CN108703971B - Application of anti-HIV (human immunodeficiency virus) medicament, namely, cobicistat, in preparation of antithrombotic medicament - Google Patents

Abstract

An anti-HIV drug, cobistat, is used in the preparation of an antithrombotic drug, and the drug can effectively inhibit platelet aggregation and inhibit artificial blood vessel thrombus formation in vitro. The present invention relates to drug cobisstat, which has the effect of inhibiting New Zealand rabbit platelet aggregation induced by platelet inducer ADP and thrombosis in vitro of artificial blood vessels; itself can effectively inhibit APJ endogenous ligands Elabela, Apelin-12, Apelin-13 and Apelin-13 36-induced platelet aggregation in New Zealand rabbits and in vitro thrombosis of artificial blood vessels. Cobistat is used as a new type of anti-platelet aggregation and thrombosis drug in the basic research of thrombotic diseases.

Description

Use of anti-HIV drug cobistat for preparing antithrombotic drug

technical field

The invention relates to the drug cobistat, which itself can effectively inhibit platelet aggregation and in vitro thrombus formation induced by the procoagulant ADP; it also inhibits the platelet-promoting effect of APJ endogenous ligands Elabela, Apelin-12, Apelin-17 and Apelin-36 Aggregation, in vitro thrombosis.

Background technique

APJ receptor is a transmembrane G protein-coupled receptor with high homology to angiotensin type Ⅰ receptor, and Apelin is its endogenous ligand. The human Apelin gene is located on the q25-26 segment of the X chromosome, and its encoded product is a precursor peptide containing 77 amino acids, which can be cleaved into active fragments of different amino acid lengths by peptidases: Apelin-12, Apelin-17, and Apelin-36. The Apelin/APJ system is widely distributed in tissues and expressed in cardiovascular, brain and kidney systems. ELABELA is a new endogenous ligand of APJ receptor discovered in 2013. The mature ELABELA contains 32 amino acids and has high homology with each subtype of Apelin. Apelin has the functions of lowering blood pressure and enhancing heart contraction in cardiovascular regulation; it has the functions of regulating pituitary hormone release, regulating food intake, water intake, and body fluid balance in the central nervous system; it can also promote angiogenesis, development and Mature. APJ receptors are regarded as new targets for the treatment of various diseases, and blockers or agonists targeting APJ receptors may become new therapeutic drugs for related diseases.

Thrombotic disease is a kind of disease caused by the two pathological processes of thrombosis and thromboembolism. Excessive activation or aggregation of platelets in the body will accelerate the occurrence of thrombus formation and thrombotic disease. Platelet aggregation is an important link in the formation of thrombosis and thrombotic diseases, and the natural components in the body's coagulation system (such as: ADP, collagen, arachidonic acid, thrombin, etc.) can act on various receptors on the platelet membrane to regulate Platelet function. Among them, ADP activates the ADP receptor on the platelet membrane, inhibits the activity of ATPase to cause platelet aggregation; thrombin, as a key enzyme in the coagulation process, plays a role in the injured blood vessel; collagen activates the platelet to make it adhere to the wound, and Promote the further release of active substances such as ADP and promote platelet aggregation; arachidonic acid can promote the release of thromboxane A2 and accelerate platelet aggregation and thrombus formation. Previous studies have found that APJ receptor endogenous ligands ELABELA, Apelin-12, Apelin-17 and Apelin-36 can effectively induce platelet aggregation in New Zealand rabbits and the in vitro thrombosis of artificial simulated blood vessels, suggesting that APJ receptors regulate platelet aggregation and an important target for thrombosis. Therefore, accelerating the development of drugs targeting APJ receptor blockers or agonists is of great significance for the treatment of thrombotic diseases.

Cobisistat is an anti-HIV synergist approved by the US FDA in 2012. The drug itself has no anti-HIV activity, but it can improve the blood concentration and pharmacokinetic parameters of anti-HIV drugs by inhibiting the main enzyme that metabolizes drugs in the human body-CYP3A, thereby improving the efficacy of the drug. In 2015, the FDA approved the anti-HIV drug Atazanavir and Cobisistat (Atazanavir-Cobisistat) compound preparation for marketing, which is used in combination with other antiretroviral drugs to treat adult patients with HIV-1 infection. At present, there is no report on whether cobistat has new targets and functions; whether it will participate in the regulation of platelet function other than anti-HIV and the treatment of thrombotic diseases. In addition, the human dose of cobistat as an anti-HIV drug is generally 15 mg/kg, and the maximum concentration used on platelets in this study is 1 μmol/ml, which is far less than the human dose after conversion.

This patent mainly discusses the functional research of cobistat, platelet aggregation and thrombus formation. It was found that cobistat can inhibit the platelet aggregation and in vitro thrombus formation induced by the coagulant ADP; at the same time, it can effectively inhibit the platelet aggregation and in vitro thrombosis of APJ endogenous ligands Elabela and Apelin subtypes.

Contents of the invention

The invention relates to the drug cobistat, which has the effect of inhibiting platelet aggregation and in vitro thrombus formation in New Zealand rabbits induced by a platelet inducer ADP;

The present invention has discovered the new pharmacological function of cobisstat, which itself can effectively inhibit the platelet aggregation of New Zealand rabbits induced by APJ endogenous ligands Elabela, Apelin-12, Apelin-13 and Apelin-36 and the in vitro thrombosis of the artificial blood vessel model effect;

The invention relates to a drug cobistat; it is used for preventing and improving platelet aggregation function; or the drug is expected to treat thrombotic diseases caused by platelet aggregation.

Beneficial technical effect of the present invention

The drug cobistat of the present invention has an ideal anti-platelet aggregation effect, therefore, it provides evidence for the side effects of anti-HIV treatment, and will be effectively used in the treatment of platelet aggregation and thrombosis diseases.

Description of drawings:

Figure 1A shows that cobistat alone cannot induce the platelet aggregation rate of New Zealand rabbits

Figure 1B shows that cobistat inhibits ADP-induced platelet aggregation

Figure 2 shows that cobicistat inhibits platelet aggregation induced by various subtypes of Apelin

Figure 3. Cobistat inhibits ADP-induced thrombosis in artificial blood vessels in vitro

Figure 4 shows that cobistat inhibits Elabela-induced artificial blood vessel thrombosis in vitro

Figure 5 shows that cobistat inhibits Apelin-12-induced thrombosis in artificial blood vessels in vitro

Figure 6 shows that cobistat inhibits Apelin-17-induced thrombosis in artificial blood vessels in vitro

Figure 7 shows that cobicistat inhibits Apelin-36-induced artificial blood vessel thrombosis in vitro

in:

In Figure 2, A is Apelin12, B is Apelin17, C is Apelin36 and D is Elabela;

In Fig. 3-Fig. 7, A is the size of the thrombus formed in vitro, B is the wet weight, C is the dry weight, and D is the length of the thrombus.

Detailed ways

Preparation Example 1

Test its function, as shown in the following example. The following implementations are used to illustrate the present invention, but should not be used to limit the scope of the present invention.

Example 1 Determination of the impact of cobistat on platelet aggregation

The platelet aggregation rate was measured with a platelet aggregation coagulation factor analyzer (LG-PABER-I, Beijing Shidi Scientific Instrument Company). The instrument adopts photoelectric turbidimetry to test platelet aggregation: platelet poor plasma (PPP) is used as the substrate, and platelet rich plasma (PRP) is used for measurement. Under the stirring of the magnetic beads, the inducer is added to the PRP, the platelets aggregate, and the transmittance of the PRP increases or the turbidity decreases. The change of light and turbidity is converted into the change of electrical signal, so as to calculate the aggregation rate of platelets.

Aggregation rate = (measured voltage value - PPP photoelectric voltage value) / (PRP photoelectric voltage value - PPP photoelectric voltage value) * 100%

1. Determination of the effect of different concentrations of cobistat on platelet aggregation

Blood was collected from the middle artery of New Zealand rabbit ears, anticoagulated with sodium citrate, and then centrifuged, first centrifuged at 800r/min for 10min, and the supernatant was obtained to obtain PRP, and then centrifuged at 3000r/min for 10min, and the supernatant was obtained to obtain PPP. Accurately add 300μl of PPP into the test cup, put it into the test channel, press the PPP key to measure the substrate and take it out. In another test cup, accurately add 300 μl of PRP. After prewarming at 37°C for 1 min, use a bead adder to add 1 test bead into the test cup. After starting the test, add different concentrations ( 0.001, 0.01, 0.1, 1 μmol/ml) can be tested for 5 minutes with bisstat, and record the maximum aggregation rate of platelets (see Figure 1A).

2. Determination of cobistat's inhibition of platelet aggregation induced by procoagulant ADP

Collect PPP and PRP as above, add coagulant ADP to PRP and incubate for 5 minutes, then put it into the test area where the baseline has been adjusted, add test beads, after starting the test, add cobisstat for 5 minutes within three seconds, and record platelets Observe the test results to detect the effect of cobistat on ADP-promoted platelet aggregation in New Zealand rabbits. Based on this, the platelet aggregation inhibiting effect of cobistat was judged (see FIG. 1B ).

3. Determination of different concentrations of cobistat on Apelin-induced platelet aggregation in New Zealand rabbits

Collect PPP and PRP as above, add APJ receptor endogenous ligands ELABELA, Apelin-12, Apelin-17 and Apelin-36 to PRP and incubate for 5 minutes, then put them into the test area where the baseline has been adjusted, and add test beads After starting the test, add the drug Cobistat for 5 minutes within three seconds, record the maximum aggregation rate of platelets, and observe the test results to detect that Cobistat can promote platelet aggregation in New Zealand rabbits on ELABELA, Apelin-12, Apelin-17 and Apelin-36 effect. Based on this, the platelet aggregation inhibiting effect of cobistat was judged (see Figure 2).

Example 2 Measuring the Effect of Cobistat on Thrombosis in Vitro

The thrombus model in vitro was established with a thrombus detector (LMK-12, Zhengzhou Mingju Technology Co., Ltd.). This instrument uses the chandler method: blood is injected into a rotating ring outside the body to simulate the blood flow state in the body to form thrombus.

1. Determination of the effect of cobistat on inhibiting the thrombus formation of artificial blood vessels induced by the coagulant ADP in vitro

New Zealand rabbit ear middle artery blood collection (without any anticoagulant, 1ml of venous blood was taken directly, and the bubble in the syringe should be removed, and different concentrations (0.001, 0.01, 0.1, 1 μmol/ml) of cobistat and After being treated with 10μmol/ml coagulant ADP, the blood sample is filled into the ring, and 1m1 blood sample is slowly injected into the tube along one end of the plastic ring, and connected to form a ring at any time, and placed in the corresponding turntable. The middle speed is 20 ±2rpm, at 37°C, after 10 minutes of circulation, take out the thrombus, measure and record the wet weight and length of the thrombus. Put the above-mentioned thrombus whose wet weight has been weighed into an oven at 60°C±1°C for drying treatment, and after 30 minutes , weigh the dried thrombus again, and record the dry weight of the thrombus (see Figure 3).

2. Determination of the effects of different concentrations of cobistat on the thrombosis of artificial blood vessels induced by Apelin subtypes in vitro

The in vitro thrombus model was constructed by the Chandler method. Different concentrations (0.001, 0.01, 0.1, 1 μmol/ml) of cobistat were mixed with 1.0 μmol/ml ELABELA (see Figure 4), 1.0 μmol/ml Apelin-12 (see Figure 5), 10-4 μmol/ml Apelin-12, respectively. 17 (see Figure 6) and 0.01 μmol/ml Apelin-36 (see Figure 7) were added to 1ml of New Zealand rabbit blood. According to the test method in 1, take out the thrombosis and record the experimental results. And observe the effect of cobistat on rabbit thrombus formation induced by procoagulant ADP (see FIG. 5 ).

In summary, the drug Cobistat of the present invention has an ideal anti-platelet aggregation effect, therefore, it provides evidence for the side effects of its anti-HIV treatment, and will be effectively used in the treatment of platelet aggregation and thrombotic diseases.

Claims (8)

1. The use of an anti-HIV drug cobistat for the preparation of antithrombotic drugs, characterized in that it is used for the treatment of thrombotic diseases. 2. The use of the anti-HIV drug cobistat according to claim 1 for the preparation of antithrombotic drugs, characterized in that it is used for the treatment of thrombotic diseases caused by platelet aggregation. 3. The use of the anti-HIV drug cobistat according to claim 1 for the preparation of antithrombotic drugs, characterized in that it is used to inhibit platelet aggregation induced by the platelet inducer ADP. 4. The anti-HIV drug cobistat according to claim 1 is used for the preparation of antithrombotic drugs, which is characterized in that it is used to inhibit the induction of APJ endogenous ligands Elabela, Apelin-12, Apelin-13 and Apelin-36 platelet aggregation. 5. The use of the anti-HIV drug cobistat according to claim 1 for the preparation of antithrombotic drugs, characterized in that it is used for the treatment of thrombosis. 6. The use of the anti-HIV drug cobistat according to claim 1 for the preparation of antithrombotic drugs, which is characterized in that it is used to inhibit the in vitro thrombus formation induced by the platelet inducer ADP. 7. The anti-HIV drug cobistat according to claim 1 is used for the preparation of antithrombotic drugs, which is characterized in that it is used to inhibit the induction of APJ endogenous ligands Elabela, Apelin-12, Apelin-13 and Apelin-36 thrombosis in vitro. 8. The use of any one of the anti-HIV drugs Cobistat in claims 1-7 for the preparation of antithrombotic drugs, characterized in that it is used for treating thrombotic diseases caused by platelet aggregation; or preventing platelet aggregation in the body.

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