CN112274510A - Application of montelukast in preparation of medicines for preventing and treating thrombotic diseases - Google Patents
Application of montelukast in preparation of medicines for preventing and treating thrombotic diseases Download PDFInfo
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Abstract
The invention discloses an application of montelukast in preparing a medicine for preventing and treating thrombotic diseases. The invention discovers that the montelukast has the inhibitory activity of the blood coagulation factor XIa (FXIa) for the first time, so the montelukast can be used for preparing and developing medicines for treating or preventing thrombotic diseases and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to application of montelukast in preparation of medicines for preventing and treating thrombotic diseases.
Background
Thrombotic cardiovascular and cerebrovascular diseases are a major cause of death worldwide, and thrombotic diseases are mainly divided into Venous Thrombosis (VT) and arterial thrombosis (ArT). One quarter of the worldwide causes of hospitalized death are reported to be associated with thrombotic disease, with one patient presenting with VT-related disease every 16 seconds and one patient dying from VT-related disease every 37 seconds worldwide.
Thrombotic diseases are the result of imbalance of procoagulant, anticoagulant and fibrinolytic processes, and clinical drugs currently used for preventing and treating thrombotic diseases include anticoagulants, antiplatelet drugs and thrombolytics, of which anticoagulant drugs account for the majority. The anticoagulant is mainly used for preventing thrombosis, such as postoperative deep vein thrombosis, heart and brain thrombosis, pulmonary embolism and the like. The oral anticoagulant is used for preventing the occurrence and development of acute thrombotic diseases, such as cerebral apoplexy, myocardial infarction and the like, by being taken by patients daily for a long time. Currently clinically approved oral anticoagulants are classified as Vitamin K Antagonists (VKAs) or Direct Oral Anticoagulants (DOACs). VKAs inhibit the coagulation pathway by competitively inhibiting vitamin K, which is essential for the modification and activation of coagulation factors. DOACs achieve the purpose of anticoagulation by directly inhibiting the activity of thrombin (thrombin) or activated coagulation factor X (FXa). Currently, the highest-market-sales DOACs worldwide are the FXa inhibitor Apixaban (Apixaban) jointly developed by feverfew and behme schnobao corporation. However, since thrombin and FXa play both a critical and immediate role in the normal coagulation (e.g., wound healing) and thrombosis (thrombotic disease), patients taking either VKAs or DOACs for extended periods of time are at risk of potentially bleeding due to impaired normal coagulation function. For example, patients taking Warfarin (Warfarin), a VKA anticoagulant, for a long period of time, are stopped for 1-2 weeks or more before undergoing dental care to prevent excessive bleeding during dental surgery. In addition, vitamin K plays an important role in other important physiological processes besides the coagulation process, so VKAs also has the defects of narrow therapeutic window, serious food-drug interaction and the like. Therefore, a perfect oral anticoagulant should be able to treat or prevent intravascular thrombosis without affecting the normal coagulation system and without the risk of bleeding.
In recent years, more and more studies have shown that activated coagulation factor XI (FXIa) is a safer therapeutic target for thrombus than thrombin and FXa. Since FXIa plays an important role in the process of thrombosis but has less impact on normal coagulation function. Clinically, patients with FXIa gene deletions present less or even no bleeding risk. Clinical data indicate that FXI levels in blood of patients with thrombus (deep vein thrombosis, arterial thrombosis, etc.) are significantly higher than normal. A large number of animal model research works prove that the FXI gene knockout, FXI gene knock-down or FXIa activity inhibition can effectively inhibit the formation of thrombus, but has little influence on the normal blood coagulation process.
Montelukast (english name Montelukast, its structural formula is:
) Is a leukotriene receptor antagonist and was originally widely used as an anti-asthma drug for long-term daily administration. With the progress of research, montelukast is found to be also used for relieving seasonal allergic rhinitis, eosinophilic esophagitis in airways, refractory chronic urticaria and other diseases. However, to date, no relevant report on montelukast antithrombotic disease has been found. The invention discovers the inhibition effect of montelukast on FXIa for the first time, and proposes that montelukast is used as an anticoagulant for preparing a medicament for preventing and treating thrombotic diseases.
Disclosure of Invention
Aiming at the problem of bleeding risk in the treatment and prevention of antithrombotic diseases, the invention provides the application of montelukast in preparing medicines for preventing and treating thrombotic diseases by utilizing the characteristic that montelukast has the inhibitory activity of blood coagulation factor xia (fxia), so that a safe and effective small-molecule compound is provided for the clinical treatment of thrombus, and the application prospect is wide.
In order to achieve the purpose, the invention adopts the following technical scheme:
the application of montelukast in preparing the medicines for preventing and treating thrombotic diseases utilizes the characteristic that montelukast has FXIa inhibitory activity, and montelukast or pharmaceutically acceptable salts thereof are used as pharmaceutically active ingredients to prepare any pharmaceutically acceptable dosage form.
The pharmaceutically acceptable dosage forms comprise tablets, capsules, granules, oral liquid or injections.
Through biological activity determination of montelukast, enzyme inhibition activity experiments show that montelukast with different concentrations has good FXIa inhibition activity, and the half inhibition concentration is 0.17 +/-0.04 mu M; in vitro thrombolytic experiment results show that montelukast can obviously prolong activated partial thromboplastin time (aPTT), but the influence on the Partial Thromboplastin Time (PTT) of human plasma is not obvious; electric current or ferric chloride (FeCl)3) The experimental result of an animal model for inducing carotid thrombosis shows that montelukast can obviously improve the formation of mouse thrombus; bleeding risk evaluation experimental results show that oral administration of montelukast only slightly prolongs the tail bleeding time of mice, and does not affect the whole blood coagulation of mice. Therefore, montelukast or a pharmaceutically acceptable salt thereof can be used as an FXIa inhibitor for preparing a medicament for treating or preventing thrombotic diseases.
Due to the application of the technical scheme, compared with the prior art, the invention has the following advantages:
1. the invention discovers for the first time that montelukast has the function of an FXIa inhibitor, can inhibit thrombus formation, has no obvious inhibition effect on thrombin or FXa, has small bleeding risk caused by the inhibition effect, can be used for preparing a medicament for treating or preventing thrombotic diseases, and provides a new application for montelukast.
2. Since the montelukast comes on the market, the long-term oral safety of the montelukast is proved by a large amount of practice, the montelukast has definite clinical data such as pharmacokinetics and the like, and if the montelukast is used as an antithrombotic treatment or preventive medicine, the clinical test time can be obviously reduced, and a large amount of research and development cost can be saved.
3. The montelukast has high oral availability, can be used for preventing thrombotic cardiovascular and cerebrovascular diseases by daily oral administration, and can be used for preventing postoperative deep vein thrombosis by intracumnant injection.
4. The montelukast has both anti-inflammatory and anticoagulant functions, and can play a dual role in treating inflammation-induced thrombosis (such as disseminated intravascular coagulation caused by acute sepsis).
Drawings
FIG. 1 is a graph of concentration-dependent inhibition and median Inhibition Concentrations (IC) of FXIa enzyme activity by Montelukast50）。
Figure 2 is a graph comparing the time to reach plateau for clotting by different concentrations of montelukast in vitro thrombolytic experiments.
FIG. 3 is a graph of the time to current-induced carotid thrombosis for different drugs.
Fig. 4 is a graph comparing the risk of bleeding caused by different drugs.
Detailed Description
For a better understanding of the present invention, reference is made to the following detailed description taken in conjunction with the accompanying drawings and specific examples, but the scope of the invention as claimed should not be limited to the scope of the examples. Unless otherwise indicated, reagents and materials used in the following examples are commercially available. In the following examples, various procedures and methods not described in detail are conventional methods well known in the art.
Example 1: determination of inhibition of FXIa and homologous serine protease Activity by Montelukast
The experimental method comprises the following steps:
the activity of FXIa and 9 homologous serine proteases is determined by a reported chemiluminescence method (Chromogenic assay), which comprises the steps of incubating FXIa or homologous serine proteases with final concentration of 10 nM and montelukast with final concentration of 0-100 mu M for 15 minutes in a 100 mu L reaction system (containing 20 mM Tris-HCl pH 7.4, 150 mM NaCl and 0.1% BSA), adding luminescent substrates with final concentration of 200 mu M (produced by Chromogenix, see Table 1 for luminescent substrates corresponding to the proteases), mixing uniformly, immediately placing the mixture into a BioTek Synergy 4 enzyme-labeling instrument, and detecting absorbance at 405 nM and 30 s/read for 30 min. At least a repetition of each test3 times. Montelukast IC was performed on the measured data using non-linear regression (sigmoidal) in GraphPad Prism 5 software50Fitting.
The experimental results are as follows:
the half inhibitory concentrations of montelukast on recombinant FXIa and other 9 homologous serine proteases are shown in table 1. As can be seen from Table 1, the half-inhibitory constant of Montelukast on FXIa is 0.17. + -. 0.04. mu.M, the half-inhibitory concentration on plasma kallikrein and tissue plasminogen activator is 26.7. + -. 0.5. mu.M and 6.1. + -. 0.3. mu.M, respectively, and at a concentration of 100. mu.M, it has no significant inhibitory effect on other 7 serine proteases including thrombin and coagulation factors Xa, XIIa, VIIa, etc. (IC50 >100 μ M). As can be seen from the combination of FIG. 1, montelukast has a remarkable concentration-dependent inhibition effect on the catalytic activity of recombinant FXIa.
TABLE 1 half inhibitory concentration of Montelukast on FXIa and homologous serine proteases
Example 2: in vitro thrombolysis experiment of montelukast
The experimental method comprises the following steps:
clot lysis experiments were performed in 96-well transparent plates and human blood was collected from healthy donors, collected in sodium citrate vacuum blood collection tubes and centrifuged to obtain clear plasma. 20 nM recombinant FXIa was preincubated with 0-25. mu.M montelukast in buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% DMSO) at 37 ℃ for 15 min, followed by addition of 25% (v/v) plasma and immediate addition of 10 mM CaCl2Coagulation is initiated. The formation and dissolution of the clot was then monitored in real time by measuring the absorbance at 405 nm with a microplate reader at 37 ℃. Finally, a clot coagulation curve was plotted using GraphPad Prism 5 software.
The experimental results are as follows:
as shown in figure 2, sodium citrate vacuum blood collection tube in the plasma with 10 mM CaCl2Post-coagulation, fibrin formation and 15 minutesA plateau is reached. The clotting plateau time was shortened to 5 minutes after addition of 20 nM recombinant FXIa. Whereas the time for blood coagulation to plateau was prolonged to 7, 9 or 14 minutes in the presence of 1, 5 or 25 μ M montelukast, respectively, demonstrating that montelukast can effectively inhibit the FXIa driven coagulation process.
Example 3: effect of Montelukast on electrically induced arterial thrombosis
The experimental method comprises the following steps:
ICR mice were randomly divided into six groups of 6 mice, and first gavage was performed, wherein montelukast and apixaban were administered in two gradients of 2 mg/kg and 10 mg/kg, respectively, and warfarin was administered in an amount of 4mg/kg, and a saline-administered group was used as a control. 3 hours after dosing, mice were anesthetized (1.5% sodium pentobarbital, 30 mg/kg, i.p.) and left common carotid arteries exposed, stimulated with 0.1 mA of current to disrupt the vessel wall, thereby forming a mixed thrombus in the vessel by a YLS-14B animal thrombometer, recording the rate of occlusion of carotid blood flow every 4 seconds by an infrared detector, and recording the average time to form an occlusive thrombus in the carotid arteries (occlusion rate of 95%).
The experimental results are as follows:
as shown in fig. 3, the carotid artery of the saline group mice was occluded within 82 seconds after the electrical stimulation, and the 2 mg/kg and 10 mg/kg montelukast groups extended the vessel occlusion time to 135 seconds and 202 seconds, respectively; the 4mg/kg warfarin group prolonged the vessel occlusion time to 160 seconds; the apixaban groups at 2 mg/kg and 10 mg/kg extended the vessel occlusion time to 145 seconds and 210 seconds, respectively. The experiment shows that the montelukast can effectively prolong the thrombus formation time in the carotid artery thrombus electrically stimulated model, and the anticoagulation effect of the montelukast is equivalent to that of apixaban.
Example 4: evaluation of the risk of hemorrhage due to montelukast by tail-breaking experiments
The experimental method comprises the following steps:
the divided administration was similar to the method in example 3, in that Kunming mice were randomly divided into six groups, each group containing 6 mice, and first gavage administration was performed, in which montelukast and apixaban were performed in two gradients of 10 mg/kg and 50 mg/kg, respectivelyWarfarin was administered at 4mg/kg, and a saline-administered group was used as a control. 3 hours after administration, the mice were anesthetized (1.5% sodium pentobarbital, 30 mg/kg, i.p.), and the tail of the mice was cut off 10 mm from the tip, immediately immersed in 10 mL of isotonic saline at 37 ℃, and the length of bleeding was recorded after stopping bleeding. The amount of blood lost was quantified by measuring the amount of hemoglobin collected in 10 mL of isotonic saline, collecting the red blood cells after centrifugation at 1500 rpm, and lysing with 2 mL of lysis buffer (8.3 g/L NH)4Cl, 1 g/L KHCO30.037 g/L EDTA) and finally the absorbance at 570 nm of each sample was determined by a microplate reader.
The experimental results are as follows:
as shown in FIG. 4, the amount of bleeding did not increase significantly in the groups of 10 mg/kg and 50 mg/kg montelukast compared to the saline group, which was only 1.36 and 1.32 times that of the saline group. The 4mg/kg warfarin group had a bleeding amount 3.72 times that of the physiological saline group. The bleeding amounts of the apixaban groups of 10 mg/kg and 50 mg/kg were 2.88 and 3.44 times those of the saline group. The experimental results show that compared with warfarin and apixaban used clinically, the risk of hemorrhage caused by montelukast is obviously lower.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (3)
1. Application of montelukast in preparing medicines for preventing and treating thrombotic diseases.
2. The use of claim 1, wherein: the medicine for preventing and treating the thrombotic diseases is any pharmaceutically acceptable dosage form prepared by taking montelukast or pharmaceutically acceptable salts thereof as a medicinal active ingredient.
3. Use according to claim 2, characterized in that: the pharmaceutically acceptable dosage forms comprise tablets, capsules, granules, oral liquid or injections.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113229213A (en) * | 2021-05-14 | 2021-08-10 | 福州大学 | Method for realizing pulmonary embolism modeling and noninvasive quantitative detection by marking thrombus with near-infrared fluorescent probe |
| CN113243338A (en) * | 2021-05-14 | 2021-08-13 | 福州大学 | Construction and evaluation method of mouse ischemic stroke model |
-
2020
- 2020-11-23 CN CN202011322512.1A patent/CN112274510A/en active Pending
Non-Patent Citations (2)
| Title |
|---|
| JEROME DUCHEMIN等: "Acquired thrombopathia related to montelukast therapy", 《THROMB HAEMOST》 * |
| RENE SCHMIDT等: "Effect of montelukast on platelet activating factor- and tachykinin induced mucus secretion in the rat", 《JOURNAL OF OCCUPATIONAL MEDICINE AND TOXICOLOGY》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113229213A (en) * | 2021-05-14 | 2021-08-10 | 福州大学 | Method for realizing pulmonary embolism modeling and noninvasive quantitative detection by marking thrombus with near-infrared fluorescent probe |
| CN113243338A (en) * | 2021-05-14 | 2021-08-13 | 福州大学 | Construction and evaluation method of mouse ischemic stroke model |
| CN113229213B (en) * | 2021-05-14 | 2022-06-14 | 福州大学 | Method for realizing pulmonary embolism modeling and noninvasive quantitative detection by marking thrombus with near-infrared fluorescent probe |
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