Detoxifying peptide

Abstract

The invention relates to a detoxifying peptide, characterised in that the sequence thereof comprises the following general amino acid sequence: N-(Val-Gly-X1-X2-Pro-Gly)n-OH, wherein: N and OH correspond to the N-terminal and C-terminal ends of the peptide, respectively; X1 and X2 are selected from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gin), alanine (Ala), leucine (Leu) and isoleucine (Ile); n is an integer between 1 and 3; for use in the treatment and / or prevention of disorders related to the glycation and / or glycoxidation of proteins and / or lipids.

Description

Description Title of the invention: Detoxifying peptide

[0001] [The present invention relates to a peptide which will find its application in particular in the pharmaceutical field.

[0002] The peptide according to the invention consists more specifically of a peptide that can be described as detoxifying, and will find an application mainly in the field of prevention and / or treatment of certain pathologies involving a transcription factor, NRF2 (nuclear factor erythroid-2-related factor 2), the peptide of the invention being capable of acting on the expression and / or translocation of NRF2, resulting, in particular, in an increase in autophagy and, consequently, in a reduction of toxic molecular compounds, including in particular advanced glycation end products, or AGEs for "Advanced Glycation End Products", or even ROS (reactive oxygen species).

[0003] The process of glycoxidation results from a combined effect of oxidation and glycation.

[0004] Glycation is a complex process involving a spontaneous, slow, non-enzymatic reaction that occurs between free amino groups in proteins and a reducing sugar, such as glucose or ribose, to form an unstable Schiff base.

[0005] These reversible reaction products then undergo irreversible oxidation, polymerization, dehydration, and crosslinking reactions to generate advanced glycation end products, or AGEs. These products include carboxyethyl-lysine (CEL), carboxymethyl-lysine (CML), glyoxal-lysine dimer (GOLD), 3-deoxyglucosone lysine dimer (DOLD), pyrroline, fructose-lysine, glucosepane, pentosidine, Argpyrimidine, pyrraline, hydroxymethylfurfural (HMF), N5-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1), fructosyl-lysine (FL).

[0006] Antiglycation research is a rapidly growing area of ​​interest in health and medicine, as it is increasingly recognized that AGEs may play an important role in the development of various chronic diseases, such as diabetes, cardiovascular disease (ischemia, cardiac fibrosis, arteriopathies, etc.) cancer, organ fibrosis (liver, kidney, heart, etc.), Alzheimer's disease and other neurodegenerative diseases (such as Parkinson's disease, amyotrophic lateral sclerosis, or multiple sclerosis).

[0007] Numerous studies have also shown the involvement of AGEs in certain skin-related pathologies such as psoriasis or dermatitis.

[0008] It would therefore be particularly advantageous to identify active ingredients and to be able to propose compositions capable of countering the long-term effects of AGE formation and preventing or treating pathologies linked to AGEs.

[0009] Current therapeutic approaches to combat glycation, AGEs, and their effects are numerous and varied. Some strategies include the use of anti-glycation agents, such as glycation inhibitors, metal chelating agents, and AGE-degrading enzymes. However, research in this area is still in its infancy, and researchers are still striving to understand the underlying mechanisms of glycation and the role that specific molecules can play in inhibiting or reversing this process. Therefore, there remains a need for anti-glycation compounds that are specific, effective, safe, and bioavailable.

[0010] For example, some treatments for fibrosis are limited and focus primarily on controlling symptoms, reducing complications, and slowing disease progression.

[0011] Therefore, these various treatments do not address the underlying causes of organ fibrosis, hence the need for a strategy capable of treating or preventing the onset of fibrotic disease.

[0012] Various compounds from plant extracts, mineral extracts, or other natural extracts have been proposed to counteract the accumulation of AGEs. However, although natural extracts may be a potential source of anti-glycation compounds, obtaining the right extracts is a time-consuming and expensive process.

[0013] Indeed, plant extracts can contain many different compounds, some of which may have adverse effects or even impair antiglycation activity. Furthermore, the concentration of each compound can vary considerably depending on growing conditions, season, and geographic regions, as plants must be grown under specific conditions to obtain optimal quality and quantity of antiglycation compounds.

[0014] It should also be noted that plant extracts are expensive to produce because large quantities of raw materials are required to obtain sufficient quantities of active compounds.

[0015] Indeed, some plants contain only a small number of these compounds, meaning that large quantities of plants must be harvested to obtain enough compounds for effective use. Partly because of this variability in compounds found in plant extracts, it can be difficult to standardize formulations, reproduce results, and ensure consistent quality from batch to batch.

[0016] Additionally, extracting the desired compounds from the plant is often a complex process. Specific methods must be used to extract the compounds in a way that preserves their integrity and effectiveness. This often requires the use of expensive solvents and sophisticated equipment. Once the extracts are obtained, they must be tested to ensure they contain the desired compounds and are free of contaminants. These tests can also be expensive and time-consuming.

[0017] These drawbacks highlight the need to find alternatives to natural extracts to target specific molecules involved in glycoxidation processes and / or in detoxification mechanisms allowing the elimination and / or increase in the degradation of AGEs.

[0018] Chemical synthesis of active compounds is an alternative to plant extracts to combat glycation. However, obtaining anti-glycation chemical compounds is difficult for several reasons.

[0019] On the one hand, advanced glycation end products (AGEs) are heterogeneous and complex, making it difficult to understand their formation. Therefore, the search for compounds capable of specifically neutralizing AGEs is a challenge. AGEs and glycated molecules can be difficult, particularly because of the wide variety of possible molecular structures.

[0020] Furthermore, glycation can occur at different locations within proteins, which can affect their structure and function in different ways. This makes it difficult to obtain anti-glycation compounds that target glycation sites.

[0021] Therefore, the object of the invention is to obtain, by chemical synthesis, an anti-glycation compound which could target specific molecules involved in the glycoxidation process and / or in the degradation of AGEs, such as, for example, glycated proteins.

[0022] Additionally, plant extracts have another disadvantage: they are not physiological compounds. Physiological compounds are preferred because they are better tolerated by the body and generally have high bioavailability, meaning they are easily absorbed and utilized by the body.

[0023] The invention therefore aims to obtain a compound which would be well tolerated by the body and which would still overcome the drawbacks mentioned above.

[0024] Despite all the difficulties mentioned above which had to be overcome, the inventors succeeded in identifying and synthesizing, by chemical synthesis, a peptide having the effect of modulating the activity of the pathway of a specific nuclear transcription factor, noted as NRF2 or "nuclear factor erythroid-2-related factor 2" or nuclear factor related to erythroid 2, mentioned above.

[0025] The NRF2 pathway plays a crucial role in cellular redox homeostasis, and activation of this pathway is one of the defense mechanisms against glycoxidative stress (Motohashi and Yamamoto, 2004), notably by modulating autophagy pathways (via the autophagosome) and protein degradation, particularly via the ubiquitin / proteasome, lysosome, neddylation, and proteasome pathways.

[0026] However, it is also worth noting that the NRF2 pathway is known to be a complex and regulated pathway by multiple mechanisms, involving many different biochemical components, such as enzymes, receptors, and transcription factors.

[0027] Therefore, this can make it extremely difficult to identify peptides or other molecules capable of modulating the activity of the Nrf2 pathway specifically and efficiently.

[0028] Furthermore, the NRF2 pathway plays an important role in many different physiological processes, making it essential to design peptides specific enough to target the NRF2 pathway only in the specific context of combating glycoxidation and / or glycation of biological molecules such as, in particular, proteins and lipids.

[0029] Thus, through the present invention, the inventors have succeeded in overcoming these difficulties in order to propose a peptide capable of modulating the activity of the NRF2 pathway in an efficient and specific manner in the context of glycation and / or glycoxidation of proteins and / or lipids, by modulation of degradation pathways (autophagy, neddylation, ubiquitin / proteasome system, lysosome, etc.), so as to stimulate the degradation of advanced glycation end products, or AGEs.

[0030] Such a peptide can thus be considered as a peptide exhibiting detoxifying properties, and may therefore be designated, in the remainder of the description, as a detoxifying peptide.

[0031] For the purposes of the present invention, the term "detoxifying" should be understood as referring to the cellular processes involved in the degradation and / or elimination of molecular compounds, potentially toxic due to a glycation or glycoxidation process (proteins, lipids, nucleic acids, etc.), via the autophagosome, lysosome, ubiquitin / proteasome, neddylation, or antioxidant pathways.

[0032] Thus, the present invention relates more specifically to a detoxifying peptide comprising the following general amino acid sequence: N-(Val-Gly-X1-X2-Pro-Gly)n-OH in which: - N and OH correspond respectively to the N-terminal and C-terminal ends of said peptide; - X1 and X2 are chosen from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gin), alanine (Ala), leucine (Leu) and isoleucine (Ile); - n is an integer between 1 and 3; for use in the treatment and / or prevention of pathologies linked to the glycation and / or glycoxidation of proteins and / or lipids, such pathologies being due to an accumulation of advanced glycation end products, or AGEs for “Advanced Glycation End Products” in the tissues of an organ, said detoxifying peptide being capable of allowing a reduction of AGEs in said tissues and / or of promoting the degradation of AGEs, by activation of the pathway of a specific nuclear transcription factor, namely the NRF2 factor.

[0033] Preferably, the detoxifying peptide has the following general amino acid sequence: N-(Val-Gly-X1 -X2-Pro-Gly)n-Arg-X3-Arg-X4-X5-X6-X7-OH in which: - N and OH correspond respectively to the N-terminal and C-terminal ends of said peptide; - X1 and X2 are chosen from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gin), alanine (Ala), leucine (Leu) and isoleucine (Ile); - X3 and X4 are chosen from valine (Val), serine (Ser), tyrosine (Tyr), alanine (Ala), glycine (Gly), arginine (Arg) and leucine (Leu); - X5, X6 and X7 are chosen from isoleucine (Ile), leucine (Leu), valine (Val), alanine (Ala), glycine (Gly) and phenylalanine (Phe) - n is an integer between 1 and 3.

[0034] In a particularly preferred embodiment, the peptide of the invention has SEQ ID NO: 12, namely N-(Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-lle-Leu-OH, with N and OH corresponding respectively to the N-terminal and C-terminal ends of said peptide, and with n being equal to 3.

[0035] The sequence of the peptide of the invention may also be chosen from the group consisting of the sequences SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 13 to SEQ ID NO: 37.

[0036] The present detoxifying peptide may have use in increasing the expression of the transcription factor NRF2 (nuclear factor erythroid-2-related factor 2” or nuclear factor erythroid 2) and / or to activate the NRF2 transcription factor pathway and / or to activate the nuclear translocation of NRF2, resulting in the activation of genes involved in the lysosome and / or ubiquitin / proteasome and / or autophagy and / or neddylation pathways.

[0037] The detoxifying peptide of the invention may, therefore, have a use for increasing the degradation of advanced glycation end products or AGEs, via the NRF2 factor pathway, for example for increasing the degradation of carboxyethyl lysine (CEL), carboxymethyl lysine (CML), glyoxal lysine dimer (GOLD), 3-deoxyglucosone lysine dimer (DOLD), pyrroline, fructose-lysine.

[0038] Said detoxifying peptide may also have use in increasing the degradation of glycated vimentin, i.e. vimentin which is modified by CML and / or CEL.

[0039] In other words, the peptide of the invention acts on NRF2, and one of the consequences of this action, which will be demonstrated later in the description, is an increase in autophagy, resulting, among other things, in a reduction of molecular compounds that can be described as "toxic" for cells and tissues, and which include AGEs.

[0040] The detoxifying peptide of the invention therefore has a particularly interesting use in pathologies involving the NRF2 factor, and in which an abnormal accumulation of AGEs is observed in particular. Indeed, said peptide will have the effect of allowing an increase in the expression and / or translocation of NRF2 and thus improve the condition of a patient suffering from such a pathology.

[0041] Thus, the detoxifying peptide may have use in the treatment and / or prevention of side effects of radiotherapy and / or chemotherapy in cancer patients.

[0042] The detoxifying peptide may also have use in the treatment and / or prevention of skin fibrosis and / or liver fibrosis and / or non-alcoholic fatty liver disease (or NASH for “Non-Alcoholic Steato Hepatitis”) and / or hepatocarcinoma.

[0043] The detoxifying peptide which is the subject of the present invention can also have a use in the following pathologies, in which are involved, in particular, advanced glycation end products AGEs, in particular an accumulation of AGEs and / or a decrease in the degradation of these AGEs, in a person suffering from one of these pathologies, in comparison with a person not suffering from one of these pathologies:

[0044] - in the treatment and / or prevention of skin pathologies such as psoriasis and / or dermatitis and / or eczema;

[0045] - in the treatment and / or prevention of diabetes and its complications, such as diabetic neuropathies, diabetic nephropathy, glaucoma, cataracts and diabetic retinopathy;

[0046] - in the treatment and / or prevention of cardiovascular pathologies;

[0047] - in the treatment and / or prevention of neurodegenerative pathologies such as Alzheimer's disease and / or Parkinson's disease and / or multiple sclerosis (MS) and / or amyotrophic lateral sclerosis (ALS);

[0048] The detoxifying peptide can advantageously be obtained by chemical synthesis.

[0049] The present invention also relates to a pharmaceutical composition comprising at least one peptide as defined above, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier for use in the treatment and / or prevention of pathologies linked to the glycation and / or glycoxidation of proteins and / or lipids.

[0050] The peptide in accordance with the invention is therefore particularly interesting in that it constitutes an anti-glycation and anti-glycoxidation compound, by targeting the NRF2 factor in the context of the glycoxidation process to limit the latter, and in the degradation of glycated proteins, so that said peptide ultimately makes it possible to limit the accumulation of AGEs and / or to promote the degradation of AGEs which would have accumulated in the tissues of an organism.

[0051] Furthermore, said peptide advantageously comprises parts of physiological compounds released during the remodeling of the extracellular matrix (ECM), in particular during the degradation of ECM proteins by proteolytic cleavage.

[0052] Therefore, such a peptide has the advantage of being better tolerated by the body, compared for example with a plant extract, while having high bioavailability.

[0053] So, in other words, the peptide in question is not toxic in itself since its sequence is partly derived from that of a natural compound in the human body (elastin). Thus, not only is it detoxifying, but it also has minimal or no intrinsic toxicity, which is an advantage over other compounds.

[0054] It should be noted here that the role of such compounds released by ECM degradation, notably elastokines and matrikines, in certain processes such as tissue homeostasis or cell signaling has been previously studied, which is however not the case for the involvement of these compounds in the pathways targeted in this application.

[0055] Thus, the peptide which is the subject of the present invention, derived from elastin, is already known from the state of the art, given that it has already been the subject of a French patent application published under number FR 2 986 233.

[0056] In this application, the ability of the peptide to bind to an elastin-binding receptor protein, called EBP for "Elastin Binding Protein" in its English name, and located on the cell surface, is described to stimulate the synthesis of type I and type II collagen.

[0057] The action of the elastin-derived peptide as a competitive inhibitor of a protease, urokinase, which is one of the activators of the plasminogen-plasmin system, is also described. By binding to the active site of the urokinase protease, the elastin-derived peptide limits the activity of urokinase on plasminogen, this activity being responsible, indirectly, for the degradation of collagen, by an activation of enzymes, the Matrix Metallo-Proteinases, or MMPs, which notably exhibit collagenolytic activity.

[0058] Finally, in patent application FR 2 986 233, it is also explained how the peptide derived from elastin is responsible, this time directly, for the inhibition of type I MMPs, a part of this peptide being capable of occupying a part of the active site of MMP-1, and of acting as a competitive inhibitor of this enzyme.

[0059] As a result, the peptide finds application for the treatment of chronic healing diseases, including the treatment of bedsores or ulcers, for the repair and / or regeneration of dermal tissue, including the treatment of skin aging.

[0060] Therefore, document FR 2 986 233 teaches a mode of action of the peptide directly linked to the interaction of enzymes, such as proteases, or with extracellular receptors, which have physiological functions very different from a transcription factor, resulting in equally very different applications, which are the subject of the present invention.

[0061] Other aims and advantages of the present invention will appear during the description which follows relating to embodiments which are given only as indicative and non-limiting examples.

[0062] Understanding of this description will be facilitated by referring to the attached drawings in which:

[0063] [Fig.1] comprises two graphs, a first graph (FIG.1 A at the top of the figure) illustrating the intensity of detection of carboxymethyl-lysine (CML) by immunofluorescence on histological slides, in fibroblasts previously subjected in vitro to a glycation inducer in the form of increasing doses of glyoxal, in the absence (white bars) and in the presence (grey bars) of the peptide in accordance with the invention (denoted BFP in the figure), and a second graph (FIG.1 B at the bottom of the figure) illustrating, under similar conditions, the intensity of glycation of a protein, vimentin.

[0064] [Fig.2] includes three graphs:

[0065] - the first graph, on the upper part of the figure (FIG. 2A), illustrates the results of concentration measurement tests, by ELISA (Enzyme-Linked ImmunoSorbant Assay) technique, of total vimentin, obtained on skin explants cultured ex vivo, under different culture conditions, namely without being subjected to oxidative stress (“no stress”, white rectangle), by being subjected to oxidative stress via 0.5 mM glyoxal without being treated with the peptide of the invention (“Glyoxal 0.5 mM -BFP >>, light gray rectangle) or by being treated with the peptide of the invention (“Glyoxal 0.5 mM +BFP >>, dark gray rectangle);

[0066] - the second graph, in the middle of the figure (FIG. 2B), illustrates the results of tests for detecting the intensity of glycation of vimentin, obtained by immunofluorescence on histological slides on skin explants cultured ex vivo, under the culture conditions described in connection with FIG. 2A;

[0067] - the third graph, at the bottom of the figure (FIG. 2C) illustrates the results of nuclear detection tests of the transcription factor NRF2 obtained by immunofluorescence on histological slides on skin explants cultured ex vivo, under the culture conditions described in connection with FIG. 2A.

[0068] [Fig.3] illustrates the results of tests carried out in vivo, in humans, for the detection of vimentin on a first graph, at the top of the figure (FIG.3A) and for the detection of CML on a second graph, at the bottom of the figure (FIG.3B), by immunofluorescence on histological slides of skin explants from volunteers who underwent a surgical facelift after a 28-day treatment with creams with the peptide (grey bars, “Vimentin intensity BFP” and “CML BFP”) or without (white bars, “Vimentin intensity Placebo” and “CML Placebo”).

[0069] [Fig.4] is a table showing the genes involved in the processes of detoxification by autophagy, mitophagy, neddylation, degradation by the ubiquitin / proteasome system and whose transcription is induced by the peptide of the invention.

[0070] [Fig.5] illustrates the results of RT-qPCR carried out on skin explants cultured ex vivo, under different culture conditions, namely: without being subjected to oxidative stress (“no stress”), by being subjected to oxidative stress via 0.5 mM glyoxal without being treated with the peptide of the invention (“Glyoxal 0.5 mM -BFP”) or by being treated with the peptide of the invention (“Glyoxal 0.5 mM +BFP”), in order to analyze the regulation of genes differentially regulated by the action of the peptide of the invention (denoted BFP in the figures) after induction of glycation identified by genomic analysis, the results of which are illustrated in Figure 4.

[0071] For preliminary and informative purposes, this description uses either the three-letter code to designate the amino acids that form the sequence of the detoxifying peptide, or the one-letter code.

[0072] Thus, Ala or A corresponds to alanine, Cys or C to cysteine, Asp or D to aspartic acid, Glu or E to glutamic acid, Phe or F to phenylalanine, Gly or G to glycine, His or H to histidine, Ile or I to isoleucine, Lys or K to lysine, Leu or L to leucine, Met or M to methionine, Asn or N to asparagine, Pro or P to proline, Gln or Q to glutamine, Arg or R to arginine, Ser or S to serine, Thr or T to threonine, Val or V to valine, Trp or W to tryptophan and Tyr or Y to tyrosine.

[0073] It should also be noted that "glycoxidation" includes a process called glycation, where a sugar molecule (such as glucose) reacts with the amino group of proteins or the amino group of lipids (in the case of glycolipids). This initial reaction, often reversible, results in the formation of an unstable Schiff base, which subsequently undergoes a molecular rearrangement, known as Amadori. In later stages of the reaction, the Amadori products undergo additional modifications such as cleavage or cyclization, which generate a group of heterogeneous compounds called "advanced glycation endproducts" (AGEs), which are highly reactive and irreversible and can persist for long periods. Because these late stages include many oxidative phenomena, this process is often referred to as "glycoxidation."

[0074] The terms "AGEs" or "AGEs" (plural) refer to advanced glycation end products, which are harmful molecules that form when proteins or lipids in the body are glycated, that is, they bind to sugar molecules. AGEs can accumulate in various tissues throughout the body, including the skin, arteries, and kidneys. They can damage these tissues by promoting inflammation, oxidative stress, and other harmful processes, leading to cellular damage and dysfunction.

[0075] It should be noted that AGEs can be mainly detected by immunochemistry, using a specific antibody, which could be done to obtain the results which will be described below in the examples in connection with the figures, in particular with the use of an anti-CML antibody. This technique is well known to those skilled in the art.

[0076] "Physiological compounds" are molecules produced naturally by the body. These compounds can include a wide range of molecules such as enzymes, hormones, neurotransmitters, metabolites, etc. The terms "physiological compounds released by the ECM" correspond to compounds such as matrikines and elastokines.

[0077] The term "synthetic peptide" refers to peptides obtained by chemical synthesis and not from extraction such as plant extracts.

[0078] The subject of the present invention is a detoxifying peptide whose sequence comprises at least the following general amino acid sequence: N- (Val-Gly-X1 -X2-Pro-Gly)n-OH in which:

[0079] - N and OH correspond respectively to the N-terminal and C-terminal ends of said peptide

[0080] - X1 and X2 are chosen from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gin), alanine (Ala), leucine (Leu) and isoleucine (Ile);

[0081] - n is an integer between 1 and 3;

[0082] for use in the treatment and / or prevention of pathologies linked to the glycation and / or glycoxidation of proteins and / or lipids.

[0083] Most preferably, X1 corresponds to valine (Val) and X2 corresponds to alanine (Ala), so that the peptide has the sequence N-(Val-Gly-Val-Ala-Pro-Gly)n-OH.

[0084] Even more preferably, the number n is equal to 3.

[0085] Thus, when n is equal to 1, the peptide of the invention has the general sequence SEQ ID NO: 1, and, most preferably, the sequence SEQ ID NO: 2 (VGVAPG).

[0086] When n is equal to 2, the peptide of the invention has the general sequence SEQ ID NO: 3, and, most preferably, the sequence SEQ ID NO: 4 (VGVAPGVGVAPG).

[0087] When n is equal to 3, the peptide of the invention has the general sequence SEQ ID NO: 5, and, most preferably, the sequence SEQ ID NO: 6 (VGVAPGVGVAPGVGVAPG).

[0088] Advantageously, the present invention relates more particularly to a detoxifying peptide having the following general amino acid sequence:

[0089] N-(Val-Gly-X1 -X2-Pro-Gly)n-Arg-X3-Arg-X4-X5-X6-X7-OH

[0090] In this general sequence:

[0091] - N and OH correspond respectively to the N-terminal and C-terminal ends of said peptide;

[0092] - residues X1 and X2 each consist of an amino acid chosen from the following amino acids: valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gin), alanine (Ala), leucine (Leu) and isoleucine (Ile);

[0093] - residues X3 and X4 each consist of an amino acid chosen from the following amino acids: valine (Val), serine (Ser), tyrosine (Tyr), alanine (Ala), glycine (Gly), arginine (Arg) and leucine (Leu);

[0094] - residues X5, X6 and X7 each consist of an amino acid chosen from the following amino acids: isoleucine (Ile), leucine (Leu), valine (Val), alanine (Ala), glycine (Gly) and phenylalanine (Phe);

[0095] - n is an integer between 1 and 3.

[0096] When n is equal to 1, the peptide of the invention has the general sequence SEQ ID NO: 7.

[0097] When n is equal to 2, the peptide of the invention has the general sequence SEQ ID NO: 8.

[0098] When n is equal to 3, the peptide of the invention has the general sequence SEQ ID NO: 9.

[0099] The peptide having such a general sequence is more particularly intended, according to the present invention, for use in the treatment and / or prevention of pathologies linked to the glycation and / or glycoxidation of proteins and / or lipids.

[0100] Most preferably, the sequence of the detoxifying peptide consists of the sequence SEQ ID NO: 12 of the sequence listing, namely N-(Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-lle-Leu-OH, with N and OH corresponding respectively at the N-terminal and C-terminal ends of said peptide, and with n being equal to 3.

[0101] The sequence of the detoxifying peptide of the invention may also consist, for example, of one of the sequences among: SEQ ID NO: 10 (corresponding to n=1), SEQ ID NO: 11 (corresponding to n=2) and SEQ ID NO: 13 to SEQ ID NO: 37.

[0102] The detoxifying peptide of the present invention is particularly interesting for use aimed at increasing the expression and / or, preferentially, the nuclear translocation of the transcription factor NRF2 (nuclear factor erythroid-2-related factor 2) and / or for activating the NRF2 transcription factor pathway leading to the activation of genes involved in the lysosome and / or ubiquitin / proteasome and / or autophagy and / or neddylation pathways, and this in the specific and particular context of the glycoxidation and glycation pathways, as will be demonstrated in the remainder of the description, in connection with the examples and figures illustrating results of tests carried out with said peptide.

[0103] Consequently, the detoxifying peptide of the invention, in particular having the sequence SEQ ID NO: 12, through the modulation of the NRF2 factor pathway, itself acting on the modulation of certain degradation pathways by activation of genes involved in these (autophagy, lysosome, ubiquitin / proteasome, or even neddylation), increases the degradation of advanced glycation end products, AGEs, such as in particular carboxyethyl-lysine (CEL), carboxymethyl-lysine (CML), glyoxal lysine dimer (GOLD), 3-deoxyglucosone lysine dimer (DOLD), pyrroline, fructose-lysine.

[0104] Furthermore, the detoxifying peptide according to the invention promotes the degradation of glycated proteins.

[0105] Advantageously, the peptide promotes the degradation of glycated vimentin.

[0106] The detoxifying peptide therefore presents, in a particularly interesting way, several functions, allowing both a detoxifying action on glycated proteins, while allowing modulation at different levels of the cascades of the NRF2 pathway.

[0107] Glycation of biological molecules is likely to modify their biomechanical and functional properties.

[0108] Among them, vimentin is a protein involved in the regulation of cell shape and migration, as well as in the response to cellular stress.

[0109] Although essential for normal cell function, high levels of vimentin and its glycation can contribute to the development of diseases, which is why the inventors are considering vimentin as a therapeutic target for several treatments.

[0110] Therefore, the invention also relates to the peptide for use in the treatment and / or prevention of side effects of radiotherapy and / or chemotherapy in cancer patients.

[0111] Glycation of extracellular matrix proteins induces changes in the shape and distribution of fibroblasts.

[0112] Thus, the invention also relates to this detoxifying peptide for use in the treatment and / or prevention of fibrosis, whether cutaneous or involving other tissues and organs such as the liver.

[0113] Indeed, it has been proposed that vimentin levels are correlated with a pro-fibrotic profile. Indeed, vimentin levels have been shown to be elevated in renal fibrosis (Lin et al., 2023). In the cutaneous context, vimentin is decreased by negative pressure wound therapy (Wu et al., 2022). Thus, therapeutic treatments leading to a decrease in vimentin levels or expression could be considered to have anti-fibrotic properties.

[0114] In particular, the neddylation pathway is particularly involved in hepatic fibrosis, NASH (“Non-Alcoholic Steatosis Hepatitis”), or even hepatocarcinoma.

[0115] Indeed, oxidative stress is recognized as a major factor in the progression of NASH. The transcription factor NRF2 and its negative regulator KEAP1 are master regulators of redox, metabolic and protein homeostasis, as well as detoxification, and therefore appear to be attractive targets for the treatment of NASH. Disruption of the KEAP1-NRF2 interaction triggers the increase in antioxidant response and the coordinated regulation of a wide range of genes involved in NASH progression, which ultimately leads to the reduction of NASH progression and liver fibrosis in mice (Seedorf et al., 2023).

[0116] The detoxifying peptide is therefore also of interest for use in the treatment and / or prevention of such pathologies.

[0117] In another particular aspect, the invention relates to the peptide described above, used as an anti-glycation peptide, an antioxidant peptide and an anti-inflammatory peptide.

[0118] Accumulation of glycation-modified vimentin can be observed in skin fibroblasts from aged donors in vivo, providing a close link between changes in AGEs in human tissues, such as skin, and loss of organ contractile functions.

[0119] Intracellular vimentin filaments found within fibroblasts undergo extensive glycation, and these filaments then redistribute into aggregates. The modification of these filaments results in a decrease in the contraction property of fibroblasts.

[0120] The invention therefore also relates to this peptide intended to be used in the treatment and / or prevention of loss of contractile functions of organs.

[0121] The inventors consider that activation of NRF2 is a treatment and / or prevention option for various skin diseases.

[0122] Indeed, under pathological conditions, NRF2 activation appears to be a promising therapeutic option for various skin diseases. Atopic dermatitis (AD) and eczema, as well as psoriasis, are common chronic inflammatory skin diseases caused by a defective skin barrier, dysregulated immune responses, genetic predispositions, and environmental factors (Ogawa and Ishitsuka, 2022). Psoriasis is accompanied by an intense process of glycoxidation (Damasiewicz-Bodzek and Nowak, 2022).

[0123] Atopic dermatitis and eczema, which are two very similar diseases, reveal compromised activation of the NRF2-antioxidant pathway in the affected epidermal regions (Koch et al., 2023).

[0124] Psoriasis is a chronic, relapsing, and often severe skin disease that is frequently associated with metabolic disturbances and an increased risk of cardiovascular complications.

[0125] One of the postulated links is the intensification of the process of glycation and / or advanced glycoxidation of proteins.

[0126] In patients with psoriasis, the concentrations of CML, CEL and sRAGE (soluble receptor for AGEs) are significantly higher than in healthy individuals.

[0127] Disease remission was followed by a significant decrease in CML, CEL, and sRAGE concentrations compared to active patients. Psoriasis is accompanied by an intense glycoxidation process, and elevated sRAGE levels appear to reflect a permanent overstimulation of RAGE, the receptor for AGEs (Damasiewicz-Bodzek and Nowak, 2022).

[0128] Therefore, the present invention also relates to the detoxifying peptide for use in the treatment and / or prevention of skin-related pathologies such as psoriasis or atopic dermatitis.

[0129] Indeed, as will be demonstrated subsequently in the examples of the present application, the peptide of the invention allows, in particular, a significant reduction in the CML level.

[0130] The peptide of the invention could also be used in the treatment and / or prevention of radiodermatitis, a disease occurring after irradiation.

[0131] Indeed, irradiation by ionizing radiation generates ROS and glycation. Thus, NRF2 has emerged as a crucial regulator of cellular defense mechanisms activating complex transcriptional programs that facilitate the detoxification of reactive oxygen species, repair oxidative damage, and prevent toxicity (Cohen et al., 2022).

[0132] Advanced glycation end products (or AGEs) are associated with the pathogenesis of diabetic complications.

[0133] Nrf2 activators combat oxidative stress and improve the wound healing process in several pathophysiological conditions, including diabetes and its complications such as diabetic foot ulcer, chronic kidney disease, and diabetic nephropathy. Several bioactive compounds have been reported to reduce cellular stress and thereby accelerate cell proliferation, neovascularization, and repair of damaged tissues through activation of the Nrf2 transcription factor (Victor et al., 2020).

[0134] Numerous studies have shown that oxidative stress plays an important role in the pathophysiology of diabetic foot ulcer (DFU). The nuclear factor erythroid-related factor 2 (Nrf2) signaling pathway and advanced glycation end products (RAGE) biochemical pathways play a critical role in regulating oxidative stress in the body. By targeting these pathways to restore redox balance, it is possible to control and mitigate the onset and development of pathologies following severe diabetes (Song et al., 2022).

[0135] The invention relates to this peptide for use in the treatment and / or prevention of diabetes and its complications, such as diabetic neuropathies, diabetic nephropathy, glaucoma, cataracts, or even diabetic retinopathy.

[0136] Furthermore, acute activation of NRF2 is cardioprotective (Collins et al., 2009; Howden, 2013; Howden et al., 2013; Li et al., 2022; Tanase et al., 2022).

[0137] Thus, the invention also relates to this peptide for use in the treatment and / or prevention of cardiovascular diseases.

[0138] NRF2 dysregulation is associated with several neurological diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

[0139] In these diseases, activation of NRF2 signaling pathways may provide neuroprotection by reducing oxidative stress and inflammation.

[0140] Furthermore, these pathologies are known to be linked to an accumulation of AGEs (Reddy et al., 2022).

[0141] Thus, the invention also relates to this peptide for use in the treatment and / or prevention of neurological diseases, in particular in the group of Alzheimer's disease, Parkinson's disease, Huntington's, multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS).

[0142] Another aspect of the invention relates to pharmaceutical compositions comprising at least one peptide for the uses which have been listed previously in the treatment and / or prevention of pathologies linked to glycation and / or glycoxidation, a mixture of several peptides, and / or its or their pharmaceutically acceptable salts, with or without pharmaceutically acceptable adjuvants.

[0143] In a non-limiting manner, the present invention covers a peptide which may be in solution or in cream, combined or not with carrier molecules to obtain such a composition.

[0144] In a particular embodiment, the peptide is used in the composition at a concentration substantially between 10 pg / ml and 1 mg / ml.

[0145] Indeed, no toxicity of the peptide according to the invention was observed on fibroblasts treated with peptide concentrations of up to 1 mg / ml.

[0146] More preferably still, the concentration recommended for the use of the detoxifying peptide of the invention, in particular in pharmaceutical compositions, is between 50 and 100 pg / mL.

[0147] It should also be noted that the detoxifying peptide of the invention can, in a particularly advantageous manner, be obtained by chemical synthesis, using any technique suitable for this purpose and known to those skilled in the art.

[0148] The following examples, describing in detail the operating mode and the results of tests conducted with the detoxifying peptide of the invention, make it possible to illustrate, in connection with the figures of the attached drawings, in particular the effects of said peptide on certain markers characteristic of glycation and glycoxidation, and its action on the NRF2 factor pathway.

[0149] Example 1: In vitro evaluation of SMCs and glycated vimentin in cultured fibroblasts

[0150] Sample preparation:

[0151] Cells were seeded at 10,000 cells per well (96-well plate) at 37°C, 5% CO2, 10% FBS (fetal bovine serum) and maintained under optimal growth conditions for 3 days.

[0152] After 3 days, glycation was induced with glyoxal at different doses (0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM) for 24 hours. The detoxifying peptide (or BFP peptide in the figures) was then added at 50 pg / mL for an additional 24 hours.

[0153] The peptide that was tested in this example has the sequence SEQ ID NO: 12, namely VGVAPGVGVAPGVGVAPGRVRLGIL or N-(Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-lle-Leu-OH, with n being equal to 3.

[0154] The control group (cells incubated with glyoxal at different doses) received no treatment except for renewal of the culture medium.

[0155] Cell labeling and image acquisition:

[0156] After treatments, cells were washed, saturated in non-specific sites (PierceTM in PBS (Phosphate Buffered Saline, pH 7.4 solutions)) and fixed on the plate, and incubated for 1 hour with the primary antibody (Rabbit monoclonal anti-Vimentin - Abeam (ab92547) or mouse monoclonal anti-CML - R&D Systems (MAB3247)) in PierceTM solution in PBS.

[0157] After incubation, cells were washed with PBS and incubated for 1 hour with the fluorochrome-coupled secondary antibody (Goat antimouse AlexaFluor 488 - Invitrogen (A11001) or Goat anti-Rabbit AlexaFluor 647 - Invitrogen (A21244)) and the nuclei were labeled with DAPI (4',6-diamino-2-phenylindole). After incubation, cells were washed with PBS and a series of images were acquired with an epifluorescence microscope (ThermoFisher, Evos M5000, 40X objective).

[0158] Images were collected and analyzed with ImageJ software (Rasband, NIH).

[0159] Quantification on each image was acquired by integration of the specific fluorescence signal. The level of vimentin was quantified by evaluating the area occupied above a fluorescence intensity threshold, then normalized to the number of cells.

[0160] The CML level was quantified by integration of the signal intensity above a threshold, normalized by the area of ​​the evaluation zone and then by the number of cell nuclei.

[0161] Glycated vimentin levels were obtained as the ratio of quantified CML intensity signal (RFU / cell) to vimentin occupancy (area / cell), then normalized to the control group. Statistical analyses were performed using the t-test for binary comparisons (p-value = 0.05).

[0162] The results obtained illustrated in Figure 1 demonstrate that the detoxifying peptide of the invention (BFP) promotes the degradation of CML (Figure 1 A) and glycated vimentin (Figure 1 B) in vitro.

[0163] For the record and in summary, human fibroblasts, incubated with increasing concentrations of glyoxal for 24 hours, were treated or not with the detoxifying peptide for an additional 24 hours.

[0164] As shown in Figure 1 A, CML detection increases compared to control (no stress) and is correlated with glyoxal concentration (white bars in the histogram).

[0165] Treatment with the detoxifying peptide significantly reduced the detection of CML, particularly at the highest concentrations of glyoxal tested, namely 0.6 mM and 0.8 mM (grey bars in the histogram).

[0166] Interestingly, as shown in Figure 1 B, vimentin-specific glycation is decreased in fibroblasts stressed by 0.6 mM glyoxal after BFP treatment (compared to non-treatment).

[0167] The detoxifying peptide of the invention therefore makes it possible to reduce in vitro cellular levels of glycation, and more particularly the glycation of vimentin, which is a preferred target of glycation.

[0168] Example 2: Ex vivo evaluation of CML, glycated vimentin and NRF2 in skin explants

[0169] Sample preparation:

[0170] Skin explants were obtained with informed consent from a 32-year-old Caucasian woman (phototype ll / ll, Ref DISC1 D10J025) from abdominal surgery. Explants were kept alive by culture on metal grids in a medium at 37°C in humidified air with 5% CO2. The culture medium was renewed every 24 hours, or 48 hours after glyoxal treatment.

[0171] After receipt, the explants were divided into 3 experimental groups (n=3 per group). On day 0, day 2, day 4, the active ingredient was applied topically at the concentration of 50pg / mL.

[0172] The peptide that was tested in this example has the sequence SEQ ID NO: 12, namely VGVAPGVGVAPGVGVAPGRVRLGIL or N-(Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-lle-Leu-OH, with n being equal to 3.

[0173] Glycation inducer (glyoxal, 500pM) was applied on day 1 and day 3 in the culture medium.

[0174] The control group received no treatment except for medium renewal.

[0175] On day 5, 24 hours after the 3rd treatment with the active ingredient, the explants were collected: ! > from each explant embedded in OCT for cryopreservation and stored at -80°C until analyses and ! > from each explant frozen in liquid nitrogen, stored at -80°C until analyses.

[0176] CML detection:

[0177] In situ analysis of vimentin glycation (via CML detection) was performed by epifluorescence microscopy via specific immunofluorescence detection of independent signals (vimentin, CML) as described above. CML levels (RFU / surface) were quantified on the co-localized vimentin surface above the threshold to allow assessment of vimentin glycation.

[0178] Anti-vimentin ELISA:

[0179] Proteins were extracted from the explants and assessment of vimentin levels was performed following the manufacturer's instructions (colorimetric method; ab246526, Abeam).

[0180] Quantification of total protein was performed by the Bradford assay and equal amounts of total protein were used for each measurement. Duplicate measurements per explant were performed, for a total of 6 measurements per condition (2 per explant). Absorbance was interpolated from the vimentin standard curve.

[0181] The results illustrated in Figure 2 demonstrate that the BFP peptide promotes the decrease of total (Figure 2A) and glycated (Figure 2B) vimentin by activation of the NRF2 pathway (Figure 2C).

[0182] For the record and in summary, skin explants were cultured ex vivo in different media by inducing or not glycation with glyoxal, with or without treatment with the peptide of the invention. The skin explants were homogenized and the total concentration of vimentin was assessed by a specific ELISA test (Figure 2A). In parallel, the same samples were labeled with anti-CML and anti-vimentin antibodies to determine the levels of glycated vimentin (Figure 2B). The same treated extracts were labeled with the anti-NRF2 antibody to detect the nuclear translocation of NRF2 (Figure 2C).

[0183] As shown in Figure 2A, total vimentin concentration is increased in glyoxal-treated explants (light gray bar) and is decreased by peptide treatment (dark gray bar), implying degradation of total vimentin by the action of the peptide.

[0184] As shown in Figure 2B, vimentin glycation is increased in glyoxal-treated explants (light gray bar) compared to control (white bar) and is decreased by BFP treatment (dark gray bar).

[0185] As shown in Figure 2C, nuclear detection of NRF2 is significantly increased in the glyoxal + BFP peptide condition (dark gray bar) compared to the untreated condition (white bar).

[0186] In addition to confirming previous results demonstrating that the detoxifying peptide of the invention leads to a degradation of glycated vimentin, these results also provide evidence that the detoxifying peptide of the invention acts by activating the NRF2 factor pathway.

[0187] Example 3: In vivo study - Clinical trial

[0188] Clinical study design:

[0189] The clinical trial was conducted in a single-blind, multicenter, randomized, controlled fashion on 22 women (aged 45 to 70 years) who had to apply the peptide of the invention twice a day for 28 days before their facelift, at during which skin samples were taken for genomic and proteomic quantification analysis.

[0190] The product (cream containing the BFP peptide) was applied to one half-face in comparison with the placebo (cream without the BFP peptide).

[0191] The peptide that was tested in this example has the sequence SEQ ID NO: 12, namely VGVAPGVGVAPGVGVAPGRVRLGIL or N-(Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-lle-Leu-OH, with n being equal to 3.

[0192] Collection and processing of skin extracts after facelift:

[0193] The surgical procedure is performed using the surgeon's standard technique. The surgical waste, removed during the procedure, corresponds to the excess skin resulting from the laxity of the skin in the periauricular area. A piece of excess skin from each side of the face was removed and divided into 2 fragments for genomic and proteomic analyses:

[0194] Processing of skin explants for proteomic analyses:

[0195] Pieces of skin on each side of the face (> 1 cm 2) are immersed in 4% formalin solution at room temperature for 24 hours, then transferred to 70% ethanol solution and stored at room temperature before paraffin embedding and analysis. Paraffin sections approximately 4 µm thick are cut and placed on Superfrost Plus glass slides, left to dry overnight at 45°C (+ / - 3°C) in an air oven before microscopy imaging.

[0196] Proteomic analyses by immunohistochemistry:

[0197] Vimentin labeling on paraffin sections was performed with the OPALTM4-color IHC (immunohistochemistry) kit from Perkin Elmer® with the anti-Vimentin antibody (Rabbit polyclonal - Abeam, ab45939). The antibody-labeled paraffin sections were then incubated with the fluorophore OPAL 690 (2018 Akoya Biosciences). Nuclei were labeled with DAPI. After complete ICH and fluorescent labeling, the paraffin sections were scanned by the Vectra Polaris (PerkinElmer) with the X20 objective. Visualization, analysis, validation, and quantification of the images were performed with HALO® software (Indicalabs). Thresholds were selected to quantify the area positive of each marker and the individual data were generated by the software in Excel.

[0198] SMC labeling on paraffin sections was performed with the SMC-specific primary antibody (anti-SMC MAB3247, R&D systems) in a 0.1% Tween-20 solution in PBS. Excess antibody was removed by a series of washes, and then the explants were incubated with the fluorochrome-coupled secondary antibody (Goat anti-mouse AlexaFluor 555 Invitrogen A21422). Nuclei were labeled using DAPI (4',6-diamidino-2-phenylindole). Finally, antibodies and excess DAPI were removed by a series of washes with 0.1% Tween-20 in PBS. A series of images was acquired with an epifluorescence microscope (ThermoFisher, Evos M5000) using strictly the same acquisition time and resolution (40x objective). Images were collected with all fluorescence signal intensity levels (raw .TIFF 16bit format), then analyzed using ImageJ software (Rasband, NIH).Quantification was performed on each image by integrating the specific fluorescence signal above the threshold, then normalized to the evaluation area. Three (3) images per experimental batch were analyzed. One mean value and one standard deviation per experimental batch and per condition (placebo vs. treated). CML levels were then normalized to each "placebo" batch to obtain the ratio of relative changes (fold changes) upon treatment with the peptide.

[0199] The normality of the data distribution was verified by the Shapiro-Wilk test, at a risk of 5%. The data were analyzed with the t-Student test for a normal distribution and with a Wilcoxon test otherwise (p-value < 0.05).

[0200] The results of the immunohistochemical analysis of the skin explants, depending on the treatment with the peptide (grey bars) or placebo (white bars) obtained and illustrated in Figure 3 demonstrate that the peptide of the invention induces the reduction in the detection of vimentin (Figure 3A) and CML (Figure 3B) in vivo in humans.

[0201] More particularly, a significant decrease in the detection of vimentin is observed in volunteers treated with the peptide of the invention, as well as a significant decrease in the detection of CML.

[0202] The detoxifying peptide of the invention therefore makes it possible to induce a reduction in glycation levels in humans.

[0203] Example 4: Genomic analysis - study of genes expressed in the presence of the detoxifying peptide

[0204] After treatment of cultured fibroblasts with a detoxifying peptide of the invention BFP or with a control peptide, a 5-step RNAseq analysis was performed to identify genes differentially expressed between the two conditions:

[0205] STEP 1: RNA Extraction: RNA extractions were performed on the samples, each representing an experimental condition, using the miRNeasy mini kit (QIAGEN) following the manufacturer's protocol on an automated workstation (QIAcube - QIAGEN). Briefly, cells were harvested and homogenized in 700 µl of QIAzol® Lysis Reagent in a 2 ml SafeLock microcentrifuge tube. Samples were stored at -80°C until use. A 2 mm stainless steel bead was added to each thawed sample and cells were disrupted by mechanical shaking using the TissueLyzer (Qiagen) 2 x 2 min at 20 Hz. Samples were then incubated for 5 minutes at room temperature. 140 µl of chloroform was added to the homogenate. The tube was shaken vigorously for 15s and returned to the bench for another 2 to 3 min.The lysate was centrifuged at 12,000 xg for 15 minutes at 4°C in a microcentrifuge. The upper aqueous phase (approximately 350 μl) was carefully transferred to a clean 2 ml microcentrifuge. The remainder of the procedure was performed according to the manufacturer's protocol using the QIAcube automated workstation (QIAcube - QIAGEN) to optimize reproducibility and standardization of RNA extraction (according to the pre-installed protocol). At the end of the procedure, each RNA was eluted with 30 μl of H2O. A 3 μl aliquot was pipetted into a new microcentrifuge tube for quality validation and all tubes were stored in an ultra-low freezer immediately (-80°C).

[0206] STEP 2: RNA Quality Control: The concentration and purity of the RNA sample were measured using the NanoDrop ND-1000 spectrophotometer (Thermo Scientific). RNA integrity was assessed using the Agilent 2200 TapeStation RNA ScreenTape (Agilent Technologies) and the RINe (RNA Integrity Number equivalent) score was checked (RINe should be > 7).

[0207] STEP 3: Construction of RNA-Seq libraries using the KAPA mRNA Hyperprep Kit (KAPA). RNA samples were used for the library construction procedure.

[0208] STEP 4: Sequencing of libraries on Illumina Novaseq (S1 run cartridge -100 cycles Loading of libraries at 1.2nM)

[0209] STEP 5: Bioinformatics processing; For this analysis, the human genome was used as a reference (version GRCh38.p13). RNA Seq data were mapped using the Ensembl database version 103 (https: / / www.ensembl.org). All pipeline steps are listed below: a. Sequence cleaning and trimming using FASTP software; (https: / / www.ncbi.nlm.nih.ciov / Dmc / articles / PMC6129281 / ); b. Quality control using FASTQC software (httD: / / www.bioinformatics.babraham.ac.uk / Droiects / fastqc / ); c. Human genome mapping with STAR software (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3530905 / ) With the reference genome "Homo_sapiens.GRCh38.dna.primary_assembly.fa" and with the annotation "Homo_sapiens.GRCh38.103.chr.gtf" from Ensembl.; d. Counting the number of reads for each gene with STAR software.

[0210] The peptide that was tested in this example has the sequence SEQ ID NO: 12, namely VGVAPGVGVAPGVGVAPGRVRLGIL or N-(Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-lle-Leu-OH, with n being equal to 3.

[0211] Figure 4 shows the main genes identified and their expression value.

[0212] In conclusion, the results of this genomic analysis demonstrate that, in vitro, the peptide of the invention induces the expression of genes involved in the fight against oxidative stress, in autophagy, in neddylation and in degradation by the ubiquitin / proteasome system.

[0213] Indeed, as shown in the table in Figure 4, a large number of genes could be identified from RNAseq data on cultured fibroblasts, demonstrating the impact of the peptide in triggering NRF2-mediated cellular defense reactions.

[0214] Example 5: RT-qPCR results performed on skin explants

[0215] Following the identification in cultured fibroblasts of genes differentially regulated by the action of the peptide, as explained in example 4 above and in connection with the attached figure 4, the analysis of their regulation on skin explants was carried out by a different technique, namely RT-qPCR.

[0216] The skin explants were subjected or not to the action of the glycation agent (glyoxal).

[0217] Skin explants were obtained with informed consent from a 32-year-old Caucasian woman who had undergone abdominal surgery.

[0218] Explants were kept alive by culture on metal grids in medium at 37°C in humidified air with 5% CO2. The culture medium was renewed every 24 hours, or 48 hours after glyoxal treatment.

[0219] After receipt, the explants were divided into 3 experimental groups (n=3 per group). On day 0, day 2, day 4, the active ingredient was applied topically at the concentration of 50pg / mL.

[0220] The active ingredient that was applied is the peptide (denoted BFP in the graphs of Figure 5) which has the sequence SEQ ID NO: 12, namely VGVAPGVGVAPGVGVAPGRVRLGIL or N-(Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-lle-Leu-OH, with n being equal to 3.

[0221] The glycation inducer (glyoxal, 500pM) was applied on day 1 and day 3 in the culture medium. The control group received no treatment except for medium renewal. On day 5, 24 hours after the 3rd treatment with the active ingredient (the peptide according to the invention), the explants were collected: one half of each explant embedded in OCT (“Optimal Cutting Temperature”) for cryopreservation and stored at -80°C until analyses and one half of each explant frozen in liquid nitrogen, stored at -80°C until analyses.

[0222] cDNA synthesis was performed using ReadyScript™ cDNA Synthesis Mix (Sigma-Aldrich) in a final reaction volume of 20 μl according to the manufacturer's protocol. For each sample, 550 ng of RNA was used as RNA template for the reverse transcriptase enzyme. cDNA synthesis was performed using 96-well plates in a final reaction volume of 20 μl using a Bio-Rad C1000 thermal cycler according to the following thermal program: 5 minutes at 25°C, 30 minutes at 42°C, 5 minutes at 85°C. Pure cDNAs were stored at -20°C until use.

[0223] All qPCR reactions were performed on 384-well plates in 3 technical replicates. qPCR reactions were performed on the LightCycler 480 System II (Roche) in a final reaction volume of 10 μl. The reaction mix consisted of 5 μl of LightCycler 480 SYBR Green I Master Mix 2X (Roche Diagnostics), 4 μl of primers (final concentration of 0.7 μM), and 1 μl of cDNA diluted 1:10.

[0224] The qPCR cycling program consists of a polymerase activation step at 95°C for 10 minutes followed by 45 cycles of qPCR reaction at 95°C for 3 seconds for denaturation, 60°C for 15 seconds for annealing and 72°C for 15 seconds for extension with fluorescence acquisition at the end of the extension step. At the end, a melting curve was performed from 65°C to 97°C with continuous fluorescence acquisition to control the amplicon specificity. The Ct value measured for each gene was repeated 3 times and the standard deviation of the repeat must be strictly lower than 0.5 Ct. The NTC (No Template Control) reaction was performed in the same way but replacing the cDNA template with PCR grade water (one technical replicate). Raw Ct data were acquired using the 2-derivative maximum method on the LightCycler480 System II instrument (Roche).

[0225] Relative gene expression analysis using real-time quantitative PCR data and statistical calculations were performed by the 2-DeltaDeltaCt method, considering YWHAZ, PPIB, and B2M as housekeeping genes.

[0226] The results obtained are illustrated in Figure 5 of the attached drawings.

[0227] As shown in Figure 5, glyoxal induced an increase in the expression of CXCL1 (p<0.01) and DNAJC10 (p<0.05) genes, and a decrease in the expression of DNAJB14 (p<0.05), UBE2D3 (p<0.05), PSMD12 (p<0.05) and RNF19A (p<0.001).

[0228] The BFP peptide counteracted the effects of glyoxal, downregulating the expression of CXCL1 (p<0.0001) and DNAJC10 (p<0.01), while upregulating the expression of DNAJB14 (p<0.01), UBE3A (p<0.05), UBE2D3 (p<0.05), DRAM2 (p<0.05), PSMD12 (p<0.05), TRIM23 (p<0.05) and RNF19A (p<0.05), which are involved in the complex process of autophagy and / or proteasomal degradation allowing cells to degrade toxic compounds.

[0229] Note that when analyzing RNA extracted from the same samples, no difference in VIM expression was observed.

[0230] In conclusion, these results confirm the protective action of the peptide against protein glycation, in particular by activation of autophagy pathways, ubiquitin-mediated protein degradation. By counteracting gene expression induced by glycation agents (glyoxal in our study) via the activation of NRF2 and by promoting autophagy, the peptide of the invention exhibits antiglycation and antifibrotic properties.

[0231] This experiment demonstrates that the decrease in vimentin protein detection (Figures 2A) is not due to a decrease in its gene expression (VIM = gene coding for vimentin), but rather to an increase in its degradation by autophagy and / or its ubiquitin-mediated protein degradation, due to its higher glycation.

[0232] It should be noted that the tests carried out and repeated in the examples above were conducted using the peptide of the invention having the particularly preferred sequence SEQ ID NO: 12, i.e. the sequence VGVAPGVGVAPGVGVAPGRVRLGIL taking into account the one-letter code for the designation of the amino acid residues.

[0233] That being said, the peptide according to the invention may have other sequences, insofar as certain residues are conserved, without this affecting its properties, in particular the residues Val, Gly, Pro and Gly in positions 1, 2, 5 and 6 of the hexapeptide repeated n times, as well as the first Arg residue immediately following this hexapeptide and the second Arg residue separated from the first by an amino acid residue must be conserved.

[0234] In particular, in addition to the amino acids to be retained and mentioned above, the other amino acid residues of the sequence SEQ ID NO: 12 may be, for example, substituted by amino acids considered to be chemically equivalent, i.e. having equivalent physicochemical characteristics.

[0235] Thus, substitutions between equivalent amino acids are made, for example, between the apolar aliphatic amino acids Ala, Val, Ile and Leu, or between the polar amino acids carrying a hydroxyl group Ser and Thr, between the amino acids Asn and Gln or between the amino acids carrying two acid functions Asp and Glu.

[0236] In particular, the detoxifying peptide of the invention can therefore most preferably have one of the sequences chosen from the sequences SEQ ID NO: 10 to SEQ ID NO: 37

[0237] Bibliographic references - Cohen, D., Portugal-Cohen, M., Oron, M., Frusic-Zlotkin, M., Soroka, Y., Ma’or, Z., Amar, D., Kohen, R., 2022. Cutaneous Nrf2-Keap1 pathway modulation by environmental factors: The Dead Sea area as a test case. Biofactors. https: / / doi.orq / 10.1002 / biof.1926 - Collins, A.R., Lyon, C.J., Xia, X., Liu, J.Z., Tangirala, R. K., Yin, F., Boyadjian, R., Bikineyeva, A., Praticé, D., Harrison, D.G., Hsueh, W.A., 2009. Age- accelerated atherosclerosis correlates with failure to upregulate antioxidant genes. Circ Res 104, e42-54. https: / / doi.org / 10.1161 / CIRCRESAHA.108.188771 - Damasiewicz-Bodzek, A., Nowak, A., 2022. Concentrations of N6- Carboxymethyllysine (CML), N6-Carboxyethyllysine (CEL), and Soluble Receptor for Advanced Glycation End-Products (sRAGE) Are Increased in Psoriatic Patients. Biomolecules 12, 1870. https: / / doi.Org / 10.3390 / biom12121870 - Howden, R., 2013. Nrf2 and cardiovascular defense. Oxid Med Cell Longev 2013, 104308. https: / / doi.org / 10.1155 / 2013 / 104308 - Howden, R., Gougian, E., Lawrence, M., Cividanes, S., Gladwell, W., Miller- DeGraff, L., Myers, P.H., Rouse, D.C., Devlin, R.B., Cho, H.-Y., Kleeberger, S.R., 2013. The influence of Nrf2 on cardiac responses to environmental stressors. Oxid Med Cell Longev 2013, 901239. https: / / doi.Org / 10.1155 / 2013 / 901239 -Koch, M., Kockmann, T., Rodriguez, E., Wehkamp, U., Hiebert, P., Ben- Yehuda Greenwald, M., Stôlzl, D., Beer, H.-D., Tschachler, E., Weidinger, S., Werner, S., Auf dem Keller, U., 2023. Quantitative Proteomics Identifies Reduced NRF2 Activity and Mitochondrial Dysfunction in Atopic Dermatitis. J Invest Dermatol 143, 220-231 ,e7. https: / / doi.Org / 10.1016 / i.jid.2022.08.048 - Li, H., Zhuang, W., Xiong, T., Park, W.S., Zhang, S., Zha, Y., Yao, J., Wang, F., Yang, Y., Chen, Y., Cai, L., Ling, L., Yu, D., Liang, J., 2022. Nrf2 deficiency attenuates atherosclerosis by reducing LOX-1 -mediated proliferation and migration of vascular smooth muscle cells. Atherosclerosis 347, 1-16. https: / / doi.Org / 10.1016 / j. atherosclerosis.2022.02.025 - Lin, Y., Cai, F., Wang, X., Yang, Y., Ren, Y., Yao, C., Yin, X., Zhuang, H., Hua, Z., 24 Oct 2022, FADD phosphorylation contributes to development of renal fibrosis by accelerating epithelial-mesenchymal transition. Cell Cycle 22, 580-595. https: / / doi.Org / 10.1080 / 15384101 .2022.2136463 - Motohashi, H., Yamamoto, M., 2004. Nrf2-Keap1 defines a physiologically important stress response mechanism. Trends Mol Med 10, 549-557. https: / / d0i.0rg / l 0.1016 / j. molmed.2004.09.003 - Ogawa, T., Ishitsuka, Y., 2022. The Role of KEAP1 -NRF2 System in Atopic Dermatitis and Psoriasis. Antioxidants (Basel) 11 , 1397. https: / / doi.Org / 10.3390 / antiox11071397 - Reddy, V. Prakash, Puspa Aryal, and Emmanuel K. Darkwah. 2022. "Advanced Glycation End Products in Health and Disease" Microorganisms 10, no. 9: 1848. https: / / doi.Org / 10.3390 / microorganisms10091848 - Seedorf, K., Weber, C., Vinson, C., Berger, S., Vuillard, L.-M., Kiss, A., Creusot, S., Broux, O., Géant, A., Hic, C., Lemaitre, K., Richard, J., Hammoutene, A., Mahieux, J., Martiny, V., Durand, D., Melchiore, F., Nyerges, M., Paradis, V., Provost, N., Duvivier, V., Delerive, P., 2023. Selective disruption of NRF2-KEAP1 interaction leads to NASH resolution and reduction of liver fibrosis in mice. JHEP Rep 5, 100651. https: / / doi.Org / 10.1016 / j.jhepr.2022.100651 - Song, J., Liu, A., Liu, B., Huang, W., Jiang, Z., Bai, X., Hu, L., Zheng, S., Guo, S., Wu, J., Chen, Q., 2022. Natural Biologies Accelerate Healing of Diabetic Foot Ulcers by Regulating Oxidative Stress. Front Biosci (Landmark Ed) 27, 285. https: / / doi.Org / 10.31083 / i.fbl2710285 - Tanase, D.M., Apostol, A.G., Costea, C.F., Tarniceriu, C.C., Tudorancea, I., Maranduca, M.A., Floria, M., Serban, I.L., 2022. Oxidative Stress in Arterial Hypertension (HTN): The Nuclear Factor Erythroid Factor 2-Related Factor 2 (Nrf2) Pathway, Implications and Future Perspectives. Pharmaceutics 14, 534. https: / / doi.Org / 10.3390 / pharmaceutics14030534 - Victor, P., Sarada, D., Ramkumar, K.M., 2020. Pharmacological activation of Nrf2 promotes wound healing. Eur J Pharmacol 886, 173395. https: / / doi.orci / 10.1016 / i.ejphar.2020.173395 - Wu, M., Matar, D.Y., Yu, Z., Chen, Z., Knoedler, S., Ng, B., Darwish, O.A., Sohrabi, S., Friedman, L., Haug, V., Murphy, G.F., Rinkevich, Y., Orgill, D.P., Panayi, A.C., 2022. Continuous NPWT Regulates Fibrosis in Murine Diabetic Wound Healing. Pharmaceutics 14, 2125. https: / / doi.Org / 10.3390 / pharmaceutics14102125

Claims

Claims

1. (Detoxifying peptide characterized in that its sequence comprises the following general amino acid sequence: N-(Val-Gly-X1-X2-Pro-Gly)n-OH in which: N and OH correspond respectively to the N-terminal and C-terminal ends of said peptide; X1 and X2 are selected from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gin), alanine (Ala), leucine (Leu) and isoleucine (Ile); n is an integer between 1 and 3; for use in the treatment and / or prevention of pathologies linked to the glycation and / or glycoxidation of proteins and / or lipids linked to an accumulation of advanced glycation end products, or AGEs for "Advanced Glycation End Products" in the tissues of an organ, said detoxifying peptide being capable of allowing a reduction of AGEs in said tissues and / or of promoting the degradation of AGEs, by activation of the pathway of a specific nuclear transcription factor, noted NRF2 or "nuclear factor erythroid-2-related factor 2", or nuclear factor related to erythroid 2.

2. A detoxifying peptide for use according to claim 1 by activating the transcription factor NRF2 resulting in the activation of genes involved in the autophagy and / or lysosome and / or ubiquitin / proteasome and / or neddylation pathways.

3. Detoxifying peptide for use according to claim 1 or claim 2 in the treatment and / or prevention of pathologies linked to the glycation and / or glycoxidation of proteins linked to an accumulation of AGES of the glycated vimentin and / or carboxymethyl-lysine (CML) type, in the tissues of an organ, said detoxifying peptide being capable of allowing a reduction of glycated vimentin and / or carboxymethyl-lysine (CML) by activation of the NRF2 factor pathway.

4. Detoxifying peptide for use according to one of claims 1 to 3, characterized in that it has the following general amino acid sequence: N-(Val-Gly-X1 -X2-Pro-Gly)n-Arg-X3-Arg-X4-X5-X6-X7-OH in which: N and OH correspond respectively to the N-terminal and C-terminal ends of said peptide; X1 and X2 are selected from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gin), alanine (Ala), leucine (Leu) and isoleucine (Ile); X3 and X4 are selected from valine (Val), serine (Ser), tyrosine (Tyr), alanine (Ala), glycine (Gly), arginine (Arg) and leucine (Leu); X5, X6 and X7 are selected from isoleucine (Ile), leucine (Leu), valine (Val), alanine (Ala), glycine (Gly) and phenylalanine (Phe) n is an integer between 1 and 3.

5. Detoxifying peptide for use according to one of claims 1 to 4 characterized in that it has SEQ ID NO: 12 namely N- (Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-lle-Leu-OH, with N and OH corresponding respectively to the N-terminal and C-terminal ends of said peptide, and with n being equal to 3.

6. Detoxifying peptide for use according to one of claims 1 to 4, characterized in that it has a sequence chosen from the group consisting of the sequences SEQ ID NO: 10, SEQ ID NO: 11, and SEQ ID NO: 13 to SEQ ID NO:

37.

7. A detoxifying peptide for use according to any one of claims 1 to 6 in the treatment and / or prevention of side effects of radiotherapy and / or chemotherapy in cancer patients.

8. Detoxifying peptide for use according to any one of claims 1 to 6 in the treatment and / or prevention of skin fibrosis and / or liver fibrosis and / or non-alcoholic fatty liver disease (or NASH for “Non Alcoholic Steato Hepatitis”) and / or hepatocarcinoma.

9. Detoxifying peptide for use according to any one of claims 1 to 6 in the treatment and / or prevention of skin pathologies such as psoriasis and / or dermatitis and / or eczema.

10. A detoxifying peptide for use according to any one of claims 1 to 6 in the treatment and / or prevention of diabetes and its complications, such as diabetic neuropathy, diabetic nephropathy, glaucoma, cataracts and diabetic retinopathy.

11. Detoxifying peptide for use according to any one of claims 1 to 6 in the treatment and / or prevention of cardiovascular pathologies.

12. Detoxifying peptide for use according to any one of claims 1 to 6 in the treatment and / or prevention of neurodegenerative pathologies such as Alzheimer's disease and / or Parkinson's disease and / or multiple sclerosis (MS) and / or amyotrophic lateral sclerosis (ALS).

13. Detoxifying peptide for use according to any one of the preceding claims, characterized in that it is obtained by chemical synthesis.

14. Pharmaceutical composition comprising at least one peptide as defined in any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier for use in the treatment and / or prevention of pathologies linked to the glycation and / or glycoxidation of proteins and / or lipids.]

Images