Application of SHP2 inhibitors in the preparation of drugs for the treatment of chronic thromboembolism

Abstract

This invention discloses the application of an SHP2 inhibitor in the preparation of drugs for treating chronic thromboembolism, particularly the application of a nanoliposome-encapsulated SHP2-targeting inhibitor for targeted therapy of chronic thromboembolism. The main structure includes: SHP2-targeting inhibitor SHP099 particles, targeting ligand CREKA, and DSPE-PEG. This invention's SHP2-targeting inhibitor can target macrophages in thrombus tissue, exhibiting good thrombolytic and recanalizing functions, with few toxic side effects and good therapeutic efficacy.

Description

Technical Field

[0001] This invention relates to the application of an SHP2 inhibitor in the preparation of a drug for treating chronic thromboembolism, and more particularly to the application of an SHP2-targeting inhibitor in the preparation of a drug for treating thrombotic diseases such as pulmonary thromboembolism, chronic pulmonary thromboembolism, and deep vein thrombosis of the lower extremities. Background Technology

[0002] (a) Chronic thromboembolism has a high mortality rate and poor prognosis.

[0003] Venous thromboembolism, including deep vein thrombosis (DVT) and pulmonary embolism (PE), is a prevalent vascular disease affecting nearly 10 million people worldwide each year. High-risk factors for venous thromboembolism include major surgery, active cancer, and immobilization (Khan F, Tritschler T, Kahn SR, Rodger MA. Venous thromboembolism. Lancet 2021, 398(10294):64-77.). PE is the most dangerous form of venous thromboembolism; undiagnosed or untreated PE can be fatal. Acute PE is associated with right ventricular dysfunction and can lead to arrhythmias, hemodynamic failure, and shock. Furthermore, even with aggressive anticoagulation therapy, acute PE can lead to long-term complications: chronic thromboembolic vascular disease (CTED) and chronic thromboembolic pulmonary hypertension (CTEPH), which can further increase pulmonary vascular resistance and progressive right heart failure, resulting in a severe decline in patients' quality of life, disability, and death (Huisman MV, Barco S, Cannegieter SC, Le Gal G, Konstantinides SV, Reitsma PH, Rodger M, Vonk Noordegraaf A, Klok FA. Pulmonary embolism. Nat Rev Dis Primers 2018, 4:18028.). A prospective, long-term follow-up study to assess the incidence of symptomatic CTPH in patients with consecutive acute pulmonary embolism but without venous thromboembolism found that the cumulative incidence of symptomatic CTPH was 1.0% at six months, 3.1% at one year, and 3.8% at two years (Pengo V, Lensing AW, Prins MH, Marchiori A, Davidson BL, Tiozzo F, Albanese P, Biasiolo A, Pegoraro C, Iliceto S, Prandoni P, Thromboembolic Pulmonary Hypertension Study G. Incidence of chronic thromboembolic pulmonary hypertension after pulmonary embolism. N Engl J Med 2004, 350(22):2257-2264.).Meta-analysis found that the incidence of CTEPH after acute pulmonary embolism is as high as 5.08% in Asian populations (Pang W, Zhang Z, Wang Z, Zhen K, Zhang M, Zhang Y, Gao Q, Zhang S, Tao X, Wan J, Xie W, Zhai Z. Higher incidence of Chronic Thromboembolic Pulmonary Hypertension After Acute Pulmonary Embolismin Asians Than in Europeans: A Meta-Analysis. Front Med (Lausanne) 2021, 8:721-294.).

[0004] (II) Treatment methods and existing problems for chronic thrombosis

[0005] Systemic anticoagulation therapy is the primary treatment for chronic thrombosis. Current medications include heparin, low molecular weight heparin, warfarin, rivaroxaban, edoxaban, apixaban, and dabigatran. Bleeding is the most significant adverse reaction to systemic anticoagulants. However, anticoagulation therapy cannot completely remove thrombi, and even with aggressive anticoagulation, complications associated with chronic thrombosis cannot be avoided.

[0006] (III) The therapeutic basis of this invention

[0007] Single-cell RNA sequencing (scRNAseq) of tissue removed during pulmonary thromboendothelial resection (PEA) identified multiple cell types containing CTEPH thrombi, and different macrophage subsets were identified. Compared with the control group, macrophages in CTEPH thrombi promoted the upregulation of inflammatory signals and facilitated pulmonary vascular remodeling (Viswanathan G, Kirshner HF, Nazo N, Ali S, Ganapati A, Cumming I, Zhuang Y, Choi I, Warman A, Almeida-Peters S, Haney J, Corcoran D, Yu YR, Rajagopal S. Single-cell analysis reveals Distinct Immune and Smooth Muscle Cell Populations that Contribute to Chronic Thromboembolic Pulmonary Hypertension. Am J Respir Crit Care Med 2023.).

[0008] Venous thromboembolism, including deep vein thrombosis (DVT) and pulmonary embolism (PE), is a vascular disease with a high incidence. Chronic thromboembolic vascular disease (CTED) and chronic thromboembolic pulmonary hypertension (CTEPH) can further lead to increased pulmonary vascular resistance and progressive right heart failure, resulting in a severe decline in patients' quality of life, disability, and death.

[0009] SHP2 is an important phosphatase that participates in physiological and pathological processes such as cell proliferation, migration, apoptosis, and inflammation by regulating multiple signaling pathways. We have found that SHP2 is involved in the formation of chronic thrombosis. This invention uses an SHP2 inhibitor encapsulated in nanoliposomes, which, after intravenous administration to mice, accumulates locally in the thrombus (see...). Figure 4 This allows for organ-specific targeting. Further analysis revealed that SHP2 inhibitors can be phagocytosed by macrophages in thrombus tissue (see...). Figure 5 It inhibits macrophage phosphatase SHP2, regulates macrophage differentiation and function, and promotes thrombus absorption. Summary of the Invention

[0010] This invention provides an SHP2 inhibitor that can target macrophages in chronic thrombotic tissues, with the advantages of good efficacy and low toxicity.

[0011] This invention is achieved through the following technical solution:

[0012] This invention provides the application of an SHP2 inhibitor in the preparation of a drug for treating chronic thromboembolism.

[0013] Furthermore, the drug for treating chronic thromboembolism is composed of an SHP2 inhibitor or a pharmaceutically acceptable salt, stereoisomer, solvate thereof, and a pharmaceutically acceptable carrier.

[0014] The pharmaceutically acceptable carrier is one or more of the following: targeting carrier, diluent, filler, binder, humectant, disintegrant, absorption enhancer, surfactant, adsorbent, lubricant, and thickener. Flavoring agents, sweeteners, etc., may also be added if necessary. The pharmaceutically acceptable carrier refers to conventional drug carriers in the pharmaceutical field, including conventional pharmaceutical diluents such as water, fillers such as starch, binders such as cellulose derivatives and gelatin, humectants such as glycerin, disintegrants such as agar and calcium carbonate, absorption enhancers such as quaternary ammonium compounds, surfactants such as hexadecyl alcohol, adsorbents such as kaolin and soap clay, lubricants such as talc, and thickeners such as sodium carboxymethyl cellulose.

[0015] This invention particularly recommends pharmaceutically acceptable carriers as targeted carriers, such as nanoliposomes using the tumor homing peptide Cys-Arg-Glu-Lys-Ala as a targeting ligand. Targeted carriers not only enhance therapeutic efficacy but also effectively reduce toxic side effects.

[0016] In one embodiment of the present invention, the targeting carrier further includes a fluorescent reagent. More specifically, the fluorescent reagent is 1,1'-octadecyl-3,3,3',3'-tetramethyl-indocyanine perchlorate, which can assist in observing drug localization.

[0017] In one embodiment of the present invention, the drug for treating chronic thromboembolism is prepared as follows: DSPE-PEG-Maleimide and tumor homing peptide Cys-Arg-Glu-Lys-Ala are dissolved in phosphate buffer at pH 7.4 at a molar ratio of 1:1, reacted at room temperature for 4 hours, and then dialyzed for the first time using deionized water as the dialysate to obtain DSPE-PEG2K-CREKA solution;

[0018] 1,2-diacyl-sn-glycerol-3-phosphocholine, cholesterol, and SHP099 were dissolved in chloroform at a mass ratio of 1:2:10 to obtain a film-forming solution. The solution was then rotary evaporated to form a film, and the DSPE-PEG2K-CREKA solution was added.

[0019] Add deionized water, and pass the resulting preparative solution through ultrasound and a liposome extruder to obtain a liposome solution; the mass ratio of cholesterol to DSPE-PEG-Maleimide is 1:10;

[0020] The liposome solution was dialyzed a second time using deionized water as the dialysate. The resulting retentate was added to a 10% sucrose solution of equal volume and then freeze-dried to obtain the drug for treating chronic thromboembolism.

[0021] Furthermore, the volume of the phosphate buffer is 0.15 mL / mg based on the mass of the DSPE-PEG-Maleimide.

[0022] Furthermore, the volume of the chloroform is 3 mL / mg based on the mass of the cholesterol.

[0023] Furthermore, the ultrasonic power is 180W, and the ultrasonic duration is 3-5 minutes. The filter membrane of the liposome extruder has a pore size of 200nm.

[0024] Furthermore, the volume of the preparation solution is 4 mL / mg based on the mass of the cholesterol.

[0025] Furthermore, the freeze-drying operation is as follows: freezing at -80℃ and sublimation at -50℃ under high vacuum.

[0026] In one embodiment of the present invention, a fluorescent reagent is also added to the film-forming solution, wherein the mass ratio of the fluorescent reagent to the cholesterol is 1:2.

[0027] Furthermore, the aforementioned medicament for treating chronic thromboembolism is a pharmaceutical preparation suitable for administration via any appropriate route, such as oral (including sublingual or sublingual), rectal, nasal, local (including sublingual, sublingual, or transdermal), or parenteral (including subcutaneous, intramuscular, intravenous, or intradermal injection). These preparations can be prepared by any method known in the field of pharmaceutics, such as by mixing the active ingredient with a carrier or excipient.

[0028] The medication for treating chronic thromboembolism is administered via intravenous injection, and it is recommended that the medication for treating chronic thromboembolism be an intravenous injection formulation using PBS as a dispersant.

[0029] The SHP2-targeting inhibitor encapsulated in nanoliposomes prepared in this invention mainly comprises: ① a core component of SHP099 particles, which can inhibit the activity of the SHP2 phosphatase and further regulate monocyte-macrophage differentiation; ② an outer layer encapsulating DSPE-PEG: 1,2-distearate-sn-glycerol-3-phosphoethanolamine-polyethylene glycol, a commonly used amphiphilic biomaterial for constructing nanoliposomes, with one hydrophilic end and one hydrophobic end, the hydrophobic end being inside the drug to encapsulate the hydrophobic component. The drug is located in the core, with the hydrophilic end extending to the outside. Nanoparticles are formed through the self-assembly of the material, exhibiting both hydrophilic and lipophilic properties; ③ CREKA: The hemagglutinin-binding peptide Cys-Arg-Glu-Lys-Ala can recognize fibrin and target fibrin in thrombi; ④ Dil: 1,1'-octadecyl-3,3,3',3'-tetramethyl-indocyanine perchlorate, a small molecule organic fluorescein, provides stable fluorescent tracer for NanoSHP099. (See...) Figure 1 , Figure 3 )

[0030] Compared with the prior art, the beneficial effects of the present invention are as follows: the targeted SHP2 inhibitor of the present invention can target macrophages in thrombus tissue, exhibiting good thrombolytic and recanalization functions of organized thrombi, with fewer toxic side effects and better therapeutic effects.

[0031] The core component of this formulation is SHP099 particles. Nanoliposomes, using DSPE-PEG, encapsulate a hydrophobic drug at the core and a hydrophilic end on the outside, forming nanoparticles through self-assembly. CREKA, embedded in DSPE-PEG, specifically recognizes fibrin and targets fibrin within thrombi. Dil is distributed on the surface of NanoSHP099, providing stable fluorescent tracer for the entire nanodrug. Therefore, NanoSHP099 exhibits good water solubility through encapsulation in nanoliposomes. CREKA helps NanoSHP099 bind to fibrin, precisely targeting thrombi, inhibiting SHP2 phosphatase activity, regulating macrophage function and differentiation, promoting the dissolution of organized thrombi, and facilitating local vascular recanalization.

[0032] This invention utilizes SHP2 inhibitors to successfully prepare drugs for the treatment of chronic thromboembolism, particularly a targeted therapy for chronic thromboembolism.

[0033] This invention uses an SHP2-targeting inhibitor encapsulated in nanoliposomes, which can be locally enriched in the thrombus after intravenous administration, thereby increasing safety and ensuring efficacy.

[0034] This invention utilizes a SHP2-targeting inhibitor encapsulated in nanoliposomes. After local thrombus aggregation, this inhibitor exhibits amphiphilic and lipophilic properties, facilitating its penetration into the thrombus tissue and phagocytosis by macrophages. Subsequently, it targets and inhibits macrophage phosphatase SHP2, regulating macrophage differentiation and function, and promoting thrombus absorption. This provides a novel drug treatment option for chronic venous thrombolysis and recanalization, effectively reducing the incidence of CTEPH and improving patient survival and quality of life. Attached Figure Description

[0035] Figure 1 Schematic diagram of the construction of .CREKA-DiI-Lipo@SHP099liposomes (NanoSHP099 liposomes): The core component of NanoSHP099 is the SHP2 targeting inhibitor SHP099 particles. The hydrophobic drug is placed in the core and the hydrophilic end is placed on the outside of the drug using DSPE-PEG. The nanoparticles are formed through the self-assembly of the material. The nanoparticles are amphiphilic. CREKA is embedded in DSPE-PEG and can specifically recognize fibrin and target fibrin in thrombi. DiI is distributed on the surface of NanoSHP099, so that the entire nanodrug has stable fluorescent tracer.

[0036] Figure 2 This is a comparison image of Masson staining of thrombus cross sections in mice with DVT model and thrombosis treated with different drug concentrations.

[0037] Figure 3NanoSHP099 is amphiphilic.

[0038] Electron microscopy revealed that NanoSHP099 exhibited a characteristic circular structure, demonstrating the successful and complete self-assembly of the amphiphilic phospholipid molecule.

[0039] Figure 4 NanoSHP099 can target and enter thrombus tissue.

[0040] In a mouse model of thrombosis, NanoSHP099 was administered intravenously. Immunofluorescence staining of pathological sections revealed that NanoSHP099 (red fluorescence) aggregated within the thrombus. The uptake of NanoSHP099 in the thrombus showed significant specificity and a higher uptake level compared to that in the heart, liver, spleen, lungs, and kidneys (uptake / organ mass).

[0041] Figure 5 NanoSHP099 targets macrophages

[0042] Analysis of neutrophils, macrophages, epithelial cells, and fibroblasts in the thrombus showed that NanoSHP099 was mostly taken up by macrophages, demonstrating the relative specificity of NanoSHP099 for macrophages. Detailed Implementation

[0043] Example 1

[0044] This invention provides the above-mentioned Figure 1 The method for preparing shp099 drug particles encapsulated in nanoliposomes, as shown, includes the following steps:

[0045] (1)DSPE-PEG2K-CREKA: Take 10 mg of phosphatidylethanolamine-polyethylene glycol 2000-maleimide (DSPE-PEG2K-MAL) and 3 mg of tumor homing peptide (CREKA) and dissolve them in 1.5 mL of PBS (pH 7.4) at a molar ratio of 1:1. React at room temperature for 4 hours, and then dialyze to deionized water to remove unreacted DSPE-PEG2K-MAL and CREKA to obtain about 2 mL of DSPE-PEG2K-CREKA solution.

[0046] (2) Dissolve HSPC (1,2-diacyl-sn-glycerol-3-phosphocholine) / cholesterol / DIL / SHP099 in 3 mL of chloroform at a mass ratio of 1:2:1:10 (total 7 mg), and transfer to a round-bottom flask. Rotary evaporate the solution to form a film at the bottom of the flask. Add all of the DSPE-PEG2K-CREKA solution from step (1), and add deionized water to a final volume of 4 mL. Then, process the solution using sonication (180 W for 3–5 minutes) and a liposome extruder (200 nm filter membrane).

[0047] (3) Dialyze using a nanodialysis device (polycarbonate membrane with a pore size of 50 nm, and deionized water as the dialysate) to remove unloaded DSPE-PEG2K-CREKA and SHP099. Finally, add deionized water to bring the volume to 10 mL (for easy calculation).

[0048] (4) Take 10 μL of liposome solution, add Triton X-100 to break the membrane, and measure the SHP099 content (8%) in the liposomes using an ELISA reader (with SHP099 as the standard curve).

[0049] (5) Lyophilization: Add an equal volume (10 mL) of 10% sucrose solution as a preservative to the liposomes and lyophilize. Before use, resuspend in PBS at the required concentration for in vivo injection.

[0050] This invention provides Figure 1 The application of shp099 particles encapsulated in nanoliposomes to promote the dissolution of organized thrombi is shown.

[0051] Six- to eight-week-old male C57BL / 6 mice were randomly divided into five groups of four: control group, SHP099 granule group 1 (15 mg / kg), SHP099 granule group 2 (30 mg / kg), and NanoSHP099 group 1.

[0052] Group 2 (15 mg / kg) and NanoSHP099 (30 mg / kg). The control group comprised 50% of the total.

[0053] The SHP099 particles were dissolved in a solvent of 50% DMSO + 20% PEG + 30% PBS. NanoSHP099 was prepared as described above.

[0054] Weigh the mice to calculate the required drug mass, then adjust the concentration with physiological saline to ensure that the solution for each tail vein injection is controlled within 200 microliters.

[0055] Mouse Inferior Vena Cava Thrombosis Model: After deep anesthesia with isoflurane gas, mice underwent routine abdominal disinfection. The skin and muscle layers were incised to fully expose the abdominal cavity. The intestines were carefully removed using saline-soaked cotton swabs, and the inferior vena cava was isolated. A 5-0 ligation suture was placed parallel to the surface of the inferior vena cava, and a 7-0 suture was wrapped around the inferior vena cava and the ligation suture once below the left renal vein, then gently tied with two surgical knots. The ligation suture was then removed, and the downstream veins at the ligation site were observed to be dilated due to obstructed blood return. The intestines were returned to their original position, and the peritoneum, muscle, and skin layers on both sides were aligned, followed by the muscle layer and abdominal skin. After disinfection, sterile dressings were applied. The SHP099 granule group and the NanoSHP099 group (15 mg / kg and 30 mg / kg) received the drug via tail vein injection every other day. Injections were administered on days 3, 5, 7, 9, and 11 after model establishment, for a total of 5 injections. Figure 2 (Masson staining of thrombus tissue) shows that compared with the solvent-treated control group, SHP099 administration can effectively reduce the area of ​​thrombus organization. Compared with bare SHP099, NanoSHP099, whether in high-dose or low-dose groups, can more significantly reduce thrombus organization and reduce thrombus area, demonstrating the effect of promoting thrombolysis.

[0056] Nanoliposomes can target thrombus tissue (see...) Figure 2 , Figure 4 After entering the thrombus, nanoliposomes can target and enter the macrophages within the thrombus (see...). Figure 5 It inhibits the activity of macrophage phosphatase SHP2, regulates macrophage function and differentiation, promotes the dissolution of organized thrombi, and facilitates local vascular recanalization (see...). Figure 1 ).

[0057] This invention provides an application that can maintain a high drug concentration in thrombus tissue. Through intravenous administration to animals, the drug is recruited into the thrombus tissue and can target macrophages within the thrombus. By inhibiting SHP2 in macrophages, it regulates macrophage function, thereby promoting thrombus absorption and achieving a therapeutic effect. Simultaneously, it reduces the drug's impact on other tissues and minimizes side effects.

Claims

1. The use of NanoSHP099 liposomes in the preparation of drugs for treating chronic thromboembolism, characterized in that... The chronic thromboembolism mentioned above refers to chronic venous thrombosis; The NanoSHP099 liposomes were prepared as follows: DSPE-PEG-Maleimide and tumor homing peptide Cys-Arg-Glu-Lys-Ala were dissolved in phosphate buffer at pH 7.4 at a molar ratio of 1:1, reacted at room temperature for 4 h, and then dialyzed for the first time with deionized water to obtain DSPE-PEG2K-CREKA solution. 1,2-Diayl-sn-glycerol-3-phosphocholine, cholesterol, and SHP099 were dissolved in chloroform at a mass ratio of 1:2:10 to obtain a film-forming solution. The solution was then rotary evaporated to form a film. The DSPE-PEG2K-CREKA solution was added, and deionized water was replenished. The resulting preparative solution was subjected to ultrasonication and a liposome extruder to obtain a liposome solution. The mass ratio of cholesterol to DSPE-PEG-Maleimide was 1:

10. The liposome solution was dialyzed a second time using deionized water as the dialysate. The resulting retentate was then added to a 10wt% sucrose solution of equal volume and freeze-dried.

2. The application as described in claim 1, characterized in that: The film-forming solution also includes a fluorescent reagent, and the mass ratio of the fluorescent reagent to the cholesterol is 1:

2.

3. The application as described in claim 1, characterized in that: The volume of the phosphate buffer solution is 0.15 mL / mg based on the mass of the DSPE-PEG-Maleimide; The volume of chloroform is 3 mL / mg based on the mass of the cholesterol. The ultrasonic power is 180W, the ultrasonic time is 3-5 minutes, and the filter membrane pore size of the liposome extruder is 200nm. The volume of the preparative solution is 4 mL / mg based on the mass of the cholesterol.

4. The application as described in claim 1, characterized in that: The medication for treating chronic thromboembolism is an intravenous injection prepared with PBS as a dispersant.

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