Endothelium-targeting peptide and application thereof in prevention and treatment of diabetic lower limb ischemia

Abstract

The invention relates to a peptide targeting endothelium, which is a polypeptide prepared by mutual covalent cross-linking of GLP-1 (32-36), RGD cyclic peptide, a polymer and an ROS-responsive polymer. Through verification, the peptide has relatively high activity and relatively strong enzymolysis stability in plasma, can be combined with a receptor on the surface of a vascular endothelial cell in a targeting manner, can be maintained in an effective treatment window for a long time, gives full play to the treatment advantage of the peptide, 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] The present invention belongs to the field of medical inventions, and specifically relates to the use of an endothelial-targeted long-acting peptide in the preparation of a drug for preventing and / or treating diabetic lower limb ischemia. Through the molecular structure of the drug, targeted binding to endothelial cells is achieved, thereby achieving long-term treatment of diabetic lower limb ischemia. Background Art

[0002] With the changes in lifestyle and living environment and the growing elderly population, the number of patients with diabetes is increasing year by year. It is estimated that by the middle of this century, more than 700 million people in the world will suffer from diabetes, and a series of acute and chronic complications caused by poor blood sugar control will follow. In addition to the acute metabolic disorders caused by high blood sugar, long-term chronic high blood sugar can also cause damage and dysfunction of various tissues and organs throughout the body, and can also cause disability or even life-threatening serious clinical conditions such as myocardial infarction, stroke, amputation, renal failure, and blindness.

[0003] Diabetic lower limb ischemia is the most common chronic vascular complication in diabetic patients. The cause of the disease is: in a high-sugar environment, reactive oxygen species (ROS) are overproduced in endothelial cell mitochondria, which activate a series of downstream signaling pathways, causing endothelial cell and microvascular dysfunction, manifested as stenosis and occlusion of lower limb arteries. Although diabetic lower limb ischemia usually manifests as claudication, the long-term lack of effective treatment often leads to severe limb ischemia and even requires amputation. In addition to surgical treatment, no drugs have been found to effectively improve diabetic lower limb ischemia. However, due to reasons such as anatomical structure, the symptoms of lower limb ischemia in some patients cannot be effectively improved after surgical treatment, and the condition will inevitably worsen. Therefore, there is an urgent need for an efficient and low-toxic drug preparation to effectively treat diabetic lower limb ischemia.

[0004] Recent studies have shown that incretin hormones such as glucagon-like peptide-1 (GLP-1) have a role in regulating angiogenesis. As a naturally occurring hormone, GLP-1 plays a vital role in regulating glucose homeostasis by stimulating insulin secretion. Early studies believed that only GLP-1 could play a role in regulating blood sugar in vivo and improve the prognosis of diabetes-related complications. As the research deepened, various metabolites of GLP-1 were also found to have various beneficial biological effects. GLP-1 (32-36), as the main end product of GLP-1 proteolysis, has been found to reduce the apoptosis of pancreatic β cells, increase energy consumption, and reduce the body weight of obese mice. However, its role in improving diabetic lower limb ischemia has rarely been reported. The team's recent research found that GLP-1 (32-36) has a direct effect on alleviating diabetic lower limb ischemia independent of insulinotropic effects. By regulating metabolic reprogramming of glycolytic flux, GLP-1 (32-36) can improve angiogenesis in endothelial progenitor cells and type 1 diabetic mouse models under high glucose exposure, which provides a certain theoretical basis for its subsequent application in patients with diabetic lower limb ischemia. However, as a short peptide composed of 5 amino acids, the half-life of GLP-1 (32-36) in the body (less than 5 minutes) is much shorter than the half-life of insulin (2 hours). When treating diabetic lower limb ischemia, it often fails to exert a therapeutic effect and has been degraded. Multiple injections are usually required to ensure the biological effect of the peptide. In addition, the short peptide has a short metabolic time in the body and obvious off-target effects. It is difficult to act on the damaged vascular endothelium for a long time, which greatly reduces the therapeutic effect. Therefore, a method is needed to effectively solve the short half-life of GLP-1 (32-36) to achieve the long-term sustained release of short peptides and the effect of targeting the vascular endothelium.

[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 integrin αvβ3 receptors and play an important role in the mutual conduction of cell signals. This receptor is strongly expressed on the membrane of newborn vascular endothelial cells, but is rarely expressed in mature vascular endothelial cells and most normal organ systems. Based on this, the treatment of endothelial cells based on RGD cyclic peptides has become a hot topic in recent years. At present, there have been reports on the use of RGD cyclic peptide complexes for targeted tumor therapy, but the above compounds mainly achieve targeted killing of tumors by biocoupling anti-tumor drugs. Based on the outstanding advantages of RGD cyclic peptides and the current research status, there is a lot of room for the development of RGD cyclic peptide complexes for targeted treatment of damaged endothelial cells, but there are no reports on RGD cyclic peptide complexes for the treatment of diabetic lower limb ischemia. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides an endothelial-targeted long-acting peptide, which is a long-acting sustained-release polypeptide for treating diabetic lower limb ischemia.

[0007] The endothelial targeted long-acting peptide of the present invention is made of GLP-1 (32-36), RGD cyclic peptide, polymer and ROS responsive polymer. Suitable polymers such as, but not limited to, polyethylene glycol (PEG), polyacrylamide, polyhydroxyethyl aspartic acid, polysulfobetaine, polycarboxylate betaine, povidone, chitosan, etc., are applied to the present invention alone or in combination. Among them, polyethylene glycol is more preferred. Suitable ROS responsive polymers such as polyborates, oligoproline peptides, polysaccharides, peroxypolythioketones, propylene sulfide, selenane, aminoacrylate, alkyl selenium telluride, etc., are applied to the present invention alone or in combination. Among them, polyborates are more preferred.

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

[0009] The present invention also provides a use of an endothelial cell targeting peptide in preparing a drug for preventing and / or treating diabetic lower limb ischemia. The molecular structure of the compound is shown in the following formula, and compounds with more than 90% homology thereof.

[0010]

[0011] Furthermore, the drug is a preparation prepared with an endothelial-targeted long-acting peptide as an active ingredient and pharmaceutically acceptable excipients.

[0012] Furthermore, the preparation is an injection preparation.

[0013] The present invention discovers for the first time that the endothelial-targeted long-acting peptide with the molecular structure shown above can be quickly and safely delivered to the damaged area in the form of a compound, meeting the clinical requirements for long-term and continuous treatment of diabetic lower limb ischemia.

[0014] The present invention also discovered for the first time that the endothelial-targeted long-acting peptide with the molecular structure shown above can effectively improve diabetic lower limb ischemia. After constructing a diabetic lower limb ischemia model and intervening with this compound, the blood perfusion on the affected side of the mice was significantly improved, and the damage to various tissues and organs of the mice under high sugar stimulation was also significantly alleviated. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram of the molecular structure of endothelial-targeted long-acting peptides and non-endothelial-targeted long-acting peptides, wherein A is a long-acting peptide targeting the endothelium, and B is a long-acting peptide targeting the non-endothelial cells.

[0016] Figure 2 High performance liquid chromatography-mass spectrometry detection data of the molecular structure of endothelial targeting long-acting peptide.

[0017] Figure 3 Figure 1 is the weight and blood sugar changes of each group of experimental mice over time. A is the weight change of each group of experimental mice over time; B is the blood sugar change of each group of experimental mice over time.

[0018] Figure 4 The molecular structure and pharmacodynamics of endothelial-targeted long-acting peptides are studied. A is a schematic diagram of the molecular structure of endothelial-targeted long-acting peptides, non-endothelial-targeted long-acting peptides and modified GLP-1 (32-36); B is a Doppler ultrasound image of lower limb blood perfusion in each group of experimental mice; C is a curve of lower limb blood perfusion changes in each group of experimental mice.

[0019] Figure 5 This is a diagram showing the safety evaluation results of endothelial-targeted long-acting peptides in organs such as the kidney, liver, lung, spleen and heart.

[0020] Figure 6 This is a pharmacodynamic study of endothelial targeting structures with or without long peptides. A is the Doppler ultrasound image of lower limb blood perfusion of each group of experimental mice; B is the change curve of lower limb blood perfusion of each group of experimental mice. DETAILED DESCRIPTION

[0021] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. In addition, the relevant terms and laboratory procedures such as protein and medicinal chemistry, molecular biology, tissue staining, animal experiments, etc. used herein are terms and conventional procedures widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0022] As used herein, "at least one" or "one or more" may mean 1, 2, 3, 4, 5, 6, 7, 8 or more.

[0023] As used herein, the expressions "comprises," "comprising," "containing," and "having" are open ended, meaning the inclusion of the listed elements, steps, or components but not the exclusion of other unlisted elements, steps, or components. The expression "consisting of" excludes any element, step, or component not specified. The expression "consisting essentially 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 basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of" and "consisting of" are encompassed within the meaning of the expression "comprising."

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

[0025] Unless otherwise indicated, any numerical value or numerical range, such as concentration or concentration range, is understood to be modified by the term "about" in any case. Therefore, numerical values ​​generally include ± 10% of the 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 the range, including integers and fractions within the range, unless the context clearly indicates otherwise.

[0026] The specific implementation modes of the present invention are described in detail below, but they are only preferred embodiments of the present invention and do not limit the present invention. For those skilled in the art, the preparation process, materials used, and the amounts of materials used in the present invention may be subject to various changes and variations. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

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

[0028] Experimental Example 1: Preparation of endothelial-targeted long-acting peptides and non-endothelial-targeted long-acting peptides

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

[0030] according to Figure 2 The HPLC-MS / MS detection data of the molecular structure of the endothelial-targeted long-acting peptide were shown, and the polypeptide conjugate (Mass=2689Da) was successfully prepared.

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

[0032] 1. Experimental methods:

[0033] Male C57BL / 6 mice aged 6-8 weeks (weight>20g) were selected as model animals. The mice were housed in an incubator with a 12-hour light-dark cycle and received a standard laboratory diet. A diabetic mouse model was established by intraperitoneal injection of streptozotocin (70mg / (kg.d)). After 2 weeks of continuous injection, blood was drawn from the tail vein of the mice to measure blood glucose. If the fasting blood glucose of the experimental mice was >11.1mmol / l, the animal model of type 1 diabetes (Type 1Diabetes Mellitus, T1DM) was successfully established.

[0034] After the blood sugar of T1DM mice was stable for 4 weeks, the diabetic lower limb ischemia animal model was established. The mice were placed in an anesthesia chamber and inhaled with isoflurane for induction of anesthesia. After the righting reflex of the mice disappeared, 1-3% isoflurane was vaporized in pure oxygen and maintained by mask inhalation at a continuous low flow rate of 1L / min. The anesthetized mice were placed in a supine position on the modeling table, the tails of the four limbs of the mice were fixed with tape, and the hair in the right inguinal area and surrounding areas was shaved with a shaver, and then a scalpel was used to cut a wound of about 5mm in the skin of the right inguinal area of ​​the mice. Then, a sterilized pointed cotton swab was used to separate the subcutaneous fat tissue, fully expose the femoral artery and femoral vein, carefully peel off the fascia tissue on the surface, and separate the femoral artery and femoral vein with curved forceps. The femoral artery and femoral vein were ligated at the distal end and proximal segment with fine thread, and then the femoral vein and femoral artery were cut between the two ligature points with scissors, and then the wound was sutured with fine thread. The left limb was not treated and kept as a control. The modeling effect was evaluated by measuring the blood perfusion of the lower limbs on the operated side using Doppler ultrasound. The interruption of blood flow at the ligation site was considered as the successful construction of the diabetic hind limb ischemia (DHLI) animal model.

[0035] Experimental Example 3: Changes in body weight and blood sugar in patients with endothelial-targeted long-acting peptides

[0036] 1. Experimental samples:

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

[0038] 2. Experimental methods:

[0039] A total of 24 T1DM mice (male, 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 every day for 28 consecutive days), a non-endothelial targeted long-acting peptide group (each mouse received 10ug intraperitoneal injection every day for 28 consecutive days), and a GLP-1 (32-36) group (each mouse received 10ug intraperitoneal injection every day for 28 consecutive days), with 6 mice in each group. The weight and blood sugar of mice in each group were measured using a scale and a blood glucose meter. The first day of administration of each group of mice was recorded as day 0, and then the mice were placed on a scale at room temperature every 7 days to weigh their body weight, and blood glucose was measured using a blood glucose meter by drawing blood from the tail vein of the mice. The weight and blood sugar of each group of mice were recorded until the 63rd day, and the weight and blood sugar of each group of mice were plotted over time and compared.

[0040] 3. Results and Discussion

[0041] pass Figure 3 A It can be observed that there was no significant difference in the body weight of mice in each group before administration. With the continuous intraperitoneal injection, the tEC-cRGD-(32-36) group and the c-EKEK-(32-36) group showed a more significant weight loss effect than the control group and the GLP-1(32-36) group. After stopping the administration, the mice in each group gradually returned to the same level. Figure 3 B shows that there is no significant difference in the changes in blood glucose levels of each group of mice before and after continuous administration.

[0042] Experimental Example 4: Pharmacodynamic study of endothelial-targeted 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] A total of 24 DHLI mice (male, 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 every day for 28 consecutive days), a non-endothelial targeted long-acting peptide group (each mouse received 10ug intraperitoneal injection every day for 28 consecutive days), and a GLP-1 (32-36) group (each mouse received 10ug intraperitoneal injection every day for 28 consecutive days), with 6 mice in each group. The hair on the back of the mice was shaved with a shaver, and the blood perfusion of the right lower limb of each group of mice before modeling was measured using a lower limb blood flow Doppler, and the blood perfusion of the healthy (left) and affected (right) lower limbs of the mice was measured at predetermined time points (0th, 3rd, 7th, 14th, 21st, 28th days) and the blood perfusion ratio of the affected / healthy lower limbs was calculated, and the improvement of lower limb blood perfusion in different groups of mice over time was compared.

[0047] 3. Results and Discussion

[0048] pass Figure 4 B observed that after the operation, the 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 shows that the blood perfusion of the endothelial targeted long-acting peptide group, the non-endothelial targeted long-acting peptide group and the GLP-1 (32-36) group was significantly improved compared with the control group, indicating that GLP-1 (32-36) has an effective effect in treating diabetic lower limb ischemia symptoms. The blood perfusion ratio recovery of the endothelial targeted long-acting peptide group was significantly better than that of the non-endothelial targeted long-acting peptide group and the GLP-1 (32-36) group, proving that the long-term effect of the endothelial targeted long-acting peptide and the characteristics of targeting endothelial cells can maximize the therapeutic effect of the drug.

[0049] Experimental Example 5: Safety evaluation of endothelial-targeted 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] A total of 12 T1DM mice (male, 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 an intraperitoneal injection of 10ug per day for 28 consecutive days), a non-endothelial targeted long-acting peptide group (each mouse received an intraperitoneal injection of 10ug per day for 28 consecutive days), and a GLP-1 (32-36) group, with 3 mice in each group. On the 28th day after administration, the mice in each group were killed by dislocation of the neck and five tissues of the liver, kidney, lung, spleen and heart of each group of mice were collected. The above tissues were placed in a 4% paraformaldehyde solution for overnight treatment, and then cut into 5um slices, and then the slices of each tissue and organ of each group of mice were obtained by hematoxylin-eosin (H&E) staining. Finally, high-resolution digital images were collected at the same magnification using an inverted white light microscope, and the tissue differences of each group of mice were compared.

[0053] 3. Results and Discussion:

[0054] pass Figure 5 A large number of apoptotic and necrotic cells can be seen in the kidneys and livers of T1DM mice, the pulmonary lobules in the lungs are filled with a large number of necrotic cells, and a large number of immune cells in the spleen are necrotic and apoptotic, indicating that high glucose stimulation has caused significant damage to the tissues and organs of mice. The degree of damage to various tissues and organs in the drug treatment group under high glucose stimulation has been improved to a certain extent, among which the improvement effect of the endothelial targeted long-acting peptide group is the most obvious, and the number of necrotic and apoptotic cells in the tissues has been significantly reduced, proving that the endothelial targeted long-acting peptide can effectively protect the damage to various tissues and organs of mice under high glucose stimulation.

[0055] Experimental Example 6: Pharmacodynamic study of endothelial targeting structures with or without long peptide linkage.

[0056] 1. Experimental samples:

[0057] Endothelial targeted long-acting peptide (tEC-cRGD-(32-36)) and targeted empty load (tEC-cRGD) prepared according to Experimental Example 1

[0058] 2. Experimental methods:

[0059] A total of 18 DHLI mice (male, 27±1g) constructed according to Experimental Example 2 were randomly divided into a pbs group (each mouse received an intraperitoneal injection of the same volume of pbs every day for 28 consecutive days), an endothelial targeted long-acting peptide group (each mouse received an intraperitoneal injection of 10ug every day for 28 consecutive days), and an endothelial targeted empty load group (each mouse received an intraperitoneal injection of 10ug every day for 28 consecutive days), with 6 mice in each group. The hair on the back of the mice was shaved with a shaver, and the blood perfusion of the right lower limb of each group of mice before modeling was measured using a lower limb blood flow Doppler, and the blood perfusion of the healthy (left) and affected (right) lower limbs of the mice was measured at predetermined time points (0th, 3rd, 7th, 14th, 21st, 28th days) and the blood perfusion ratio of the affected / healthy lower limbs was calculated, and the improvement of lower limb blood perfusion in different groups of mice over time was compared.

[0060] 3. Results and Discussion

[0061] pass Figure 6 A observed that after surgery, the blood flow in the right lower limb of the mouse was significantly reduced, proving that the diabetic lower limb ischemia model was successfully established. 7-28 days after surgery, the blood perfusion of the endothelial-targeted long-acting peptide group was significantly improved compared with the pbs group and the endothelial-targeted empty group, and there was no significant difference in blood perfusion between the pbs group and the targeted empty group, indicating that endothelial-targeted GLP-1 (32-36) has an effective effect in treating diabetic lower limb ischemia and its therapeutic effect is unrelated to the targeting structure itself.

Claims

1. A peptide targeting endothelium, characterized in that: The endothelial targeted long-acting peptide is formed by sequentially connecting GLP-1 (32-36), RGD cyclic peptide, polymer and ROS-responsive polymer.

2. The peptide according to claim 1, characterized in that: The polymer is selected from one or more of polyethylene glycol, polyacrylamide, polyhydroxyethyl aspartic acid, polysulfide betaine, polycarboxylate betaine, povidone and chitosan.

3. The peptide according to claim 1, characterized in that: The ROS-responsive polymer is selected from one or more of polyboric acid ester, oligoproline peptide, polysaccharide, peroxy polythioketone, propylene sulfide, selenane, amino acrylate, and alkyl selenium telluride.

4. The peptide according to claim 1, characterized in that: The GLP-1 (32-36), RGD cyclic peptide, polymer and ROS-responsive polymer are synthesized by covalent cross-linking under specific preparation conditions.

5. The peptide according to claim 1, characterized in that: Prepared by solid phase synthesis.

6. The peptide according to claim 1, characterized in that: The molecular structure of the compound is shown below, and its homology is more than 90%.

7. Use of the peptide according to claim 1 in the preparation of a medicament for preventing and / or treating diabetic lower limb ischemia.

8. The use according to claim 7, characterized in that The compounds are shown below.

9. The use according to claim 7 or 8, characterized in that The medicine is a preparation prepared with polypeptide as active ingredient and pharmaceutically acceptable excipients.

10. The use according to claim 9, characterized in that The preparation is an injection preparation.

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