Peptides targeting endothelium and use in the prevention and treatment of diabetic lower limb ischemia

Abstract

The application discloses a peptide targeting endothelium, which is a polypeptide made by covalently cross-linking GLP-1(32-36), an RGD cyclic peptide, a polymer and a ROS-responsive polymer with each other. It is verified that the peptide has high activity and strong enzymatic stability in blood plasma, can be targeted to combine with receptors on the surface of vascular endothelial cells, can make the peptide maintain in an effective treatment window for a long time, can maximize the treatment advantages, has the characteristics of strong treatment targeting and long-acting slow release, and can be used for long-term treatment of diabetic lower limb ischemia complications.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical inventions, specifically relating to the application of an endothelial-targeting long-acting peptide in the preparation of a drug for the prevention and / or treatment of diabetic lower limb ischemia. Through the molecular structure of the drug, it achieves targeted binding to endothelial cells, providing long-term treatment for diabetic lower limb ischemia. Background Technology

[0002] With changes in lifestyle and living environment, and the increasing aging population, the number of people with diabetes is rising year by year. It is estimated that by the middle of this century, more than 700 million people worldwide will have diabetes, along with a series of acute and chronic complications caused by poor blood sugar control. In addition to acute metabolic disorders caused by hyperglycemia, long-term chronic hyperglycemia can also lead to damage and dysfunction of various tissues and organs throughout the body, and may subsequently cause disability or even endanger life due to serious clinical conditions such as myocardial infarction, stroke, amputation, kidney failure, and blindness.

[0003] Lower limb ischemia in diabetic patients is the most common chronic vascular complication. Its cause is the excessive production of reactive oxygen species (ROS) in the mitochondria of endothelial cells under high glucose conditions. This ROS activates a series of downstream signaling pathways, leading to endothelial cell and microvascular dysfunction, manifesting as stenosis and occlusion of the lower limb arteries. Although lower limb ischemia in diabetic patients usually presents as claudication, a lack of effective treatment over a long period often results in severe limb ischemia, even requiring amputation. Currently, no drugs have been found to effectively improve lower limb ischemia in diabetic patients besides surgical treatment. However, due to anatomical limitations, the symptoms of lower limb ischemia in some patients do not improve effectively after surgical treatment, inevitably leading to disease progression. Therefore, there is an urgent need for a highly effective and low-toxicity drug formulation to effectively treat lower limb ischemia in diabetic patients.

[0004] Recent studies have shown that incretins such as glucagon-like peptide-1 (GLP-1) play a role in regulating angiogenesis. GLP-1, a naturally occurring hormone, plays a crucial role in regulating glucose homeostasis by stimulating insulin secretion. Early research suggested that only GLP-1 could regulate blood glucose and improve the prognosis of diabetes-related complications in vivo. However, with further research, various metabolites of GLP-1 have also been found to exert various beneficial biological effects. GLP-1(32-36), as the main end product of GLP-1 proteolysis, has been found to reduce pancreatic β-cell apoptosis, increase energy expenditure, and reduce body weight in obese mice. However, its role in improving diabetic lower limb ischemia has been rarely reported. Our recent research found that GLP-1(32-36) has a direct effect on alleviating diabetic lower limb ischemia independently of insulin stimulation. By metabolically reprogramming the glycolytic flux, GLP-1(32-36) improved angiogenesis in endothelial progenitor cells and type 1 diabetic mouse models under high glucose exposure, providing a theoretical basis for its subsequent application in diabetic patients with lower limb ischemia. However, as a short peptide composed of 5 amino acids, GLP-1(32-36) has a much shorter half-life in vivo (less than 5 minutes) than insulin (2 hours). In treating diabetic lower limb ischemia, it often degrades before exerting its therapeutic effect, requiring multiple injections to ensure the peptide's biological action. Furthermore, the short metabolic time of the short peptide leads to significant off-target effects, making it difficult to exert long-term effects on damaged vascular endothelium, thus greatly reducing the therapeutic efficacy. Therefore, a method is needed to effectively address the short half-life of GLP-1(32-36) to achieve long-acting sustained release and endothelial targeting.

[0005] RGD cyclic peptide is a five-membered cyclic peptide composed of arginine, glycine, aspartic acid, D-phenylalanine, and lysine. Previous studies have found that RGD cyclic peptide can specifically bind to the integrin αvβ3 receptor and play an important role in the intertransmission of cell signaling. This receptor is strongly expressed on the membranes of neovascular endothelial cells, but rarely expressed in mature vascular endothelial cells and most normal organ systems. Therefore, targeted therapy based on RGD cyclic peptide for endothelial cells has become increasingly popular in recent years. Currently, research on the use of RGD cyclic peptide complexes for targeted tumor therapy has been reported, but these compounds mainly achieve targeted tumor killing through bio-coupled antitumor drugs. Based on the significant advantages of RGD cyclic peptide and the current research status, there is great potential for the development of RGD cyclic peptide complexes for targeted therapy of damaged endothelium. However, to date, there are no reports on the use of RGD cyclic peptide complexes for the treatment of diabetic lower limb ischemia. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides an endothelial-targeting long-acting peptide, which is a long-acting sustained-release polypeptide used to treat diabetic lower limb ischemia.

[0007] The endothelial-targeting long-acting peptide of this invention is made from GLP-1 (32-36), RGD cyclic peptide, polymers, and ROS-responsive polymers. Suitable polymers include, but are not limited to, polyethylene glycol (PEG), polyacrylamide, polyhydroxyethyl aspartic acid, polythiobetaine, polycarboxylate betaine, povidone, chitosan, etc., which are used alone or in combination in this invention. PEG is preferred. Suitable ROS-responsive polymers include, polyboronic acid esters, oligoproline peptides, polysaccharides, peroxypolythioketones, propylene sulfate, selenoalkyl, aminoacrylates, alkyl selenide telluride, etc., which are used alone or in combination in this invention. Polyboronic acid esters are preferred.

[0008] Furthermore, the endothelial-targeting long-acting peptide is prepared by solid-phase synthesis.

[0009] The present invention also provides the use of an endothelial cell-targeting peptide in the preparation of a medicament for the prevention and / or treatment of diabetic lower limb ischemia, wherein the molecular structure of the compound is shown in the following formula, and compounds thereof having more than 90% homology.

[0010]

[0011] Furthermore, the drug is a formulation prepared with endothelial-targeting long-acting peptides as the active ingredient and pharmaceutically acceptable excipients.

[0012] Furthermore, the formulation is an injectable formulation.

[0013] This invention is the first to discover that the endothelial-targeting long-acting peptide with the molecular structure shown above can be rapidly and safely delivered to the damaged site in a compound manner, meeting the clinical requirements for long-acting and continuous treatment of diabetic lower limb ischemia.

[0014] This invention also discovers for the first time that the endothelial-targeting long-acting peptide with the molecular structure shown above can effectively improve diabetic lower limb ischemia. After constructing a diabetic lower limb ischemia model, intervention with this compound significantly improved blood perfusion on the affected side of the mice, and the damage to various tissues and organs of the mice was also significantly alleviated under high glucose stimulation. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the molecular structure of endothelial-targeting long-acting peptides and non-endothelial-targeting long-acting peptides, where A is a long-acting peptide targeting the endothelium and B is a long-acting peptide not targeting the endothelium.

[0016] Figure 2 High-performance liquid chromatography-mass spectrometry (HPLC-MS) detection data for the molecular structure of endothelial-targeting long-acting peptides.

[0017] Figure 3 The graph shows the changes in body weight and blood glucose levels of mice in each group over time. Figure A shows the changes in body weight of mice in each group over time; Figure B shows the changes in blood glucose levels of mice in each group over time.

[0018] Figure 4 This study investigates the molecular structure and pharmacodynamics of endothelial-targeting long-acting peptides. A shows schematic diagrams of the molecular structures of the endothelial-targeting long-acting peptide, the non-endothelial-targeting long-acting peptide, and the modified GLP-1(32-36); B shows Doppler ultrasound images of lower limb blood perfusion in each group of experimental mice; and C shows the lower limb blood perfusion curves in each group of experimental mice.

[0019] Figure 5 The figure shows the safety evaluation results of the endothelial-targeting long-acting peptide in organs such as the kidney, liver, lung, spleen, and heart.

[0020] Figure 6 This study investigates the pharmacodynamics of endothelial targeting structures with and without long peptide linkages. A shows Doppler ultrasound images of hind limb blood perfusion in mice from each group; B shows the hind limb blood perfusion curves of mice from each group. Detailed Implementation

[0021] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and medicinal chemistry, molecular biology, tissue staining, animal experiments, etc., used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0022] As used herein, “at least one species” or “one or more species” can mean 1, 2, 3, 4, 5, 6, 7, 8 or more species.

[0023] As used herein, the expressions “comprising,” “including,” “containing,” and “having” are open-ended, meaning they include the listed elements, steps, or components but do not exclude other unlisted elements, steps, or components. The expression “composed of” excludes any unspecified elements, steps, or components. The expression “essentially composed of” means that the scope is limited to the specified elements, steps, or components, plus optional elements, steps, or components that do not significantly affect the essential and novel nature of the claimed subject matter. It should be understood that the expressions “essentially composed of” and “composed of” are encompassed within the meaning of the expression “comprising.”

[0024] As used herein, the connecting term “and / or” between multiple referred elements should be understood to include both individual and combined options. In other words, “and / or” includes both “and” and “or”. For example, A and / or B includes A, B, and A+B. A, B, and / or C includes A, B, C, and any combination thereof, such as A+B, A+C, B+C, and A+B+C. Further elements qualified by “and / or” are understood in a similar manner and include any one of them and any combination thereof.

[0025] Unless otherwise stated, any numerical value or range, such as concentration or concentration range, shall in any case be understood to be modified by the term “about”. Thus, numerical values ​​typically include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). As used herein, the use of numerical ranges explicitly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions within that range, unless the context clearly indicates otherwise.

[0026] The following describes in detail the specific embodiments of the present invention, but these are only preferred embodiments and do not limit the present invention. For those skilled in the art, the preparation process, the substances used, and the amount of substances used in the present invention can be modified and varied in various ways. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

[0027] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0028] Experimental Example 1: Preparation of Endothelial-Targeting Long-Acting Peptides and Non-Endothelial-Targeting Long-Acting Peptides

[0029] according to Figure 1 The polypeptide molecular structure shown was used to synthesize an endothelial-targeting long-acting peptide ( ) using conventional methods in the art. Figure 1 A) and non-endothelial-targeting long-acting peptides ( Figure 1 B). The purity of the polypeptide is greater than 98%.

[0030] according to Figure 2 The high-performance liquid chromatography-mass spectrometry (HPLC-MS) data showing the molecular structure of the endothelial-targeting long-acting peptide led to the successful preparation of the peptide conjugate (Mass = 2689 Da).

[0031] Example 2: Establishment of animal models of type 1 diabetes and diabetic lower limb ischemia

[0032] 1. Experimental Methods:

[0033] Male C57BL / 6 mice (weight >20g) aged 6-8 weeks were selected as model animals. Mice were housed in a 12-hour light-dark cycle incubator and fed a standard laboratory diet. A diabetic mouse model was established by intraperitoneal injection of streptozotocin (70mg / (kg·d)). After two weeks of continuous injection, blood glucose was measured by drawing blood from the tail vein. A fasting venous blood glucose level >11.1 mmol / L was considered a successful establishment of a type 1 diabetes mellitus (T1DM) animal model.

[0034] Four weeks after blood glucose levels stabilized in T1DM mice, a diabetic lower limb ischemia animal model was established. Mice were placed in an anesthesia chamber and anesthesia was induced by inhalation of isoflurane. After the righting reflex disappeared, 1-3% isoflurane was vaporized in pure oxygen and maintained via face mask inhalation at a continuous low flow rate of 1 L / min. The anesthetized mice were placed supine on the modeling table, their tails and limbs were secured with adhesive tape, and the hair in the right groin area and surrounding region was shaved. A 5 mm incision was then made in the skin of the right groin area. Subcutaneous adipose tissue was separated using sterile pointed cotton swabs to fully expose the femoral artery and vein. The overlying fascia was carefully dissected, and the femoral artery and vein were separated using curved forceps. The femoral artery and vein were ligated distally and proximally with fine sutures, and then cut between the two ligation points with scissors. The wound was then sutured with fine sutures. The left limb was left untreated and used as a control. The modeling effect was evaluated by measuring the blood perfusion of the operated lower limb using lower limb blood flow Doppler ultrasound. The interruption of blood flow at the ligation site was considered as the successful establishment of a diabetic hind limb ischemia (DHLI) animal model.

[0035] Experiment Example 3: Changes in body weight and blood glucose levels of endothelial-targeting long-acting peptides

[0036] 1. Experimental Samples:

[0037] Endothelial-targeting long-acting peptide (tEC-cRGD-(32-36)) and non-endothelial-targeting long-acting peptide (c-EKEK-(32-36)) were prepared according to Experimental Example 1.

[0038] 2. Experimental Methods:

[0039] Twenty-four male T1DM mice (27±1g) constructed according to Experimental Example 2 were randomly divided into four groups: a control group, an endothelial-targeted long-acting peptide group (each mouse received 10ug intraperitoneally daily for 28 consecutive days), a non-endothelial-targeted long-acting peptide group (each mouse received 10ug intraperitoneally daily for 28 consecutive days), and a GLP-1 (32-36) group (each mouse received 10ug intraperitoneally daily for 28 consecutive days), with six mice in each group. The weight and blood glucose levels of the mice in each group were measured using a weighing scale and a blood glucose meter. The first day of administration was recorded as day 0. Every 7 days thereafter, the mice were weighed at room temperature using a weighing scale, and blood glucose levels were measured using a blood glucose meter by drawing blood from the tail vein. The weight and blood glucose levels of each group were recorded until day 63, and the changes in weight and blood glucose levels over time were plotted and compared.

[0040] 3. Results and Discussion:

[0041] pass Figure 3 As observed, there was little difference in body weight among the groups of mice before administration. With continued intraperitoneal injection, the tEC-cRGD-(32-36) and c-EKEK-(32-36) groups showed more significant weight loss than the control and GLP-1(32-36) groups. After administration was discontinued, the mice in each group gradually returned to the same weight. Figure 3 B indicates that there was no significant difference in blood glucose levels among the mice before and after continuous administration.

[0042] Experimental Example 4: Pharmacodynamic Study of Endothelial-Targeting Long-Acting Peptides

[0043] 1. Experimental Samples:

[0044] Endothelial-targeting long-acting peptide (tEC-cRGD-(32-36)) and non-endothelial-targeting long-acting peptide (c-EKEK-(32-36)) prepared according to Experimental Example 1

[0045] 2. Experimental Methods:

[0046] Twenty-four male DHLI mice (27±1g) constructed according to Experimental Example 2 were randomly divided into four groups: a control group, an endothelial-targeted long-acting peptide group (each mouse received 10ug intraperitoneal injection daily for 28 consecutive days), a non-endothelial-targeted long-acting peptide group (each mouse received 10ug intraperitoneal injection daily for 28 consecutive days), and a GLP-1 (32-36) group (each mouse received 10ug intraperitoneal injection daily for 28 consecutive days), with six mice in each group. The back hair of the mice was shaved. Blood perfusion in the right hind limb was measured using a hind limb Doppler ultrasound before modeling. Blood perfusion in the healthy (left) and affected (right) hind limbs was measured at predetermined time points (days 0, 3, 7, 14, 21, and 28), and the blood perfusion ratio of the affected / healthy hind limbs was calculated to compare the improvement in hind limb blood perfusion over time in different groups.

[0047] 3. Results and Discussion:

[0048] pass Figure 4 B observed that after surgery, blood flow in the right lower limb of the mouse was significantly reduced, proving that the diabetic lower limb ischemia model was successfully established. Figure 4 B and Figure 4 C indicates that the endothelial-targeted long-acting peptide group, the non-endothelial-targeted long-acting peptide group, and the GLP-1(32-36) group showed significant improvement in blood perfusion compared to the control group, demonstrating that GLP-1(32-36) has an effective therapeutic effect on lower limb ischemia symptoms in diabetic patients. The blood perfusion recovery rate in the endothelial-targeted long-acting peptide group was significantly better than that in the non-endothelial-targeted long-acting peptide group and the GLP-1(32-36) group, proving that the long-acting nature of the endothelial-targeted long-acting peptide and its endothelial cell-targeting characteristics can maximize the therapeutic effect of the drug.

[0049] Experimental Example 5: Safety Assessment of Endothelial-Targeting Long-Acting Peptides

[0050] 1. Experimental samples: Endothelial-targeting long-acting peptide (tEC-cRGD-(32-36)) and non-endothelial-targeting long-acting peptide (c-EKEK-(32-36)) prepared according to Experimental Example 1.

[0051] 2. Experimental Methods:

[0052] Twelve male T1DM mice (27±1g) constructed according to Experimental Example 2 were randomly divided into a control group, an endothelial-targeted long-acting peptide group (each mouse received 10ug intraperitoneal injection daily for 28 consecutive days), a non-endothelial-targeted long-acting peptide group (each mouse received 10ug intraperitoneal injection daily for 28 consecutive days), and a GLP-1 (32-36) group, with three mice in each group. On day 28 after drug administration, mice in each group were sacrificed by cervical dislocation, and liver, kidney, lung, spleen, and heart tissues were collected from each group. The tissues were treated overnight in 4% paraformaldehyde solution, then cut into 5µm sections, and then stained with hematoxylin and eosin (H&E) to obtain sections of each tissue and organ of each group of mice. Finally, high-resolution digital images were acquired at the same magnification using an inverted white light microscope, and the tissue differences between the groups of mice were compared.

[0053] 3. Discussion of Results:

[0054] pass Figure 5 It was observed that the kidneys and livers of T1DM mice contained a large number of apoptotic and necrotic cells, the lung lobules were filled with necrotic cells, and a large number of immune cells in the spleen underwent necrosis and apoptosis, indicating that high glucose stimulation caused significant damage to various tissues and organs in mice. In contrast, the drug treatment group showed some improvement in the degree of damage to various tissues and organs under high glucose stimulation, with the endothelial-targeting long-acting peptide group showing the most significant improvement. The number of necrotic and apoptotic cells in the tissues was significantly reduced, demonstrating that the endothelial-targeting long-acting peptide can effectively protect against damage to various tissues and organs in mice under high glucose stimulation.

[0055] Example 6: Pharmacodynamic study of endothelial targeting structures with and without long peptide linkages.

[0056] 1. Experimental Samples:

[0057] Endothelial-targeting long-acting peptide (tEC-cRGD-(32-36)) and targeting empty vector (tEC-cRGD) were prepared according to Experimental Example 1.

[0058] 2. Experimental Methods:

[0059] Eighteen male DHLI mice (27±1g) constructed according to Experimental Example 2 were randomly divided into three groups: a PBS group (each mouse received an equal volume of PBS intraperitoneally daily for 28 consecutive days), an endothelial-targeted long-acting peptide group (each mouse received 10ug intraperitoneally daily for 28 consecutive days), and an endothelial-targeted empty vector group (each mouse received 10ug intraperitoneally daily for 28 consecutive days), with six mice in each group. The back hair of the mice was shaved. Blood perfusion in the right hind limb was measured using a hind limb Doppler ultrasound before modeling. At predetermined time points (days 0, 3, 7, 14, 21, and 28), blood perfusion in the healthy (left) and affected (right) hind limbs was measured, and the affected / healthy hind limb perfusion ratio was calculated to compare the improvement in hind limb blood perfusion over time in different groups.

[0060] 3. Results and Discussion:

[0061] pass Figure 6 A observed a significant reduction in blood flow in the right hind limb of mice after surgery, confirming the successful establishment of a diabetic lower limb ischemia model. 7-28 days post-surgery, the endothelial-targeted long-acting peptide group showed significantly improved blood perfusion compared to the PBS group and the endothelial-targeted empty vector group. There was no significant difference in blood perfusion between the PBS group and the empty vector group, indicating that endothelial-targeted GLP-1 (32-36) has an effective therapeutic effect on diabetic lower limb ischemia, and its therapeutic effect is independent of the target structure itself.

Claims

1. A peptide targeting endothelium, characterized in that: The structure is as follows: 。 2. The peptide according to claim 1, characterized in that: Prepared by solid-phase synthesis.

3. Use of the peptide of claim 1 in the preparation of a medicament for the prevention and / or treatment of diabetic lower limb ischemia.

4. The use according to claim 3, characterized in that, The drug also includes pharmaceutically acceptable excipients.

5. The use according to claim 4, characterized in that, The drug is an injectable preparation.

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