preferably, the determination is carried out at the beginning of treatment. In one embodiment, the dosage is adjusted for later administration(s) depending on the surface area of the affected body part at the time of such later administration(s). In an alternative embodiment, dosing is not adjusted for later administration(s) so that the dose of administration depends solely on the size of the affected body surface area to be treated at the start of administration (first dosing) and subsequent doses correspond to the first dose.

In one embodiment, the dose/each dose is from 0.3 to 6 μg of the polypeptide per mm ² of the treated surface of the affected body (from 0.3 to 6 μg/mm ² ).

Optionally, in all embodiments according to the present invention, the dose is calculated based on the actual size of the affected body surface (eg, ulcer) at the time of treatment. In other words, the dose can be calculated (recalculated) at each time point of administration based on the actual size of the affected body surface (eg, ulcer) at that time point.

The process of obtaining a polypeptide

In one embodiment, the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 can be obtained from a biological source. Optionally, the polypeptide of SEQ ID NO: 3 and the polypeptide of SEQ ID NO: 4 can be obtained by recombinant expression. For this purpose, an open reading frame encoding the corresponding polypeptide is introduced into a recombinant protein producer, such as a host cell or a cell-free protein expression system. Indeed, given that human NGF is only produced in small amounts in vivo , murine NGF is typically produced as a heterogeneous mixture of different proteins (see WO 2000/022119 A1) and the polypeptides of the present invention are non-natural and therefore not produced in vivo at all. , the most significant opportunity for obtaining the polypeptide of the present invention provides recombinant expression in accordance with equivalent approaches for wild-type NGF prior art (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 of sufficient purity for administration to a mammal has been a persistent problem. This problem has been overcome by the inventors of the present invention, as detailed herein (see also Examples 1 and 2).

Preferably, the polypeptide according to the present invention can be obtained by recombinant expression in bacteria. More preferably, the polypeptide according to the present invention can be obtained by cytosolic recombinant expression in bacteria. In general, bacterial cells, in particular E. coli, are capable of recombinant production of a significant amount of recombinant proteins, but, as with 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 (the so-called inclusion bodies (IB)) (WO 2000/022119 A1; US2018/0086805 A1). Unlike NGF, pro-NGF is known to be a rather unstable protein and requires considerable effort to refold and purify at a low recovery rate, making the process of NGF production via pro-NGF in bacteria relatively complex and costly. Thus, the main difficulties associated with bacterially produced NGF and related bacterially produced polypeptides through the appropriate proforms relate to folding, processing and purification of the recombinant protein. These difficulties 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 a purity suitable for administration to a mammal, including a human.

Preferably, the polypeptide of the present invention is expressed together with the prosequence. Without limitation, a suitable prosequence is a wild-type human NGF prosequence (amino acid positions 18 to 121 of SEQ ID NO: 1), usually fused to the N-terminus of a polypeptide of SEQ ID NO: 3 or 4. For wild-type NGF, although the prosequence is not is part of mature NGF and therefore not required for the biological function of NGF, the presence of a covalently attached prosequence has been shown to promote refolding of recombinant NGF from inclusion bodies, with concomitant formation of a disulfide bond in the mature portion (beta-NGF). Thus, the presence of a covalently attached prosequence has a positive effect on the yield and rate of refolding compared to the in vitro refolding of mature NGF from inclusion bodies (Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, pp. 3296-3303 ). Without being bound by any particular theory, the same is plausible and is postulated here for the polypeptides of SEQ ID NOS: 3 and 4.

Thus, when a polypeptide of the present invention is produced in inclusion bodies, correct folding is required and this is usually achieved post-translationally, as is cleavage of the covalently attached prosequence; sophisticated folding, digestion and purification methods have been proposed in the past, in particular for wild-type human NGF. Notably, most of the published NGF studies use the general refolding regimen previously established by Rattenholl et al. (2001, Eur. J. Biochem, vol. 268, pp. 3296-3303). As part of this original study, several parameters of protein refolding (eg, temperature, refolding time period, pH of the refolding reaction, and concentrations of protein, arginine, and glutathione) were examined in detail and their influence on the efficiency of refolding was evaluated. The protocol of Rattenholl et al. based on re-naturation of a proform that has very poor solubility, can be obtained from inclusion bodies after recombinant production in prokaryotes, whereby pro-NGF is solubilized in a solution of a denaturing agent at a denaturing concentration, transferred to a solution that is not denaturing in principle, or slightly denatures so that solubility is maintained and thus dissolved denatured pro-NGF can assume a biologically active conformation, including disulfide bonding, as in native NGF, after which NGF is purified and the prosequence is removed proteolytically (WO 2000/022119 A1; Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, pp. 3296-3303). Notably, as part of this study, it was found that a low protein concentration resulted in a higher specific yield of properly folded product compared to a higher protein concentration. Approximate protein concentrations of about 50 mg per liter of the refolding reaction resulted in a specific yield of approximately 25% of correctly folded pro-NGF, while this fraction dropped to 10% at protein concentrations of 500 mg per liter. Based on this, Rattenholl et al. suggest that the protein concentration in the refolding solution should be very low: according to Rattenholl et al., a yield of 15-20 mg of correctly folded protein per liter of the refolding reaction is expected. However, it is necessary to scale up the process (eg, beyond laboratory scales) to purify even a few hundred mg of recombinant protein.

Although human pro-NGF contains a native cleavage site for the Furin protease (Arg ¹ -Ser ² -Lys ³ -Arg ⁴ ; R ¹ S ² K ³ R ⁴ ), and Furin cleaves pro-NGF at this site in vivo , furin is not commercially available in significant purity or quantity. According to the present invention, the polypeptide of the present invention, when expressed together with a prosequence, for example in E. coli, is preferably cleaved by the protease trypsin (EC 3.4.21.4), which is commercially available. Indeed, for wild-type NGF, trypsin has been reported to produce a satisfactorily biologically active mature NGF that can eventually be purified (Rattenholl et al., Eur. J. Biochem, 2001, vol. 268, p. 3296-3303) and proteolysis of recombinantly expressed pro-NGF by trypsin has meanwhile received support from other researchers (eg, D'Onofrio et al., 2011, PLoS One, vol. 6, e20839). However, it was later shown that the cleavage of wild-type pro-NGF by trypsin to form beta-NGF is associated with several disadvantages, since low amounts of trypsin can lead to inefficient cleavage, while high amounts of trypsin further reduce the selectivity of cleavage, since trypsin is able to cleave the C-terminus of any residue at arginine and lysine (R and K residues) so that pro-NGF containing R ¹ S ² K ³ R ⁴ can be digested with trypsin to produce several alternative cleavage products; thus, the use of trypsin as the cleavage enzyme would lead to very low yields of properly cleaved NGF, as well as purification and yield problems, since these various cleavage products are economically uneconomical to separate under standard conditions. One solution has been to express a pro-NGF variant where the R ¹ S ² K ³ R ⁴ protease cleavage site in the propeptide contains substitutions with other amino acids at least at positions R ¹ and K ³ corresponding to positions 101 and 103 of the pro- Wild type human NGF (SEQ ID NO: 1) (WO 2013/092776 A1). In one example, R ¹ and K ³ , respectively, are replaced by valine (V) and alanine (A), transforming the original furin cleavage site R ¹ S ² K ³ R ⁴ into V ¹ S ² A ³ R ⁴ , where trypsin is able to specifically cleave only C-terminal R ⁴ ; Trypsin-mediated cleavage of the corresponding pro-NGF may also be referred to as the "VSAR method". Although WO 2013/092776 A1 says nothing about the polypeptides of SEQ ID NO: 3 or 4 according to the present invention, the VSAR method was originally proposed for application to certain variants of pro-NGF muteins, although it was reported that proteolysis conditions need to be balanced with care (US 2018/ 0086805A1). During the development of the present invention, the authors of the present invention found that the VSAR technology, contrary to previous assumptions, does not satisfactorily solve the purification problems associated with obtaining a recombinant polypeptide of the present invention of a satisfactory degree of purity. Indeed, purification of recombinantly expressed beta-NGF or muteins thereof not only from host cell proteins (HCP) but also from trypsin (or other protease used for digestion) is still a problem; of course, a proteolytic enzyme (such as trypsin) is required to be absent from the final pharmaceutical protein preparation to avoid proteolysis during storage of the polypeptide, so that the polypeptide is kept substantially pure and without degradation until such time as it is administered to a subject according to the present invention. The authors of the present invention have solved this problem, as set forth in the description. Thus, the present invention makes the polypeptide according to SEQ ID NO: 3 or 4 available in high purity and thus substantially free of trypsin and/or degradation products of the polypeptide. Although certain methods for producing NGF (e.g., WO2013092776 A1) and a polypeptide of SEQ ID NO: 4 (e.g., Malerba et al., 2015, PLOS One, vol. 10, e0136425) have been described previously, the inventors of the present invention surprisingly found that previously published methods are not sufficient to obtain the corresponding high purity polypeptide. As a solution to this drawback, the authors of the present invention proposed a new process and related aspects, as described in detail in this document.

The method of obtaining a polypeptide of SEQ ID NO: 3 and a polypeptide of SEQ ID NO: 4 by recombinant expression, for example, in a host cell according to the present invention may include purification. Purification in the broadest sense means that the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 must be separated from other molecules, including other proteins, such as proteins of the host cell. Thus, purification may include separating the polypeptide from one or more other molecules, including other proteins such as host cell proteins, proteases (eg, trypsin), and/or degradation products of the polypeptide of the invention.

The method for producing a polypeptide of SEQ ID NO: 3 and a polypeptide of SEQ ID NO: 4 according to the present invention preferably includes the following steps:

(a) obtaining a precursor of the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4,

(d) cleaning,

where cleaning in stage (d) usually includes cleaning on the stationary phase in a mixed mode of the stationary phase. Thus, in one embodiment, a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 can be obtained by recombinant expression and purification, where the purification comprises a mixed stationary phase purification. The term "in stationary phase mixed mode" should be understood in a broad sense, where it means that a mixture containing a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 or a precursor of either, together with other types of molecules, is exposed to stationary phase in a mixed mode, for example at the stage of chromatography or other suitable technological stage. Indeed, preferably a mixture containing a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 or a precursor of either of these, together with other kinds of molecular compounds, is subjected to chromatography, such that purification in step (d) includes purification by mixed chromatography. Preferably, mixed mode chromatography involves the use of a stationary phase having a charged group, preferably a negatively charged group, an aromatic group and/or a hydrophobic group.

Purification in the broadest sense in accordance with the present invention means that the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 according to the invention is at least partially separated from other types of molecular compounds, including other proteins, such as proteins of the host cell , precursors and/or degradation products. As a result, a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 can be obtained that is at least partially purified. While other molecular entities may optionally be removed or saved, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is preferably recovered and kept purified.

Preferably, mixed mode chromatography involves the use of a stationary (stationary) phase having a charged group, preferably a negatively charged group, an aromatic group and/or a hydrophobic group.

Each of these stages may itself include several activities, which may also be referred to as stages for simplicity. For illustrative purposes and as detailed below, step (d) may include more than one purification step, such as on more than one stationary phase.

Any letter or number used here to refer to one or more process steps, e.g. (a), (b), (c), (d), (d1), (d2) should not be taken as a limitation, but rather as provided for your reference. It should not be understood that the sequence of events in the process or use according to the present invention may be limited to an alphabetical sequence (letters) or a numerical sequence (numbers). Notwithstanding the foregoing, it is strongly recommended that the sequence of events in the process or use of the present invention be that described herein.

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

Optionally, the polypeptide of SEQ ID NO: 3 or the polypeptide of SEQ ID NO: 4 can be obtained in a process that includes (re)folding and/or chromatographic purification and/or cleavage with a protease, and optionally bringing the protein to the final concentration and/preparation of the desired drug.

Thus, administration of a SEQ ID NO: 3 or SEQ ID NO: 4 polypeptide to a subject in need as disclosed herein is also possible due to the industrially significant degree of purification and yield of the SEQ ID NO: 3 or SEQ ID polypeptide. NO: 4, the implementation of which, based on the disclosure in this document, becomes possible for a person skilled in the art. Thus, the present disclosure also describes the process for obtaining a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4.

The method for producing a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 according to the present invention preferably includes 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) scrubbing, where scrubbing includes scrubbing in a stationary phase in a mixed mode.

In the present invention, it is also preferred that the precursor polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is subjected to the step

(c) exposure to protease.

Said exposure is usually carried out before step (d).

The method of the present invention is preferably also characterized in that no chromatographic purification is carried out prior to exposure to the protease. Indeed, the present inventors surprisingly found that protease digestion works well and efficiently also in the crude fraction derived from the host cell, ie. when chromatographic purification is not performed prior to exposure to the protease.

Preferably, step (a) comprises expression of the precursor polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4, preferably recombinant expression. More preferably, recombinant expression is carried out in a host cell. Upon completion of culturing the host cells, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is obtained in the cell culture fraction. The fraction may consist of host cells, i.e. in the case where the protein is essentially not secreted from the host cells. This is true, for example, when the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is produced in inclusion bodies and/or otherwise finds its way into an intracellular compartment, 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 Rosetta (DE3). In one embodiment, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is produced in a conformation other than the native conformation and/or in aggregates, most preferably in inclusion bodies. Then, preferably, the method of the present invention includes step (b) of (re)folding the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. Preferably, step (c) is performed after step (b).

Preferably, in step (c), the protease is a protease capable of cleaving the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 such that the (mature) polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is released. In a specific embodiment implementation of the specified protease is trypsin, preferably porcine trypsin, optionally expressed recombinantly.

Preferably, purification step (d) comprises the following steps, preferably in sequential order:

(d1) capture,

(d2) polishing.

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

Preferably, the polishing step (d2) is carried out by chromatography, preferably column chromatography. More preferably, said polishing step is carried out using stationary phase cation exchange chromatography. In an even more preferred embodiment, said capture step (d1) is carried out using SP-Sepharose, preferably SP-Sepharose with a small particle size. SP is an abbreviation for the name sulfopropyl.

Optionally, the method according to the present invention includes the additional step of bringing the protein to the final concentration and preparation of the desired drug. The result is a composition according to the invention.

In other words, the present invention provides a method for performing mixed mode chromatography to obtain a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4. Mixed mode chromatography is useful in obtaining a polypeptide of SEQ ID NO: 3 or SEQ. ID NO: 4. In preferred embodiments, the polypeptide precursor of SEQ ID NO: 3 or SEQ ID NO: 4 is exposed to a protease to cleave, and mixed mode chromatography is used in the step following the exposure to the protease. In preferred embodiments, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 is not chromatographically purified prior to said exposure to the protease.

Polypeptide purity

The polypeptide of the invention is largely or substantially free of the components that would normally accompany it in its native state. The polypeptide of the invention is isolated prior to administration. In one embodiment, an "isolated polypeptide" refers to a polypeptide that has been purified from the cellular and extracellular environment, such as the tissue that naturally surrounds it, e.g., isolated from the cell in which it was expressed, e.g., from a host cell. . In another embodiment, "isolated polypeptide" refers to a polypeptide isolated and/or purified in vitro , respectively, and to the isolation of such from its natural cellular environment, as well as purification from association with other components of the environment in which the polypeptide is normally found.

Preferably, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for use in the present invention is substantially free of impurities. Such predominantly pure polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 can be obtained as described in this document.

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

In a particularly preferred embodiment of the present invention, the polypeptide according to the present invention is obtained essentially free from degradation products of said polypeptide. In particular, the present inventors have observed that, in contrast to prior art reports of human wild-type NGF, exposure of the precursor of SEQ ID NO: 4 to trypsin characteristically results in partial C-terminal cleavage of said precursor at the arginine (Arg, R) Residue 9 of SEQ ID NO: 4, either before or after purification, if purification does not completely remove trypsin (des-nona variant, data not shown). Using a special purification method provided in the present invention, the polypeptide in accordance with the present invention can be obtained essentially free from trypsin and/or variant des-nona.

Preferably, the polypeptide obtained as described above is substantially free of degradation products. In particular, the present disclosure makes the polypeptide of the invention available in a novel, improved purity, and it is preferred that the polypeptide be administered at the same purity. Preferably, the polypeptide of the invention for use according to the invention is at least 90% pure. More preferably, the polypeptide of the invention for use according to the invention is at least 91% pure. More preferably, the polypeptide of the invention for use according to the invention is at least 92% pure. More preferably, the polypeptide of the invention for use in the invention is at least 93% pure. More preferably, the polypeptide of the invention for use according to the invention is at least 94% pure. More preferably, the polypeptide of the invention for use in the invention is at least 95% pure. More preferably, the polypeptide of the invention for use in the invention is at least 96% pure. More preferably, the polypeptide of the invention for use in the invention is at least 97% pure. More preferably, the polypeptide of the invention for use according to the invention is at least 98% pure. Even more preferably, the polypeptide of the invention for use according to the invention is at least 99% pure.

Most preferably, the polypeptide of the invention for use according to the invention is characterized by a purity of at least 99.0%, eg a purity of greater than 99.1%, greater than 99.2%, greater than 99.3%, greater than 99.4%. %%, over 99.5%, over 99.6%, over 99.7%, over 99.8%, over 99.9%.

Here, "purity" generally refers to the percentage (wt.) of the polypeptide of the present invention relative to the mass (wt.) of biological material other than the polypeptide of the present invention. To illustrate, typically at 99.0% purity, a polypeptide of the present invention is present in a relative amount (weight) of 99.0 units (e.g., 1.0 mg), and the sum of the weights of all biological materials except the polypeptide of the present invention, is 1.0 units (for example, 1.0 mg). Such biological material other than the polypeptide of the present invention includes, without limitation, host cell proteins, nucleic acids, proteases such as, for example, trypsin, inactivated or not, degradation products of the polypeptide of the invention, and other macromolecules of biological origin. In a specific embodiment, the degree of "purity" refers to purity compared to polypeptides other than the polypeptides of the invention. To illustrate, in this embodiment, at 99.0% purity, 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 that are not identical to the polypeptide of the present invention is 1 .0 units (for example, 1.0 mg). Degradation products of a polypeptide of the present invention are, for the avoidance of doubt, included in "polypeptides not identical to a polypeptide of the present invention". A specific degradation product is the des-nona variant (see Examples 1 and 2).

In particular, essentially do not contain des-nona variant of the polypeptide. The des-nona variant is a previously uncharacterized degradation product of a polypeptide of the present invention that is associated with the production of certain NGF variants, including the polypeptide of the present invention, except when the polypeptide is produced by the novel method described herein (see, for example, Examples 1 and 2). "Substantially free" in this context means that the polypeptide of the invention for use in accordance with the invention is characterized by a purity compared to the des-nona variant of more than 99.0%, for example, a purity of more than 99.1%, more than 99, 2%, more than 99.3%, more than 99.4%, more than 99.5%, more than 99.6%, more than 99.7%, more than 99.8%, more than 99.9%, in relation to option des -nona. In the most preferred embodiment, the des-nona variant is not defined and/or absent.

It is also preferred that the polypeptide of the present invention is substantially free of proteases (eg, trypsin). "Substantially free" in this context means that the polypeptide of the invention for use in accordance with the invention is characterized by a purity relative to the sum of the weights of all proteases (including trypsin) of more than 99.0%, for example, a purity of more than 99.1%, more 99.2%, more than 99.3%, more than 99.4%, more than 99.5%, more than 99.6%, more than 99.7%, more than 99.8%, more than 99.9% in relation to the amount weights of all proteases (including trypsin). In the most preferred embodiment, trypsin is not detected and/or absent.

Such a high degree of purity in the embodiments described above makes the formulation more acceptable to regulatory authorities and qualifies the polypeptide of the present invention as a drug for use in mammals, including humans in particular. Thus, the degree of purity according to the present invention allows for the first time to use this polypeptide for administration/application to the affected surface of the body, including the affected surface of the human body and, in particular, ulcers, in a safe and reliable way. The high purity grade for protease (trypsin) in particular allows the storage of the polypeptide also in non-frozen form.

Compositions

In some embodiments, the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 described herein is contained in a composition further comprising one or more carriers and/or one or more excipients. The term "carrier" as used herein refers to an organic or inorganic component of a natural or synthetic nature that is combined with an active ingredient to enable, improve or facilitate the use of the active ingredient. As used herein, the term "filler" is intended to refer to all substances that may be present in the pharmaceutical composition of the present invention and which are not active ingredients.

Preferably, the composition according to the present invention contains at least water as a filler. In some embodiments, the implementation of the composition according to the present invention contains an aqueous medium, and, more preferably, the composition of the present invention is in the form of an aqueous solution. In one embodiment, the polypeptide is contained in an aquatic environment, and the aquatic environment is administered to a mammal. The aqueous medium may be, for example, an aqueous solution. Aqueous ingredient solutions and other appropriate compositions, in some embodiments, may be prepared directly after NGF purification in an aqueous medium. For example, when the agent of the present invention is obtained by purification from a biological source, the corresponding aqueous compositions can be obtained directly from the last stage of purification, for example, by elution from the last chromatographic column (usually at the stage of polishing) and/or filtration. Alternatively, appropriate formulations are available through the additional step of adjusting the final protein concentration and/or preparing the desired formulation. Such an additional step may 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. Examples of compositions applicable in the present invention, without limitation, are described in this document.

Thus, a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 described herein may be present in a composition, eg in a pharmaceutical formulation. The compositions described herein are preferably sterile and preferably contain the polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 as the pharmaceutically active peptide or protein, and optionally other agents mentioned or not mentioned herein. The compositions can be in any state, for example, be liquid, frozen, lyophilized, etc.

The compositions described herein may include salts, buffer compounds, preservatives, carriers, diluents and/or excipients, all of which are preferably pharmaceutically acceptable. The term "pharmaceutically acceptable" describes something that is non-toxic and/or does not interfere with the activity of the active ingredient of a pharmaceutical composition.

Suitable buffer compounds for use in the invention include acetic acid salts, citric acid salts, boric acid salts and phosphoric acid salts. For example, it is preferred that the polypeptide of the invention, as a result of the various aspects of the present invention, be prepared in a buffer having a pH of 4.5 to 6.5, preferably 5.0 to 6.0. In one embodiment, an acetate buffer is a suitable buffer for such purposes and is therefore particularly preferred. Thus, in one embodiment, the polypeptide of the invention is prepared in an acetate buffer having a pH of 4.5 to 6.5, preferably 5.0 to 6.0.

Suitable preservatives for use in the compositions of the present invention include preservatives known in the art, including, for illustrative purposes and without limitation, benzyl alcohol, benzalkonium and salts thereof, M-cresol, phenol, chlorobutanol, paraben, and thimerosal.

Thus, the present invention provides a polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 for therapeutic use, that is, for use in a method of treating a human or animal body through therapy. Therapy may include prevention and/or treatment of the condition. Taking into account the possibility of therapeutic use, the specified polypeptide can also be called a pharmaceutically active protein or peptide.

Optionally, the administration according to the present invention is accompanied by the administration of at least one antimicrobial agent, such as an antibiotic. The antimicrobial agent may be part of a composition containing a polypeptide of the present invention, or, alternatively, may be administered to the subject separately, at the same or different site, by the same or different route of administration.

Industrial Applicability

The polypeptide of SEQ ID NO: 3 or SEQ ID NO: 4 described herein is suitable for various purposes, for example, for use in therapy as described here.

The following examples and drawings are intended to illustrate certain preferred embodiments of the invention and should not be interpreted as limiting the scope of the invention, which is defined by the claims.

EXAMPLES

Materials and methods used for more than one example

Unless otherwise noted, the following experimental examples specifically refer to a polypeptide of SEQ ID NO: 4 characterized against wild-type human NGF by substitutions P61S R100E (denoted "NGF P61S R100E", Malerba et al. PLOS One, 2015, vol. 10, e0136425, SEQ ID NO: 4), as well as pro forma thereof, etc. The polypeptide of SEQ ID NO: 4 may also be referred to as "NGF mutein", but it should be understood that the therapeutic utility specific to this protein, in accordance with this invention and as demonstrated in experimental examples, especially in Examples 3 and 4, is markedly different on the suitability of wild-type human NGF. Similarly, as described in Example 2, the purification of the polypeptide of SEQ ID NO 4 differs from the published purification protocols for wild-type NGF, and the specific method for obtaining said polypeptide according to the present invention is suitable for achieving a high degree of purity, in the absence of the des-nona variant and trypsin, in particular.

The polypeptide of SEQ ID NO: 4 was recombinantly expressed as a precursor. To this end, SEQ ID NO: 4 was fused to a wild-type human NGF propeptide (positions 1-121 of SEQ ID NO: 1). In other words, the polypeptide precursor of SEQ ID NO: 4 consisted of the wild-type human NGF precursor (SEQ ID NO: 1), except for the P61S R100E substitutions in the mature portion of wild-type human NGF (in addition, for accuracy, the two most C-terminal amino acids from SEQ ID NO: 1 that do not form part of the wild-type human NGF polypeptide sequence). Expression was performed in E. coli Rosetta (DE3) (strain: E. coli Rosetta (DE3)/pET11a-hpro NGF P61S R100E) to form insoluble inclusion bodies.

Equipment

Device Device part number Serial number Provider 1 L bioreactors (including sensors and pumps) E023, E024 07462/09, 07463/09 Sartorius Stedim Bioreactor 10 l E082 - Sartorius Stedim Power supply 300 V E018; E019 - VWR Äkta Explorer100a E011 001054 GE Healthcare Äkta Explorer100a E054 18111241 GE Healthcare Autoclave Systec VX-120 E050 2512 Systec GmbH Centrifuge Galaxy 14D E016 904090 VWR Centrifuge Sorvall Evolution RC F683 - Sorvall clean table E006 40970929 Thermo Scientific Novex Mini Cell Electrophoresis Chamber - - Invitrogen High pressure homogenizer APV 2000 F688 5-07.791 APV HPLC, 1100 Series E053 - Agilent Magnetic stirrer MR Hei-Mix S E013 30948231 Heidolph Magnetic Stirrer PC-620D 686 - Corning pH meter laboratory pH720 E017 9080718 W.T.W. Photometer Genesys 10uv E051 2L9Q013008 Thermo Spectronics Pipettes Pipetus® - - Hirschmann Laborgerate Pump VL 1000 F606 0208004 Verder Scales F651 - Sartorius Scales E030 W092934 Kern Scales E009 - Mettler Shaker IKA KS 4000ic E049 - IKA Shaker Vortexer E012 40934086 VWR

Table 1. List of used equipment

Protein parameters of the proteins and peptides described herein

Theoretical values of the protein parameters of the corresponding proteins were calculated using the ExPASy's ProtParam-Tool program available at http://web.expasy.org/protparam. They are shown in Table 2 below:

Predecessor
SEQ ID NO: 4 SEQ ID NO: 4 Pig trypsin MW,
monomer 24.8 kDa 13.23 kDa 24.4 kDa pi 9.7 8.2 7.0 ε 25168 l/mol/cm 19668 l/mol/cm 34295 l/mol/cm

Table 2 : Theoretically derived properties of the respective proteins/polypeptides

Analytical Methods

SDS-PAGE and Western blotting

SDS-PAGE and Western blot were performed using standard procedures. For this, 12% Bis-TRIS NuPAGE SDS-PAGE gels (Article No. NP0342BOX, Thermo Fisher) were placed under reducing conditions at constant voltage (175 V) in NuPAGE MES running buffer (Article No. NP0002, Thermo Fisher). Primary antibodies for Western blotting were purchased from Santa Cruz Biotechnology (NGF(H-20) sc-548). Examples of results are shown, for example, in FIG. 9A and FIG. 10.

Analytical Chromatography CEX-HPLC

CEX-HPLC was performed using ProPac SCX-10 from Dionex. The column was run with 50 mM citrate buffer, pH 5.5, at 1 ml/min. For elution, 1 M NaCl (B) was added and eluted in a linear gradient from 0 to 100% (B) over 50 minutes. An example of the results is shown in Fig. 9B .

SE-HPLC

SE-HPLC was performed using Superdex 200 Increase 10/300 GL from GE Healthcare. The column worked for PBS. The product was detected at 280 nm.

Endotoxin, DNA and HCP

Endotoxin, DNA and host cell proteins (HCP) were determined according to standard protocols.

Example 1: Expression of a polypeptide of SEQ ID NO: 4 as a precursor protein

Production Strain

The gene encoding pro-NGF was cloned into the pET11a expression plasmid. The gene is derived from H. sapiens and two point mutations (namely P61S and R100E) have been introduced into the open reading frame. Subsequently, chemically induced Rosetta (DE3) competent cells were transformed with an expression plasmid and a single colony was selected (the resulting strain was named E5901 STRAIN (= E. coli Rosetta (DE3)/pET11a-pro NGF P61S R100E

NGF RCB C-151101)). Aliquots were stored at <-60 °C in 1.0 ml.

Example 1 describes the initial development of a fermentation based on strain E5901 STRAIN.

Equipment

Device Device part number Serial number Provider Autoclave Systec VX-120 E050 2512 Systec GmbH Centrifuge Galaxy 14D E016 904090 VWR Centrifuge Sorvall Evolution RC F683 - Sorvall clean table E006 40970929 Thermo Scientific Magnetic stirrer MR Hei-Mix S E013 30948231 Heidolph Magnetic Stirrer PC-620D 686 - Corning pH meter laboratory pH720 E017 9080718 W.T.W. Photometer Genesys 10uv E051 2L9Q013008 Thermo Spectronics Pipettes Pipetus - - Hirschmann Laborgerate Shaker IKA KS 4000ic E049 - IKA Scale Kern 572 E030 W092934 Kern Scales Mettler AE160 E009 - Mettler 1 L bioreactors (including sensors and pumps) E023, E024 07462/09, 07463/09 Sartorius Stedim

Cultural environment

Complete Fermentation Environment

The complex medium used for fermentation consisted of: 49.3 g/l yeast extract, 0.61 g/l MgSO ₄ *7H ₂ O, 0.5 g/l NH ₄ Cl, 14.2 g/l K ₂ HPO ₄ *3H ₂ O and 10 g/l glucose. The nutrient used for this fermentation consisted of 263 g/l yeast extract and 133 g/l glucose.

Minimal Media (MM, Minimal Media) for fermentation

Components MM I - Final conc. [mm] MM II - Ultimate conc. [mm] Aluminum chloride, hexahydrate No 0.000063 Ammonium sulfate 39.4 No Boric acid 0.005 0.000125 Calcium chloride, dihydrate 2 0.000875 Citric acid, monohydrate 25.2 10 Cobalt(II) chloride, hexahydrate No 0.00075 Cobalt(II) sulfate, heptahydrate 0.014 No Copper(II) sulfate, pentahydrate 0.032 0.00425 Diammonium Phosphate No 35 potassium phosphate No 45 Sodium hydrogen phosphate 7.5 No Ferric chloride, hexahydrate 0.37 0.17 Kanamycin 0.103 0.103 Magnesium sulfate, heptahydrate 4 3 Manganese(II) sulfate, monohydrate 0.142 0.00375 Polypropylene Glycol 2000 No No potassium chloride 53.6 No Sodium chloride 8.5 40 Sodium dihydrogen phosphate, monohydrate 31.9 No Sodium molybdate, dihydrate 0.001 0.00005 Zinc sulfate, heptahydrate 0.073 0.000375

At the stage of cell cultivation, 30 g/l of glucose was added to both basic media. Unless otherwise noted, the nutrient had the same composition as the corresponding media in this series, but contained 300 g/l of the appropriate carbon source.

LB agar plates with ampicillin and chloramphenicol

The dishes were filled with fresh medium based on LB-agar. The medium consisted of 10 g/l peptone, 5 g/l yeast extract, 5 g/l NaCl and 15 g/l agar. After autoclaving, 100 μg/ml ampicillin and 30 μg/ml chloramphenicol were added to the medium.

Fermentation

Unless otherwise indicated, the fermentation in this Example 1 was carried out in 1 liter glass bioreactors with a stirrer, controlled by the Biostat B device from Sartorius. Typically, pO ₂ was controlled at 30%, the culture temperature was maintained at 37°C, and the pH was adjusted to 7 using 2M phosphoric acid and 25% ammonium hydroxide. Unless otherwise indicated, the cell culture step was followed by exponential feeding with F0=6 g/l/h and μ=0.25/h. For practical reasons, all exponential feeds were approximated by two linear feeds. Typically, product expression was induced by adding 1 mM IPTG, and after induction, a constant feed rate of 10 g/l/h was used. 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.

Determination of the relative amount of the product in biomass samples

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

The relative accumulation of the product was calculated from the increase in the band corresponding to the precursor of the polypeptide of SEQ ID NO: 4, before and after induction. Notably, the measured value is the specific output (ie, normalized to OD ₆₀₀ =10). To obtain an absolute yield value for a given fermentation, the actual cell density must be taken into account (see below).

Quantification of absolute product yield in biomass samples

The polypeptide precursor standard SEQ ID NO: 4 was obtained from the European Brain Research Institute (EBRI, Rome, Italy). The standard was diluted to a concentration of 65 μg/ml in Laemmli's buffer. The declared protein concentration was determined according to the EBRI protocol. A standard curve was generated for the 260, 520, 780, 1040, and 1300 ng polypeptide precursor standard SEQ ID NO: 4. Samples were run on the same gel as the standard curve samples and the sample dilution factor was used to calculate the absolute specific point in time.

Brief description and conclusions of Example 1

Based on the foregoing, it is concluded that the producer strain (E5901 STRAIN, see above) was successfully used for the 1 L scale fermentation. Although various media formulations were evaluated for their ability to stimulate bacterial growth and product expression, MM I minimum medium supplemented with 5 g/L yeast extract was found to be favorable in terms of achieving expression and obtaining the desired cell density. In terms of product formation, we did not observe significant differences when the main culture was performed with or without antibiotics (ampicillin and chloramphenicol, data not shown).

Example 1 can be scaled up to produce a polypeptide on an industrial scale.

Example 2: Cleaning in the laboratory, creation of Capto MMC

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

Starting point for prior art optimization

Initially, the authors of the present invention came to the conclusion that the development of the process can, in the absence of indications to the contrary, follow the basic concepts of purification of NGF, previously described in the literature. However, it was also taken into account that for efficient production on a large scale, it is necessary to consider the possibility of adaptation suitable for later scaling. Thus, we concluded based on Rattenholl et al. (see above), WO2013092776 A1 and other publications that the polypeptide of SEQ ID NO: 4 can similarly be obtained, at least in the laboratory, by non-specific cleavage of its precursor with trypsin and subsequent purification. It has been suggested that a high purity mature polypeptide of SEQ ID NO: 4 can be obtained in this way. However, it was only through the specific adaptations and modifications described in this example that the mature polypeptide of SEQ ID NO: 4 was obtained in high purity. Therefore, administration of the polypeptide of SEQ ID NO: 4 to a subject in need thereof is possible, in particular, in view of the high purity of the polypeptide of SEQ ID NO: 4, as described herein.

Equipment for the production of polypeptide SEQ ID NO: 4

Device Device part number Serial number Provider 1 L bioreactors (including sensors and pumps) E023, E024 07462/09, 07463/09 Sartorius Stedim Power supply 300 V E018; E019 - VWR Äkta Explorer100a E011 001054 GE Healthcare Äkta Explorer100a E054 18111241 GE Healthcare Autoclave Systec VX-120 E050 2512 Systec GmbH Centrifuge Galaxy 14D E016 904090 VWR Centrifuge Sorvall Evolution RC F683 - Sorvall clean table E006 40970929 Thermo Scientific Novex Mini Cell Electrophoresis Chamber - - Invitrogen High pressure homogenizer APV 2000 F688 5-07.791 APV HPLC, 1100 Series E053 - Agilent Magnetic stirrer MR Hei-Mix S E013 30948231 Heidolph Magnetic Stirrer PC-620D 686 - Corning pH meter laboratory pH720 E017 9080718 W.T.W. Photometer Genesys 10uv E051 2L9Q013008 Thermo Spectronics Pipettes Pipetus - - Hirschmann Laborgerate Pump VL 1000 F606 0208004 Verder Scales F651 - Sartorius Scales E030 W092934 Kern Scales E009 - Mettler Shaker IKA KS 4000ic E049 - IKA Shaker Vortexer E012 40934086 VWR

List of equipment used in Example 2.

Details of the manufacturing process according to this example, including the improvements described in example 2B, are given in the process overview of FIG. 1.

Unless otherwise indicated, analytical methods were as described in the "Analytical Methods" section above.

Example 2A: Purification According to Previously Described Protocols

E. coli cells expressing the polypeptide precursor of SEQ ID NO: 4 ("biomass") were prepared as described in Example 1 and lysed by the addition of lysozyme followed by sonication in an ice bath. Inclusion bodies ("IB") were (1) extracted from host cells and washed with 6% Triton X100 solution (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. IB was solubilized for 2 hours at room temperature. After that, the pH was lowered to 3-4 by adding 37% HCl. The solution thus obtained, containing the solubilized precursor polypeptide of SEQ ID NO: 4 ("solubilizate" (solubilizate)), was subjected to dialysis against 6 M gHCl (pH 3-4).

Refolding of the precursor polypeptide of SEQ ID NO: 4 was performed 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 at +4°С. For this, protein was added every hour in the amount of 50 μg of protein per ml of refolding buffer. After refolding, the reaction mixture was dialyzed against 50 mM sodium phosphate, pH 7.0. When changing the buffer, a significant precipitate was formed.

The polypeptide precursor of SEQ ID NO: 4 was purified using successive steps of cation exchange chromatography (SP Sepharose HP, set with 50 mM sodium phosphate, pH 7.0 and eluted with a NaCl gradient) and subsequent hydrophobic interaction chromatography (Phenyl Sepharose HP, set with 50 mM phosphate sodium, 1 M ammonium sulfate, pH 7.0). Thereafter, another dialysis was used to replace the sample buffer with 50 mM sodium phosphate buffer, pH 7.0 (note that this second dialysis, however, could have been omitted in the process described in Example 5). Again, precipitation of significant amounts of product was observed during the decrease in buffer conductivity.

The polypeptide precursor of SEQ ID NO: 4 thus obtained was subjected to limited proteolysis by the addition of 1 mg trypsin per 250 mg pro-NGF. The impact of the protease on the precursor of the polypeptide of SEQ ID NO: 4 occurred within 14 hours at 2-8°C.

Mature NGF was finally purified ("polished") on a cation exchanger (SP Sepharose XL, equilibrated with 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.

Example 2B: Innovation

The following describes several optimizations compared to example 2A, which were tested and implemented by the authors of the present invention during the creation of the present invention. Unless the context dictates otherwise, all those details not explicitly stated were the same as described above for Example 2A.

IB Solubilization Optimization

Although it has been previously reported that small amounts of IB (e.g. obtained from shake flask cultures) readily dissolve in solubilization buffer (6M gHCl, 0.1 M Tris-HCL pH 8.0, 1 mM EDTA, 100 mM (fresh) DTT ), it turned out that IBs obtained from high cell density fermentation could not be completely dissolved. The present inventors were able to overcome this problem by adding 2M urea to said solubilization buffer, which proved to significantly improve the solubilization yield (data not shown). For the avoidance of doubt: 2 M urea was present in addition to 6 M gHCl and other ingredients.

Refolding optimization

A decision was made, initially based on Rattenholl et al . (2001, Eur. J. Biochem, vol. 268, p. 3296-3303; Rattenholl, 2001, Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.), Martin-Luther-Universität Halle-Wittenberg ( Germany)), but taking into account possible later scalability, refold the precursor polypeptide of SEQ ID NO: 4 in an amount of 200 to 500 mg per liter of the refolding reaction, preferably in an amount of 200 to 300 mg of the precursor polypeptide of SEQ ID NO: 4 per liter of refolding reaction. This results in a relatively good yield of the solubilized precursor polypeptide of SEQ ID NO: 4. In particular, it is important to consider that due to the increased amount of NGF compared to the volume of the refolding reaction, and given that the refolding reaction includes relatively expensive ingredients such as glutathione and arginine, relatively more of the precursor polypeptide of SEQ ID NO: 4 per volume of refolding reaction can be refolded, which should make refolding economically viable, also on a production scale.

Purification of the precursor polypeptide SEQ ID NO: 4

Purification of the precursor polypeptide of SEQ ID NO: 4 after refolding was carried out using an approach that uses a fairly high pro-NGF isoelectric point and uses a cation exchange stationary phase (namely SP-sepharose) for purification. For this type of chromatography, for technical reasons, the refolding buffer had to be replaced with a low conductivity buffer. A significant amount of the precursor polypeptide of SEQ ID NO: 4 was observed to precipitate (data not shown). This observation can be explained by a decrease in the concentration of arginine in the buffer.

Therefore, some attempts have been made to replace the capture column with a different type of column (column with a different selectivity) that would be more tolerant of the presence of arginine in the refolding reaction. In the first attempt at implementation, the effectiveness of several stationary phases was evaluated, but none of the approaches gave satisfactory results (see Table 6). Thus, the stationary phase used for the capture column was retained as described in the previous process. However, due to the high isoelectric point (pI) of the precursor polypeptide of SEQ ID NO: 4, an increase in running buffer conductivity (by adding 250 mM L-arginine) without affecting performance was possible. Through this, the refolded precursor of the polypeptide of SEQ ID NO: 4 could be stabilized to a certain extent, and the amount of precipitated precursor was reduced (data not shown).

Possible Capture Stage Short description Grade Hydrophobic interaction chromatography Hydrophobic interaction chromatography is independent of the conductivity and salt composition of the loaded sample. If ammonium sulfate or sodium chloride was added to the product present in the refolding buffer, significant protein precipitation occurred. Even the addition of a small amount of ammonium sulfate (up to a concentration of 0.25 M) led to precipitation. It was also not possible to add sodium chloride without causing precipitation of the product. Therefore, a capture step based on phenyl or butyl Sepharose is not suitable. Mixed Mode Chromatography Capto MMC is a mixed mode stationary phase combining hydrophobic properties with cation exchange stationary phase properties. Since binding is not only mediated by ionic interactions, this stationary phase is more salt tolerant than the classical cation exchange stationary phase. In the initial setup, a pH 5.5 phosphate buffer was supplemented with 0.25 M L-arginine and the elution was carried out with a NaCl gradient. However, no significant amounts of product (precursor) were recovered with this approach. size exclusion chromatography Size exclusion chromatography provides good resolution and is independent of sample buffer. The typical bottleneck of this type of chromatography (i.e., its limited throughput) can be ignored because relatively small amounts of product are required. Although the preparative chromatogram looked promising, separation of the precursor polypeptide of SEQ ID NO: 4 from impurities could not be achieved. This may be due to the existence of this specific precursor in the form of a polydisperse mixture under the conditions under study.

Table above : Alternative possibilities tested to capture the precursor polypeptide of SEQ ID NO: 4 and their evaluation.

With regard to mixed mode chromatography, the inventors, without wishing to be bound by a particular theory, understand that in mixed mode chromatography, the precursor polypeptide of SEQ ID NO: 4 is not efficiently eluted, while the mature polypeptide of SEQ ID NO: 4 is effectively eluted.

Protease Cleavage to Obtain Mature NGF

For the production of the polypeptide of SEQ ID NO: 4, a protease (trypsin) is essential and therefore it has been suggested that ideally the particular trypsin selected should meet the following criteria:

1. Derived from a recombinant source. Certification of animal-free raw materials is critical to ensure that the process is GMP compliant.

2. Possesses low side activity. Notably, trypsin can undergo autolysis. This process can lead to the so-called pseudotrypsin, which has an extended spectrum of substrates and exhibits chymotrypsin-like activity. Ca ²⁺ (eg 1 mM CaCl ₂ ) may be added to reduce autolysis. However, at present, a "modified trypsin" is usually used for each protocol, requiring high sequence specificity (for example, for peptide fingerprinting). This modified trypsin is usually prepared by acylation of the trypsin-exposed ε-amino groups of lysine residues.

3. Low variability from batch to batch, which ensures the reproduction of the production process. On the other hand, the selected enzyme must have a certificate stating the specific activity of the respective lot. The amount of enzyme required may depend on activity rather than weight.

Despite an intensive search, trypsin fulfilling criteria 1 and 2 has not been found on the commercial market. The view was expressed that criterion 1 was more important than all the others. The addition of CaCl ₂ may be sufficient to reduce autolysis. As a result, recombinant trypsin "GMP grade" from Roche (Roche 06369880103, lot: 11534700) was chosen as the raw material for the process. The sequence of this enzyme, which is expressed in Pichia pastoris, was obtained from Sus scrofa . According to the certificate, the batch of used trypsin has a specific activity of 4997 U/mg (determined according to USP).

Skip the second purification step before trypsinization

As part of the initial screening to find the optimal ratios of enzyme/substrate for putative trypsinization used the precursor polypeptide SEQ ID NO: 4, obtained on the capture column (see above). In contrast to the previously established process (European Brain Research Institute (EBRI), details not published, process based on Rattenholl et al., supra ), the present inventors chose not to use an additional hydrophobic interaction chromatography step prior to trypsinization. The decision to omit this second column purification step before trypsinization was made on the basis of two considerations: on the one hand, the product obtained after column capture was already practically pure according to SDS-PAGE. On the other hand, trypsinization itself could improve the impurity profile by digesting the remaining host cell proteins (HCPs).

Table 7 presents the main conditions tested in the first round. The results of trypsinization in 12% SDS-PAGE were examined (data not shown). The results (data not shown) show that trypsinization reproducibly produces a stable polypeptide of SEQ ID NO: 4 over a fairly wide range of enzyme/substrate ratios (i.e., 1 to 5 μg trypsin per 375 μg of SEQ ID NO: 4 polypeptide precursor). Digestion time doesn't really matter. Therefore, stopping the reaction and the time required to load the reaction volume into the column for cleaning ("polishing"), obviously, are not limiting. This discovery is of particular importance because the reaction cannot be stopped in a suitable or economical manner on a preparative scale.

Some additional experiments were carried out to clarify the optimal enzyme/substrate ratio for putative trypsinization, and it was found that at an enzyme/substrate ratio of 1/100 to 1/200 (protein weight/protein weight), a good yield of the polypeptide of SEQ ID NO: 4, on the one hand, and small quantities of truncated products, on the other hand, can be obtained reproducibly. It should be noted that under the conditions used (i.e. in phosphate-arginine buffer (pH 7.0) at 2-8°C and incubated (for protease treatment) for two to six hours), the quality of digestion was not strongly dependent on enzyme/substrate ratio. This finding is of particular importance because basic enzyme digestion is dependent on minor changes in the experimental setup (e.g. change in trypsin activity due to batch-to-batch variability or enzyme storage; time and temperature of the incubation step (protease exposure); error in determination of protein concentration). Moreover, this is also the reason why extended fine-scale fine-tuning to further reduce potential truncated products seems impractical. If the "optimal" condition can be identified on a small scale, then there is still a good chance of slightly changing the production scheme the next time the actual digest is repeated on a larger scale.

"Polishing" by chromatography to obtain a pure polypeptide after trypsinization

In contrast to the previously established process (European Brain Research Institute (EBRI), details not published, process based on Rattenholl et al., supra), which used a stationary phase of SP-Sepharose to polish the mature polypeptide (Note: SP-Sepharose is a cation exchange stationary phase), the authors searched for a more suitable stationary phase, based on the following considerations: for efficient loading into a column with SP-Sepharose, reducing the conductivity of the solution containing the precursor of the polypeptide of SEQ ID NO: 4, for example, by changing the buffer, is not required. However, it is known (eg Example 2A) that lowering the ionic strength of the solution does lead to precipitation of the target molecule, and therefore buffer replacement with a low conductivity buffer should be avoided. Moreover, a cation exchange stationary phase has already been used to capture the precursor polypeptide of SEQ ID NO: 4, and orthogonal selectivity is preferred to achieve better separation of remaining impurities. The third and final argument against using the SP stationary phase to purify the trypsin reaction is that the potentially remaining precursor polypeptide of SEQ ID NO: 4 will bind to this column and can be separated from the mature polypeptide of SEQ ID NO: 4 simply by elution selectivity, and not by selectivity of binding.

To create such a polishing column with orthogonal selectivity for purification of the mature polypeptide of SEQ ID NO: 4, it was first proposed to use a column with hydrophobic interaction (HIC). This stationary phase was chosen not only because it has orthogonal selectivity, but also because a buffer exchange with a low conductivity buffer is not required. Despite testing several stationary phases and HIC conditions (e.g., phenyl and butyl sepharoses operating with 1 M (NH4) 2SO4 and 0.5 M (NH4) 2SO4, respectively), the authors concluded that a satisfactory HIC-based polishing step cannot be implemented (data not shown).

However, the Capto MMC mixed-mode stationary phase was tested in the next pilot plant for the polishing step and could be successfully implemented. It was found that under optimized conditions the stationary phase binds reversibly to the polypeptide of SEQ ID NO: 4 and the product can be eluted by increasing the pH (data not shown). In contrast, the polypeptide precursor of SEQ ID NO: 4 binds irreversibly to the stationary phase and can only be eluted using 1M NaOH as the mobile phase (data not shown). In addition, it can be demonstrated that trypsin does not bind at all to the column operating under these conditions (data not shown). These results provide clear evidence that the Capto MMC stationary phase is able to efficiently separate the mature polypeptide of SEQ ID NO: 4 from trypsin and from the remaining precursor polypeptide of SEQ ID NO: 4.

Installation of additional membrane chromatography

For more efficient depletion of endotoxin and DNA impurities, an additional anion exchange membrane was used in the process. Typically, as is 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 through a normally charged membrane. For this purpose, in this case, a STIC membrane (Sartorius, Goettingen, Germany) was included in the system at the positions indicated in FIG. 1. The polypeptide of SEQ ID NO: 4 was determined not to bind to the membrane, and thus provided a proof of concept that membrane chromatography is suitable for the purification of the polypeptide of SEQ ID NO: 4. For an illustration of the entire process, including membrane chromatography, see Fig. 1.

Reproducibility of the process described in Example 2

To test the reliability of the process, the process was carried out five times and the resulting fractions were analyzed for their yield and purity. Throughout these cycles, steady optimization of process details and buffer composition, gradients, etc. was carried out. were tested until the final, optimized details of the process were established (see Fig. 1). The results show that approximately 50 to 100 mg of the polypeptide of SEQ ID NO: 4 can be obtained from a single successive production run on a laboratory scale. (Less than five percent of host cell contaminating proteins and a trace amount of truncated NGF are present, data not shown).

For the precursor polypeptide of SEQ ID NO: 4, a satisfactory SE-HPLC method could not be established. In contrast, the SE-HPLC analysis for the mature polypeptide of SEQ ID NO: 4 was simple and resulted in a peak of homogeneous product in the region of 16 kDa, which corresponds to the monomeric form of the polypeptide of SEQ ID NO: 4 n (data not shown).

Brief description and conclusions

In this process, the refolded polypeptide precursor of SEQ ID NO: 4 was captured using SP-sepharose FF ("FF" stands for "Fast Flow", i.e. relatively large particle stationary phase) and subsequently trypsinized to produce mature NGF. To this end, the concentration of arginine in the refolding reaction was reduced from 1 M (as recommended by the prior art) to 350 mm.

The control of proteolytic cleavage of the precursor polypeptide of SEQ ID NO: 4 to produce the mature polypeptide of SEQ ID NO: 4 is regarded as the most important process parameter. Here, conditions have been determined that reproducibly facilitate cleavage with high efficiency on the one hand and prevent the formation of NGF degradation products on the other hand. The experimental data presented here has shown that a reasonably stable manufacturing process can be established over a fairly wide range of enzyme/substrate ratios. As far as trypsinization is concerned, the yields at this stage appear to be good and no significant losses are expected at this stage of the process. The product sample obtained does not appear to be strongly dependent on the reaction conditions used (in terms of enzyme/substrate ratio and incubation time (protease exposure time)). Notably, even if the yield is good enough for the enzyme polishing step, at least 2*x grams of the SEQ ID NO: 4 polypeptide must be processed to obtain x grams of the mature polypeptide of SEQ ID NO: 4.

Purification according to this example is an economical process consisting of only two chromatographic purification steps. The existing purification process has been further optimized and several aspects of the process have been modified to scale up (see FIG. 1). Examples of previously used cell disruption techniques were replaced by high pressure homogenization and all dialysis steps could be replaced by tangential flow filtration. The process thus established allows the production of the polypeptide of SEQ ID NO: 4 in high purity.

Despite the problems identified, it appears that the overall process produces a product of acceptable quality.

The complete process, including optimizations according to Example 2, including membrane chromatography, is schematically depicted in Fig.1.

Example 2 can be scaled up to produce a polypeptide on an industrial scale.

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

The present invention is based in part on experiments with two animal models of skin ulcers. In these models, skin ulcers are induced in diabetic mice by circular biopsy or pressure cycles and the polypeptide of the present invention is applied topically.

This document reports a study on the administration of a polypeptide of SEQ ID NO: 4 to non-human animals. 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.

The purpose of this example is to investigate the efficacy of topical application of the polypeptide of SEQ ID NO: 4 in the context of wound healing in diabetic mice, consistent with histopathology, pain threshold, and human NGF (hNGF) plasma levels. Reference compounds (human NGF (SEQ ID NO: 2) and murine NGF, public domain amino acid sequence) were also used in the study.

Animals treated with the polypeptide of SEQ ID NO: 4 exhibited skin disease as described herein. The animals were an animal model of a human suffering from diabetes mellitus or having a predisposition to diabetes mellitus, eg type 1 diabetes mellitus or type 2 diabetes mellitus.

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 can be administered to subjects with diabetic ulcers and animals in the diabetic neuropathic foot ulcer (DFU) model.

This example includes the following sections:

Example 3A: In vitro PC12 neurite extension test . The purpose of this experiment was to establish the efficacy of the polypeptide of SEQ ID NO: 4. For this purpose, a conventional in vitro neurite extension test in NGF-responsive cells (PC12) was used.

Example 3B: in vivo efficacy study . The purpose of this section was to determine whether topical application of the polypeptide of SEQ ID NO: 4 improves wound healing (surgical injury) in diabetic mice.

The following groups were included:

- db/db, intact (intact) N=8, each time

- db/db, wound+carrier N=8, each time point

- db/db, wound + polypeptide SEQ ID NO: 4, 1 μg/day; N=8, every time

- db/db, wound + polypeptide SEQ ID NO: 4, 10 μg/day; N=8, every time

- db/db, wound + polypeptide SEQ ID NO: 4, 30 μg/day; N=8, every time

- db/db, wound+hNGF, 10 mcg/day N=8, each time point

- db/db, wound+mNGF, 10mcg/day N=8, each time point

(dose in mcg refers to the corresponding doses administered to the wound (each animal has one wound)

In this study experiment, animals were sacrificed 7 and 30 days after wound induction with the following endpoints determined: time to closure; histology (N=4) and immunohistochemistry of lesions (N=4).

Example 3C: mechanism: exploratory study . The aim of this section was to study the molecular mechanisms supporting the positive effect of the SEQ ID NO: 4 polypeptide on wound healing in diabetic mice, focusing on inflammation, extracellular matrix deposition, innervation and angiogenesis.

The following groups were included:

- db/db, intact (intact) N=6

- db/db, wound+vehicle N=6

- db/db, wound + polypeptide SEQ ID NO: 4, 1 μg/day N=6

- db/db, wound + polypeptide SEQ ID NO: 4, 10 μg/day N=6

- db/db, wound + polypeptide SEQ ID NO: 4, 30 μg/day N=6

- db/db, wound+hNGF, 10 mcg/day N=6

- db/db, wound+mNGF, 10 mcg/day N=6

(dose in mcg refers to the corresponding doses administered to the wound (each animal has one wound)

In this section of the study, animals were sacrificed 14 days after wound induction, which corresponds to 50% wound healing, with the following endpoint determined: study of possible mechanisms responsible for the therapeutic effect of the polypeptide of SEQ ID NO: 4, taking into account the expression and regulation of mRNA (N= 252), encoding proteins involved in the processes of extracellular matrix biology (N=84), angiogenesis (N=84), growth factors and neurotrophin biology (N=84).

Materials and methods in this example

Animals and monitoring

Mice homozygous for the spontaneous mutation of diabetes (Leprdb) (genetically C57BL/6J) and mice matching 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 "Introduction" for information on grouping and killing animals.

Animals were housed in single cages with pelleted chow and water ad libitum and with a 12 hour dark cycle. All animal protocols described here have been performed in accordance with the European Community Council Directives 2010/63/EU, approved by the Ministry of Health (No. 350/2015-PB), and comply with the recommendations published in the NIH Guidelines for Care and Use. laboratory animals (NIH Guide for the Care and Use of Laboratory Animals).

Blood glycemia was measured before treatment, the day after the last treatment and before sacrifice (Contour XT, Bayer, Basel, Switzerland).

Experiment schedule for an 8-day cohort:

Experiment schedule for a 30-day cohort:

First week: same as 8-day cohort

Next: photo twice a week until mortification.

Glycemic level on day 28

Mortification on the 29th day

In this example, the experiments are labeled as "8 days" and "30 days" cohorts, for which the days of testing or killing are indicated, as indicated in the schedule of experiments.

Defeat, drugs and observation.

A round wound of even thickness with a diameter of 6 mm was created by puncture dermal biopsy on the middle part of the back of a mouse. Briefly, animals were anesthetized with isofluorane (+2 L/min O2). The skin of the back was dehaired by waxing and disinfected with Clorexidina 4% ("Clorexyderm" I.C.F. srl Industria Chimica Fine - CR-Italy) or Iodopovidone 10% ("Poviderm" Nuova Farmec srl - VR - Italy). A sterile 6 mm diameter needle biopsy instrument was used to create an open wound of a certain thickness on the back. The wound area was immediately covered with Tegaderm semi-occlusive preparation ((Tegaderm Roll -3M Health Care, St. Paul, MN, USA), and a 1.5 cm thick protective band was placed around the chest to prevent the mice from removing the wound dressing. A 26 gauge needle was used to inject 50 µl of drug through the tegaderm into the wound bed on days 0 to 6 post-lesion. Notably, the tegaderm dressing completely prevented leakage of the solution from the lesion.

A full thickness round wound with a diameter of 6 mm had an area of 28.26 mm ² .

Based on this, the following doses were administered to the animals:

Dose 1 µg: 0.0035 µg/ ^mm2

Dose 10 µg: 0.35 µg/ ^mm2

Dose 30 mcg: 1.05 mcg/ ^mm2

The animals were observed daily, checking the integrity of the bandage and the absence of infections. Tegaderm was changed weekly in all animals until the wounds were completely healed.

Photographs of the wound were taken, including a ruler, and the area of the lesion was measured by computer image analysis (NIS Elements, Nikon) three times during the first week and then twice a week until the end of the experiment.

Introduction of the polypeptide SEQ ID NO: 4.

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

Human NGF (hNGF, Recombinant, E. Coli Cat N°: N-245, Alomone, Jerusalem, Israe) and recombinant mouse NGF (mNGF, Cat N°: 1156-NG, R&D System) were used as NGF controls.

The test compounds were administered daily for 7 days, starting from the day the wound was formed. 50 μl of test compound solution at each concentration was injected under the Tegaderm dressing on the wound area using a 26-gauge needle. The elasticity of the tegaderm makes it possible to seal the needle hole after the needle is withdrawn, without leakage of the liquid solution.

Pain threshold monitoring

hyperalgesia was assessed in freely moving animals by the Hargreave method using a thermal plantar tester (Ugo Basile - Comerio, Varese). The animals were allowed to acclimatize in a Plexiglas instrument box for 15 min. A radiant heat source of constant intensity (beam diameter 0.5 cm and intensity 25 I.R.) was placed under the hindpaw and withdrawal time (seconds) was recorded as the time from the start of radiant heat application to paw withdrawal. For statistical analysis, the average of four indicators was used. Animals were tested with a plantar test on day -3 (before surgery) and on Day 7 (24 hours after the last application of test compounds).

Collection and processing of tissues

On the day of sacrifice, mice were subjected to deep anesthesia (isoflurane+2 L/min O ₂ ) and skin samples (1 cm×1 cm) were taken from the wound area. For Study B, 4 immunohistochemistry samples and 4 histology samples were collected in each group. For Study C, a 6 mm area of skin (at the wound area) was taken with an excision punch, an 8 mm ring around it (at the perimeter of the wound) and a 6 mm area of intact skin were taken.

Samples collected for histology were embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E); samples collected for immunohistochemistry were postfixed, washed with sucrose PBS, cryosectioned and prepared for indirect immunofluorescence.

Immunohistochemistry and quantitation

The skin was immersed in 4% paraformaldehyde (w/v) and saturated aqueous picric acid in 0.1 M Sorensen's buffer, pH 7 for 24 hours, then washed for at least 48 hours with 5% sucrose in 0.1 M phosphate buffer. After freezing in CO ₂ sections (14 μm thick) were cut using a cryostat (HM550 Microm, Bio-Optica). Sections were collected on gelatin-coated slides first incubated in 0.1 M PBS at room temperature for 20 min, followed by overnight incubation at 4°C in a humid atmosphere with primary antibodies diluted in 0.3% PBS-Triton. X-100, rev./rev. 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 minutes (2×10 min), sections were incubated at 37°C for 30 minutes in a humid atmosphere with secondary antiserum conjugated with Rhodamine Red™-X-conjugated - affinity-pure Donkey anti-Rabbit IgG ( Jackson Immunoresearch) were diluted in newt PBS 0.3%. The sections were then washed with PBS (as above) and placed in glycerol containing 1,4-phenylenediamine (0.1 g/l).

Immunohistological images were obtained using a Nikon Eclipse E600 microscope equipped with a CCD Q Imaging Retiga-2000RV digital camera (Q Imaging, Surrey, BC, Canada). The analysis was carried out using the Nis-Elements AR 3.2 software. The immunoreactive area of laminin and PGP9.5 was calculated as a proportion (percentage) in the epidermal layer at 7 days and 30 days after skin lesion induction. The germination index was assessed by the number of sites in which PGP9.5 IR approached the border of the canker. For the total morphological analysis, five images were analyzed for each animal and at two levels/per animal. All analyzes were performed blind. The mean value per animal was used for statistical analysis.

Quantification of hNGF

Blood was collected in Vacuntainer tubes with EDTA-K2 and centrifuged for 30 min at 3000×g for 10 min at 4°C. Plasma was collected, aliquoted into polypropylene tubes and stored at -80°C until use.

The Human Adipokine Magnetic Bead Panel 2 Kit (HADK2MAG-61K, EMD Millipore Corporation, Billerica, MA, 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 with monoclonal antibodies specific to a particular protein, which allows the simultaneous capture and detection of specific analytes with high sensitivity from a small sample volume. We used a simple version of the kit that included only one population of beads conjugated to the human monoclonal antibody NGF-β. The analysis was performed according to the manufacturer's specifications with minor modifications.

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

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

Culture of PC12 and processing (treatment) thereof

Cells were maintained under standard culture media conditions (DMEM, 10% horse serum, 5% FBS and pen/strep 1x) in T25 cm ² (NUNC) flasks. After at least two passages, the cells were plated (1000 cells/well) in 96-well flat-bottomed (NUNC) cell culture plates. After 1 day in vitro (DIV), the culture medium was removed and the cells were maintained in deprivation medium (DMEM, 1% horse serum, 0.5% FBS and pen/strep 1x). Cells were treated with three different concentrations of all test compounds (50, 100 and 200 nM) 24 hours after serum deprivation. Following medium refresh at 2 DIV and at 7 DIV, cells were fixed and stained for beta-III-tubulin antigen using indirect immunofluorescence (FIG. 1).

Immunocytochemistry

At 7 DIV, cells were fixed with cold 4% paraformaldehyde for 20 minutes. After treatment for 1 hour with blocking solution (PBS, Triton X-100 0.3%, BSA 1%, and normal donkey serum 1%), cells were incubated with primary antiserum (Mouse Anti-beta-III-tubulin, 1:1000; R&D) overnight at 4°C. Cells were then incubated with anti-mouse secondary antibodies (Donkey Anti-mouse Alexa-488 conjugated; 1:500; Jackson) at 37° C. for 30 minutes. Finally, cells were incubated with Hoechst33258 nuclear dye at room temperature for 20 minutes.

Screening assay at high cell counts (high throughput screening)

Neurite elongation analysis was performed with Cell Insight™ CX5 High Content Screening (HCS; Thermo Scientific) using the Neuronal Profiling BioApplication. The software is able to recognize each cell in each well by nuclear dye fluorescence. Each nucleus is identified as an object, and each object corresponds to a separate cell. The system recognizes green fluorescence (beta-III-tubulin immunoreactivity) around the nucleus, identifying the cell body. This neural profiling device is capable of recognizing and tracking all neurites that originate in each cell body. This allows the neurites in each cell to be counted and measured. Cell aggregates were not recognized as a single cell object and were excluded from the analysis.

Individual cells were analyzed at 2000-4000 per well and 6 wells/per treatment.

RT-PCR (RT-PCR)

An exploratory study of possible mechanisms underlying the beneficial effect of the SEQ ID NO: 4 polypeptide on wound healing in diabetic mice was performed using an exploratory strategy (analysis of gene expression focusing on coupled cascades using RT2 Profiler PCR arrays), with a focus on the main molecular cascades of reactions involved in wound healing in 50% of the healing process, for example, including angiogenesis, extracellular matrix and adhesion proteins, growth factors. Samples were collected from the center of the lesion (diameter 6 mm) and RNA was extracted from all animals (6 animals per group), quantified (Nanodrop 2000 spectrophotometer) and pooled (100 ng per animal). Thus, 600 ng of RNA per group was used for reverse transcription.

One single PCR array was performed for each group using a CFX96 real-time PCR instrument (BioRad). The same threshold value was used for all plates and relative gene expression was calculated by the comparative 2-ΔΔCq method. Rt2 Profiler™ (QIAGEN) PCR templates for angiogenesis, extracellular matrix and adhesion proteins (ECM), and mouse growth factors (GF) were used to profile the expression of 250 key genes involved in angiogenesis, ECM and GF (84 genes each) and synthesized cDNA using the RT2 First Strand kit (QIAGEN) according to the manufacturer's instructions.

Results

The results are presented in the following order:

- PC12 in vitro analysis

- Efficiency, 8 days

- Efficiency, 30 days

- Mechanism, 14 days

Example 3A : In vitro PC12 neurite extension test.

The potency of the SEQ ID NO: 4 polypeptide was tested in vitro on PC12 cells (see Experimental Design in Fig. 1), measuring neurite elongation by high-throughput cell screening using the following parameters:

- Mean neurite length: represents the average neurite length per cell;

- Average total neurite length: represents the total neurite length per cell;

- % of cells with the highest neurite length (% High Neurite): represents the percentage of cells in which the length of the neurite is equal to or greater than the length of the cell body.

First, all doses of test compounds were analyzed and compared with a control (vehicle) (data not shown). This report only shows the results for the most effective dose of each test compound (FIG. 1). Cells exposed to effective doses of all tested compounds showed an increase in mean mean neurite length (A; mNGF, P=0.0394; hNGF, P=0.0196; polypeptide SEQ ID NO: 4, P=0.0033) and mean total neurite length (B; mNGF, P=0.0338; hNGF, P=0.0006; polypeptide SEQ ID NO: 4, P=0.0211; Aloe, P<0.0001) compared to vehicle-treated cells . Only the polypeptide of SEQ ID NO: 4 (P=0.0367) was able to increase the percentage of cells showing extended neurite.

Example 3B: Efficacy study, 8-day cohort.

Animal monitoring

In the period before the formation of the wound in animals was observed for the level of glucose in the blood. The results are presented in Fig. 3. Glycemia was higher in diabetic mice than in control animals, according to a pilot study. No differences were observed between animals assigned to different experimental groups.

Weight gain between day 0 and day 7 after injury is shown in FIG. 4. No differences were observed in either time (day 0 vs. day 7) or treatment.

Hyperalgesia

Thermal hyperalgesia was assessed in freely moving animals by the Hargreave method using a thermal plantar tester on Day -3 (prior to skin lesions) and the day after the last application of test compound (Day 7). The results are illustrated in FIG. 5. No difference between groups was observed on day 0. Comparing the mean paw withdrawal latency measured in the same treatment group on Day 0 and Day 7, hNGF-treated animals showed a significant decrease by day 7. In other groups, hyperalgesia to thermal stimuli was not observed. Notably, a higher threshold was observed in mice treated with the polypeptide of SEQ ID NO: 4 on day 7, indicating a higher pain threshold in this group.

Time to wound healing

Wound healing was assessed by macroscopic observation of photographs taken from day 0 (day of surgery) every two days for the first week. "Time to heal" was measured on these wound images using NIS (Nikon) elements. The results are presented in Fig. 6, which shows graphs of both outer and inner regions. Two-tailed analysis of variance shows a time effect (F(4203)=41.25, p<0.0001) and a group effect (F(5203)=2.565, P=0.0282).

The time to healing at day 7 is shown in Fig. 7. No differences between the groups were observed, despite the fact that in groups treated with the polypeptide of SEQ ID NO: 4, there was a dose-dependent trend towards accelerated wound healing.

Plasma NGF levels

Blood was collected at sacrifice, ie 48 hours after the last application of the test compounds. Plasma NGF levels were determined using an antibody-based assay using antibodies capable of detecting human NGF, mouse NGF, as well as the polypeptide indicated in SEQ ID NO: 4. In this document, plasma NGF levels determined in this way are referred to as "total level NGF in Plasma. The results are presented in Fig. 8. In treated mice, a dose-dependent increase in total plasma NGF levels was observed reaching values in excess of 350 pg/ml. Moreover, 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% amino acid sequence identity with human NGF and is recognized by the same antibody.

Example 3C : efficacy study, 30-day cohort.

Animal monitoring

Animals were monitored for blood glucose levels prior to wound formation, after completion of test compound administration, and at sacrifice. The results are presented in Fig. 9. We observed a gradual increase in blood glucose levels during the 28 days of observation in all experimental groups. No differences were observed between treatment groups at different times.

The weight gain between days 0 and 28 after skin lesion induction is shown in FIG. 10. No differences were observed either by time or by treatment.

Hyperalgesia

Thermal hyperalgesia was assessed in freely moving animals by the Hargreave method using a thermal plantar tester on Day -3 (before skin lesions) and on Day 7, 24 hours after the last application of NGF. The results are illustrated in FIG. 11. No difference between groups was observed at Day 0. When comparing Day 0 and Day 7 latency in the same treatment group, there was a non-significant trend towards reduced paw withdrawal latency at Day 7 in hNGF-treated animals. However, when data from the 8-day and 30-day cohorts were pooled, hNGF-treated animals showed a significant reduction in pain threshold, suggesting that it is this formulation of hNGF that causes hyperalgesia. No differences were observed in the other groups. The results are presented in Fig. 12.

Time to healing

Evaluation of wound healing was performed by macroscopic observation, photographing the wounds every two days during the first week, starting from day -3 (day of surgery), then twice a week until sacrifice. "Time to heal" was measured on these wound images using NIS (Nikon) elements.

Individual data for the assessment of "time to healing" are presented in tabular form (Fig. 13). Sequential measurements of external wound area are plotted against time in FIG. 14. Two-tailed analysis of variance shows time effect (F(50434)=417.3, p<0.0001), treatment effect (F(5434)=21.45, p<0.0001) and correlation between treatment effects and time (F (50,434) = 1.638, p=0.0055).

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

The time to healing on day 8 is shown in Fig. 15, which combines data for both 8-day and 30-day cohorts. A significant reduction in wound healing time already at this time is observed in the group treated with the polypeptide of SEQ ID NO: 4 at a dose of 30 μg/day, compared with the group treated with the carrier.

Plasma NGF levels

Blood was collected at the time of sacrifice. Plasma NGF levels were determined by an antibody-based assay using antibodies capable of detecting human NGF, mouse NGF, as well as the polypeptide of SEQ ID NO: 4. In this document, plasma NGF levels thus determined are referred to as "total NGF level in plasma. The results are presented in Fig. 16. Total plasma levels of NGF are very low (compared to Fig. 8), do not exceed 10 pg/ml and are the same in all groups.

Histology (after 8 and 30 days) (see also pilot study report)

Skin samples containing an ulcer area including a 5 mm margin of intact skin were excised, embedded in paraffin, and serially sectioned according to the scheme shown in FIG. 17b. Sections were then stained (H&E) and representative images at different levels of wound depth at low magnification are shown in FIG. 17A. High magnification micrographs illustrate the process of re-epithelialization at the wound margin (Fig. 17D), showing migrating epidermal tongue (MET), extensive granulation tissue in the dermis below the epidermal layer, characterized by inflammation, cell proliferation, matrix deposition ( Fig. 17E) and angiogenesis (Fig. 17F). Re-epithelialization was assessed by measuring the thickness of the epidermal layer. Representative group images of intact animals, vehicle, polypeptide of SEQ ID NO: 4, mice treated with 1 μg/day, and mice treated with 30 μg/day of the polypeptide of SEQ ID NO: 4 are shown in FIG. 18. The polypeptide of SEQ ID NO: 4 causes a dose-dependent thickening of the epidermal layer, which is much higher than in intact skin. The basal layer of the epidermis is characterized by hypercellularity of the basal and spinous layers, possibly reflecting increased cell proliferation. In addition, the dermis is also thickened and strongly colored, indicative of higher extracellular matrix deposition, and enriched in skin structures (glands and hair follicles) also in a dose dependent manner. The thickness of the epidermis in all groups is presented in the graph. The polypeptide of SEQ ID NO: 4 causes a dose-dependent thickening of the epidermal layer, which becomes much thicker than in the carrier group. Also, mNGF and hNGF produced the same effect, comparable to a group treated with a similar dose of the polypeptide of SEQ ID NO: 4.

Immunohistochemistry (after 8 and 30 days)

Skin reinnervation was analyzed by immunostaining for the PGP9.5 protein, a highly sensitive neuroectodermal marker that is widely used to visualize cutaneous innervation. The anatomy of the cutaneous innervation is shown in Fig. 19 showing PGP9.5-IR fibers in intact mouse skin. In particular, subcutaneous, deep dermal and subepidermal plexuses are visualized; the subepidermal plexus provides the epidermis with free epidermal nerve endings.

The effect of topical application of the polypeptide of SEQ ID NO: 4 on nerve regrowth in regenerating skin was analyzed using the "outgrowth index" at the lesion margin on day 8 post-lesion and PGP9.5-IR at the lesion in the epidermis and dermis of the regenerated skin area . Typical images are shown in Fig. 20. Panels A, B and C show plots of PGP9.5-IR at 30 days in intact animals when mice are treated with vehicle and SEQ ID NO: 4 polypeptide at 30 μg/day, respectively. The results of morphometric analysis are presented in Fig. 21. Treatment with the polypeptide of SEQ ID NO: 4 at 30 μg/day causes a significant increase in germination after 8 days, the same as with hNGF. After 30 days, while the animals treated with the carrier had not yet recovered innervation, and no differences were observed between intact and treated (NGF) animals that were injected with the SEQ ID NO: 4 polypeptide (all doses) and mNGF. On the contrary, when using hNGF, hyperinnervation is observed.

The effect of topical application of the polypeptide of SEQ ID NO: 4 on angiogenesis was assessed by the method using laminin as a marker. Laminin is a basement membrane marker, thus marking several skin structures, including endothelial cells. Other endothelial markers such as PECAM (also known as CD31), von Willebrand factor, collagen provided staining unsuitable for quantification under the fixation conditions used in this study. Representative images are shown in Fig. 22. Panels A illustrate the epidermal layer visualized using conventional histology (H&E). The arrows indicate the basal layer. Panel B illustrates the basement membrane underlying the epidermis (arrows); Panel C illustrates sensory innervation of the epidermis originating from the subependymal plexus; Panel D illustrates the wound boundary and associated innervation at 8 days (E) and after skin repair (F). Panels G-I illustrate angiogenesis at 8 days (G, EE; H, laminin-IR) and at 30 days (I).

The results of morphometric analysis are presented in Fig. 23. The use of the polypeptide of SEQ ID NO: 4 at a dose of 30 μg/day causes a significant increase in laminin-IR after 8 days, such as in hNGF, which is still observed after 30 days, possibly reflecting angiogenesis.

Mechanism: Exploratory Research

Regulation of gene expression

The aim of the study was to investigate the molecular mechanisms that support the positive effect of the SEQ ID NO: 4 polypeptide on wound healing in diabetic mice, with particular attention to inflammation, extracellular matrix deposition, innervation, angiogenesis. Research strategy (analysis of gene expression oriented to cascades of reactions using RT2 Profiler PCR arrays) was used to determine the main molecular pathway (cascade of reactions) in the wound at the time point corresponding to 50% healing. Mouse angiogenesis, extracellular matrix and adhesion protein (ECM ), and growth factors (GF) analyzed with Rt2 Profiler™ were used to profile the expression of 252 key genes involved in angiogenesis, ECM and GF (84 genes each).

Expression analysis for each panel (angiogenesis, ECM, growth factor) includes the following:

- list of genes;

a heat map providing a graphical representation of folding regulation data between the two groups superimposed on a PCR array layout;

- a scatterplot (scatterogram) comparing the normalized expression of each gene in the PCR matrix between two groups by comparing them with each other to quickly visualize significant changes in gene expression, and a list of genes whose expression changes are greater than the selected threshold value (≥ 3).

Scatterplots show comparison of the following groups:

- db/db compared to WT mice (WT as controls)

- vehicle db/db versus db/db intact mice (db/db intact mice were used as controls)

- db/db NGF (polypeptide SEQ ID NO:, mNGF, hNGF) vs. db/db vehicle (db/db vehicle group was used as control group)

- db/db NGF (polypeptide SEQ ID NO:, mNGF, hNGF) compared to db/db intact mice (db/db intact mice were used as controls)

The results were analyzed taking into account the extracellular matrix and adhesion molecules, growth factors and neurotrophins (drawings not shown).

The main results and conclusions of this analysis are as follows:

Genotype effect:

- Comparison between intact db/db and intact WT mice shows that numerous angiogenesis and ECM genes are differently regulated according to genotype, while very few GF genes are differently expressed, suggesting that ECM and angiogenesis are processes that, in the context of wound healing, are mainly affected by the diabetic condition.

- Damage effect in db/db:

- The lesion induced downregulation of numerous angiogenesis and ECM genes and upregulation of several GF genes, suggesting that ECM and angiogenesis are the main processes driving wound healing in diabetic mice.

- Effect of the polypeptide SEQ ID NO: 4 in db/db (compared to the vehicle):

- polypeptide SEQ ID NO: 4 suppresses several genes, including: angiogenesis genes: akt, Ccl2 (chemokine (C-C motif) ligand 2), Ctgf (connective tissue growth factor), Hif1a, MMP14, thbs2 (thrombospondin 2);

- the polypeptide of SEQ ID NO: 4 activates several genes, and only some of them are also regulated by hNGF and mNGF

- polypeptide SEQ ID NO: 4 does not regulate GF genes, and only some of them are regulated by hNGF and mNGF

STRING analysis was also performed (data not shown). STRING is a biological database and web resource of known and predicted protein-protein interactions that is widely used to search for interaction relationships between differentially expressed genes. "Cluster" analysis using STRING software is based on all genes that are regulated in different matrices.

CONCLUSIONS

The main takeaways from this example are:

1. PC12 cells, in combination with HCS as a method of analytical approach, is a suitable tool for evaluating the effectiveness of the polypeptide SEQ ID NO: 4 in vitro ;

2. polypeptide of SEQ ID NO: 4 improves healing in a dose-dependent manner.

3. The polypeptide of SEQ ID NO: 4 significantly increases the recovery of the epidermal layer and causes a strong increase in its thickness; the polypeptide of SEQ ID NO: 4 also has a positive effect on reinnervation and angiogenesis (as assessed using laminin-IR). All of these parameters are higher in mice treated with the polypeptide of SEQ ID NO: 4 than in intact control mice, suggesting that the wound reconfiguration phase of healing should be assessed in a further study.

4. A preliminary study suggests that the AKT-mTOR cascade may be mediated by the polypeptide of SEQ ID NO: 4. Since Akt and mTOR are considered promoters of survival and cell growth, it has been suggested that short-term pharmacological activation of the PI3K-Akt-mTOR signaling cascade may represent a new strategy for clinical intervention to accelerate healing. Notably, a disrupted AKT-mTOR cascade has been pointed out as a possible cause of impaired wound healing in diabetic mice, and the AKT-mTOR cascade has been shown to be involved in improved healing through several molecules, such as notoginsenoside Ft1 in diabetic mice; acemannan; SR-0379; miRNA-99 family.

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 makes it painless (hNGFp) and therapeutically effective.

Table above: Experimental design of the in vitro neurite extension test in PC12 cells. See Methods for more information

Example 4: Proof of concept: mouse pressure ulcer model

This document reports a study on the administration of a polypeptide of SEQ ID NO: 4 to non-human animals. 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.

Methods

The experiment included control (C57BL6, albino) and genetically diabetic mice C57BL/KsJ-m +/+ Leprdb (db/db), Jackson laboratories, 8-10 weeks old. Under gas anesthesia, the backs of the animals were shaved and the shaved area thoroughly cleaned to prevent skin irritation. The skin fold was lifted and two magnetic ceramic discs, 12 mm in diameter and 5.0 mm thick, with an average weight of 2.4 g and a magnetic field strength of 1000 G (Magnetic Fountain, Castle Rock, CO), were applied to the skin, leaving " bridge” made of approximately 5.0 mm thick leather between two magnets. This process creates a compression pressure of 50 mmHg. between two plates, which has been documented as necessary for local tissue ischemia (Peirce et al., 2000, Wound Repair Regen., Vol. 8, p. 68-76). Three ischemia-reperfusion (I/R) cycles were performed to induce 2 ulcers of uniform severity. A single I/R cycle consists of a 12-hour magnet application period starting at 8:00 AM followed by a 12-hour magnet-free rest period. Treatment with vehicle and test compounds was started 3 days after the end of the I/R cycles to allow surgical curettage of the ulcers, including removal of fibrin exudate and necrotic tissue.

Some mice were subjected to test treatment with the polypeptide of SEQ ID NO: 4, which may also be referred to as painless recombinant human nerve growth factor mutant (hNGFp). The following experimental groups have been studied in detail:

- db/db, media

- db/db, polypeptide SEQ ID NO: 4, 1 μg/cm ² /day

- db/db, polypeptide SEQ ID NO: 4, 10 μg/cm ² /day

- db/db, polypeptide SEQ ID NO: 4, 100 μg/cm ² /day.

Treatment (treatment) was continued daily for 14 consecutive days, and then twice a week until healing at the same dosage (the dosage was calculated depending on the size of the ulcer at the time of treatment). Pressure ulcers were monitored by visual inspection to establish the day of closure. In addition, pictures of the bedsores were taken, including a ruler, and the area of the lesion was measured by computer image analysis, during the period when the bedsores were treated with drugs twice a week. Pressure ulcers were assessed using a standard scale and by measuring the area of pressure ulcers using computer image analysis.

The effect of the compound on the pain threshold was assessed in freely moving animals at the site of injury using an electronic von Frey Bioseb device, which allows determining the threshold of mechanical pain sensitivity in rodents.

Lesion histology, innervation, and angiogenesis were analyzed using histology and immunohistochemistry, as well as computer image analysis.

Results

Placing magnets on a skin fold on the back of diabetic mice caused irreversible damage to the entire dermoepidermal tissue below the compressive site. Both visual examination (presence of a necrotic and hemorrhagic area) and histological analysis confirmed that 3 cycles of I/R were capable of producing pressure sore-like lesions.

In all groups treated with the polypeptide of SEQ ID NO: 4, the rate of wound healing was increased compared to animals treated with the carrier. Closing of bedsores in animals treated with the polypeptide of SEQ ID NO: 4 was evident from days 17 and 21, while vehicle treated animals were cured from day 23.

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

In addition to the positive effect on wound healing, histological and immunohistochemical data provide additional evidence that the polypeptide of SEQ ID NO: 4 has a biological ability to improve the degree of wound healing in diabetic mice with impaired healing. At the histological level, the repaired area showed complete re-epithelialization and restoration of normal skin anatomy after treatment with the polypeptide of SEQ ID NO: 4. Immunohistochemistry showed that the polypeptide of SEQ ID NO: 4 also had a positive effect on reinnervation (as measured by PGP 9.5 immunoreactivity in the epidermis) and neoangiogenesis ( as measured by PECAM immunoreactivity in the dermis) (FIGS. 26 A and B).

Since wild-type NGF has been reported to increase pain sensitivity at the injection site, mechanical pain threshold was assessed after 14 consecutive days of treatment with the polypeptide of SEQ ID NO: 4 by applying a mechanical stimulus at the pressure ulcer margin. No changes in pain mechanical threshold were observed compared to vehicle-treated diabetic mice, suggesting that the SEQ ID NO: 4 polypeptide after chronic topical treatment may have a positive trophic effect on the skin over a wide dose range without causing nociceptor sensitization (Fig. 27).

Example 5: Randomized, Double-Blind, Placebo-Controlled Study to Study the Safety, Tolerability, Pharmacokinetic, and Pharmacodynamic Profiles of the Polypeptide of SEQ ID NO: 4 after Single and Multiple Dose Escalations in Subjects with Diabetic Neuropathic Foot Ulcers (DFU)

This document reports a study on the administration of a polypeptide of SEQ ID NO: 4 at single and multiple escalating doses to participants with diabetic neuropathic foot ulcers (DFU). The participants were people.

The study described in this example has been ethically approved by the relevant authorities.

The polypeptide of SEQ ID NO: 4 may also be referred to as recombinant mutant human painless nerve growth factor (hNGFp). The polypeptide of SEQ ID NO: 4 is alternatively referred to as "RECOMBINANT HUMAN NERVE GROWTH FACTOR (RHNGF)" or "SUB77552" and the full molecular formula of the same is C580H895N163O176S8. The polypeptide of SEQ ID NO: 4 may be of biological/biotechnological origin (except Advanced Therapy IMP (ATIMP). This is a recombinant drug product (see also Example 1). In particular, the polypeptide of SEQ ID NO: 4 can be obtained by expression as described in Example 1, and purification as described in Example 2. In particular, the high purity that can be achieved as described in Example 2, preferably in accordance with GMP standards, allows the use of the polypeptide of SEQ ID NO: 4 as a drug.

In this example, the polypeptide of SEQ ID NO: 4 is an investigational drug (Investigational Medicinal Product (IMP)). According to Directive 2001/20/EC, "IMP" is "a pharmaceutical form of the active substance or placebo tested or used as a reference in clinical trials, including products already marketed but used or assembled (formulated or packaged) in a manner other than the authorized form, or when used for unauthorized use, or when used to obtain additional information about an authorized form." In this case, the polypeptide of SEQ ID NO: 4 is an IMP for use in the first human clinical trial. Thus, this example describes the first human clinical trial. No risk factors were identified according to the "first use in humans" guidelines.

The IMP used in this example is a clear, colorless solution of hNGFp prepared at a concentration of 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. Solution concentration - 1 mg/ml.

The polypeptide of SEQ ID NO: 4 is administered to people in need thereof. The specified polypeptide of SEQ ID NO: 4 is administered as a skin solution. This is not a special pediatric formulation.

Humans in need of administration of the polypeptide of SEQ ID NO: 4 are subjects with diabetic neuropathic foot ulcers (DFU). No risk factors have been identified according to the "first use in human" guidelines.

The polypeptide of SEQ ID NO: 4 is administered topically (externally). Thus, the polypeptide of SEQ ID NO: 4 is intended for topical use (not currently).

The polypeptide of SEQ ID NO: 4 is administered at a total dose of 0.3 to 6 μg/mm ² . "mm ² " refers to the area of the ulcer. The indicated amount (in µg) refers to the amount of polypeptide that is administered per day.

The polypeptide of SEQ ID NO: 4 was administered twice a day for 14 consecutive days. After that, the reception is stopped.

There is a placebo in this study. The placebo is designated as PL1. The placebo is a skin solution. The placebo is for topical use (not currently). The placebo is a placebo according to the polypeptide of SEQ ID NO: 4 (PR1). Otherwise placebo is identical to IMP (PR1). The placebo is administered identically to the polypeptide of SEQ ID NO: 4.

PR1 and placebo 1 were prepared for study at Klifo A/S, Smedeland 36, 2600 Glostrup, Denmark.

There is no other (comparative) drug in this study.

Subjects participating in this study suffer from, are affected by, or are predisposed to a disease, which is a disease of the skin and connective tissue. In particular, subjects suffering from diabetic neuropathic foot ulcer (DFU) participate in this study. A diabetic foot ulcer is a serious complication of diabetes mellitus, a non-healing or poorly healing full-thickness wound that affects the dermis below the ankle in a person with diabetes.

MedDRA version, system organ class, level, term and classification code (EMEA EudraCT website (http://eudract.ema.europa.eu/)): Version System Organ Class Classification code Term Level 20.0 100000004858 10012664 diabetic foot ulcer LLT

An independent data monitoring committee was formed for this study.

The initial estimate of the trial duration is 2 years 1 month.

The planned number of subjects is 92 (of which 60 are in the age range of 18-64 years; 32 of which are in the age range of 65 years and older). The study group consists of patients and does not include healthy volunteers. Specific vulnerable populations included.

Treatment or care after the subject has completed their participation in the study is standard care.

Ethics approval obtained. A positive conclusion was issued.

Purpose of the study:

Primary Objective: To evaluate the safety and tolerability of the SEQ ID NO: 4 polypeptide in DFU subjects at single and multi-day topical dosing.

Secondary Goals:

(a) Assess the pharmacokinetic profile of a systemically available drug after one- and many-day topical administration of the polypeptide of SEQ ID NO: 4 in subjects with DFU;

(b) To evaluate the pharmacodynamic effects of multi-day topical dosing of the polypeptide of SEQ ID NO: 4 on DFU healing over a 12 week period.

No additional studies have been conducted.

Main inclusion criteria:

Part 1 SD and Part 2 MD:

Subjects must meet all of the following criteria to be eligible to participate in the study:

1. Written informed consent of the subject obtained prior to any study-related procedure;

2. Male or female subject aged 18 to 80 years (inclusive) diagnosed with type I or II diabetes mellitus with glycosylated hemoglobin (HbA1c) ≤10%.

3. Female subjects of childbearing potential (WONCBP): - females must report surgical sterilization (performed at least 6 months prior to screening) or - menopause (must have had no regular menstrual bleeding for at least one year prior to screening , age ≥45 years, and FSH levels at screening ≥40 mIU/mL).

4. Female Subjects of Childbearing Potential (WOCBP): Women must use one or more of the following reliable methods of contraception during the study period and for at least 90 days after the last administration of study drug, such as: a) insertion of an intrauterine device spirals (IUD) or intrauterine system (IUD); b) hormonal contraception (implantable, patch, oral); c) barrier methods of contraception: condom or closure cap (diaphragm or cervical vaults/caps) with spermicidal foam/gel/film/cream/suppository; d) sterilization of the male partner (with appropriate documentation after vasectomy of the absence of spermatozoa in the ejaculate).

5. Male subjects must use two effective methods of contraception throughout the study period and not donate semen for 90 days after the last administration of study drug.

6. Presence of at least one diabetic foot ulcer that meets the following criteria:

a) Diagnosed with full-thickness neuropathic DFU located at or distal to the ankle (excluding ulcers between the toes, but including ulcers on the heel).

b) SD: The ulcer is present for 6 weeks to 12 months and has an area of 3-5 cm ² after excision of the ulcer, confirmed by screening.

MD: The ulcer is present for 6 weeks to 12 months and has an area of 3-5 cm ² after excision of the ulcer, confirmed at screening after a 2-week preparatory period.

c) A minimum distance of 2 cm between an ulcer suitable for examination and any other ulcers on the specified foot.

d) Depth ≥5 mm and Grade 1A according to The University of Texas Staging System for Diabetic Foot Ulcers (22) with no exposed capsules, tendons or bones, and without tunnels, ruptures, or sinuses found after initial excision of the ulcer.

7. The subject must be able to hold the target ulcer in such a position and orientation that the investigational drug can be applied without significant loss of substance due to runoff until a dressing is applied.

8. Adequate vascular perfusion of the affected limb, demonstrated within 30 days prior to screening, as determined by at least one of the following criteria:

a) Ankle-brachial index (ABI) ≥0.9 and ≤1.2, confirmed by transcutaneous oxygen partial pressure (TcPO2) > 50 mmHg.

b) Toe pressure (plethysmography) > 50 mmHg.

c) Doppler ultrasound (biphasic or triphasic) of at least two vessels in the ankle, where the result is consistent with adequate blood flow to the affected limb, as determined by SoC.

Main exclusion criteria:

Part 1 SD and Part 2 MD:

Subjects must not meet any of the following criteria to be eligible to participate in the experiment:

1. For female subjects only: Pregnancy confirmed by a positive serum pregnancy test result at screening and urinalysis performed on Day-1 or breastfeeding.

2. The subject has:

a) Ulcer(s) with infected cellulitis, osteomyelitis, or clinical signs or symptoms of infection as recommended by the Infectious Diseases Society of the America's Guidelines (IDSA) (19).

b) Gangrene or necrosis of any part of the affected limb.

c) Active or chronic Charcot foot on the limb under study.

d) Planned vascular surgery, angioplasty, thrombolysis, or revascularization procedure performed within 1 month prior to enrollment in the study.

e) Ulcers with exposure of the tendon, bone or joint capsule (ulcers extending through the dermis and subcutaneous tissue with granulation tissue are acceptable).

f) Ulcer(s) of non-diabetic etiology.

g) Previous major amputations of the same target foot.

h) Current or recent (3 weeks) antibiotic therapy for any reason.

i) as well as bedridden subjects or subjects with a life expectancy of less than one year.

3. Use of any other growth factor therapy within 6 months prior to screening.

4. History of malignancy in the 5 years prior to screening, or individuals with a strong family history of cancer (eg, familial cancers), excluding squamous cell or basal cell skin cancer that has been successfully treated.

5. Clinically significant cardiovascular, pulmonary, renal, endocrine, hepatic, neurological, psychiatric, immunological, gastrointestinal, hematological, or metabolic disease which, in the opinion of the Investigator, cannot be stabilized or may otherwise affect the safety of the subject or results of the study (in case of doubt, the doctor of the clinical trial appointed by the sponsor should be consulted).

6. Subject on hemodialysis or peritoneal dialysis, or with chronic renal failure (plasma creatinine > 2 mg/dL).

7. Subject with significantly abnormal key laboratory parameters affecting patient safety as assessed by PI.

Scope of studies / parts of the study:

The study consists of two parts:

- Part 1 SD - single incremental dose

- Part 2 MD - multiple incremental dose

Dosages:

SD: 0.3, 1, 3 and 6 µg/ ^mm2

MD: 1 and 3 µg/ ^mm2

Endpoints of the experiment:

Primary Endpoint:

Part 1 SD:

Safety:

Adverse Events (AE) and Adverse Drug Reactions (ADR)

Vital signs: systolic (SBP) and diastolic (DBP) blood pressure.

12-Lead Holter ECG Parameters (HR, PR, QRS, QTcF, QT)

Clinical laboratory studies (chemistry, hematology and urinalysis).

Part 2 MD:

Safety:

AE and ADR;

Vital Signs: SBP, DBP; temperature;

Triple ECG in 12 leads;

(In the event that any ECG/cardiovascular findings appear in Part 1 of the study, the SAC may also apply Holter monitoring for part or all of Part 2 as indicated);

12-lead ECG parameters obtained by Holter (HR, PR, QRS, QTcF, QT);

Abnormalities on 24-hour Holter ECG (general pauses > 2.5 seconds, atrial fibrillation and atrial flutter, ventricular starts, premature atrial contractions (PAC), premature ventricular contractions (PVC), aberrant morphology); heart rate 0-24 hours (according to 24-hour Holter ECG) and average hourly heart rate;

Clinical laboratory studies (chemistry, hematology and urinalysis).

Time point(s) of time to characterize this end point of the experiment: indicated above.

Secondary Endpoint:

Part 1 SD:

Pharmacokinetic (PK) variables:

The following PK parameters will be derived based on serum concentrations of the polypeptide of SEQ ID NO: 4:

AUC 0-12h, AUC00-24h, AUC0-t, AUC0-∞, Cmax, tmax, t½, CL/F, Vd/F;

AUC 0-12h DN, AUC00-24h DN, AUC0-t DN, AUC0-∞ DN, Cmax DN.

Immunogenicity variables: ADA Ct

Part 2 MD:

Pharmacokinetic (PK) variables:

The following PK parameters will be derived from serum concentrations of the polypeptide of SEQ ID NO: 4:

Day 1: AUC 0-12 h, Cmax and tmax;

Day 2 to Day 13: Ctrough

Last day of dosing (Day 14): AUC 0-12 h, AUC0-t, AUC0-∞, Ctrough, Cmax , Cmin, tmax, tmin, Cav and Rac, t½, CL/F, and Vd/F.

Variable parameters of immunogenicity:

Serum anti-drug antibody (ADA) concentrations 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 day (12th week).

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 area and ulcer volume. Cure will be defined as "complete recovery". Different definitions of "cure" will also apply (partial reduction: 50%, 66%, 75%).

Time points for evaluation of this end point of the experiment: as above.

Single incremental dose (SAD) is carried out at the following dosages: Cohort A: 0.3 μg/mm ² ; Cohort B: 1 µg/mm ² ; CA cohort: 3 µg/mm ² ; Cohort D: 6 µg/mm ² . Each cohort consists of subjects with a native sequence of SEQ ID NO: 4 to avoid the possibility of transferring effects between cohorts. This is especially important in relation to the hyperalgesia known for wild-type human NGF. Dose levels are adjusted as necessary, and washout periods may be included as necessary to achieve study objectives.

At single ascending dose (SAD), Standard of Care (SOC) was applied at screening and at each subsequent visit until the end of the follow-up period unless complete re-epithelialization/healing persisted for 2 consecutive visits. In this case, the SoC could be terminated and the subject's leg treated according to the investigator's judgment/decision. SoC consisted of the following procedures:

treatment of the target ulcer (any possible bleeding caused by the treatment of the wound was controlled only by squeezing and raising the leg),

dressing the lesion with paraffin gauze and applying a protective dressing from sterile gauze wipes,

use of unloading removable walkers (not removed during the observation period) after dressing.

In the event of a lesion becoming infected during the study, a sample for microbiological culture was to be taken from the lesion and systemic empiric antibiotic therapy was administered to the subject, as determined by the investigator, who should adjust therapy according to the result of the culture study. Every infection, especially a severe one, should be assessed for severity according to its intensity.

With multiple ascending dose (MAD), the study is conducted sequentially in two cohorts, each with native subjects. Target daily dose levels are 1 μg/mm ² and 3 μg/mm ² for polypeptide SEQ ID NO: 4 (20) or placebo (10). After screening, eligible subjects are treated according to standard of care (SOC; lead-in period); registration is confirmed after measuring the size of the ulcer. If the ulcer area decreases by 50% or more during this preparatory period, patients will not be included in the study.

Scope of study :

Determination of safety, pharmacokinetics, pharmacodynamics and other (tolerability) IMPs in humans.

results

Single incremental dose (SOC)

Single dose incremental dose (SAD) was used in four consecutive cohorts at the following dosages: Cohort A: 0.3 μg/mm ² ; Cohort B: 1 µg/mm ² ; CA cohort: 3 µg/mm ² ; Cohort D: 6 µg/mm ² above standard treatment. In all of these cohorts, quantifiable levels of the polypeptide of SEQ ID NO: 4 were detected in the systemic circulation of the respective subjects. Thus, the administered polypeptide is present in the subjects after administration.

Subjects were monitored for adverse events for 28 days following single dose administration. No adverse events associated with the administration of the polypeptide of SEQ ID NO: 4 were observed. Thus, administration of the polypeptide of SEQ ID NO: 4 was not associated with any observed adverse drug reaction. As a result of the absence of adverse drug reactions, it can be concluded that the polypeptide of SEQ ID NO: 4 is safe and well tolerated by humans.

prospects

The biological activity of the polypeptide of SEQ ID NO: 4 stems from its ability to stimulate growth, as well as to ensure the maintenance, proliferation and survival of cells, especially nerve cells.

As a result of this example, the profiles of safety, tolerability, pharmacokinetics and pharmacodynamics of the polypeptide of SEQ ID NO: 4 are investigated and confirmed after administration of single and multiple doses to a person.

Brief description of the drawings

Fig. 1. Process scheme according to Example 2, including modifications (optimizations) described in Example 2B.

Fig. 2. NEURITE EXTENSION TEST IN VITRO PC12.

Illustration of mean neurite length (A; mean length per well), total neurite length (B; mean total length per cell), and percentage of cells whose neurite length exceeds cell body length (C).

Bars represent the mean ± standard error of the mean. Statistical analysis: One-way ANOVA followed by Tukey's post hoc method compared to vehicle-treated group (*P<0.05; **P<0.01; ***P<0.001)

CHF6467=polypeptide SEQ ID NO: 4

Fig. 3. EFFICACY STUDY, 8-DAY COHORT

Blood glucose levels in db/db mice measured during skin biopsy. No differences were observed between animals assigned to different experimental groups.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 4. EFFICACY STUDY, 8-DAY COHORT.

Weight gain during the experiment (Day 0 and Day 7 after skin lesions).

CHF6467=polypeptide SEQ ID NO: 4

Fig. 5. EFFICACY STUDY, 8-DAY COHORT.

Thermal threshold. Delayed paw withdrawal on a plantar test performed on Days 3 and 7 after skin biopsy and NGF administration. Data are presented as mean+SEM. Statistical analysis was performed using Student's t-test, *p <0.05.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 6. EFFICACY STUDY, 8-DAY COHORT.

"Time to heal" is measured as the outer and inner area of the ulcer (see the pilot study report for details) up to day 8 after skin biopsy. Results are expressed as a percentage of the affected area on Day 0; data are presented as mean+SEM. See text for two-way ANOVA statistical analysis.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 7. EFFICACY STUDY, 8-DAY COHORT.

Time to ulcer healing (outer area) on Day 8, expressed as percentage of ulcer closure compared to Day 0. Data are presented as mean+SEM.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 8. EFFICACY STUDY, 8-DAY COHORT

Plasma NGF levels at sacrifice. Data are mean+SEM; Statistical Analysis: One-way ANOVA followed by Dunnett's post hoc test. *p<0.05, **p<0.01.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 9. EFFICACY STUDY, 30-DAY COHORT

Blood glucose concentration was measured at skin biopsy (d-1), after treatment with the polypeptide of SEQ ID NO: 4 (d-8) and at sacrifice (d28). See text for details.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 10. EFFICACY STUDY, 30-DAY COHORT

Increase in body weight during the experimental time. No differences were observed between the experimental groups.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 11. EFFICACY STUDY, 30-DAY COHORT

Plantar test results 0 and 7 days after skin biopsy and polypeptide administration of SEQ ID NO: 4 (N=8 in each group). Data from the 30-day cohort are presented as mean+SEM; statistical analysis was performed using Student's t-test.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 12. EFFICACY STUDY, 30-DAY COHORT

Thermal hyperalgesia measured on days 0 and 7 after needle biopsy. This graph combines data from both 8-day and 30-day experiments (N=16 in each group); data are means+SEM. Statistical analysis: Student's t-test, *p<0.05.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 13. EFFICACY STUDY, 30-DAY COHORT.

The table shows the day of healing during the follow-up, obtained from clinical observations. "No" means that healing was not observed.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 14. EFFICACY STUDY, 30-DAY COHORT.

Dynamics of the healing process, measured as the outer area of the ulcer, up to 29 days after skin biopsy. Results are expressed as a percentage of the affected area on Day 0; data are means+SEM. See text for statistical analysis.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 15. EFFICACY STUDY, cohorts 8+30 days

Time-to-heal of ulcer (outer area) on Day 8. The data presented in the graph was obtained by combining the values for both the 8-day and 30-day experiments; N=16. Data are presented as mean+SEM. Statistical Analysis: One-way analysis of variance followed by Dunnett's multiple comparison post hoc test: F(5, 77)=3.007, P=0.0156.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 16. EFFICACY STUDY, 30-DAY COHORT.

Plasma NGF levels were measured on day 30. Data are presented as mean+SEM; statistical analysis: one-way analysis of variance and Dunnett's post hoc test.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 17. EFFICACY STUDY, 30-DAY COHORT.

A. Photomicrographs illustrating the histology of the wound (H&E stain) after 7 days, taken according to the scheme presented in B.

D-F. Micrographs of the wound border illustrating MET (migrating epidermal tongue, migrating epidermal tongue) (D, E) and angiogenesis (F).

Fig. 18. EFFICACY STUDY, 30-DAY COHORT.

Representative photomicrographs of the re-epithelialization process in mice treated with vehicle, SEQ ID NO: 4 (1, 10 and 30 μg/day). For comparison, a micrograph of intact skin is provided. The thickness of the epidermal layer in different groups is shown in the graph. Data are presented as mean+SEM; statistical analysis: one-way analysis of variance followed by Tukey's post hoc test, **p<0.01; ****p<0.0001.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 19. EFFICACY STUDY, 30-DAY COHORT.

Mouse dorsal skin innervation anatomy visualized with PGP-9.5 immunostaining.

Fig. 20. EFFICACY STUDY, COHORT 8+30 DAYS.

PGP9.5-IR in the skin of intact animals (A), mice treated with vehicle (B) and SEQ ID NO: 4 (30 μg/day) (C), 30 days after induction of the lesion. D, E: ROI (region of interest, D) and cutoff setting (E) for a computer-aided image analysis procedure used to evaluate skin innervation after 30 days.

Fig. 21. EFFICACY STUDY, 8+30 DAY COHORS.

Morphometric analysis of PGP9.5-IR in skin 8 (A) and 30 (B) days after ulcer 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 SEQ ID NO: 4

Fig. 22. EFFICACY STUDY, 8+30 DAY COHORS.

Histological (H&E staining) and immunohistochemical analysis of healed (repaired) skin in the group treated with SEQ ID NO: 4 (30 μg/day). A-B: Repaired skin layers on basement membrane as indicated by laminin staining (B, arrows); reinnervation originates from the subepidermal plexus and projects upward through the basement membrane (C). MET (D) is significantly innervated both 8 days (E) and 30 (F) days after the lesion; angiogenesis is observed in MET as visualized by H&E (G), laminin-IR staining at low (H) and high (I) magnifications.

Abbreviations: ep, epidermal layer; MET- migratory epidermal tongue

Fig. 23. EFFICACY STUDY, 8+30 DAY COHORS.

Morphometric analysis of Laminin-IR in skin 8 days (A) and 30 days (B) after ulcer induction. The gray horizontal bar in plot B represents 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.

FIG. 24. POLYPEPTIDE SEQUENCES. Asterisk (*) = POSITION 61 IN MATURE HUMAN NGF; CROSS (+): POSITION 100 IN MATURE HUMAN NGF.

A : SEQ ID NO: 1: pre-pro-human NGF sequence encoded by the corresponding open reading frame of the human gene.

Pre peptide: amino acid positions 1-18; pro peptide: amino acid positions 19-121; mature NGF: amino acid positions 122-239; C-terminal dipeptide: amino acid positions 240-241.

Disulfide bonds (in a properly folded mature part): linking positions of amino acids 136 ↔201, 179 ↔ 229, 189 ↔ 231.

Furin cleavage site (RSKR): amino acid positions 118-121.

B : Schematic overview of prepeptide, propeptide and mature NGF.

C : SEQ ID NO: 2: mature human NGF sequence.

D : SEQ ID NO: 3

E : SEQ ID NO: 4

Fig. 25. The rate of wound healing after treatment with vehicle or polypeptide of SEQ ID NO: 4 (1-10-100 μg/cm ² /day) in diabetic mice is presented as the "Proportion of mice with a non-healed surface" during the entire course of the study. Statistical Analysis: Kaplan-Meier Survival Curve Estimation.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 26.Morphometric analysis of PGP9.5-IR (A) and PECAM1-IR (A) in repaired skin 28 days after ulcer induction. Data are expressed as mean ± standard error of the mean (n=15-18). Statistical analysis: one-way analysis of variance followed by Dunnett's post hoc test; *p<0.05; **p<0.01.

CHF6467=polypeptide SEQ ID NO: 4

Fig. 27.The effect of the polypeptide SEQ ID NO: 4 on the mechanical pain threshold in the area surrounding the ulcer margin was assessed after 14 days of chronic topical treatment using the Bioseb von Frey electronic apparatus. Data are expressed as mean ± standard error of the mean (n=15-18). Statistical analysis: one-way analysis of variance.

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SEQUENCE LIST

<110> Kiesi Farmacukici S.p.A.

<120> Drug for the treatment of dermatological diseases

<130> D411P2

<140>

<141>

<150>EP18194930.6

<151> 2018-09-17

<150>EP18203665.7

<151> 2018-10-31

<160> 4

<170> BiSSAP 1.3.6

<210> 1

<211> 241

<212> PROTEIN

<213> Homo sapiens

<220>

<223> Human pre-pro-NGF sequence,

encoded by the corresponding human open reading frame.

<400> 1

Met Ser Met Leu Phe Tyr Thr Leu Ile Thr Ala Phe Leu Ile Gly Ile

1 5 10 15

Gln Ala Glu Pro His Ser Glu Ser Asn Val Pro Ala Gly His Thr Ile

20 25 30

Pro Gln Ala His Trp Thr Lys Leu Gln His Ser Leu Asp Thr Ala Leu

35 40 45

Arg Arg Ala Arg Ser Ala Pro Ala Ala Ala Ile Ala Ala Arg Val Ala

50 55 60

Gly Gln Thr Arg Asn Ile Thr Val Asp Pro Arg Leu Phe Lys Lys Arg

65 70 75 80

Arg Leu Arg Ser Pro Arg Val Leu Phe Ser Thr Gln Pro Pro Arg Glu

85 90 95

Ala Ala Asp Thr Gln Asp Leu Asp Phe Glu Val Gly Gly Ala Ala Pro

100 105 110

Phe Asn Arg Thr His Arg Ser Lys Arg Ser Ser Ser His Pro Ile Phe

115 120 125

His Arg Gly Glu Phe Ser Val Cys Asp Ser Val Ser Val Trp Val Gly

130 135 140

Asp Lys Thr Thr Ala Thr Asp Ile Lys Gly Lys Glu Val Met Val Leu

145 150 155 160

Gly Glu Val Asn Ile Asn Asn Ser Val Phe Lys Gln Tyr Phe Phe Glu

165 170 175

Thr Lys Cys Arg Asp Pro Asn Pro Val Asp Ser Gly Cys Arg Gly Ile

180 185 190

Asp Ser Lys His Trp Asn Ser Tyr Cys Thr Thr Thr His Thr Phe Val

195 200 205

Lys Ala Leu Thr Met Asp Gly Lys Gln Ala Ala Trp Arg Phe Ile Arg

210 215 220

Ile Asp Thr Ala Cys Val Cys Val Leu Ser Arg Lys Ala Val Arg Arg

225 230 235 240

Ala

<210> 2

<211> 118

<212> PROTEIN

<213> Homo sapiens

<220>

<223> SEQ ID NO: 2: Mature human NGF sequence

<400> 2

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95

Ala Ala Trp Arg Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110

Ser Arg Lys Ala Val Arg

115

<210> 3

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SEQ ID NO: 3

<400> 3

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Pro Asn Pro Val

50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95

Ala Ala Trp Glu Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110

Ser Arg Lys Ala Val Arg

115

<210> 4

<211> 118

<212> PROTEIN

<213> Artificial sequence

<220>

<223> SEQ ID NO: 4

<400> 4

Ser Ser Ser His Pro Ile Phe His Arg Gly Glu Phe Ser Val Cys Asp

1 5 10 15

Ser Val Ser Val Trp Val Gly Asp Lys Thr Thr Ala Thr Asp Ile Lys

20 25 30

Gly Lys Glu Val Met Val Leu Gly Glu Val Asn Ile Asn Asn Ser Val

35 40 45

Phe Lys Gln Tyr Phe Phe Glu Thr Lys Cys Arg Asp Ser Asn Pro Val

50 55 60

Asp Ser Gly Cys Arg Gly Ile Asp Ser Lys His Trp Asn Ser Tyr Cys

65 70 75 80

Thr Thr Thr His Thr Phe Val Lys Ala Leu Thr Met Asp Gly Lys Gln

85 90 95

Ala Ala Trp Glu Phe Ile Arg Ile Asp Thr Ala Cys Val Cys Val Leu

100 105 110

Ser Arg Lys Ala Val Arg

115

<---

Claims (12)

1. A method of treating skin ulcers in a mammalian subject, comprising administering a polypeptide of SEQ ID NO: 4, wherein the polypeptide is administered topically and applied to the surface of an injured body part, and wherein the dose/each dose has an amount of 0.3 to 6 μg of the polypeptide per mm ² of the treated damaged body surface, which is from 0.3 to 6 μg / mm ² . 2. The method of claim 1 wherein the mammalian subject is a human. 3. The method according to any one of the preceding claims, wherein the mammal, preferably a human, is diabetic or has a predisposition to diabetes. 4. The method of claim 3 wherein the diabetes mellitus is selected from type 1 diabetes mellitus and type 2 diabetes mellitus. 5. The method of any one of the preceding claims, wherein the polypeptide is administered once. 6. The method of any one of claims 1 to 4, wherein the polypeptide is administered multiple times from one to five times a day. 7. The method of claim 6 wherein the polypeptide is administered twice daily. 8. The method of claim 6 or 7 wherein the polypeptide is administered repeatedly over a period of three to 30 days, preferably seven to 14 days, or alternatively until the affected body surface has healed. 9. The method of any one of the preceding claims, wherein the polypeptide is administered to subjects with diabetic foot ulcers (DFU), preferably subjects with neuropathic diabetic foot ulcers (DFU). 10. The method of any one of the preceding claims, wherein the treatment and/or prophylaxis does not cause hyperalgesia in the mammal. 11. The method of any one of the preceding claims, wherein the polypeptide is contained in an aqueous medium and the aqueous medium is administered to the mammal. 12. The method of any one of the preceding claims, wherein the polypeptide can be obtained by recombinant expression and purification, wherein the purification comprises a mixed stationary phase purification.

Images