Application of tinapanole in medicine for resisting platelet aggregation and arterial thrombosis

Abstract

The invention relates to an application of a patent medicine Tenapanor approved by FDA in preparing a medicament for resisting platelet and arterial thrombosis. The medicament can be used for effectively inhibiting platelet activation and aggregation and arterial thrombosis block formation. The invention relates to an FDA approved patent medicine Tanipanol capable of effectively inhibiting platelet activation and aggregation induced by a natural agonist ADP and carotid artery and mesenteric artery thrombosis. Tanipamox is used as a novel candidate drug for resisting formation of platelets and arterial thrombosis blocks in fundamental research of thrombotic diseases, and has potential application value in prevention and treatment of arterial thrombosis related diseases.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical research, and specifically relates to a new medical use of the FDA-approved drug tenapanor for platelet aggregation and arterial thrombosis. Background Art

[0002] Platelet activation and thrombosis are closely related to cardiovascular and cerebrovascular diseases such as ischemic heart disease and ischemic stroke. Rupture of atherosclerotic plaques can lead to platelet activation, adhesion, and aggregation within blood vessels. More importantly, plaque rupture activates a large number of platelets, promoting coronary artery thrombosis and cerebral thrombosis, ultimately leading to vascular occlusion in ischemic heart disease and ischemic stroke, as well as various acute cardiovascular and cerebrovascular accidents. Therefore, exploring the mechanisms of platelet activation and arterial thrombosis will help identify new antiplatelet targets and is of great significance for the development of new potential antiplatelet drugs.

[0003] Tenapanor was first approved by the US FDA on September 12, 2019, and is mainly used to relieve irritable bowel syndrome with constipation (IBS-C) in adults. On April 3, 2023, the FDA expanded the approval of Tenapanor for the control of hyperphosphatemia in patients with chronic kidney disease (CKD) on dialysis. The trade name is Tenapanor is an adjunct to diet and phosphate binders to lower serum phosphorus levels. It is a sodium / hydrogen exchanger isoform 3 (NHE3) inhibitor that indirectly reduces phosphorus absorption by inhibiting intestinal sodium absorption. However, its potential role in the treatment of platelet aggregation and thrombotic disorders has not been reported.

[0004] This patent mainly explores the functional research of FDA-approved drug Tenapano and platelet aggregation and arterial thrombosis. After repeated experiments, the inventor found that Tenapano can effectively inhibit the function of SLC39A8 and reduce the Mn 2+ level, inhibiting platelet activation and aggregation, and significantly inhibiting arterial thrombosis. Compared with the current antiplatelet drug clopidogrel, tenapanor shows comparable anti-arterial thrombotic effect at a lower dose. This discovery has important clinical significance: (1) As an FDA-approved drug, tenapanor has passed Phase I, Phase II, and Phase III clinical trials for safety and efficacy, thus saving more initial human and material investment. In the next step of research, it is only necessary to observe the antiplatelet and antithrombotic effects of tenapanor and whether there are corresponding complications in a large population sample. This is very valuable for the development of new antiplatelet and antithrombotic drugs. (2) Targeting SLC39A8 may provide a new strategy for antithrombotic treatment, especially for patients who are resistant to existing antiplatelet drugs. Summary of the Invention

[0005] The present invention aims to provide a new medical use of tenapanor, a patented drug approved by the U.S. Food and Drug Administration (FDA). Tenapanor can act as an inhibitor of SLC39A8, blocking the influx of manganese ions in platelets, thereby inhibiting platelet activation, aggregation, and arterial thrombosis. Therefore, tenapanor, an FDA-approved patented drug, is expected to become a new candidate drug for the prevention and treatment of arterial thrombosis-related diseases.

[0006] The technical solution of the present invention:

[0007] The application of tenapanor in antiplatelet aggregation and arterial thrombosis drugs.

[0008] Furthermore, the anti-arterial thrombosis is achieved by targeting and antagonizing the SLC39A8-mediated platelet manganese ion influx, thereby inhibiting platelet activation, aggregation and arterial thrombosis.

[0009] Furthermore, the dosage forms of the drug include powders, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.

[0010] The present invention relates to tenapanor, which is used for preventing and improving platelet activation, aggregation and thrombosis function, or the drug has good application prospects in treating arterial thrombosis-related diseases caused by platelet aggregation.

[0011] Beneficial technical effects of the present invention

[0012] This study first discovered that tenapanor has desirable anti-platelet activation, aggregation, and arterial thrombosis effects, and validated its beneficial effects from multiple perspectives. Tenapanor, an FDA-approved drug, significantly inhibits platelet activation, aggregation, spreading, and retraction, and mitigates carotid and mesenteric artery thrombosis, making it a potential new drug candidate for the treatment of arterial thrombotic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Tenapanor significantly inhibits platelet aggregation;

[0014] Figure 2 Tenapanor significantly inhibited clot retraction;

[0015] Figure 3 Tenapanor significantly increased the platelet spreading area on fibrinogen and pseudopodia formation;

[0016] Figure 4 Tenapanor significantly inhibited carotid artery thrombosis, mesenteric artery thrombosis, and pulmonary thrombosis in mice;

[0017] Figure 5 Tenapano targets SLC39A8 to inhibit Mn 2+ Induced thrombosis. DETAILED DESCRIPTION

[0018] The present invention first discovered, through a series of in vitro cell experiments and in vivo animal experiments, that the FDA-approved drug tenapanor has a new pharmacological function of inhibiting platelet aggregation and thrombosis. Platelet activation and aggregation are the key links in arterial thrombosis. Therefore, in in vitro experiments, the present invention first verified the effect of tenapanor on activation and aggregation: First, blood was collected from the abdominal aorta of mice to separate and extract platelet-rich plasma, which was then divided into different treatment groups, including a control group (no drug added), a tenapanor group (only tenapanor added), a manganese-treated group (only manganese dichloride added), and a tenapanor + manganese-treated group. By pre-treating the platelet-rich plasma with tenapanor (20 μmol / L), then adding the natural agonist ADP for stimulation, and then using turbidimetry to detect the platelet aggregation curve and maximum aggregation rate, the effect of tenapanor on platelet aggregation was observed to determine the effect of tenapanor on platelet aggregation in vitro. The results showed that tenapanor has a new effect of inhibiting platelet aggregation. In addition, platelet-rich plasma was pre-treated with tenapanor and then stimulated by the addition of manganese (72.8 μmol / L) and the natural agonist ADP. The platelet aggregation curve and maximum aggregation rate were then measured using turbidimetry to observe the effect of tenapanor on platelet aggregation. This was to determine the effect of tenapanor on the aggregation of manganese-treated platelets in vitro. The results showed that tenapanor has a new role in inhibiting manganese-induced platelet aggregation.

[0019] A retraction experiment was used to observe the effect of tenapanor on platelet retraction: Platelet-rich plasma (PRP) was pre-treated with tenapanor and then divided into groups. Fibrinogen (150 μg / mL) and thrombin (0.012 U / mL) were then added to the drug-treated EP tubes and mixed thoroughly. The tubes were then quickly transferred to clean glass aggregation tubes and incubated at 37°C. Blood clot retraction was recorded at 10, 15, 20, and 25 minutes. The results revealed a novel effect of tenapanor in inhibiting platelet retraction.

[0020] Then, a fluorescence experiment was used to verify the effect of tenapanor on platelet spreading and pseudopodia formation in vitro: blood was collected from the abdominal aorta of mice to separate, extract and wash the platelet suspension. After grouping and processing, the platelets were labeled with phalloidin, and the platelet spreading area and the number of pseudopodia formed were observed by laser confocal microscopy to evaluate the effect of tenapanor on platelet spreading and pseudopodia formation, and verify the anti-platelet activation of tenapanor in vitro from another perspective. The results showed that tenapanor has a new effect of inhibiting platelet spreading and pseudopodia formation. The above in vitro cell experimental results show that tenapanor has a new effect of inhibiting platelet aggregation, platelet spreading and pseudopodia formation.

[0021] Secondly, the present invention uses in vivo animal experiments to further clarify the effects of platelet spreading and pseudopodia formation on thrombosis: C57BL / 6JJ mice were selected, and different treatment groups were set up at the same time. Tenapano (0.2, 2, 20 mg / kg) or clopidogrel (20 mg / kg) were given intraperitoneally. A carotid artery thrombosis model was established by applying filter paper soaked with 15% ferric chloride to one side of the common carotid artery of the mouse. Laser speckle blood flow imaging technology was then used to observe the changes in intravascular blood flow after carotid artery thrombosis. The severity of carotid artery thrombosis in mice in different treatment groups was compared, and the inhibitory effect of tenapano on carotid artery thrombosis in mice was analyzed, thereby verifying the anti-thrombotic effect of tenapano in vivo. The results showed that tenapano can effectively alleviate the formation of arterial thrombosis, and tenapano (2 mg / kg) can significantly inhibit carotid artery thrombosis in mice, with an effect close to that of clopidogrel (20 mg / kg); Next, a muscular artery thrombosis model was constructed using the mouse mesenteric artery. A mesenteric artery thrombosis model was established by applying filter paper soaked in 10% ferric chloride to the appendicular artery. Laser speckle blood flow imaging was then used to observe blood flow in the blood vessels after mesenteric thrombosis. The results showed that tenapanor significantly inhibited mesenteric artery thrombosis in mice. Next, a mixed solution of collagen, thrombin, and epinephrine was used to establish a pulmonary thrombosis model in mice. The results showed that tenapanor (2 mg / kg) significantly inhibited pulmonary thrombosis in mice and reduced the area of ​​pulmonary infarction. The above results indicate that the FDA-approved drug tenapanor has great potential in preventing arterial thrombosis.

[0022] In addition, C57BL / 6JJ mice were intraperitoneally injected with tenapanor and then intraperitoneally injected with Mn 2+ (0.5mg / kg), 15% ferric chloride soaked filter paper was applied to one side of the common carotid artery of mice to establish a carotid artery thrombosis model and a mouse mesenteric artery thrombosis model. The results showed that tenapanor significantly alleviated Mn 2+ Induced carotid artery thrombosis in mice and mesenteric artery thrombosis in mice. Next, a mixed solution of collagen, thrombin and epinephrine was used to construct a pulmonary thrombosis model in mice. The results showed that tenapanor significantly alleviated the Mn 2+ The above results show that tenapanor significantly alleviates the Mn 2+ Induced carotid and mesenteric artery thrombosis in mice, and inhibited pulmonary thrombosis. Therefore, combined with in vitro cell experiments and in vivo animal experiments, it was confirmed that tenapanor can effectively alleviate arterial thrombosis by inhibiting platelet aggregation, spreading, and pseudopodia formation, and is expected to become a new candidate drug for the prevention and treatment of thrombotic diseases.

[0023] The technical solution of the present invention is further described below with reference to the embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention shall be included in the scope of protection of the present invention. The process equipment or devices not specifically noted in the following examples are all conventional equipment or devices in the art. Unless otherwise specified, the raw materials used in the examples of the present invention can be obtained commercially; unless otherwise specified, the technical means used in the examples of the present invention are all conventional means well known to those skilled in the art.

[0024] The dosage of tenapanor in the present invention is 20 μmol / L in in vitro experiments and 0.2, 2, and 20 mg / kg in in vivo animal experiments, and 2 mg / kg can achieve the best anti-arterial thrombotic effect.

[0025] Example 1

[0026] Determination of the effect of tenapanor on platelet aggregation

[0027] Turbidimetry was used to detect the platelet aggregation curve and maximum aggregation rate to determine the inhibitory effect of tenapanor on platelet aggregation. The specific steps are as follows:

[0028] Blood was collected from the abdominal aorta of mice and slowly mixed with 1:1 normal saline, and centrifuged at 200g / min for 10 minutes at room temperature to separate PRP; the upper layer of platelet-poor plasma (PPP) was aspirated after the upper layer of PRP was centrifuged at 1000g / min for 10 minutes at room temperature; then, based on the principle of turbidimetry, platelet-poor plasma (PPP) was used as a blank control, and stirring magnetic beads were added to PRP to record the turbidity changes. Calculation formula: Aggregation rate = (A sample - A blank) / (A maximum - A blank) × 100%. The results showed that the FDA-approved drug tenapanor itself significantly inhibited platelet aggregation and significantly reversed manganese-induced platelet aggregation ( Figure 1 ), the above results indicate that the FDA-approved drug tenapanor has a new anti-platelet aggregation effect.

[0029] Example 2

[0030] The platelet retraction reaction was detected by the retraction test method to determine the effect of tenapanor on inhibiting platelet retraction. The specific steps are as follows:

[0031] Blood was collected from the abdominal aorta of mice and centrifuged at 200 g / min for 15 minutes at room temperature. After centrifugation, the upper PRP layer was carefully aspirated. The PRP was centrifuged at 3000 g / min for 5 minutes at room temperature, and the supernatant was removed, leaving the platelet pellet. The platelets were resuspended and washed twice with preheated CGS solution. The platelets were resuspended with modified benchtop solution (3 × 10 7 / mL), and placed in a 37℃ water bath for 2 hours; according to the grouping situation, the platelets (3×107 / mL) in an EP tube, and after drug treatment, place it in a 37°C incubator for 10 minutes; fibrinogen (150μg / mL) and thrombin (0.012U / mL) were added to the drug-treated EP tube and mixed, then quickly transferred to a clean glass aggregation tube and incubated at 37°C; photos were taken at 10 minutes, 15 minutes, 20 minutes, and 25 minutes, and the retraction of the blood clot in the tube was recorded. The results showed that the FDA-approved drug tenapanor itself significantly inhibited platelet retraction and significantly reversed Mn 2+ Induced platelet retraction ( Figure 2 ), the above results indicate that the FDA-approved drug tenapanor has a new anti-platelet retraction effect.

[0032] Example 3

[0033] Determination of the effects of tenapanor on platelet spreading and pseudopodia formation

[0034] Platelets were labeled with phalloidin and the platelet spreading area and pseudopodia number were observed under a laser confocal microscope to determine the effects of tenapanor on platelet spreading and pseudopodia formation. The specific steps are as follows:

[0035] Coat the round glass slides in the well plate with 500 μL of 1× polylysine for 30 minutes, then remove the remaining liquid and dry the 12-well plate at room temperature overnight; treat the platelet suspension (3×107 / mL) with different drugs and add it to the 12-well plate containing the round cover glass and mark it; fix the platelets with preheated 4% paraformaldehyde at room temperature for 30 minutes; wash the platelets fixed at room temperature with PBS three times, and penetrate the platelets with 0.5% TritonX-100 solution for 5 minutes. Wash with PBS three times, incubate with rhodamine-labeled phalloidin at room temperature for 30 minutes (be careful to avoid light); wash with PBS three times, and seal the slides with a sealing agent. Adjust the parameters according to the wavelength of the fluorescent dye, and use a confocal microscope for image acquisition. The results showed that tenapanor significantly inhibited the spreading area of ​​platelets on fibrinogen and pseudopodia formation ( Figure 3 ), the above results indicate that the FDA-approved drug tenapanor has a new effect of inhibiting platelet spreading and pseudopodia formation.

[0036] Example 4

[0037] Measuring the Effect of Tenapanor on Arterial Thrombosis

[0038] C57BL / 6JJ male mice (8-10 weeks, approximately 25 g) were weighed and intraperitoneally injected with drugs or an equal amount of normal saline (control). After anesthesia, they were fixed on a 37°C temperature-controlled plate. One common carotid artery was isolated, and a filter paper soaked in 10% ferric chloride solution was applied to a 0.5 cm long common carotid artery for approximately 3 minutes to construct a carotid artery thrombosis model. The blood flow in the common carotid artery was recorded using a laser speckle blood flow imager. Figure 4 AB results showed that the FDA-approved drug tenapanor (2 mg / kg) can significantly inhibit carotid artery thrombosis in mice, with an effect close to that of clopidogrel (20 mg / kg). C57BL / 6JJ male mice (8-10 weeks, approximately 25 g) were weighed and intraperitoneally injected with the drug or an equal amount of normal saline (control). After anesthesia, they were fixed on a 37°C temperature-controlled plate. The abdomen was cut open, and the cecum and appendix were gently pulled out (be careful not to pull excessively to ensure normal blood supply). Filter paper soaked in 10% ferric chloride solution was then applied to the appendicular artery for 3 minutes to create a mesenteric artery thrombosis model. The mesenteric artery blood flow was recorded using a laser speckle blood flow imager. Figure 4 CD results showed that the FDA-approved drug tenapanor (2 mg / kg) significantly inhibited mesenteric artery thrombosis in mice. C57BL / 6JJ male mice, 8-10 weeks old, weighing about 25g, were treated with a mixture of collagen (3 μg / g), epinephrine (0.6 μg / g) and thrombin (0.05u / g) dissolved in an appropriate amount of normal saline and injected with the drug (0.01 mL / g) through the mouse tail vein to construct a pulmonary thrombosis model. Subsequently, after anesthesia, 500 μL of 2% Evans blue solution was slowly injected into the right atrium for staining, and the lung staining was observed and photographed. The effect of tenapanor on thrombosis was determined. Figure 4 Results E showed that the FDA-approved drug tenapanor significantly inhibited pulmonary thrombosis in mice and reduced the area of ​​pulmonary infarction. These results indicate that the FDA-approved drug tenapanor has a new effect in alleviating arterial thrombosis.

[0039] In addition, C57BL / 6JJ mice were intraperitoneally injected with FDA-approved tenapanor (2 mg / kg), and then intraperitoneally injected with Mn 2+ (0.5mg / kg), 15% ferric chloride soaked filter paper was applied to one side of the common carotid artery of mice to establish a carotid artery thrombosis model. The results showed that the FDA-approved drug tenapanor (2mg / kg) significantly alleviated the Mn 2+ Induced carotid artery thrombosis in mice ( Figure 5 AB). The FDA-approved drug tenapanor (2 mg / kg) also significantly alleviated Mn 2+ Induced mesenteric artery thrombosis in mice ( Figure 5 CD). Next, a mixed solution of collagen, thrombin, and epinephrine was used to establish a mouse model of pulmonary thrombosis. The results showed that the FDA-approved drug tenapanor significantly alleviated the Mn 2+ Induced pulmonary thrombosis in mice and reduced pulmonary infarct size ( Figure 5 E). The above results show that the FDA-approved drug tenapanor significantly alleviates Mn 2+ Induced carotid and mesenteric artery thrombosis in mice and inhibited pulmonary thrombosis.

[0040] In summary, tenapanor of the present invention possesses novel and desirable antiplatelet aggregation, retraction, spreading, pseudopodia formation, and arterial thrombosis effects. Therefore, the FDA-approved tenapanor is expected to be effective in treating diseases involving platelet aggregation and arterial thrombosis.

Claims

1. The use of tenapanor in drugs for preventing platelet activation, aggregation and thrombus formation, characterized in that: The tenapanor is a patent medicine approved by the U.S. Food and Drug Administration (FDA).

2. The use according to claim 1, characterized in that The anti-platelet activation and aggregation is achieved by targeting and antagonizing the influx of manganese ions mediated by SLC39A8, thereby inhibiting the formation of arterial thrombus.

3. The use according to claim 1, characterized in that The dosage forms of the drug include powders, tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.

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