Use of lipophilic bisphosphonates in the preparation of anti-platelet drugs

Abstract

The application discloses application of a lipophilic bisphosphonate in preparation of an anti-platelet drug, and further discloses an anti-platelet drug which can be used for preventing or treating platelet activation and thrombosis, and the drug comprises an effective dose of the lipophilic bisphosphonate. The lipophilic bisphosphonate comprises one or both of TH-Z93 and TH-Z145. The application finds that the lipophilic bisphosphonate TH-Z93 and TH-Z145 compound can be applied to preparation of the anti-platelet drug, can significantly inhibit human platelet aggregation and activation, platelet-induced neutrophil NETs release, microvascular thrombus injury, block a double pathological process of sepsis inflammation and thrombosis, and significantly improve the survival rate of sepsis.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to antiplatelet drugs. Background Technology

[0002] Sepsis is a systemic inflammatory response syndrome in which the body responds uncontrollably to infection. Severe sepsis is often accompanied by disseminated intravascular coagulation (DIC), abnormal platelet activation, neutrophil-platelet complex (neutrophil-platelet complex) formation, and microvascular thrombosis in multiple organs, leading to organ damage such as the liver and lungs, thrombocytopenia, and significantly increasing the risk of death. Among these, abnormal platelet activation is the core initiating step of the thrombotic cascade in sepsis: activated platelets highly express P-selectin and PAC1, adhere to and aggregate with neutrophils and monocytes, induce neutrophils to release NETs, ​​further amplify the coagulation and inflammatory cascade, and form a vicious cycle of "inflammation-thrombosis".

[0003] Conventional anticoagulants only block the coagulation cascade and cannot inhibit the interaction between platelets and immune cells or the release of NETs; moreover, conventional antiplatelet drugs have a high bleeding risk and are not suitable for patients with sepsis and bleeding tendencies. Currently, there are no specific drugs in clinical practice that simultaneously target inflammation and thrombosis.

[0004] Bisphosphonates are a core class of drugs used to treat various bone diseases. Traditional hydrophilic nitrogenous bisphosphonates (pamidronate, zoledronic acid) specifically inhibit farnesyl pyrophosphate synthase (FPPS) and are clinically used to target and inhibit osteoclasts to treat osteoporosis. In vitro experiments have confirmed that pamidronate and zoledronic acid, due to their high hydrophilicity, cannot target human platelets and have no antiplatelet or antiseptic thrombotic activity, thus they cannot be used for sepsis thrombosis intervention. Lipophilic bisphosphonates TH-Z93 and TH-Z145 are currently only research-grade chemical reagents. TH-Z93 is an FPPS inhibitor, and TH-Z145 is a GGPPS (geranyl-geranyl pyrophosphate synthase) inhibitor; their research applications are focused on their use as vaccine adjuvants to enhance immune responses. There are currently no reports of bisphosphonates being used to block platelet activation, NETs (neutrophil extracellular traps) formation, or microthrombi. Summary of the Invention

[0005] One of the technical problems to be solved by the present invention is to provide an antiplatelet drug that can prevent or treat thrombotic diseases.

[0006] To solve the above-mentioned technical problems, the antiplatelet drug of the present invention contains an effective dose of lipophilic bisphosphonate.

[0007] The second technical problem to be solved by this invention is to provide the application of lipophilic bisphosphonates in the preparation of antiplatelet drugs.

[0008] The antiplatelet drug may also include a pharmaceutically acceptable carrier and / or excipients. The excipients preferably include mannitol.

[0009] The antiplatelet drug can be in the form of injection, subcutaneous injection, lyophilized powder for injection, intravenous solution, oral tablets or capsules. Intravenous administration is preferred for patients with sepsis; oral prophylactic administration can be used for high-risk groups of sepsis.

[0010] The third technical problem to be solved by the present invention is to provide a method for preparing the antiplatelet drug, the method comprising the following steps: taking 500 mg of lipophilic bisphosphonate and 5 g of mannitol, making up to 1000 mL with water for injection, aseptically filtering, dispensing, and freeze-drying to prepare 100 vials of lyophilized powder for injection.

[0011] The lipophilic bisphosphonate preferably includes one or both of TH-Z93 and TH-Z145.

[0012] The antiplatelet drugs can inhibit platelet activation, aggregation, NETs and thrombosis, and can be used in particular to prevent or treat sepsis, severe sepsis, sepsis complicated with disseminated intravascular coagulation, and platelet activation, aggregation, NETs and thrombosis induced by multi-organ damage in sepsis.

[0013] Compared with existing antiplatelet drugs, the antiplatelet drug of the present invention has the following advantages and beneficial effects: (1) It can significantly inhibit thrombin or collagen-induced human platelet aggregation and inhibit sepsis-induced excessive platelet activation; (2) It can significantly downregulate platelet P-selectin expression, PAC1 and JON / A activation marker levels; (3) It can significantly inhibit the generation of NETs in the platelet-neutrophil co-culture system, block the release of platelet-induced NETs, ​​and inhibit the vicious cycle of inflammation and thrombosis; (4) It can significantly reduce microvascular thrombosis, vascular occlusion and organ pathological damage in sepsis-affected liver and lung tissues; (5) It can significantly improve the survival rate of sepsis and reduce the mortality rate of sepsis.

[0014] (6) Bisphosphonate skeleton is a clinically mature drug skeleton. Lipophilic bisphosphonates can retain the drug properties and ensure the stability of clinical efficacy, and have high safety potential. Attached Figure Description

[0015] Figure 1The results of in vitro platelet aggregation and activation experiments in the control group, TH-Z93 group, and TH-Z145 group of Example 1 are shown. Figure A shows the thrombin-induced human platelet aggregation rate in each group; Figure B shows the collagen-induced human platelet aggregation rate in each group; Figure C shows the expression level of the platelet activation marker P-selectin in each group; and Figure D shows the expression level of the platelet activation marker PAC1 in each group.

[0016] Figure 2 These are the immunofluorescence staining results of the in vitro co-culture experiment in Example 2. Image A is the negative control, image B is the control group, image C is the TH-Z145 group, and image D is the TH-Z93 group. The abbreviations NEUTs, PLTs, and Thr are neutrophils, platelets, and thrombin, respectively.

[0017] Figure 3 This is a statistical analysis result graph showing the ratio of the number of neutrophils generated per field to the total number of neutrophils after immunofluorescence staining in the in vitro co-culture experiment of Example 2.

[0018] Figure 4 This is the 7-day Kaplan-Meier survival curve of CLP mice in Example 3.

[0019] Figure 5 This is a graph showing the results of flow cytometry analysis of the expression level of P-selectin, a marker of platelet activation, in CLP mice in Example 3.

[0020] Figure 6 This is a graph showing the results of flow cytometry detection of the expression level of the platelet activation marker JON / A in CLP mice in Example 3.

[0021] Figure 7 These are HE-stained pathological sections of liver and lung tissue from septic mice, as described in Example 3. Statistical notation: mean ± standard error. p<0.05; p<0.01; p<0.001; ns showed no statistically significant difference, so one-way and two-way ANOVA were used. Detailed Implementation

[0022] To provide a more detailed understanding of the technical content, features, and effects of the present invention, the technical solution of the present invention will now be described in further detail with reference to the accompanying drawings.

[0023] The TH-Z93 and TH-Z145 used in the examples were purchased from MedChemExpress (MCE), with product numbers HY-150167 and HY-150168, respectively.

[0024] P-selectin was purchased from BD Biosciences, catalog number 555524.

[0025] PAC1 was purchased from Thermo, part number MA5-28564.

[0026] Example 1: In vitro platelet aggregation and activation experiment I. Experimental Grouping The in vitro experiments in this embodiment were divided into a control group, a TH-Z93 group, and a TH-Z145 group.

[0027] II. In vitro experiments Thrombin and collagen were used as stimulants.

[0028] 1. In vitro platelet aggregation experiment (1) Prepare human washed platelets and adjust the cell concentration to 3×10⁻⁶. 8 / mL, rest in a 37℃ water bath for 1 hour; (2) TH-Z93 (10 μM) and TH-Z145 (10 μM) were added to the washed platelets of the corresponding groups after resting. After incubation at 37°C, the platelet activity of different groups was evaluated. (3) The washed platelets of different groups were placed in the aggregation channel in sequence, and the platelets were activated by stimulating them with 2 μg / mL collagen and 0.05 U / mL thrombin. The aggregation curves were analyzed and statistically analyzed.

[0029] The results are as follows Figure 1 As shown in Figures A and B, the platelet aggregation rates in the TH-Z93 and TH-Z145 groups were significantly lower than those in the control group, demonstrating that TH-Z93 and TH-Z145 can significantly reduce the level of collagen- or thrombin-induced platelet aggregation.

[0030] 2. In vitro platelet activation experiment The expression levels of platelet activation markers P-selectin and PAC1 in different groups were detected by platelet flow cytometry. The specific method steps are as follows: (1) Prepare human washed platelets and adjust the cell concentration to 3×10⁻⁶. 8 / mL, rest in a 37℃ water bath for 1 hour; (2) TH-Z93 (10 μM) and TH-Z145 (10 μM) were added to the washed platelets of the corresponding groups after resting. After incubation at 37°C, the activity of platelets in the control group, TH-Z93 group and TH-Z145 group was evaluated.

[0031] (3) Dilute the platelet concentrations of different groups to 1×10⁻⁶. 7 / mL, 100μl of platelets are taken as one sample. Each group of platelets is divided into resting state and activated state. The activated state is the one with the corresponding concentration of thrombin added. Add 1μL of P-selectin or 2μL of PAC1 antibody to 100μL of platelets, incubate at 37℃ for 30 minutes, and then perform flow cytometry analysis.

[0032] The results are as follows Figure 1 As shown in Figures C and D, the release levels of the activation marker P-selectin and the binding levels of PAC1 in the TH-Z93 and TH-Z145 groups were significantly lower than those in the control group.

[0033] The in vitro platelet aggregation and activation experiments in this embodiment demonstrate that the lipophilic bisphosphonates TH-Z93 and TH-Z145 can significantly inhibit human platelet activation and aggregation in vitro; while zoledronic acid and pamidronate at the same concentration, due to their high hydrophilicity, cannot penetrate the platelet membrane and therefore do not have the above-mentioned inhibitory activity.

[0034] Example 2: In vitro co-culture experiment I. Cell Model The cell model in this embodiment uses a co-culture system of human primary neutrophils and activated platelets.

[0035] II. Experimental Grouping The in vitro co-culture experiment in this embodiment was divided into a control group, a TH-Z93 group, and a TH-Z145 group.

[0036] III. In vitro co-culture experiment 1. Prepare human washed platelets and adjust the cell concentration to 1×10⁻⁶. 8 / mL, add 10μM TH-Z93 and 10μM TH-Z145 to the platelets of the TH-Z93 group and TH-Z145 group respectively, mix well and incubate at 37℃ for 30 minutes, then use for later use; 2. Draw whole blood from healthy volunteers for later use. Take Polymorphprep neutrophil separation solution out of the 4℃ refrigerator in advance and bring it to room temperature. Add 5mL of separation solution to a 15mL centrifuge tube at a ratio of separation solution:whole blood = 1:1, and then slowly add 5mL of fresh whole blood to the top of the separation solution. 3. Place a 15mL centrifuge tube in a centrifuge and centrifuge at a relative centrifugal force of 500×g for 30 minutes. After centrifugation, two distinct layers of white blood cells can be seen, with the lower layer being neutrophils. 4. Transfer the neutrophil layer to a clean centrifuge tube and centrifuge at 400×g for 10 minutes. After centrifugation, resuspend the neutrophils in PBS (phosphate-buffered saline) and centrifuge again at 400×g for 10 minutes. Discard the supernatant and resuspend the cells in RPMI-1640 medium (containing 1% FBS (fetal bovine serum)) and adjust the neutrophil concentration to 5×10⁻⁶ cells / mL. 5 / mL for later use.

[0037] 5. Place cell smears in 24-well plates in advance, add 900 μl of neutrophils adjusted to the desired concentration to each well, then add platelets from each group that have completed incubation, along with thrombin. Mix thoroughly using a pipette. Place the 24-well plates with the added samples in a 37°C, 5% CO2 incubator for incubation. After incubation, aspirate the supernatant and fix the cells overnight with 4% paraformaldehyde. After fixation, perform immunofluorescence staining (CitH3) to observe the NET network structure and count the ratio of the number of neutrophils generating NETs per field to the total number of neutrophils.

[0038] The results are as follows Figure 2-3 As shown, compared with the control group, both TH-Z93 and TH-Z145 compounds can significantly inhibit platelet-mediated NETs generation in vitro and reduce inflammatory thrombus formation.

[0039] Example 3: In vivo CLP sepsis mouse survival, platelet activation and pathological experiments I. Animal Models This embodiment uses cecal ligation (CLP) to induce a mouse sepsis model. The modeling method is as follows: (1) Place the experimental mice in a supine position on the mask of the anesthesia machine, turn on isoflurane and oxygen, and fix the mice with medical tape.

[0040] (2) Disinfect the skin of the mouse abdomen with 75% alcohol, cut a window of about 1 cm on the lower right side of the midline of the abdomen, and carefully find and expose the cecum through the window with blunt forceps.

[0041] (3) After locating the cecum, gently remove it from the window with tweezers and place it on sterile gauze moistened with PBS. After ligating the cecum at the designated location, puncture it with a 22g needle and squeeze out the corresponding intestinal contents at the puncture site, keeping the amount of intestinal contents squeezed out consistent for each mouse.

[0042] (4) Use forceps to put the cecum back into the mouse's abdominal cavity. Then, use sutures to suture the inner mucosa and outer skin layers of the mouse in sequence. After suturing, inject 1 mL of warm saline subcutaneously to further replenish the fluid in the experimental mice.

[0043] II. Experimental Grouping The experiment in this embodiment was divided into a CLP model control group, a TH-Z93 (2.5 mg / kg) administration group, and a TH-Z145 (2.5 mg / kg) administration group.

[0044] III. In vivo CLP sepsis mouse survival experiment Survival curves were recorded for 7 consecutive days after mice in the TH-Z93 and TH-Z145 administration groups were administered via tail vein injection.

[0045] The results are as follows Figure 4 As shown, the survival rate of mice in the treatment group was significantly improved after 7 days, and the number of peripheral blood platelets was significantly higher than that in the control group. This demonstrates that the lipophilic bisphosphonates TH-Z93 and TH-Z145 can improve the survival rate of CLP sepsis in animals and inhibit the mortality rate of sepsis mice.

[0046] IV. In vivo platelet activation experiment in CLP-infected septic mice At 0h and 24h after CLP modeling, peripheral blood was collected from the orbits of mice in each group, and the expression levels of platelet activation markers P-selectin and JON / A were detected by flow cytometry (the detection method is the same as in Example 1).

[0047] The results are as follows Figure 5-6 As shown, TH-Z93 and TH-Z145 can significantly reduce platelet activation levels and decrease immune cell-platelet aggregation.

[0048] V. In vivo CLP sepsis mouse pathological experiment Liver and lung tissue sections from CLP mice were fixed and stained with hematoxylin and eosin (H&E) as follows: (1) Place the paraffin slices in a glass staining jar and add xylene three times, each reaction lasting 10 minutes.

[0049] (2) After the xylene dewaxing is complete, continue to add 100% alcohol twice, 95% alcohol twice, 85% alcohol, 75% alcohol and double distilled water in sequence, with each reaction time being 5 minutes.

[0050] (3) After alcohol dewaxing is completed, hematoxylin staining solution is added to stain the cell nuclei. Then, the slide is rinsed with PBS. The staining of the cell nuclei is then observed under a microscope. If the color of the cell nuclei is relatively dark, 1% hydrochloric acid alcohol is used to decolorize them to remove non-specific staining. After decolorization is completed, the hydrochloric acid alcohol separation solution is immediately poured out.

[0051] (4) Place the slide in double-distilled water to further restore its blue color, allowing the cell nuclei to gradually return to their blue hue. After restoration, add 75% alcohol and 85% alcohol to the slide sequentially, allowing the reaction time to be 10 minutes. Then add eosin staining solution to further stain the cytoplasm. After staining for 1 minute, remove the slide and observe it under a microscope. The color contrast of eosin and hematoxylin should be moderate.

[0052] (5) Further dehydration with alcohol is carried out by adding 95% alcohol twice and 100% alcohol twice in sequence, with each reaction time being 10 minutes.

[0053] (6) After the alcohol dehydration is complete, add xylene three times, each time for 10 minutes. Then take out the slide, add neutral resin to the slide, and then cover it with a coverslip along one side of the slide. Carefully squeeze out the air bubbles between the coverslip and the tissue with tweezers. After the slide is sealed, let it air dry at room temperature.

[0054] (7) Depending on the experimental requirements, select different objective magnifications under the microscope to observe the slide and take pictures.

[0055] The results are as follows Figure 7 As shown, the control group of the CLP model had a large number of thrombi in the liver and lungs; the drug-treated group had a significantly reduced thrombus area, and the inflammatory infiltration and tissue necrosis of organs were significantly relieved, which proves that TH-Z93 and TH-Z145 can inhibit the level of intravascular thrombus formation in septic mice and reduce microvascular occlusion, thrombosis and inflammatory infiltration damage in the liver and lungs.

[0056] Example 4: Preparation of antiplatelet drugs The antiplatelet drug in this embodiment mainly includes TH-Z93 and the excipient mannitol, and the dosage form is a lyophilized powder for injection.

[0057] The formula and preparation method for 100 vials of lyophilized powder injection are as follows: Mix 500mg TH-Z93 and 5g mannitol, then dilute to 1000mL with water for injection, and then perform sterile filtration, dispensing, and freeze-drying to prepare lyophilized powder injection.

[0058] The drug in this embodiment can be administered clinically via intravenous drip for patients with thrombosis-related diseases and severe sepsis.

[0059] The above embodiments are merely feasible or preferred embodiments of the present invention and are used to illustrate the present invention. They are not intended to limit the scope of the patent application of the present invention. Therefore, all equivalent changes and modifications made in accordance with the scope of the patent application of the present invention should fall within the scope of the patent application of the present invention.

Claims

1. Application of lipophilic bisphosphonates in the preparation of antiplatelet drugs.

2. The application according to claim 1, characterized in that, The lipophilic bisphosphonate includes one or both of TH-Z93 and TH-Z145.

3. The application according to claim 1, characterized in that, The antiplatelet drugs include those used to prevent or treat sepsis-induced platelet activation, aggregation, NETs, ​​and thrombosis.

4. An antiplatelet drug, characterized in that, It contains lipophilic bisphosphonates.

5. The drug according to claim 4, characterized in that, The lipophilic bisphosphonate includes one or both of TH-Z93 and TH-Z145.

6. The drug according to claim 5, characterized in that, The drug also includes pharmaceutically acceptable carriers and / or excipients.

7. The drug according to claim 6, characterized in that, The excipients include mannitol.

8. The medicament according to any one of claims 4-7, characterized in that, The drugs include those used to prevent or treat sepsis-induced platelet activation, aggregation, NETs, ​​and thrombosis.

9. The medicament according to any one of claims 4-7, characterized in that, The dosage forms of the drug include injections, subcutaneous injections, lyophilized powder injections, intravenous solutions, oral tablets, or capsules.

10. A method for preparing the drug according to any one of claims 4-7, characterized in that, The steps include: Take 500 mg of lipophilic bisphosphonate and 5 g of mannitol, dilute to 1000 mL with water for injection, filter aseptically, dispense, freeze dry, and prepare 100 vials of lyophilized powder for injection.

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