Application of RCL-M1A and / or RCL-M8H in preparation of medicine for preventing and / or treating venous thrombosis and embolism

Abstract

The invention provides an application of RCL-M1A and / or RCL-M8H in preparation of drugs for preventing and / or treating venous thrombosis and embolism, the RCL-M1A and / or RCL-M8H is adopted to simulate the occurrence and development of VTE by applying a mouse inferior vena cava thrombosis model, and it is found that the RCL-M1A and / or RCL-M8H inhibits venous thrombosis by inhibiting platelet and neutrophil extracellular traps (NETs). Meanwhile, the bleeding risk is not increased, and the compound can be applied to preparation of drugs for preventing and / or treating venous thrombosis and embolism.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to an application of RCL-M1A and / or RCL-M8H in the preparation of a drug for preventing and / or treating venous thrombosis and embolism. Background Art

[0002] Venous thrombosis and embolism (VTE) include deep vein thrombosis (DVT) and pulmonary embolism (PE), which are manifestations of the same disease at different stages of onset and in different tissues and organs. In the United States, the incidence of VTE is about 1.17 / 1000 person-years, with about 350,000 new cases of VTE each year. The incidence of VTE in my country is also showing a clear upward trend. A study of newly diagnosed VTE patients between 2004 and 2016 showed that the incidence of VTE increased from 28.1 / 100,000 person-years to 48.3 / 100,000 person-years. Another study of 105,723 VTE patients in 90 hospitals between 2007 and 2016 showed that the hospitalization rate for VTE increased from 3.2 / 100,000 to 17.5 / 100,000. At the same time, the risk of VTE in cancer patients is 6 times that of non-cancer patients, and VTE is an important cause of death in cancer patients. Although the mortality rate of VTE is declining, VTE is still the third leading cause of death worldwide, and it imposes a huge economic and resource burden on individuals and society.

[0003] The components of venous thrombosis mainly include red blood cells and fibrin, so it is also called "red thrombosis". Venous thrombosis is loosely adhered to the blood vessel wall and is easy to fall off. After falling off, the thrombus is easy to block the lungs and cause pulmonary embolism. If rescue is not timely, it is very easy to cause the patient's death in a short period of time. In terms of the pathogenesis of venous thrombosis, it is currently believed that the three classic factors that constitute the risk of VTE are endothelial damage, blood stasis and turbulence, and blood hypercoagulability, that is, Virchow's triangle. In the case of slow blood flow, endothelial damage causes tissue factor exposure. Tissue factor is the starting link of the extrinsic coagulation pathway. Therefore, tissue factor activates the extrinsic coagulation pathway, and the hypercoagulable state further causes overactivation of the coagulation system.

[0004] From the perspective of pathogenesis, the overactivation of the coagulation system in which coagulation factors participate has become the main pathogenesis of venous thrombosis and embolism. In recent years, the formation process of venous thrombosis has been considered to be an inflammatory process involving multiple factors such as coagulation factors, leukocytes, and platelets, but the overactivation of the coagulation system is still the core pathogenesis of venous thrombosis and embolism. Therefore, in terms of treatment, anticoagulation strategy is currently the main choice for the treatment and prevention of VTE. Currently, commonly used anticoagulant drugs in clinical practice include heparin, low molecular weight heparin, warfarin, and new oral anticoagulants such as rivaroxaban and apixaban. These drugs will inevitably cause a higher risk of bleeding while effectively inhibiting the coagulation system. While inhibiting venous thrombosis, it is difficult to avoid bleeding side effects, which has an adverse effect on the patient's prognosis.

[0005] Therefore, developing new treatment strategies for venous thrombosis and embolism without increasing the risk of bleeding has become a promising direction. Summary of the invention

[0006] The present invention aims to provide a new use of small molecule chemical RCL-M1A and / or RCL-M8H in the preparation of drugs for preventing and / or treating venous thrombosis and embolism.

[0007] According to the first aspect of the present invention, there is provided a use of RCL-M1A and / or RCL-M8H in the preparation of a drug for preventing and / or treating venous thrombosis and embolism, wherein the chemical structural formula of the RCL-M1A is shown in Formula I, and the chemical structural formula of the RCL-M8H is shown in Formula II;

[0008]

[0009] As an optional embodiment, the prevention and / or treatment of venous thrombosis and embolism is to inhibit venous thrombosis by inhibiting platelet and neutrophil extracellular traps (NETs) without increasing the risk of bleeding.

[0010] As an optional embodiment, the venous thrombosis and embolism include deep vein thrombosis, pulmonary embolism and thrombosis in other parts of the venous system.

[0011] According to a second aspect of the present invention, an antagonist is provided, comprising the aforementioned RCL-M1A and its structural derivatives and / or RCL-M8H and its structural derivatives.

[0012] According to a third aspect of the present invention, a pharmaceutical composition is provided, comprising the aforementioned RCL-M1A and its structural derivatives and / or RCL-M8H and its structural derivatives.

[0013] As an optional embodiment, the pharmaceutical composition further comprises other anticoagulant drugs.

[0014] As an optional embodiment, the anticoagulant drug includes warfarin and / or rivaroxaban.

[0015] As an optional embodiment, the pharmaceutical composition is a solid dosage form, a liquid preparation or a semisolid preparation.

[0016] As an optional embodiment, the pharmaceutical composition is an oral preparation or an injection.

[0017] The present invention proposes the use of RCL-M1A and / or RCL-M8H in the preparation of drugs for the prevention and / or treatment of venous thrombosis and embolism. RCL-M1A and / or RCL-M8H are used to simulate the occurrence and development of VTE using a mouse inferior vena cava thrombosis model. It is found that RCL-M1A and / or RCL-M8H inhibit venous thrombosis by inhibiting platelet and neutrophil extracellular traps (NETs) without increasing the risk of bleeding, and can be used to prepare drugs for the prevention and / or treatment of venous thrombosis and embolism. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a statistical diagram of the effect of each group on thrombus length in a venous thrombosis and embolism model of the present invention.

[0019] Figure 2 This is a statistical diagram showing the effect of each group on thrombus weight in a venous thrombosis and embolism model according to the present invention.

[0020] Figure 3 This is a diagram showing the HE staining results of thrombus sections after thrombus formation in each group in the venous thrombosis and embolism model according to the example of the present invention.

[0021] Figure 4 This is a diagram showing the histochemical results of CD41 (representing platelets) in thrombus sections of each group after thrombus induction in a venous thrombosis and embolism model according to the present invention.

[0022] Figure 5 This is an example of the present invention, showing the histochemical results of MPO (representing NETs formation) in thrombus slices after thrombus formation in each group in the venous thrombosis and embolism model.

[0023] Figure 6 It is a statistical diagram of the MPO content in plasma before and after the establishment of the venous thrombosis and embolism model (representing NETs) according to the example of the present invention.

[0024] Figure 7 is a statistical diagram of an exemplary bleeding model of the present invention.

[0025] Figure 8 FIG. 1 is a diagram showing an example of a bleeding model according to an embodiment of the present invention. DETAILED DESCRIPTION

[0026] In order to better understand the technical content of the present invention, specific embodiments are given and described as follows in conjunction with the accompanying drawings.

[0027] Various aspects of the present invention are described in this disclosure with reference to the accompanying drawings, in which many illustrative embodiments are shown. The embodiments of the present disclosure are not necessarily intended to include all aspects of the present invention. It should be understood that the various concepts and embodiments introduced above, as well as those described in more detail below, can be implemented in any of many ways.

[0028] RCL-M1A and RCL-M8H belong to a type of compound of formula III. The compound of formula III can specifically bind to the SH3 domain of Src kinase, interfere with the interaction between integrin αIIbβ3 and Src kinase, and then selectively inhibit the outside-in signal transduction of platelets, without affecting the inside-out signal transduction of platelets, and has the effect of inhibiting arterial thrombosis (such as myocardial infarction and cerebral infarction). However, the main component of arterial thrombosis is platelets. Therefore, antiplatelet is the main treatment strategy. Therefore, the therapeutic effect of antiplatelet drugs and small molecules on arterial thrombosis is very critical.

[0029]

[0030] Arterial thrombotic diseases mainly include myocardial infarction and stroke (cerebral infarction, transient cerebral ischemia). Thrombi mainly come from the detachment of emboli from the heart or aorta. The site of thrombosis is located in the arteries with faster blood flow, mostly in the coronary arteries and cerebral arteries. Platelets need to resist the high blood flow shear force and high blood flow impact force caused by faster blood flow. The basis of its pathogenesis is excessive platelet activation and a series of functions such as adhesion, stretching and aggregation lead to pathological thrombosis, causing ischemic necrosis of heart and brain tissues in the affected blood vessel distribution area, which can seriously endanger the patient's life. The main component of arterial thrombosis is a firm thrombus formed by platelets and fibrin, also known as a "white thrombus", which adheres to the damaged arterial endothelium and causes stenosis or even blockage of the arterial lumen. Therefore, antiplatelet drugs have become the first choice for the treatment of arterial thrombotic diseases such as myocardial infarction and cerebral infarction. Classic antiplatelet drugs, such as aspirin, clopidogrel, and eptifibatide, block the bidirectional signal of platelet surface integrin αIIbβ3, which simultaneously affects thrombosis and hemostasis. While exerting antithrombotic effects, they also have a serious risk of bleeding. Therefore, the development of new antiplatelet drugs that do not increase the risk of bleeding is a key strategy for the treatment of arterial thrombotic diseases.

[0031] Venous thrombosis mainly comes from the blockage of venous return throughout the body, mainly occurring in veins with slower blood flow, such as deep vein thrombosis (DVT) in the lower limbs. Because there are more venous valves in the lower limbs and the veins themselves have weak vascular walls and poor elasticity, long-term sitting can lead to slow venous return in the lower limbs. Blood flow is easily stagnant at the venous valves, which can easily lead to deep vein thrombosis in the lower limbs. The characteristic of venous thrombosis is that it is not firmly bonded to the venous wall and is very easy to fall off. The detached thrombus can easily block the capillary-rich lungs with the blood flow, causing pulmonary embolism. Therefore, deep vein thrombosis (DVT) of the lower limbs and pulmonary embolism (PE) are the two main disease phenotypes of venous thrombosis and embolism.

[0032] The main components of venous thrombosis are red blood cells and fibrin. The pathogenesis is slow blood flow, hypercoagulable state and endothelial damage. Virchow's triangle is the main pathogenesis of venous thrombosis and embolism. The treatment strategy is mainly anticoagulant drugs, such as warfarin and rivaroxaban.

[0033] Although both are called thrombosis, venous thrombosis and embolism and arterial thrombosis are two completely different diseases. The difference between the two is that the thrombus has different sources, different sites of occurrence, different thrombus components, different pathogenesis and different treatment strategies. Therefore, arterial thrombosis and venous thrombosis and embolism are two completely different types of diseases from pathogenesis to treatment strategy. Drugs that can inhibit arterial thrombosis are not necessarily effective for venous thrombosis. At present, there have not been a large number of clinical trials to confirm the exact role of antiplatelet drugs in the treatment of venous thrombosis and embolism, nor have antiplatelet drugs for the treatment of venous thrombosis and embolism been included in the guidelines.

[0034] In an exemplary embodiment of the present invention, there is provided a use of RCL-M1A and / or RCL-M8H in the preparation of a drug for preventing and / or treating venous thrombosis and embolism, the chemical structure of RCL-M1A is shown in Formula I, and the chemical structure of RCL-M8H is shown in Formula II;

[0035]

[0036]

[0037] Among them, the preparation of RCL-M1A and RCL-M8H is prior art, and can be prepared according to the method of prior art, which will not be described in detail here.

[0038] The present invention constructs a method for screening candidate compounds for resisting venous thrombosis and embolism, and unexpectedly finds that RCL-M1A and RCL-M8H have an inhibitory effect on venous thrombosis and embolism.

[0039] In one exemplary embodiment, a method for screening candidate compounds for anti-venous thrombosis and embolism is provided, the method comprising the steps of:

[0040] (a) A mouse model of venous thrombosis and embolism was constructed. In the test group, in the presence of the test compound (RCL-M1A, RCL-M8H), the mice were compared with the solvent group to detect the effect of the test compound on whether venous thrombosis was formed and on venous thrombosis and embolism.

[0041] (b) A mouse model of venous thrombosis and embolism was constructed. In the control group, in the presence of the control substance, i.e., the anticoagulant drug rivaroxaban (0.25, 0.5, 1 mg / kg) and the antiplatelet drug aspirin (5.4 mg / kg), the control group was compared with the test compound group to detect the effect of the control substance on whether venous thrombosis was formed and on venous thrombosis and embolism.

[0042] As an optional example, the prevention and / or treatment of venous thrombosis and embolism is to inhibit venous thrombosis by inhibiting platelet and neutrophil extracellular traps (NETs) without increasing the risk of bleeding.

[0043] As optional examples, venous thrombosis and embolism include deep vein thrombosis and pulmonary embolism and thrombosis in other parts of the venous system.

[0044] In a more preferred example, the venous thrombosis and embolism is selected from the group consisting of deep vein thrombosis and embolism (usually in the lower limbs), pulmonary embolism, or a combination thereof.

[0045] In another exemplary embodiment of the present invention, an antagonist is further provided, comprising the aforementioned RCL-M1A and its structural derivatives and / or RCL-M8H and its structural derivatives.

[0046] In another exemplary embodiment of the present invention, a pharmaceutical composition is also provided, comprising the aforementioned RCL-M1A and its structural derivatives and / or RCL-M8H and its structural derivatives.

[0047] As an optional example, the pharmaceutical composition further includes other anticoagulant drugs.

[0048] As optional examples, anticoagulant drugs include warfarin and / or rivaroxaban.

[0049] As optional examples, the pharmaceutical composition is a solid dosage form, a liquid preparation or a semisolid preparation.

[0050] As an optional example, the pharmaceutical composition is an oral preparation or an injection.

[0051] In a more preferred example, the pharmaceutical composition is a tablet, granule, capsule, injection, infusion, paste, gel, solution, microsphere or film.

[0052] In other optional examples, the subjects of the drug for preventing and / or treating venous thrombosis and embolism are humans and non-human mammals (rodents, rabbits, monkeys, livestock, dogs, cats, etc.).

[0053] For better understanding, the present invention is further described below in conjunction with several specific examples, but the preparation process is not limited thereto, and the content of the present invention is not limited thereto.

[0054] Unless otherwise specified, the materials in the examples were prepared according to existing methods or directly purchased from the market.

[0055] Example 1

[0056] [Constructing a venous thrombosis and embolism model and testing the inhibitory effects of small molecules on venous thrombosis and embolism]

[0057] Thirty-three 8-week-old wild-type (WT) male mice were divided into a DMSO group (6 mice), an RCL-M1A (10 mg / kg) group (6 mice), an RCL-M8H group (10 mg / kg) (6 mice), a rivaroxaban (0.25, 0.5, 1 mg / kg) group (9 mice) and an aspirin (5.4 mg / kg) group (6 mice). Mice were anesthetized with aflototin according to their weight, and the abdomen was shaved; the abdominal skin, subcutaneous tissue, and peritoneum of the mice were then incised in turn, the abdominal tissue was bluntly separated, the inferior vena cava was exposed, and the inferior vena cava was separated; a silicone thread with a diameter of 0.35 mm was placed next to the inferior vena cava, and the silicone thread and the lower limb vein were ligated together above the renal vein branch with 5-0 medical surgical sutures, and then the silicone thread was removed to prepare a partially ligated venous thrombosis model; the abdominal organs were put back, and the peritoneum, subcutaneous tissue, and skin were sutured layer by layer; physiological saline was used throughout the process to keep the incision and abdominal cavity moist; the mice were then placed on a warming pad and observed for 2 hours.

[0058] The DMSO, RCL-M1A and RCL-M8H groups were injected with 10 mg / kg of the drug via tail vein 1 hour before the operation; the rivaroxaban group was injected with different doses of the drug via tail vein before the operation; the aspirin group was given 5.4 mg / kg of the drug by oral gavage 24 hours and 2 hours before the operation.

[0059] After 2 hours, the abdominal cavity was opened, the inferior vena cava was completely separated, and the inferior vena cava was ligated above the iliac vein branch (the fixed length of the ligature in each group was 9 cm), and then the blood vessels were cut above and below the ligatures at both ends. The length and weight of the thrombus formation were measured in each group, and then the thrombus tissue was placed in a tissue fixative, embedded and sliced, and HE staining and immunohistochemistry experiments were performed. Blood was collected from mice before and after modeling, and plasma was taken to determine the MPO content. The results are shown in Figure 1-6 shown.

[0060] from Figure 1 It can be seen that RCL-M1A and RCL-M8H can reduce the length of thrombus, among which RCL-M1A and RCL-M8H can achieve the effect of the clinical dose of positive control rivaroxaban; Figure 2 It showed that RCL-M1A and RCL-M8H could reduce the weight of thrombus, and both could achieve the effect of positive control rivaroxaban; Figure 3 The presence of RCL-M1A or RCL-M8H in the lumen greatly reduced thrombus formation or even almost eliminated thrombus formation; Figure 4 It showed that RCL-M1A or RCL-M8H could be seen in the lumen, which reduced the aggregation of platelets at the thrombus (CD41 is a platelet marker); Figure 5 It showed that RCL-M1A or RCL-M8H could be seen in the lumen, which reduced the formation of NETs (MPO is a marker of NETs); Figure 6 It was shown that RCL-M1A or RCL-M8H reduced the production of MPO and inhibited the formation of NETs.

[0061] Therefore, it can be demonstrated that RCL-M1A and RCL-M8H greatly reduced the formation of venous thrombus and decreased platelet aggregation and NETs formation.

[0062] Example 2

[0063] [Effects of small molecules RCL-M1A and RCL-M8H on bleeding time]

[0064] Thirty-two 8-week-old wild-type (WT) male mice were randomly divided into DMSO group (8 mice), RCL-M1A-10 mg / kg group (8 mice), RCL-M8H-10 mg / kg group (8 mice) and rivaroxaban-0.25 mg / kg group (8 mice). 10 mg / kg of RCL-M1A and RCL-M8H were injected into the tail vein 2 hours before the experiment, and 0.25 mg / kg of rivaroxaban was injected before the experiment. Then, the tail tip was cut off 5 mm from the tail of the mouse with sharp scissors, and blood was dripped on the filter paper every 15 seconds, and it was ensured that the injured area would not be touched until the mouse stopped bleeding (when the bleeding reached 10 minutes, the experiment was stopped to reduce the pain of the experimental animals).

[0065] The results are as follows Figure 7 and Figure 8 As shown in the results, it can be seen that compared with the control group DMSO, RCL-M1A and RCL-M8H do not increase bleeding time, while the oral anticoagulant rivaroxaban has severe bleeding at clinical doses, and some mice have continuous bleeding. Therefore, RCL-M1A and RCL-M8H do not increase the risk of bleeding.

[0066] In the present invention, we found that the small molecule compound RCL-M1A and / or RCL-M8H can effectively inhibit venous thrombosis, wherein the effect of RCL-M1A is equivalent to that of the oral anticoagulant rivaroxaban at 5 times the clinically commonly used dose, and the effect of RCL-M8H is equivalent to that of the clinically commonly used dose of rivaroxaban, including the inhibition of thrombus length and thrombus weight ( Figure 1 , Figure 2 ).

[0067] In terms of mechanism, RCL-M1A and / or RCL-M8H mainly exert their effects by inhibiting the formation of platelet-neutrophil extracellular traps (NETs) ( Figure 3 , Figure 4 , Figure 5 , Figure 6 ).

[0068] In the tail-cut bleeding model, RCL-M1A and / or RCL-M8H did not increase the risk of bleeding at high doses, and the bleeding time was comparable to that of the DMSO control group ( Figure 6 ), while the oral anticoagulant rivaroxaban caused severe bleeding at clinical doses, with an average bleeding time of about 8 minutes, which was significantly longer than the control group DMSO (about 3 minutes). Figure 7 ).

[0069] In the results of using filter paper to record the size and number of blood drops (blood drops were placed on the filter paper every 15 seconds, and the larger the number, the longer the bleeding time), the size and number of blood drops of RCL-M1A and / or RCL-M8H were comparable to those of the control group DMSO, while the size and number of blood drops of the oral anticoagulant rivaroxaban at clinical doses, especially the number, were significantly greater than those of the control group ( Figure 8 ), suggesting that bleeding is already quite significant at clinical doses of rivaroxaban.

[0070] Therefore, compared with the oral anticoagulant rivaroxaban, RCL-M1A and / or RCL-M8H can effectively inhibit venous thrombosis without increasing the risk of bleeding.

[0071] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. A person with ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention shall be determined by the definition of the claims.

Claims

1. A use of RCL-M1A and / or RCL-M8H in the preparation of a drug for preventing and / or treating venous thrombosis and embolism, wherein the chemical structure of the RCL-M1A is shown in Formula I, and the chemical structure of the RCL-M8H is shown in Formula II; 2. The use according to claim 1, characterized in that: The prevention and / or treatment of venous thrombosis and embolism is to inhibit venous thrombosis by inhibiting platelet and neutrophil extracellular traps (NETs) without increasing the risk of bleeding.

3. The use according to claim 1, characterized in that: The venous thrombosis and embolism include deep vein thrombosis, pulmonary embolism and thrombosis in other parts of the venous system.

4. An antagonist, characterized in that The invention comprises RCL-M1A and its structural derivatives and / or RCL-M8H and its structural derivatives as described in any one of claims 1 to 3.

5. A pharmaceutical composition, characterized in that The invention comprises RCL-M1A and its structural derivatives and / or RCL-M8H and its structural derivatives as described in any one of claims 1 to 3.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition also includes other anticoagulant drugs.

7. The pharmaceutical composition according to claim 6, characterized in that The anticoagulant drugs include warfarin and / or rivaroxaban.

8. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition also includes a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is in the form of a solid dosage form, a liquid preparation or a semisolid preparation.

10. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is an oral preparation or an injection.

Images