antidote peptide

Abstract

The present invention relates to a 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 end of the peptide; X1 and X2 are chosen from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gln), alanine (Ala), leucine (Leu) and isoleucine (Ile); n is an integer between 1 and 3; for use in the treatment and / or prevention of a disorder associated with glycation and / or glycoxidation of proteins and / or lipids.

Description

[0001] This invention relates to peptides that will be particularly used in the pharmaceutical field.

[0002] The peptides according to the invention are more specifically composed of peptides that can be characterized as “detoxifying” and will be primarily used in the prevention and / or treatment of certain pathologies involving the transcription factor NRF2 (“nuclear factor erythrocyte 2-associated factor 2”). The peptides of the invention are capable of acting on the expression and / or translocation of NRF2, particularly leading to an increase in autophagy, and thus resulting in a reduction of toxic molecular compounds (particularly including advanced glycation end products (or AGEs)) or ROS (reactive oxygen species).

[0003] Sugar oxidation is the result of a combination of oxidation and glycation.

[0004] Glycation is a complex process involving a spontaneous, slow, non-enzymatic reaction between the free amino groups of a protein and reducing sugars such as glucose or ribose, forming an unstable Schiff base.

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

[0006] Anti-glycation research is a rapidly expanding area of ​​focus in the health and medicine field, as it is increasingly recognized that AGEs can play a significant role in the development of various chronic diseases such as diabetes, cardiovascular diseases (ischemia, cardiac fibrosis, arterial disease, 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] Many studies have also shown that AGEs are involved in certain skin-related pathologies, such as psoriasis or dermatitis.

[0008] Therefore, it would be particularly advantageous to identify active ingredients and to develop compositions that can counteract the long-term effects of AGE formation and prevent or treat AGE-related pathologies.

[0009] Currently, there is a variety of treatments available for combating glycation, AGEs, and their effects. Some strategies involve the use of anti-glycation agents, such as glycation inhibitors, metal chelators, and AGE-degrading enzymes. However, research in this field is still in its early stages, and researchers are still working to understand the underlying mechanisms of glycation and the roles that specific molecules may play in inhibiting or reversing this process. Therefore, there remains a need for anti-glycation compounds that are specific, effective, safe, and have good bioavailability.

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

[0011] Therefore, these various treatments do not address the underlying causes of organ fibrosis, and strategies that can treat or prevent the onset of fibrotic diseases are still needed.

[0012] Various compounds derived from plant extracts, mineral extracts, or other natural extracts have been proposed to combat AGEs. However, while natural extracts may be a potential source of anti-glycation compounds, obtaining the right extracts remains a lengthy and expensive process.

[0013] This is because plant extracts may contain many different compounds, some of which may have undesirable effects or even impair anti-glycation activity. Furthermore, the concentration of each compound can vary significantly depending on growing conditions, season, and geographical region, as plants must grow under specific conditions to obtain the optimal quality and quantity of anti-glycation compounds.

[0014] It should also be noted that producing plant extracts is expensive because it requires a large amount of raw materials to obtain a sufficient quantity of active compounds.

[0015] Specifically, some plants contain only small amounts of these compounds, meaning that a large number of plants must be harvested to obtain enough compounds for effective use. This variability in the compounds present in plant extracts can make it difficult to standardize formulations, reproduce results, and ensure batch-to-batch consistency in quality.

[0016] Furthermore, extracting desired compounds from plants is typically a complex process. Specific methods must be used to extract the compounds while preserving their integrity and efficacy. This often requires 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 costly and time-consuming.

[0017] These drawbacks highlight the need to find alternatives to natural extracts that target specific molecules involved in sugar oxidation processes and / or detoxification mechanisms, thereby eliminating AGEs and / or increasing AGE degradation.

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

[0019] First, advanced glycation end products (AGEs) are heterogeneous and complex, making their formation difficult to understand. Therefore, finding compounds that can specifically neutralize AGEs and glycation molecules can be challenging, especially given the wide variety of possible molecular structures.

[0020] Furthermore, glycation can occur at different locations within a protein, thus affecting its structure and function in various ways. Therefore, it is difficult to obtain anti-glycation compounds that target glycation sites.

[0021] Therefore, the objective of this invention is to obtain an anti-glycation compound through chemical synthesis that can target specific molecules involved in the sugar oxidation process and / or AGE degradation, such as glycated proteins.

[0022] Furthermore, plant extracts have another drawback: 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] Therefore, the object of the present invention is to obtain a compound that will be well tolerated by the body and will also overcome the above-mentioned disadvantages.

[0024] Despite having to overcome all the difficulties mentioned above, the inventors successfully identified and synthesized peptides that regulate the activity of specific nuclear transcription factor (NRF2 factor or "nuclear factor erythrocyte-associated factor 2" mentioned above) pathways through chemical synthesis.

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

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

[0027] Therefore, this may make it extremely difficult to identify peptides or other molecules that can specifically and effectively regulate Nrf2 pathway activity.

[0028] Furthermore, the NRF2 pathway plays an important role in many different physiological processes, which makes it necessary to design peptides with sufficient specificity to target the NRF2 pathway only in the specific context of resisting the oxidation and / or glycation of biomolecules (especially proteins and lipids).

[0029] Therefore, through this invention, the inventors have successfully overcome these difficulties and proposed a peptide that can effectively and specifically regulate the activity of the NRF2 pathway, thereby stimulating the degradation of advanced glycation end products (AGEs) by regulating degradation pathways (autophagy, ubiquitin-like glycosylation, ubiquitin / proteasome system, lysosomes, etc.) in the context of protein and / or lipid glycation and / or glucose oxidation.

[0030] Therefore, this peptide can be considered a peptide with detoxification properties, and thus can be referred to as a detoxification peptide in the remainder of the specification thereafter.

[0031] For the purposes of this invention, the term "detoxification" should be understood as referring to the cellular process of degrading and / or eliminating potentially toxic molecular compounds (proteins, lipids, nucleic acids, etc.) through autophagosomes, lysosomes, ubiquitin / proteasomes, ubiquitination or antioxidant pathways.

[0032] Therefore, the present invention more specifically relates to a detoxifying peptide comprising the following universal amino acid sequence: N-(Val-Gly-X1-X2-Pro-Gly)n-OH, wherein: - N and OH correspond to the N-terminus and C-terminus of the peptide, respectively; - X1 and X2 are selected from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gln), 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 associated with the glycation and / or oxidation of proteins and / or lipids, which are attributed to the accumulation of advanced glycation end products (AGEs) in organ tissues, the detoxifying peptide is capable of reducing AGEs in said tissues and / or promoting AGE degradation by activating a pathway of a specific nuclear transcription factor (i.e., NRF2 factor).

[0033] Preferably, the detoxifying peptide has the following universal amino acid sequence: N-(Val-Gly-X1-X2-Pro-Gly)n-Arg-X3-Arg-X4-X5-X6-X7-OH in: - N and OH correspond to the N-terminus and C-terminus of the peptide, respectively; - X1 and X2 are selected from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gln), 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.

[0034] In a particularly preferred embodiment, the peptide of the present invention has SEQ ID NO: 12, namely N-(Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-Ile-Leu-OH, wherein N and OH correspond to the N-terminus and C-terminus of the peptide, respectively, and wherein n equals 3.

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

[0036] The detoxification peptides of the present invention can be used to increase the expression of NRF2 (“Nuclear Factor Erythrocyte 2-Associated Factor 2”) transcription factor and / or to activate the NRF2 transcription factor pathway and / or to activate the nuclear translocation of NRF2, thereby causing activation of genes involved in lysosomal and / or ubiquitin / proteasome and / or autophagy and / or ubiquitin-like pathways.

[0037] Therefore, the detoxifying peptides of the present invention can be used to increase the degradation of advanced glycation end products (AGEs) via the NRF2 factor pathway, for example, to increase the degradation of carboxyethyl lysine (CEL), carboxymethyl lysine (CML), glyoxal-lysine dimer (GOLD), 3-deoxyglucuronide lysine dimer (DOLD), pyrrolidone, and fructose lysine.

[0038] The detoxifying peptide can also be used to increase the degradation of glycated vimentin (i.e. vimentin modified with CML and / or CEL).

[0039] In other words, the peptides of the present invention act on NRF2, and one of the consequences of this action (which will be demonstrated later in the specification) is an increase in autophagy, which in particular leads to a reduction in molecular compounds that can be characterized as “toxic” to cells and tissues, including AGEs.

[0040] Therefore, the detoxifying peptides of the present invention are particularly advantageous for use in pathologies involving the NRF2 factor, in which abnormal accumulation of AGEs is particularly observed. This is because the peptides will have the effect of increasing NRF2 expression and / or translocation, and thus improving the condition of patients suffering from such pathologies.

[0041] Therefore, detoxifying peptides can be used to treat and / or prevent the side effects of radiotherapy and / or chemotherapy in patients with cancer.

[0042] Detoxifying peptides can also be used to treat and / or prevent skin fibrosis and / or liver fibrosis and / or non-alcoholic steatohepatitis (or NASH) and / or liver cancer.

[0043] The detoxifying peptides, which are the subject of this invention, can also be used in the following pathologies (which, in people who have one of these pathologies, particularly involve advanced glycation end products (AGEs), especially the accumulation of AGEs and / or reduced degradation of these AGEs, compared to people who do not have one of these pathologies): - Used for the treatment and / or prevention of skin pathologies such as psoriasis and / or dermatitis and / or eczema; - Used to treat and / or prevent diabetes and its complications, such as diabetic neuropathy, diabetic nephropathy, glaucoma, cataracts and diabetic retinopathy; - Used for the treatment and / or prevention of cardiovascular pathology; - Used for 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). Advantageously, detoxifying peptides can be obtained through chemical synthesis.

[0044] The present invention also relates to pharmaceutical compositions 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 associated with glycation and / or oxidation of proteins and / or lipids.

[0045] Therefore, the peptide according to the invention is particularly advantageous because it is an anti-glycation and anti-glycation compound that limits the glycation process by targeting the NRF2 factor in the context of the glycation process, and in the degradation of glycated proteins, the result is that the peptide ultimately enables the limitation of AGE accumulation and / or the promotion of the degradation of AGE that may have already accumulated in biological tissues.

[0046] Furthermore, the peptide advantageously comprises a portion of physiological compounds released during extracellular matrix (ECM) remodeling, particularly during the degradation of ECM proteins via proteolytic cleavage.

[0047] Therefore, compared to plant extracts, for example, this peptide has the advantage of being better tolerated by the body, while also having high bioavailability.

[0048] Therefore, in other words, the peptide in question is not toxic itself because its sequence is partially derived from the sequence of a natural human compound (elastin). Thus, it not only has detoxifying properties but also exhibits extremely low or even zero inherent toxicity, a superior advantage over other compounds.

[0049] It should be noted that the roles of such compounds (particularly elastomer factors and matrix factors) released from ECM degradation have been previously studied in certain processes (such as tissue homeostasis or cell signaling); however, this is not the case with the pathways targeted in this application.

[0050] Therefore, peptides derived from elastin, which are the subject of this invention, are known in the prior art because they were the subject of a French patent application published under number FR 2 986 233.

[0051] This application describes the ability of the peptide to bind to an elastin-binding receptor protein (called EBP, or "elastin-binding protein") located on the cell surface to stimulate the synthesis of type I and type II collagen.

[0052] It also describes the role of an elastin-derived peptide as a competitive inhibitor of the protease urokinase (one of the activators of the plasminogen-plasmin system). This elastin-derived peptide limits the activity of urokinase against plasminogen by binding to the active site of the protease urokinase, which indirectly leads to collagen degradation by activating matrix metalloproteinases (MMPs), enzymes that exhibit collagen-degrading activity.

[0053] Finally, patent application FR 2 986 233 also explains how this elastin-derived peptide directly leads to the inhibition of type I MMPs—a portion of which occupies a portion of the MMP-1 active site, thereby acting as a competitive inhibitor of this enzyme.

[0054] Therefore, this peptide is used in the treatment of chronic wound healing disorders, especially pressure sores or ulcers, for the repair and / or regeneration of skin tissue, and particularly for the treatment of skin aging.

[0055] Therefore, FR 2 986 233 describes the mechanism of action of this peptide as being directly related to the interaction with enzymes (such as proteases) or extracellular receptors, whose physiological functions are very different from those of transcription factors, resulting in very different applications, which is the subject of this invention.

[0056] Other objects and advantages of the invention will become apparent in the following description of embodiments, which are given by way of indicative and non-limiting examples only.

[0057] Referring to the accompanying drawings will make this specification easier to understand, in which: [Figure 1] includes two figures, the first figure (the one at the top of the figure) Figure 1A The first figure shows the detection intensity of carboxymethyl lysine (CML) obtained by immunofluorescence on a histological slide in the absence (white bar) and presence (gray bar) of the peptide according to the invention (denoted as BFP in the figure), while the second figure (the bottom of the figure) shows the detection intensity of carboxymethyl lysine (CML) obtained by immunofluorescence on a histological slide in the absence (white bar) and presence (gray bar) of the peptide according to the invention. Figure 1B The results show the glycation intensity of the protein vimentin under similar conditions.

[0058] [Figure 2] includes three figures: - The first diagram at the top of this diagram ( Figure 2A The results of a test for measuring the concentration of total vimentin obtained from in vitro cultured skin explants under different culture conditions using ELISA (enzyme-linked immunosorbent assay) technology are shown, namely, those without oxidative stress ("no stress", white rectangles), those subjected to oxidative stress by means of 0.5 mM glyoxal but not treated with the peptide of the present invention ("glyoxal 0.5 mM-BFP", light gray rectangles), or those treated with the peptide of the present invention ("glyoxal 0.5 mM+BFP", dark gray rectangles). - The second diagram in the middle of this diagram ( Figure 2B This demonstrates the combination Figure 2A The test results obtained by detecting the glycation intensity of vimentin on a histological slide of skin explants cultured in vitro under the described culture conditions; - The third image at the bottom of this chart ( Figure 2C This demonstrates the combination Figure 2A The results of intranuclear detection of the NRF2 transcription factor were obtained by immunofluorescence on histological slides of skin explants cultured in vitro under the described culture conditions.

[0059] [Figure 3] shows the immunofluorescence assay performed on histological slides of skin explants from volunteers who underwent surgical facelift 28 days after treatment with either a peptide-containing cream (grey bars, “Vimentin Strength BFP” and “CMLBFP”) or a peptide-free cream (white bars, “Vimentin Strength Placebo” and “CML Placebo”). (The first image at the top of this figure is the first image.) Figure 3A )) and the second figure at the bottom of the figure ( )) and used to detect CML ( Figure 3B The results of the test.

[0060] [ Figure 4 [This is a table listing genes involved in detoxification processes via autophagy, mitophagy, ubiquitination, and degradation via the ubiquitin / proteasome system, the transcription of which is induced by the peptides of this invention.]

[0061] [ Figure 5 The results of RT-qPCR on in vitro cultured skin explants under the following different culture conditions: no oxidative stress (“no stress”), oxidative stress with 0.5 mM glyoxal but without treatment with the peptide of the present invention (“glyoxal 0.5 mM-BFP”), or treatment with the peptide of the present invention (“glyoxal 0.5 mM+BFP”), were presented to analyze the differential regulation of genes identified by genomic analysis after glycation induction by the peptide of the present invention (denoted as BFP in the figure). The results are shown in […]. Figure 4 middle.

[0062] For illustrative purposes, this specification uses three-letter or single-letter codes to identify the amino acids that form the detoxification peptide sequence.

[0063] Therefore, Ala or A corresponds to alanine, Cys or C corresponds to cysteine, Asp or D corresponds to aspartic acid, Glu or E corresponds to glutamic acid, Phe or F corresponds to phenylalanine, Gly or G corresponds to glycine, His or H corresponds to histidine, Ile or I corresponds to isoleucine, Lys or K corresponds to lysine, Leu or L corresponds to leucine, Met or M corresponds to methionine, Asn or N corresponds to asparagine, Pro or P corresponds to proline, Gln or Q corresponds to glutamine, Arg or R corresponds to arginine, Ser or S corresponds to serine, Thr or T corresponds to threonine, Val or V corresponds to valine, Trp or W corresponds to tryptophan, and Tyr or Y corresponds to tyrosine.

[0064] It should also be noted that “glycemic oxidation” involves a process called glycation, in which sugar molecules (such as glucose) react with the amino groups of proteins or, in the case of glycolipids, the amino groups of lipids. This initially reversible reaction results in the formation of an unstable Schiff base, which subsequently undergoes a molecular rearrangement (called the Amadori rearrangement). Later in the reaction, the Amadori product undergoes additional cleavage or cyclization modifications, generating a set of highly reactive and irreversible heterogeneous compounds known as advanced glycation end products (or AGEs), which can persist for a long time. Because these late stages involve numerous oxidation phenomena, the process is often referred to as “glycemic oxidation.”

[0065] The term "AGE" and its plural form refer to advanced glycation end products, which consist of harmful molecules formed when proteins or lipids in the body undergo glycation (i.e., when they bind to sugar molecules). AGEs can accumulate in various tissues of the body, especially 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.

[0066] It should be noted that AGEs can be detected primarily by immunochemistry using specific antibodies, which is achieved to obtain the results described below in conjunction with the accompanying figures (particularly the results using anti-CML antibodies). This technique is well known to those skilled in the art.

[0067] "Physiological compounds" are molecules that are naturally produced by the body. These compounds can include a wide range of molecules, such as enzymes, hormones, neurotransmitters, and metabolites. The term "physiological compounds released by the ECM" corresponds to compounds such as matrix factors and elasticity factors.

[0068] The term "synthetic peptide" refers to peptides obtained through chemical synthesis rather than extraction (such as plant extracts).

[0069] The subject of this invention is a detoxifying peptide whose sequence contains at least the following universal amino acid sequence: N-(Val-Gly-X1-X2-Pro-Gly)n-OH, wherein: - N and OH correspond to the N-terminus and C-terminus of the peptide, respectively; - X1 and X2 are selected from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gln), 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 related to glycation and / or oxidation of proteins and / or lipids.

[0070] Very preferably, X1 corresponds to valine (Val) and X2 corresponds to alanine (Ala), such that the peptide has the sequence N-(Val-Gly-Val-Ala-Pro-Gly)n-OH.

[0071] Even more preferentially, the number n equals 3.

[0072] Therefore, when n equals 1, the peptide of the present invention has the universal sequence SEQ ID NO: 1, and very preferably has the sequence SEQ ID NO: 2 (VGVAPG).

[0073] When n equals 2, the peptide of the present invention has the universal sequence SEQ ID NO: 3, and very preferably has the sequence SEQ ID NO: 4 (VGVAPGVGVAPG).

[0074] When n equals 3, the peptide of the present invention has the universal sequence SEQ ID NO: 5, and very preferably has the sequence SEQ ID NO: 6 (VGVAPGVGVAPGVGVAPG).

[0075] Advantageously, the present invention relates more particularly to detoxification peptides having the following universal amino acid sequence: N-(Val-Gly-X1-X2-Pro-Gly)n-Arg-X3-Arg-X4-X5-X6-X7-OH In this general sequence: - N and OH correspond to the N-terminus and C-terminus of the peptide, respectively; - Residues X1 and X2 are each selected from the following amino acid compositions: valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gln), alanine (Ala), leucine (Leu), and isoleucine (Ile); - Residues X3 and X4 are each selected from the following amino acid compositions: valine, serine, tyrosine, alanine, glycine, arginine, and leucine. - Residues X5, X6 and X7 are each selected from the following amino acid compositions: isoleucine (Ile), leucine (Leu), valine (Val), alanine (Ala), glycine (Gly) and phenylalanine (Phe); - n is an integer between 1 and 3.

[0076] When n equals 1, the peptide of the present invention has a universal sequence SEQ ID NO: 7.

[0077] When n equals 2, the peptide of the present invention has a universal sequence SEQ ID NO: 8.

[0078] When n equals 3, the peptide of the present invention has a universal sequence SEQ ID NO: 9.

[0079] According to the present invention, peptides having such a universal sequence are more specifically intended for the treatment and / or prevention of pathologies related to glycation and / or oxidation of proteins and / or lipids.

[0080] Preferably, the sequence of the detoxifying peptide consists of the sequence listed in the sequence listing, SEQ ID NO: 12, namely N-(Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-Ile-Leu-OH, wherein N and OH correspond to the N-terminus and C-terminus of the peptide, respectively, and wherein n equals 3.

[0081] The sequence of the detoxifying peptide of the present invention may also consist of one of the following sequences, for example: 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.

[0082] The detoxification peptides of the present invention are particularly advantageous for use in increasing the expression of the NRF2 transcription factor (“Nuclear Factor Erythrocyte 2-Associated Factor 2”) and / or preferentially increasing its nuclear translocation and / or activating pathways of the NRF2 transcription factor, thereby causing activation of genes involved in lysosomal and / or ubiquitin / proteasome and / or autophagy and / or ubiquitination pathways, in the specific context of the glucose oxidation and glycation pathways, as demonstrated in the figures, which will be illustrated in the remainder of the specification in conjunction with examples and figures showing the results of tests performed with the said peptides.

[0083] Therefore, the detoxification peptide of the present invention (specifically having the sequence SEQ ID NO: 12) increases the degradation of advanced glycation end products (AGEs) such as carboxyethyl lysine (CEL), carboxymethyl lysine (CML), glyoxal-lysine dimer (GOLD), 3-deoxyglucuronide lysine dimer (DOLD), pyrrolidine, and fructose lysine by regulating the NRF2 factor pathway. The NRF2 factor itself acts on the regulation of certain degradation pathways by activating genes involved in these pathways (autophagy, lysosomes, ubiquitin / proteasomes, or ubiquitin-like substances).

[0084] Furthermore, the detoxifying peptides according to the present invention promote the degradation of glycosylated proteins.

[0085] Advantageously, the peptide promotes the degradation of glycated vimentin.

[0086] Therefore, this detoxifying peptide has several advantageous functions, enabling it to both detoxify glycosylated proteins and regulate at different levels of the NRF2 pathway cascade.

[0087] Glycosylation of biomolecules can alter their biomechanical and functional properties.

[0088] Among these, vimentin is a protein involved in regulating cell morphology and migration, as well as in cellular stress responses.

[0089] Although it is essential for normal cell function, high levels of vimentin and its glycation can contribute to disease progression, which is why the inventors believe that vimentin is a therapeutic target for several treatments.

[0090] Therefore, the present invention also relates to peptides for use in the treatment and / or prevention of side effects of radiotherapy and / or chemotherapy in patients with cancer.

[0091] Glycation of proteins in the extracellular matrix can induce changes in the morphology and distribution of fibroblasts.

[0092] Therefore, the present invention also relates to such detoxifying peptides for use in the treatment and / or prevention of fibrosis, whether the fibrosis is fibrosis of the skin or involves other tissues and organs such as the liver.

[0093] This is because vimentin levels have been proposed to be associated with profibrotic properties. Specifically, elevated vimentin levels have been found in renal fibrosis (Lin et al., 2023). In the context of skin, vimentin can be reduced by negative pressure wound therapy (Wu et al., 2022). Therefore, therapeutic treatments that induce a decrease in vimentin levels or expression can be considered to have antifibrotic properties.

[0094] In particular, the ubiquitination pathway is involved in liver fibrosis, NASH (non-alcoholic steatohepatitis), or liver cancer.

[0095] In fact, oxidative stress is considered a major factor in the progression of NASH. The NRF2 transcription factor and its negative regulator KEAP1 are key regulators of redox, metabolism, protein homeostasis, and detoxification, thus appearing as attractive targets for NASH treatment. Disruption of the KEAP1-NRF2 interaction triggers increased antioxidant responses and coordinated regulation of a wide range of genes involved in NASH progression, ultimately leading to reduced NASH and liver fibrosis progression in mice (Seedorf et al., 2023).

[0096] Therefore, the use of detoxifying peptides in the treatment and / or prevention of such pathologies is also of interest.

[0097] In another particular aspect, the present invention relates to the aforementioned peptides used as anti-glycation peptides, antioxidant peptides, and anti-inflammatory peptides.

[0098] Accumulation of glycated vimentin can be observed in fibroblasts of aged donor skin (in vivo), thus establishing a close link between changes in AGEs in human tissues (such as skin) and loss of organ contractile function.

[0099] Intracellular vimentin filaments within fibroblasts undergo extensive glycosylation, and these filaments then redistribute to form aggregates. This modification of the filaments leads to a decrease in the contractile properties of fibroblasts.

[0100] Therefore, the present invention also relates to such peptides intended for use in the treatment and / or prevention of loss of organ contractile function.

[0101] The inventors believe that activation of the NRF2 factor is a treatment and / or preventative option for various skin diseases.

[0102] This is because, under pathological conditions, the activation of NRF2 presents as a promising treatment option for various skin diseases. Atopic dermatitis (AD), eczema, and psoriasis are common chronic inflammatory skin diseases caused by skin barrier defects, dysregulation of the immune response, genetic susceptibility, and environmental factors (Ogawa and Ishitsuka, 2022). Psoriasis is accompanied by a strong process of glycation oxidation (Damasiewicz-Bodzek and Nowak, 2022).

[0103] Atopic dermatitis and eczema are two very similar diseases, revealing impaired activation of the NRF2-antioxidant pathway in the affected epidermal regions (Koch et al., 2023).

[0104] Psoriasis is a chronic, relapsing, and often severe skin disease that is frequently associated with an increased risk of metabolic disorders and cardiovascular complications.

[0105] One putative association is the enhancement of late-stage glucose oxidation and / or glycation processes in proteins.

[0106] In patients with psoriasis, the concentrations of CML, CEL, and sRAGE (soluble receptors for AGE) are significantly higher than in healthy individuals.

[0107] Compared with patients in the active phase, the concentrations of CML, CEL, and sRAGE were significantly reduced after disease remission. Psoriasis is accompanied by a strong process of glucose oxidation, and the high level of sRAGE reflects long-term overstimulation of RAGE (the receptor for AGE) (Damasiewicz-Bodzek and Nowak, 2022).

[0108] Therefore, the present invention also relates to detoxifying peptides for use in the treatment and / or prevention of skin-related pathologies such as psoriasis or atopic dermatitis.

[0109] Specifically, as will be demonstrated in the examples of this application below, the peptides of the present invention are particularly capable of significantly reducing CML levels.

[0110] The peptides of the present invention can also be used in the treatment and / or prevention of radiation dermatitis, a disease that occurs after irradiation.

[0111] Specifically, ionizing radiation irradiation produces ROS and glycation. Therefore, NRF2 has become a key regulator of cellular defense mechanisms, activating complex transcriptional programs, promoting the detoxification of reactive oxygen species, repairing oxidative damage, and preventing toxicity (Cohen et al., 2022).

[0112] Advanced glycation end products (AGEs) are associated with the pathogenesis of diabetic complications.

[0113] Nrf2 activators combat oxidative stress and improve wound healing processes in several pathophysiological conditions, including diabetes and its complications such as diabetic foot ulcers, chronic kidney disease, and diabetic nephropathy. Several bioactive compounds have been reported to reduce cellular stress by activating the Nrf2 transcription factor, thereby accelerating cell proliferation, angiogenesis, and damaged tissue repair (Victor et al., 2020).

[0114] Numerous studies have demonstrated the crucial role of oxidative stress in the pathophysiology of diabetic foot ulcers (DFU). The nuclear factor erythrocyte-associated factor 2 (Nrf2) signaling pathway and the biochemical pathways of advanced glycation end products (AGEs) play vital roles in regulating oxidative stress in vivo. Targeting these pathways to restore redox balance can control and mitigate the onset and progression of pathologies secondary to severe diabetes (Song et al., 2022).

[0115] This invention relates to such peptides for use in the treatment and / or prevention of diabetes and its complications such as diabetic neuropathy, diabetic nephropathy, glaucoma, cataracts or diabetic retinopathy.

[0116] In addition, acute activation of NRF2 has a cardioprotective effect (Collins et al., 2009; Howden, 2013; Howden et al., 2013; Li et al., 2022; Tanase et al., 2022).

[0117] Therefore, the present invention also relates to such peptides for use in the treatment and / or prevention of cardiovascular diseases.

[0118] NRF2 dysregulation is associated with many neurological diseases, particularly Alzheimer's disease, Parkinson's disease, Huntington's disease, and amyotrophic lateral sclerosis (ALS).

[0119] In these diseases, activation of the NRF2 signaling pathway can provide neuroprotection by reducing oxidative stress and inflammation.

[0120] Furthermore, these pathologies are known to be associated with the accumulation of AGEs (Reddy et al., 2022).

[0121] Therefore, the present invention also relates to such peptides for use in the treatment and / or prevention of neurological diseases, particularly those from the following group of neurological diseases: Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis (MS), and amyotrophic lateral sclerosis (ALS).

[0122] Another aspect of the invention relates to pharmaceutical compositions for use in the treatment and / or prevention of pathologies associated with glycation and / or sugar oxidation as listed above, the pharmaceutical composition comprising at least one peptide, a mixture of multiple peptides, and / or a pharmaceutically acceptable salt thereof, with or without a pharmaceutically acceptable adjuvant.

[0123] In a non-limiting manner, the peptides covered by this invention may be in the form of a solution or cream, and optionally combined with a carrier molecule to obtain such a composition.

[0124] In a particular embodiment, the peptide was used in the composition at a concentration substantially between 10 μg / ml and 1 mg / ml.

[0125] This is because no peptide toxicity was observed in fibroblasts treated with peptides according to the invention at concentrations up to 1 mg / ml.

[0126] More preferably, the recommended concentration of the detoxifying peptide of the present invention (particularly in pharmaceutical compositions) is between 50 μg / ml and 100 μg / ml.

[0127] It should also be noted that the detoxifying peptides of the present invention can be obtained, in a particularly advantageous manner, by chemical synthesis, using any technique suitable for this purpose and known to those skilled in the art.

[0128] The following examples describe in detail the procedures and test results performed using the detoxification peptides of the present invention, so that the effects of the peptides on certain characteristic markers of glycation and sugar oxidation, and their role in the NRF2 factor pathway can be particularly illustrated in conjunction with the accompanying figures.

[0129] Example 1: In vitro assessment of CML and glycosylated vimentin in cultured fibroblasts Sample preparation: Cells were inoculated at a rate of 10,000 cells per well (96-well plate) at 37°C, 5% CO2, and 10% FBS (fetal bovine serum) and maintained under optimal growth conditions for 3 days.

[0130] Three days later, glycation was induced with different doses (0.1 mM, 0.2 mM, 0.4 mM, 0.6 mM, and 0.8 mM) of glycated peptides for 24 hours. Then, 50 μg / ml of detoxifying peptide (or peptide BFP shown in the figure) was added, and the treatment continued for another 24 hours.

[0131] The peptide 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-Ile-Leu-OH, where n equals 3.

[0132] Apart from changing the culture medium, the control group (cells incubated with different doses of glyoxal) received no treatment.

[0133] Cell labeling and image acquisition: After treatment, cells were washed, nonspecific sites were blocked (Pierce™ in PBS (phosphate-buffered saline, pH 7.4), and cells were fixed onto plates and incubated for 1 hour with primary antibodies (rabbit monoclonal anti-Vimentin - Abcam (ab92547) or mouse monoclonal anti-CML - R&D Systems (MAB3247)) in Pierce™ solution in PBS.

[0134] After incubation, cells were washed with PBS and incubated for 1 hour with a secondary antibody conjugated with a fluorescent dye (goat anti-mouse AlexaFluor 488 - Invitrogen (A11001) or goat anti-rabbit AlexaFluor 647 - Invitrogen (A21244)). The cell nuclei were then labeled with DAPI (4',6-diamino-2-phenylindole). Following incubation, cells were washed with PBS, and a series of images were acquired using an epifluorescence microscope (Thermo Fisher Scientific, Evos M5000, 40X objective).

[0135] Images were collected and analyzed using ImageJ software (Rasband, NIH).

[0136] Quantitative data for each image were acquired by integrating specific fluorescence signals. Vimentin levels were quantified by assessing the area of ​​the occupied surface above a fluorescence intensity threshold, and then normalized by cell number.

[0137] CML levels are quantified by integrating signal intensities above a threshold, normalized by the area of ​​the assessment region, and then normalized by the number of cell nuclei.

[0138] Glycated vimentin levels were obtained by comparing the quantitative intensity signal (RFU / cell) of CML with the vimentin-positive area (region area / cell), and then normalized to a control group. Statistical analysis of binary comparisons was performed using a t-test (p = 0.05).

[0139] As shown in Figure 1, the results obtained demonstrate that the detoxifying peptide (BFP) of the present invention promotes CML in vitro. Figure 1A ) and glycated vimentin ( Figure 1B The degradation of ).

[0140] For ease of review and summary, human fibroblasts incubated with increasing concentrations of glyoxal for 24 h were then treated with detoxifying peptides (or not treated) for 24 h.

[0141] like Figure 1A As shown, compared with the control (no stress), the detection of CML increased and was correlated with glyoxal concentration (white bars in the histogram).

[0142] Treatment with detoxifying peptides can significantly reduce the detection of CML, especially at the highest glyoxal concentrations tested, namely 0.6 mM and 0.8 mM (gray bars in the histogram).

[0143] Interestingly, it was noted that, Figure 1B As shown, in fibroblasts subjected to 0.6 mM glyoxal stress, specific glycation of vimentin decreased after treatment with BFP (compared to untreated cells).

[0144] Therefore, the detoxifying peptide of the present invention enables the reduction of cellular glycation levels in vitro, and more particularly reduces the glycation level of vimentin, which is a preferred target of glycation.

[0145] Example 2: In vitro assessment of CML, glycosylated vimentin, and NRF2 in skin explants Sample preparation: Skin explants were obtained surgically via abdominal surgery in a 32-year-old Caucasian woman (skin type II / II, reference number DISC1D10J025) with informed consent. Explant viability was maintained by culturing in culture medium on a metal grid at 37°C, 5% CO2, and humidified air. The culture medium was changed every 24 hours, or 48 hours after treatment with glyoxal.

[0146] After receiving the explants, they were divided into three experimental groups (n = 3 / group). The active ingredient was applied topically at a concentration of 50 µg / ml on days 0, 2, and 4.

[0147] The peptide 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-Ile-Leu-OH, where n equals 3.

[0148] The glycation inducer (glyoxal, 500 μM) was applied to the culture medium on days 1 and 3.

[0149] Apart from changing the culture medium, the control group received no treatment.

[0150] On day 5, 24 hours after the third active ingredient treatment, explants were collected: half of each explant was embedded in an OCT compound and cryopreserved at -80°C until analysis; and half of each explant was frozen in liquid nitrogen and stored at -80°C until analysis.

[0151] CML detected: As described above, in situ analysis of vimentin glycation (via CML detection) is performed by specific detection of independent signals (vimentin, CML) using epifluorescence microscopy. CML levels (RFU / region area) are quantified in regions co-localized with vimentin above a threshold to assess vimentin glycation.

[0152] Anti-vimentin ELISA: Proteins were extracted from explants and vimentin levels were assessed according to the manufacturer’s instructions (colorimetric method; ab246526, Abogen).

[0153] Total protein was quantified using the Bradford assay, with equal amounts of total protein used for each measurement. Two copies of each explant were measured, resulting in a total of six measurements per condition (two per explant). Absorbance values ​​were interpolated based on the vimentin standard curve.

[0154] The results shown in Figure 2 demonstrate that peptide BFP activates the NRF2 pathway ( Figure 2C ) promotes total vimentin ( Figure 2A ) and glycated vimentin ( Figure 2B The decrease of ).

[0155] For ease of review and summary, skin explants were cultured in vitro in various culture media, with or without glycine-induced saccharification, and with or without treatment using the peptides of this invention. Skin explants were homogenized, and the total vimentin concentration was assessed using a specific ELISA test. Figure 2A In parallel, the same samples were labeled with anti-CML and anti-vimentin antibodies to determine glycated vimentin levels. Figure 2B Extracts treated with the same method were labeled with anti-NRF2 antibody to detect NRF2 nuclear translocations. Figure 2C ).

[0156] like Figure 2A As shown, the total concentration of vimentin increased in explants treated with glyoxal (light gray bar) and decreased by peptide treatment (dark gray bar), which means that the total vimentin was degraded by the action of peptides.

[0157] like Figure 2B As shown, compared with the control (white bar), glycan glycosylation of vimentin increased in explants treated with glyoxal (light gray bar) and decreased by treatment with BFP (dark gray bar).

[0158] like Figure 2C As shown, compared with the untreated condition (white bar), the intranuclear detection of NRF2 was significantly increased under the glyoxal + peptide BFP condition (dark gray bar).

[0159] These results not only confirm previous findings demonstrating that the detoxifying peptides of the present invention cause degradation of glycated vimentin, but also demonstrate that the detoxifying peptides of the present invention function by activating the NRF2 factor pathway.

[0160] Example 3: In vivo studies - clinical trials Clinical study design: This clinical trial was a single-blind, multicenter, randomized controlled trial involving 22 women (aged 45 to 70) who were required to receive the peptide of this invention twice daily for 28 days prior to a facelift, during which time skin samples were collected for quantitative genomic and proteomic analysis.

[0161] In a half-face test, the product (a cream containing peptide BFP) was applied and compared to a placebo (a cream without peptide BFP).

[0162] The peptide 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-Ile-Leu-OH, where n equals 3.

[0163] Collection and processing of skin extracts after a facelift: The surgery was performed using standard surgical techniques. Surgical waste removed during the procedure corresponded to excess skin around the ears caused by skin laxity. A piece of excess skin was removed from each side of the face and divided into two portions for genomics and proteomics analysis. Treatment of skin explants for proteomics analysis: ≥ 1 cm from each side of the face 2 Skin samples were immersed in 4% formalin solution at ambient temperature for 24 hours, then transferred to 70% ethanol solution and stored at ambient temperature before paraffin embedding and analysis. Paraffin sections approximately 4 μm thick were cut and placed on Superfrost Plus glass slides and dried overnight in an air oven at 45°C (+ / -3°C) before microscopic imaging.

[0164] Proteomics analysis using immunohistochemistry: Vimentin on paraffin sections was labeled with an anti-vimentin antibody (rabbit polyclonal antibody - Abogen, ab45939) using the OPAL™ 4-color IHC kit from PerkinElmer®. The antibody-labeled paraffin sections were then incubated with the fluorophore OPAL 690 (2018 Akoya Biosciences). Cell nuclei were labeled with DAPI. Following complete IHC and fluorescent labeling, the paraffin sections were scanned using a Vectra Polaris (PerkinElmer) with an X20 objective lens. Images were visualized, analyzed, validated, and quantified using HALO® software (Indicalabs). Thresholds were selected for quantifying the positive area of ​​each label, and the data were generated in Excel using the software.

[0165] CML was labeled on paraffin sections with a CML-specific primary antibody (anti-CMLMAB3247, R&D Systems) in 0.1% Tween-20 solution in PBS. Excess antibody was removed by a series of washes, and explants were then incubated with a fluorescently conjugated secondary antibody (goat anti-mouse Alexa Fluor 555, Ingenium A21422). Nuclei were labeled with DAPI (4',6-diamidindo-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 were acquired using an epifluorescence microscope (Thermo Fisher Scientific, Evos M5000) at exactly the same acquisition time and resolution (40x objectives). Images were collected at all fluorescence intensity levels (16-bit .TIFF raw format) and then analyzed using ImageJ software (Rasband, NIH). Each image was quantified by integrating specific fluorescence signals above a threshold and then normalized by the area of ​​the evaluated region. Three (3) images were analyzed for each experimental batch. A mean and a standard deviation were obtained for each experimental batch and for each condition (placebo control treatment). CML levels were then normalized for each "placebo" batch to obtain the relative rate of change (fold change) during peptide treatment.

[0166] The normality of the data distribution was verified using the Shapiro-Wilk test at a 5% risk threshold. The normality of the data distribution was analyzed using the Student's t-test, and other analyses were performed using the Wilcoxon test (p-value ≤ 0.05).

[0167] Immunohistochemical analysis of the obtained skin explants, shown in Figure 3, grouped according to whether they were treated with peptide (gray bars) or placebo (white bars), demonstrated that the peptide-induced vimentin in the human body (…) of the present invention… Figure 3A ) and CML ( Figure 3B The detection rate of ) decreased.

[0168] More specifically, a significant reduction in the detection of vimentin was observed in volunteers treated with the peptides of the present invention, and a significant reduction in the detection of CML was also observed.

[0169] Therefore, the detoxifying peptide of the present invention enables the induction of a decrease in glycation levels in humans.

[0170] Example 4: Genomics Analysis - Study of Genes Expressed in the Presence of Detoxification Peptides After treating cultured fibroblasts with the detoxification peptide BFP or the control peptide of this invention, a 5-step RNA-seq analysis was performed to identify differentially expressed genes between the following two conditions: Step 1: RNA Extraction: Using the miRNeasy mini kit (QIAGEN), RNA was extracted from samples representing various experimental conditions on an automated workstation (QIAcube, Qiagen) following the manufacturer's instructions. In short, cells were harvested and homogenized in 700 μl of QIAzol® lysis reagent in 2 ml SafeLock microcentrifuge tubes. Samples were stored at -80°C until use. A 2 mm stainless steel bead was added to each thawed sample, and cells were lysed using a TissueLyzer (QIAGEN) by mechanical shaking at 20 Hz (2 × 2 min). The samples were then incubated at ambient temperature for 5 min. 140 μl of chloroform was added to the homogenate. The tubes were shaken vigorously for 15 s and returned to the lab bench for another 2–3 min. The lysates were centrifuged in microcentrifuge tubes at 4°C, 12000 xg for 15 min. Carefully transfer the upper aqueous phase (approximately 350 μl) to a clean 2 ml microcentrifuge tube. Using a QIAcube automated workstation (QIAcube - Qiagen), perform the remainder of the procedure according to the manufacturer's protocol to optimize the reproducibility and standardization of RNA extraction (according to a pre-established protocol). At the end of the procedure, elute each RNA sample with 30 μl of H2O. Aliquot 3 μl of the sample into new microcentrifuge tubes for quality verification, and immediately store all tubes in an ultra-low temperature freezer (-80°C).

[0171] Step 2: RNA Quality Control: The concentration and purity of RNA samples were measured using a NanoDrop ND-1000 spectrophotometer (Thermo Scientific). RNA integrity was assessed using an Agilent 2200 TapeStation RNAScreenTape (Agilent Technologies), and the RINe (RNA Integrity Number Equivalent) score was verified (RINe must be >7).

[0172] Step 3: Construct an RNA-seq library using the KAPA mRNA Hyperprep Kit (KAPA). Use the RNA sample in the library construction procedure.

[0173] Step 4: Sequencing the library on Illumina NovaSeq (S1 execution cartridge - 100 cycles, library loaded at 1.2 nM). Step 5: Bioinformatics processing; for this analysis, the human genome was used as a reference (version GRCh38.p13). RNA Seq data were aligned using the Ensembl database version 103 (https: / / www.ensembl.org). All steps of the pipeline are listed below: a. Using FASTP software ( https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC6129281 / Perform sequence cleaning and pruning; b. Use FASTQC software ( http: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / Quality control is carried out. c. Using STAR software (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC3530905 / ), the human genome was aligned with the reference genome “Homo_sapiens.GRCh38.dna.primary_assembly.fa” and the annotation file “Homo_sapiens.GRCh38.103.chr.gtf”; d. Count the readings for each gene using STAR software.

[0174] The peptide 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-Ile-Leu-OH, where n equals 3.

[0175] Figure 4 The main genes identified and their expression values ​​are listed.

[0176] In summary, the results of this genomic analysis demonstrate that the peptides of the present invention induce the expression of genes involved in resisting oxidative stress, autophagy, ubiquitination, and degradation by the ubiquitin / proteasome system in vitro.

[0177] Specifically, such as Figure 4 As shown in the table, a large number of genes were identified from RNAseq data of cultured fibroblasts, demonstrating the role of this peptide in triggering NRF2-mediated cellular defense responses.

[0178] Example 5: Results of RT-qPCR on skin explants As shown in Example 4 above and in conjunction with the appendix Figure 4 The explanation is that after identifying genes that are differentially regulated by peptides in cultured fibroblasts, the regulation of these genes in skin explants is analyzed using different techniques (i.e., RT-qPCR).

[0179] Skin explants may or may not be subjected to glycation agents (glyoxal).

[0180] The skin explant was obtained with the informed consent of a 32-year-old Caucasian woman who had undergone abdominal surgery.

[0181] Explant viability was maintained by culturing in a medium on a metal grid at 37°C, 5% CO2, and humidified air. The medium was changed every 24 hours, or 48 hours after treatment with glyoxal.

[0182] After receiving the explants, they were divided into three experimental groups (n = 3 / group). The active ingredient was applied topically at a concentration of 50 µg / ml on days 0, 2, and 4.

[0183] The applied active ingredient is a peptide having the sequence SEQ ID NO: 12 (in Figure 5 The figure is denoted as BFP), that is, VGVAPGVGVAPGVGVAPGRVRLGIL or N-(Val-Gly-Val-Ala-Pro-Gly)n-Arg-Val-Arg-Leu-Gly-Ile-Leu-OH, where n equals 3.

[0184] On days 1 and 3, the glycation inducer (glyoxal, 500 μM) was applied to the culture medium. The control group received no treatment except for a change of culture medium. On day 5, 24 hours after the third treatment with the active ingredient (the peptide according to the invention), explants were collected: half of each explant was sealed in an OCT (optimal cutting temperature) compound and cryopreserved at -80°C until analysis; and half of each explant was frozen in liquid nitrogen and stored at -80°C until analysis.

[0185] cDNA synthesis was performed using the ReadyScript™ cDNA Synthesis Mixture (Sigma-Aldrich) in a final reaction volume of 20 μl, following the manufacturer's protocol. For each sample, 550 ng of RNA was used as the RNA template for reverse transcriptase. Using a Bio-Rad C1000 thermal cycler, cDNA synthesis was performed in 96-well plates using the following thermal program in a final reaction volume of 20 μl: 25°C for 5 min, 42°C for 30 min, and 85°C for 5 min. The purified cDNA was stored at -20°C until use.

[0186] All qPCR reactions were performed in 384-well plates (with 3 technical replicates). qPCR reactions were performed on a LightCycler 480 System II (Roche) with a final reaction volume of 10 μl. The reaction mixture consisted of 5 μl LightCycler 480 SYBR Green I master mix 2X (Roche Diagnostics), 4 μl primers (final concentration 0.7 μM), and 1 μl cDNA diluted 1:10.

[0187] The qPCR cycling program consisted of the following: a polymerase activation step at 95°C for 10 minutes, followed by 45 qPCR reaction cycles: denaturation at 95°C for 3 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 15 seconds (fluorescence was collected at the end of the extension step). Finally, melting curves from 65°C to 97°C were generated with continuous fluorescence collection to check the specificity of the amplicon. Ct values ​​measured for each gene were repeated three times, with the standard deviation between replicates strictly less than 0.5 Ct. NTC (template-free control) reactions were performed in the same manner, but the cDNA template was replaced with PCR-grade water (one technical replicate). Raw Ct data were acquired using the second derivative maxima method on a LightCycler 480 System II instrument (Roche).

[0188] YWHAZ, PPIB, and B2M were considered housekeeping genes, and their relative gene expression was analyzed and statistically calculated using real-time quantitative PCR data via the 2-ΔΔCt method.

[0189] The results are shown in the attached figures. Figure 5 middle.

[0190] like Figure 5 As shown, glyoxal induces increased expression of genes CXCL1 (p<0.01) and DNAJC10 (p<0.05), and decreased expression of DNAJB14 (p<0.05), UBE2D3 (p<0.05), PSMD12 (p<0.05) and RNF19A (p<0.001).

[0191] The peptide BFP can counteract the effects of glyoxal by downregulating the expression of CXCL1 (p<0.0001) and DNAJC10 (p<0.01), while upregulating the expression of genes 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). These genes are involved in the complex process of autophagy and / or proteasome degradation, enabling cells to degrade toxic compounds.

[0192] It should be noted that no difference in VIM expression was observed by analyzing RNA extracted from the same samples.

[0193] In summary, these results confirm the protective effect of this peptide against protein glycation, particularly by activating autophagy and ubiquitin-mediated protein degradation pathways. By counteracting gene expression induced by glycation agents (glyoxal in our study) via NRF2 activation and by promoting autophagy, the peptide of this invention exhibits anti-glycation and anti-fibrotic properties.

[0194] This experiment demonstrates that the detection of the protein vimentin is reduced ( Figure 2A The decrease is not due to a decrease in its gene expression (VIM = gene encoding vimentin), but rather to an increase in its degradation via autophagy and / or its ubiquitin-mediated protein degradation due to its greater degree of glycosylation.

[0195] It should be noted that the tests performed and presented in the above examples were performed using the peptide of the present invention having a particularly preferred sequence SEQ ID NO: 12 (i.e., sequence VGVAPGVGVAPGVGVAPGRVRLGIL), with amino acid residues identified by single-letter codes.

[0196] That is, the peptide according to the invention may have other sequences, provided that certain residues are retained without affecting its properties; in particular, the residues Val, Gly, Pro and Gly at positions 1, 2, 5 and 6 of the hexapeptide are repeated n times, and the first Arg residue immediately following the hexapeptide and the second Arg residue separated from the first Arg residue by one amino acid residue should also be retained.

[0197] In particular, apart from the amino acids to be retained mentioned above, other amino acid residues in sequence SEQ ID NO: 12 can be replaced by amino acids that are chemically equivalent (i.e. have equivalent physicochemical characteristics), for example.

[0198] Therefore, substitutions can be made between equivalent amino acids, for example, between aliphatic nonpolar amino acids Ala, Val, Ile, and Leu, or between polar amino acids containing hydroxyl groups, such as Ser and Thr, between amino acids Asn and Gln, or between amino acids containing two acidic functional groups, such as Asp and Glu.

[0199] In particular, the detoxifying peptide of the present invention can therefore very preferably have one of the sequences selected from SEQ ID NO: 10 to SEQ ID NO: 37.

[0200] References - Cohen, D., Portugal-Cohen, M., Oron, M., Frusic-Zlotkin, M.,Soroka, Y., Ma'or, Z., Amar, D., Kohen, R., 2022. Cutaneous Nrf2-Keap1pathway modulation by environmental factors: The Dead Sea area as a testcase. Biofactors. https: / / doi.org / 10.1002 / biof.1926 - Collins, AR, Lyon, CJ, Xia, https: / / doi.org / 10.1161 / CIRCRESAHA.108.188771 - Damasiewicz-Bodzek, A., Nowak, A., 2022. Concentrations of N6-Carboxymethyllysine (CML), N6-Carboxyethyllysine (CEL), and Soluble Receptorfor Advanced Glycation End-Products (sRAGE) Are Increased in PsoriaticPatients. Biomolecules 12, 1870. https: / / doi.org / 10.3390 / biom12121870 - Howden, R., 2013. Nrf2 and cardiovascular defense. Oxid Med CellLongev 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 toenvironmental 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., Stölzl, D., Beer, H.-D., Tschachler, E., Weidinger,S., Werner, S., Auf dem Keller, U., 2023. Quantitative Proteomics IdentifiesReduced NRF2 Activity and Mitochondrial Dysfunction in Atopic Dermatitis. JInvest Dermatol 143, 220-231.e7. https: / / doi.org / 10.1016 / j.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-mediatedproliferation and migration of vascular smooth muscle cells. Atherosclerosis347, 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 todevelopment of renal fibrosis by accelerating epithelial-mesenchymaltransition. Cell Cycle 22, 580-595. https: / / doi.org / 10.1080 / 15384101.2022.2136463 - Motohashi, H., Yamamoto, M., 2004. Nrf2-Keap1 defines aphysiologically important stress response mechanism. Trends Mol Med 10, 549-557. https: / / doi.org / 10.1016 / j.molmed.2004.09.003 - Ogawa, T., Ishitsuka, Y., 2022. The Role of KEAP1-NRF2 System inAtopic 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., Geant, A., Ilic, 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 andreduction 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 Biologics AccelerateHealing of Diabetic Foot Ulcers by Regulating Oxidative Stress. Front Biosci(Landmark Ed) 27, 285. https: / / doi.org / 10.31083 / j.fbl2710285 - Tanase, D.M., Apostol, A.G., Costea, C.F., Tarniceriu, C.C.,Tudorancea, I., Maranduca, M.A., Floria, M., Serban, I.L., 2022. OxidativeStress 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. Pharmacologicalactivation of Nrf2 promotes wound healing. Eur J Pharmacol 886,173395. https: / / doi.org / 10.1016 / j.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 inMurine Diabetic Wound Healing. Pharmaceutics 14, 2125. https: / / doi.org / 10.3390 / pharmaceutics14102125.

Claims

1. A detoxifying peptide, characterized in that... Its sequence contains the following universal amino acid sequence: N-(Val-Gly-X1-X2-Pro-Gly)n-OH, where: -N and OH correspond to the N-terminus and C-terminus of the peptide, respectively; -X1 and X2 are selected from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gln), 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 associated with the glycation and / or oxidation of proteins and / or lipids, which are related to the accumulation of advanced glycation end products (AGEs) in organ tissues, the detoxifying peptide is capable of reducing AGEs in the tissues and / or promoting the degradation of AGEs by activating a pathway of a specific nuclear transcription factor, designated as NRF2 factor or "Nuclear Factor Erythrocyte 2-Associated Factor 2".

2. The detoxification peptide of claim 1, which, by activating the NRF2 transcription factor, induces activation of genes involved in autophagy and / or lysosomes and / or ubiquitin / proteasomes and / or ubiquitin-like pathways.

3. The detoxifying peptide as described in claim 1 or claim 2, for use in the treatment and / or prevention of pathologies associated with protein glycation and / or glucose oxidation, which are related to the accumulation of AGEs of the glycated vimentin and / or carboxymethyl lysine (CML) type in organ tissues, wherein the detoxifying peptide is capable of reducing glycated vimentin and / or carboxymethyl lysine (CML) by activating the NRF2 factor pathway.

4. The detoxifying peptide for use as described in any one of claims 1 to 3, characterized in that... It has the following universal amino acid sequence: N-(Val-Gly-X1-X2-Pro-Gly)n-Arg-X3-Arg-X4-X5-X6-X7-OH in: -N and OH correspond to the N-terminus and C-terminus of the peptide, respectively; -X1 and X2 are selected from valine (Val), glycine (Gly), threonine (Thr), asparagine (Asn), glutamine (Gln), 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. The detoxifying peptide for use as described in any 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-Ile-Leu-OH, wherein N and OH correspond to the N-terminus and C-terminus of the peptide, respectively, and wherein n equals 3.

6. The detoxifying peptide for use as described in any one of claims 1 to 4, characterized in that... It has sequences selected from the group consisting of the following sequences: SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 13 to SEQ ID NO:

37.

7. The detoxifying peptide as described in any one of claims 1 to 6, for use in the treatment and / or prevention of side effects of radiotherapy and / or chemotherapy in patients with cancer.

8. The detoxifying peptide as described in any one of claims 1 to 6, for use in the treatment and / or prevention of skin fibrosis and / or liver fibrosis and / or non-alcoholic steatohepatitis (or NASH) and / or liver cancer.

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

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

11. The detoxifying peptide as described in any one of claims 1 to 6, for use in the treatment and / or prevention of cardiovascular pathology.

12. The detoxifying peptide as described in any one of claims 1 to 6, for use 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. The detoxifying peptide for use as described in any of the preceding claims, characterized in that... It is obtained through chemical synthesis.

14. A pharmaceutical composition comprising at least one peptide as described 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 associated with glycation and / or oxidation of proteins and / or lipids.

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