HIP / PAP protein or derivatives thereof for treating peripheral neuropathy

Abstract

The present invention relates to the use of HIP / PAP proteins, or derivatives thereof, for treating or preventing peripheral neuropathy in an individual, particularly diabetic peripheral neuropathy in diabetic or pre-diabetic individuals. The present invention also relates to the use of HIP / PAP proteins, or derivatives thereof, for preventing complications of peripheral neuropathy in an individual, particularly diabetic foot wounds, particularly diabetic foot ulcers, and the outcomes thereof.

Description

[Technical field]

[0001] FIELD OF THEINVENTION

[0002] The present invention relates to the use of HIP / PAP proteins, or derivatives thereof, in the treatment and prevention of peripheral neuropathy in individuals, particularly diabetic peripheral neuropathy in individuals, and in the prevention of disorders directly caused by such peripheral neuropathy, such as diabetic foot lesions, particularly diabetic foot ulcers, and their outcomes.

[0003] prior art

[0004] Peripheral neuropathy is a condition that involves damage to the peripheral nervous system, the vast communication network that sends signals between the central nervous system (brain and spinal cord) and every other part of the body.

[0005] Symptomatic acquired peripheral neuropathy can result from several different causes: physical injury (trauma), such as car accidents, falls, sports, and medical procedures or arthritis; diabetes; vascular and blood problems; systemic autoimmune diseases; autoimmune diseases that attack only the nerves; hormone imbalances; nutritional or vitamin imbalances; alcoholism; exposure to toxins (such as some medications, such as chemotherapy drugs); some cancers or benign tumors; or infections.

[0006] Diabetic peripheral neuropathy is the most common complication of diabetes, affecting up to 50% of diabetic patients. It occurs in both type 1 and type 2 diabetic patients, with a higher prevalence in type 2 diabetic patients.

[0007] Diabetic peripheral neuropathy is characterized by progressive distal-to-proximal degeneration of peripheral nerve axons, pain and loss of sensation as a result of nerve damage caused by chronically high blood sugar levels. It leads to numbness, loss of sensation, and sometimes pain in the feet, legs, or hands. As a result, diabetic peripheral neuropathy worsens the quality of life of diabetic patients and is the leading cause of death in diabetic patients.

[0008] Furthermore, diabetic peripheral neuropathy can cause a wide range of complications, including chronic pain; foot deformities; foot injuries, and especially foot ulcers; foot infections; and amputations. Indeed, damage to the innervation of the intrinsic muscles of the foot leads to an imbalance between flexion and extension of the affected foot. This produces an anatomical foot deformity that creates abnormal bony prominences and pressure points that gradually cause skin breakdown and ulcers. Furthermore, autonomic neuropathy leads to decreased sweating, which leads to overlying skin drying, increased susceptibility to cracking, and subsequent development of infection. Loss of sensation as part of peripheral neuropathy exacerbates the development of ulcers. Because trauma occurs to the affected area, patients are unable to detect damage to their lower extremities. As a result, many injuries go unnoticed and progressively worsen because the affected area is continually subjected to repetitive pressure and shear forces from walking and weight bearing. Furthermore, autonomic plexus transection leads to bone demineralization through impairment of vascular smooth muscle, which leads to an increase in blood flow to bone with eventual osteolysis (Sharad P. Pendsey. Int J Diabetes Dev Ctries. 2010 Apr-Jun;30(2): 75-79).

[0009] The estimated global prevalence of diabetic foot ulcers is 6%, with major risk factors including larger size, older age, lower body mass index, longer duration of diabetes, hypertension, diabetic retinopathy, and smoking (Global epidemiology of diabetic foot ulceration: a systematic review and meta-analysis. Zhang P. et al. Ann Med. 2017 Mar;49(2):106-116.). Approximately 25% of people with diabetes will develop foot ulcers during their lifetime, which can progress to infection and, in severe cases, amputation. Of all amputations in diabetic patients, 85% are preceded by foot ulcers, which subsequently deteriorate to severe gangrene or infection. For example, in the United States, where the highest prevalence of diabetic foot ulcers is reported, diabetes accounts for 83% of major amputations. The total healthcare costs for treating diabetic foot disease in the United States range from $9 billion to $13 billion, an additional cost associated with diabetes (Rice JB et al. Burden of diabetic foot ulcers for medicare and private insurers. Diabetes Care. 2014;37:651-8).

[0010] Because the correlation between hyperglycemia and progression of neuropathy is well established and many studies have depicted a high prevalence of diabetic peripheral neuropathy in diabetic patients with poor glycemic control, controlling blood glucose is recognized as the primary method of preventing diabetic peripheral neuropathy (N Engl J Med 1993;329:977-86). However, this method has demonstrated its limitations, and most diabetic patients are unable to maintain control of their blood glucose.

[0011] Other solutions have been tested in the form of therapeutic agents, such as alpha lipoic acid, aldose reductase inhibitors, and gamma linoleic acid. α-Lipoic acid (thioctic acid) is an antioxidant and has been identified as reducing hyperglycemia-induced oxidative stress leading to neuronal death, thereby blocking the pathogenesis of diabetic neuropathy. Aldose reductase inhibitors prevent glucose from entering the polyol pathway in neuronal cells, thereby inhibiting the accumulation of sorbitol, leading to neuronal damage, and blocking the progression of diabetic neuropathy. However, the development of most of these active ingredients has been discontinued midway due to side effects and insufficient efficacy. Y-linolenic acid is both a component of nerve membrane phospholipids and a component of prostaglandin E, plays an important role in the homeostasis of blood flow in neuronal cells, and has been found to ameliorate some symptoms of diabetic neuropathy.

[0012] However, as indicated by the Centers for Disease Control and Prevention, controlling blood sugar in diabetic patients only helps slow the progression of nerve damage in established neuropathy, especially peripheral neuropathy.

[0013] It is widely believed that the damage caused to nerves in neuropathy, and particularly diabetic neuropathy, is irreversible (see, e.g., Richard AC Hughes, BMJ. 2002 Feb 23;324(7335): 466-469 and G. Sloan et al., Nature Reviews Endocrinology volume 17, pages 400-420 (2021)).

[0014] Sloan et al. (Pathogenesis, diagnosis and clinical management of diabetic sensorimotor peripheral neuropathy, Nature Reviews Endocrinology volume 17, pages 400-420 (2021)) show that holistic management of patients with distressing painful neuropathic symptoms (DSPN) not only includes strategies aimed at treating symptoms (e.g. neuropathic pain, comorbid mood disorders, insomnia, autonomic symptoms, unsteadiness, and falls), preventing the progression of DSPN (managing cardiometabolic risk factors), and addressing foot complications.

[0015] Similarly, the UK Prospective Diabetes Study (UKPDS) in 3,867 patients with type 2 diabetes found no effect of glucose control on the prevalence of neuropathy (approximately a 0.9% reduction in HbA1c), whereas there was a significant reduction in the risk of retinopathy and nephropathy (UKPDS Group 1998).

[0016] Thus, the treatment of diabetes in diabetic individuals does not make it possible to treat already existing neuropathic damage and in particular does not treat / repair damaged nerves, in particular damaged terminal nerves, i.e. does not make it possible to restore the activity of damaged nerve endings.

[0017] Furthermore, as shown above, a significant proportion of individuals suffering from or at risk of suffering from peripheral neuropathy are non-diabetic, i.e., obese or non-obese, pre-diabetic or normoglycemic individuals. BC Callaghan et al. (Mayo Clin Proc. 2020 Jul;95(7):1342-1353) showed, for example, that after recruiting 138 obese individuals and 46 lean individuals, the prevalence of neuropathy was determined to be 2.2% in lean controls, 12.1% in obese participants with normoglycemia, 7.1% in obese participants with pre-diabetes, and 40.8% in obese participants with diabetes. OH Nienov (Rev Assoc Med Bras (1992). 2017 Apr;63(4):324-331) further evaluated the prevalence of peripheral polyneuropathy (PPN) in subjects with grades II and III obesity (Ob-II, III) and determined that a total of 24 out of 218 non-diabetic obese grades II and III metabolic syndrome (MetS) patients had PPN.

[0018] As the number and prevalence of peripheral neuropathy cases, particularly those associated with diabetes, have been steadily increasing worldwide over the past few decades, and with the estimated number of patients suffering from diabetes estimated by the World Health Organization at well over 420 million (with over 1.6 million deaths each year directly attributable to diabetes), finding new solutions to prevent and treat peripheral neuropathy, particularly diabetic peripheral neuropathy, remains an important goal from a public health perspective.

[0019] There is therefore an increasing need for new active substances which are able to prevent and / or treat peripheral neuropathy, in particular diabetic peripheral neuropathy.

[0020] Furthermore, there is an increasing need for the prevention of complications of peripheral neuropathy, more particularly diabetic peripheral neuropathy, particularly foot wounds, and in particular foot ulcers; foot infections; and amputations thereof.

[0021] There is therefore an increasing need for new active substances which are able to prevent and / or treat damaged nerves, in particular damaged sensory and / or motor nerves, more particularly damage to nerve endings, in particular damage to sensory and / or motor nerve endings, in particular in individuals with or susceptible to peripheral neuropathy.

[0022] Certainly, there is a need for new active substances capable of repairing nerve endings, in particular sensory and / or motor nerve endings, especially in individuals with peripheral neuropathy.

[0023] Certainly, there is a need for new active substances capable of restoring the activity of nerve endings, in particular of sensory and / or motor nerve endings, in particular in individuals with peripheral neuropathy.

[0024] Summary of the Invention

[0025] The Applicant has surprisingly demonstrated that the HIP / PAP protein, or a derivative thereof, makes it possible to prevent and / or treat peripheral neuropathy in patients, in particular to prevent and / or treat diabetic peripheral neuropathy in diabetic or pre-diabetic patients. The HIP / PAP protein may therefore be advantageously used to: - Treating or preventing peripheral neuropathy in a patient, particularly diabetic peripheral neuropathy in a diabetic or pre-diabetic patient; and - Preventing complications of peripheral neuropathy, particularly diabetic peripheral neuropathy, particularly diabetic foot wounds, more particularly diabetic foot ulcer formation.

[0026] The present invention therefore relates to the following:

[0027] Item 1: A HIP / PAP protein, or a derivative thereof, for use in treating and / or preventing peripheral neuropathy in an individual.

[0028] Item 2: A HIP / PAP protein, or a derivative thereof, for use according to item 1 in treating and / or preventing diabetic peripheral neuropathy, particularly in diabetic or prediabetic individuals, more particularly in diabetic individuals.

[0029] Item 3: A HIP / PAP protein, or a derivative thereof, for use in treating and / or preventing damage to nerve endings, in particular sensory and / or motor nerve endings, in an individual.

[0030] Item 4: A HIP / PAP protein, or derivative thereof, for use in preventing peripheral neuropathy complications in an individual, particularly in preventing peripheral neuropathy complications in diabetic or pre-diabetic individuals, more particularly in preventing diabetic peripheral neuropathy complications in diabetic or pre-diabetic individuals, particularly in diabetic individuals.

[0031] Item 5: The HIP / PAP protein or derivative thereof according to item 4, wherein the complications of diabetic peripheral neuropathy are selected from the group consisting of foot deformities; diabetic foot wounds, particularly diabetic foot ulcers; foot infections; and amputations.

[0032] Item 6: A HIP / PAP protein or a derivative thereof for use according to any one of items 1 to 5, characterized in that the individual is a mammal, in particular a human.

[0033] Item 7: A HIP / PAP protein, or a derivative thereof, for use according to any one of items 1 to 6, wherein the diabetic individual is characterized in that he has type II diabetes.

[0034] Item 8: A HIP / PAP protein or a derivative thereof for use according to any one of Items 1 to 7, characterized in that the HIP / PAP protein comprises an amino acid sequence selected from the group consisting of the sequences shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0035] Item 9: A HIP / PAP protein or a derivative thereof for use according to any one of Items 1 to 8, wherein the derivative comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and a biological activity of the same nature as an amino acid sequence selected from the group consisting of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0036] Item 10: A HIP / PAP protein or derivative thereof for use according to any one of items 1 to 9, wherein the HIP / PAP protein or derivative thereof is administered to the individual in combination with at least one agent known to be useful in the prevention and / or treatment of peripheral neuropathy, in particular selected from the group consisting of steroids, anti-inflammatory drugs, immunosuppressants and vitamin D.

[0037] Item 11: A HIP / PAP protein or derivative thereof for use according to any one of items 1 to 10, wherein the HIP / PAP protein or derivative thereof is administered to the individual in combination with at least one antidiabetic agent, in particular selected from the group consisting of insulin, metformin, dipeptidyl peptidase 4 inhibitors, glucagon-like peptide 1 receptor agonists, sodium-glucose cotransporter 2 inhibitors and sulfonylureas and thiazolidinediones.

[0038] Item 12: A HIP / PAP protein or a derivative thereof for use according to any one of items 1 to 11, characterized in that the HIP / PAP protein or a derivative thereof is in a composition comprising a physiologically acceptable medium.

[0039] Item 13: A HIP / PAP protein, or a derivative thereof, for use according to item 12, wherein the composition is for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal or rectal administration.

[0040] Item 14: A HIP / PAP protein or derivative thereof for use according to item 12 or 13, wherein the composition is for oral, subcutaneous, intravenous, topical or local administration.

[0041] Item 15: The HIP / PAP protein or derivative thereof for use according to any one of items 12 to 14, wherein the composition further comprises at least one agent known to be useful in the prevention and / or treatment of peripheral neuropathy, in particular selected from the group consisting of steroids, anti-inflammatory drugs, immunosuppressants and vitamin D.

[0042] Item 16: HIP / PAP protein or a derivative thereof for use according to any one of items 12 to 15, wherein the composition further comprises at least one antidiabetic drug, in particular selected from the group consisting of insulin, metformin, dipeptidyl peptidase 4 inhibitors, glucagon-like peptide 1 receptor agonists, sodium-glucose cotransporter 2 inhibitors and sulfonylureas and thiazolidinediones. [Brief description of the drawings]

[0043] [Figure 1] Figure 1 is a table depicting the severity of infection assessed according to the International Consensus on Diabetic Foot Classification System. [Diagram 2] FIG. 2 depicts the protocol carried out and detailed in Example 2. [Diagram 3] Figure 3 shows hot plate latency in placebo vs. ALF-5755 treated GK rats. Vertical axis: hot plate latency (sec). Horizontal axis (from left to right): placebo and ALF-5755 treated rats (1.25 mg / kg). Unpaired t-test *p<0.05. [Figure 4] Figure 4 shows CMAP amplitudes in placebo vs. ALF-5755 treated GK rats. Vertical axis: Amplitude (mV) Horizontal axis (from left to right): Placebo and ALF-5755 treated rats (1.25mg / kg). Unpaired t-test *p<0.05.

[0044] Detailed Description of the Invention

[0045] definition

[0046] In the context of the present invention, the terms "prevent", "prevention" and "preventing" refer to the reduction of the risk or probability of the occurrence of a given phenomenon to a lower degree, i.e., in the present invention, the prevention of the occurrence of peripheral neuropathy and / or its complications, in particular complications selected from the group consisting of diabetic foot ulcers, foot infections and amputations. The terms "prevent", "prevention" and "preventing" also include preventing the worsening of the given phenomenon, slowing down the rate of progression or preventing its recurrence.

[0047] As used herein, the terms "treating," "treatment," or "treat" include the reduction of symptoms and / or elimination of symptoms associated with a particular disorder or condition, i.e., in the present invention, the treatment of diabetic peripheral neuropathy.

[0048] A "diabetic" individual refers to an individual suffering from the generally accepted definition of the term, i.e., a progressive disease of carbohydrate metabolism involving insufficient production or utilization of insulin and often characterized by hyperglycemia and urination.

[0049] By "pre-diabetic" individuals we refer to individuals who do not have the characteristics, symptoms, and the like typically observed in diabetes, but who have characteristics, symptoms, and the like that may progress to diabetes if left untreated. The presence of these conditions can be determined, for example, using either a fasting plasma glucose (FPG) test or an oral glucose tolerance test (OGTT). In an FPG test, a subject's blood glucose is measured after the end of fasting; typically, the subject fasts overnight, and blood glucose is measured in the morning, before the subject eats. A healthy subject will typically have an FPG concentration between about 90 mg / dL and about 100 mg / dL, a subject with "pre-diabetes" will typically have an FPG concentration between about 100 mg / dL and about 125 mg / dL, and a subject with "diabetes" will typically have an FPG level above about 126 mg / dL. In an OGTT, a subject's blood glucose is measured after fasting and again two hours after drinking a glucose-rich drink. Two hours after ingestion of a glucose-rich beverage, healthy subjects typically have a blood glucose below about 140 mg / dL, pre-diabetic subjects typically have a blood glucose between about 140 mg / dL and about 199 mg / dL, and diabetic subjects typically have a blood glucose of about 200 mg / dL or higher.

[0050] "Complications of peripheral neuropathy" according to the present invention are medical problems caused by peripheral neuropathy. Examples of such complications of peripheral neuropathy are skin and limb trauma or burns. Because an individual has altered sensitivity to temperature or pain, he or she may have, in particular, diabetic skin and limb trauma or burns; foot deformities; heart and blood circulation problems; foot wounds, in particular diabetic foot wounds, more specifically diabetic foot ulcers; gangrene; and foot infections and limb amputations, in particular lower limb amputations.

[0051] In accordance with the present invention, a "foot wound" is a wound on the skin of the foot resulting from either trauma (i.e., as a result of an external force, e.g., an accident (e.g., a fall) due to damage to motor and / or sensory nerves), or gradual impairment of tissue due to an underlying (thus, internal) etiology / pathology (such as, for example, in the case of diabetic foot ulcers described above).

[0052] A "diabetic foot wound" according to the invention is a foot wound as defined above which occurs in diabetic individuals and is a consequence of diabetic peripheral neuropathy as explained above. A diabetic foot wound according to the invention is in particular a diabetic foot ulcer.

[0053] The development of diabetic foot ulcers usually occurs in three stages. The initial stage is the development of a callus, which results from diabetic peripheral neuropathy. The neuropathy causes physical deformation and sensory loss in the foot, which leads to ongoing trauma. Dry skin due to neuropathy is also another contributing factor. Finally, frequent trauma to the callus leads to subcutaneous bleeding, which erodes and becomes an ulcer.

[0054] A "diabetic foot infection" according to the present invention is a foot infection, i.e., infiltration of tissue by a diabetic individual with microbial proliferation causing tissue damage with or without an associated inflammatory response.

[0055] The diagnosis of diabetic foot infection is clinical. The diagnosis of infection is based on the presence of at least two of the following signs: swelling, induration, perilesional erythema, localized tenderness or pain, localized warmth, or the presence of pus. The severity of the infection is assessed according to the International Consensus on Diabetic Foot Classification System (see Figure 1).

[0056] An "amputation" according to the present invention is the surgical removal of a body part of a diabetic patient, particularly the amputation of a lower limb.

[0057] Amputation surgery occasionally remains the only option in cases of severe deep infection and / or wounds. The choice of the level of amputation depends on the vascular status, with all efforts made to preserve weight bearing of the heel with a prosthesis. The most common amputations in people with diabetes are toes, feet, and lower legs. In particular, major amputations are required in patients with severe sepsis or deep compartment abscesses with extensive forefoot gangrene or imminent loss of toes. The aim of amputation is to preserve limb length, since the longer the stump, the better the rehabilitation outcome.

[0058] The term "antidiabetic drug" refers to a substance that helps a person with diabetes manage the level of glucose in the blood. Examples of antidiabetic drugs can be, for example, insulin, metformin, dipeptidyl peptidase 4 inhibitors, glucagon-like peptide 1 receptor agonists, sodium-glucose cotransporter 2 inhibitors, and antidiabetic drugs selected from the group consisting of sulfonylureas and thiazolidinediones.

[0059] Dipeptidyl peptidase 4 (DPP-4) inhibitors are a class of drugs that lower high blood glucose levels and can be used in the treatment of type 2 diabetes. Mention may be made of saxagliptin, sitagliptin, alogliptin, and linagliptin.

[0060] Glucagon-like peptide-1 receptor agonists, also known as GLP-1 receptor agonists or incretin mimetics, are agonists of the GLP-1 receptor used for the treatment of diabetes mellitus type 2. Mention may be made of exenatide, lixisenatide, liraglutide, albiglutide, dulaglutide, and semaglutide.

[0061] Sodium-glucose cotransporter 2 inhibitors are a class of drugs used to lower high blood glucose levels in people with type 2 diabetes. Mention may be made of ertugliflozin, canagliflozin, empagliflozin, and dapagliflozin.

[0062] The term "physiologically acceptable medium" is intended to mean a medium that is compatible with the body of the individual to which the composition must be administered. It is, for example, a non-toxic solvent, such as water. In particular, the medium is suitable for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal or rectal administration, more particularly for oral, subcutaneous, intravenous, topical or local administration.

[0063] As used herein, the "percentage of identity" between two amino acid sequences is determined by comparing both optimally aligned sequences over a comparison window.

[0064] Thus, a portion of the amino acid sequence in the comparison window may contain additions or deletions (e.g., "gaps") compared to a reference sequence (not including these additions or these deletions) such that optimal alignment is obtained between both sequences.

[0065] The terms "sequence homology" or "sequence identity" or "homology" or "identity" are used interchangeably herein. For the purposes of the present invention, to determine the percentage of sequence homology or sequence identity of two amino acid sequences, it is defined herein that the sequences are aligned for optimal comparison purposes. To optimize the alignment between the two sequences, gaps may be introduced in either of the two sequences being compared. Such alignment can be performed over the entire length of the sequences being compared. Alternatively, the alignment can be performed over a shorter length, for example, over about 20, about 50, about 100 or more nucleic acids / bases or amino acids. Sequence identity is the percentage of identical matches between the two sequences over the reported alignment region.

[0066] The comparison of sequences and the determination of the percentage of sequence identity between two sequences can be accomplished using a mathematical algorithm. Those skilled in the art will recognize the fact that several different computer programs are available for aligning two sequences and determining the identity between two sequences (Kruskal, JB (1983) An overview of sequence comparison In D. Sankoff and JB Kruskal, (ed.), Time warps, string edits and macromolecules: the theory and practice of sequence comparison, pp. 1-44 Addison Wesley).

[0067] The percent sequence identity between two amino acid sequences can be determined using the Needleman and Wunsch algorithm for alignment of two sequences (Needleman, SB and Wunsch, CD (1970) J. Mol. Biol. 48, 443-453). Both amino acid sequences can be aligned by the algorithm. The Needleman-Wunsch algorithm is implemented in the computer program NEEDLE.

[0068] For the purposes of the present invention, the NEEDLE program from the EMBOSS package was used (version 2.8.0 or later, EMBOSS: The European Molecular Biology Open Software Suite (2000) Rice, P. Longden J. and Bleasby, A. Trends in Genetics 16, (6) pp276-277, http: / / emboss.bioinformatics.nl / ). For protein sequences, EBLOSUM62 is used for the substitution matrix. Optional parameters used are gap opening penalty of 10 and gap extension penalty of 0.5. No end gap penalty is added. In the output section, "Yes" is indicated in response to the question "Simple identity and similarity" and "SRS pairwise" is indicated as the output alignment format.

[0069] After alignment by the program NEEDLE described above, the percentage of sequence identity between the query sequence and the sequence of the invention is calculated as follows: the number of corresponding positions in the alignment that show identical amino acids in both sequences, divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. Identity as defined herein can be obtained from NEEDLE by using the NOBRIEF option and is labeled as "longest identity" in the program output.

[0070] Amino acid sequence similarity, i.e. percentage of sequence identity, can be determined using several other algorithms known in the art, preferably using the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), using hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or using the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80) (available e.g. at https: / / www.ebi.ac.uk / Tools / msa / clustalo / ) or the GAP program (mathematical algorithm of the University of Iowa) or the mathematical algorithm of Myers and Miller (1989 - Cabios 4: 11-17) or the Clone Manager. 9 can be used to determine via sequence alignment. The preferred parameters used are the default parameters, as they are set out at https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0071] The grade of sequence identity (sequence matching) can be calculated, for example, using BLAST, BLAT, or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASTN and BLASTP programs of Altschul et al (1990) J. Mol. Biol. 215, 403-410. BLAST polynucleotide searches are performed using the BLASTN program, score=100, wordlength=12, to obtain polynucleotide sequences that are homologous to nucleic acids encoding related proteins.

[0072] BLAST protein searches are performed with the BLASTP program, score=50, wordlength=3 to obtain amino acid sequences homologous to the SHC polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al (1997) Nucleic Acids Res. 25, 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis can be supplemented with established homology mapping techniques such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1: 154-162) or Markov Random Fields. When percentages of sequence identity are referred to in this application, these percentages are calculated with respect to the full length of the longer sequence unless otherwise specifically indicated.

[0073] In certain embodiments, the percent identity between two sequences is determined using CLUSTAL O (version 1.2.4).

[0074] As used herein, the term "polypeptide" refers to a molecule that contains amino acid residues linked by peptide bonds and that contains more than five amino acid residues. Amino acids are identified by either a one-letter or three-letter designation. The term "protein" as used herein is synonymous with the term "polypeptide" and can refer to two or more polypeptides. Thus, the terms "protein," "peptide," and "polypeptide" can be used interchangeably. Polypeptides can optionally be modified (e.g., glycosylated, phosphorylated, acylated, farnesylated, prenylated, sulfonated, etc.) to add functionality.

[0075] HIP / PAP PROTEINS AND DERIVATIVES THEREOF ACCORDING TO THE PRESENT INTENTION

[0076] The HIP / PAP protein is known for its anti-apoptotic and mitogenic activity on hepatocytes (US13 / 032,521, WO2004 / 112824, Simon et al., FASEB J. 2003 Aug;17(11):1441-50).

[0077] Also, HIP (Human proIslet Peptide) peptide, a 15 amino acid peptide derived from the Reg IIIa family (HIP / PAP), has been shown to have regenerative activity for pancreatic islets and thus stimulate insulin production (US2010 / 0093605).

[0078] A HIP / PAP protein according to the invention can comprise an amino acid sequence selected from the group consisting of the sequences shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4.

[0079] The amino acid sequence SEQ ID NO:1 corresponds to the HIP / PAP protein of sequence SEQ ID NO:4 from which the N-terminal 26 amino acid signal peptide of the protein has been deleted.

[0080] In a specific embodiment, a HIP / PAP protein according to the invention comprises or consists of the amino acid sequence shown as SEQ ID NO:4.

[0081] In a specific embodiment, a HIP / PAP protein according to the invention comprises or consists of the amino acid sequence shown as SEQ ID NO:1.

[0082] The amino acid sequence SEQ ID NO:2 corresponds to a truncated form of the HIP / PAP protein, which has an 11 amino acid propeptide at the N-terminal position deleted compared to the amino acid sequence SEQ ID NO:1.

[0083] In a specific embodiment, a HIP / PAP protein according to the invention comprises or consists of the amino acid sequence shown in SEQ ID NO:2.

[0084] Sequence SEQ ID NO:3 corresponds to sequence SEQ ID NO:1, to which a methionine has been added at the N-terminal position. The HIP / PAP derivative of sequence SEQ ID NO:3 is also called rcHIP / PAP or ALF5755. This derivative can be produced recombinantly, in particular in E. coli cells. The 12 amino acid N-terminal propeptide (11 amino acids plus an additional methionine propeptide) may be truncated to obtain a shorter form of the HIP / PAP protein (SEQ ID NO:2).

[0085] In a specific embodiment, a HIP / PAP protein according to the invention comprises or consists of the amino acid sequence shown as SEQ ID NO:3.

[0086] According to the present invention, the short or long form of the HIP / PAP protein or its derivatives may be used without distinction.

[0087] A derivative of a HIP / PAP protein according to the present invention designates a biologically active derived form of a HIP / PAP protein of any one of the sequences SEQ ID NOs: 1 to 4. The term "biologically active" means that a derivative of a HIP / PAP protein has the same biological activity as a HIP / PAP protein of any one of the sequences SEQ ID NOs: 1 to 4.

[0088] A derivative of a HIP / PAP protein according to the present invention comprises or consists of an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, and a biological activity of the same nature as an amino acid sequence selected from the group consisting of the sequences SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4.

[0089] The biological activity of the same nature of the HIP / PAP protein according to the present invention is as previously described, namely the ability to treat and / or prevent peripheral neuropathy, in particular diabetic peripheral neuropathy, in an individual and therefore to prevent complications of peripheral neuropathy in an individual, in particular the complications detailed elsewhere in this text.

[0090] As described herein, an amino acid sequence having at least 80% amino acid identity to a reference amino acid sequence includes amino acid sequences having at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% amino acid identity to the reference amino acid sequence, and also amino acid sequences having the same qualitative biological activity as the reference amino acid sequence.

[0091] In certain embodiments, the HIP / PAP protein, or derivative thereof, may be non-covalently associated or bound to a non-HIP / PAP moiety. For example, the HIP / PAP protein, or derivative thereof, may be associated with a liposome particle. Depending on the type of liposome or the production process, the HIP / PAP protein, or derivative thereof, may be associated with the surface of the liposome or encapsulated within the liposome.

[0092] The HIP / PAP protein, or derivatives thereof, may also be covalently associated with non-HIP / PAP moieties, which may be selected from proteins or non-proteinaceous compounds, such as polyethylene glycol, thus forming a PEGylated HIP / PAP derivative.

[0093] Derivatives of HIP / PAP proteins in accordance with the present invention also include derivatives that become biologically active only when they are administered to a patient.

[0094] Finally, derivatives of HIP / PAP proteins also include chimeric or fusion proteins. Such proteins are fused to a non-HIP / PAP polypeptide. The latter may be fused at the N- or C-terminal part. Typically, HIP / PAP proteins or derivatives thereof may be fused at the level of their C-terminal part with GST sequences to facilitate the purification of recombinant proteins.

[0095] In a specific embodiment of the invention, the HIP / PAP proteins according to the invention, or derivatives thereof, are recombinantly produced in bacterial cells or animal cells, including insect cells and mammalian cells, according to techniques known to those of skill in the art.

[0096] In other embodiments, the HIP / PAP proteins, or derivatives thereof, in accordance with the present invention can be isolated from cells or tissues by known purification techniques.

[0097] HIP / PAP proteins and derivatives thereof can also be produced by chemical synthesis.

[0098] As used herein, the term "HIP / PAP protein" encompasses the HIP / PAP protein itself, and also its derivatives as described above.

[0099] composition

[0100] The present invention also relates to the packaging of the previously defined HIP / PAP protein, or a derivative thereof, in a composition comprising a physiologically acceptable medium.

[0101] The physiologically acceptable medium has been defined hereinbefore, but may be selected from the usual excipients known to those skilled in the art according to the desired pharmaceutical form and mode of administration (see Remington's Pharmaceutical Sciences, 16th edition, Osol, A ed., 1980).

[0102] By way of example, a composition according to the invention may comprise, according to the therapeutic indication and the HIP / PAP protein or derivative thereof: a) a HIP / PAP protein or a derivative thereof; and b) a buffer capable of maintaining the pH in the maximum stable range, preferably between 1 and 9, more particularly between 4 and 8, even more particularly between 6 and 7.5; and / or c) a detergent or surfactant that stabilizes the protein or polypeptide against agitation-induced aggregation; and / or d) isotonic; and / or e) a preservative selected from the group consisting of, for example, phenol, benzyl alcohol, benzoterium halide and chloride; and / or f) Water.

[0103] If the detergent or surfactant used is non-ionic, it may be selected from polysorbates, PLURONIC™, polyethylene glycols (PEG), or poloxamers.

[0104] Isotonicity agents allow the composition to maintain its isotonicity and typically include polyhydric alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, or mannitol, used alone or in combination. Alternatively, sodium chloride and / or other inorganic salts may be used as isotonicity agents.

[0105] The buffer can be, for example, acetate, citrate, succinate, phosphate buffers, or other inorganic buffers, depending on the desired pH.

[0106] Phenol, benzyl alcohol, benzoterium halide and chloride type preservatives are known antimicrobial agents.Typical preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol.

[0107] Additional excipients may also include antioxidants such as ascorbic acid and methionine, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol.

[0108] The HIP / PAP protein or its derivatives according to the present invention may be in the form of pharma- ceutically acceptable salts. This is intended to mean salts prepared from pharma-ceutically acceptable non-toxic acids or pharma-ceutically acceptable non-toxic bases, including organic and inorganic salts and acids. By way of example, mention may be made of alkali metal salts (sodium and potassium salts), alkaline earth metal salts (calcium and magnesium salts), ammonium salts, salts of organic bases (pyridine or triethylamine salts), salts of inorganic acids (hydrochlorides, sulfates, nitrates) and salts of organic acids (acetates, oxalates, p-toluenesulfonates).

[0109] Compositions implemented according to the invention may be for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal or rectal administration, in particular for oral, subcutaneous, intravenous, topical or local administration.

[0110] According to a preferred embodiment, the HIP / PAP protein is administered in an effective amount, i.e., the amount required to obtain the anticipated effect of the invention. Such amount of HIP / PAP protein will generally be empirically determined according to the subject being treated and its pathological condition. The effective amount will also depend on the envisaged mode of administration. The adjustments required to determine the effective amount to obtain the maximum therapeutic effect represent routine skill for the clinician.

[0111] An effective amount of a HIP / PAP protein, or derivative thereof, can be, for example, between 0.1 μg / day / kg and 100 mg / day / kg body weight of the individual to whom it must be administered. In certain embodiments, an effective amount of a HIP / PAP protein, or derivative thereof, can reach more than 10 mg / kg, although an effective amount of a HIP / PAP protein, or derivative thereof, according to the present invention will generally be less than 5 mg / kg body weight, including amounts less than 4.5 mg / kg, 4 mg / kg, 3.5 mg / kg, 3 mg / kg, 2.5 mg / kg, or 2000 μg / kg. More specifically, an effective amount of the HIP / PAP protein or derivative thereof according to the present invention is at least 1 μg / kg, 2 μg / kg, 3 μg / kg, 4 μg / kg, 5 μg / kg, 6 μg / kg, 7 μg / kg, 8 μg / kg, 9 μg / kg, 10 μg / kg, 15 μg / kg, 20 μg / kg, 25 μg / kg, 30 μg / kg, 40 μg / kg, 50 μg / kg, or more, based on the body weight of the individual to whom it is or should be administered. g, 60μg / kg, 70μg / kg, 80μg / kg, 90μg / kg, 100μg / kg, 150μg / kg, 200μg / kg, 250μg / kg, 300μg / kg, 350μg / kg, 400μg / kg, 4 Contains amounts of 50μg / kg, 500μg / kg, 600μg / kg, 700μg / kg, 800μg / kg, 900μg / kg, 1mg / kg, 2mg / kg, 3mg / kg, 4mg / kg, 5mg / kg or more.

[0112] According to a particular embodiment, the HIP / PAP protein, or a derivative thereof, is administered according to a dosage of between 10 and 5000 μg / kg, preferably between 100 and 2000 μg / kg of body weight.

[0113] In compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal or intranasal or rectal administration, the active ingredient (HIP / PAP protein or derivative thereof) may be administered in a unit dosage form, in admixture with a pharmaceutical excipient.

[0114] When the composition is for oral administration, the composition may be selected from the group consisting of a food, a beverage, a pharmaceutical, a dietary supplement, a food additive, a dietary supplement, and a dairy product.

[0115] Preferred modes of administration are oral, subcutaneous, intravenous, topical or local routes, more particularly subcutaneous, intravenous, topical or local routes.

[0116] Administration of a compound or composition of the invention may be accomplished, for example, through the use of a sheath, patch, pad, compress, bandage, tape, gauze-based dressing, woven or non-woven sponge, or syringe.

[0117] The HIP / PAP protein, or derivative thereof, can be sterilized prior to in vivo administration. Sterilization may be obtained by filtration over sterile filtration membranes, before or after lyophilization or reconstitution. HIP / PAP protein, or derivative thereof, administered systemically, can be advantageously lyophilized or stored in solution. In lyophilized form, the HIP / PAP protein, or derivative thereof, will generally be formulated in combination with excipients allowing for reconstitution with a suitable diluent at the time of use.

[0118] The HIP / PAP protein, or derivative thereof, can be administered in one daily intake or in a divided fashion (e.g., two to three times daily) until the desired therapeutic effect is achieved. It can also be administered chronically.

[0119] The HIP / PAP protein, or derivatives thereof, may also be administered in the form of a course, for example, in a course ranging from 15 days to 3 months, optionally repeated 1 to 6 times at given doses and time intervals.

[0120] The HIP / PAP protein, or derivatives thereof, according to the present invention may be combined in the context of a polytherapy with other compounds to treat and / or prevent peripheral neuropathy, in particular diabetic peripheral neuropathy, or to prevent complications of peripheral neuropathy in individuals, in particular to prevent complications of diabetic peripheral neuropathy in diabetic individuals.

[0121] Thus, the HIP / PAP protein, its derivatives or compositions comprising same according to the invention may be administered to individuals as defined above, in particular pre-diabetic or diabetic individuals, in particular diabetic individuals, in combination with at least one agent known to be useful in the prevention and / or treatment of peripheral neuropathy, in particular in the prevention and / or treatment of diabetic peripheral neuropathy.

[0122] Such drugs are known by those skilled in the art and can be selected, for example, from the group consisting of steroids, anti-inflammatory drugs, immunosuppressants, vitamin D, antiepileptic drugs such as gabapentin and carbamazepine, amitriptyline, pregabalin, duloxetine, venlafaxine, notriptyline, desipramine, anticholinergic drugs, anticonvulsants, more specifically, from the group consisting of steroids, anti-inflammatory drugs, immunosuppressants and vitamin D.

[0123] Furthermore, as previously mentioned, the HIP / PAP protein according to the invention, its derivatives or compositions comprising same may be administered to individuals as previously defined, in particular to pre-diabetic or diabetic individuals, more particularly to diabetic individuals, in combination with at least one anti-diabetic agent.

[0124] The antidiabetic agent may be selected from the group consisting of insulin, metformin, dipeptidyl peptidase 4 inhibitors, glucagon-like peptide 1 receptor agonists, sodium-glucose cotransporter 2 inhibitors, and sulfonylureas and thiazolidinediones.

[0125] The dipeptidyl peptidase 4 (DPP-4) inhibitor may be selected from the group consisting of saxagliptin, sitagliptin, alogliptin, and linagliptin.

[0126] The glucagon-like peptide 1 receptor agonist may be selected from the group consisting of exenatide, lixisenatide, liraglutide, albiglutide, dulaglutide, and semaglutide.

[0127] The sodium-glucose cotransporter 2 inhibitor may be selected from the group consisting of ertugliflozin, canagliflozin, empagliflozin and dapagliflozin.

[0128] By administered in combination, it is meant that the HIP / PAP protein according to the invention, a derivative thereof, or a composition comprising same may be administered simultaneously or sequentially compared to other compounds, in particular compared to at least one antidiabetic agent. If they are in separate compositions, the composition comprising the HIP / PAP protein according to the invention, a derivative thereof, and the composition comprising at least one antidiabetic agent may be administered via the same route or via different routes.

[0129] The HIP / PAP protein, or derivative thereof, according to the present invention and the at least one anti-diabetic agent may be administered in the same composition or in separate compositions.

[0130] It is understood that the compositions can be administered simultaneously, or on the same day or up to several days.

[0131] It will be appreciated that sequentially, the compositions can be administered on at least several days, for example, on at least two different days.

[0132] Implementation of HIP / PAP proteins and / or their derivatives

[0133] As previously mentioned, the HIP / PAP protein, or derivatives thereof, as well as compositions comprising same, are for use in: - the treatment and / or prevention of peripheral neuropathy, in particular diabetic peripheral neuropathy, in an individual; and / or - prevention of complications of peripheral neuropathy, particularly diabetic peripheral neuropathy, in an individual; and / or - preventing and / or treating damaged nerves, in particular damaged sensory and / or motor nerves, more particularly damage to nerve endings, in particular damage to sensory and / or motor nerve endings, especially in individuals with or prone to peripheral neuropathy; and / or - repairing nerve endings, especially sensory and / or motor nerve endings, especially in individuals with peripheral neuropathy; and / or - restoring the activity of nerve endings, especially sensory and / or motor nerve endings, especially in individuals with peripheral neuropathy; Particularly in diabetic or pre-diabetic individuals, more particularly in diabetic individuals.

[0134] As previously indicated, complications of peripheral neuropathy, particularly diabetic peripheral neuropathy, may be selected from the group consisting of skin and limb trauma or burns, particularly diabetic skin and limb trauma or burns; foot deformities; heart and blood circulation problems; foot wounds, particularly diabetic foot wounds, more particularly diabetic foot ulcers; gangrene; and foot infections and limb amputations, particularly lower limb amputations.

[0135] Complications of diabetic peripheral neuropathy according to the invention may in particular be selected from the group consisting of foot deformities, diabetic foot wounds, in particular diabetic foot ulcers, foot infections and amputations.

[0136] The individual, particularly the diabetic individual, to whom the HIP / PAP protein, derivatives thereof or compositions according to the present invention are administered may in particular be a mammal, particularly a human.

[0137] The HIP / PAP protein, derivatives thereof, or compositions according to the invention may be administered in combination with standard medical care for peripheral neuropathy and / or complications of peripheral neuropathy, more specifically diabetic peripheral neuropathy and / or complications of diabetic peripheral neuropathy.

[0138] The present invention also relates to a method for treating and / or preventing peripheral neuropathy in an individual, in particular for treating and / or preventing diabetic peripheral neuropathy in a diabetic individual, comprising administering to said individual a HIP / PAP protein, a derivative thereof, or a composition comprising same.

[0139] The present invention also relates to a method for treating and / or preventing damaged nerves, particularly in individuals with or susceptible to peripheral neuropathy, particularly damaged sensory and / or motor nerves, more particularly for preventing and / or treating damage to nerve endings, particularly damage to sensory and / or motor nerve endings, comprising administering to said individual a HIP / PAP protein, a derivative thereof, or a composition comprising same.

[0140] The present invention also relates to a method for repairing nerve endings, particularly sensory and / or motor nerve endings, in an individual with peripheral neuropathy, comprising administering to the individual a HIP / PAP protein, a derivative thereof, or a composition comprising same.

[0141] The present invention also relates to a method for restoring activity of nerve endings, particularly sensory and / or motor nerve endings, in an individual, particularly with peripheral neuropathy, comprising administering to said individual a HIP / PAP protein, a derivative thereof, or a composition comprising same.

[0142] The present invention further relates to the use of HIP / PAP protein, a derivative thereof, or a composition comprising same, for treating and / or preventing peripheral neuropathy in an individual, in particular for treating and / or preventing diabetic peripheral neuropathy in diabetic individuals.

[0143] The present invention further relates to the use of the HIP / PAP protein, a derivative thereof or a composition comprising same for treating and / or preventing damaged nerves, more particularly for preventing and / or treating damage to nerve endings, particularly damage to sensory and / or motor nerve endings, especially in individuals with or susceptible to peripheral neuropathy, especially damaged sensory and / or motor nerves.

[0144] The present invention further relates to the use of the HIP / PAP protein, a derivative thereof or a composition comprising same for repairing nerve endings, in particular sensory and / or motor nerve endings, in particular in individuals with peripheral neuropathy.

[0145] The present invention further relates to the use of HIP / PAP protein, a derivative thereof or a composition comprising same for restoring the activity of nerve endings, in particular for restoring the activity of sensory and / or motor nerve endings, particularly in individuals with peripheral neuropathy.

[0146] The present invention further relates to the use of a HIP / PAP protein, a derivative thereof, or a composition comprising same for the manufacture of a medicament for treating and / or preventing peripheral neuropathy in an individual, in particular for treating and / or preventing diabetic peripheral neuropathy in a diabetic individual.

[0147] The present invention further relates to the use of the HIP / PAP protein, a derivative thereof or a composition comprising same for the manufacture of a medicament for treating and / or preventing damaged nerves, more particularly for preventing and / or treating damage to nerve endings, especially damage to sensory and / or motor nerve endings, especially in individuals with or susceptible to peripheral neuropathy, especially damaged sensory and / or motor nerves.

[0148] The present invention further relates to the use of a HIP / PAP protein, a derivative thereof or a composition comprising same for the manufacture of a medicament for repairing nerve endings, in particular sensory and / or motor nerve endings, in particular in individuals with peripheral neuropathy.

[0149] The present invention further relates to the use of a HIP / PAP protein, a derivative thereof or a composition comprising same for the manufacture of a medicament for restoring the activity of nerve endings, in particular for restoring the activity of sensory and / or motor nerve endings, in particular in individuals with peripheral neuropathy.

[0150] The present invention also relates to a method for preventing complications of peripheral neuropathy in an individual, in particular for preventing complications of diabetic peripheral neuropathy in a diabetic or pre-diabetic individual, comprising administering to said individual a HIP / PAP protein, a derivative thereof, or a composition comprising same.

[0151] The present invention further relates to the use of the HIP / PAP protein, a derivative thereof, or a composition comprising same, for preventing complications of peripheral neuropathy in an individual, in particular for preventing complications of diabetic peripheral neuropathy in diabetic or pre-diabetic individuals.

[0152] The present invention further relates to the use of a HIP / PAP protein, a derivative thereof, or a composition comprising same for the manufacture of a medicament for preventing complications of peripheral neuropathy in an individual, in particular for preventing complications of diabetic peripheral neuropathy in diabetic or pre-diabetic individuals.

[0153] The invention will now be described in more detail by way of the following examples, given by way of illustration only.

[0154] All references to percentages are by weight unless otherwise indicated.

[0155] Working Example

[0156] Example 1

[0157] The effect of two months of systemic treatment with ALF-5755 (compound of sequence SEQ ID NO:3) on peripheral neuropathy in the db / db model (Janvier Labs), a mouse model of type II diabetes, is evaluated.

[0158] 10 non-diabetic and 50 diabetic mice are tested per period. Food and water are provided ad libitum.

[0159] In group A (10 non-diabetic mice), the glycemia level is assessed only once: no treatment is performed in these mice.

[0160] Fifty diabetic mice are divided into four groups of 10-15 animals that are either (i) treated subcutaneously with placebo or (ii) treated subcutaneously with ALF-5755 (1.25 mg / kg): Group B: 15 diabetic mice treated with placebo from D0 to D56; Group C: 15 diabetic mice treated with ALF-5755 from D0 to D56; Group D: 10 diabetic mice treated with placebo from D0 to D24; Group E: 10 diabetic mice treated with ALF-5755 from D0 to D24.

[0161] Animals have no access to food for 2 hours prior to glycemia measurements, which are performed once a week to check for diabetes levels. Glycemic levels are obtained by venipuncture from the tail vein and are classically assayed using a glucometer (AccuCheck, Roche).

[0162] Animals are administered ALF-5755 or placebo daily.

[0163] Neuropathy is assessed through tactile allodynia using Von Frey filaments and through observation of the response to mechanical stimulation of the animals' hind paws.

[0164] Initial evaluations are performed in groups A to E and for animals from groups AC before treatment (D-1), on D20 and 55 days before euthanasia.

[0165] Animals are placed in individual plexiglass cylinders with mesh floors. Animals undergo a 30-minute habituation session the day before behavioral assessment. Animals do not stay on the cylinders for more than 1 hour to avoid dehydration.

[0166] On the day of testing, animals are left undisturbed for a minimum of 15 min to habituate to the wire grid and observation area. Tactile allodynia testing is performed using Von Frey filaments applied to the mid-plantar surface of the hind paw (up-down Von Frey technique).

[0167] Using the "up-down" Von Frey method, LD 50 Based on the statistical formula used to determine s, the mechanical force required to elicit a paw withdrawal response in 50% of the animals is determined (Dixon, 1980, Annu. Rev. Pharmacol. Toxicol. 20, 441-462; Chaplan et al., 1994, J. Neurosci. Methods 53, 55-63).

[0168] This method is well known to those skilled in the art, as exemplified, for example, in JR Deuis, LS Dvorakova, and I. Vetter (Methods Used to Evaluate Pain Behaviors in Rodents; Front Mol Neurosci. 2017;10: 284), and Mills et al. (Estimating efficacy and drug ED50's using von Frey thresholds: impact of Weber's law and log transformation; J Pain. 2012 Jun;13(6):519-23).

[0169] Responses to mechanical stimulation are also assessed at D55 for animals in groups A, B, and C using an algometer with calibrated forceps (Rodent Pincher Analgesia Meter, Bioseb).

[0170] Animals undergo an habituation session the day before behavioral assessment.

[0171] On the day of testing, animals are placed in the experimental room 2 hours before evaluation. Calibrated forceps are then applied gradually by hand at a constant rate (200 g every 2 seconds) until a nociceptive response occurs. Measurements of the nociceptive threshold are repeated 3 times for each hind paw with an interval of approximately 5 seconds.

[0172] The appearance and behavior of the animals are evaluated at least daily from the beginning to the end of the in vivo phase. Any abnormal findings are recorded.

[0173] Furthermore, all animals are euthanized and tissues are collected on D24 for groups D and E and on D56 for groups B and C. Skin samples from the plantar surface of the two hind paws from each animal (groups B-E) are collected and placed in formalin for immunohistochemical analysis of intradermal nerve fiber density of the paw.

[0174] The epidermal innervation of these tissues is then assessed.

[0175] Histology is processed by NOVAXIA (Saint-Laurent-Nouan, France). The skin of the plantar surface of the hind paw is included in a paraffin block. After paraffin embedding, one section is performed and PGP9.5 staining is performed to evaluate the epidermal innervation.

[0176] Example 2

[0177] Effect of 2-month sustained treatment with ALF-5755 on peripheral neuropathy in Goto-Kakizaki (GK) rats (housed in Metabrain), a rat model of non-obese type II diabetes mellitus

[0178] animal

[0179] Animals are kept in the Metabrain animal facility. All rats are fed with normal diet 113 from SAFE (Scientific Animal Food and Engineering - Route de Saint Bris - 89290 AUGY - France) and allowed to drink water ad libitum. During the 50-day study, ALF-5755 is delivered to rats at a dose of 1.25 mg / kg using an Alzet® osmotic pump (reference: 2ML4 batch 10405-19 - 2.5 μL per hour).

[0180] Animals are weighed once a week for follow-up. Plasma is collected in non-fasted rats before pump implantation (D-1 or D27) and before sacrifice (D46) for measurement of glycemia and insulinemia.

[0181] A. Pain Sensitivity – Hot Plate Test

[0182] The test is performed in a hot plate analgesia apparatus. Rats are placed individually on a hot plate maintained at 52-55°C. The latency to first lick or withdraw the hind paw is taken as an index of the nociceptive threshold. The animal is then immediately removed from the apparatus. A cut-off time, typically 50 seconds, is established to minimize the risk of the animal sustaining tissue damage from prolonged exposure to the heated surface.

[0183] result

[0184] Figure 3 shows the results of the hot plate test, which measured the time it took rats to withdraw their paws from the hot plate. Results are presented as mean ± SEM. Unpaired t-test in Figure 3 shows that after 47 days of continuous subcutaneous treatment with ALF-5755, GK rats exhibited improved heat pain perception compared to placebo-treated rats, with a 27% improvement in pain sensitivity (t(18) = 2.4, *p = 0.027) (unpaired t-test *p < 0.05).

[0185] The results demonstrate that treatment with molecules according to the invention improves peripheral neuropathy.

[0186] B. Electrophysiological assessment – ​​compound muscle action potential

[0187] Electrophysiological recordings of compound muscle action potentials (CMAPs) have been performed.

[0188] After anesthesia, the sciatic nerve of the rat is percutaneously stimulated with a bipolar needle electrode placed at the hip and popliteal levels. The recording bipolar needle electrode is placed in the gastrocnemius belly. A common reference (ground electrode) is placed based on the tail.

[0189] The electrical stimulation is a square pulse with a frequency of 1 Hz and a duration of 0.2 m / s. The stimulation current intensity is gradually increased up to a supramaximal intensity (current intensity 30% above the value that induces the maximum CMAP). The stimulation is repeated five times for every measurement and the average value is recorded.

[0190] result

[0191] To assess the physiological effects of treatment on nerve function, we stimulated the sciatic nerve using needle electrodes and measured compound muscle action potentials (CMAPs) in the gastrocnemius muscle.

[0192] Figure 4 shows the results of the CMAP test where peak-to-peak amplitude was measured. Results are presented as mean ± SEM. The unpaired t-test in Figure 4 shows that after 2 months of treatment with ALF-5755, CMAP amplitude in GK rats is significantly increased compared to placebo (t(16) = 2.76, *p = 0.014).

[0193] The results demonstrate that treatment with molecules according to the invention improves muscle response to electrical signals, indicating amelioration of peripheral neuropathy.

[0194] Sequence Listing

[0195] SEQ ID NO:1 is the amino acid sequence of the HIP / PAP protein lacking the N-terminal 26 amino acid signal peptide. [ka]

[0196] SEQ ID NO:2 is the amino acid sequence of the HIP / PAP protein lacking the N-terminal 26 amino acid signal peptide and the 11 amino acid propeptide in the N-terminal position. [ka]

[0197] SEQ ID NO:3 is the amino acid sequence of the HIP / PAP protein with the N-terminal 26 amino acid signal peptide deleted and a methionine added at the N-terminal position. [ka]

[0198] SEQ ID NO:4 is the amino acid sequence of the complete HIP / PAP protein. [ka]

[0199] The amino acid sequences are disclosed herein and serve as a reference. The same sequences are also presented in a sequence listing formatted according to standard requirements for patent purposes. In the case of any sequence discrepancy with the standard sequence listing, reference shall be made to the sequences described herein.

Claims

1. A HIP / PAP protein, or a derivative thereof, for use in treating and / or preventing peripheral neuropathy in an individual.

2. 2. A HIP / PAP protein, or a derivative thereof, for use according to claim 1 in treating and / or preventing diabetic peripheral neuropathy, particularly in diabetic or pre-diabetic individuals, more particularly in diabetic individuals.

3. A HIP / PAP protein, or a derivative thereof, for use in treating and / or preventing damage to nerve endings, particularly sensory and / or motor nerve endings, in an individual.

4. 1. A HIP / PAP protein, or derivative thereof, for use in preventing peripheral neuropathy complications in an individual, particularly in preventing peripheral neuropathy complications in diabetic or pre-diabetic individuals, more particularly in preventing diabetic peripheral neuropathy complications in diabetic or pre-diabetic individuals, particularly in diabetic individuals.

5. 5. The HIP / PAP protein, or derivative thereof, for use according to claim 4, wherein the complications of diabetic peripheral neuropathy are selected from the group consisting of foot deformities; diabetic foot wounds, and in particular diabetic foot ulcers; foot infections; and amputations.

6. HIP / PAP protein, or a derivative thereof, for use according to any one of claims 1 to 5, characterized in that the individual is a mammal, in particular a human being.

7. A HIP / PAP protein, or derivative thereof, for use according to any one of claims 1 to 5, wherein the diabetic individual is characterized in that it has type II diabetes.

8. A HIP / PAP protein, or a derivative thereof, for use according to any one of claims 1 to 5, characterized in that the HIP / PAP protein comprises an amino acid sequence selected from the group consisting of the sequences shown as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:

4.

9. A HIP / PAP protein or a derivative thereof for use according to any one of claims 1 to 5, wherein the derivative comprises an amino acid sequence having at least 80% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, and having the same biological activity as an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:

4.

10. 6. The HIP / PAP protein, or derivative thereof, for use according to any one of claims 1 to 5, wherein said HIP / PAP protein, or derivative thereof, is administered to said individual in combination with at least one agent known to be useful in the prevention and / or treatment of peripheral neuropathy, in particular selected from the group consisting of steroids, anti-inflammatory drugs, immunosuppressants, and vitamin D.

11. 6. The HIP / PAP protein, or derivative thereof, for use according to any one of claims 1 to 5, wherein said HIP / PAP protein, or derivative thereof, is administered to said individual in combination with at least one antidiabetic drug, in particular selected from the group consisting of insulin, metformin, dipeptidyl peptidase 4 inhibitors, glucagon-like peptide 1 receptor agonists, sodium-glucose cotransporter 2 inhibitors and sulfonylureas and thiazolidinediones.

12. A composition comprising a HIP / PAP protein, or a derivative thereof, for use as claimed in any one of claims 1 to 5, and a physiologically acceptable medium.

13. 13. The composition of claim 12, wherein the composition is for oral, sublingual, subcutaneous, intramuscular, intravenous, topical, local, intratracheal, intranasal, or rectal administration.

14. The composition of claim 13, wherein the composition is for oral, subcutaneous, intravenous, topical, or local administration.

15. A kit comprising a HIP / PAP protein, or a derivative thereof, for use as claimed in any one of claims 1 to 5, further comprising at least one agent known to be useful in the prevention and / or treatment of peripheral neuropathy, in particular selected from the group consisting of steroids, anti-inflammatory drugs, immunosuppressants, and vitamin D.

16. A kit comprising a HIP / PAP protein, or a derivative thereof, for use as claimed in any one of claims 1 to 5, further comprising at least one antidiabetic drug, in particular selected from the group consisting of insulin, metformin, dipeptidyl peptidase 4 inhibitors, glucagon-like peptide 1 receptor agonists, sodium-glucose cotransporter 2 inhibitors and sulfonylureas and thiazolidinediones.

Images