Treatment of conditions involving hypoglycemia associated with hyperinsulinemia

Abstract

The present disclosure relates to modulators and / or antagonists of the insulin-insulin receptor signaling complex, as well as methods for selecting such modulators and / or antagonists. Such modulators and / or antagonists can be used to treat mammalian subjects suffering from, or to prevent the onset of, disease states and conditions characterized by, for example, type 2 diabetes, obesity, hyperglycemia, hyperinsulinemia, insulin overdose, chronic kidney disease, type 1 diabetes, insulin resistance, and insulin resistance. The present disclosure provides novel modulators and / or antagonists of the insulin-insulin receptor signaling complex, methods for selecting such modulators and / or antagonists, and disease states and conditions characterized by abnormally increased production and / or utilization of insulin. Use of such modulators and / or antagonists for the treatment or prevention of endogenous hyperinsulinism, preferably congenital hyperinsulinism (CHI), is also provided.

Description

[Technical Field]

[0001] This application relates generally to pharmaceuticals and treatments for conditions associated with, for example, hyperinsulinemia. Insulin secretion-related disorders represent a variety of serious conditions typically characterized by a lack of treatment and a significant burden on patients. Novel therapeutic strategies are urgently needed to address the unmet needs typical of insulin secretion-related disorders. Promising approaches rely on the use of peptides that target specific relevant pathways. Sequences derived from C-peptide have shown promise in modulating insulin secretion and other processes, such as angiogenesis. This application also relates to substances and methods for treating or preventing hypoglycemia in hyperinsulinemia (particularly in congenital hyperinsulinism in children, the most common cause of persistent hypoglycemia). [Background technology]

[0002] Insulin secretion-related disorders (ISRDs) are a heterogeneous group of conditions characterized by altered insulin secretion, resulting in severely debilitating conditions. ISRDs are a rapidly growing global problem, with a collective prevalence exceeding 6% of the global adult population (T1DM patients account for 5–15%) and increasing in number over the past few decades. Due to the large patient population and high disease burden, ISRDs have significant societal and health-economic impacts worldwide. Insulin, a peptide hormone that maintains normal blood glucose levels by promoting intracellular glucose uptake, is central to these disorders. Pancreatic β cells finely regulate insulin secretion to maintain plasma glucose levels within a narrow physiological range (3.5–5.5 mmol / L). Hyperinsulinemic hypoglycemia (HH) is the inappropriate secretion of insulin in the presence of low plasma glucose levels, resulting in severe and persistent hypoglycemia in neonates and children. Mutations in 12 different key genes involved in regulating insulin secretion from pancreatic β-cells ( ABCC8 , KCNJ11 , GLUD1 , GCK , HADH , SLC16A1 , UCP2 , HNF4A , HNF1A , HK1 , PGM1 , and PMM2 ) have been described to be involved in the underlying molecular mechanisms leading to congenital HH.

[0003] For example, excessive insulin secretion, as observed in congenital hyperinsulinism (CHI), can lead to a severe condition characterized by acute hypoglycemia. On the other hand, defective insulin secretion, such as in type 1 diabetes mellitus (T1DM), can cause severe hyperglycemia. Both conditions can potentially lead to life-threatening situations. In the case of T1DM, the discovery of insulin replacement therapy represented a major breakthrough in treatment, benefiting millions of patients to date. However, even with insulin replacement therapy, diabetic patients still face a significant disease burden, manifested in particular by disease-related complications, such as metabolic dysregulation leading to vascular damage. Insulin is the most important hormone for controlling blood glucose levels. When food is ingested, blood glucose levels rise, and the pancreas secretes insulin to maintain blood glucose within a normal range. Insulin acts by transporting glucose into the body's cells. This action of insulin has two functions: 1) maintaining blood glucose levels and 2) storing glucose, particularly in the liver as glycogen. When food intake is completed and glucose level falls, insulin secretion is stopped, and glucose storage in glycogen is released into bloodstream, so that blood glucose level remains normal.In addition, stopping insulin secretion allows access to protein and lipid storage, and can be used as fuel source instead of glucose.In this way, regardless of whether eating or fasting, blood glucose level remains within normal range, and the body always has access to energy.

[0004] In less common hyperinsulinemic ISRDs, such as CHI (Therapies and outcomes of congenital hyperinsulinism-induced hypoglycemia. Diabet Med. 2019 Jan;36(1):9-21), the immediate treatment goal is to stabilize plasma glucose levels and achieve normoglycemia. This is usually achieved by administering additional dextrose. Oral dextrose does little to prevent severe hypoglycemia, and in many cases, high concentrations of dextrose must be administered through a central venous catheter. To increase blood glucose, glucagon promotes hepatic glucose output through a coordinated multi-mechanism approach by increasing glycogenolysis and gluconeogenesis and decreasing glycogen production and glycolysis. Children with CHI respond readily to glucagon treatment, which allows de novo glucose production, and continuous intravenous glucagon infusion can reduce dependence on large fluid volumes, especially in cases where central venous catheter access is difficult. Therefore, early use of glucagon is recommended to ensure rapid achievement of normoglycemia without inducing excessive fluid infusion and complications such as pulmonary edema, heart failure, and electrolyte imbalance. Glucagon has been used by continuous subcutaneous infusion in long-term CHI treatment; however, fibrillation of native glucagon in slow-moving solutions often results in catheter blockage, making treatment unsafe and ineffective. Newer glucagon formulations that are soluble and stable in saline offer a long-term treatment option. Glucagon is generally effective in CHI, but side effects, including the potential for necrotizing migratory erythema, must be monitored.

[0005] The first step in insulin action is hormone binding to the insulin receptor (INSR), an integral membrane glycoprotein also known as CD220 or HHF5. INSR belongs to the tyrosine kinase growth factor receptor superfamily and consists of two extracellular α subunits that bind insulin and two transmembrane β subunits with intrinsic tyrosine kinase activity. The amino acid sequence of INSR is listed in U.S. Patent No. 4,761,371 as NCBI Reference Sequence NP_000199.2. INSR is expressed as two isoforms, INSR-A and INSR-B. INSR isoforms also form INSR-A / INSR-B and hybrid INSR / IGF-1R receptor heterodimers, but their roles in physiology and disease remain incompletely understood.

[0006] Upon binding of insulin to the INSR receptor, the receptor is activated by tyrosine autophosphorylation, and the INSR tyrosine kinase phosphorylates various effector molecules, including insulin receptor substrate-1 (IRS-1), resulting in the hormone's actions (Ullrich et al., Nature 313: 756-761, 1985; Goldfine et al., Endocrine Reviews 8: 235-255, 1987; White and Kahn, Journal Bio 26: 1-4, 1994). IRS-1 binding and phosphorylation ultimately increase the abundance of high-affinity glucose transporter (Glut4) molecules on the outer membrane of insulin-responsive tissues, including muscle cells and adipose tissue, leading to increased glucose uptake from the blood. Glut4 is transported from intracellular vesicles to the cell surface, where it can mediate glucose transport into cells. Increased INSR signaling leads to increased cellular glucose uptake, hypoglycemia (decreased circulating glucose), and all of its consequential sequelae.

[0007] Endogenous hyperinsulinemia (HI) is an abnormal clinical condition involving excessive insulin secretion. Fifty-five percent of cases are associated with insulinoma. Other possible causes of endogenous hyperinsulinemia include pancreatic islet cell hyperplasia, nesidioblastosis, or antibodies to insulin or the insulin receptor. Distinguishing between these different predisposing factors can be difficult, especially in cases where morphological testing is negative. Approximately 1 in 50,000 infants are born with congenital hyperinsulinism (CHI). A defect, likely inherited, means that the beta cells in these children's pancreas produce too much insulin. As a result, children are at constant risk for low blood glucose levels (hypoglycemia), which can cause severe brain damage. Without prompt intervention, most children will die.

[0008] Congenital hyperinsulinism (Horm Res Paediatr 2018;89:82-89) is characterized by persistent hypoglycemia due to dysregulated and excessive insulin secretion. It constitutes a heterogeneous group of disorders, and an underlying genetic predisposition has been identified in approximately 40% of patients. To date, 11 different causative mutations have been described. These mutations often appear to be related to ATP-sensitive K+ channels (KATP channels), which sense metabolic changes in pancreatic β cells, thereby coupling metabolism to electrical activity and ultimately insulin secretion. Opening of KATP channels hyperpolarizes β cells and inhibits insulin secretion. In CHI, the opening of these channels appears to be dysfunctional, resulting in continued insulin secretion and even a state of (persistent) hypoglycemia. Congenital hyperinsulinism is a rare genetic disorder affecting 1 in 50,000 births and caused by mutations in the gene responsible for insulin production. Patients with CHI are characterized by abnormally high insulin secretion by beta cells in the pancreas, resulting in frequent episodes of low blood sugar (hypoglycemia). Because the brains of newborns and infants have a higher glucose consumption requirement compared with adults, newborns are particularly vulnerable to CHI. The goal of emergency treatment for hyperinsulinemic hypoglycemia (HH) is to rapidly achieve euglycemia and maintain plasma glucose levels within a safe range (>3.5 mmol / L), while simultaneously identifying underlying predisposing factors and determining long-term treatment.

[0009] If hypoglycemia is not responsive to oral intake, glucose should be administered intravenously. Patients with HH usually require very high glucose infusion rates to achieve and maintain euglycemia. Glucagon may also be administered urgently to maintain adequate blood glucose levels.

[0010] Repeated hypoglycemic episodes due to undiagnosed and / or untreated CHI in infants can lead to life-threatening complications associated with hypoglycemic brain injury and an increased risk of severe and permanent brain damage, often resulting in irreversible neurodevelopmental disabilities. Diffuse CHI affects all pancreatic islets, while focal CHI affects only specific regions. Treatment of focal CHI relies on surgical removal of the affected area, while patients with diffuse CHI rely on permanent glucose intake and tightly controlled drug therapy. However, side effects can be severe, and treatment is not always effective for patients. As a result, the only existing solution for these patients is surgical removal of the pancreas, which results in extensive side effects, particularly digestive and hormone-related problems and diabetes, which requires strict lifelong medication and an overall reduced quality of life. Therefore, more effective and less invasive treatment strategies for patients with CHI are urgently needed. Hyperinsulinism in infants is one of the most challenging management problems in modern pediatric endocrinology. Early recognition and accurate management of CHI are crucial to prevent brain damage and resulting neurodevelopmental problems. Although diagnosis can usually be achieved without incident, it presents daunting daily management challenges for pediatricians. Despite recent advances in understanding the pathophysiology of hyperinsulinism, the neurological prognosis remains poor, and treatment options are often unsatisfactory, leaving children and their families with lifelong sequelae. In limited-stage CHI, overfunctioning pancreatic beta cells are localized to a discrete area of the pancreas as a focal entity that can be detected by [F18]FDOPA-PET / CT. Treatment options for limited-stage CHI are surgical resection of the affected area. Conversely, in patients with diffuse-stage CHI, treatment options consist of glucose replacement and pharmacotherapy. However, even when treatment is available and timely, it is not always a perfect solution. More than three-quarters of patients do not respond to medication, leaving surgical removal of the entire pancreas as the only option—a major operation that can save lives.However, it can lead to lifelong diabetes and chronic digestive problems, common complications in very young children undergoing major surgery.

[0011] Congenital hyperinsulinism with hypoglycemia is also known by a variety of different names, including childhood "idiopathic" hypoglycemia, leucine-sensitive hypoglycemia, neonatal insulinoma, pancreatic microadenomatosis, nesidioblastosis, persistent hyperinsulinemic hypoglycemia of infancy, and congenital hyperinsulinism. Both sporadic and familial forms of the disease are recognized, with the former occurring in an estimated 1 in 50,000 live births in Western Europe. In isolated areas of Europe, including parts of Finland, the incidence is much higher, with the highest rates found in societies with high rates of consanguinity. In these cultural settings, particularly in the Arabian Peninsula, the incidence can be as high as 1 in 2,500 live births. Most infants with CHI develop symptoms within the first day of life, while others develop symptoms within the first year. Rarely, older children develop de novo hypoglycemia. However, postprandial hyperinsulinemic hypoglycemia after bariatric surgery (post-gastric bypass hypoglycemia) has been described as a new entity, characterized by severe neurohypoglycemic symptoms such as coma and seizures that can lead to patient disability (J Clin Endocrinol Metab 2007;92:4678-4685).

[0012] Prompt treatment of hypoglycemia due to hyperinsulinism (HI) is essential to prevent brain damage. In contrast to other hypoglycemic conditions, in which alternative fuels such as ketones or lactate may be available to the brain during periods of hypoglycemia, HI blocks the production of these fuels, leaving the brain without an energy source. Hypoglycemia can be treated by oral administration of fast-acting carbohydrate-containing fluids or, in severe cases, by intravenous glucose or glucagon injections. Tube-fed children may also receive glucose through a tube. The goal of treatment is to prevent hypoglycemia while simultaneously allowing the child to follow a normal, age-appropriate eating pattern with some degree of safety. For example, a 1-year-old child who normally fasts for 10–12 hours at night should be able to fast for at least 14–15 hours during a successful medical regimen.

[0013] Pharmacological treatment options for CHI are limited and often complicated by ineffectiveness and adverse events. No currently available medications are approved for children with CHI. Pharmacological treatment guidelines vary between institutions, and most treatment decisions are based primarily on expert opinion. The recommended first-line pharmacologic treatment consists of diazoxide in combination with the diuretic chlorothiazide. Diazoxide acts on ATP-sensitive K+ (K-ATP) channels to reduce cellular depolarization and thereby reduce insulin secretion. However, not all patients respond to this treatment, particularly those with mutations in the K-ATP channel genes (ABCC8, KCNJ11). Diazoxide therapy is often complicated by side effects, such as fluid retention, thrombocytopenia, hirsutism, and gastrointestinal dysmotility, which can lead to treatment discontinuation. The calcium antagonist nifedipine was previously prescribed as a second-line treatment when children did not respond to diazoxide. However, nifedipine is rarely used in clinical practice because the clinical response to this drug is generally unsatisfactory. Continuous subcutaneous glucagon is another treatment option and has been utilized not only for the initial treatment of hypoglycemia but also for the long-term treatment of CHI. However, commercially available glucagon preparations precipitate in a slow-moving solution within the indwelling catheter, causing blockage and unreliable drug delivery. Therefore, continuous glucagon is not currently intended for the long-term treatment of CHI.

[0014] Somatostatin is a hormone that preserves the electrical stability of pancreatic beta-cell membranes and thus inhibits insulin release. Octreotide, a short-acting somatostatin analog, is widely used as a second-line treatment for CHI, preferring nifedipine or continuous glucagon over nifedipine. Octreotide's half-life is relatively short, approximately 100 minutes, and therefore octreotide must be administered by continuous intravenous infusion, frequent subcutaneous injections, or continuous subcutaneous pump therapy. Long-term subcutaneous octreotide treatment is burdensome for patients and families, and home-managed multiple daily injections or pump therapy is not always feasible. Side effects of octreotide treatment include gastrointestinal dysmotility and tachyphylaxis, necessitating increased drug dosage to maintain efficacy.

[0015] Randomized clinical trials of novel treatments for CHI are limited, which may be due to several factors, including the limited number of patients worldwide and the complexity of organizing multicenter trials. The first phase 3 clinical trial using long-acting subcutaneous glucagon failed to meet its primary endpoint. In a phase 1 clinical study, a single infusion of the antibody XOMA 358 (also known as RZ358) resulted in a dose-dependent reduction in insulin sensitivity in healthy adults. (MAbs. 2018 Jul;10(5):796-802) and results presented at the Pediatric Endocrine Society's 2022 Annual Meeting on May 1, 2022, demonstrated a reduction in hypoglycemia in infants over the age of 2. However, monoclonal antibodies are administered intravenously (injected into a vein). Because antibodies are proteins, administering them can sometimes cause allergic or immunological reactions. Potential adverse effects include hypersensitivity reactions, infusion reactions, and persistent immunogenicity, which may be exacerbated in infants. Besides CHI medications that attempt to increase plasma glucose levels (dextrose, glucagon, and analogs) or reduce insulin secretion or release from beta cells (diazoxide, somatostatin receptor analogs, nifedipine), there are currently no additional approved treatments except for partial or complete removal of the pancreas.

[0016] Congenital hyperinsulinism (CHI) is the most common cause of severe, persistent hypoglycemia (low blood glucose, or sugar) in newborns and children. This disorder is characterized by high blood insulin levels, which leads to decreased glucose (sugar). Recurrent and severe hypoglycemia can adversely affect brain function. Despite currently available treatments, CHI can result in serious neurological and developmental complications, including recurrent seizures, learning disabilities, and focal brain lesions. The condition is currently managed by having patients follow a regular high-carbohydrate diet and treating them with medications that reduce insulin secretion, such as diazoxide, chlorothiazide, nifedipine, glucagon, and octreotide. Treatment may include pancreatic surgery (pancreatectomy). However, these procedures are challenging because these patients require constant monitoring, and the medications have undesirable adverse effects. Furthermore, although a rare disorder, CHI has been found to pose a substantial economic burden to the NHS.

[0017] In short, there is a significant unmet medical need to develop therapeutics, preferably oral, aimed at preventing hypoglycemia in HI, particularly in the most common cause of persistent hypoglycemia in children. In the words of Congenital Hyperinsulinism International (congenitalhi.org), we need to better understand congenital hyperinsulinism, identify new means to improve the lives of those with HI, reduce the incidence of irreversible brain damage, detect genetic predispositions for currently unknown types of HI, and find a cure for all types of HI. Regardless of the numerous and diverse genetic predispositions to HI, therapeutics directly aimed at reducing hypoglycemia caused by hyperinsulinemia (also known as hyperinsulinemic hypoglycemia (HH); J Clin Res Pediatr Endocrinol. 2017 Dec 30;9(Suppl 2):69-87) would be preferred. Summary of the Invention

[0018] The present invention discloses novel findings regarding the regulation of blood glucose by malaria parasites (Plasmodium spp.) during the blood stage of the malaria infection cycle. Essentially (Binh et al., Glucose metabolism in severe malaria: minimal model analysis of the intravenous glucose tolerance test incorporating a stable glucose label. Metabolism. 1997 Dec;46(12):1435-40), basal plasma glucose is typically elevated in uncomplicated malaria, suggesting insulin resistance. As infection progresses, the risk of hypoglycemia increases as host glucose production becomes insufficient for host / parasite needs. The malaria data indicate that basal plasma glucose utilization increases by approximately 50% in severe malaria. Prevention and treatment of early hypoglycemia should be based on appropriate glucose replacement therapy. Strategies to reduce insulin secretion or action are thought to be less important in malaria. White et al. (Severe hypoglycemia and hyperinsulinemia in falciparum malaria. N Engl J Med. 1983 Jul 14;309(2):61-6) observed that in Plasmodium falciparum malaria, kinin-induced insulin secretion is involved in hypoglycemia, but other factors, including the malaria parasite's large glucose requirements, also contribute. Recent findings (Kumar et al., Mol Microbiol. 2021 May;115(5):891-900) link nutrient sensing to gene expression in Plasmodium falciparum blood-stage parasites, particularly in merozoites that spread to infected host erythrocytes during the parasite's intraerythrocytic developmental cycle (IDC).Typically, merozoites express erythrocyte membrane protein 1 (PfEMP1) on the surface of infected erythrocytes, and this is accompanied by increased production of a set of proteins belonging to the serine-rich antigen (SERA) family, such as SERA 5. During IDC and to accommodate rapid parasite growth, the glucose requirements of the parasite are high, necessitating manipulation of the host's blood glucose levels by the parasite.

[0019] Detailed analysis of blood-stage malaria parasite proteins (EMP1 and Sera 5, see Example 1 herein) has shown that these proteins extensively mimic long tandem repeats of peptides with the specific elastin receptor complex (ERC)-binding motif XGXXPG (where G represents the amino acid glycine, P represents proline, and X represents any amino acid). Very specifically, malaria parasites (Plasmodium) that infect primates have been found to specifically express long stretches of repeats of the chemotaxis domain VGVAPG, while malaria parasites (Plasmodium) that infect rodents have been found to specifically express long stretches of repeats of the chemotaxis domain PGAVPG. Each of these domains is capable of activating the ERC, many of which contain the motif XGXXPG (see, e.g., Table 2 in Heinz et al., Elastases and elastokines: elastin degradation and its significance in health and disease. Crit Rev Biochem Mol Biol. 2020 Jun;55(3):252-273). Repeats of this motif result in the formation of additional structures that closely resemble type VIIIb turns, and when they occur multiple times in the sequence, are thought to facilitate peptide-cell interactions with the ERC.

[0020] In this regard, the present invention discloses peptide modulators of insulin-insulin receptor signaling affected by the ERC (for a diagrammatic illustration, see e.g., Figure 1 in Haxho F, Alghamdi F, Neufeld RJ, Szewczuk MR (2014) Novel Insulin Receptor-Signaling Platform. Int J Diabetes Clin Res 1:005) that have reduced insulin-receptor signaling and allow for the promotion of insulin resistance through modulation of the insulin receptor (IR), for use in treating mammalian, preferably primate, more preferably human, or alternatively rodent, subjects suspected of having episodes of hypoglycemia associated with hyperinsulinemia. The mechanism of action of the peptides is to produce insulin resistance in hyperinsulinemic patients, preferably HH patients, with a concomitant reduction in glucose uptake (mediated by GLUT4) and a promotion of increased blood glucose levels. Blaise et al. reported that chronic administration of elastin-derived peptides (EDPs) in mice promotes insulin resistance through modulation of the insulin receptor (IR) by the elastin receptor complex (ERC; Diabetes (2013) 62:3807-16). In chow-fed C57Bl / 6J male mice, acute or chronic intravenous injection of EDPs induced reduced glucose uptake and hyperglycemic effects associated with insulin resistance in skeletal muscle, liver, and adipose tissue. Based on in vivo, in vitro, and in silico approaches, Blaise et al. proposed that this insulin resistance results from an interaction between the insulin receptor (IR) and the neuraminidase-1 subunit of the elastin receptor complex, triggered by elastin-derived peptides (EDPs), such as VGVAPG and κ-elastin (kE or kappa-elastin). This interaction correlated with reduced sialic acid levels in the β-chain of the IR and reduced IR signaling.This is the first study to show that EDPs, which accumulate primarily with aging, are involved in the hidden development of insulin resistance that leads to type 2 diabetes. Blaise et al., however, did not relate their work to the treatment of either hypoglycemia or hyperinsulinism, much less the hyperinsulinemic hypoglycemia that is the subject of the present application.

[0021] In a preferred embodiment, the present invention discloses said modulator comprising or provided with a chemotactic domain structure of an elastin receptor complex for use in the treatment of a primate, preferably a human subject, suspected of having episodes of hypoglycemia associated with hyperinsulinemia, such as, for example, that seen in congenital hyperinsulinemia (CHI) and post-gastric bypass hypoglycemia, most preferably an infant exhibiting symptoms of said CHI, most preferably an infant having or repeatedly exhibiting symptoms of said CHI, or an adult having post-gastric bypass hypoglycemia and exhibiting episodes of plasma glucose levels below 4 mmol / L (below 72 mg / dL), more preferably below 3.5 mmol / L (below 63 mg / dL), more preferably below 3 mmol / L (below 54 mg / dL), more preferably below 2.5 mmol / L (below 45 mg / dL), more preferably below 2 mmol / L (below 36 mg / dL). The present invention discloses a modulator of insulin-insulin receptor signaling with reduced insulin receptor signaling, which can promote insulin resistance and thereby reduce insulin-mediated blood glucose uptake by the subject's fat or muscle cells, resulting in beneficially higher plasma glucose levels and avoiding the dire neurological pathology and other negative prognoses of CHI. The modulator can reduce insulin-induced glucose transporter 4 (GLUT4) translocation to the plasma membrane of the subject's fat or muscle cells, and more preferably induce whole-body (systemic) insulin resistance. In a preferred embodiment, the present invention discloses a modulator according to the present invention that can promote insulin resistance, wherein the modulator is provided with a molecule having an elastin receptor-binding motif (the motif is also identified herein as the chemotactic domain of elastin). Preferably, the modulator is provided with or comprises a molecule having an elastin receptor binding motif that is at least functionally equivalent to a peptide having the motif XGXXPG (where G represents the amino acid glycine, P represents proline, and X represents any amino acid).In a preferred embodiment, the modulator comprises a peptide or peptidomimetic molecule having an elastin receptor-binding motif at least functionally equivalent to a peptide having the motif XGXXPG (where G represents the amino acid glycine, P represents proline, and X represents any amino acid). The motif is preferably derived from the group of primate, preferably human, elastin-derived peptides, primate, preferably human C-peptide, or primate, preferably human galectin-3. In a preferred embodiment, the inventors disclose peptide modulators according to the invention having retro-inverso Pg domain (all D amino acids) peptides, such as gagpggl, gagpggal, agpggl, gpgggpa, gpggal, gpggg, gpgag, gpvagp, gpavgv, which were found to fit a model of EBP similarly designed to dock the chemotactic domain VGVAPG.

[0022] It should be noted that, except for glycine, all standard α-amino acids can exist in two optical isomers that are mirror images of each other, and these are referred to as L-amino acids and D-amino acids. As used herein, when referring to the amino acid sequence of a peptide in one-letter code, lowercase letters indicate D-amino acids, while uppercase letters indicate L-amino acids, with the notable exception of glycine, where g and / or G indicate the amino acid glycine, which has no optical isomers and therefore does not differ between D- and L-forms. Generally, all-L amino acid-containing peptides are functionally equivalent to their all-D counterparts, with the notable exception that all-D amino acid peptides are generally more resistant to general proteolysis, such as that found in the intestinal system. As used herein, lowercase letters indicate D-amino acids, while uppercase letters indicate L-amino acids, with the notable exception of glycine, where g and / or G indicate the amino acid glycine, which has no optical isomers and therefore does not differ between D- and L-forms.

[0023] The L-amino acid peptides GGGPG and GAGPG also fit the model. EBP-related biological activity is thought to depend on whether a peptide with a PG domain (having a GXXP or pxxg motif) can adopt a type VIII beta-turn configuration at the proline (P / p). In one embodiment, the peptide with this motif is preferably at least functionally equivalent to the peptides VGVAPGVGVAPGVGVAPG, QVGQVELGGGPGAGSLQP, or GAYPGAPGAYPGAPAPGV. The peptide with this motif is also preferably derived from a group of primate, preferably human, elastin-derived peptides, e.g., kappa-elastin-derived peptides, with the motif XGXXPG. In a preferred embodiment, and based on the malaria mimetic modulators of Example 1, the peptide with this motif is at least functionally equivalent to the peptides VGVAPGVGVAPGVGVAPG or AVPGAVPGAVPG. Typically, the use of the VGVAPG-based modulators in primate subjects and the PGAVPG-based modulators in rodent subjects, respectively, is disclosed; however, for comparison purposes, the reverse is also provided. In a further embodiment, the modulator comprises the peptide VGVAPG(n) or AVPG(n), where n represents the number of repeat occurrences of the motif and is selected from the group of 1, 2, 3, 4, 5, 6, 7, and 8; preferably, n is selected from the group of 1, 2, 3, 4, 5, 6; and more preferably, n is selected from the group of 1, 2, 3, 4, 5, 6. In another embodiment said modulator comprises a peptide having the motif gpva(n) or gpavgv(n), where n indicates the number of (repeat) occurrences of said motif and is selected from the group of 1, 2, 3, 4, 5, 6, 7 and 8, more preferably from 2, 3, 4, 5, 6, 7, 8, preferably n is selected from the group of 1, 2, 3, 4, 5, 6, more preferably from 2, 3, 4, 5, 6, more preferably n is selected from the group of 1, 2, 3, 4, more preferably from 2, 3, 4. Preferably such repeats include at least one tandem repeat.The present invention also provides pharmaceutical compositions comprising the modulators according to the present invention. In one embodiment, such pharmaceutical compositions are provided for parenteral administration. Specifically, rapidly degradable all-L amino acid peptides, such as VGVAPGVGVAPGVGVAPG, AVPGAVPGAVPG, QVGQVELGGGPGAGSLQP, or GAYPGAPGAYPGAPAPGV, and AQGVAPG (n=1-8), LQGVAPG (n=1-8), VGVAPG (n=1-8), or AVPG (n=1-8), where n indicates the number of occurrences of the motif, are most suitable for parenteral administration. In another embodiment, such pharmaceutical compositions are provided for oral administration. Preferred dosage requirements for parenteral administration (preferably intravenous or intraperitoneal administration) of such modulators range from 1 to 1000 mg / kg, preferably 5 to 500 mg / kg, and more preferably 10 to 100 mg / kg. Specifically, stable all-D amino acid (reverse inversion) peptides, such as gpavgvgpavgvgpavgv, gpvagpvagpva, pqlsgagpggglevqgvq, or vgpapagpyagpagpgpyag, and gpavgqa (n=1-8), gpavgql (n=1-8), gpva (n=1-8), or gpavgv (n=1-8), are most suitable for oral administration. Preferred dosage requirements for oral administration of such modulators range from 1 to 10,000 mg / kg, preferably 5 to 5,000 mg / kg, and more preferably 10 to 1,000 mg / kg. Dosage requirements can be adjusted based on the resulting blood glucose levels of the treated subject. The present invention also discloses a method for the treatment of a primate, preferably a human subject, suspected of having an episode of hypoglycemia associated with hyperinsulinemia, comprising the step of treating said subject with a modulator or a pharmaceutical composition according to the present invention.

[0024] The present disclosure relates to modulators and / or antagonists of the insulin-insulin receptor signaling complex, preferably peptides, and methods for selecting such modulators and / or antagonists. Such modulators and / or antagonists can be used to treat mammalian subjects suffering from disease states and conditions, or to prevent the onset of diseases in subjects at risk. The facilitative glucose transporter (GLUT) family is involved in regulating tissue-specific glucose uptake and metabolism in the liver, skeletal muscle, and adipose tissue to ensure homeostatic control of blood glucose levels. Reduced glucose transport activity leads to abnormal utilization of energy substrates and is associated with insulin resistance and type 2 diabetes. It is well established that GLUT2, the master regulator of hepatic hexose flux, and GLUT4, the main force behind insulin- and contraction-stimulated glucose uptake in skeletal muscle, are important contributors to the control of systemic blood glucose. Carbohydrate ingestion results in an immediate increase in circulating blood glucose levels after glucose absorption from the intestine. As a direct response, pancreatic beta cells sense elevated blood glucose levels via a GLUT2-dependent process and increase insulin secretion. Consequently, insulin binding to its receptor enhances glucose transport to skeletal muscle, adipose tissue, and the heart, primarily driven by the rapid translocation of GLUT4 transporter vesicles to the plasma membrane, and also inhibits hepatic gluconeogenesis. Both regulatory pathways combine to result in the clearance of glucose from the bloodstream (GLUT4 exocytosis. J Cell Sci. 2011 Dec 15;124(Pt 24):4147-59). GLUT4 is an insulin-regulated glucose transporter responsible for insulin-regulated glucose uptake into adipocytes and muscle cells. In the absence of insulin, GLUT4 is primarily found in intracellular vesicles called GLUT4 storage vesicles (GSVs).In response to insulin stimulation, GSVs rapidly translocate to and fuse with the plasma membrane. In the continued presence of insulin, GLUT4 molecules are internalized and recycled to the plasma membrane in vesicles distinct from GSVs, likely of endosomal origin. Without wishing to be bound by theory, insulin stimulates glucose transport into muscle and adipose tissue 10-30-fold with a half-life of 2-5 minutes. The major glucose transporter expressed in these tissues is GLUT4. In the absence of insulin, the majority of GLUT4 is stored in intracellular vesicles called GLUT4 storage vesicles (GSVs) or insulin-responsive vesicles (IRVs). After a meal, insulin is secreted by the pancreas and engages its receptors on the surface of muscle and adipocytes, thereby activating the canonical PI3K-AKT pathway. Activation of this pathway is necessary and sufficient to trigger exocytosis of GSVs to the plasma membrane. Reduced GLUT4 transport is believed to be one of the earliest factors contributing to insulin resistance in primates, preferably humans, and disruption of GLUT4 translocation to the plasma membrane in muscle or adipose tissue induces insulin resistance by reducing glucose uptake by said muscle cells or adipocytes, and is provided herein as a treatment for the benefit of subjects who have experienced episodes of hypoglycemia associated with hyperinsulinemia. Specifically, reduced translocation of GLUT4 in adipose tissue contributes to the development of whole-body insulin resistance.

[0025] Insulin resistance describes a state of relative inability of peripheral tissues to respond to increased amounts of circulating insulin, resulting in chronically elevated blood glucose levels. The present invention provides a novel means of treating hypoglycemia driven by hyperinsulinemia, utilizing the phenomenon of reduced GLUT4 translocation to the plasma membrane in muscle or adipose tissue, resulting in insulin resistance.

[0026] The present disclosure provides novel modulators and / or antagonists of the insulin-insulin receptor signaling complex, methods for selecting such modulators and / or antagonists, and disease states and conditions characterized by abnormally increased production and / or utilization of insulin. Use of such modulators and / or antagonists for the treatment or prevention of conditions of endogenous hyperinsulinism (HI), preferably congenital hyperinsulinism (CHI), is also provided.

[0027] Methods for treating a subject with, suspected of having, or suspected of having a condition involving episodes of hypoglycemia associated with hyperinsulinemia (HI) are provided, comprising treating the subject with a modulator or pharmaceutical composition provided with a molecule having an elastin receptor-binding motif at least functionally equivalent to a peptide having the motif XGXXPG (where G represents the amino acid glycine, P represents proline, and X represents any amino acid). Such peptides provided herein preferably have the canonical XGXXPG or gpxxgx motif, allowing for the formation of a type VIII β-turn required for binding to its cognate receptor, the elastin receptor complex (ERC, Blanchevoye et al., J Biol Chem. 2013 Jan 11; 288(2):1317-28). As used herein, a condition involving episodes of hypoglycemia associated with hyperinsulinemia (abbreviated hyperinsulinemic hypoglycemia) describes the state and effects of low blood glucose caused by excess insulin. Such conditions are often biochemically characterized by dysregulated secretion of insulin from pancreatic beta cells in the presence of low blood glucose levels. Under normal physiological conditions, beta cells synthesize, store, and secrete insulin in a precisely controlled manner so that fasting blood glucose levels are maintained within a narrow range, generally between 3.5 and 5.5 mmol / L. Symptoms of hyperinsulinemic hypoglycemia typically develop in otherwise healthy individuals at plasma glucose levels below 55 mg / dL (3.0 mmol / L). At glucose levels below 55 mg / dL (3.0 mmol / L), insulin secretion is usually almost completely suppressed, and detection of insulin under these conditions is highly indicative of hyperinsulinemic hypoglycemia.

[0028] In a preferred embodiment, there is provided a method for the treatment of a mammalian, preferably a primate, more preferably a human subject having, suspected of having, or suspected of having a condition involving episodes of hypoglycemia associated with hyperinsulinemia (HI), and having a blood glucose level of 55 mg / dL (3.0 mmol / L), preferably a level less than 50 mg / dL (2.7 mmol / L), and preferably also having a glucose to insulin ratio of less than 3, preferably less than 2, the method comprising the step of treating said subject with a modulator or pharmaceutical composition provided with a molecule having an elastin receptor binding motif that is at least functionally equivalent to a peptide having the motif XGXXPG or gpxxgx (where G / g represent the amino acid glycine, P / p represent proline, and X / x represent any l or D amino acid, respectively).

[0029] In a preferred embodiment, there is provided a method for the treatment of a primate, preferably a human subject, having, suspected of having, or suspected of having a condition involving episodes of hypoglycemia associated with hyperinsulinemia (HI), and having a blood glucose level of 55 mg / dL (3.0 mmol / L), preferably a level less than 50 mg / dL (2.7 mmol / L), and preferably also having a glucose to insulin ratio of less than 3, preferably less than 2, wherein said subject is administered a medicament containing one or more of the motifs VGVAPG(n), vGVAPG(n), vGvAPG(n), vGVaPG(n), vGvaPG(n), gpavgv(n), PGAVPG(n), pGvAPG(n), pGVaPG(n), pGvaPG(n), gpavgp(n), LGGGPG(n), lGGGPG(n), gpgggl(n), P The method includes the step of treating a cell with a modulator or pharmaceutical composition provided with a molecule having an elastin receptor binding motif that is at least functionally equivalent to any of the peptide molecules, wherein the peptide molecule is a peptide having a motif selected from the group consisting of GAYPG(n), pGAYPG(n), pGaYPG(n), pGAyPG(n), pGayPG(n), pgyagp(n), AQGVAPG(n), gpavgqa(n), LQGVAPG(n), or gpavgql(n) (n represents the number of occurrences of the motif, and n varies from 1 to 8, preferably 1 to 6, more preferably 1 to 4, and most preferably 2 to 4), wherein the method allows the modulator to be targeted to elastin receptors on the surface of the cell.

[0030] In another embodiment, the peptide is selected from the group VGVAPG(n), gpavgv(n), PGAVPG(n), gpvagp(n), LGGGPG(n), gpgggl(n), AQGVAPG(n), gpavgqa(n), LQGVAPG(n), gpavgql(n), PGAYPG(n), and gpyagp(n), where n varies from 1 to 8, preferably 1 to 6, more preferably 1 to 4, and most preferably 2 to 4, and allows for targeting of the modulator to elastin receptors on the surface of cells.

[0031] In one other embodiment, the peptide is selected from the group VGVAPG(n), gpavgv(n), PGAVPG(n), gpvagp(n), PGAYPG(n), and gpyagp(n), where n varies from 1 to 8, preferably from 1 to 6, more preferably from 1 to 4, and most preferably from 2 to 4, and allows targeting of the modulator to elastin receptors on the surface of cells.

[0032] In one other embodiment, the peptide is selected from the group VGVAPG(n), gpavgv(n), PGAVPG(n), gpvagp(n) (n varies from 1 to 8, preferably from 1 to 6, more preferably from 1 to 4, most preferably from 2 to 4) and allows targeting of the modulator to elastin receptors on the surface of cells.

[0033] In one other embodiment, the peptide is selected from the group of VGVAPG(n), gpavgv(n), PGAVPG(n), and gpvagp(n), AQGVAPG(n), gpavgqa(n), LQGVAPG(n), and gpavgql(n) (n varies from 1 to 8, preferably from 1 to 6, more preferably from 1 to 4, most preferably from 2 to 4), and allows targeting of the modulator to elastin receptors on the surface of cells.

[0034] Proteins and most naturally occurring peptides are generally composed of L-configuration amino acids.However, D-amino acids have been found in various peptides synthesized in animal cells.Examples include antimicrobial peptides derived from opiates and frog skin, neuropeptides derived from snails, hormones derived from crustaceans, and venom derived from spiders.These D-amino acid forms are formed when L-amino acids undergo post-translational modification.Many proteins or peptides composed of L-configuration amino acids are easily degraded or tagged for selective destruction in cells or intestinal tract.Peptides made at least partially from D-amino acids show strong resistance to proteolysis and have improved intestinal uptake, making them suitable for oral administration.

[0035] In another preferred embodiment there is provided a method for the treatment of a primate, preferably a human subject, having, suspected of having or suspected of having a condition involving episodes of hypoglycemia associated with hyperinsulinemia (HI), and having a blood glucose level of 55 mg / dL (3.0 mmol / L), preferably a level less than 50 mg / dL (2.7 mmol / L), and preferably also having a glucose to insulin ratio of less than 3, preferably less than 2, the method comprising the step of treating said subject with a modulator or pharmaceutical composition provided with a molecule having an elastin receptor binding motif that is at least functionally equivalent to any of the peptide molecules, wherein the peptide has a motif selected from the group consisting of peptides having the motifs gpavgv, gpavgV, gpaVgV, gpAvgV, gpAVgV, gpgggl, gpgggL, gpyagp, gpaygP, gpaYgP, gpAygP, or gpAYgP. As used herein, being functionally equivalent preferably means having functionally equivalent ERC reactivity or biological activity.

[0036] Low plasma glucose levels occur without symptoms or signs in healthy individuals during prolonged fasting when alternative fuels, such as ketones, are used. Due to this variability, no single plasma glucose concentration defines hypoglycemia. Recurrent hypoglycemia in newborns, infants, and children is not normal. Healthy newborns who experience a drop in blood glucose levels typically do not fall below 50 mg / dL (2.7 mmol / L). Newborns and infants with blood glucose levels below 50 (2.7 mmol / L) or whose blood glucose levels fall below the normal neonatal minimum should be evaluated and managed according to hypoglycemia guidelines. A glucose-to-insulin ratio of less than 3, preferably less than 2, and low concentrations of free fatty acids and ketones during hypoglycemia are highly suggestive of hyperinsulinemic hypoglycemia.

[0037] Hypoglycemia due to insulin excess is the most common type of severe hypoglycemia. It can be due to endogenous insulin or insulin injections. Symptoms of hyperinsulinemic hypoglycemia vary with age and the severity of the hypoglycemia. In general, most signs and symptoms can be attributed to (1) the effects of glucose deprivation on the brain (neurohypoglycosis) or (2) the adrenergic response of the autonomic nervous system to hypoglycemia. Some other symptoms are difficult to attribute to either of these causes. In most cases, all effects are reversed when normal glucose levels are restored.

[0038] More lasting harm from this type of severe hypoglycemia is rare, and death is extremely rare. One reason the risk of hypoglycemia from excess (exogenous) insulin may be high is that insulin reduces the availability of most alternative brain fuels, such as ketones. Various types of brain damage can occur, ranging from localized effects like stroke to memory and thinking disorders. Children with prolonged or recurrent hyperinsulinemic hypoglycemia in infancy may suffer brain harm and may be developmentally delayed. Hypoglycemia from endogenous insulin can be congenital or acquired and may manifest in the neonatal period or many years later. Hypoglycemia can be severe and life-threatening or a mild, occasional discomfort. Currently, the most common type of severe but transient hyperinsulinemic hypoglycemia occurs unintentionally in people with type 1 diabetes who take (high doses of) insulin, and can also occur when people with type 2 diabetes overdose insulin. People with hyperinsulinemic hypoglycemia due to excess or excessive insulin or overdose of medications that cause hyperinsulinism often respond well to oral glucose.

[0039] There are numerous cases of hypoglycemia due to endogenous insulin. First, congenital hyperinsulinism may be transient neonatal hyperinsulinism (the mechanism is unknown). It may also be focal hyperinsulinism (due to KATP channelopathy), such as paternal SUR1 mutations with clonal loss of heterozygosity at 11p15 or paternal Kir6.2 mutations with clonal loss of heterozygosity at 11p15. It may also manifest as diffuse hyperinsulinism due to KATP channelopathy, SUR1 mutations, Kir6.2 mutations, glucokinase gain-of-function mutations, hyperammonemic hyperinsulinism (glutamate dehydrogenase gain-of-function mutations), short-chain acyl-coenzyme A dehydrogenase deficiency, carbohydrate-deficient glycoprotein syndrome (Jaeken's disease), and Beckwith-Wiedemann syndrome (suspected to be due to hyperinsulinism, but the pathophysiology is uncertain: 11p15 mutations or IGF2 excess).

[0040] In a preferred embodiment, a method is provided for treating a primate, preferably a human subject, who has, is suspected of having, or is suspected of having congenital HI, an extremely rare genetic endocrine disorder occurring in various populations in 1 in 2,500 to 1 in 50,000 live births. Congenital HI (CHI) is characterized by excessive insulin secretion, which causes recurrent episodes of low blood sugar, or hypoglycemia. This condition often goes unrecognized in infants, who are at risk for complications from recurrent hypoglycemic events, including developmental delay, seizures, coma, and death.

[0041] Insulin resistance refers to the state that physiological amount of insulin is insufficient to obtain normal insulin response from cell or tissue.Severe insulin resistance is associated with type 2 diabetes, while milder insulin resistance is also associated with many disease states (for example, acute inflammation or atherosclerosis) and physiological states (for example, pregnancy or adolescence) that exist in many individuals who do not have type 2 diabetes (Woods. et al., End, Metab & Immuno Disorders-Drug Targets 9: 187-198, 2009). [Brief explanation of the drawings]

[0042] [Figure 1-1]Figures 1a and 1b show that a preference for the xGxxPG motif was detected in Plasmodium proteins relative to merozoite blood-stage proteins, which correlated with the development of insulin resistance in the blood stage. After clustering internal dipeptide preferences at the x1x2 position of the Gx1x2P elastin receptor complex (ERC) motif occurrence in Plasmodium merozoite surface proteins and / or SERA5 proteins, initial results were detected in A) Plasmodium proteins from the primate-infecting malaria parasites P. vivax (n=13) or P. falciparum (n=11) present in the Uniprot database considered in October 2022, and B) Plasmodium proteins from the rodent-infecting malaria parasites P. yoelii yoelii (n=4) or P. berghei (n=3) present in the Uniprot database considered in October 2022. Of the 400 dipeptide sequences analyzed, clustering revealed a preference for the dipeptide VA in the motif GVAP in the analyzed primate-infecting Plasmodium proteins (1A), and a preference for the dipeptide AV in the motif GAVP in the analyzed rodent-infecting Plasmodium proteins (1B). The rodent and primate Plasmodium protein entries in Uniprot were then probed by peptide searches using various peptide motifs containing the GVAP and GAVP motifs found. In particular, GVAP was found in the VGVAPG motif, which was found to be recurrently present in Plasmodium proteins from primate-infecting Plasmodium parasites, and which is also found recurrently in primate elastin (see also Tables 1-4). [Figure 1-2] Same as above. [Figure 2-1]Blaise et al. (Retrieved from Diabetes. 2013 Nov; 62(11): 3807-3816. Notably, Blaise et al. do not relate to the treatment of either hypoglycemia or hyperinsulinism, much less the treatment of hyperinsulinemic hypoglycemia, which is the subject of the present application). A: Blood glucose 30 minutes after a single intravenous injection of various doses of kE (a collection of peptides with an XGXXPG motif derived from elastin and binding to the ERC, e.g., peptides with the bioactive motifs PGAIPG, GAVPG, GVLPG, GGVPG, and GVVPG, VGVAPG, or VVGPGA) (n=7 mice). B: Glucose uptake measured in isolated soleus muscles incubated with kE with (dark gray bars) or without (black bars) insulin. White and light gray bars correspond to the no-kE insulin condition at the indicated concentrations (n=5 mice per group). C: Time course study of blood glucose after a weekly intravenous injection of kE compared to fed mice injected with PBS (n=10 mice per group). D: Food intake at 1, 8, and 11 weeks (n=5 mice per group). [Figure 2-2]E: Time course study of blood glucose after a weekly intravenous injection of kE (n = 5 mice per group). F: Blood glucose levels in mice fasted for 6 hours and treated with kE or PBS for 7 weeks (n = 9 mice per group). G: Glucose tolerance test (GTT) results at 7 weeks post-injection in mice after a 6-hour fast (n = 10 mice per group). H: Bar graphs represent the mean area under the curve (AUC) of GTT results. I: Glycogen quantification in mice fasted for 6 hours and treated with kE or PBS for 7 weeks (n = 5 mice per group). J: Expression of glucose 6-phosphatase (G6Pase) and phosphoenolpyruvate carboxykinase (PEPCK) by quantitative real-time PCR in the liver of fasted mice with or without 7 weeks of kE treatment. Target mRNA levels were normalized to 36B4 mRNA levels (n = 6–9 mice). Results are mean ± SEM. Statistically significant difference (*P<0.05, Mann-Whitney). Blood glucose conversion: mg / dL to mmol / L conversion factor: 1 mg / dL = 0.0555 mmol / L [Figure 3]Retrieved from International Publication Nos. WO2018 / 141969 and WO2018 / 141970. Of note, neither International Publication Nos. WO2018 / 141969 nor WO2018 / 141970 is related to the treatment of hypoglycemia or hyperinsulinism, nor to the treatment of hyperinsulinemic hypoglycemia, the subject of this application. Various GxxP hexapeptides were docked into the peptide-binding site of elastin-binding protein (EBP) using Vina / Autodock and PyMOL (1, 2, 3). The binding conformation of each peptide was selected from the top 20 best-scoring configurations. A homology model of EBP (4) was used as the receptor in the docking procedure. The peptides tested were: VGVAPG (prototype PG domain, GXXP-peptide ligand of EBP (4)), LGGGPG (selected from the C-peptide (5)), QGQLPG (immunomodulating peptide provided herein), PGAYPG (selected from galectin-3 (6)), and QGVLPA (selected from loop 2 of beta-hCG (7)). Similarly, the inverted PG-domain (all D amino acid) peptides gagpgggl, gagpggal, agpggl, gpgggpa, gpggal, gpggg, gpgag, gpvagp, and gpavgv fit similarly into the model of EBP designed to dock VGVAPG. The L amino acid peptides GGGPG and GAGPG also fit similarly. EBP-related biological activity and therefore functional equivalence appears to depend on whether peptides containing PG domains (with GXXP or pxxg motifs) can adopt a type VIII beta-turn configuration at proline (P / p) ( 4 ).References for Figure 3: 1 Trott, O & Olson, AJ (2010) AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization and multithreading, J Comp Chem 31: 455-461. L SEP2 Seeliger, D & de Groot, BL (2010) Ligand docking and binding site analysis with PyMOL and Autodock / Vina. J Comput-Aided Mol Des 24 :417-422.3 www.pymol.org4 Blanchevoye, C et al. (2013) Interaction between the elastin peptide VGVAPG and human elastin binding protein, J Biol Chem 288:1317-28.5 Ido, Y et al. (1997) Prevention of vascular and neural dysfunction in diabetic rats by C-peptide Science 277:563-6.6 De Boer, R et al. (2011) Plasma Galectin-3 Is Associated with Near-Term Rehospitalization in Heart Failure: A Meta-Analysis Journal of Cardiac Failure Vol 17, Issue 8, S93 L SEP7 Khan, NA et al. (2010) Mitigation of septic shock in mice and rhesus monkeys by human chorionic gonadotrophin-related oligopeptides, Clin Exp Immunol 160:466-478.。 [Figure 4-1]Figure 4a: ERC is a heterotrimeric receptor complex of human elastin-binding protein (EBP), neuraminidase-1 (Neu-1), and protective protein cathepsin A (PPCA). EBP consists of an alternatively spliced variant of beta-galactosidase. It binds to the hexapeptide X-Gly-XX-Pro-Gly (XGXXPG) motif in extracellular matrix proteins, such as elastin and fibrillin-1 (proteolytic fragments). The best-known form of this motif is the hexapeptide VGVAPG found in (tropo)elastin, but numerous other biologically active peptides conforming to the signature sequence xGxxPG, commonly referred to as elastin peptides, have been reported as agonists. The minimal essential sequence for biological activity is GxxP, with the peptide in P forming a type VIII beta turn. ERC specifically binds to peptides with the motif XGXXPG (herein also identified as GXXP-peptides) through its receptor-binding site located on EBP. The minimal essential sequence for biological activity of peptides with the binding motif is GXXP, which is considered to be fulfilled whenever the peptide in P takes a type VIII beta-turn and the amino acid following the proline (to its N-terminal end) is a glycine. The V14 peptide VVGSPSAQDEASPL, which corresponds to the peptide-binding site in the EBP portion of the receptor, is used to antagonize elastin peptide binding. Various galactosides, such as lactose and chondroitin sulfate, can antagonize ERC-mediated signaling through binding to the beta-galactoside binding site or the carbohydrate recognition domain (CRD) on EBP. Notably, the galactose-binding site, which has a similar affinity for galactose sugars, is also shared by galectins.Galectins are subdivided into prototypic galectins, which contain one carbohydrate recognition domain (CRD) and can form homodimers; tandem-repeat galectins, which contain two different CRDs connected in tandem by a linker of up to 70 amino acids; and the unique chimeric type galectin-3, which consists of a unique tandem repeat of a proline- and galectin-rich short stretch fused to a CRD, whereby the tandem repeat is characterized by a repeated GXXP motif, which is a ligand for ERCs. [Figure 4-2]Figure 4b: Taken from Hinek et al., (Lysosomal sialidase (neuraminidase-1) is targeted to the cell surface in a multiprotein complex that facilitates elastic fiber assembly. J Biol Chem. 2006 Feb 10;281(6):3698-710, which is incorporated herein by reference. For clarity, Hinek et al. is not related to the treatment of hypoglycemia or hyperinsulinism, much less the hyperinsulinemic hypoglycemia that is the subject of this application.) The top panels are representative photomicrographs of cultured ASMCs immunostained with anti-tropoelastin antibodies showing the three stages of elastin production: accumulation of tropoelastin in endosomes and the Golgi apparatus (A), transport in secretory vesicles (B), and initial assembly into microfibrillar scaffolds occurring in grooves on the cell surface (C). The bottom panel shows a proposed model of elastinogenesis in which all three subunits of a cell surface-targeted multiprotein complex (S-gal / EBP, Neu1, and PPCA) act in concert in the process of extracellular assembly of elastic fibers. During the early secretory pathway, a key component of this multiprotein complex binds to the XGXXPG motif of tropoelastin in the endoplasmic reticulum (ER), and the entire complex is then transported to the Golgi compartment and the cell surface, where the action of two other components of this complex ensures the proper release of tropoelastin from its chaperones. During the assembly of tropoelastin into the microfibrillar scaffold, Neu1 (activated by PPCA) removes terminal sialic acid residues from the carbohydrate chains protruding from the microfibrillar glycoprotein. This leads to the exposure of the penultimate galactose sugar, which interacts with the galactolectin site of S-gal / EBP, inducing the release of the transported tropoelastin molecule from its chaperone to the vicinity of the acceptor site on the microfibrillar scaffold.This ensures the proper assembly and mutual orientation of multiple tropoelastin molecules, which is a prerequisite for their final crosslinking (by lysyl oxidase) into insoluble polymerized elastin. During the recycling secretory pathway, S-gal / EBP molecules that have temporarily exited the plasma membrane after unloading their tropoelastin cargo return to the endosomal compartment after reattachment to the plasma membrane-anchored Neu1·PPCA complex. Residual proteolytic fragments of elastin peptides containing the XGXXPG motif can return bound to the ERC. In the recycling endosome, this molecular complex, having released the bound XGXXPG fragments, again binds to new tropoelastin molecules delivered from the endoplasmic reticulum and escorts them (chaperones) to the cell surface. MAGP, microfibril-associated glycoprotein. [Figure 4-3]Figure 4c: Cell surface EBP activity: The XGXXPG peptide binds to EBP (Blanchevoy) and initiates endocytosis of the entire ERC complex (Hinek et al., ibid.), thus at least temporarily internalizing Neu-1 and PPCA, and thus reducing their normal physiological surface activity. This may be a normal aspect of vascular repair. However, chronic excess of circulating XGXXPG peptide fragments, such as those derived from circulating elastin-derived peptides, C-peptide, and galectin-3 peptides, leads to the internalization of Neu-1 and PPCA along with XGXXPG-bound EBP, and thus reduced activity of cell surface-bound Neu-1 and PPCA, leading to various features of metabolic syndrome, such as insulin resistance, increased LDL cholesterol, vascular cell proliferation with intimal thickening, and vasoconstriction with elevated blood pressure (hypertension). Key cell surface Neu-1 activity is inhibited by ERC internalization: Binding of the GXXP peptide to ERC and subsequent internalization of ERC results in reduced Neu-1-induced receptor maturation, which may contribute to the increased insulin resistance due to inactive insulin receptors, reduced LDL uptake and increased circulating LDL levels due to inactive LDL receptors, and increased endothelial proliferation with intimal thickening due to active growth factor receptors, which are hallmarks of human vascular disease in metabolic syndrome. At the cell surface, Neu-1 is involved in desialylation of cell surface receptors, thereby regulating cell surface receptor signaling (Pshezhetsky AV, Ashmarina LI. Desialylation of surface receptors as a new dimension in cell signaling. Biochemistry (Mosc). 2013 Jul;78(7):736-45), among other things, increasing insulin sensitivity and LDL uptake while simultaneously reducing growth factor receptor-induced proliferation through the removal of sialic acid from various growth factor receptors.Neu-1, among other things, regulates the maturation and activation of the insulin receptor (Alghamdi et al., A novel insulin receptor-signaling platform and its link to insulin resistance and type 2 diabetes. Cell Signal. 2014 Jun;26(6):1355-68). DANA, an inhibitor of NEU1, blocks both the interaction between insulin receptor subunits and its activation (Zhang et al., Lipids Health Dis. 2019;18:173). As an integral membrane glycoprotein, the LDL receptor also has terminal sialic acid residues. A significant inhibition of LDL internalization in human fibroblasts was observed after neuraminidase treatment of these receptors, which was associated with a decrease in the number of active cell surface LDL receptor sites (Sprague et al., Stimulation of receptor-mediated low density lipoprotein endocytosis in neuraminidase-treated cultured bovine aortic endothelial cells. J Cell Physiol. 1988 Nov;137(2):251-62). Furthermore, EDP binding to ERCs was found to modulate CD36 sialylation levels, regulating oxidized LDL uptake through Neu-1 (Kaweckie et al., Identification of CD36 as a new interaction partner of membrane NEU1: potential implication in the pro-atherogenic effects of the elastin receptor complex. Cell Mol Life Sci. 2019 Feb;76(4):791-807).With regard to growth factor activity, NEU-1 induces growth factor receptor inactivation (Hinek et al., Neuraminidase-1, a subunit of the cell surface elastin receptor, desialylates and functionally inactivates adjacent receptors interacting with the mitogenic growth factors PDGF-BB and IGF-2. Am J Pathol. 2008 Oct;173(4):1042-56). Briefly, GXXP peptide binding and the subsequent Neu-1-induced effect on receptor maturation can result in increased insulin resistance due to inactivated insulin receptors, reduced LDL uptake and increased circulating LDL levels due to inactivated LDL receptors, and increased endothelial proliferation due to still-inactivated (desialylated) growth factor receptors. It is further known that active, desialylated NEU-1 inhibits angiogenesis, and angiogenic signaling can be enhanced when NEU-1 activity is blocked by treatment with DANA (reported herein in Figure 4) (Lee et al., "NEU1 sialidase regulates the sialylation state of CD31 and disrupts CD31-driven capillary-like tube formation in human lung microvascular endothelia." J Biol Chem. 2014 Mar 28;289(13):9121-35). The activity of other cell surface PPCAs is inhibited by ERC internalization: binding of GXXP peptides to ERCs and subsequent ERC internalization results in a reduction of PPCA-induced proteolysis of circulating vasoconstrictor peptides, leading to increased levels of circulating vasoconstrictor peptides and the associated increase in blood pressure, another hallmark of metabolic syndrome.PPCA expressed on the cell surface regulates blood pressure through the proteolysis of bioactive peptides such as angiotensin (Timur et al., Lysosomal Cathepsin A Plays a Significant Role in the Processing of Endogenous Bioactive Peptides. Front Mol Biosci. 2016 Oct 25;3:68) and endothelin-1 (Seyrantepe et al., Enzymatic activity of lysosomal carboxypeptidase (cathepsin) A is required for proper elastic fiber formation and inactivation of endothelin-1. Circulation. 2008 Apr 15;117(15):1973-81). [Figure 5]Angiogenesis, the formation of capillaries from pre-existing microvessels, is important in many processes. Activation of the elastin receptor complex (ERC), along with elevated blood glucose levels (see Figure 1), accelerates angiogenesis (J Cell Sci. 2005 Jan 15;118(Pt 2):343-56), whereby elevated blood glucose stimulates vascular endothelial growth factor (VEGF) to promote angiogenesis. Microvascular angiogenesis assays of human pulmonary microvascular endothelial cells grown in Matrigel® were performed to determine ERC reactivity and in response to increasing concentrations of GXXP peptides 3, 6, and 8 (VGVAPGVGVAPGVGVAPG, QVGQVELGGGPGAGSLQP, and GAYPGAPGAYPGAPAPGV, derived from the exon 24 region of human elastin, the central region of human C-peptide, and the N-terminal portion of human galectin-3, respectively; Figures 4a, 4b, and 4c). Data shown are total vessel length and total branch length measurements at t = 24 h. *p<0.05, **p<0.01, ***p<0.001. At 4 h, no angiogenic activity of the various peptides was observed. Also, as expected, DANA significantly enhanced the angiogenic effect of peptide 3 at t = 16 h, 24 h, and 40 h (P = 0.48, 0.011, and 0.032, respectively). [Figure 6] EC50 plot with pharmacological profiles of peptides 3, 6, and 8 tested in the assay in Figure 4, showing functionally equivalent ERC reactivity profiles of peptides 3, 6, and 8. [Figure 7]Figure 7a: Alignment of human, mouse, and rat C-peptide amino acid sequences, and the localization of the PG domain and pentapeptide domain therein. Figure 7b: Retrieved from Ohtomo et al., (Differential effects of proinsulin C-peptide fragments on Na+, K+-ATPase activity of renal tubule segments. Diabetologia. 1998 Mar;41(3):287-91. doi: 10.1007 / s001250050905. PMID: 9541168, incorporated herein by reference. For clarity, Ohtomo et al. is not concerned with the treatment of either hypoglycemia or hyperinsulinism, and in particular not with the treatment of hyperinsulinemic hypoglycemia, which is the subject of the present application as described below and above). Glucose metabolism initiates stimulus-secretion coupling in beta cells by increasing the ATP:ADP ratio and closing ATP-regulated potassium (K+ ATP)1 channels. These channels are the primary regulators of beta cell resting membrane potential, and their closure initiates membrane depolarization and opening of voltage-gated L-type Ca2+ channels, thereby increasing cytosolic free Ca2+ concentration ([Ca2+]i) and insulin exocytosis (Ashcroft FM, Harrison DE, Ashcroft SJ. Glucose induces closure of single potassium channels in isolated rat pancreatic beta-cells. Nature. 1984 Nov 29-Dec 5;312(5993):446-8. doi: 10.1038 / 312446a0. PMID: 6095103).Opening of K ATP channels results in beta cell hyperpolarization and inhibits insulin secretion (Koster JC, Permutt MA, Nichols CG. Diabetes and insulin secretion: the ATP-sensitive K channel (K ATP) connection. Diabetes. 2005 Nov;54(11):3065-72. doi: 10.2337 / diabetes.54.11.3065. PMID: 16249427). Na+,K+-ATPase is responsible for maintaining Na+ and K+ gradients across the beta cell plasma membrane. It pumps three Na+ ions in exchange for two K+ ions, generating a purely outward cation flux through the plasma membrane. This makes the pump electrogenic and produces a hyperpolarizing effect on the membrane potential. Consequently, inhibition of Na+,K+-ATPase activity (e.g., by ouabain) causes beta-cell membrane depolarization and Ca2+ influx. Therefore, it has been hypothesized that a decrease in the Na+,K+-ATPase-mediated ion gradient may be a mechanism contributing to insulin secretion (Owada et al., Glucose decreases Na+,K+-ATPase activity in pancreatic beta-cells. An effect mediated via Ca2+-independent phospholipase A2 and protein kinase C-dependent phosphorylation of the alpha-subunit. J Biol Chem. 1999 Jan 22;274(4):2000-8. doi: 10.1074 / jbc.274.4.2000. PMID: 9890957). Thus, ATP-sensitive K+ channels (KATP channels) and Na+K+ATPase channels can sense metabolic changes in pancreatic beta cells, thereby coupling metabolism to modulation of electrical activity and ultimately insulin secretion.In accordance with previous observations by Othomo et al., who observed stimulation of Na+,K+-ATPase activity in rat renal tubule segments by C-peptide (and fragments), it is hypothesized herein that the PG domain XGXXPG or gpxxgx-peptide, as well as its mixed D / L amino acid variants, such as those derived from C-peptide, elastin-peptide, galectin-3, and particularly their fragments, may contribute to the activation of Na+,K+-ATPase channel activity, thereby helping to restore β-cell membrane polarization and inhibit insulin secretion. The amino acid sequence of rat C-peptide 1 is shown in Figures 7a and 7b. Full-length C-peptide was found to elicit Na+,K+-ATPase activity when tested in rat renal tubule segments. Full-length C-peptide activity, tested at 5 × 10 ± 7 mol / L, was set to 100%. The numbers in Figure 7 indicate the percentage of stimulatory activity retained by each fragment, as indicated by the line, relative to the total molecule. All peptide fragments were tested at 5 × 10 ± 7 mol / L. The possibility that C-peptide fragments, such as the PG domain or pentapeptide domain fragments, might exert a direct stimulation of Na+,K+-ATPase, independent of membrane-mediated intracellular activation, was also examined by Othomo et al.

[19] . A purified preparation of Na+,K+-ATPase was incubated with rat C-peptide 1 and its pentapeptide domain fragment EVARQ or the PG-domain peptide ELGGGPEAG. Neither of these peptides produced any measurable activation of the isolated ATPase, indicating that this effect is likely not exerted directly on the ATPase but rather indirectly, possibly via a receptor.Zhong et al. (C-peptide stimulates Na+,K+-ATPase via activation of ERK1 / 2 MAP kinases in human renal tubular cells. CMLS, Cell. Mol. Life Sci. 61, 2782-2790 (2004)) determined the molecular mechanism by which C-peptide stimulates Na+,K+-ATPase in primary human renal tubular cells (HRTCs). Incubating cells with 5 nM human C-peptide for 10 minutes at 37°C stimulated 86Rb+ uptake by 40% (p<0.01). The carboxy-terminal human pentapeptide EGSLQ was found to elicit 57% of the activity of the intact molecule. In parallel with ouabain-sensitive 86Rb+ uptake, C-peptide increased the phosphorylation of the α subunit and the basement membrane (BLM) abundance of the Na+,K+-ATPase α1 and β1 subunits. The increased abundance of Na+,K+-ATPase α1 and β1 subunits in BLM was accompanied by their loss from the endosomal compartment. The effect of C-peptide on Na+,K+-ATPase was ERK1 / 2 dependent in HRTCs. C-peptide-stimulated Na+,K+-ATPase activation, α1 subunit phosphorylation, and translocation of α1 and β1 subunits to BLM were abolished by the MEK1 / 2 inhibitor (20 μM PD98059). C-peptide-stimulated 86Rb+ uptake was also abolished by preincubating HRTCs with a PKC inhibitor (1 μM GF109203X). C-peptide stimulated phosphorylation of the human Na+,K+-ATPase α subunit at the Thr-Pro amino acid motif, which forms a specific ERK substrate. In conclusion, C-peptide and its pentapeptide EGSLQ stimulate sodium pump activity via ERK1 / 2-induced phosphorylation of Thr residues on the α-subunit of Na+,K+-ATPase.Because C-peptide and some of its fragments have been shown to be ligands for the ERC, it is disclosed herein that ERC-mediated activation of Na+,K+-ATPase by C-peptide and related peptides containing the PG domain or pentapeptide domain, or both the PG domain and pentapeptide domain fragments, provides a feedback mechanism that reduces beta-cell insulin exocytosis and insulin secretion. DETAILED DESCRIPTION OF THE INVENTION [Example]

[0043] [Example 1] Existing malaria parasite molecular mimicry patterns are utilized to identify important host receptor binding motifs in silico. The present invention discloses an in silico bioinformatics approach on the malarial proteome of various blood-stage proteins encoded by a multigene family, using publicly available tools and databases to identify best-fit amino acid motifs of the human elastin receptor complex (ERC), which is currently difficult to study for functional expression and molecular characterization. Essentially, the present invention discloses a method for identifying amino acid motifs that bind to mammalian cell receptors (such motifs are also identified herein as chemotactic domains), comprising the following steps in sequential order: (i) selecting a candidate amino acid motif; (ii) determining the frequency of occurrence of each of the candidate amino acid motifs in the proteome of a pathogen, wherein the pathogen is a pathogen capable of infecting a mammal; and iii) identifying candidate amino acid motifs with the highest frequency of occurrence in the pathogen proteome as amino acid motifs that bind to mammalian cell receptors;

[0044] The binding motif for ERC resides in short peptide motifs with the sequence xGxxPx, preferably xGxxPG, the prototype motif VGVAPG of which is herein found to be repeatedly expressed in Plasmodium species that infect primates. These sequences have distinct vascular pathogenicity in humans. The present invention discloses a cluster map of internal dipeptides that show the best fit to ERC, which required in silico examination of 400 dipeptides contained in xGxxPG found in blood-stage proteins of Plasmodium.

[0045] background. The acquisition of the ability to molecularly mimic host-like proteins and therefore their functions has been observed in many bacterial and viral pathogens (Cell. 2006 Feb 24;124(4):767-82). In parasitic protozoa, there are cases where stretches of amino acids present on cell surface proteins encoded by the parasite match regions of host proteins (Mol Biochem Parasitol. 1998 Aug 1;94(2):185-96). In these and other cases, the matches correspond to common amino acid repeats shared between them (Nature. 1988 Sep 1;335(6185):82-5; Mol Biochem Parasitol. 1992 Jul;53(1-2):105-12).

[0046] Malaria parasites (Plasmodium spp.) invade both liver cells and red blood cells (RBCs) in vertebrate hosts. Typically, Plasmodium spp. vary their gene expression profiles depending on the host in which they reside and their developmental stage. During the so-called blood stage, they actively remodel infected RBCs (iRBCs) by exporting various proteins encoded by multigene families and transporting them to the RBC plasma membrane (PLoS Pathog. 2016 Nov 16;12(11):e1005917).

[0047] Such proteins are known to have potential immunomodulatory roles, either as functional homologs of host molecules or by binding to host antigen-presenting cells (Proc Natl Acad Sci U S A. 2001 Sep 11;98(19):10829-32, Nature. 1999 Jul 1;400(6739):73). Paine et al. (Nature. 2008 Oct 9;455(7214):799-803) provided the first observation of such a type in a malarial protein, indicating the acquisition of host peptide sequences that are likely on the surface of infected cells and therefore may interact with the host. Given the wide and diverse genomic repertoire of Plasmodium spp. (Genes (Basel). 2021 May 30;12(6):843), it is likely that many more such acquired host-like protein sequences will be found. Furthermore, given that different Plasmodium species have evolved together with their respective preferred hosts for hundreds of years, the minimal essential sequences for binding to different host receptors of the preferred hosts, as well as the best-fit sequences, are likely enriched in the repertoire. Studying the repertoire of Plasmodium species infecting primates for their preferred xGxxPG motifs will elucidate human ERC preferences. Because rodents have truncated elastin-binding proteins in their ERCs, controlled studies using rodent-infecting Plasmodium species will inevitably yield different results (unpublished). Mapping preferred agonist peptides of the elastin-receptor complex associated with insulin resistance.

[0048] result The xGxxPG motif preference was detected in Plasmodium proteins related to merozoite blood-stage proteins, in association with the development of insulin resistance at the blood stage. Initial results after clustering internal dipeptide preferences at the x1x2 positions of Gx1x2P elastin receptor complex (ERC) motif occurrence in Plasmodium merozoite surface proteins and / or the SERA5 protein showed that A) Plasmodium proteins from the primate-infecting malaria parasites P. vivax (n=13) or P. falciparum (n=11) present in the Uniprot database considered in October 2022; B) Plasmodium proteins from the rodent-infecting malaria parasites P. yoelii yoelii (n=4) or P. berghei (n=3) present in the Uniprot database considered in October 2022 was detected in

[0049] As shown in Figure 1, of the 400 dipeptide sequences analyzed, clustering revealed a preference for the dipeptide VA in the motif GVAP, a preferred chemotaxis domain that binds to the ERC, in the primate-infecting Plasmodium proteins analyzed (1A), and a preference for the dipeptide AV in the motif GAVP in the rodent-infecting Plasmodium proteins analyzed (1B).

[0050] The rodent and primate Plasmodium protein entries in Uniprot were then probed by peptide searches using various peptide motifs containing the GVAP and GAVP motifs found. In particular, GVAP was found in the VGVAPG motif, which was found to be recurrently present in Plasmodium proteins from primate-infecting Plasmodium parasites, and which is also found recurrently in primate elastin (see also Tables 1 and 2).

[0051] [Table 1]

[0052] The VGVAPG peptide is typically considered the prototype ERC-binding peptide and is commercially available (Bachem product number 4010536, described as: chemotactic domain of elastin; VGVAPG stimulated human dermal fibroblast proliferation and was chemotactic for fibroblasts and monocytes. The palmitoylated form is commercially available as a cosmetic ingredient).

[0053] The VGVAPG peptide domain is largely encoded and expressed by exon 24 of primate elastin. In contrast, exon 24 of rodent elastin neither encodes nor expresses VGVAPG (see Table 1), and furthermore, typically lacks the presence of the preferred clustered peptide(s) with alanine (A) and the motif GAVP shown in Figure 1B. Several xGxxPG peptide motifs have been found in mouse exon 24 sequences, which have been found together in 1143 Uniprot entries, 420 of which are elastin protein entries (ranging from primates to rodents, and from fish and reptiles to birds), and in 24 malaria parasite protein entries, although none of these 24 are malaria parasite proteins infecting rodents (see Table 2 for details). Furthermore, the long tandem repeat sequence, VGVAPG (2) For example, VGVAPG (3) , and VGVAPG (6)(VGVAPGVGVAPG, VGVAPGVGVAPGVGVAPG, and VGVAPGVGVAPGVGVAPGVGVAPGVGVAPGVGVAPG, respectively, are found abundantly in primate elastin and in malaria parasite proteins that infect primates, allowing us to conclude that primate Plasmodium species specifically mimic the chemotactic domain of elastin: VGVAPG.

[0054] From the above findings, we conclude that the mouse exon 24 sequence (in contrast to the primate exon 24 sequence with VGVAPG) did not specifically represent the chemotactic domain of elastin available in the malaria parasite protein infecting rodents.

[0055] The structural requirements for ERCs with the elastin-binding protein EBP have been well described in humans (see, e.g., Uniprot entry P16278 isoform 2 and Hinek et al., Am J Hum Genet. 2000 Jul;67(1):23-36; see also Blanchevoye et al (2013) Interaction between the elastin peptide VGVAPG and human elastin binding protein, J Biol Chem 288:1317-28).

[0056] However, little is known about the structural requirements of ERCs with elastin-binding protein EBP in rodents. Herein, we share previously unpublished information that rodent EBP has a significantly shortened N-terminus compared to human EBP, which results in an affected preferred chemotaxis domain of rodent protein. Specifically, rodent EBP is missing the first approximately 80 N-terminal amino acids of the human version, but still contains the typical VVGSPSAQDEASPL binding site of XGXXPG motif peptides. This change is likely to have some effect on the binding of XGXXPG peptides to rodent EBP when compared to human EBP. Furthermore, the human elastin gene has two fewer exons than the mouse, due to the sequential loss of exons 34 and 35 during primate evolution (Szabo et al., (1999) Sequential loss of two neighboring exons of the tropoelastin gene during primate evolution. J. Mol. Evol. 49, 664-671). In addition, although still included in the human gene, exon 22 is never included in human elastin transcripts, but is found in mouse transcripts.

[0057] When the mouse and rat sequences were searched for predicted GAVP motifs found to cluster in rodent malaria parasite proteins, it was exon 22 (and not 34 or 35) that contained the GAVP motif in the preferred chemotaxis domain for binding to the ERC (Table 2).

[0058] Specifically, GAVP was found in the PGAVPG motif, which was found to occur repeatedly in Plasmodium proteins from malaria parasites that infect rodents and which was also found repeatedly in rodent elastin (see also Tables 1 and 2), whereas crossover analysis showed neither the occurrence of PGVAPG tandem repeats in primates nor the occurrence of VGAVPG tandem repeats in rodents. Furthermore, the long tandem repeat sequence AVPG (2) , e.g., AVPG (3) and AVPG (6) (AVPGAVPG, AVPGAVPGAVPG, and AVPGAVPGAVPGAVPGAVPGAVPG, respectively, are found abundantly in rodent elastin and rodent malaria parasite proteins, allowing us to conclude that rodent malaria parasite (Plasmodium) species preferentially and specifically mimic another chemotactic domain of elastin: PGAVPG.)

[0059] [Table 2]

[0060] [Table 3-1]

[0061] [Table 3-2]

[0062] [Table 3-3]

[0063] [Table 4]

[0064] [Example 2] Peptide synthesis and suitable peptides with the required chemotactic domain When describing the composition, structure, and function of proteins or peptides herein, reference is made to amino acids. Amino acid residues are represented herein using the following abbreviations. Also, unless explicitly stated otherwise, the amino acid sequences of peptides and proteins are specified from N-terminus to C-terminus, left to right, with the N-terminus being the first residue: Ala: alanine residue; Asp: aspartic acid residue; Glu: glutamic acid residue; Phe: phenylalanine residue; Gly: glycine residue; His: histidine residue; Lie: isoleucine residue; Lys: lysine residue; Leu: leucine residue; Met: methionine residue; Asn: asparagine residue; Pro: proline residue; Gin: glutamine residue; Arg: arginine residue; Ser: serine residue; Thr: threonine residue; Val: valine residue; Trp: tryptophan residue; Tyr: tyrosine residue; Cys: cysteine residue. Amino acids may also be referred to by their conventional single-letter code abbreviations: A=Ala, T=Thr, V=Val, C=Cys, L=Leu, Y=Tyr, I=Ile, N=Asn, P=Pro, Q=Gln, F=Phe, D=Asp, W=Trp, E=Glu, M=Met, K=Lys, G=Gly, R=Arg, S=Ser, and H=His.

[0065] The modulator peptides for the treatment of insulin secretion-related disorders (ISRD) provided herein preferably have a motif selected from the group at least functionally equivalent to any of the peptides having the motifs VGVAPG, vGVAPG, vGvAPG, vGVaPG, vGvaPG, LGGGPG, lGGGPG, PGAYPG, pGAYPG, pGaYPG, pGAyPG, or pGayPG, which allow targeting to elastin receptors on the surface of cells. Typical examples are peptides such as RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR, PGAIPG, GAVPG, GVLPG, GGVPG, GVVPG, VGVAPG, VVGPGA, VGVAPG, LGGGPG, QGQLPG, PGAYPG, QGVLPA, AQGVAPG, LQGVAPG, AQGVLPG, LQGVLPG, AQGVAPGQ, LQGVAPGQ, AQGVLPGQ, L QGVLPGQ, VGVAPGVGVAPG, LGGGPGAGSLQP and PGAYPGAPAPGV, VGVAPGVGVAPGVGVAPGVGVAPG, QVGQVELGGGPG and GAYPGAPGAYPG, VGVAPGVGVAPGVGVAPG, QVGQVELGGGPGAGSLQP and GAYPGAPGAYPGAPAPGV, VGVAPG, GVAPGV, VAPGVG, APGVGV, PGVGVA, GVGVAP, PGAIPG, LGTIPG, LGGGPGAG, GGGPGAG, GGGP, GGGPG, GAGPG, GGGPE, GAIPG, GGVPG, GVAPG, YTTGKLPYGYGPGG, YGARPGVGVGIP, PGFGAVPGA, GVYPG, GFGPG, GVLPG, GAIPG, PGAIPG, PGAVPG, VGAMPG, VGSLPG, VGMAPG, VPGVG, IPGVG, V GSLPG, VGVAPG, VGVPG, AGAIPG, VPGV, LGITPG, GDNP, GAIP, GKVP, GVQP, GVGP, GFGP, GGIP, GVAP, GIGP, GAGP, GGIPP, GQ FP, GLSP, GGPQP, GPQPG, GGPQPG, GIPP, GGIPP, GIPPA, GGIPPA, EAEDLQVGQVELGGGPGAGSLQPL, DLQVGQVELGGGPGAGSLQP,LVGQVELGGGPGAGSLQPL, QVGQVELGGGPGAGSLQPL, ELGGGPGAGSLQPL, DLQVGQVELGGGPGAGSLQPLALEGSLQ, GQVELGGGPGAGSLQPLALEGSLQ, LGGGPGAGSLQPLALEGSLQ, LVGQVELGGGPGAGSLQPL, LGGGPGAGSLQPL, and LQVGQVELGGGPG, LQVGQVELGG and / or GGPGAGSLQPL, PQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRM, AGGYPGASYPGAYPGQAP PGAYPGQAPPGAYP, MMRVLQAVLPPLPQVVCTYR, GAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPY and GAPAGPLIVPYNLPLPGGVVP, GAYPGAPGAYPGAPAPGV and PGAYPG, PGAYPGQAPPGAYPG, PGAYPGQA and GQAPPGAYPG, vGVAPGVGVAPGVGVApG, vGVAPGVGVAPGEGSLQa, qVGQVELGGGPGAGSLQp, qVGQVELGGGPGEGSLQa, GaYPGAPGAYPGEGSLQav, GaYPGAPGAYPGAPAPGv, vGvAPGVGVAPGVGVapG, qvGQVELGGGPGAGSLqp, GayPGAPGAYPGAPApGv vGvaPGVGVAPGVGvapG, qvGqVELGGGPGAGSlqp, and GaypGAPGAYPGAPapgV, gpavgvgpavgvgpavgv, pqlsgagpggglevqgvq, vgpapagpyagpagpyay, aqlsgegpavgvgpavgv, aqlsgegpggglevqgvq, aqlsgegpyagpagpyay, vGVAPGVGVApG, vGVAPGEGSLQa, qGGGPGAGSLQp, qGGGPGEGSLQa, GaAYPGEGSLQav, GaYPGAAPAPGv, vGvAPGVGVapG, qvGGGPGAGSLqp, GayPGAAPApGv vGvaPGVGvapG,qvGqVELGGGPGqp and GaypGAAPapgV, gpavgvgpavgv, pqlsgegpggglvq, vgpapgpagpyay, aqlsgegpavgv, aqlsgegpggqgvq, aqlsgegpagpyay, or their functional equivalents.

[0066] It should be noted that, with the exception of glycine, all standard alpha amino acids can exist in either of two optical isomers that are mirror images of each other, and these are referred to as L- and D-amino acids. When defining amino acid sequences of peptides in single-letter code herein, lowercase letters indicate D-amino acids, while uppercase letters indicate L-amino acids, with the notable exception of glycine, where either g and / or G indicate the amino acid glycine, which has no optical isomers and therefore no distinction exists between the D- or L-form.

[0067] GxxP motif peptides containing a PG domain ERC binding motif, e.g., VGVAPG, GVAPGV, VAPGVG, APGVGV, PGVGVA, GVGVAP, PGAIPG, LGTIPG, LGGGPGAG, GGGPGAG, GGGP, GGGPG, GAGPG, GGGPE, GAIPG, GGVPG, GVAPG, YTTGKLPYGYGPGG, YGARPGVGVGIP, PGFGAVPGA, GVYPG, GFGPG, GVLPG, GAIPG, PGAIPG, PGAVPG, VGAMPG, VGSLPG, VGMAPG, VPGVG, IPGVG, VGSLPG, VGVAPG, VGVPG, AGA IPG, VPGV, LGITPG, GDNP, GAIP, GKVP, GVQP, GVGP, GFGP, GGIP, GVAP, GIGP, GAGP, GGIPP, GQFP, GLSP, GGPQP, GPQPG, GGPQPG, GIPP, GGIPP, GIPPA, GGIPPA, AQGVAPG, LQGVAPG, AQGVLPG, LQGVLPG, AQGVAPGQ, LQGVAPGQ, AQGVLPGQ, LQGVLPGQ, or their reverse or reverse or reverse or mixed L / D chemotaxis domain variants are synthesized by classical solid-phase synthesis or alternative methods known in the art.V14 peptide, which is the peptide that reproduces the sequence of S-Gal that interacts with elastin peptides with motif GxxP, is obtained from Neosystem (Strasbourg, France). Alternatively, V14 peptide and its variants are synthesized as described herein. The purity of the peptides is confirmed by high performance liquid chromatography and fast atom bombardment mass spectrometry.

[0068] Traditionally, peptides are defined as molecules consisting of 2 to 50 amino acids, while proteins are made up of 50 or more amino acids. Additionally, peptides tend to have less well-defined structures than proteins, which can adopt complex conformations known as secondary, tertiary, and quaternary structures. Functional distinctions can also be made between peptides and proteins. Peptides, however, can be subdivided into peptides with a small number of amino acids (e.g., 2 to 30-50) and polypeptides with a large number of amino acids (more than 50). Proteins are formed from one or more polypeptides joined together. Thus, proteins are essentially very large peptides. In practice, most researchers, as well as this application, use the term peptide to specifically refer to peptides or otherwise relatively short amino acid chains (fewer than 51 amino acids), while the term polypeptide is used to describe proteins or chains of more than 50 or even more amino acids.

[0069] [Example 3] Elastin Receptor Complex (ERC, also identified herein as the elastin receptor) Elastin receptors are involved in leukocyte chemotaxis and activation of matrix metalloproteinases, endothelial cell migration and angiogenesis, and proliferation of fibroblasts and vascular smooth muscle cells. The receptors are activated by (proteolytic) fragments of the extracellular matrix in granulation tissue after tissue injury or inflammation, driving the repair process to tissue repair.

[0070] This receptor consists of an alternatively spliced variant of beta-galactosidase. It is known to bind to any peptide with the hexapeptide X-Gly-XX-Pro-Gly (XGXXPG) motif in extracellular matrix proteins, such as elastin and fibrillin-1 (proteolytic fragments thereof). The most well-known form of this motif is the hexapeptide VGVAPG found in (tropo)elastin, but many other biologically active peptides that fit the signature sequence XGXXPG, commonly referred to as elastin peptides, have been reported as agonists. International Publication No. WO2018141970 identifies various other peptides and proteins, such as C-peptide, particularly its central portion, and galectin-3, particularly its N-terminal fragment, as ligands for ERC. The minimal sequence required for biological activity is GXXP, and this requirement is always met when the peptide in P forms a type VIII beta turn and the amino acid following the proline (to its N-terminal end) is glycine. The V14 peptide VVGSPSAQDEASPL, which corresponds to the lactose and / or peptide-binding site of the receptor, is used to antagonize elastin peptide binding.

[0071] The elastin receptor consists of a complex of elastin-binding protein (EBP) on the cell surface with neuraminidase (Neu-1) and protective protein-cathepsin A (PPCA). Upon binding to its ligand, the complex is internalized into the endosomal compartment of the cell and triggers numerous cellular responses. In mice, exogenous elastin peptides with the motif GXXP enhance atherosclerosis through Neu-1 and regulate insulin resistance due to the interaction between Neu-1 and the insulin receptor. Furthermore, in mice, PPCA is required for elastic fiber assembly and endothelin-1 inactivation, and impaired endothelin-1 activation leads to hypertension.

[0072] A proof-of-concept study was conducted in Aquilo BV by Resiliun BV to evaluate the efficacy of elastin fragments and related C-peptide and galectin-3-derived GXXP peptide fragments on Neu-1-mediated angiogenesis and insulin receptor sensitivity. Active desialylated NEU1 inhibits angiogenesis (J Biol Chem. 2014 Mar 28;289(13):9121-35), and this activity is blocked by treatment with DANA.

[0073] Sialidases, also known as neuraminidases (NEUs), are a family of enzymes responsible for regulating sialic acid expression on cell surfaces by removing sialic acid from endogenous glycoconjugates (Xiao et al., Proc Natl Acad Sci U S A. 2016 Sep 13; 113(37):10304-9). In addition, terminal sialic acid promotes endothelial barrier integrity (Cioffi et al., Am J Physiol Lung Cell Mol Physiol. 2012 May 15; 302(10):L1067-77). The most well-known enzyme in this family is influenza neuraminidase, which was first discovered in the 1950s. Neuraminidases are a large family found in many organisms, including viruses, bacteria, fungi, protozoa, birds, and mammals (Pshezhetsky and Ashmarina, Biochemistry (Mosc). 2013 Jul; 78(7):736-45). Neuraminidases are found in many mammalian organs. NEU1 is most highly expressed in the kidney, pancreas, skeletal muscle, liver, lung, placenta, and brain; NEU2 is found primarily in muscle tissue; NEU3 is most highly expressed in the adrenal gland, skeletal muscle, heart, testis, and thymus; and NEU4 is most highly expressed in the brain, skeletal muscle, heart, pancreas, and liver (Pshezhetsky and Ashmarina, ibid.). In cells, NEU1 is localized to lysosomes and plasma membranes and is involved in exocytosis, immune response, phagocytosis, and elastic fiber assembly. Because neuraminidase 1 (NEU1) cleaves terminal sialic acids from glycoconjugates, it also modulates the structure and activity of cell surface receptors affecting a number of diverse pathways, among others. Dridi et al. (Positive Regulation of Insulin Signaling by Neuraminidase 1, Diabetes 2013 Jul;62(7):2338-2346) show that NEU1 activates the insulin receptor. Insulin signaling is a key event in the regulation of glucose homeostasis, and the signaling cascade is initiated by insulin binding to the cell surface insulin receptor kinase (IRK). This receptor is rapidly activated, autophosphorylates at specific tyrosine residues, and is internalized into endosomes. Activated IRK phosphorylates substrates, including IRS-1-4, which bind to and activate effector molecules such as phosphatidylinositol 3-kinase (PI3K) (reviewed in Taniguchi et al., Critical nodes in signaling pathways: insights into insulin action. Nat Rev Mol Cell Biol 2006;7:85-96). Dridi et al. have shown that Neu-1 desialylates and activates the human insulin receptor kinase (IRK) through desialylation of glycan chains attached to the β-chain of the IRK, thus providing a feedback mechanism for the regulation of glucose uptake. Pretreatment with the Neu-1 inhibitor 2,3-dehydro-2-deoxy-N-acetylneuraminic acid (DANA) significantly reduced IRK desialylation, confirming that Neu1 indeed removes sialic acid residues from the glycan chains of the receptor's β-subunit.

[0074] Angiogenesis is a tightly orchestrated process in which pro- and anti-angiogenic factors are released and bind to their cognate endothelial cell (EC) surface receptors, such as the elastin receptor complex. Pro-angiogenic receptor-ligand interactions are associated with EC disengagement from neighboring ECs, protease-mediated degradation of the underlying EC extracellular matrix (ECM), and release of elastin-derived peptides from the disrupted ECM. These signaling and proteolytic events enable ECs to migrate through the ECM toward pro-angiogenic stimuli generated from elastin-derived peptides, particularly those containing the XGXXPG motif. Proliferative signals propagate to the EC population, which, coupled with ECM remodeling, leads to EC-EC reengagement with capillary-like tube formation. Some of these pro-angiogenic processes involve altered glycosylation patterns and specific carbohydrate-mediated recognition events.

[0075] Endothelial cell surfaces are highly sialylated, and changes in sialylation status affect angiogenesis. Human NEU1 is the most abundant sialidase found in matrix gel systems, which inhibits angiogenesis (Lee et al., Biol Chem. 2014 Mar 28; 289(13):9121-35). It is also the first sialidase described as an angiogenesis regulator (Glanz et al., Eur J Pharmacol. 2019 Jan 5; 842():345-350). The highly sialylated vascular endothelial surface undergoes changes in sialylation when it adopts a migratory / angiogenic phenotype. Cross et al. established the expression of NEU1 sialidase on the cell surface of endothelial cells (ECs) (2012; EU1 and NEU3 sialidase activity expressed in human lung microvascular endothelia. NEU1 restrains endothelial cell migration, whereas NEU3 does not. J. Biol. Chem. 287, 15966-15980). They further found that NEU1 regulates EC capillary-like tube formation on Matrigel substrate in human lung microvascular ECs (HPMECs). Pre-silencing of NEU1 did not alter tube formation, indicating that quiescent Neu-1 is not involved.

[0076] Elastin-induced atherosclerosis depends on sialidase activity and the EBP-cathepsin A-Neu-1 elastin receptor complex (ERC). Therefore, elastin can be used as an enhancer of atherosclerosis (Glanz et al., Eur J Pharmacol. 2019 Jan 5; 842():345-350). The ERC is also essential for the ability of fibroblasts to respond to elastin degradation. The combination of elastin-derived peptides and the ERC activates an intracellular signaling cascade, including activation of extracellular-regulated protein kinase (ERK) 1 / 2 and production of pro-MMP-1 (Parker and Kohler, ACS Chem Biol. 2010 Jan 15; 5(1):35-46). Sialic acid content in the endothelial glycocalyx plays an important role in the development of atherosclerosis, as well as in the regulation of leukocyte and platelet adhesion, mechanotransduction, and endothelial cell uptake of low-density lipoproteins (Frontiers of Pharmacology 2020; 11: 590614).

[0077] [Example 4] Because measuring insulin resistance in vitro is challenging, we instead evaluated the effects of three elastin fragment peptides and the related GXXP peptide on endothelial cell sprouting. Endothelial cells (Hpmec, n = 4) were serum-starved for 24 hours, trypsinized, and seeded on growth factor-reduced Matrigel. Cells were treated with vehicle or increasing concentrations of peptides, and digital images were captured after 4 hours of incubation. A second evaluation was performed 24 hours after incubation. Sprouting was quantified using ImageJ software using the mean total tube length and mean total branch length per field.

[0078] method cell culture Human pulmonary microvascular endothelial cells (Hpmec) were cultured in EBM-2 medium (Lonza, CC-3156) supplemented with 10% FBS and all components included in a bullet kit (Lonza, CC-4147) containing human FGF-B, human recombinant VEGF, human recombinant R3-IGF-1, ascorbic acid, human recombinant EGF, and GA-1000 (gentamicin sulfate-amphotericin). Twenty-four hours prior to the assay, Hpmec were starved in EBM-2 containing 0.5% FBS instead of 10% FBS. To prepare the angiogenesis assay, endothelial cells were washed twice with warm PBS, trypsinized, and resuspended in serum-free EBM-2 medium.

[0079] Angiogenesis assay Matrigel (Fisher Scientific, Landsmeer, The Netherlands #11523550) was diluted 1:1 with serum-free EBM-2 medium and plated (50 μL) in a 96-well plate. After polymerization, cells were combined with experimental compounds and added on top of Matrigel (100 μL). Photographs of the microvascular networks were taken with a 4x objective after 4 and 24 hours of incubation.

[0080] analysis The photographs were analyzed using the Angiogenesis Analyzer plug-in in ImageJ. For analysis, the total vessel length and total branch length of peptides 3, 6, and 8, which are 18-mer GXXP peptide fragments (VGVAPGVGVAPGVGVAPG, QVGQVELGGGPGAGSLQP, and GAYPGAPGAYPGAPAPGV) obtained from the exon 24 region of human elastin, the central portion of human C-peptide, and the N-terminal portion of galectin-3, respectively, were examined.

[0081] Results and Discussion See Figure 4. Microvascular angiogenesis assay of human pulmonary microvascular endothelial cells grown in Matrigel in response to increasing concentrations of elastin fragment peptides 3, 6, and 8 (Figures 4a, 4b, and 4c, respectively). Data shown are total tube length and total branch length measurements at t = 24 h. *p<0.05, **p<0.01, ***p<0.001. No different results were obtained at 4 h. Also, as expected, DANA significantly enhanced the angiogenic effect of peptide 3 at t = 16 h, 24 h, and 40 h (P = 0.48, 0.011, and 0.032, respectively).

[0082] It is interesting to note that a biphasic concentration-response curve was evident for these biologically active peptides, with maximal activity typically occurring at approximately 4,000–40,000 nM, and less potent at higher concentrations (400,000 nM). Statistical analysis supports this conclusion. Three possible explanations for this biphasic nature are suggested: a) desensitization / internalization of the target receptor resulting in loss of activity at higher concentrations; b) the presence of a second receptor with lower affinity and functionally opposing activity; or c) toxicity of the peptide at the highest concentrations. Because the biphasic nature was strongest for the peptides with the most potent functional activity, explanation A appears to be the most likely. Toxic activity would be expected to be more ubiquitous, while functional activity of a second receptor is unlikely given the highly structurally diverse nature of the peptides, which reduces the likelihood of such a common off-target effect.

[0083] Na + ,K +Na+,K(+)-ATPase is a ubiquitous membrane enzyme that allows the export of three sodium ions from cells and two potassium ions from the extracellular fluid. Na+,K(+)-ATPase activity is reduced in the erythrocyte membranes of individuals with type 1 diabetes, regardless of the degree of diabetic control. It is less impaired or even normal in those with type 2 diabetes. Vague et al. (Na+,K(+)-ATPase, and diabetes. Exp Diabesity Res. 2004 Jan-Mar;5(1):37-50. doi: 10.1080 / 15438600490424514. PMID: 15198370; PMCID: PMC2478626) showed that Na+,K(+)-ATPase activity in red blood cells from type 2 diabetic patients was strongly correlated with circulating C-peptide levels in both non-insulin-treated patients (where C-peptide concentrations mirror those of insulin) and insulin-treated patients. Short-term C-peptide infusion in type 1 diabetic patients restores normal Na+,K(+)-ATPase activity. Pancreatic islet transplantation, which restores endogenous C-peptide secretion, enhances Na+,K(+)-ATPase activity proportional to the increase in C-peptide. This C-peptide effect is not indirect. Indeed, incubation of diabetic erythrocytes with physiological concentrations of C-peptide results in an increase in Na+,K(+)-ATPase activity. In isolated rat proximal tubules or medullary thick ascending limbs of the kidney, C-peptide stimulates Na+,K(+)-ATPase activity in a dose-dependent manner.

[0084] [Example 5] PG domain C-peptide, a cleavage product of the proinsulin molecule, has long been considered biologically inactive and merely serves as a surrogate marker of insulin release. Recent discoveries indicate both physiological and protective roles for C-peptide when administered to individuals with type 1 diabetes. Data indicate that C-peptide appears to bind to cell surface receptors at nanomolar concentrations, most likely G protein-coupled. C-peptide binding initiates multiple cellular effects, including elevation of intracellular calcium, increased PI-3 kinase activity, and Na+. + / K + It induces ATPase stimulation, increased eNOS transcription, and activation of the MAPK signaling pathway. These cell signaling actions have been studied in multiple cell types derived from multiple tissues. Overall, these observations raise the possibility that C-peptide may serve as a promising therapeutic agent for the treatment or prevention of long-term complications associated with diabetes. Clearly, if PG domain C-peptide has peptide hormone-like effects, the existence of a receptor must be postulated. In fact, 125 Specific and displaceable binding of I-labeled C-peptide was first demonstrated by Flatt et al. (Biosci Rep. 1986 Feb;6(2):193-9), who derived a curved Scatchard plot for specific C-peptide binding to pancreatic islet B cells.

[0085] Subsequently, using the C-peptide enantiomer with reversed sequence and all D amino acids, Ido et al. (Science. 1997 Jul 25; 277(5325):563-6) demonstrated that Na + ,K +These results suggest that C-peptide-induced improvements in neuronal function and vascular permeability in diabetes through increased C-ATPase activity were not due to C-peptide binding to its receptor in a stereospecific manner. Rather, we hypothesized that the biological activity of C-peptide relied on poorly defined membrane interactions resulting from structural features related to the C-peptide sequence but independent of its orientation or chirality. The central sequence of C-peptide, which is largely conserved and contains a high proportion of nonpolar amino acids adjacent to the C16 proline, was responsible for this activity.

[0086] Other investigators have also reported that Na+ in rat renal tubule segments + ,K +Using α-ATPase activity as a readout, the biological activity of C-peptide fragments has been tested (Diabetologia. 1998 Mar; 41(3):287-91). Two segments of C-peptide demonstrated functional importance. EVARQ, a carboxy-terminal pentapeptide of rat C-peptide, elicited 100% of the activity of intact C-peptide, whereas the remainder of the molecule, lacking the five amino acid terminal sequence, was completely inactive. In this rat system, the terminal pentapeptide of human C-peptide (EGSLQ) elicited 75% of the activity. Notably, the glutamic acid at position 1 and the glutamine at position 5 are generally conserved in mammals. Similar well-defined C-terminal functional sequences have also been found in gastrin and cholecystokinin. Overall, the behavior of the C-terminal pentapeptides in these studies was typical of peptide ligands interacting with specific receptors. In contrast, sequences from the central region of some C-peptides were able to partially reproduce the activity of the intact molecule but displayed rather different properties. Activity associated with this region was absent for des-(27-31)-C-peptide, and sequences containing several unnatural D amino acids showed some activity. The activity of these segments was reduced when their length exceeded nine amino acids. This behavior, previously not thought to be reminiscent of a peptide-receptor interaction, resembled the nonspecific type of interaction of C-peptides with the plasma membrane postulated by Ido et al. (ibid.).

[0087] How the balance of activity provided by these two domain regions of the C-peptide molecule manifests itself in vivo is not fully understood at present. Very recent studies suggest that efficient activation of signaling pathways requires the presence of conserved glutamic acid residues at positions 3, 11, and 27 of the C-peptide as well as helix-promoting residues in the N-terminal segment (Henriksson et al., Cell Mol Life Sci. 2005 Aug; 62(15):1772-8). Together with data related to its N-terminus, which are not discussed here, the currently emerging picture of the structure-activity relationship of the C-peptide molecule is one of a tripartite structure, with the terminal portions involved in functional interactions and the central portion of the PG-domain forming the ERC-binding segment.

[0088] The present invention provides another emerging class of drugs: peptide modulators of mTOR that act as autophagy-inhibiting compounds for use in inducing the activity required to establish glycemic control that counteracts hyperinsulinism, even in patients with otherwise adequate glycemic control. When autophagy is inhibited and mTOR is activated following amino acid consumption by adipocytes or muscle cells derived from these peptide modulators provided herein, sustained stimulation of downstream S6K1 increases IRS1 Ser307 phosphorylation. This reduces their activity and insulin responsiveness, thereby rendering adipocytes or muscle cells insulin-resistant. Similarly, treatment of endothelial cells, such as vascular endothelial beta cells, with these peptide modulators of mTOR provided herein sustained stimulation of downstream S6K1 increases IRS1 Ser307 phosphorylation and induces cell proliferation. This proliferation can be experimentally measured, for example, by detecting Ki-67 activity in these cells. Provided is an autophagy-inhibiting mTOR peptide modulator for use in inducing increased blood glucose in the prevention or treatment of hyperinsulinism in a subject, wherein the modulator comprises a source of amino acids, preferably peptides, and the amino acids are selected from the group consisting of alanine (single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N) for at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95%.To target such peptide modulators to cells, the peptides are provided with the xGxxPG motif, as discussed above.In a preferred embodiment, there is provided an autophagy-inhibiting mTOR modulator for use in inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, said modulator comprising a source of amino acids, preferably peptides, wherein at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). In a preferred embodiment, there is provided an autophagy-inhibiting mTOR modulator for use in inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, said modulator comprising a source of amino acids, preferably peptides, wherein at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), and leucine (L). In a preferred embodiment, there is provided an autophagy-inhibiting mTOR modulator for use in inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, said modulator comprising a source of amino acids, preferably peptides, wherein at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and valine (V).In a preferred embodiment, there is provided an autophagy-inhibiting mTOR modulator for use in inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, said modulator comprising a source of amino acids, preferably peptides, wherein at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and leucine (L). In a preferred embodiment, there is provided an autophagy-inhibiting mTOR modulator for use in inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, said modulator comprising a source of amino acids, preferably peptides, wherein at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), leucine (L), and proline (P). In a preferred embodiment, an autophagy-inhibiting mTOR modulator is provided for use in inducing increased blood glucose in the prevention or treatment of hyperinsulinism in a subject, the modulator comprising a source of amino acids, preferably peptides, wherein at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group consisting of alanine (single-letter code: A), valine (V), leucine (L), and proline (P). Use of such an autophagy-inhibiting mTOR peptide modulator according to the present invention is particularly preferred when the subject is also treated to achieve or maintain glycemic control, particularly when the subject is also treated with insulin to achieve or maintain glycemic control.Provided is a method for identifying a source of preferably L-proteinogenic amino acids, preferably peptides, that can induce an increase in blood glucose in preventing or treating hyperinsulinism in a subject, the method comprising the steps of: providing a cell with a peptide containing L-proteinogenic amino acids, wherein at least 50% of the amino acids are selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N); and determining the angiogenic activity in a sprouting assay.The angiogenic activity can be determined by assessing capillary tube formation and capillary branch formation as provided herein. It is preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N).It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). It is even more preferred that the amino acids are selected from the group alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) for at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%.It is more preferred that at least 50%, more preferably at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of leucine (single letter code: L), glutamine (Q), glycine (G), and valine (V). It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and valine (V). It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and leucine (L). It is even more preferred that the amino acids are selected from the group of alanine (single letter code: A), valine (V), leucine (L), and proline (P) for at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95%. The present invention also provides a method for inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, comprising providing to cells a source of amino acids, preferably L-proteinogenic, wherein the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N) for at least 50%, more preferably at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95%.It is more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P).It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L). It is even more preferred that the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and valine (V) for at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%. It is even more preferred that the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and valine (V) for at least 50%, more preferably at least 60%, more preferably at least 80%, more preferably at least 85%. It is more preferred that at least 90%, more preferably at least 95%, of the amino acids are selected from the group consisting of leucine (single letter code: L), glutamine (Q), glycine (G), and valine (V). It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), and leucine (L). It is even more preferred that at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group consisting of alanine (single letter code: A), valine (V), leucine (L), and proline (P). The present invention also provides a method for inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, comprising the step of providing to cells a source of amino acids, preferably L-proteinogenic, wherein at least 50% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N), and more preferably at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), proline (P), arginine (R), and asparagine (N).It is more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P).It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L). It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and valine (V).It is even more preferred that at least 50%, more preferably at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of leucine (single letter code: L), glutamine (Q), glycine (G), and valine (V).It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and leucine (L). It is even more preferred that the amino acids are selected from the group of alanine (single letter code: A), valine (V), leucine (L), and proline (P) for at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%.The present invention also provides a method for inducing increased blood glucose in the prevention or treatment of hyperinsulinism in a subject, comprising providing cells with a source of amino acids, preferably L-proteinogenic, wherein at least 50% of the amino acids are selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N). More preferably, at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), and leucine (L).It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group alanine (single letter code: A), glutamine (Q), glycine (G), and valine (V).It is even more preferred that at least 50%, more preferably at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group leucine (single letter code: L), glutamine (Q), glycine (G), and valine (V).It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and leucine (L).It is even more preferred that at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), valine (V), leucine (L), and proline (P). The present invention also provides a method for inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, comprising the step of providing to cells a source of amino acids, preferably L-proteinogenic, wherein at least 50% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N). The present invention also provides a method for inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, comprising providing a peptide to a cell, preferably as a source of L-proteinogenic amino acids, wherein the peptide consists of at least 50%, more preferably at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of amino acids selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), proline (P), arginine (R), and asparagine (N).It is more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P).It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L). It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and valine (V).It is even more preferred that at least 50%, more preferably at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of leucine (single letter code: L), glutamine (Q), glycine (G), and valine (V).It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and leucine (L). It is even more preferred that the amino acids are selected from the group of alanine (single letter code: A), valine (V), leucine (L), and proline (P) for at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%.The present invention also provides a method for inducing increased blood glucose in the prevention or treatment of hyperinsulinism in a subject, comprising providing cells with a source of amino acids, preferably L-proteinogenic, wherein at least 50% of the amino acids are selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N). More preferably, at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95% of the amino acids are selected from the group alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L). It is even more preferred that at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and valine (V). It is even more preferred that at least 50%, more preferably at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of leucine (single letter code: L), glutamine (Q), glycine (G), and valine (V). It is even more preferred that at least 60%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and leucine (L). It is even more preferred that the amino acids are selected from the group of alanine (single letter code: A), valine (V), leucine (L), and proline (P) for at least 60%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95%. The present invention also provides a method for inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, comprising providing to cells a source of, preferably L-proteinogenic amino acids, wherein the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N) for at least 50%. The present invention also provides a method of treatment according to the present invention, wherein the source of, preferably L-proteinogenic amino acids, preferably peptides, is identifiable by the methods provided herein.The present invention specifically provides a method of treatment according to the present invention, in which the source or peptide has an amino acid sequence derived from a peptide or protein that has been shown to have or is suspected to have an activity. Often, in the proteome of an organism, such an angiogenic sequence in a protein is located adjacent to or between arginine (R) or lysine (K) residues located at the N- and C-terminus, and an enzyme, such as a converting enzyme, can cleave the amino acid sequence that can induce an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, making it possible to use it as an autophagy-inhibiting mTOR peptide modulator. It is preferable to correlate the use of the peptide with the species of cells in which it is used. For example, when a peptide is used as an autophagy-inhibiting mTOR peptide modulator in human cells, it is preferable that the peptide is derived from the human proteome. Similarly, in cells of another species, such as species X, it is preferable to use a peptide derived from the proteome of species X. In a preferred embodiment, the autophagy-inhibiting peptide has an amino acid sequence derived from chorionic gonadotropin (CG), a protein involved in activity during pregnancy, preferably derived from the beta chain of CG, preferably loop 2 of the beta chain. Human CG (hCG) has the amino acid sequence MTRVLQGVLPALPQVVCNYR, which can induce an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, where the core sequence is located between the arginine (R) residues at the N- and C-termini. Furthermore, this core contains the ERC-binding motif xGxxPx. Splitting this motif facilitates the use of hCG derivatives LQGV, VLPALP AQGV, LAGV, LQAV, LQGA, ALPALP, VAPALP, VLAALP, VLPAAP, and VLPALA as autophagy-inhibiting peptide modulators, particularly in combination with fast- and short-acting insulin preparations to exert both blood glucose induction and blood glucose control.For example, the peptide VLQGVLPALPQVV is more suitable for use in combination with intermediate- and long-acting insulin preparations, where it is released more slowly and in a manner more in line with the release rate of insulin. In another preferred embodiment, the autophagy-inhibiting peptide is provided, wherein the peptide has an amino acid sequence derived from C-peptide, which can induce an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject. In the preproinsulin molecule, human C-peptide has the sequence RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR, and the core sequence is actually flanked by arginine (R) or lysine (K) residues. Furthermore, this core contains the ERC-binding motif xGxxPG. Because ERC-binding motifs are involved in coacervation through oligomerization of peptides with these sequences, such autophagy-inhibitory peptides with ERC-binding motifs typically have a slower release rate when injected and are typically more useful and preferred in situations where such slow release is desired, such as in combination formulations with medium- or more preferably long-acting insulin. Coacervation typically involves the aggregation of colloidal droplets of peptides held together by electrostatic attraction, which explains the slow release properties of such ERC-motif-containing peptides when injected, at least compared to peptides that do not have ERC motifs and do not exhibit coacervation (which typically have faster release characteristics).Preferred C-peptide fragment peptides for use as autophagy-inhibiting mTOR peptide modulators in the treatment of angiogenic dysfunction, particularly in the setting of C-peptide deficiency, are preferably isolated and / or synthetic, and preferably non-PEGylated. C-peptide fragments EAEDLQVGQVELGGGPGAGSLQPL, DLQVGQVELGGGPGAGSLQP, LVGQVELGGGPGAGSLQPL, QVGQVELGGGPGAGSLQPL, ELGGGPGAGSLQPL, DLQVGQVELGGGPGAGSLQPLALEGSLQ, GQVELGGGPGAGSLQPLALEGSLQ, and LGGGPGAGSLQPLALEGSLQ, LVGQVELGGGPGAGSLQPL, LGGGPGAGSLQPL, and LQVGQVELGGGPG, and functional equivalents all containing the xGxxPG motif, are therefore most suitable and preferred for inclusion in or combination with intermediate or long-acting insulins. LQVGQVELGG and / or GGPGAGSLQPL, and functional equivalents that do not contain the xGxxPG motif, are therefore most suitable and preferred for inclusion in or combination with fast- or short-acting insulins.

[0089] In another preferred embodiment, the autophagy-inhibiting peptide has an amino acid sequence derived from galectin-3, which can induce an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject. The mature N-terminal fragment of human galectin-3, PQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRM, is typically characterized by multiple ERC-binding motifs, xGxxPG, and the core sequence can also be released by hydrolysis. Typically (Pineda et al., Trypanosoma cruzi cleaves galectin-3 N-terminal domain to suppress its innate microbicidal activity. Clin Exp Immunol. 2020 Feb;199(2):216-229), the human pathogen T. cruzi not only binds but also hydrolyzes human N-terminal galectin-3. Remarkably, this mechanism prevents galectin-3-mediated parasite death, suggesting that T. cruzi may have evolved a complex strategy to modulate galectin-3 function to successfully infect, survive, and reproduce in its mammalian host. Indeed, nonpathogenic T. rangeli binds galectin-3 but does not modify its protein structure. Thus, T. cruzi cleaves the N-terminal collagen-like domain, rendering C-terminal galectin-3 unable to oligomerize by coacervation, allowing the N-terminal fragment bearing the motif xGxxPG to interact with ERC and modulate mTOR in a manner favorable to the parasite.Specifically, the N-terminal sequences of the three fragments obtained through hydrolysis of the parasite are band 1: AGGYPGASYPG, band 2: GAPGAYPGAP, and band 3: GAPAGPLIVP, demonstrating the mTOR modulation characteristics of the N-terminal peptide fragments of galectin-3 derived from AGGYPGASYPGAYPGQAPPGAYPGQAPPGAYP, GAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPY, and GAPAGPLIVPYNLPLPGGVVP in humans. For example, the fragments GAYPGAPGAYPGAPAPGV and PGAYPG possess core activity as shown herein, making PGAYPGQAPPGAYPG, PGAYPGQA, and GQAPPGAYPG useful autophagy-inhibitory peptides for use in humans, particularly when included in or with intermediate- or long-acting insulin. The present invention also provides a method of treatment and its use, wherein the peptide has an amino acid sequence derived from lutropin (LH), a protein involved in monthly activity in women, and preferably the peptide is derived from the beta chain of LH, preferably loop 2 of the beta chain. The beta-2 loop of human LH (hLH) has an amino acid sequence, MMRVLQAVLPPLPQVVCTYR, which can induce an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, where the core sequence is located between the arginine (R) residues located at the N- and C-termini. Furthermore, this core does not contain the ERC-binding motif xGxxPx, facilitating the use of LH-derived sequences, such as VLQAVLPPLPQVV, VLQAVLP, VLQA, LQAVLP, LQAV, PLPQVV, and PPLQV, and further derivatives, as mTOR modulators in rapid- and short-acting insulin formulations. The present invention also provides methods of treatment and uses thereof, wherein the peptide has an amino acid sequence derived from extracellular matrix proteins (ECM). ECM proteins contain a rich source of autophagy-inhibiting amino acids and are useful as templates for designing autophagy-inhibiting peptides.Proline, as a substrate, is stored in elastin and collagen in the extracellular matrix, connective tissue, and bone, and is rapidly released from this reservoir by the sequential actions of matrix metalloproteinases, peptidases, elastases, and prolidases. As the only proteinogenic secondary amino acid, proline has special biological effects, acting as a regulator of all protein-protein interactions and responses to metabolic stress, initiating various downstream metabolic activities, including autophagy (Kadowaki et al. Nutrient control of macroautophagy in mammalian cells. Mol Aspects Med. 2006 Oct-Dec;27(5-6):426-43). The present invention also provides a treatment method and use thereof, in which the extracellular matrix protein is elastin. Exemplary core activities are represented herein by the peptides VGVAPG and VGVAPGVGVAPGVGVAPG, both derived from the exon 24 region of human elastin. The present invention also provides a method of treatment according to the present invention and uses therein, wherein the extracellular matrix protein is collagen, which is rich in potentially useful PGP sequences. Some collagens typically contain a core peptide flanked by R or K, such as RRAQGVPQIAVLVTHR (an example from human COL6A5 is shown here), and the core sequence AQGVPQIAVLV, as well as derivatives such as AQGVPQ, AQGVPQI, AQGVPQIA, GVPQIAVLV, PQIAVLV, IAVLV, and others, have been identified.Further such sequences can be found in collagen stretches such as (in human COL6A5) RGAPGQYGEKGFPGDPGNPGQNNNIKGQKGSKGEQGRQGRSGQKGVQGSPSSRGSRGREGQRGLRGVSGEPGNPGPTGTLGAEGLQGPQGSQGNPGRKGEKGSQGQKGPQGSPGLMGAKGSTGRPGLLGKKGEPGLPGDLGPVGQTGQRGRQGDSGIPGYGQMGRKGVKGPRGFPGDAGQK, which have been found to show repeated occurrences of a core peptide sequence flanked by R or K repeats, from which desirable mTOR peptide modulators can be selected. Another angiogenic protein for which peptides are provided herein is human alpha-fetoprotein, for which a peptide with the sequence KDLCQAQGVALQTMK has been observed, containing the antigenic core QAQGVALQ. Derivatives AQGV, AQGVA, AQGVAL, QGVALQ, and GVALQ have been found, all of which are useful as autophagy-inhibiting mTOR peptide modulators, preferably in combination with fast- or short-acting insulin. Typically, autophagy inhibition by dipeptide modulators of mTOR AQ has recently been shown to have improved modulation characteristics in piglets compared with amino acids A+Q (Zhang et al., Alanyl-glutamine supplementation regulates mTOR and ubiquitin proteasome proteolysis signaling pathways in piglets. Nutrition. 2016 Oct;32(10):1123-31).

[0090] Meanwhile, as provided herein, an autophagy-inhibiting mTOR peptide modulator is provided for the purpose of blood glucose control. Also provided is a growth factor formulation (preferably a formulation herein, including a pharmaceutical formulation or pharmaceutical composition), such as an insulin formulation, an IGF formulation, a PDGF formulation, a VEGF formulation, or another growth factor formulation useful in the treatment of vascular diseases, particularly blood glucose control. Also provided is a formulation of a growth factor and an autophagy-inhibiting mTOR modulator, wherein the modulator comprises a source of amino acids, preferably peptides, and the amino acids are selected from the group consisting of alanine (single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N), for at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95%. In a preferred embodiment, an insulin formulation having an autophagy-inhibiting mTOR modulator is provided, wherein the modulator comprises a source of amino acids, preferably peptides, wherein at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). In a preferred embodiment, an insulin formulation having an autophagy-inhibiting mTOR modulator is provided, wherein the modulator comprises a source of amino acids, preferably peptides, wherein at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), and leucine (L).In a preferred embodiment, an insulin formulation having an autophagy-inhibiting mTOR modulator is provided, wherein the modulator comprises a source of amino acids, preferably a peptide, and the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and valine (V) for at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95%. In a preferred embodiment, an insulin formulation having an autophagy-inhibiting mTOR modulator is provided, wherein the modulator comprises a source of amino acids, preferably a peptide, and the amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), and leucine (L) for at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95%. In a preferred embodiment, an insulin formulation having an autophagy-inhibiting mTOR modulator is provided, wherein the modulator comprises a source of amino acids, preferably peptides, wherein at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group consisting of alanine (single-letter code: A), glutamine (Q), leucine (L), and proline (P). In a preferred embodiment, an insulin formulation having an autophagy-inhibiting mTOR modulator is provided, wherein the modulator comprises a source of amino acids, preferably peptides, wherein at least 50%, preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and more preferably at least 95% of the amino acids are selected from the group consisting of alanine (single-letter code: A), valine (V), leucine (L), and proline (P).Such autophagy-inhibiting mTOR peptide modulators and their use in growth factor formulations, as provided herein, typically include peptides and / or amino acid sources that target the nutrient-sensing system of the mechanistic target of rapamycin (mTOR), particularly the amino acid sensing system, to inhibit autophagy and, therefore, induce its activity in patients in need thereof. Typically, in cases where glycemic control is achieved through the use of insulin formulations, insulin requirements (units / kg / day) are determined based on the patient's age, weight, and remaining pancreatic insulin activity. Patients typically require a total daily insulin dose of 0.4-1.0 units / kg / day, with a typical starting dose for metabolically stable patients being 0.5 units / kg / day. However, as previously mentioned, glycemic control using insulin does not replace C-peptide deficiency.

[0091] Typically, for purposes of this disclosure, a peptide is defined herein as having 50 or fewer amino acids, and a protein is defined as having more than 50 amino acids. An autophagy-inhibiting mTOR peptide modulator is defined herein as a linear, branched, or cyclic string of no more than 50 amino acids, comprising a peptide sequence in which at least 50%, more preferably at least 75%, and most preferably 100% of the amino acids are selected from the group consisting of the autophagy-inhibiting amino acids alanine (single-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), proline (P), isoleucine (I), arginine (R), and asparagine (N). The molecular mode of action (MoA) of this group of peptides is not dependent on their exact sequence. Instead, these constitutive amino acids provide an autophagy-inhibiting signal to the mTOR nutrient-sensing system, resulting in the inhibition of autophagy and protein synthesis associated with disease resolution.

[0092] As discussed in Sciarretta et al. (New Insights into the Role of mTOR Signaling in the Cardiovascular System. Circ Res. 2018 Feb 2;122(3):489-505), the mechanistic (previously called mammalian) target of rapamycin (mTOR) is an atypical serine / threonine kinase belonging to the phosphoinositide kinase-related kinase (PIKK) family. It is an evolutionarily conserved protein that plays a central role in regulating cellular physiology, metabolism, and stress response. It has been referred to by some as a cellular master switch. mTOR interacts with specific adaptor proteins to form two distinct macromolecular complexes, named mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Previous paradigms in mTOR biology included the involvement of mTOR in regulating protein synthesis, cell growth, and ribosome biogenesis, as well as the sensing and integration of various upstream inputs, such as growth factors, nutrients, amino acids, starvation, and hypoxia. However, it is now known that the mTOR pathway also controls other important cellular processes, including cell survival, mitochondrial biogenesis and function, lipid synthesis, and autophagy. The mTORC2 signaling network is less well characterized than that of mTORC1. Several studies suggest that the activities of mTORC1 and mTORC2 are strongly interconnected, and that mTORC2 is less sensitive to acute rapamycin treatment than mTORC1. Previous studies have also shown that mTORC2 is involved in regulating cell survival, growth, and proliferation, and controls cellular architecture and polarity. Amino acids are typically not only the building blocks of proteins but also signaling molecules, as well as regulators of gene expression, metabolic processes, and developmental changes in the body. Therefore, amino acids and their metabolism play important roles in health and disease. Substantial evidence suggests that amino acids play a fundamental role in the vascular system.While amino acids serve as basic building blocks for protein synthesis and constitute an important energy source, select groups have been extensively studied in the context of vascular disease. mTOR, particularly mTORC1, is a crucial kinase that regulates cell growth and proliferation by sensing nutrients such as amino acids and glucose. Alanine and glutamine are the most abundant amino acids circulating in the blood. Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signaling, which controls major metabolic pathways. Among other pathways, autophagy is responsible for the degradation of long-term proteins and the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in predisposition to several pathologies. Notably, only certain amino acids can modulate autophagy, and their function is highly cell-specific.

[0093] Discovery in the malaria parasite Plasmodium identifies the VGVAPG(n) motif, which is involved in producing insulin resistance in primates, particularly humans.

[0094] As discussed by Eze et al. (Asymptomatic Plasmodium infection and glycemic control in adults: Results from a population-based survey in south-central Côte d'Ivoire, Diabetes Research and Clinical Practice, Volume 156, 2019, 107845), the inflammatory response to repeated infection with malaria parasites (Plasmodium species) can result in persistent physiological changes in insulin sensitivity and increased susceptibility to diabetes. Plasmodium infection (PI) induces systemic inflammatory pathways that are also characteristic of type 2 diabetes. Proinflammatory blood markers, such as circulating C-reactive protein, IL-1β, IL-6, IL-8, and IL-10, have been associated with both malaria severity and insulin resistance. IL-6 levels were higher in adults from malaria endemic settings than in their western counterparts, adjusted for age. Intrauterine malaria is thought to induce physiological pathways in the fetus that lead to insulin resistance and impaired fasting glucose (FG) in adolescence.

[0095] Abidha, CA, Amoako, YA, Nyamekye, RK et al. (Fasting blood glucose in a Ghanaian adult is causally affected by malaria parasite load: a mechanistic case study using convergent cross-mapping. Malar J 21, 93 (2022)) conducted a study aimed at testing the hypothesis that increased fasting blood glucose (FG) promotes parasite growth, as reflected by increased parasite density. However, the opposite was found. Seven adults without DM were recruited from rural Ghana to determine the relationship between FG and malaria parasite load. Socioeconomic data were recorded using a questionnaire-based survey. Over a 6-week period, FG and Plasmodium sp. infection were measured in peripheral blood samples by photometric and polymerase chain reaction (PCR) assays, respectively. Daily physical activity and weather data were documented using smartphone recording. We applied experimental dynamic modeling to the complex natural system of homeostatic glucose regulation and the life cycle of the malaria parasite Plasmodium sp. At baseline, four men and three women (median age 33 years, interquartile range 30-48) showed a median FG of 5.5 (5.1-6.0 mmol / L), and one participant had an asymptomatic Plasmodium sp. infection (parasite density: 240 / µL). In this participant, convergent cross-mapping (CCM) over 34 consecutive days showed that FG (and therefore insulin resistance) was necessarily influenced by parasite density (p<0.02), but no inverse relationship was found (p>0.05).

[0096] In addition to the embodiments described above, further embodiments are disclosed herein below.

[0097] Further embodiment 1. 1. A method for treating a human subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia, comprising treating the subject with a modulator or pharmaceutical composition provided with a molecule comprising an elastin receptor binding motif that is at least functionally equivalent to a PG-domain peptide having the motif XGXXPG (where G represents the amino acid glycine, P represents proline, and X represents any amino acid). Further embodiment 2. A method for treating a human subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia, comprising treating the subject with a modulator or pharmaceutical composition provided with a molecule having a Na+K+ATPase activation motif that is at least functionally equivalent to a pentapeptide domain peptide having the motif EXXXQ (E represents the amino acid glutamic acid, Q represents glutamine, and X represents any amino acid). Further embodiment 3. 1. A method for treating a human subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia, comprising treating the subject with a modulator or pharmaceutical composition comprising a molecule comprising an elastin receptor binding motif at least functionally equivalent to a PG-domain peptide having the motif XGXXPG (where G represents the amino acid glycine, P represents proline, and X represents any amino acid), and further comprising a molecule having a Na+K+ ATP-ase activation domain at least functionally equivalent to a pentapeptide domain motif EXXXQ (where E represents the amino acid glutamic acid, G represents glutamine, and X represents any amino acid). Further embodiment 4. 4. The method of further embodiment 1 or 3, wherein said molecule comprises a peptide having a motif selected from the group XGXXPG, xGXXPG, xGxXPG, xGXxPG, and xGxxPG. Further embodiment 5. 5. The method of further embodiment 4, wherein the molecule comprises a peptide having a motif selected from the group of peptides at least functionally equivalent to any of the peptide motifs VGVAPG, vGVAPG, vGvAPG, vGVaPG, vGvaPG, LGGGPG, lGGGPG, PGAYPG, pGAYPG, pGaYPG, pGAyPG or pGayPG, AQGVAPG(n), gpavgqa(n), LQGVAPG(n), gpavgql(n). Further embodiment 6. 6. The method of further embodiment 5, wherein said peptide is selected from the group VGVAPGVGVAPGVGVAPG, QVGQVELGGGPGAGSLQP, and GAYPGAPGAYPGAPAPGV, or functional equivalents thereof. Further embodiment 7. 4. The method of further embodiment 1 or 3, wherein said molecule comprises a peptide having a motif selected from the group gpxxgx, gpxxgX, gpxXgX, gpXxgX, and gpXXgX. Further embodiment 8. 8. The method of further embodiment 7, wherein said molecule comprises a peptide having a motif selected from the group that is at least functionally equivalent to any of the peptide motifs gpavgv, gpavgV, gpaVgV, gpAvgV, gpAVgV, gpgggl, gpgggL, gpyagp, gpaygP, gpaYgP, gpAygP, or gpAYgP. Further embodiment 9. 9. The method of further embodiment 8, wherein said peptide is selected from the group gpavgvgpavgvgpavgv, pqlsgagpggglevqgvq, vgpapagpyagpagpyay, or functional equivalents thereof. Further embodiment 10. 4. The method of further embodiment 2 or 3, wherein said molecule comprises a peptide having a motif selected from the group EXXXQ, ExXXQ, EXXXQ, EXXxQ, ExxXQ, EXxxQ, ExXxQ, and ExxxQ. Further embodiment 11. 11. The method of further embodiment 10, wherein said peptide is selected from the group EGSLQ, EVPPQ, EGALQ, EGPLQ, EVAQQ, EGPPQ, EAPLQ, EMALQ, EVARQ, or functional equivalents thereof. Further embodiment 12. 4. The method of further embodiment 2 or 3, wherein said molecule comprises a peptide having a motif selected from the group qxxxe, qxxXe, qxXxe, qXxxe, qxXXq, qXXxe, qXxXe, and qXXXe. Further embodiment 13. 13. The method of further embodiment 12, wherein said peptide is selected from the group qlsge, qppve, qlage, qlpge, qqave, qppge, qlpae, qlame, qrave, or functional equivalents thereof. Further embodiment 14. 14. The method of any one of further embodiments 4 to 13, wherein the peptide provides improved resistance to proteolysis by providing up to three D amino acids at each of the N-terminus and C-terminus flanking portions of the peptide. Further embodiment 15. 15. The method of further embodiment 14, wherein said peptide is selected from synthetic. Further embodiment 16. 16. The method of any one of further embodiments 1 to 15, wherein said hyperinsulinemia is endogenous hyperinsulinemia. Further embodiment 17. 17. The method of any one of further embodiments 1-16, wherein said hyperinsulinemia comprises congenital hyperinsulinemia. Further embodiment 18. 18. The method of any one of further embodiments 1-17, wherein said treatment comprises modulating membrane polarization of beta cells. Further embodiment 19. 19. The method of any one of further embodiments 1 to 18, wherein said treatment comprises modulating NA+ K+ ATPase activity. Further embodiment 20. 20. The method of any one of further embodiments 1-19, wherein said treating comprises modulating insulin secretion. Further embodiment 21. A molecule having an elastin receptor binding motif that is at least functionally equivalent to the PG-domain peptide motif XGXXPG (where G represents the amino acid glycine, P represents proline, and X represents any amino acid) for use in treating a human subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia. Further embodiment 22. A molecule having a Na+K+ATPase activation motif that is at least functionally equivalent to the pentapeptide domain peptide motif EXXXQ (where E represents the amino acid glutamic acid, G represents glutamine, and X represents any amino acid) for use in treating a human subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia. Further embodiment 23. A molecule having an elastin receptor binding motif at least functionally equivalent to the PG-domain peptide motif XGXXPG (where G represents the amino acid glycine, P represents proline, and X represents any amino acid), and having a Na+ K+ ATPase activating motif at least functionally equivalent to the pentapeptide domain peptide motif EXXXQ (where E represents the amino acid glutamic acid, G represents glutamine, and X represents any amino acid), for use in treating a human subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia. Further embodiment 24. 24. The molecule according to further embodiments 21 and 23, which is a peptide having a motif selected from the group XGXXPG, xGXXPG, xGxXPG, xGXxPG, and xGxxPG. Further embodiment 25. 25. The molecule of further embodiment 24, which is a peptide having a motif selected from the group that is at least functionally equivalent to any of the peptide motifs VGVAPG, vGVAPG, vGvAPG, vGVaPG, vGvaPG, LGGGPG, lGGGPG, PGAYPG, pGAYPG, pGaYPG, pGAyPG, or pGayPG. Further embodiment 26. 25. The molecule of further embodiment 24, wherein said peptide is selected from the group VGVAPGVGVAPGVGVAPG, QVGQVELGGGPGAGSLQP, and GAYPGAPGAYPGAPAPGV, or functional equivalents thereof. Further embodiment 27. 24. The molecule according to further embodiments 21 and 23, which is a peptide having a motif selected from the group gpxxgx, gpxxgX, gpxXgX, gpXxgX, and gpXXgX. Further embodiment 28. 28. The molecule of further embodiment 27, which is a peptide having a motif selected from the group that is at least functionally equivalent to any of the peptide motifs gpavgv, gpavgV, gpaVgV, gpAvgV, gpAVgV, gpgggl, gpgggL, gpyagp, gpaygP, gpaYgP, gpAygP, or gpAYgP. Further embodiment 29. 24. The molecule of further embodiments 22 and 23, comprising a peptide having a motif selected from the group EXXXQ, ExXXQ, EXXXQ, EXXxQ, ExxXQ, EXxxQ, ExXxQ, and ExxxQ. Further embodiment 30. 30. The method of further embodiment 29, wherein said peptide is selected from the group EGSLQ, EVPPQ, EGALQ, EGPLQ, EVAQQ, EGPPQ, EAPLQ, EMALQ, EVARQ, or functional equivalents thereof. Further embodiment 31. 24. The molecule of further embodiments 22 and 23, comprising a peptide having a motif selected from the group qxxxe, qxxXe, qxXxe, qXxxe, qxXXq, qXXxe, qXxXe, and qXXXe. Further embodiment 32. 32. The method of further embodiment 31, wherein said peptide is selected from the group qlsge, qppve, qlage, qlpge, qqave, qppge, qlpae, qlame, qrave, or functional equivalents thereof. Further embodiment 33. 33. The molecule of any one of further embodiments 24 to 32, wherein the peptide provides improved resistance to proteolysis by providing up to three D amino acids at each of the N- and C-terminal flanks of the motif. Further embodiment 34. The peptides may be selected from the group vGVAPGVGVAPGVGVApG, vGVAPGVGVAPGEGSLQa, qVGQVELGGGPGAGSLQp, qVGQVELGGGPGEGSLQa, GaYPGAPGAYPGEGSLQav, GaYPGAPGAYPGAPAPGv, vGvAPGVGVAPGVGVapG, qvGQVELGGGPGAGSLqp, GayPGAPGAYPGAPApGv vGvaPGVGVAPGVGvapG, qvGqVELGGGPGAGSlqp, and GaypGAPGAYPGAPapgV, gpavgvgpavgvgpavgv, pqlsgagpggglevqgvq, vgpapagpyagpagpyay, aqlsgegpavgvgpavgv, aqlsgegpggglevqgvq, aqlsgegpyagpagpyay, vGVAPGVGVApG, vGVAPGEGSLQa, qGGGPGAGSLQp, qGGGPGEGSLQa, GaAYPGEGSLQav, GaYPGAAPAPGv, vGvAPGVGVapG, qvGGGPGAGSLqp, GayPGAAPApGv vGvaPGVGvapG, qvGqVELGGGPGqp and GaypGAAPapgV, gpavgvgpavgv, pqlsgegpggglvq, vgpapgpagpyay, aqlsgegpavgv, aqlsgegpggqgvq, aqlsgegpagpyay, or their functional equivalents 34. The molecule of further embodiment 33, selected from: Further embodiment 35. The molecule of any one of further embodiments 21 to 34, wherein said hyperinsulinemia is endogenous hyperinsulinemia. Further embodiment 36. The molecule of further embodiment 35, wherein said hyperinsulinemia comprises congenital hyperinsulinemia. Further embodiment 37. 37. The molecule according to any one of embodiments 21 to 36, which is obtainable by peptide synthesis. Further embodiment 38. 38. The molecule of any one of embodiments 21 to 37, comprising at least one D amino acid, preferably at least two, more preferably at least three D amino acids. Further embodiment 39. A modulator or pharmaceutical composition comprising a molecule according to any of embodiments 21 to 38. Further embodiment 40. The composition of further embodiment 39, comprising a pharmaceutically acceptable excipient. Further embodiment 41. A cell provided with a molecule according to any one of embodiments 21 to 38. Further embodiment 42. 42. The cell of further embodiment 41, wherein an elastin receptor complex (ERC) is provided. Further embodiment 43. 43. The cell of further embodiment 41 or 42, wherein an insulin receptor kinase (IRK) is provided. Further embodiment 44. 44. The cell of any of further embodiments 41 to 43, wherein DANA is provided. Further embodiment 45. A cell culture comprising at least one cell according to any one of embodiments 41 to 44. Further embodiment 46. 46. Use of the cell culture of further embodiment 45 for screening molecules and determining the effect of said molecules on modulating NA+K+ATPase activity. Further embodiment 47. Use of the cell culture according to further embodiment 45 for screening molecules and determining the effect of said molecules on membrane polarization. Further embodiment 48. Use of the cell culture of further embodiment 45 for screening molecules and determining the effect of said molecules on modulating insulin secretion. Further embodiment 49. A method for identifying a source of, preferably a peptide, an L-proteinogenic amino acid that can induce an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, the method comprising the steps of: providing a cell with a peptide comprising an L-proteinogenic amino acid, wherein the amino acid is selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R) for at least 50% of the amino acid; and determining angiogenic activity in a sprouting assay. Further embodiment 50. 1. A method for inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, comprising the step of providing cells with a source of amino acids, preferably L-proteinogenic, wherein the amino acids are selected for at least 50% from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R). Further embodiment 51. 1. A method for inducing an increase in blood glucose in the prevention or treatment of hyperinsulinism in a subject, comprising the step of providing a peptide to a cell, preferably as a source of L-proteinogenic amino acids, wherein the peptide consists of at least 50% amino acids selected from the group consisting of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), and arginine (R). Further embodiment 52. 52. The method according to further embodiment 50 or 51, wherein said source of preferably L-proteinogenic amino acids, preferably peptides, is identifiable by the method according to further embodiment 49. Further embodiment 53. 53. The method of any one of further embodiments 49 to 52, wherein the amino acids are selected for at least 50% from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), and proline (P). Further embodiment 54. 54. The method of any one of further embodiments 50 to 53, wherein said source or peptide has an amino acid sequence derived from a peptide or protein shown to have or suspected to have an activity. Further embodiment 55. 55. The method of further embodiment 54, wherein the peptide has an amino acid sequence derived from chorionic gonadotropin. Further embodiment 56. 55. The method of further embodiment 54, wherein said peptide has an amino acid sequence derived from C-peptide. Further embodiment 57. 55. The method of further embodiment 54, wherein the peptide has an amino acid sequence derived from galectin-3. Further embodiment 58. 55. The method of further embodiment 54, wherein the peptide has an amino acid sequence derived from luteotropin. Further embodiment 59. 55. The method of further embodiment 54, wherein the peptide has an amino acid sequence derived from an extracellular matrix protein. Further embodiment 60. 60. The method of further embodiment 59, wherein the extracellular matrix protein is elastin or collagen. Further embodiment 61. 61. The method of any one of further embodiments 49 to 60, wherein said peptide comprises fewer than 31 amino acids, preferably fewer than 26 amino acids, more preferably fewer than 21 amino acids, more preferably fewer than 16 amino acids, more preferably fewer than 13 amino acids. Further embodiment 62. 62. The method of any one of further embodiments 49 to 61, wherein said peptide comprises more than 3 amino acids, preferably more than 5 amino acids, more preferably more than 7 amino acids, more preferably at least 9 amino acids. Further embodiment 63. 63. The method of further embodiment 62, wherein said source comprises peptides comprising more than 6 amino acids and fewer than 26 amino acids. Further embodiment 64. 63. The method of further embodiment 62, wherein said source comprises peptides comprising more than 7 amino acids and fewer than 31 amino acids. Further embodiment 65. 65. The method of any one of further embodiments 49 to 64, wherein the peptide is provided as a salt of an organic acid, preferably the peptide is provided as a salt of maleic acid, more preferably acetic acid, more preferably tartaric acid, most preferably citric acid. Further embodiment 66. 66. The method of any one of further embodiments 49 to 65, wherein said cells are of human origin. Further embodiment 67. 67. The method of any one of further embodiments 49 to 66, wherein said peptide is of human origin. Further embodiment 67. A further embodiment according to embodiments 49 to 66, wherein the peptide is provided as a salt of an organic acid, preferably the peptide is provided as a salt of maleic acid, more preferably acetic acid, more preferably tartaric acid, most preferably citric acid. Further embodiment 68. 1. A method for the treatment of a human subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia, comprising the step of treating said subject with a modulator or pharmaceutical composition provided with a molecule comprising an elastin receptor binding motif that is at least functionally equivalent to the peptide motif XGXXPG (where G represents the amino acid glycine, P represents proline, and X represents any amino acid). Further embodiment 69. A method for treating a human subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia, comprising treating the subject with a modulator or pharmaceutical composition provided with a molecule having a Na+K+ATPase activation motif that is at least functionally equivalent to the pentapeptide domain peptide motif EXXXQ (where E represents the amino acid glutamic acid, G represents glutamine, and X represents any amino acid). Further embodiment 70. 1. A method for treating a human subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia, comprising treating the subject with a modulator or pharmaceutical composition comprising a molecule comprising an elastin receptor binding motif at least functionally equivalent to the PG-domain peptide motif XGXXPG (where G represents the amino acid glycine, P represents proline, and X represents any amino acid), and further comprising a molecule having a Na+K+ ATP-ase activation domain at least functionally equivalent to the pentapeptide domain motif EXXXQ (where E represents the amino acid glutamic acid, G represents glutamine, and X represents any amino acid). Further embodiment 71. 1. A method of treating a subject diagnosed with an episode of hypoglycemia associated with hyperinsulinemia, comprising: a. administering to a subject a modulator or pharmaceutical composition comprising a molecule having an elastin receptor binding motif that is at least functionally equivalent to the peptide motif XGXXPG (where G represents glycine, P represents proline, and X represents any amino acid); thereby reducing episodes of hypoglycemia associated with hyperinsulinemia, method. Further embodiment 72. 1. A method of treating a subject diagnosed with an episode of hypoglycemia associated with hyperinsulinemia, comprising: b. administering to the subject a modulator or pharmaceutical composition comprising a molecule having a Na+K+ATPase activating motif that is at least functionally equivalent to the pentapeptide domain peptide motif EXXXQ (where E represents glutamic acid, G represents glutamine, and X represents any amino acid); thereby reducing episodes of hypoglycemia associated with hyperinsulinemia, method. Further embodiment 73. 1. A method of treating a subject diagnosed with an episode of hypoglycemia associated with hyperinsulinemia, comprising: c. To the subject, i. a molecule having an elastin receptor binding motif that is at least functionally equivalent to the PG-domain peptide motif XGXXPG (where G represents glycine, P represents proline, and X represents any amino acid); and ii. Molecules with a Na+K+ ATP-ase activating motif that is at least functionally equivalent to the pentapeptide domain motif EXXXQ (where E represents glutamic acid, G represents glutamine, and X represents any amino acid). wherein the method comprises administering a modulator or pharmaceutical composition comprising: method. Further embodiment 74. 1. A method for identifying amino acid motifs that bind to mammalian cell receptors, comprising the steps of: (i) selecting a candidate amino acid motif; (ii) determining the frequency of occurrence of each of the candidate amino acid motifs in the proteome of a pathogen, wherein the pathogen is a pathogen capable of infecting a mammal; and iii) identifying candidate amino acid motifs with the highest frequency of occurrence in the pathogen proteome as amino acid motifs that bind to mammalian cell receptors; a method including, in consecutive order, Further embodiment 75. 75. The method of further embodiment 74, wherein the mammal is a primate. The method of further embodiment 74 or 75, wherein the mammal is a human. Further embodiment 76. 76. The method of any one of embodiments 73 to 75, wherein the cellular receptor is a receptor expressed in an immune cell. Further embodiment 77. 76. The method of any one of embodiments 73 to 75, wherein the cellular receptor is an elastin receptor complex. Further embodiment 78. 78. The method of any one of embodiments 73 to 77, wherein in step (i), the candidate amino acid motifs are selected based on known characteristics of amino acid motifs that bind to mammalian cell receptors. Further embodiment 79. 79. The method of embodiment 78, wherein the known feature is one or more known amino acids of an amino acid motif that binds to a mammalian cell receptor. Further embodiment 80. 79. The method of embodiment 78, wherein the known feature is a known secondary structure of an amino acid motif that binds to a mammalian cell receptor. Further embodiment 81. 81. The method of any one of embodiments 73 to 80, wherein in step (i) at least 100 candidate amino acid motifs are selected, preferably at least 200 candidate amino acid motifs are selected in step (i). Further embodiment 82. 82. The method of any one of embodiments 78 to 81, wherein the cellular receptor is an elastin receptor complex and the candidate amino acid motif comprises the amino acid sequence xGxxPx (where G is glycine, P is proline, and x is any naturally occurring amino acid). Further embodiment 83. 82. The method of any one of embodiments 78 to 81, wherein the cellular receptor is an elastin receptor complex and the candidate amino acid motif comprises the amino acid sequence xGxxPG (where G is glycine, P is proline, and x is any naturally occurring amino acid). Further embodiment 84. 82. The method of any one of embodiments 78 to 81, wherein the cellular receptor is an elastin receptor complex and the candidate amino acid motifs are all amino acid motifs having the amino acid sequence XGXXPG, where G is glycine, P is proline, and X is any naturally occurring amino acid. Further embodiment 85. 86. The method of any one of embodiments 73 to 85, wherein the pathogen is a virus, bacterium, or protozoan. Further embodiment 86. 187. The method of any one of embodiments 73 to 186, wherein the pathogen is a pathogen that acquires host proteins during natural infection. Further embodiment 87. 77. The method of any one of embodiments 73 to 76, wherein the pathogen is a Plasmodium spp. Further embodiment 88. 88. The method of embodiment 87, wherein the pathogen is a Plasmodium spp. that infects primates or a Plasmodium spp. that infects rodents. Further embodiment 89. 89. The method of embodiment 88, wherein the Plasmodium spp. infecting the primate is one or more Plasmodium spp. selected from the list consisting of Plasmodium reichenowi, Plasmodium malariae, Plasmodium gonderi, Plasmodium vivax, Plasmodium ovale, Plasmodium falciparum, Plasmodium coatneyi, Plasmodium sp. DRC-Itaito, and Plasmodium sp. gorilla clade. Further embodiment 90. 91. The method of any one of embodiments 73 to 90, wherein the proteome of the pathogen is a partial proteome of the pathogen. Further embodiments 91. 91. The method of embodiment 90, wherein the partial proteome of the pathogen comprises cell membrane proteins of the pathogen and secreted proteins of the pathogen. Further embodiment 92. 92. The method of any one of embodiments 73 to 91, wherein the pathogen is a Plasmodium spp. that infects primates, and the proteome of the pathogen is a partial proteome comprising blood-stage proteins of a Plasmodium spp. that infects primates.

Claims

1. A modulator of insulin-insulin receptor signaling for use in treating a mammalian subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia.

2. The modulator for use according to claim 1, wherein the subject is a primate or a rodent.

3. 3. A modulator for use according to claim 1 or 2, which is capable of reducing insulin-induced glucose transporter 4 (GLUT4) translocation to the plasma membrane of fat cells or muscle cells of said subject.

4. 4. A modulator for use according to any one of claims 1 to 3, wherein said modulator is provided with a molecule having an elastin receptor binding motif.

5. 5. A modulator for use according to any one of claims 1 to 4, wherein said modulator is provided with a molecule having an elastin receptor binding motif that is at least functionally equivalent to the peptide XGXXPG (where G represents the amino acid glycine, P represents proline and X represents any amino acid).

6. 6. A modulator for use according to any one of claims 1 to 5, comprising a peptide or peptidomimetic having an elastin receptor binding motif that is at least functionally equivalent to the peptide XGXXPG (where G represents the amino acid glycine, P represents proline and X represents any amino acid).

7. The peptides having the motif are selected from the group consisting of peptides VGVAPG(n), vGVAPG(n), vGvAPG(n), vGVaPG(n), vGvaPG(n), gpavgv(n), PGAVPG(n), pGvAPG(n), pGVaPG(n), pGvaPG(n), gpvagp(n), LGGGPG(n), lGGGPG(n), gpgggl(n), PGAYPG(n), pGAYPG(n), pGaYPG(n), pGAyP 7. The modulator for use according to claim 6, which is selected from the group of peptides which are at least functionally equivalent to any of G(n), pGayPG(n), or gpyagp(n), AQGVAPG(n), gpavgqa(n), LQGVAPG(n), gpavgql(n) (n indicates the number of occurrences of the motif, n varies from 1 to 8), and which enables the modulator to be targeted to the elastin receptor.

8. 7. The modulator for use according to claim 6, wherein the peptide having said motif is selected from the group of peptides that are at least functionally equivalent to any of the peptides VGVAPG(n), gpavgv(n), PGAVPG(n), gpvagp(n) (n indicates the number of times the motif is repeated, n varying from 1 to 8), and allows the modulator to be targeted to the elastin receptor.

9. 7. The modulator for use according to claim 6, wherein said peptide having said motif is at least functionally equivalent to the peptide VGVAPGVGVAPGVGVAPG, QVGQVELGGGPGAGSLQP, or GAYPGAPGAYPGAPAPGV.

10. 7. The modulator for use according to claim 6, wherein said peptide having said motif is at least functionally equivalent to the peptide VGVAPGVGVAPGVGVAPG or AVPGAVPGAVPG.

11. 11. A pharmaceutical composition comprising a modulator as defined in any one of claims 1 to 10.

12. 12. The pharmaceutical composition of claim 11, formulated for parenteral administration.

13. 12. The pharmaceutical composition of claim 11, formulated for oral administration.

14. 14. A method for the treatment of a mammalian subject suspected of having an episode of hypoglycemia associated with hyperinsulinemia, the method comprising administering to said subject a modulator as defined in any one of claims 1 to 10, or a pharmaceutical composition as defined in any one of claims 11 to 13.

15. 15. The method of claim 14, wherein the subject is a primate or a rodent.

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