CN106924273B

CN106924273B - Application of Militarine in preparation of heparin antagonist drugs

Info

Publication number: CN106924273B
Application number: CN201710099695.7A
Authority: CN
Inventors: 廖尚高; 何迅; 王永林; 赵菲菲; 李靖; 周孟; 徐国波; 关焕玉; 李勇军; 兰燕宇; 黄勇
Original assignee: Guizhou Medical University
Current assignee: Guizhou Medical University
Priority date: 2017-02-23
Filing date: 2017-02-23
Publication date: 2020-04-07
Anticipated expiration: 2037-02-23
Also published as: CN106924273A

Abstract

The invention discloses a new application of Militarine in the preparation of heparin antagonist drugs. The molecular formula of Militarine is C₃₄H₄₆O₁₇The chemical name is bis [4- (β -D-glucopyranosyloxy) benzyl]-2-isobutyl malate. The Militarine provided by the invention can be prepared into pharmaceutical dosage forms suitable for clinical use, and mainly comprises injections, granules, capsules, tablets, powder, dropping pills, sustained-release agents and the like. Has obvious heparin antagonism effect, similar or same pharmacological effect as protamine, no adverse reaction such as human body allergy, safety and no toxicity, can be developed into protamine substitute for clinical use, and has good application prospect.

Description

Application of Militarine in preparation of heparin antagonist drugs

Technical Field

The invention relates to application of effective components of traditional Chinese medicinal materials, in particular to novel application of Militarine in preparation of heparin antagonist drugs.

Background

With the development of surgical techniques, heparin is applied more and more frequently in operations, and is mainly used for thromboembolic diseases, myocardial infarction, cardiovascular operations, cardiac catheter examination, extracorporeal circulation, hemodialysis and the like in clinic, and is particularly more commonly used in the field of cardiac surgery. Meanwhile, the use of heparin easily causes spontaneous hemorrhage which is manifested by various mucosal hemorrhage, hemarthrosis, wound hemorrhage and the like, and when the dosage is seriously excessive, the life of a patient is also endangered, and at the moment, a heparin antidote is required to be injected.

Protamine (protamine) in the existing drugs has played an irreplaceable role in surgery since 1937 as the sole antidote for heparin. The reversion of the anticoagulation effect after heparin operation is an important milestone in surgical history, and the protamine is often prepared into injection for clinical use, but protamine has inevitable side effects and adverse reactions, for example, the rapid intravenous injection of the protamine can cause hypotension, bradycardia, pulmonary hypertension, dyspnea, transient facial flushing and warmth sensation; those who have received protamine or protamine-containing insulin (e.g., neutral protamine insulin) in the past are prone to developing anti-protamine IgE-mediated hypersensitive or allergic reactions; some of the male infertility or vasectomized patients are prone to hypersensitive reactions to protamine.

Meanwhile, protamine is an alkaline protein obtained by crushing and separating the head part of fish sperms, has certain biological activity, can only be extracted from the fish sperms in production, cannot be chemically synthesized, and limits the yield. The phenomenon of shortage often appears in the market. At present, protamine which is a frequently-prepared medicine for cardiac surgery is in a shortage state in hospitals, even the shortage of goods in China is avoided, so that many patients cannot normally perform surgery, and the recovery of the patients is seriously influenced.

Therefore, the development of a protamine substitute which is safe, has outstanding effects, extremely high clinical application value and huge market demands becomes urgent for the development of current drugs.

Disclosure of Invention

Aiming at the defects of the prior art, the application of Militarine in preparing heparin antagonist medicines, namely the new application in pharmacy is provided.

The technical scheme for realizing the invention is as follows: the application of Militarine in the preparation of heparin antagonist drugs.

Preferably, the heparin antagonist drug contains other pharmaceutical excipients or carriers.

Preferably, the pharmaceutical dosage form of the heparin antagonist comprises injection, granules, capsules, tablets, powder, dripping pills and sustained release preparations.

Preferably, the mode of administration is oral administration or injection administration.

Use of Militarine for the manufacture of a medicament for reversing the anticoagulant effect of heparin in surgery.

Compared with the prior art, the invention has the beneficial effects that: the Militarine is a monomeric compound separated from rhizoma bletillae medicinal materials which have clinical application history for many years, are safe and effective, is a pure natural component, has obvious heparin antagonism effect through pharmacodynamic verification, has similar pharmacological effect to protamine, does not cause adverse reactions such as human allergy and the like, is safe and low-toxic, and can be developed into a protamine substitute for clinical application.

Drawings

FIG. 1 shows the effect of different concentrations of Militarine on CT and BT in whole-body heparinized mice;

FIG. 2 is a graph of the effect of Militarine at various time points on whole-body heparinized mouse CT;

FIG. 3 is a graph of the effect of different concentrations of Militarine on systemic heparinization of PT, TT, FIB, PL;

FIG. 4 is a graph showing the effect of various concentrations of Militarine on systemic heparinized P-selectin, TXB2, 6-keto-PGF1 α.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The Militarine adopted in the embodiment is extracted from plant bletilla striata or obtained by synthesis, and the molecular formula of the Militarine is C₃₄H₄₆O₁₇The chemical name is bis [4- (β -D-glucopyranosyloxy) benzyl]-2-isobutyl malate.

The chemical structure is as follows:

the prepared medicine can contain a medicine excipient or a carrier, and is used for antagonizing the effect of heparin for clinical application; the medicine can be prepared into pharmaceutical preparations suitable for clinical use, including injections, granules, capsules, tablets, powders, dripping pills and sustained-release agents; the administration form of the preparation mainly comprises oral administration or injection administration.

In order to show that the Militarine has good effects in the aspect of preparing heparin antagonist drugs, the effects obtained by the Militarine are further explained by combining specific experiments.

First, the effect of different concentrations of Militarine on CT and BT in whole-body heparinized mice is shown in FIG. 1.

Generally, the mouse tail-breaking Bleeding Time (BT) and capillary blood-Clotting Time (CT) experiments are a more classical method for screening the activity of hemostatic and procoagulant Chinese medicines, and can comprehensively reflect the functions of various factors participating in the hemostatic process, such as blood vessels, platelets, blood coagulation systems, and the like. Wherein BT means the time required by blood from natural outflow to natural stop under a certain condition, and can reflect the quantity and quality of platelets and the hemostatic function of capillary vessels; CT refers to the time during which factor xii is activated when blood contacts a negatively charged surface after being isolated, and then a series of coagulation factors are activated sequentially, and finally fibrinogen is changed into fibrin to coagulate blood, and the length of CT is mainly related to the content and function of various coagulation factors. According to the invention, by considering that the Militarine has no adverse reaction to normal mice and whole-body heparinized mice at 20, 40, 80 and 160mg/kg, the Militarine has low toxicity, high safety concentration and good safety. By examining the influence of Militarine on BT and CT of the whole-body heparinized mice, the Militarine is found to be capable of obviously shortening the BT and CT values (P <0.01 or 0.05) of the whole-body heparinized mice, wherein the BT shortening rate is 22.1%, 26.3% and 31.9%, and the CT shortening rate is 8.1%, 28.7% and 35.0%, respectively. The results show that Militarine can effectively antagonize the anticoagulation effect of heparin sodium and has obvious procoagulant effect on mice with pathological bleeding.

Second, the effect of Militarine on whole-body heparinized mouse CT at different time points is shown in fig. 2.

The results demonstrate that Militarine has a significant effect on heparin antagonism, but to further understand the onset and duration of Militarine, we examined the effect of Militarine on the Clotting Time (CT) in heparinized mice 0.5, 1, 3, 6, 12, and 24h post-dose, respectively. As a result, the CT value of the heparin sodium is increased and statistically different (P is less than 0.01 or 0.05) compared with the blank mice within the time from 0.5h to 12h, and after 24h, the CT value of the heparin sodium model group is not statistically different from that of the blank mice, and the anticoagulation effect disappears. And the Militarine shortens the CT value in 0.5h to 12h relative to the sodium heparin model group, and has statistical difference (P <0.01 or 0.05), 80mg/kg of Militarine can reduce the anticoagulation effect of the sodium heparin by 70% at 0.5h, the antagonism effect reaches 152% and 178% at 6h and 12h respectively, the CT value after 24h has no statistical difference compared with the sodium heparin model group, and has no statistical difference compared with the blank group mice (P > 0.05). The experimental result shows that when the in vivo heparin sodium has obvious anticoagulation effect, the Militarine can antagonize the anticoagulation effect of the in vivo heparin sodium, and when the anticoagulation effect of the heparin sodium disappears, the Militarine can not generate the procoagulant effect on the mouse to cause thrombotic diseases.

Thirdly, the effect of different concentrations of Militarine on APTT, PT, TT, FIB, PL in whole-body heparinized mice is shown in FIG. 3.

The coagulation process is generally divided into three pathways, the intrinsic, extrinsic and common coagulation pathways. The PT value mainly reflects extrinsic coagulation pathway, and can detect various coagulation factors in extrinsic coagulation pathway, if the factors are deficient after extrinsic coagulation pathway, the PT value is prolonged, and if the PT value is shortened, it reflects that the drug may enhance the activity of various coagulation factors after extrinsic coagulation pathway. APTT can be used in assays for determining coagulation factors in the intrinsic pathway, and can detect other plasma coagulation factors other than factor VII, with an increase in value reflecting a deficiency or a decrease in activity of coagulation factors IX, XI, XII, which decrease would suggest an increase in coagulation factor activity or the possible presence of procoagulant substances in the blood. The TT value mainly reflects whether abnormal anticoagulation exists in the process of converting fibrinogen into fibrin in the common pathway of blood coagulation. The FIB value reflects the fibrinogen level in vivo, and the FIB plays an important role in maintaining normal hemostasis and blood coagulation functions in vivo, can promote blood coagulation to stop bleeding, and can prevent plasminogen from being converted into fibrinolytic enzyme, so that fibrinolysis is prevented, and the hemostasis function is played. Plasmin (PL) is proteolytic enzyme capable of specifically degrading fibrin gel, is an important component in fibrinolytic system, and two systems of in vivo coagulation and fibrinolysis are closely related to each other, once the organism generates coagulation reaction, the fibrinolytic system is almost simultaneously activated, and the activated PL degrades in vivo plasma fibrinogen by negative feedback effect, so that excessive coagulation of fibrin is avoided, but excessive damage to blood balance can be caused, and blood is in a blood activating state. Experimental results prove that Militarine can shorten the TT value of a heparinized mouse, increase the FIB content, reduce the PL content in the plasma of the heparinized mouse, and has no obvious influence on the APTT and PT values. Indicating that Militarine can activate the coagulation-fibrinolysis system, regulate the content of certain coagulation fibrinolysis factors in vivo and antagonize the anticoagulation effect of heparin.

Fourth, the effect of different concentrations of Militarine on systemic heparinized mouse P-selectin, TXB2, 6-keto-PGF1 α, as shown in FIG. 4.

The platelet is a blood cell with plasma membrane, no nucleus and irregular shape, is a tangible component in blood, participates in many links of the hemostasis process, and plays an important role in the hemostasis process. P-selectin (P-S) is the most specific molecular marker for platelet activation, and platelets deform, adhere and are stimulated by stimuli,The aggregation and release reactions are positively correlated with the expression of its granulosa membrane protein P-S. TXA₂And PGI₂The dynamic balance between them is the basis for maintaining normal hemostatic function. TXA₂Is an important metabolite of arachidonic acid epoxidase, TXA₂The generation of the compound (A) starts from phospholipid on a platelet membrane, is generated under the catalysis of thromboxane synthetase in the platelet, has strong functions of aggregating the platelet and contracting blood vessels, and has the function of stopping bleeding; PGI₂Mainly generated in vascular endothelial cells, the platelet aggregation inhibitor is a strong platelet aggregation inhibitor and has the effects of inhibiting adhesion, aggregation and release reactions of platelets, inhibiting procoagulant activity of the platelets and the like; due to TXA₂And PGI₂Unstable and therefore frequently TXA₂And PGI₂Stable metabolite TXB₂And 6-keto-PGF_1αAs an index for determining the concentration thereof. Experimental results prove that Militarine can increase the content of P-S, TXB2 in heparinized mice, namely 6-keto-PGF1_αThe generation is reduced. Militarine has been shown to act on the platelet system to regulate or promote platelet release of certain procoagulant factors, thereby antagonizing the anticoagulant effect of heparin on blood.

The experimental conclusion proves that Militarine has obvious antagonistic action on the hemorrhage caused by heparin and shows good hemostatic action. Experimental results show that Militarine can activate platelet system, activate platelet membrane glycoprotein P-S, promote platelet release to induce aggregation factor TXB₂Inhibits the action of 6-keto-PGF1 a; activating blood coagulation fibrinolysis system, shortening TT value of mouse plasma, increasing FIB content in plasma, and inhibiting PL generation, thereby accelerating blood coagulation, inhibiting degradation of fibrin, shortening BT and CT value of mouse, and playing anticoagulant effect of antagonistic heparin on blood.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.

Claims

The application of Militarine in the preparation of heparin antagonist drugs.
2. The use of Militarine according to claim 1 for the preparation of a heparin antagonist comprising other pharmaceutical excipients or carriers.
3. The use of Militarine according to claim 1 or 2 for the preparation of a heparin antagonist in pharmaceutical dosage forms including injections, granules, capsules, tablets, powders, drop pills and sustained release formulations.
4. The use of Militarine according to claim 3 for the preparation of a heparin antagonist by oral or parenteral administration.
Use of Militarine for the preparation of a medicament for reversing the anticoagulant effect of heparin during surgery.