Angiogenesis control, preferably in combination with glycemic control

Abstract

The present invention relates to a distinct and emerging class of drugs: peptide modulators of the mechanistic target of rapamycin (mTOR), which act as autophagy inhibitor compounds, target the mTOR nutrient sensing system, and induce angiogenic activity.The present invention provides formulations, methods and means for preventing or treating vasculopathy, particularly diabetic vasculopathy, to achieve or maintain angiogenic control, preferably concomitant with glycemic control.

Description

[Technical field]

[0001] The present invention relates to a distinct and newly emerging class of drugs: peptide modulators of the mechanistic target of rapamycine (mTOR), which act as autophagy inhibitory compounds, targeting the mTOR nutrient sensing system and inhibiting autophagy.The present invention provides pharmaceutical formulations, methods and means for exerting angiogenic control, both for use alone and in combination with established insulin formulations, methods and means, particularly for exerting glycemic control concomitant with said angiogenic control. [Background technology]

[0002] Diabetes mellitus is an endocrine disorder caused by global insulin deficiency or defective insulin function, resulting in hyperglycemia. Type 1 diabetes is usually seen in younger patients, accounts for 5%-10% of cases worldwide, and is thought to be secondary to autoimmune destruction of the B islet cells of the pancreas. Type 2 diabetes accounts for 90%-95% of cases worldwide, and is thought to be due to genetic and environmental factors, with resultant insulin resistance and pancreatic beta cell dysfunction. Complications resulting from hyperglycemia can be either macrovascular or microvascular. Macrovascular disease primarily affects the cardiovascular and cerebrovascular systems, while microvascular disease includes nephropathy, retinopathy, and neuropathy. Glycemic control in diabetes mellitus often requires replacing the action of the beta cells of the pancreatic islets to detect the need for insulin and ensure that insulin is administered according to the patient's body's needs. Insulin is a natural hormone and an essential drug therapy for numerous medical conditions. One of the most significant uses of insulin is in type 1 and type 2 diabetes mellitus. Insulin is one of the few medications indicated for use in the management of gestational diabetes. In patients with diabetes mellitus (Dave HD, Preuss CV. Human Insulin. [Updated 2021 Feb 17]. Published in: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK545190 / ), a single insulin preparation with a defined onset, peak and duration of action is often considered useless to reach the goal of normal glycemic control with similar 24-hour insulin activity as in healthy adults without diabetes mellitus. Therefore, there is a need for different types of insulin with different pharmacokinetics. Based on the mechanism of action, there are at least four different types of insulin analogs. Fast-acting insulin has a rapid onset of action (less than about 30 minutes), a peak action at about the first hour and a short duration of action (up to 5 hours). Insulin lispro and insulin aspart are examples of fast-acting insulins.These insulins help achieve glycemic control, especially in the postprandial state. Short-acting insulin analog activity begins in about 30-60 minutes, peaks at 2-4 hours, and activity lasts for about 8 hours. These insulin analogs, for example, insulin glulisine, must be administered approximately 20-30 minutes before a meal for effectiveness. Rapid-acting or short-acting insulin analogs (such as insulin lispro, insulin aspart, and insulin glulisine) typically act faster than regular human insulin. Intermediate-acting insulin analogs have an onset of activity around 1-2 hours, a peak effect at 6-10 hours, and a duration of action up to about 16 hours. Neutral protamine Hagedorn (NPH) and lente insulin are examples of intermediate-acting insulin analogs. Some of the long-acting insulin analogs are insulin detemir and insulin glargine. Their activity begins around 2 hours, with a peak effect at 6-20 hours, and lasts for up to about 36 hours. Intermediate-acting and long-acting (basal) insulins are recommended for patients with type 1, type 2, or gestational diabetes. They can also be used in other types of diabetes (i.e., steroid-induced). People with type 1 diabetes generally use intermediate-acting or long-acting insulin in conjunction with regular or rapid-acting insulin to achieve glycemic control. People with type 2 diabetes can use intermediate-acting or long-acting insulin in conjunction with regular or rapid-acting insulin or in conjunction with oral medication to achieve glycemic control.

[0003] However, achieving or exerting glycemic control does not resolve all diseases in patients with endogenous insulin deficiency. In particular, diabetic vasculopathy, e.g., retinopathy, peripheral neuropathy (DPN), in the overall state of insulin deficiency, is a well-known microvascular complication of diabetes mellitus that does not resolve even with the best glycemic control. DPN, for example, leads to further infections and increases the risk of diabetic foot ulcers (Figure 1), non-traumatic amputation, and death. Although hypoglycemia along with hyperglycemia plays an important role in the development of DPN, intensive glycemic control (insulin or not) does not eliminate the risk of developing DPN in diabetic patients, suggesting that factors other than insulin deficiency and / or unstable glucose metabolism may be involved in the development of DPN. C-peptide levels in peripheral blood are widely accepted as the most appropriate measure of insulin secretion and are not excreted in first-pass metabolism via the liver. Although previously considered to be an inactive by-product of insulin synthesis, C-peptide is a hormonally active peptide. In type 1 diabetes, an Italian study (Panero et al. Fasting plasma C-peptide and micro- and macrovascular complications in a large clinic-based cohort of type 1 diabetic patients. Diabetes Care. 2009;32:301-5) demonstrated a significant and negative association between C-peptide levels and microvascular complications, including neuropathy. C-peptide deficiency is typically associated with these microvascular complications, which has motivated attempts to treat such patients with exogenous C-peptide.Several studies in diabetic animal models and type 1 diabetic patients have indeed demonstrated the beneficial effects of C-peptide supplementation on both peripheral and autonomic nerve function in diabetes (Johansson et al. Beneficial effects of C-peptide on incipient nephropathy and neuropathy in patients with Type 1 diabetes mellitus. Diabetic Med. 2000;17:181-9.). In type 2 diabetes, C-peptide levels were also significantly and negatively associated with microvascular complications associated with DPN according to a Chinese study (Qiao et al. C-peptide is independent associated with diabetic peripheral neuropathy: a community-based study. Diabetol Metab Syndr 9, 12 (2017)). Endogenous C-peptide is thought to exert an angiogenic activity in our vasculature, contributing to the resolution of vasculopathy by controlling angiogenesis in the vasculature. A high prevalence of vasculopathy with retinopathy, nephropathy and neuropathy is observed in patients with low C-peptide levels. Peripheral neuropathic changes to the body result in decreased sensation in the feet and predispose the diabetic foot to mechanical or pressure injury wounds. Microvascular changes can result in reduced blood flow to the limbs and delayed wound healing. This complication can be prevented as the precipitating factor is most often minor trauma. Early identification of these cutaneous lesions can also result in improved outcomes while decreasing the risk of progression.

[0004] However, a clinical trial evaluating the efficacy and safety of long-acting (pegylated) C-peptide in subjects with type 1 diabetes and neuropathy failed to meet its primary endpoint (Wahren et al., Long-acting C-peptide and neuropathy in type 1 diabetes: A 12-month clinical trial. Diabetes Care. 2016 Apr;39(4):596-602.). A total of 250 patients with type 1 diabetes and peripheral neuropathy received long-acting (pegylated) C-peptide in weekly dosages of 0.8 mg (n=71) or 2.4 mg (n=73) or placebo (n=106) for 52 weeks. Weekly subcutaneous administration of long-acting C-peptide for 52 weeks did not improve bilateral sural nerve conduction velocity (SNCV), other electrophysiological variables, or the modified Toronto Clinical Neuropathy Score (mTCNS), but only improved vibration perception threshold (VPT) compared with placebo, and C-peptide in this formulation did not produce angiogenic control.

[0005] Infectious gangrene of the foot is a serious complication of diabetes. It usually results in a certain level of lower extremity amputation (LEA). Foot gangrene is defined as dead tissue in the foot due to inadequate blood flow supply. It is one of the end signs of critical limb ischemia. It can be caused by obstructed peripheral circulation or microbial infection. Foot ulcers in diabetic patients are at increased risk of foot gangrene initiation, mainly due to peripheral arterial disease. Foot gangrene ranges from two extremes: (Figure 1A) early signs of foot ulcer with dry gangrene development, with ischemic and necrotic tissue but no infection, to (Figure 1B) late signs of wet gangrene, with both infected and necrotic tissue. Thus, dry gangrene may lead to wet gangrene. In the more pronounced stages of dry gangrene, the affected area is often characterized by a dark green or purple, almost black, color. Wet gangrene has a wet appearance with blisters and swelling. Wet gangrene can also occur in people who have frostbite or experience severe burns. People with diabetes can eventually develop wet gangrene after experiencing minor toe or foot injuries, such as those seen in athlete's foot. Blood flow to the extremities is generally reduced in people with diabetes. This means that tissues in these areas cannot heal as quickly. As a result, infections can develop more easily. Wet gangrene can spread quickly and can be fatal if left untreated. Typically, such patients rapidly develop sepsis and die.

[0006] Treatment for all forms of gangrene involves removing dead tissue, treating and stopping possible spread of infection, and treating the condition that caused the gangrene. Treatment can include surgery (also called debridement) to remove dead tissue and prevent the infection from spreading. It may be necessary to remove the affected limb, hand, finger, foot, or toe. Sometimes, instead of surgery, fly larvae maggots are placed on the ulcer wound, where the maggots feed on the dead and infected tissue without harming healthy tissue. Antibiotics can be used to treat or prevent infection, and hyperbaric oxygen therapy can treat wet gangrene or ulcers related to diabetes or peripheral arterial disease.

[0007] Thus, infectious gangrene is both a limb- and life-threatening disease. Proper medical treatment with antibiotics and wound dressings is ineffective without timely surgery to remove necrotic and infected tissues, which may ultimately result in limb amputation. However, reports have shown that patients who undergo diabetes-related amputations still have a high risk of death with a 5-year survival rate of 40-48%, regardless of the etiology of the amputation (Huang et al., Survival and associated risk factors in patients with diabetes and amputations caused by infectious foot gangrene. J Foot Ankle Res. 2018 Jan 4;11:1.), see also Figure 2.

[0008] Patients with diabetes mellitus (type 1 or 2) have an overall lifetime risk of diabetic foot ulcer complications as high as 25% (some report 50%), with an estimated cost to the NHS of £935 million already in England alone (Diabetic foot care in England: an economic study - Marion Kerr, Insight Health Economics, 2017). The end result of ill-treated diabetic ulcers is an increase in overall morbidity (Packer CF, Ali SA, Manna B. Diabetic Ulcer. [Updated 2021 Feb 20]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK499887 / ).

[0009] As indicated, diabetic ulcer formation is exacerbated and accelerated under conditions of worsened glycemic control. Peled et al. (Association of Inpatient Glucose Measurements With Amputations in Patients Hospitalized With Acute Diabetic Foot, The Journal of Clinical Endocrinology & Metabolism, Volume 104, Issue 11, November 2019, Pages 5445-5452) find that patients experiencing any hyperglycemia and any or severe hypoglycemia were more likely to undergo any or major amputation during hospitalization. High glycemic variability was associated with major amputation. Peripheral vascular disease (PVD), high Wagner score, and hypoglycemia were independent predictors of amputation. Older age, peripheral vascular disease (PVD), previous amputation, elevated white blood cell levels, high Wagner score, and hypoglycemia were independent predictors of major amputation. In a systematic review of 60 observational studies, 47 of which were included in a meta-analysis (Lane et al., Glycemic control and diabetic foot ulcer outcomes: A systematic review and meta-analysis of observational studies. J Diabetes Complications. 2020 Oct;34(10):107638.), hyperglycemia (higher A1C and higher fasting glucose) was associated with increased likelihood of lower-limb amputation among subjects with diabetic foot ulcers. Lane et al.'s findings suggest that A1C levels of 8% or higher and fasting glucose levels of 126 mg / dl or higher are associated with increased likelihood of lower-limb amputation in patients with existing diabetic foot ulcers.

[0010] Not only are diabetic ulcers debilitating in themselves, but the risk of non-traumatic lower limb amputation due to ill-treatable foot ulcers is 15 times higher in diabetic patients compared to non-diabetic patients. Diabetic patients are at higher risk for lower limb amputation, higher medical costs, and lower quality of life (Song K, Chambers AR. Diabetic Foot Care. [Updated 2021 Jul 31]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2021 Jan-. Available from: https: / / www.ncbi.nlm.nih.gov / books / NBK553110 / ). In a systematic study of non-traumatic amputations in patients with diabetes mellitus and peripheral vascular disease (Thorud et al., Mortality After Nontraumatic Major Amputation Among Patients With Diabetes and Peripheral Vascular Disease: A Systematic Review. J Foot Ankle Surg. 2016 May-Jun;55(3):591-9), the 5-year mortality rate of patients after below-knee amputation was 40-82%, and after above-knee amputation was 40-90%. Many of these complications are preventable with thorough annual foot exams and routine foot care performed by the patient. Patient education on proper foot care practices is the current cornerstone of prevention of diabetic foot disease. Foot ulcers are a common reason for hospital stays in people with diabetes. Foot ulcers may take weeks or even months to heal. Diabetic ulcers are often painless (due to decreased sensation in the foot). Treatment of ulcerated diabetic foot is a complex problem (Lepantalo et al., Chapter V: Diabetic foot. Eur J Vasc Endovasc Surg. 2011 Dec;42 Suppl 2:S60-74.).Ischemia, neuropathy, and infection are the three pathological components that result in diabetic foot complications and frequently occur together as an etiological triad. Neuropathy and ischemia, most often occurring together as neuroischaemia, are the initiating factors, whereas infection is mostly the consequence. Furthermore, ulcer healing is impeded by microvascular dysfunction. Guidelines have largely ignored these specific demands related to the ulcerated diabetic foot. Any diabetic foot ulcer should always be considered to have vascular dysfunction unless proven otherwise. Early referral, vascular testing, imaging, and intervention are crucial to improve diabetic foot ulcer healing and prevent amputation. Timing is essential, as therapeutic opportunities to heal the ulcer and save the leg are easily lost. However, there is a paucity of data on how best to diagnose and treat these diabetic patients. The majority of studies addressing the diabetic foot are not comparable in terms of patient population, intervention, or outcome.

[0011] Diabetes mellitus may disrupt wound repair and lead to the development of chronic wounds due to impaired angiogenesis. Approximately 40% of patients with diabetes mellitus of more than 15 years duration suffer from autonomic neuropathy. This may develop through deficits, inability to regulate temperature and bladder dysfunction followed by cardiovascular dysreflexia. Late signs may include generalized sweating disorder, postural hypotension, gastrointestinal problems and reduced glycemic control. The latter condition has significant clinical implications involving various comorbidities including poor wound healing, chronic ulceration and the resulting severe complication of limb amputation. In cutaneous wound healing with distinct ordered phases, diabetes leads to improper function at all stages. The pathogenesis of chronic non-healing diabetic wounds is multifaceted, but the progression to a non-healing phenotype is closely associated with an inadequate vascular network pointing to inadequate vasculogenesis / angiogenesis. Although these terms are often used interchangeably (as they are used herein to denote various aspects of blood vessel formation and repair), strictly speaking, vasculogenesis is defined as the formation of new blood vessels from precursor cells that coalesce into primitive vascular networks, whereas angiogenesis is the formation of new capillaries from established vasculature. This distinction depicts the fact that blood vessels develop through two subsequent processes, vasculogenesis and angiogenesis, both of which are critical for vascular maintenance and repair. These processes are also involved in the development of the fetal and placental vasculature.Vascular development (neovascularization, Rafii S, Lyden D. Therapeutic stem and progenitor cell transplantation for organ vascularization and regeneration. Nat Med. 2003 Jun;9(6):702-12; Masuda, H and Asahara, T. Post-natal endothelial progenitor cells for neovascularization in tissue regeneration, Cardiovascular Research, Volume 58, Issue 2, May 2003, Pages 390-398,) is a regulated process involving proliferation, migration and remodeling of endothelial cells (ECs) driven by in situ proliferation and migration of pre-existing endothelial cells from adjacent pre-existing vessels (angiogenesis) or following differentiation of endothelial progenitor cells (EPCs) from mesodermal precursors (vasculogenesis). Vasculogenesis and angiogenesis are important and complex processes involving extensive interactions between cells and growth factors. In vasculogenesis, the earliest capillary formation is achieved by in situ differentiation of hemangiogenic stem cells derived from multipotent mesenchymal cells. The subsequent process, angiogenesis, is characterized by the development of new blood vessels through angiogenic activity, with cell proliferation arising from already existing vessels and vascular cells, and is a well-coordinated process initiated by the stimulation of various growth factors. Typically, angiogenic activity is mainly involved in neovascularization in tissue regeneration and wound repair (i.e., postnatal). Angiogenic activity is typically studied in vitro in colony assays (clonogenic assays or colony formation assays), which are in vitro cell survival assays based on the ability of single cells to grow into colonies. Angiogenic activity is typically studied in vitro in sprouting assays (tube formation assays or angiogenesis assays), which are in vitro cell survival assays based on the ability of test cells to form capillary-like structures in vitro, recapitulating angiogenesis.In our body, endothelial cells are surrounded by a thin, highly specialized extracellular matrix (ECM), the basement membrane. When endothelial cells, such as human umbilical vein cells (HUVECs), are seeded on a basement membrane-like surface (e.g., Matrigel®), they form branching tubes or spouts that resemble capillaries.

[0012] In the past decade, numerous studies in both humans and animals have demonstrated that C-peptide, while not affecting glycemic control, may play a role in preventing and potentially reversing some of the chronic complications of type 1 diabetes. Lim et al. (Proinsulin C-peptide prevents impaired wound healing by activating angiogenesis in diabetes. J Invest Dermatol. 2015 Jan;135(1):269-278) demonstrated that human C-peptide can protect against vasculopathy in diabetes and used streptozotocin-induced diabetic mice and human umbilical vein endothelial cells to investigate the potential role of C-peptide in protecting against impaired wound healing by inducing angiogenesis. Diabetes delayed wound healing in mouse skin, and C-peptide supplementation using an osmotic pump significantly increased the rate of skin wound closure in diabetic mice. Furthermore, C-peptide induced endothelial cell migration and tube formation in a dose-dependent manner, with a maximal effect at 0.5 nM. C-peptide-enhanced angiogenesis in vivo was demonstrated by immunohistochemistry and Matrigel plug assay. The findings of Lim et al. highlight the angiogenic role of C-peptide and its ability to protect against impaired wound healing, which may have significant implications in repair and therapeutic angiogenesis in diabetes, and suggest that C-peptide supplementation is a promising treatment for impaired angiogenesis and delayed wound healing in diabetes. The consistent lack of C-peptide can indeed lead to microvascular lesions that can be corrected by exogenous C-peptide, indicating that C-peptide plays an active role in the vascular repair process. When beta cells fail and little or no C-peptide is excreted, the microvascular complications can lead to retinopathy, nephropathy and neuropathy, which may be corrected by treatment with C-peptide.To investigate whether C-peptide can normalize impaired wound healing in diabetic mice, C-peptide was continuously supplemented using subcutaneously implanted osmotic pumps in streptozotocin-induced diabetic mice. Wounds of 4 mm diameter were created on the dorsal surface of the hind paws of normal, diabetic, and C-peptide-supplemented diabetic mice. Diabetic mice showed a significantly slower healing rate compared to normal mice. However, C-peptide supplementation accelerated wound closure in diabetic mice. This effect was quantitatively analyzed by Lim et al. (supra) measuring wound diameter. These results indicate that C-peptide significantly prevents impaired wound healing, including wound closure, in diabetic mice. Because angiogenesis is a crucial process for wound healing, Lim et al. (supra) investigated whether C-peptide can activate endothelial cell migration, proliferation, and tube formation in an in vitro assay, also called the sprouting assay. C-peptide has been shown to induce sprouting by activating endothelial cell migration, proliferation, and tube formation, which are essential for angiogenesis. However, these promising preclinical studies contrast starkly with human clinical trials with C-peptide, which have so far failed to bring about any progress in the control of diabetic vasculopathy.

[0013] In short, there is an urgent need for a paradigm shift in the care of patients with diabetic vasculopathy such as nephropathy, retinopathy and neuropathy in patients with C-peptide deficiency (typically in type 1 and end-phase type 2 diabetes), and thus ultimately in diabetic ulcer care as well; i.e., new approaches and classification of diabetic patients with vascular dysfunction, in clinical practice and research. Regardless of the intervention technique chosen, new strategies need to be developed and implemented for patients with diabetic vasculopathy, such as those with nephropathy, retinopathy and / or neuropathy, and ultimately for ulcer patients with vascular dysfunction, to improve healing, speed up the healing rate and avoid amputation. Intensive research on the value of predictive tests, new treatment modalities and selective and targeted strategies are needed. Specific data on the ulcerated diabetic foot are lacking, so recommendations are often low-grade. In short, beyond the existing and well-tested strategies of glycemic control, some form of angiogenic control is also explored for many of these patients.

[0014] Nutrient sensing is important for sustaining normal cell growth and proliferation. mTOR complex 1 (mTORC1) senses nutrients to regulate cell growth, autophagy, and other mTORC1-mediated processes. Growth factors such as insulin, IGF, PDGF, and VEGF, amino acids, energy status, and stress control mTORC1. Amino acids are essential for mTORC1 activation. Growth factors alone cannot achieve maximum mTORC1 activity without amino acid supplementation (Sancak et al., The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science. 2008 Jun 13;320(5882):1496-501.). Increased intracellular amino acid concentration promotes mTORC1 lysosomal localization and subsequent activation. Summary of the Invention

[0015] On the one hand, the present invention provides the use of a distinct and emerging class of drugs: peptide modulators of mTOR acting as autophagy-inhibiting compounds for use in inducing angiogenic activity required for establishing angiogenic control to combat said vascular disorders occurring even in patients with otherwise adequate glycemic control.The present invention provides an autophagy-inhibiting modulator of mTOR for use in inducing angiogenic activity in the prevention or treatment of vascular disorders in a subject, said modulator comprising a source of amino acids, preferably peptides, wherein the amino acids are selected from the group of alanine (in one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R) and asparagine (N) for 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%. In a preferred embodiment, the present invention provides an autophagy inhibitory modulator of mTOR for use in inducing angiogenic activity in the prevention or treatment of a vascular disorder 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 said amino acids are selected from the group of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) and proline (P).In a preferred embodiment, the present invention provides an autophagy inhibitory modulator of mTOR for use in inducing angiogenic activity in the prevention or treatment of a vascular disorder in a subject, said modulator comprising a source of amino acids, preferably a peptide, 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 said amino acids are selected from the group of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L). In a preferred embodiment, the present invention provides an autophagy inhibitory modulator of mTOR for use in inducing angiogenic activity in the prevention or treatment of a vasculopathy in a subject, said modulator comprising a source of amino acids, preferably a peptide, 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 said amino acids are selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G) and valine (V). In a preferred embodiment, the present invention provides an autophagy inhibitory modulator of mTOR for use in inducing angiogenic activity in the prevention or treatment of a vascular disorder in a subject, said modulator comprising a source of amino acids, preferably a peptide, 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 said amino acids are selected from the group of alanine (in single letter code: A), glutamine (Q), glycine (G) and leucine (L).In a preferred embodiment, the present invention provides an autophagy inhibitory modulator of mTOR for use in inducing angiogenic activity in the prevention or treatment of a vascular disorder 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 said amino acids are selected from the group of alanine (in single letter code: A), glutamine (Q), leucine (L) and proline (P). In a preferred embodiment, the present invention provides an autophagy inhibitory modulator of mTOR for use in inducing angiogenic activity in the prevention or treatment of vasculopathy in a subject, said modulator comprising a source of amino acids, preferably peptides, wherein said amino acids are selected from the group of alanine (in one letter code: A), valine (V), leucine (L) and proline (P) for 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%. In another preferred embodiment, the present invention provides a modulator according to the present invention for use in the prevention or treatment of nephropathy, in particular for use in the prevention of end-stage renal disease. In another preferred embodiment, the present invention provides a modulator according to the present invention for use in the prevention or treatment of retinopathy, in particular for use in the prevention of partial blindness or loss of vision. In another preferred embodiment, the present invention provides a modulator according to the present invention for use in the prevention or treatment of neuropathy, more preferably for use in the prevention or treatment of peripheral diabetic neuropathy, more preferably for use in the prevention or treatment of diabetic ulcers.It is particularly preferred that such autophagy inhibitory peptide modulators of mTOR according to the invention are used when the subject is also treated to achieve or maintain glycemic control, in particular when the subject is also treated with insulin to achieve or maintain glycemic control. The present invention provides a method for identifying a source of preferably L-proteogenic amino acids, preferably peptides, capable of inducing angiogenic activity, comprising the steps of providing a cell with a peptide comprising an L-proteogenic amino acid, said amino acids being selected from the group of alanine (in 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% of the amino acids, and determining the angiogenic activity in a sprouting assay. The angiogenic activity can be determined by assessing capillary tube formation and by assessing capillary branch formation as provided herein. It is preferred that the amino acids are selected from the group of alanine (in the one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R) and asparagine (N) 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 the amino acids are selected from the group of alanine (in the one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) and proline (P) 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 said amino acids are selected from the group of alanine (in the one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) 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 the amino acids are selected from the group of leucine (in the one-letter code: L), glutamine (Q), glycine (G) and valine (V) at least 50%, more preferably at least 60%, 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 (in the one-letter code: A), glutamine (Q), glycine (G) and valine (V) 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 (in the one-letter code: A), glutamine (Q), glycine (G) and leucine (L) 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 said amino acids are selected from the group of alanine (in the one letter code: A), valine (V), leucine (L) and proline (P) over 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 angiogenic activity, comprising the step of providing a cell with a source of amino acids, preferably L-proteogenic, wherein said amino acids are selected from the group of alanine (in the one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R) and asparagine (N) over 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%.It is more preferred that the amino acids are selected from the group of alanine (in the one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) and proline (P) 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 (in the one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) 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 (in the one-letter code: A), glutamine (Q), glycine (G) and valine (V) 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 leucine (in the one-letter code: L), glutamine (Q), glycine (G) and valine (V) at least 50%, more preferably at least 60%, 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 (in the one-letter code: A), glutamine (Q), glycine (G) and leucine (L) 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 said amino acids are selected from the group of alanine (in the one letter code: A), valine (V), leucine (L) and proline (P) 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 angiogenic activity comprising the step of providing a cell with a source of amino acids, preferably L-proteogenic, selected from the group of alanine (in the one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R) and asparagine (N) by at least 50%, more preferably selected from the group of alanine (in the one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), proline (P), arginine (R) and asparagine (N) by 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 the amino acids are selected from the group of alanine (in the one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) and proline (P) 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 (in the one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) 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 said amino acids are selected from the group of alanine (in the one letter code: A), glutamine (Q), glycine (G) and valine (V) 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 the amino acids are selected from the group of leucine (in the one-letter code: L), glutamine (Q), glycine (G) and valine (V) at least 50%, more preferably at least 60%, 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 (in the one-letter code: A), glutamine (Q), glycine (G) and leucine (L) at least 60%, more preferably at least 75%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, more preferably at least 95%. It is even more preferred that the amino acids are selected from the group of alanine (in the one-letter code: A), valine (V), leucine (L) and proline (P) 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 angiogenic activity, comprising the step of providing a cell with a source of amino acids, preferably L-proteogenic, wherein said 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) by at least 50%. It is more preferred that said amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) and proline (P) by 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 (in the one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) 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 (in the one-letter code: A), glutamine (Q), glycine (G) and valine (V) 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 leucine (in the one-letter code: L), glutamine (Q), glycine (G) and valine (V) at least 50%, more preferably at least 60%, 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 (in the one-letter code: A), glutamine (Q), glycine (G) and leucine (L) 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 (in the one-letter code: A), valine (V), leucine (L) and proline (P) 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 angiogenic activity comprising the step of providing a cell with a source of amino acids, preferably L-proteogenic, wherein said amino acids are selected from the group consisting of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R) and asparagine (N) over at least 50% of the cells. The present invention also provides a method for inducing angiogenic activity, comprising providing a cell with a peptide, preferably as a source of L-proteogenic amino acids, wherein said peptide consists of amino acids 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) for 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%. It is more preferred that said amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) and proline (P) 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 said amino acids are selected from the group of alanine (in the one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) 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 (in the one-letter code: A), glutamine (Q), glycine (G) and valine (V) 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 leucine (in the one-letter code: L), glutamine (Q), glycine (G) and valine (V) at least 50%, more preferably at least 60%, 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 (in the one-letter code: A), glutamine (Q), glycine (G) and leucine (L) 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 said amino acids are selected from the group of alanine (in the one letter code: A), valine (V), leucine (L) and proline (P) 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 angiogenic activity, comprising the step of providing a cell with a source of amino acids, preferably L-proteogenic, wherein said 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) by at least 50%. It is more preferred that said amino acids are selected from the group of alanine (single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) and proline (P) by 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 (in the one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) 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 (in the one-letter code: A), glutamine (Q), glycine (G) and valine (V) 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 leucine (in the one-letter code: L), glutamine (Q), glycine (G) and valine (V) at least 50%, more preferably at least 60%, 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 (in the one-letter code: A), glutamine (Q), glycine (G) and leucine (L) 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 (in the one-letter code: A), valine (V), leucine (L) and proline (P) 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 angiogenic activity, comprising the step of providing a cell with a source of amino acids, preferably L-proteogenic, selected over at least 50% of the cells from the group of alanine (in the one 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 of treatment according to the present invention, wherein said source of L-proteogenic amino acids, preferably a peptide, is identifiable by the methods provided herein. The present invention particularly provides a method of treatment according to the present invention, wherein said source or peptide has an amino acid sequence derived from a peptide or protein shown to have or suspected to have angiogenic activity. Often, in the proteome of an organism, such an angiogenic sequence in a protein is located near or between arginine (R) or lysine (K) residues located more N- and C-terminally, allowing an enzyme such as a convertase to cleave off said angiogenic amino acid sequence, which can then be used as an autophagy inhibitory peptide modulator of mTOR. It is preferred to correlate the peptide use with the species of cell in which it is placed and used. For example, if a peptide is used in human cells as an autophagy inhibitory peptide modulator of mTOR, it is preferred that said peptide is derived from the human proteome. Similarly, it is preferred to use a peptide derived from species X proteome in another species of cell, such as species X. In a preferred embodiment, the present invention provides said autophagy inhibitory peptide having an amino acid sequence derived from chorionic gonadotropin (CG), a protein involved in angiogenic activity during pregnancy, and preferably said peptide is derived from the beta chain of CG, preferably loop 2 of said beta chain. Human CG (hCG) has the angiogenic amino acid sequence MTRVLQGVLPALPQVVCNYR, in which the core angiogenic sequence is located between the arginine (R) residues located at the N- and C-termini. Furthermore, this angiogenic core contains the ERC-binding motif xGxxPx.This division of motifs facilitates the use of hCG derivatives LQGV, VLPALP AQGV, LAGV, LQAV, LQGA, ALPALP, VAPALP, VLAALP, VLPAAP and VLPALA as autophagy-inhibiting peptide modulators, especially in combination with fast- and short-acting insulin preparations to exert both glycemic and angiogenic control. For example, peptide VLQGVLPALPQVV finds better use in combination with intermediate- and long-acting insulin preparations, which are released slower and more in line with the release rate of insulin. In another preferred embodiment, the present invention provides said autophagy-inhibiting peptides, which have an angiogenic amino acid sequence derived from C-peptide. In the preproinsulin molecule, human C-peptide has the sequence RREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKR, and indeed the angiogenic core sequence is flanked by arginine (R) or lysine (K) residues. Moreover, this angiogenic core contains the ERC binding motif xGxxPG. Because ERC binding motifs are involved in the coacervation of peptides with these sequences by oligomerization, such autophagy-inhibiting peptides with ERG binding motifs typically have a slower release rate when injected, and are typically more useful and preferred under circumstances where such slow release is desired, such as in combination formulations with intermediate-acting or more preferably long-acting insulin. Coacervation typically involves the aggregation of colloidal droplets of peptides held together by electrostatic attraction, which explains the nature of the slow release of such ERC motifs that constitute peptides when injected, at least compared to peptides that do not have ERC motifs and do not show coacervation, which typically have faster release characteristics. Preferred C-peptide fragment peptides for use as autophagy inhibitory peptide modulators of mTOR in the treatment of angiogenic dysfunction, particularly in the setting of C-peptide deficiency, are preferably isolated and / or synthetic, preferably non-peggylated, 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, not containing the xGxxPG motif, are therefore most suitable and preferred for inclusion in or combination with fast or short acting insulins.

[0016] In another preferred embodiment, the present invention provides said autophagy inhibitor peptide, which has an angiogenic amino acid sequence derived from galectin-3. The mature N-terminal fragment of human galectin-3, PQGWPGAWGNQPAGAGGYPGASYPGAYPGQAPPGAYPGQAPPGAYPGAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPYGAPAGPLIVPYNLPLPGGVVPRM, is typically characterized by multiple ERC binding motifs xGxxPG, and the angiogenic core sequence is also 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 to human N-terminal galectin-3 but also hydrolyzes it. Remarkably, this mechanism prevents galectin-3-mediated parasite death, suggesting that T. cruzi may have developed a complex strategy to modulate galectin-3 function to successfully infect, survive and thrive within its mammalian host. Indeed, the nonpathogenic T. rangeli binds to galectin-3 but does not modify the 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 the ERC and modulate mTOR to the parasite's benefit.Specifically, the N-terminal sequences of the three fragments obtained by parasite hydrolysis are band 1: AGGYPGASYPG, band 2: GAPGAYPGAP and band 3: GAPAGPLIVP, indicating the mTOR modulating characteristics of the galectin-3 N-terminal peptide fragments derived from AGGYPGASYPGAYPGQAPPGAYPGQAPPGAYP, GAPGAYPGAPAPGVYPGPPSGPGAYPSSGQPSATGAYPATGPY and GAPAGPLIVPYNLPLPGGVVP in humans. For example, the fragments GAYPGAPGAYPGAPAPGV and PGAYPG have angiogenic core activity as demonstrated herein, making PGAYPGQAPPGAYPG, PGAYPGQA and GQAPPGAYPG also useful autophagy inhibitory peptides for use in humans, especially when included in or with intermediate- or long-acting insulin. The present invention also provides a method of treatment and use thereof according to the present invention, in which the peptide has an amino acid sequence derived from lutropin (LH), a protein that is often involved in monthly angiogenic activity in women, preferably the peptide is derived from the beta chain of LH, preferably loop 2 of said beta chain. The beta-2-loop of human LH (hLH) has the angiogenic amino acid sequence MMRVLQAVLPPLPQVVCTYR, in which the core angiogenic sequence is located between the arginine (R) residues located at the N- and C-termini. Furthermore, this angiogenic 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 fast- and short-acting insulin formulations. The present invention also provides a method of treatment and use thereof according to the present invention, in which the peptide has an amino acid sequence derived from an extracellular matrix protein (ECM). ECM proteins contain a rich source of autophagy-inhibitory amino acids and are useful as templates for the design of autophagy-inhibitory peptides.Proline as a substrate is stored in elastin and collagen in extracellular matrix, connective tissue and bone, and is rapidly released from this reservoir by the sequential action of matrix metalloproteinases, peptidases, elastases and prolidases. As the only proteinogenic secondary amino acid, proline has a special biological effect, 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 method of treatment according to the present invention and its use therein, wherein the extracellular matrix protein is elastin. Exemplary core-angiogenic activity has been demonstrated by the peptides VGVAPG and VGVAPGVGVAPGVGVAPG herein, both of which are derived from the exon 24 region of human elastin. The present invention also provides a method of treatment and use thereof according to the present invention, wherein said extracellular matrix protein is collagen. Collagen is full of PGP sequences that may be useful. Typical of some collagens (herein an example from human COL6A5 is shown) containing an angiogenic core peptide flanked by R or K, such as RRAQGVPQIAVLVTHR, which identifies the core angiogenic sequence AQGVPQIAVLV and derivatives such as AQGVPQ, AQGVPQI, AQGVPQIA, GVPQIAVLV, PQIAVLV, IAVLV and others.Many more such sequences can be found in collagen stretches such as (in human COL6A5) RGAPGQYGEKGFPGDPGNPGQNNNIKGQKGSKGEQGRQGRSGQKGVQGSPSSRGSRGREGQRGLRGVSGEPGNPGPTGTLGAEGLQGPQGSQGNPGRKGEKGSQGQKGPQGSPGLMGAKGSTGRPGLLGKKGEPGLPGDLGPVGQTGQRGRQGDSGIPGYGQMGRKGVKGPRGFPGDAGQK, which are found to demonstrate the repeated occurrence of angiogenic core peptide sequences flanked by R or K repeats, from which desirable peptide modulators of mTOR can be selected. Another angiogenic protein, whose peptides are provided herein, is human alpha-fetoprotein, where peptides with the sequence KDLCQAQGVALQTMK, including the antigenic core QAQGVALQ, have been observed, and derivatives AQGV, AQGVA, AQGVAL, QGVALQ and GVALQ have been found, all useful as autophagy-inhibiting peptide modulators of mTOR, preferably in combination with fast-acting or short-acting insulin. Typically, autophagy inhibition by dipeptide modulators of mTOR AQ has been recently demonstrated in piglets, with improved modulation characteristics over 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.).

[0017] When said vasculopathy comprises diabetic vasculopathy, and particularly when said subject has or is suspected to have C-peptide deficiency, the autophagy-inhibiting peptide modulator of mTOR of the present invention is particularly provided for use in angiogenesis control.In many cases, such patients may already be treated with insulin (on-pump with fast-acting insulin, or subcutaneously with fast-acting, short-acting, intermediate-acting or long-acting insulin, with other growth factors, such as IGF, PDGF or VEGF or growth factors related thereto) to combat either the risk of blindness, end-stage renal failure or lower limb amputation that is commonly associated with and believed to result from diabetic vasculopathy. Such mTOR modulators find particular advantageous use in patients suffering from C-peptide deficiency, said deficiency being present, for example, when said patients have preferably recurrent fasting C-peptide levels of at least less than 1 nmol / L, preferably at least less than 0.5 nmol / L, more preferably at least less than 0.3 nmol / L, more preferably at least less than 0.2 nmol / L, more preferably at least less than 0.1 nmol / L, more preferably at least less than 0.06 nmol / L. In particular, the present invention provides the use of an autophagy inhibitory peptide modulator of mTOR in the treatment of vasculopathy, preferably diabetic vasculopathy. In another embodiment, the present invention provides the use of an autophagy inhibitory peptide modulator of mTOR in the treatment of nephropathy, preferably diabetic nephropathy. In yet another embodiment, the present invention provides the use of an autophagy inhibitory peptide modulator of mTOR in the treatment of retinopathy, preferably diabetic retinopathy. In yet another embodiment, the present invention provides the use of an autophagy inhibitory peptide modulator of mTOR in the treatment of neuropathy, preferably diabetic neuropathy.

[0018] On the other hand, the present invention provides 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 useful in glycemic control, provided with an autophagy inhibitory peptide modulator of mTOR, as provided herein, for the purpose of angiogenesis control.A formulation of a growth factor and an autophagy inhibitory modulator of mTOR 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 (in one-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%, more preferably at least 95%. In a preferred embodiment, the invention provides an insulin formulation having an autophagy inhibitor modulator of mTOR, said modulator comprising a source of amino acids, preferably a peptide, 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 said amino acids are selected from the group of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) and proline (P).In a preferred embodiment, the present invention provides an insulin formulation having an autophagy inhibitory modulator of mTOR, the modulator comprising a source of amino acids, preferably a peptide, the amino acids being selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), 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%, more preferably at least 95%. In a preferred embodiment, the present invention provides an insulin formulation having an autophagy inhibitory modulator of mTOR, the modulator comprising a source of amino acids, preferably a peptide, the amino acids being selected from the group of alanine (in one 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%, more preferably at least 95%. In a preferred embodiment, the invention provides an insulin formulation having an autophagy inhibitor modulator of mTOR, said modulator comprising a source of amino acids, preferably a peptide, 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 said amino acids are selected from the group of alanine (in one letter code: A), glutamine (Q), glycine (G) and leucine (L).In a preferred embodiment, the present invention provides an insulin formulation having an autophagy inhibitory modulator of mTOR, the modulator comprising a source of amino acids, preferably a peptide, the amino acids being selected from the group of alanine (in one letter code: A), glutamine (Q), leucine (L) and proline (P) for 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%. In a preferred embodiment, the present invention provides an insulin formulation having an autophagy inhibitory modulator of mTOR, the modulator comprising a source of amino acids, preferably a peptide, the amino acids being selected from the group of alanine (in one letter code: A), valine (V), leucine (L) and proline (P) for 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%. Formulations of insulin comprising (having) an autophagy inhibitory modulator of mTOR according to the present invention are preferred for use in inducing angiogenic activity in the prevention or treatment of vasculopathy in subjects who also need to achieve or maintain glycemic control, in particular for use in the prevention or treatment of nephropathy, for use in the prevention or treatment of end stage renal disease, for use in the prevention or treatment of retinopathy, for use in the prevention or treatment of blindness or substantial loss of vision (>30% to <90%), for use in the prevention or treatment of neuropathy, for use in the prevention or treatment of peripheral diabetic neuropathy, for use in the prevention or treatment of diabetic ulcers, in particular when said subject has or is suspected of having a C-peptide deficiency as defined herein, in particular said vasculopathy includes diabetic vasculopathy.As provided herein, such autophagy-inhibiting peptide modulators of mTOR and their use in growth factor formulations typically include a source of peptides and / or amino acids that target the mechanistic rapamycin target of mTOR, in particular the amino acid sensing system, and inhibit autophagy while inducing angiogenic activity in patients in need thereof. Typically, for glycemic control with the use of insulin formulations, the insulin requirement (units / kg / day) is determined based on the patient's age, weight, and residual pancreatic insulin activity. Patients typically require a total daily insulin dose of 0.4-1.0 units / kg / day, with a typical starting dose in metabolically stable patients being 0.5 units / kg / day. However, as noted above, glycemic control, even with insulin, is no substitute for C-peptide deficiency. This is particularly provided herein for the use of an insulin formulation which also comprises an autophagy inhibitory peptide modulator of mTOR in patients suffering from C-peptide deficiency, said deficiency e.g. existing when said patient has preferably recurrent fasting C-peptide levels of at least less than 1 nmol / L, preferably at least less than 0.5 nmol / L, more preferably at least less than 0.3 nmol / L, more preferably at least less than 0.2 nmol / L, more preferably at least less than 0.1 nmol / L, more preferably at least less than 0.06 nmol / L.

[0019] The present invention therewith provides pharmaceutical formulations, methods and means for preventing or treating the occurrence of diabetic vasculopathy in patients, such as treating patients with nephropathy (predisposing to end stage renal disease), retinopathy (predisposing to blindness) and / or neuropathy, particularly peripheral diabetic neuropathy, and finally providing treatment of vascular dysfunction, particularly predisposing to lower limb amputation in humans, ulcer patients with diabetic ulcers. Typically, a peptide is defined herein for the purposes of this disclosure as having 50 amino acids or less, and a protein is defined as having >50 amino acids. Autophagy-inhibiting peptide modulators of mTOR are defined herein as linear, branched or cyclic strings of 50 or less amino acids that contain peptide sequences with at least 50%, more preferably at least 75%, and most preferably 100% amino acids selected from the group of autophagy-inhibiting amino acids alanine (in one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), proline (P), isoleucine (I), arginine (R) and asparagine (N). The molecular mechanism of action (MoA) of this group of peptides does not depend on their exact sequence. Instead, the constituent amino acids provide an autophagy-inhibiting signal to the nutrient-sensing system of mTOR; resulting in the inhibition of autophagy and resulting in proteogenesis, with resolution of the disease.

[0020] As reviewed 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 is referred to in part 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 in sensing and integrating different 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 signaling network of mTORC2 is not as well characterized as that of mTORC1. Some studies have suggested 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 indicated that mTORC2 is involved in regulating cell survival, growth, and proliferation, and controls cell architecture and polarity. Typically, amino acids are not only building blocks of proteins, but also signaling molecules and regulators of gene expression, metabolic processes, and developmental body changes, with the crucial roles of amino acids and their metabolites in health and disease. Ample evidence indicates that amino acids play a fundamental role in the vascular system.Amino acids serve as basic building blocks for protein synthesis and constitute an important energy source, but a select group has been extensively studied in the context of vascular diseases. mTOR, especially 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 are not only involved in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction, which controls major metabolic pathways. Among these is the autophagy pathway, which is responsible for the degradation of long-lived 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 the etiology of several pathologies. Clearly, only certain amino acids can modulate autophagy, and their function is highly cell-specific. However, many of the details of the relationship between certain amino acids and cellular metabolism are unclear (see further Figure 5).

[0021] Further embodiments 1. A method for identifying a source of, preferably a peptide, of preferably L-proteogenic amino acids capable of inducing angiogenic activity, comprising the steps of providing a cell with a peptide comprising an L-proteogenic amino acid, said amino acid being selected for at least 50% from the group of alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R) and determining the angiogenic activity in a sprouting assay. 2. A method for inducing angiogenic activity comprising the step of providing a cell with a source of amino acids, preferably L-proteogenic, wherein said amino acids are selected from the group consisting of alanine (in the one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R) over at least 50% of the time. 3. A method for inducing angiogenic activity comprising the step of providing a cell with a peptide as a source of amino acids, preferably L-proteogenic, wherein the peptide consists, for at least 50% of the amino acids, of alanine (in the one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R). 4. The method according to further embodiment 2 or 3, wherein said source of preferably L-proteogenic amino acids, preferably peptides, is identifiable by the method according to further embodiment 1. 5. The method according to any one of the preceding embodiments, wherein the amino acids are selected from the group consisting of alanine (in the one-letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L) and proline (P) at least 50% of the time. 6. The method of any one of further embodiments 2-5, wherein the source or peptide has an amino acid sequence derived from a peptide or protein shown to have or suspected of having angiogenic activity. 7. The method of further embodiment 6, wherein the peptide has an amino acid sequence derived from chorionic gonadotropin. 8. The method of further embodiment 6, wherein the peptide has an amino acid sequence derived from the C-peptide. 9. The method of further embodiment 6, wherein the peptide has an amino acid sequence derived from galectin-3. 10. The method of further embodiment 6, wherein the peptide has an amino acid sequence derived from luteotropin. 11. The method of further embodiment 6, wherein the peptide has an amino acid sequence derived from an extracellular matrix protein. 12. The method of further embodiment 12, wherein the extracellular matrix protein is elastin or collagen. 13. The method of any one of further embodiments 1 to 12, wherein the 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. 14. The method of any one of further embodiments 1 to 13, 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. 15. The method of any one of further embodiments 1-14, wherein said cells also contain a compound capable of glycemic control, such as insulin or a derivative thereof. 16. The method of further embodiment 15, wherein said insulin is a fast-acting insulin. 17. The method of further embodiment 16, wherein said source comprises peptides containing more than 3 amino acids and fewer than 13 amino acids. 18. The method of further embodiment 15, wherein the insulin is a short-acting insulin. 19. The method of further embodiment 18, wherein said source comprises peptides containing more than 4 amino acids and fewer than 21 amino acids. 20. The method of further embodiment 15, wherein said insulin is an intermediate-acting insulin. 21. The method of further embodiment 20, wherein said source comprises peptides containing more than 6 amino acids and fewer than 26 amino acids. 22. The method of further embodiment 15, wherein said insulin is a long-acting insulin. 23. The method of further embodiment 22, wherein said source comprises peptides containing more than 7 amino acids and fewer than 31 amino acids. 24. The method according to any one of further embodiments 1 to 23, 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 of acetic acid, more preferably of tartaric acid, most preferably of citric acid. 25. The method of any one of further embodiments 1-24, wherein said cells are of human origin. 26. The method of any one of further embodiments 1-25, wherein the peptide is of human origin. 27 Use of a source of amino acids, preferably L-proteogenic, preferably peptides, for the manufacture of a pharmaceutical composition for the treatment of a subject having or suspected of having a vasculopathy, preferably diabetic peripheral neuropathy, wherein said amino acids are selected over at least 50% from the group alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R). 28. The use according to further embodiment 26, wherein said subject has or is at risk of developing a diabetic ulcer, preferably a foot ulcer. 29. The use according to further embodiment 26 or 27, wherein said subject has or is suspected of having a C-peptide deficiency. 30. The use according to any one of further embodiments 26 to 28, wherein the subject has a fasting C-peptide level of at least less than 1 nmol / L, preferably at least less than 0.5 nmol / L, more preferably at least less than 0.3 nmol / L, more preferably at least less than 0.2 nmol / L, more preferably at least less than 0.1 nmol / L, more preferably at least less than 0.06 nmol / L. 31. The use according to any one of further embodiments 26 to 29, 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 of acetic acid, more preferably of tartaric acid, most preferably of citric acid. 32. The use according to any one of further embodiments 26 to 30, wherein said treatment comprises a method for inducing angiogenic activity according to any one of further embodiments 2 to 25. 33 A source of amino acids, preferably L-proteogenic, preferably peptides, in the treatment of subjects having or suspected of having a vasculopathy, preferably diabetic peripheral neuropathy, wherein said amino acids are selected over at least 50% from the group alanine (in one letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P) and arginine (R). 34. A source according to further embodiment 30, wherein said subject has or is at risk of developing a diabetic ulcer, preferably a foot ulcer. 35. A source according to further embodiment 30 or 31, wherein said subject has or is suspected of having a C-peptide deficiency. 36. A source according to any one of further embodiments 32-34, wherein the subject has a fasting C-peptide level of at least less than 1 nmol / L, preferably at least less than 0.5 nmol / L, more preferably at least less than 0.3 nmol / L, more preferably at least less than 0.2 nmol / L, more preferably at least less than 0.1 nmol / L, more preferably at least less than 0.06 nmol / L. 37. The source according to any one of further embodiments 32 to 35, 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 of acetic acid, more preferably of tartaric acid, most preferably of citric acid. 38. A source according to any one of further embodiments 30-33, wherein said treatment comprises a method for inducing angiogenic activity according to any one of further embodiments 2-24. 39. A method of treatment of a subject having or suspected of having a vasculopathy, preferably diabetic peripheral neuropathy, more preferably wherein said subject has or is at risk of developing a diabetic ulcer, preferably a foot ulcer, said method comprising the step of providing to said subject a source of an amino acid according to any one of further embodiments 30 to 34. [Brief description of the drawings]

[0022] [Figure 1] The need for angiogenesis control concomitant with glycemic control is particularly well illustrated in the case of diabetic vasculopathy with diabetic peripheral neuropathy (DPN), which invariably develops in a large set of patients with type 1 or type 2 diabetes with C-peptide deficiency. DPN predisposes to foot ulcers that can develop into foot gangrene. This ranges between (Figure 1A) dry gangrene, an ulcer with the development of ischemic and necrotic tissue but without infection, and (Figure 1B) wet gangrene, with both infected and necrotic tissue. Dry gangrene may lead to wet gangrene. In the more pronounced dry gangrene stage than depicted here, the affected areas are often characterized by a dark green or purple, almost black color. The skin may be dry and wrinkled due to lack of oxygen. Wet gangrene has a moist appearance. This form is characterized by blisters and swelling that point to fulminant inflammation. [Diagram 2]Similarly, the need for angiogenesis control concomitant with glycemic control is particularly well illustrated in the case of diabetic peripheral neuropathy (DPN) and diabetic vasculopathy with nephropathy that eventually develop in a large set of patients with type 1 diabetes or type 2 diabetes with C-peptide deficiency. 2A Adjusted survival curves from Cox proportional hazards models according to lower extremity amputation (LEA) status and renal function status. 2A Compared with the small LEA group, the adjusted hazard ratio for mortality was 1.80 (95% CI 1.05-3.09, P=0.03) for patients with large LEA. 2B Compared with the normal renal function group, the adjusted hazard ratio for mortality was 0.87 (95% CI 0.48-1.59, P=0.66) for CKD patients and 2.09 (95% CI 0.99-4.41, P=0.05) for patients undergoing dialysis. [Diagram 3] Glutamine inter-tissue metabolic flux, which begins in skeletal muscle, liver and intestine, continues in immune cells.Abbreviations: Glutamine, GLN; Glutamate, GLU; Aspartate, ASP; Arginine, ARG; Leucine, LEU; Alanine, ALA; Glucose, Gluc; Pyruvate, Pyr; Pyruvate dehydrogenase; PDC; Pyruvate carboxylase, PC; Malate dehydrogenase, MD; Glyceraldehyde-3-phosphate, G3-P; Lactate, Lac; Triacylglycerol, TG; Ribose 5-phosphate, R5P; Alanine aminotransferase, ALT; Glutamate dehydrogenase, GDH; Glutamine synthetase, GS; glutaminase, GLS; inducible nitric oxide synthase, iNOS; intracellular heat shock protein, iHSP; heat shock factor 1, HSF-1; heat shock element, HSE; sirtuin 1, SIRT1; hexosamine biosynthetic pathway, HBP; ammonia, NH3; glutathione, GSH; oxidized GSH, GSSG; glutathione S-reductase, GSR; protein kinase B, Akt; AMP-activated protein kinase, AMPK; mTOR complexes 1 and 2, mTORC1 / 2, extracellular signal-regulated kinase, ERK; c-Jun N-terminal kinase, JNK; gamma-aminobutyric acid, GABA. [Figure 4]The mechanism of mTOR complex activation by glutamine binding to the Pib2 complex, which directly binds glutamine, activates the TOR complex, induces cell growth, and inhibits autophagy. When glutamine is available, cells initiate growth, but when it is not available, they initiate autophagy. [Diagram 5]The elastin receptor complex (ERC) binding motif xGxxPx is present in a peptide fragment derived from approximately positions 41-57 in loop 2 of beta-human chorionic gonadotropin (beta-hCG). Left: Structure of beta-human chorionic gonadotropin (beta-hCG) with loop 2 indicated in grey, redrawn from Lapthorn AJ, Harris DC, Littlejohn A, Lustbader JW, Canfield RE, Machin KJ, et al. Crystal structure of human chorionic gonadotropin. Nature. 1994;369(6480):455-61. Top right: Amino acid sequence of loop 2 of beta-hCG. The xGxxPx motif is represented by the hexapeptide QGVLPA. Various "nicked" or fragmented peptides of beta-hCG are proteolytically derived from the peptide backbone between amino acids 40-54 (Birken S, Berger P, Bidart JM, Weber M, Bristow A, Norman R, et al. Preparation and Characterization of New WHO Reference Reagents for Human Chorionic Gonadotropin and Metabolites. Clinical Chemistry. 2003;49(1):144-54). All amino acid sequences are represented in single letter code. Bottom right: Various synthetic peptides derived from nicked fragments of loop 2, such as MTRVLQGVLPALPQ, cyclic CVLQGVLPALPQVVC, VLQGVLPALPQVVC, VLQGVLPALPQ, LQGV, AQGV, VLPALP and VLPALPQ, are biologically active when tested in immune and / or vascular cells. Receptors for these fragments have not been disclosed to date. [Figure 6]The activity of human C-peptide depends on the xGxxPG motif and is blocked by antagonists of the ERC. A: Synthetic human C-peptide (1-31, with the ERC binding motif LGGGPG) was tested in a human CD4+ lymphocyte migration assay along with negative and treatment controls: no C-peptide added, a scrambled version of human C-peptide (1-31, lacking any xGxxPx motif) and porcine C-peptide (1-29, approximately 74% similar to human C-peptide but lacking any xGxxPx motif; alignment with human C-peptide shown herein). Freshly isolated human CD4+ cells were incubated with the indicated C-peptide variants (10 nM) for 3 h to assess the effect on cell migration in a modified Boyden chamber. Results are expressed as mean ± SEM. ****P<0.001 treated vs. control. B: Freshly isolated human CD4 cells were preincubated with V14 peptide (10 μM) or lactose (10 mM) for 3 h and then stimulated with C-peptides (1–31) for 3 h as indicated in (A). Results are expressed as mean ± SEM. *P<0.05, ****P<0.001 treated vs. control, ####P<0.001 treated vs. human C-peptide (1–31). To illustrate the lack of an ERC-binding motif in porcine C-peptides versus their presence in human C-peptides, a CLUSTAL O (1.2.4) multiple sequence alignment of human and porcine C-peptides is shown. [Figure 7]Nutrient sensing is important for sustaining normal cell growth and proliferation. mTOR complex 1 (mTORC1) senses nutrients to regulate cell growth, autophagy, and other mTORC1-mediated processes. Growth factors such as insulin, IGF-1, PDGF, and VEGF, amino acids, energy status, and stress control mTORC1. Amino acids are essential for mTORC1 activation. Growth factors alone cannot achieve maximal mTORC1 activity without amino acid supplementation (Sancak et al., The Rag GTPases bind raptor and mediate amino acid signaling to mTORC1. Science. 2008 Jun 13;320(5882):1496-501.). Increased amino acid concentrations promote mTORC1 lysosomal localization and subsequent activation. Typically, some amino acids activate mTOR more than others, thereby inhibiting autophagy more than others. Autophagy-inhibiting peptide modulators of mTOR preferentially include peptides with a relative increased content of these autophagy-inhibiting amino acids to best activate mTOR. Such peptides (shown herein as QVGQVELGGGPGAGSLQP (derived from human C-peptide), QGVLPA or AQGV (both derived from hCG), as well as others disclosed in this application) can enter cells by a common mechanism of extracellular hydrolysis and uptake by various amino acid transporters, as depicted in the upper left. Intracellular glutamine also has the ability to exchange with extracellular essential amino acids (https: / / www.nature.com / articles / ncomms11457 - ref-CR14), especially leucine. Peptide entry is depicted in the upper right. Extracellular di- and tripeptides can enter via peptide transporter (PEPT1 / 2)-regulated transport. Larger peptides can enter via (receptor-mediated) endocytosis or phagocytosis.Arginine-rich cell-penetrating peptides can passively enter vesicles and live cells by inducing membrane multilamellarity and fusion. Further intracellular hydrolysis of the autophagy-inhibiting peptide liberates autophagy-inhibiting amino acids that modulate mTOR, signaling a multitude of processes that maintain a balance between metabolic synthesis (protein synthesis) or degradation (autophagy), ranging from cell survival to cell death. [Figure 8] Microvascular angiogenesis (sprouting) assay of human pulmonary microvascular endothelial cells grown in Matrigel in response to increasing concentrations of the peptides hexapeptide QGVLPA (derived from hCG) and the 18-mer peptide QVGQVELGGGPGAGSLQP (derived from human C-peptide). Data shown are total tube length measurements at t=24 hours. *p<0.05; **p<0.01. The hexapeptides VGVAPG (derived from human elastin), LGGGPG (derived from human C-peptide) and PGAYPG (derived from human galectin-3) also significantly induced angiogenic activity in this experiment (not shown). [Figure 9] Microvascular angiogenesis (sprouting) assay of human pulmonary microvascular endothelial cells grown in Matrigel in response to increasing concentrations of peptides 3 (18-mer peptide VGVAPGVGVAPGVGVAPG, derived from human elastin), 6 (18-mer peptide QVGQVELGGGPGAGSLQP, derived from human C-peptide) and 8 (18-mer peptide GAYPGAPGAYPGAPAPGV, derived from the human N-terminal fragment of galectin-3) (Figures 9a, 9b and 9c, respectively). Data shown are total tube length and branch length measurements at t=24 hours. *p<0.05; **p<0.01; ***p<0.001. [Figure 10]Determination of EC50, the pharmacological concentration required to obtain 50% angiogenic activity in tube or branch formation, of the autophagy inhibitory peptide modulators of mTOR VGVAPGVGVAPGVGVAPG (derived from human elastin), QVGQVELGGGPGAGSLQP (derived from human C-peptide) and GAYPGAPGAYPGAPAPGV (derived from human galectin-3), examined in FIG. [Figure 11] Cutaneous wound healing is a comprehensive and complex process involving inflammatory response, angiogenesis, new tissue formation and tissue remodeling, consisting of proliferation and migration of various cell types (inflammatory cells, keratinocytes, endothelial cells (ECs), fibroblasts, platelets) that ultimately restore the integrity of the skin barrier. Angiogenesis in wound healing involves modulation of angiopoetin 1 (ANG1) versus angiopoetin 2 (ANG2), along with the PI3K / AKT / mTOR pathway in angiogenesis. The PI3K / AKT / mTOR pathway modulates the expression of angiogenic factors such as nitric oxide and angiopoietin. Numerous inhibitors of the PI3K / AKT / mTOR pathway have been developed, and such agents have been shown to reduce VEGF secretion and angiogenesis, thereby impeding wound healing. On the contrary, activation of mTORC1 has actually been shown to promote angiogenesis (Karar J, Maity A. PI3K / AKT / mTOR Pathway in Angiogenesis. Front Mol Neurosci. 2011 Dec 2;4:51). The autophagy-inhibiting peptide modulators of mTOR provided herein activate mTOR, thereby increasing VEGF secretion and angiogenesis in wound healing. [Figure 12] Yet another factor that contributes to diminished wound healing includes hyperglycemia, illustrating the importance of maintaining proper glycemic control. [Figure 13]Determination of C-peptide deficiency. Qiao et al. (C-peptide is independent associated with diabetic peripheral neuropathy: a community-based study. Diabetol Metab Syndr 9, 12 (2017).) investigated the relationship between residual C-peptide and DPN in patients with type 2 diabetes. With increasing duration of diabetes, islet function gradually shrinks, resulting in reduced C-peptide and insulin levels, and the prevalence of DPN increases. Fasting C-peptide, 2-hour postprandial C-peptide, and ΔC-peptide (i.e., 2-hour postprandial C-peptide, minus fasting C-peptide) serum concentrations in the non-DPN group were significantly higher than those in the clinical DPN group (all P ≤ 0.040) and confirmed DPN groups (all P < 0.002). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] Autophagy Inhibitory Peptides 1-character code In describing protein or peptide composition, structure and function herein, reference is made to amino acids. Amino acid residues are identified herein by using the following abbreviations. Also, unless expressly indicated otherwise, the amino acid sequences of peptides and proteins are identified from N-terminus to C-terminus, left to right, with the N-terminus being identified as the first residue. Ala: alanine residue; Asp: aspartic acid residue; Glu: glutamic acid residue; Phe: phenylalanine residue; Gly: glycine residue; His: histidine residue; Ile: isoleucine residue; Lys: lysine residue; Leu: leucine residue; Met: methionine residue; Asn: asparagine residue; Pro: proline residue; Gln: glutamine residue; Arg: arginine residue; Ser: serine residue; Thr: threonine residue; Val: valine residue; Trp: tryptophane residue; Tyr: tyrosine residue; Cys: cysteine ​​residue. Amino acids may also be referred to by their conventional one-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.

[0024] peptide Peptide is intended herein to mean a natural biological or artificially produced (synthetic) short chain of amino acid monomers linked by peptide (amide) bonds. Glutamine peptide is intended herein to mean a natural biological or artificially produced (synthetic) short chain of amino acid monomers linked by peptide (amide) bonds, one of said amino acid monomers being glutamine. Chemically synthesized peptides generally have free N- and C-termini. N-terminal acetylation and C-terminal amidation reduce the overall charge of the peptide; therefore, its overall solubility may decrease. However, terminal acetylation / amidation may also increase the stability of the peptide, since it produces a more similar mimic of the native protein. These modifications can increase the biological activity of the peptide, and are also provided herein.

[0025] Peptide synthesis In this application, peptides are synthesized by classically known chemical synthesis on solid support (Ansynth BV, Roosendaal, The Netherlands) or in solution (Syncom BV, Groningen, The Netherlands and Diosynth BV, Oss, The Netherlands). Pharmaceutical peptide compositions can be synthesized using trifluoroacetate as counterion or salt, which is then exchanged by counterions such as maleate (from maleic acid), acetate (from acetic acid), tartrate (from tartaric acid) or citrate (from citric acid). The drug substance (EA-230) of AQGV for use in preclinical and clinical human studies was manufactured by Organon NV (formerly Diosynth BV) (Oss, The Netherlands), while the filling and finishing of the final product was performed by Octoplus Development, Leiden (The Netherlands). The molecular weight of EA-230 (AQGV) is 373 g / mol.

[0026] Determination of chemotactic activity Human U937 monocytic cells are purchased from the American Type Culture Collection (ATCC catalog number CRL-1593.2, Manassas, Va). Cells are maintained in suspension culture in T-75 flasks containing RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics, and cultures are split every 3-5 days. Three days prior to use in chemotaxis assays, U937 cells are stimulated to differentiate along the macrophage lineage by exposure to 1 mmol / L dibutyryl cyclic adenosine monophosphate (dbcAMP; Sigma Chemical Co) as described. Cells are washed three times to remove culture medium and then resuspended in chemotaxis medium (Dulbecco's modified essential medium supplemented with 1% lactalbumin hydrolysate) for plating into the assay chamber at a final concentration of 2.5 x 106 cells / mL. Chemotaxis assays are performed in 48-well microchemotaxis chambers (Neuro Probe, Cabin John, Md). The bottom wells of the chambers are filled with 25 mL of chemotactic stimuli (or medium alone) in triplicate. An uncoated 10 mm thick polyvinylpyrrolidone-free polycarbonate filter with a pore size of 5 mm is placed on top of the sample (Neuro Probe). A silicone gasket and the top piece of the chamber are applied, and 50 mL of the monocyte cell suspension is placed in the top well. The chambers are incubated at 37°C in a humidified 5% CO2 atmosphere for 3 hours, and non-migrating cells are gently wiped off from the top surface of the filter. The filters are immersed in a methanol-based fixative for 30 seconds, stained with a modified Wright-Giemsa technique (Protocol Hema 3 stain set; Biochemical Sciences, Inc, Swedesboro, NJ), and then mounted on glass slides. Cells that have completely migrated through the filter are counted under a light microscope, counting three random high power fields (HPF; original magnification x400) per well.Human monocytes are isolated from freshly drawn blood of healthy volunteers using sequential Ficoll / Pelastin receptor complex (ERC) oll gradient centrifugation as described elsewhere. Cells are cultured for 16 h in RPMI-1640 medium supplemented with 0.5% human serum to become quiescent after isolation. Cell purity is >95% as determined by flow cytometric analysis. Monocyte chemotaxis is assayed in serum-free medium in 48-well microchemotaxis chambers (Neuroprobe, Gaithersburg, MD). Wells in the upper and lower chambers are separated by polyvinylpyrrolidone-free polycarbonate membranes (pore size 5 μm; Costar). Freshly isolated monocytes at a density of 5 × 105 / mL are incubated with recombinant C-peptide (Sigma) for 2.5 h, after which migrated cells on the bottom surface of the filter are stained and counted under a light microscope. Maximal chemotactic activity was measured with 0.1 mmol / L N-formyl-methionyl-leucyl-phenylalanine (f-MLF; Sigma Chemical Co.) and checkerboard analysis was used to distinguish chemotaxis from chemokinesis.

[0027] Cell isolation Blood is collected from healthy volunteers into tubes containing citrate as an anticoagulant. Neutrophils are isolated by using the Polymorphprep kit (Nicomed, Oslo, Norway) according to the manufacturer's instructions; monocytes are purified using magnetic beads (Miltenyi Biotech). Cell purity is >93%, as assessed by flow cytometry (anti-CD45, 14, DR and CD66b). For each cell type, samples from two different donors are tested.

[0028] Distribution and excretion of intravenously injected [14C]-AQGV in mice The study, conducted by TNO Biosciences, Utrechtseweg, Netherlands, demonstrated the potent potency of [14C]-AQGV (Ala-Gln-[1- 14 The study was designed to provide data on the distribution and metabolism of radiolabeled AQGV ([C]Gly-Val). For this purpose, mice were sacrificed 10, 30 and 60 min and 6 and 24 h after administration of radiolabeled AQGV, counts in various tissues were determined, and the radioactivity present in urine and plasma was analyzed by HPLC.

[0029] Ten minutes after the radiolabeled peptide was injected, there was relatively little radioactivity in the blood. If all of the counts were in the intact peptide, the amount present would be 17.2 μg / g. No parent compound could be detected in the plasma after 10 minutes; 14 [C]-AQGV appeared to be very rapidly hydrolyzed. No parent compound was detected in urine either. Radioactivity in urine was mostly present as hydrophilic compounds eluting in or shortly after the dead volume of the HPLC column.

[0030] The radioactivity present in the various organs exceeded that in the blood. The highest concentrations of the "peptide" after 10 min were found in the kidneys (362 μg / g), liver (105 μg / g), testes (85.7 μg / g), lungs (75.2 μg / g) and spleen (74.7 μg / g). In general, a gradual decrease in radioactivity was observed thereafter. After 24 h, the highest concentrations were found in the kidneys (61.9 μg / g), thymus (43.1 μg / g), spleen (39.3 μg / g), liver (37.6 μg / g) and skin (37.5 μg / g).

[0031] The mean total recovery of radioactivity at 10, 30, and 60 min and at 6 and 24 h after dose administration was 83.2, 70.5, 62.9, 52.6, and 50.8%, respectively. These results suggest that the post-dose [14C]-volatiles, most likely due to exhaled air, are the source of the radioactivity. 14This strongly indicates that there is a rapid formation of C-CO2. After 24 hours, 10.2% of the administered radioactivity was excreted in the urine and 2.6% in the feces.

[0032] - Conclusion After intravenous injection, 14 [C]-AQGV was rapidly cleared from the blood, which is consistent with the results of the pharmacokinetic studies presented below. Metabolite profiles in plasma and urine revealed the absence of the parent compound, and [ 14 These results indicate a rapid metabolism of [C]-AQGV. Approximately 50% of the administered radioactivity is released into the blood as volatile substances, most likely 14 The results of this study were as follows: 14 C]-glycine 14 C]-AQGV, indicating rapid hydrolysis of [1- 14 C]-glycine is then 14 The absence of parent compound in plasma and urine indicates that the radioactivity present in tissues and organs is [ 14 These results suggest that it may exist only as a hydrolysis product of the metabolism of [C]-AQGV.

[0033] Peptide Hydrolysis The present disclosure provides that when a peptide having an autophagy-inhibiting amino acid, such as peptide AQGV, encounters a cell, the peptide is hydrolyzed at the cell's surface if extracellular, or in the case of vascular cells, for example, in the phagolysosome after endocytosis of the peptide by the cell via elastin receptor-mediated endocytosis.Many peptidases are known to exist on the cell surface or intracellularly, which can rapidly hydrolyze peptides, and continued hydrolysis necessarily produces tripeptides and dipeptides.Similarly, hydrolysis in lysosomes by tripeptidyl and dipeptidyl peptidases will equally produce single amino acids. 14Studies with C-labeled AQGV showed virtually complete hydrolysis of the peptide 15 min after its administration in mice. Tripeptides, dipeptides and single amino acids could result from hydrolysis of AQGV or its sister compound LQGV, or any other suitable oligopeptide for that matter, when presented to cells.

[0034] Peptide Transport Similarly, several studies have reported the role of p38 MAPK in the survival of different types of mature granulocytes. Granulocytes (e.g., neutrophils, eosinophils, basophils) have in common their terminal differentiation stage. These cells have fragmented nuclei and have accumulations of granules containing preformed secretory factors. Herein, it was proposed that p38 MAPK is required for the survival of neutrophils, and that inactivation of p38 MAPK is essential for the death and elimination of these cells, and that p38 MAPK is required for the contraction of endothelial cells, and that inactivation of p38 MAPK is essential for the relaxation of such vascular cells so that they can restore vascular wall integrity, and that inactivation of p38 MAPK activity is essential for calming neutrophils and other white blood cells that explore the vascular permeability of the vascular endothelial vessel wall.

[0035] Di- and tripeptides are preferentially transported via the PEPT1 / 2 transporters. Tripeptides, dipeptides and single amino acids are actively transported across the cell membrane, whereby the uptake of di- and tripeptides involves a separate mechanism from the uptake of single amino acids, i.e., via the PEPT1 and PEPT2 transporters. Potentially, a total of 400 dipeptides and 8,000 tripeptides can be transported by PepT1 and PEPT2. It has been demonstrated that enterocyte transport of amino acids in the form of peptides is a faster route of uptake per unit time than their constituent amino acids in free form (reviewed in J Anim Sci, 2008; 9, 2135-2155).

[0036] mTOR is involved Finally, we propose the involvement of the mechanistic rapamycin target, mTOR. In this perspective, the peptide enters the cell via PEPT1 / 2 or by active endocytosis or phagocytosis processing, and then the peptide is completely hydrolyzed in the phagolysosome, and the resulting autophagy-inhibiting amino acids are presented to the mTOR complex, where they cause the inhibition of cellular autophagy. The tetrapeptide, tripeptide and dipeptide activities can all reflect the final causal activity of single amino acids A, Q, G, V, selected from the group of amino acids A, Q, G, V, L and P. In this way, amino acids A, Q, G, V, L and P are food for mTOR. Indeed, when different tripeptides and dipeptides derived from AQGV are used in the FPR signaling assay, preliminary results show similar effects in inhibiting the p38 pathway. While individual amino acids can approach mTOR via the cytosol, amino acids in peptide fragments (strings such as AQGV) may enter the mTOR machinery via the phagolysosome. Thus, activation of mTOR by amino acids in sources such as peptides can be explained from two perspectives: 1) whereby endocytosis of peptide strings is highest for all phagocytic cells, e.g., neutrophils and monocytes, and 2) whereby peptide fragments enter via PEPT1 / 2.

[0037] Amino acids activate the mTOR pathway and inhibit autophagy Autophagy functions to produce amino acids for cell survival when nutrients are insufficient, and amino acids are effective inhibitors of autophagy. The mechanistic target of rapamicin (mTOR) is a crucial regulator of autophagy induction, where activated mTOR suppresses autophagy and negative regulation of mTOR promotes it. Amino acids are indeed considered to be key regulators of mTOR complex 1 or 2 activation, affecting cell proliferation, protein synthesis, autophagy and survival. These findings identify new signaling pathways used by amino acids, highlight the crucial importance of these nutrients in cellular metabolism, and propose new mechanistic insights in the development of pharma- ceutical active peptides.

[0038] Differential signaling of amino acids to the mTOR pathway Some amino acids are known to regulate protein production (mTOR kinase) or protein degradation (autophagy) more than others. Recent and earlier data identify leucine (L), valine (V), isoleucine (I), alanine (A), glutamine (Q), arginine (R), glycine (G), proline (P) and asparagine (N), alone or in combination, as more potent activators of mTOR or inhibitors of autophagy than other amino acids such as glutamic acid (E), threonine (T), serine (S), lysine (K), threonine (T), phenylalanine (F), tyrosine (Y) and methionine (M), which were reported to have no effect or opposite effects. The amino acids leucine (L), alanine (A), glutamine (Q) and proline (P) are reported to have the most significant autophagic effects in human cells (AJ Meijer et al Amino Acids 2015, 47, 2037-2063.).

[0039] Several autophagy-inhibitory peptide modulators of mTOR and organic salts of some such peptides have previously been found to be useful in treating inflammation or addressing problems of hemodynamic instability in PCT / NL2018 / 052822, PCT / NL2020 / 050535, PCT / NL2020 / 050605 or PCT / NL2021 / 050223. In the present application, it is disclosed that this class of autophagy-inhibitory peptides has angiogenic properties as shown in a human vascular endothelial cell sprouting assay and is useful in healing diabetic (foot) ulcers in humans.

[0040] Some of these peptides possess binding motifs for the human elastin receptor complex (ERC); such a binding motif xGxxPx is present, for example, in a peptide fragment derived from approximately positions 41-57 in loop 2 of β-human chorionic gonadotropin (beta-hCG) (see also FIG. 5). Small peptides derived from approximately positions 41-57 (some designate positions 40-54) in β-human chorionic gonadotropin (beta-hCG) may protect both mother and child from maternal immune responses during pregnancy (US6583109B1, US7358330B2, Khan, Immunoregulatory Properties of Break Down Products of Human Choriogonadotropin, thesis, 2010). US6583109B1 identifies LQGVLPALPQVVC as involved, Khan (supra, 2010) identifies MTRVLQGVLPALPQ as involved, and US7358330B2 identifies the consensus motif LQGVLPALPQ (recognized in the present application by the core ERC binding motif QGVLPA) as at least involved in providing said protection. Khan (supra, 2010) examines LQGV and VLPALP in type 1 diabetes (T1D): - both peptides significantly (p<0.0001) delay the onset of T1D in NOD mice. Biotempt (US7358330B2) tested LQGV, VLPALP and the respective hCG-derived alanine-supplemented peptides in an in vitro LPS study: LQGV, AQGV, LAGV, LQAV, LQGA, VLPALP, ALPALP, VAPALP, VLAALP, VLPAAP and VLPALA. All significantly inhibited LPS-activity in vitro.Van den Berg et al. (Synthetic oligopeptides related to the [beta]-subunit of human chorionic gonadotropin attenuate inflammation and liver damage after (trauma) haemorrhagic shock and resuscitation. Shock. 2009 Mar;31(3):285-91) examined LQGV, AQGV and LAGV in vivo in a rat model of hemorrhagic shock. All three significantly (p<0.001) prevented systemic release of TNF-α and IL-6. The activity of human C-peptide also depends on the xGxxPG motif and is blocked by antagonists of ERC (see also Figure 6). Figure 6A shows that CD4 + We have shown the upregulatory effect of xGxxPG-bearing human C-peptide on lymphocyte migration (Kwee et al., CD4 T-cells regulate angiogenesis and myogenesis. Biomaterials. 2018 Sep;178:109-121). We also found that the prototype xGxxPG-binding motif of the VGVAPG elastin peptide upregulates IL-4 production in CD4(+) T-cells (Lemaire et al. The elastin peptide VGVAPG increases CD4(+) T-cell IL-4 production in patients with chronic obstructive pulmonary disease. Respir Res. 2021;22(1):14). + Modulation of lymphocyte IL-4 production and similar CD4 activity of elastin-derived peptides in angiogenesis + The binding activity of Gal-3 is indicative of the binding activity of Gal-3. Scrambled C-peptide and porcine C-peptide, which do not have the xGxxPG motif, did not affect CD4+ migration. Gal-3 inhibits CD4 + The C-peptide was not tested in a similar assay because it is considered to be expressed by CD4 +We show that the migratory effect is blunted by V14 peptide and lactose, which are specific inhibitors of xGxxPG-elastin receptor binding, respectively, as demonstrated in the art for peptides with the motif VGVAPG. Thus, the binding of the central part (fragment) of human C-peptide is elastin receptor specific and dependent on the presence of the xGxxPG motif in angiogenesis, further explaining that such peptides with xGxxPG enter cells by receptor-mediated endocytosis. Indeed, Luppi et al. (Luppi et al., C-peptide is internalised in human endothelial and vascular smooth muscle cells via early endosomes. Diabetologia. 2009 Oct;52(10):2218-28.) show that C-peptide enters cells by endocytosis and signals from within endosomes. According to Luppi et al. (op. cit.), endosomes represent signaling stations where C-peptide can achieve its cellular effects. This explains the conditions necessary for amino acids to hydrolyze, activate mTOR, and inhibit autophagy. Typically, some amino acids activate mTOR more than others, thereby inhibiting autophagy more than others (see also Figure 7). Alanine has been shown to have a very specific coregulatory effect on autophagy (Meijer et al., Regulation of autophagy by amino acids and MTOR-dependent signal transduction. Amino Acids. 2015 Oct;47(10):2037-63;Kadowaki et al. Nutrient control of macroautophagy in mammalian cells. Mol Aspects Med. 2006 Oct-Dec;27(5-6):426-43).The combination of alanine and leucine at physiological concentrations induces the same inhibitory effect on autophagic protein degradation in rat hepatocytes as the complete mixture of all 20 amino acids (Meijer et al., supra). This same study also showed that alanine metabolism is required for its inhibitory effect on autophagy. Also, the role of L-glutamine (Gln) in nutrition and health has been extensively reported, but its effects in the cardiovascular system have only recently come to light (Bertero et al., The molecular rationale for therapeutic targeting of glutamine metabolism in pulmonary hypertension. Expert Opin Ther Targets. 2019 Jun;23(6):511-524). Tan et al. (Glutamine metabolism regulates autophagy-dependent mTORC1 reactivation during amino acid starvation. Nat Commun. 2017 Aug 24;8(1):33) show that glutamine metabolism is sufficient to restore mTORC1 activity during prolonged amino acid starvation in an autophagy-dependent manner. Ukai et al. (Gtr / Ego-independent TORC1 activation is achieved through a glutamine-sensitive interaction with Pib2 on the vacuolar membrane. PLoS Genet 14(4): e1007334. https: / / doi.org / 10.1371 / journal.pgen.1007334, 2018) show that two molecular mechanisms, Pib2 and Gtr / Ego, form distinct complexes with TORC1 in a mutually exclusive manner, implying exclusive functional relationships between TORC1 and Pib2 or Gtr / Rag in response to various amino acids.They also show that amino acid-dependent activation of TORC1 is achieved by Pib2 and the Gtr / Ego pathway by tethering them to the vacuolar membrane, show that glutamine directly binds to the Pib2 complex and enhances Pib2-TORC1 complex formation, and find Pib2 as an element of a putative glutamine sensor for mTOR (see also Figure 4). Intracellular glutamine also has the ability to exchange for extracellular essential amino acids (https: / / www.nature.com / articles / ncomms11457 - ref-CR14) (Nicklin et al., Bidirectional transport of amino acids regulates mTOR and autophagy. Cell. 2009 Feb 6;136(3):521-34), thereby further regulating mTOR activity (Jewell Metabolism. Differential regulation of mTORC1 by leucine and glutamine. Science. 2015 Jan 9;347(6218):194-8. doi: 10.1126 / science.1259472. Epub 2015 Jan 7). Sun et al. (Glycine Regulates Protein Turnover by Activating Protein Kinase B / Mammalian Target of Rapamycin and by Inhibiting MuRF1 and Atrogin-1 Gene Expression in C2C12 Myoblasts. J Nutr. 2016 Dec;146(12):2461-2467.) find that glycine regulates protein turnover by activating mTORC1 and inhibiting the expression of genes for protein degradation.In addition, glycine removal by decarboxylase (GLDC, an enzyme belonging to the glycine cleavage system) stimulates autophagy (Zhuang et al., Glycine decarboxylase induces autophagy and is downregulated by miRNA-30d-5p in hepatocellular carcinoma. Cell Death Dis 10, 192 (2019)). Furthermore, T2D causes endothelial dysfunction, induces abnormal angiogenesis, and alters serum amino acid metabolism, especially with regard to glycine, which is significantly decreased due to hyperglycemia. Metabolomic analysis of amino acid profiles showed that intracellular glycine was significantly lower in induced pluripotent stem cell-derived cells (iPSCs) derived from endothelial cells (ECs) from diabetic patients than in such cells derived from healthy subjects (Su et al., Diabetic Endothelial Cells Differentiated From Patient iPSCs Show Dysregulated Glycine Homeostasis and Senescence Associated Phenotypes. Front Cell Dev Biol. 2021 May 31;9:667252), pointing to glycine loss in ECs in T2D. The branched-chain amino acid (BCAA) valine, an essential amino acid in humans, plays a particularly important role in cell growth and metabolism. Several studies have shed light on the ability of valine to increase protein synthesis and milk yield by stimulating the mammalian target of rapamycin (MTOR) pathway (Long et al., Valine increases milk fat synthesis in mammary gland of gilts through stimulating AKT / MTOR / SREBP1 pathway, Biology of Reproduction, Volume 101, Issue 1, July 2019, Pages 126-137,). Recently, plasma BCAA concentrations have been considered as a novel proof of lipid metabolism homeostasis. Evidence in humans and rats has identified a strong relationship between valine metabolism and fat metabolism. In the control of autophagy, leucine appears to be the most, or at least especially prominent, inhibitory amino acid, together with arginine. In addition, its antiproteolytic effect was enhanced by insulin and cell swelling (Meijer et al., supra). Substrate proline is stored as collagen in the extracellular matrix, connective tissues and bone and is rapidly released from this reservoir by the sequential action of matrix metalloproteinases, peptidases and prolidases.As the only proteinogenic secondary amino acid, proline has special biological effects and functions 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). 1) because proline-utilizing enzymes respond to stress signaling; 2) because a large mobile pool of proline exists; 3) because proline metabolism plays a special stress function. In cancer, proline has been reported to act as a responder to nutrient and oxygen loss in autophagy. Furthermore, in the last few years, proline has been investigated for its distinct metabolic function in neuronal autophagy. It has been shown that proline inhibits neuronal autophagy, while the oxidation of proline by proline-oxidase enhances oxidative stress, which then induces autophagy. Decades of research have established the importance of L-arginine in promoting cardiovascular health through the production of the gas nitric oxide (NO) by the enzyme NO synthase (NOS). Release of NO by endothelial cells (EC) regulates blood flow and blood pressure by inhibiting arterial tone. In addition, NO maintains blood fluidity and prevents thrombosis by limiting platelet aggregation and adhesion. NO also protects against intimal hyperplasia by blocking smooth muscle cell (SMC) proliferation, migration and collagen synthesis. Recently, L-asparagine was found to signal to mTORC1 through Arf1 in the absence of Rag GTPases, and like glutamine, intracellular asparagine exchanges with extracellular amino acids. Glutamine can participate in the synthesis of different amino acids through the production of glutamate, but only asparagine requires glutamine for de novo synthesis. Asparagine synthetase (ASNS) is an enzyme that converts glutamine and aspartate to glutamate and asparagine in an ATP-dependent manner.6 ASNS expression is upregulated during amino acid starvation through activation of activating transcription factor 4 (ATF4). Asparagine was identified as an exchange amino acid factor for the serine / threonine kinase mammalian target of rapamycin complex 1 (mTORC1), regulating nucleotide biosynthesis and proliferation (Bodineau et al., Two parallel pathways connect glutamine metabolism and mTORC1 activity to regulate glutamoptosis. Nat Commun. 2021 Aug 10;12(1):4814.).

[0041] Microvascular angiogenesis (sprouting) assay of human vascular endothelial cells grown in Matrigel (see also Figures 8, 9 and 10) Human pulmonary microvascular endothelial cells (Hpmec, HUVE or other vascular ECs can also be used) were cultured in EBM-2 medium (Lonza, CC-3156) supplemented with 10% FBS and all components present in the bullet kit (Lonza, CC-4147) containing human recombinant FGF-B, human recombinant VEGF, human recombinant R3-IGF-1, ascorbic acid, human recombinant EGF and GA-1000 (gentamicin sulfate-amphotericin). 24 hours prior to the assay, Hpmec were starved in EBM-2 medium containing 0.5% FBS instead of 10% FBS. For introduction in the angiogenesis assay, endothelial cells were washed twice with warm PBS, trypsinized and resuspended in serum-free EBM-2 medium. For angiogenesis assays, Matrigel (Fisher Scientific, Landsmeer, The Netherlands #11523550) was diluted 1:1 in serum-free EBM-2 medium and plated in 96-well plates (50 uL). After polymerization, cells were combined with experimental peptide fragments and added on top of Matrigel (100 μL). After 24 hours of incubation, microvascular networks were photographed using a 4× objective and analyzed using the angiogenesis analyzer plug-in in ImageJ.

[0042] Angiogenesis in wound healing requires modulation of angiopoietin 1 (ANG1) versus angiopoietin 2 (ANG2) (see also Figure 11) The angiopoietin family (Ang-1-4) has been shown to play a critical role in modulating physiological and pathological neovascularization. VEGF and angiopoietins function together while playing independent roles in vascular development and embryonic development; VEGF acts early in angiogenesis and Ang-1 acts later in vascular remodeling, maturation and stabilization. Angiopoietins 1 and 2 have been studied in in vivo and in vitro models. Ang-1, a well-established secreted 70 KDa ligand that shares many of the pro-angiogenic properties of VEGF, protects blood vessels from increased plasma leakage by counteracting transendothelial permeability stimulated by VEGF. Ang-1 signals primarily through a transmembrane receptor tyrosine kinase (Tie2) that is ubiquitously expressed in the vascular endothelium and is phosphorylated in quiescent blood vessels. Ang-1 interacts with several cells, such as neutrophils, endothelial cells, and fibroblasts, via integrins to mediate survival, cell adhesion, and migration. Ang-1 is an essential and critical regulator of vascular development, as evidenced by embryonic lethality of Ang-1 null mice. In contrast, Ang-1 and Ang-2, an antagonist of Tie2 signaling, are generally not expressed in healthy adult tissues, but are expressed in secretory tissues undergoing inflammation and vascular remodeling, such as wound healing.

[0043] Factors contributing to decreased wound healing (see also Figure 12) Chronic diabetic ulcers are responsible for over 42,500 non-traumatic lower limb amputations in the United States each year and 27% of diabetic medical costs. Dysfunction in the cutaneous diabetic wound healing phenotype is related to several intrinsic and extrinsic factors that have been shown. Wounds in diabetic patients and in mouse models of type I and type II diabetes show defects in angiogenesis, re-epithelialization and wound closure. While early re-epithelialization is not dependent on angiogenesis, complete healing and maturation are tightly regulated by the vasculogenic response and cell-matrix interactions of several cells involved in angiogenic activity. The angiogenic deficiency was attributed to poorly controlled blood glucose levels and to associated vascular defects in both endothelial cells (ECs) and endothelial progenitor cells (EPCs). Although it is known that the defect in neovascularization in diabetes is associated with an impaired response to ischemia in wound healing as a result of metabolic derangement, beyond this vague understanding, the impaired ability of ischemic tissues to revascularize in diabetes is poorly understood. A potential mechanism to explain this dysfunction is the reduced expression of angiogenic growth factors such as vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and their receptors. Skin samples taken from the periwound area of ​​the lower limbs of type 2 diabetic subjects (skin within 1 cm of the wound margin) have significantly less expression of both VEGF (46%) and Ang-1 (36%) than skin tissue from non-diabetic subjects. Many groups have presented evidence linking impaired neovascularization to delayed closure of diabetic wounds and further demonstrated that supplementation with angiogenic growth factors, such as VEGF, Ang-1, EGF, bFGF and HIF1-α, or combinations of these factors, by recombinant growth factor therapy or gene transfer, has a positive effect on improving neovascularization and diabetic wound closure outcomes.

[0044] Determination of C-peptide deficiency (see also Figure 13) Panero et al. (Fasting plasma C-peptide and micro- and macrovascular complications in a large clinic-based cohort of type 1 diabetic patients. Diabetes Care. 2009 Feb;32(2):301-5) assessed residual β-cell function and, therefore, relative C-peptide deficiency by measuring fasting plasma C-peptide (normal 0.36-1.17 nmol / l; Diagnostics Product Corporation, Los Angeles, CA) in T1D patients with microvascular vasculopathy. They then performed logistic regression analyses to study variables independently associated with microvascular (retinopathy, micro- and macroalbuminuria, and diabetic peripheral neuropathy (DPN)) and macrovascular complications (myocardial infarction, angina, coronary artery bypass graft, stroke, and peripheral arteriopathy). The independent role of C-peptide was tested using tertiles of its distribution (<0.06, 0.06-0.10, and 0.11-2.76 nmol / l). Regarding fasting C-peptide values ​​in the lowest tertile (<0.06 nmol / l), higher values ​​were associated with a lower prevalence of microvascular complications (odds ratio [OR] 0.59 [95% CI 0.37-0.94]). No association with macrovascular complications was evident (0.77 [0.38-1.58]).

[0045] Qiao et al. (C-peptide is independent associated with diabetic peripheral neuropathy: a community-based study. Diabetol Metab Syndr 9, 12 (2017).) investigated the relationship between residual C-peptide and DPN in patients with type 2 diabetes. With increasing duration of diabetes, islet function gradually shrinks, resulting in reduced C-peptide and insulin levels, and increasing prevalence of DPN. Fasting C-peptide, 2-hour postprandial C-peptide, and ΔC-peptide (i.e., 2-hour postprandial C-peptide, minus fasting C-peptide) serum concentrations in the non-DPN group were significantly higher than those in the clinical DPN group (all P ≤ 0.040) and confirmed DPN groups (all P < 0.002). The three C-peptide parameters were independently associated with DPN (all P<0.05) after adjustment for age, sex, diabetes duration, smoking status, systolic pressure, body mass index, angiotensin-converting enzyme inhibitor / angiotensin receptor blocker use, fasting plasma glucose, HbA1c, triglycerides, and estimated glomerular filtration rate. Compared with delta C-peptide quartile 1 (reference), patients in quartile 3 (odds ratio [OR], 0.110; 95% confidence interval [CI], 0.026-0.466; P=0.003) and quartile 4 (OR, 0.012; 95% CI, 0.026-0.559; P=0.007) had a lower risk of DPN after adjustment for confounding factors. C-peptide was measured by chemiluminescence (Siemens Healthcare Diagnostics, Malvern, USA) on an ADVIA Centaur XP automated analyzer (Siemens Healthcare Diagnostics) using a sample volume of 200 μL.These studies have shown that fasting C-peptide deficiency in patients with DPN is increasingly present at values ​​below 1 nmol / l, more specifically below 0.5 nmol / l, more specifically below 0.3 nmol / L, more specifically below 0.3 nmol / l, more specifically below 0.2 nmol / L, more specifically below 0.1 nmol / l, more specifically below 0.06 nmol / L. To better distinguish such patients, further analysis can be performed by determining postprandial C-peptide levels (e.g., as provided by Qiao et al., supra), indicating that clinically relevant C-peptide deficiency can already be found after a meal at values ​​below 1 nmol / l. Determination of ΔC-peptide (i.e., 2-hour postprandial C-peptide, minus fasting C-peptide, e.g., as provided by Qiao et al., supra) can further determine whether a patient suffers from a C-peptide deficiency. In general, patients with ΔC-peptide less than 1 nmol / l are considered to be at risk for DPN. EXAMPLES

[0046] Example of formulation, variables after further dose-finding studies.

[0047] [Example 1] QAQGVALQ-Maleate To prepare 1 L of composition: QAQGVALQ-Maleate salt --1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0048] [Example 2] LQGVLPAL-Maleate To prepare 1 L of composition: LQGVLPAL-Maleate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0049] [Example 3] VLQAVLPP-Maleate To prepare 1 L of composition: VLQAVLPP-maleate --1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0050] [Example 4] PGAYPGQA-Maleate To prepare 1 L of composition: PGAYPGQA-Maleate--1.8mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0051] [Example 5] AQGV-Maleate To prepare 1 L of composition: AQGV-maleate --1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0052] [Example 6] LQGVL-Malate To prepare 1 L of composition: LQGVL-maleate -- 1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0053] [Example 7] AQGLQ-Maleate To prepare 1 L of composition: AQGLQ-Maleate--1.8mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0054] [Example 8] LQGLQ-Malate To prepare 1 L of composition: LQGLQ-Maleate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0055] [Example 9] QAQGVALQ-Acetate To prepare 1 L of composition: QAQGVALQ-Acetate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0056] [Example 10] LQGVLPAL-Acetate To prepare 1 L of composition: LQGVLPAL-Acetate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0057] [Example 11] VLQAVLPP-Acetate To prepare 1 L of composition: VLQAVLPP-Acetate--1.8mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0058] [Example 12] PGAYPGQA-Acetate To prepare 1 L of composition: PGAYPGQA-Acetate--1.8mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0059] [Example 13] AQGV - Acetate To prepare 1 L of composition: AQGV-Acetate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0060] [Example 14] LQGVL-Acetate To prepare 1 L of composition: LQGVL-Acetate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0061] [Example 15] AQGLQ-Acetate To prepare 1 L of composition: AQGLQ-Acetate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0062] [Example 16] LQGLQ-Acetate To prepare 1 L of composition: LQGLQ-Acetate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0063] [Example 17] QAQGVALQ-Tartrate To prepare 1 L of composition: QAQGVALQ-tartrate --1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0064] [Example 18] LQGVLPAL-tartrate To prepare 1 L of composition: LQGVLPAL-tartrate -- 1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0065] [Example 19] VLQAVLPP-tartrate To prepare 1 L of composition: VLQAVLPP-tartrate --1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0066] [Example 20] PGAYPGQA-tartrate To prepare 1 L of composition: PGAYPGQA-tartrate--1.8mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0067] [Example 21] AQGV-tartrate To prepare 1 L of composition: AQGV-tartrate --1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0068] [Example 22] LQGVL-tartrate To prepare 1 L of composition: LQGVL-tartrate -- 1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0069] [Example 23] AQGLQ-tartrate To prepare 1 L of composition: AQGLQ-tartrate --1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0070] [Example 24] LQGLQ-tartrate To prepare 1 L of composition: LQGLQ-tartrate --1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0071] [Example 25] QAQGVALQ-Citrate To prepare 1 L of composition: QAQGVALQ-Citrate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0072] [Example 26] LQGVLPAL-Citrate To prepare 1 L of composition: LQGVLPAL-Citrate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0073] [Example 27] VLQAVLPP-Citrate To prepare 1 L of composition: VLQAVLPP-citrate -- 1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0074] [Example 28] PGAYPGQA-Citrate To prepare 1 L of composition: PGAYPGQA-Citrate--1.8mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0075] [Example 29] AQGV-Citrate To prepare 1 L of composition: AQGV-citrate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0076] [Example 30] LQGVL-Citrate To prepare 1 L of composition: LQGVL-citrate -- 1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0077] [Example 31] AQGLQ-Citrate To prepare 1 L of composition: AQGLQ-Citrate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0078] [Example 32] LQGLQ-Citrate To prepare 1 L of composition: LQGLQ-Citrate--1.8 mol Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0079] [Example 33] QAQGVALQ-Malate and short-acting insulin To prepare 1 L of composition: QAQGVALQ-Maleate salt --1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0080] [Example 34] LQGVLPAL-Malate and intermediate-acting insulin To prepare 1 L of composition: LQGVLPAL-Maleate--1.8 mol Human intermediate-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0081] [Example 35] VLQAVLPP-Malate and Rapid-Acting Insulin To prepare 1 L of composition: VLQAVLPP-maleate --1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0082] [Example 36] PGAYPGQA-Malate and intermediate-acting insulin To prepare 1 L of composition: PGAYPGQA-Maleate--1.8mol Human intermediate-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0083] [Example 37] AQGV-Malate and Rapid-Acting Insulin To prepare 1 L of composition: AQGV-maleate --1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0084] [Example 38] LQGVL-Malate and Rapid-Acting Insulin To prepare 1 L of composition: LQGVL-maleate -- 1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0085] [Example 39] AQGLQ-Malate and Short-Acting Insulin To prepare 1 L of composition: AQGLQ-Maleate--1.8mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0086] [Example 40] LQGLQ-Malate and Rapid-Acting Insulin To prepare 1 L of composition: LQGLQ-Maleate--1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0087] [Example 41] QAQGVALQ-Acetate and short-acting insulin To prepare 1 L of composition: QAQGVALQ-Acetate--1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0088] [Example 42] LQGVLPAL-Acetate and intermediate-acting insulin To prepare 1 L of composition: LQGVLPAL-Acetate--1.8 mol Human intermediate-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0089] [Example 43] VLQAVLPP-acetate and short-acting insulin To prepare 1 L of composition: VLQAVLPP-Acetate--1.8mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0090] [Example 44] PGAYPGQA-acetate and intermediate-acting insulin To prepare 1 L of composition: PGAYPGQA-Acetate--1.8mol Human intermediate-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0091] [Example 45] AQGV-Acetate and Rapid-Acting Insulin To prepare 1 L of composition: AQGV-Acetate--1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0092] [Example 46] LQGVL-acetate and rapid-acting insulin To prepare 1 L of composition: LQGVL-Acetate--1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0093] [Example 47] AQGLQ-acetate and short-acting insulin To prepare 1 L of composition: AQGLQ-Acetate--1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0094] [Example 48] LQGLQ-acetate and rapid-acting insulin To prepare 1 L of composition: LQGLQ-Acetate--1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0095] [Example 49] QAQGVALQ-tartrate and short-acting insulin To prepare 1 L of composition: QAQGVALQ-tartrate --1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0096] [Example 50] LQGVLPAL-tartrate and intermediate-acting insulin To prepare 1 L of composition: LQGVLPAL-tartrate -- 1.8 mol Human intermediate-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0097] [Example 51] VLQAVLPP-tartrate and short-acting insulin To prepare 1 L of composition: VLQAVLPP-tartrate --1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0098] [Example 52] PGAYPGQA-tartrate and intermediate-acting insulin To prepare 1 L of composition: PGAYPGQA-tartrate--1.8mol Human intermediate-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0099] [Example 53] AQGV-tartrate and fast-acting insulin To prepare 1 L of composition: AQGV-tartrate --1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0100] [Example 54] LQGVL-tartrate and fast-acting insulin To prepare 1 L of composition: LQGVL-tartrate -- 1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0101] [Example 55] AQGLQ-tartrate and fast-acting insulin To prepare 1 L of composition: AQGLQ-tartrate --1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0102] [Example 56] LQGLQ-tartrate and short-acting insulin To prepare 1 L of composition: LQGLQ-tartrate --1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0103] [Example 57] QAQGVALQ-Citrate and short-acting insulin To prepare 1 L of composition: QAQGVALQ-Citrate--1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0104] [Example 58] LQGVLPAL-citrate and short-acting insulin To prepare 1 L of composition: LQGVLPAL-Citrate--1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0105] [Example 59] VLQAVLPP-citrate and short-acting insulin To prepare 1 L of composition: VLQAVLPP-citrate -- 1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0106] [Example 60] PGAYPGQA-citrate and intermediate-acting insulin To prepare 1 L of composition: PGAYPGQA-Citrate--1.8mol Human intermediate-acting insulin (2800U / mg) - 100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0107] [Example 61] AQGV-citrate and fast-acting insulin To prepare 1 L of composition: AQGV-citrate--1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0108] [Example 62] LQGVL-citrate and fast-acting insulin To prepare 1 L of composition: LQGVL-citrate -- 1.8 mol Human fast acting insulin (2800U / mg)--100000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0109] [Example 63] AQGLQ-citrate and short-acting insulin To prepare 1 L of composition: AQGLQ-Citrate--1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0110] [Example 64] LQGLQ-citrate and short-acting insulin To prepare 1 L of composition: LQGLQ-Citrate--1.8 mol Human short-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0111] [Example 65] VGVAPGVGVAPGVGVAPG-citrate and intermediate-acting insulin To prepare 1 L of composition: VGVAPGVGVAPGVGVAPG-citrate--1.8 mol Human intermediate-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0112] [Example 66] QVGQVELGGGPGAGSLQP-citrate and intermediate-acting insulin To prepare 1 L of composition: QVGQVELGGGPGAGSLQP-Citrate -- 1.8 mol Human intermediate-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0113] [Example 67] GAYPGAPGAYPGAPAPGV-citrate and intermediate-acting insulin To prepare 1 L of composition: GAYPGAPGAYPGAPAPGV-citrate -- 1.8 mol Mix with 100,000 U of human intermediate-acting insulin (2800 U / mg).

[0114] [Example 68] VGVAPGVGVAPGVGVAPG-citrate and long-acting insulin To prepare 1 L of composition: VGVAPGVGVAPGVGVAPG-citrate--2.4 mol Human long-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0115] [Example 69] QVGQVELGGGPGAGSLQP-citrate and long-acting insulin To prepare 1 L of composition: QVGQVELGGGPGAGSLQP-Citrate -- 2.4 mol Human long-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

[0116] [Example 70] GAYPGAPGAYPGAPAPGV-citrate and long-acting insulin To prepare 1 L of composition: GAYPGAPGAYPGAPAPGV-citrate -- 2.4 mol Human long-acting insulin (2800U / mg)--100,000U Mix M-Kreosol--25 mg, glycerol--160 mg, 0.9% NaCL, water and either 10% hydrochloric acid or 10% sodium hydroxide as needed to obtain a composition volume of 1 L and the desired final pH.

Claims

1. 1. An autophagy inhibitory modulator of mTOR for use in controlling angiogenesis, including inducing angiogenic activity, in the prevention or treatment of vascular disorders in a subject, said modulator comprising a source of amino acids, preferably peptides, wherein at least 50% of said amino acids are selected from the group consisting of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N).

2. 10. The modulator of claim 1 for use in the prevention or treatment of nephropathy.

3. 3. A modulator according to claim 1 or 2 for use in the prevention or treatment of end stage renal disease.

4. 10. The modulator of claim 1 for use in the prevention or treatment of retinopathy.

5. 10. A modulator according to claim 1 or 4 for use in the prevention or treatment of blindness.

6. 10. The modulator of claim 1 for use in the prevention or treatment of neurological disorders.

7. 10. The modulator of claim 1 or 6 for use in the prevention or treatment of peripheral diabetic neuropathy.

8. 10. The modulator of claim 1 or 6 for use in the prevention or treatment of diabetic ulcers.

9. 10. The modulator of any one of claims 1, 2, 4 and 6, wherein the subject is also treated to achieve or maintain glycemic control.

10. 10. The modulator of claim 9, wherein the subject is also treated with insulin to achieve or maintain glycemic control.

11. The modulator of any one of claims 1, 2, 4 and 6, wherein the vasculopathy comprises diabetic vasculopathy.

12. 10. The modulator of any one of claims 1, 2, 4 and 6, wherein the subject has or is suspected of having a C-peptide deficiency.

13. 1. A formulation of a growth factor and an autophagy inhibitor modulator of mTOR, wherein said modulator comprises a source of amino acids, preferably peptides, wherein at least 50% of said amino acids are selected from the group consisting of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N).

14. 14. The formulation of claim 13, wherein the growth factors comprise insulin for use in glycemic control and an autophagy inhibitor modulator of mTOR for use in angiogenesis control.

15. A pharmaceutical formulation comprising the formulation of claim 13 or 14.

16. 15. The formulation of claim 13 or 14 for use in inducing angiogenic activity in the prevention or treatment of a vascular disorder in a subject.

17. 17. The formulation of claim 16 for use in the prevention or treatment of nephropathy.

18. 17. The formulation of claim 16 for use in the prevention or treatment of end stage renal disease.

19. 17. The formulation of claim 16 for use in the prevention or treatment of retinopathy.

20. 17. The formulation of claim 16 for use in the prevention or treatment of blindness.

21. 17. The formulation of claim 16 for use in the prevention or treatment of neurological disorders.

22. 17. The formulation of claim 16 for use in the prevention or treatment of peripheral diabetic neuropathy.

23. 17. The formulation of claim 16 for use in the prevention or treatment of diabetic ulcers.

24. 15. The formulation of claim 13 or 14, wherein the vasculopathy comprises diabetic vasculopathy.

25. 15. The formulation of claim 13 or 14, wherein the subject has or is suspected of having a C-peptide deficiency.

26. 1. A method for inducing angiogenic activity in the prevention or treatment of vascular disorders in a subject, comprising the step of administering to the subject an autophagy-inhibiting modulator of mTOR, wherein said modulator comprises a source of amino acids, preferably peptides, wherein at least 50% of said amino acids are selected from the group consisting of alanine (in single letter code: A), glutamine (Q), glycine (G), valine (V), leucine (L), isoleucine (I), proline (P), arginine (R), and asparagine (N).

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