Use of HMG-CoA and its pharmaceutically acceptable salts for preparing platelet preservatives

Abstract

The present invention discloses the use of HMG-CoA and its pharmaceutically acceptable salts for preparing a platelet preservative. The present invention has confirmed that adding HMG-CoA or its pharmaceutically acceptable salts to platelets to be preserved can effectively alleviate platelet activation and apoptosis, while prolonging the in vivo survival period of platelets after platelet transfusion and protecting the hemostatic function of platelets.

Description

Technical Field

[0001] The present invention relates to the preservation technology of platelets, and specifically relates to the application of 3-hydroxy-3-methylglutaryl coenzyme A and its pharmaceutically acceptable salts in the preparation of platelet preservatives. Background Art

[0002] Platelets are small cytoplasmic fragments formed by the degranulation of megakaryocytes, and their main function is to participate in hemostasis and blood coagulation. Although there are multiple reasons for the shortage of platelet supply, the short shelf life (only 5 days) under conventional storage conditions (22 ± 2°C with continuous shaking) is one of the main factors, which directly leads to an expiration and rejection rate as high as 10-20%. Due to the high expiration and rejection rate, and considering the high cost of the special equipment and consumables required for platelet collection, it is difficult for blood centers and blood stations to carry out large-scale platelet blood reservation, resulting in shortages. Moreover, the high expiration and rejection rate will further exacerbate the shortage. Summary of the Invention

[0003] Aiming at the defects or deficiencies of the prior art, the present invention provides the application of HMG-CoA and its pharmaceutically acceptable salts in the preparation of platelet preservatives. The present invention also simultaneously provides corresponding platelets, and HMG-CoA or its pharmaceutically acceptable salt is added to the provided platelets.

[0004] Optionally, the pharmaceutically acceptable salt of HMG-CoA is 3-hydroxy-3-methylglutaryl coenzyme A disodium salt. The addition amount of the HMG-CoA or its pharmaceutically acceptable salt in platelets will not cause the content of β-hydroxybutyric acid in platelets to exceed 0.27 mmol / L. Preferably, the concentration of the HMG-CoA or its pharmaceutically acceptable salt in platelets is less than or equal to 0.2 mmol / L.

[0005] The present invention has confirmed that adding HMG-CoA or its pharmaceutically acceptable salt to platelets to be preserved can effectively alleviate platelet activation and apoptosis, while prolonging the in vivo survival period of platelets after platelet transfusion and protecting the hemostatic function of platelets. Brief Description of the Drawings

[0006] Figure 1 Effect of HMG-CoA.2Na on the externalization of phosphatidylserine (PS) during in vitro platelet preservation in Example 1 of the present invention (PS + platelet percentage statistics); n = 6, all data in the histogram are presented as mean ± SEM; NS: no statistical difference; **p < 0.01.

[0007] Figure 2 Effect of HMG-CoA.2Na on the expression of CD62P during in vitro platelet preservation in Example 2 of the present invention (CD62P +Platelet percentage statistics); n = 6, all data in the histogram are presented as mean ± SEM; NS: no statistical difference; ***p < 0.001.

[0008] Figure 3 This shows the effect of HMG-CoA.2Na on the in vivo survival period after platelet transfusion in Example 3 of the present invention (APC + / APC + &FITC + Platelet analysis statistics); n = 5, all data in the histogram are presented as mean ± SEM; NS: no statistical difference; *p < 0.05; **p < 0.01.

[0009] Figure 4 This shows the effect of HMG-CoA.2Na on the in vivo hemostatic function after platelet transfusion in Example 4 of the present invention (mouse carotid artery thrombosis formation time statistics); n = 6, NS: no statistical difference; *p < 0.05. Detailed implementation manners

[0010] Unless otherwise specified, scientific and technical terms herein are understood according to the knowledge of those of ordinary skill in the relevant fields.

[0011] As is well known, platelets will undergo platelet storage lesions (PSLs) during storage, specifically manifested as apoptosis and activation of cells with the extension of storage time, as well as the shortening of the in vivo survival period and the impairment of hemostatic function after transfusion of stored platelets. Therefore, the present invention first analyzes the effect of HMG-CoA on PS eversion of human platelets, and proves that HMG-CoA has the effect of alleviating apoptosis during in vitro storage of platelets; then analyzes the effect of HMG-CoA on the expression of CD62P in human platelets, and proves that HMG-CoA has the effect of alleviating activation during in vitro storage of platelets. On this basis, the inventors further analyze the proportion of HMG-CoA-pretreated human platelets transfused into SCID mice and the carotid artery thrombosis formation time of SCID mice transfused with HMG-CoA-pretreated human platelets through a SCID mouse platelet transfusion model, and verify that HMG-CoA can improve the in vivo survival period after platelet transfusion and protect its hemostatic function at the same time.

[0012] The present invention will be further described below through examples. It should be noted that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) is one of the intermediate products of human fat oxidative metabolism. After the action of HMG-CoA lyase and β-hydroxybutyrate dehydrogenase respectively, it can be converted into β-hydroxybutyric acid. In practice, HMG-CoA mostly exists in the form of salts, and the hydrolysis product of HMG-CoA salt is HMG-CoA. In the following examples, disodium 3-hydroxy-3-methylglutaryl coenzyme A is selected as a representative to explain the present invention. In addition, all platelets used in the following examples were collected from healthy blood donors, HMG-CoA·2Na was purchased from Sigma, Annexin V-FITC Apoptosis Staining / Detection was purchased from BD; the mice used were male SCID mice weighing 20-25 g purchased from Cyagen Biosciences, APC anti-human CD41 Antibody and FITC anti-mouse CD41 Antibody were both purchased from Biolegend; Anti-mice CD42b Antibody was purchased from Emfret, and rhodamine was purchased from Sigma.

[0013] Example 1: The inventors found through research that HMG-CoA·2Na has the effect of alleviating apoptosis during the in vitro preservation of human platelets.

[0014] 1. Preparation of platelets

[0015] Healthy adults who had not taken any medications within 10 days were recruited as volunteers, and then apheresis platelets were prepared using a blood cell separator (Amicus 4R4580, Fenwal) for standby.

[0016] 2. Treatment of platelets

[0017] 5 mL of platelets were respectively aspirated and added to three platelet storage bags. One bag was directly used as Control Group 1, another bag was added with 25 μL of normal saline (vehicle) as Control Group 2, and the third bag was added with 25 μL of normal saline dissolved with HMG-CoA·2Na (the final concentration of HMG-CoA·2Na in the platelet treatment system was 0.2 mmol / L) as the experimental group; the three groups of platelets were placed in a platelet shaking incubator for in vitro preservation (shaking continuously at 22 ± 2 °C).

[0018] 3. Detection of PS-positive platelets

[0019] Appropriately sized platelets were taken on the 3rd, 5th, and 7th days of storage, and PS was labeled with reference to the Annexin V-FITC Apoptosis Staining / Detection instruction manual. Then, the percentage of Annexin V-positive platelets was detected using a flow cytometer (BD FACS Canto).

[0020] After detection, the statistical analysis of the percentage of Annexin V-positive platelets is as Figure 1 shown. The percentages of Annexin V-positive platelets in Control Group 1 on the 3rd, 5th, and 7th days were 6.2%, 8.5%, and 10.6% respectively, and those in Control Group 2 on the 3rd, 5th, and 7th days were 6.2%, 8.4%, and 10.7% respectively, showing no significant statistical differences; this result indicates that adding a certain amount of normal saline does not affect the percentage of Annexin V-positive platelets.

[0021] The percentages of Annexin V-positive platelets in the experimental group on the 3rd, 5th, and 7th days were 4.2%, 5.3%, and 8.0% respectively, all significantly lower than those in Control Group 1 on the 3rd, 5th, and 7th days (6.2%, 8.5%, and 10.6% respectively). Also, the 8.0% on the 7th day in the experimental group was not significantly different from the 8.5% on the 5th day in Control Group 1; this result indicates that adding a certain amount of HMG-CoA.2Na reduces the percentage of Annexin V-positive platelets, and the percentage of Annexin V-positive platelets on the 7th day in the experimental group is similar to that on the 5th day in Control Group 1.

[0022] Annexin V is a fluorescent probe for PS, and the externalization of PS is a classical indicator of cell apoptosis. Therefore, the above results of this example confirm that HMG-CoA.2Na has the effect of alleviating the apoptosis of human platelets during in vitro storage, and may extend the storage period of platelets from the conventional 5 days to 7 days.

[0023] Moreover, using the enzyme kinetic continuous monitoring method for detection, when the final concentration of HMG-CoA.2Na added to the platelets in the experimental group of this example was 0.2 mmol / L, the β-hydroxybutyric acid in the platelet storage system increased to 0.22 mmol / L, not exceeding the upper limit of the normal human value of 0.27 mmol / L. Therefore, by adding a certain amount of HMG-CoA.2Na to preserve platelets, the present invention will not cause harm to the body.

[0024] Example 2: The inventors' research found that HMG-CoA.2Na has the effect of alleviating the activation of human platelets during in vitro storage

[0025] The preparation and treatment of platelets were the same as in Example 1. Appropriate amounts of platelets were taken on the 3rd, 5th, and 7th days of storage respectively. After labeling with FITC anti - human CD62P, the percentage of CD62P - positive platelets was detected using a flow cytometer (BD FACS Canto).

[0026] After detection, the statistical analysis of the percentage of CD62P - positive platelets was as Figure 2 shown. On the 3rd, 5th, and 7th days, the percentages of CD62P - positive platelets in Control Group 1 were 11.8%, 15.8%, and 25.9% respectively, and those in Control Group 2 on the 3rd, 5th, and 7th days were 11.9%, 15.9%, and 25.8% respectively. There were no statistical differences between the two groups. This result indicates that adding a certain amount of normal saline does not affect the percentage of CD62P - positive platelets.

[0027] On the 3rd, 5th, and 7th days, the percentages of CD62P - positive platelets in the experimental group were 8.6%, 10.3%, and 16.9% respectively, which were significantly lower than those in Control Group 1 on the 3rd, 5th, and 7th days (11.8%, 15.8%, and 25.9% respectively). And there was no statistical difference between 16.9% on the 7th day in the experimental group and 15.8% on the 5th day in Control Group 1. This result indicates that adding a certain amount of HMG - CoA.2Na can reduce the percentage of CD62P - positive platelets, and the percentage of CD62P - positive platelets on the 7th day in the experimental group is similar to that on the 5th day in Control Group 1.

[0028] The generation of CD62P is a classic indicator of platelet activation. Therefore, the above results of this example confirm that HMG - CoA.2Na has the effect of delaying the activation of human platelets during in vitro storage, and may extend the storage period of platelets.

[0029] Example 3: The inventor's research found that pretreatment with HMG - CoA.2Na can extend the in - vivo survival period of stored platelets after transfusion

[0030] The above Examples 1 and 2 confirmed that adding a certain amount of HMG - CoA.2Na would affect platelet apoptosis and activation, while normal saline would not. Therefore, in this example, Control Group 2 was reduced on the basis of Example 1. The preparation and treatment of platelets were the same as in Example 1;

[0031] Then, 4×10 8 platelets of Control Group 1 were taken on the 5th day of storage, and 4×10 8, it was intravenously injected into SCID mice via the tail vein. Then, 50 μl of peripheral blood was collected from the tail tip at 0.5, 2, and 5 hours respectively, and after lysing red blood cells, it was labeled with APC anti-human CD41 Antibody and FITC anti-mouse CD41 Antibody, and detected by flow cytometry (BD FACS Canto) for the APC + / APC + &FITC + percentage.

[0032] After detection, the APC + / APC + &FITC + percentage statistical analysis was as Figure 3 shown. After the platelets stored on the 5th day in the experimental group were injected into mice, the APC + / APC + &FITC + at 0.5, 2, and 5 hours were 41.2%, 32.0%, and 25.4% respectively, which were all significantly higher than 26.0%, 18.9%, and 17.5% at 0.5, 2, and 5 hours after the platelets stored on the 5th day in the control group 1 were injected into mice;

[0033] And when it was the 7th day of storage in the experimental group, the APC + / APC + &FITC + of 24.5%, 19.2%, and 16.3% had no statistical difference from 26.0%, 18.9%, and 17.5% of the control group 1 stored on the 5th day.

[0034] Among them, the APC + / APC + &FITC + percentage represents the proportion of human platelets injected into SCID mice, which is proportional to the in vivo survival period after platelet transfusion. Therefore, the results of this example show that HMG-CoA.2Na can extend the in vivo survival period after the transfusion of stored platelets; at the same time, it is confirmed that HMG-CoA.2Na pretreatment can extend the storage period of platelets.

[0035] Example 4: The inventor's research found that HMG-CoA.2Na pretreatment can protect the hemostatic function of platelets

[0036] 1. Knockout of SCID mouse platelets

[0037] Prepare SCID mice, and inject Anti-mice CD42b Antibody monoclonal antibody into the tail vein according to the instructions to pre-clear autologous platelets in the mice.

[0038] 2. Platelet Transfusion

[0039] Except for reducing control group 2, the preparation and treatment of platelets in this example were the same as those in Example 1; then, an appropriate amount of platelets from control group 1 was taken on the 5th day of storage, and an appropriate amount of platelets from the experimental group was taken on the 5th and 7th days of storage, 1×10 for each mouse 9 They were intravenously infused into the corresponding mice through the tail vein.

[0040] 3. Establishment of Mouse Fecl3 Carotid Artery Thrombosis Model

[0041] ① Prepare a 0.5 mg / mL rhodamine 6G solution and store it in the dark; ② Prepare a 100 mg / mL FeCl3 solution for later use; ③ After intraperitoneally anesthetizing the mice treated in step 2 with a 10 g / L pentobarbital sodium solution, inject 200 μL of rhodamine 6G solution into the medial canthus vein of the mice; ④ Place the mice in the supine position, cut the skin of the mice along the midline of the neck, separate the sublingual gland under a stereomicroscope, turn it up, and fully expose the carotid artery of the mice by peeling; ⑤ Dip a filter paper with a length and width of 1 mm in a 100 mg / mL FeCl3 solution and apply it to the carotid artery of the mice for 1 min; ⑥ Remove the filter paper, rinse it with normal saline, observe the thrombus formation process under a stereoscopic fluorescence microscope, and record the thrombus formation time.

[0042] The statistical analysis of the thrombus formation time in the carotid artery of mice is as Figure 4 shown. The average thrombus formation time on the 5th day in the experimental group was 490 seconds, which was significantly shorter than 898 seconds on the 5th day in control group 1. And there was no statistical difference between the average thrombus formation time of 905 seconds on the 7th day in the experimental group and 898 seconds on the 5th day in control group 1. The thrombus formation time is inversely proportional to the platelet function. Therefore, the results of this example indicate that HMG-CoA.2Na can protect the hemostatic function of platelets; at the same time, it further confirms that HMG-CoA.2Na pretreatment can extend the storage period of platelets.

Claims

1. Use of HMG-CoA and its pharmaceutically acceptable salts for preparing a platelet preservative, wherein the added amount of the HMG-CoA or its pharmaceutically acceptable salt in platelets does not cause the content of β-hydroxybutyric acid in platelets to exceed 0.27 mmol / L, and the concentration of the HMG-CoA or its pharmaceutically acceptable salt in platelets is less than or equal to 0.2 mmol / L.

2. The application according to claim 1, wherein The pharmaceutically acceptable salt of HMG-CoA is monosodium 3-hydroxy-3-methylglutaryl coenzyme A disodium salt.

3. A platelet preservative, characterized in that, The platelet preservative is prepared by dissolving HMG-CoA or its pharmaceutically acceptable salt in physiological saline; the added amount of the HMG-CoA or its pharmaceutically acceptable salt in platelets does not cause the content of β-hydroxybutyric acid in platelets to exceed 0.27 mmol / L, and the concentration of the HMG-CoA or its pharmaceutically acceptable salt in platelets is less than or equal to 0.2 mmol / L.

Images