CN108703971B

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

Info

Publication number: CN108703971B
Application number: CN201810776447.6A
Authority: CN
Inventors: 陈临溪; 李兰芳; 胡昊良; 何璐; 伍乐乐; 刘佳琪
Original assignee: University of South China
Current assignee: University of South China
Priority date: 2017-07-11
Filing date: 2018-07-10
Publication date: 2023-04-07
Anticipated expiration: 2038-07-10
Also published as: CN108703971A

Abstract

Use of an anti-HIV drug cobicistat for the preparation of an antithrombotic drug effective in inhibiting platelet aggregation and in inhibiting in vitro thrombosis of an artificial blood vessel. The invention relates to a medicine which can inhibit platelet inducer ADP induced new Zealand rabbit platelet aggregation and artificial blood vessel in vitro thrombosis; the compound can effectively inhibit platelet aggregation of New Zealand rabbits induced by APJ endogenous ligands Elabela, apelin-12, apelin-13 and Apelin-36 and the thrombus formation in vitro of artificial blood vessels. The cobicistat is researched on the basis of thrombotic diseases to be used as a novel medicament for resisting platelet aggregation and thrombosis.

Description

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

Technical Field

The invention relates to a medicine which can be used for inhibiting platelet aggregation and in-vitro thrombosis induced by coagulation accelerator ADP effectively; it also inhibits platelet aggregation and in vitro thrombosis of APJ endogenous ligands Elabella, apelin-12, apelin-17 and Apelin-36.

Background

The APJ receptor is a transmembrane G protein coupled receptor with high homology with an angiotensin I type receptor, and Apelin is an endogenous ligand of the receptor. The human Apelin gene is located in the q25-26 segment of the X chromosome, and the encoded product is a precursor peptide containing 77 amino acids, which can be cleaved by peptidase into active fragments of different amino acid lengths: apelin-12, apelin-17, apelin-36, and the like. The Apelin/APJ system is widely distributed in tissues and is expressed in cardiovascular, brain, kidney and other systems. The ELABELA is a novel endogenous ligand of an APJ receptor discovered in 2013, and the mature ELABELA contains 32 amino acids and has high homology with various Apelin subtypes. Apelin has the functions of reducing blood pressure and enhancing cardiac contraction in cardiovascular regulation; has effects in regulating pituitary hormone release, regulating ingestion, water intake, and fluid balance in central nervous system; can also promote angiogenesis, development and maturation in the aspect of blood vessel function regulation. The APJ receptor is regarded as a novel target for treating various diseases, and a blocker or an agonist targeting the APJ receptor can become a novel therapeutic drug for related diseases.

Thrombotic diseases are diseases caused by two pathological processes of thrombosis and thromboembolism, and excessive activation or aggregation of platelets in vivo can accelerate the occurrence of thrombosis and thrombotic diseases. Platelet aggregation is an important link in the formation of thrombus and thrombotic diseases, and natural components (such as ADP, collagen, arachidonic acid, thrombin and the like) in the blood coagulation system of the body can act on various receptors on platelet membranes to regulate the functions of platelets. Wherein, ADP activates ADP receptor on platelet membrane, and inhibits ATPase activity to cause platelet aggregation; thrombin, a key enzyme in the coagulation process, acts at injured blood vessels; the collagen activates the blood platelet to be adhered to the wound, and promotes ADP and other active substances to be further released and promote the blood platelet aggregation; arachidonic acid promotes the release of thromboxane A2 to accelerate platelet aggregation and thrombosis. The early research finds that: the 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, and the APJ receptor is an important target point for regulating and controlling platelet aggregation and thrombosis. Therefore, the development of drugs for blocking agents or agonists targeting the APJ receptor is accelerated, and the drugs have great significance for the treatment of thrombotic diseases.

Cobicistat (Cobisistat) is a synergist approved by FDA in us to be marketed against HIV in 2012. The medicine has no anti-HIV activity, but can improve the blood concentration and the pharmacokinetic parameters of the anti-HIV medicine by inhibiting CYP3A which is a main enzyme of the metabolism medicine in a human body, thereby improving the medicine effect. In 2015, the FDA approved anti-HIV drug Atazanavir and Cobisstat (Atazanavir-costastat) compound preparation to be marketed, and combined with other anti-retroviral drugs for the treatment of HIV-1 infected adult patients. At present, whether the comparastat has a new target point and a new function or not is judged; whether the polypeptide can be involved in platelet function regulation and thrombotic disease treatment besides HIV resistance is not reported at present. In addition, the human dose of Cobicistrit used as an anti-HIV drug is generally 15mg/kg, and the maximum concentration used on blood platelets in the study is 1 mu mol/ml, which is far less than the human dose after conversion.

This patent mainly discusses the functional research of comparastat and platelet aggregation and thrombosis. It was found that cobicistat inhibits coagulant ADP-induced platelet aggregation and in vitro thrombosis; and simultaneously effectively inhibits platelet aggregation and in-vitro thrombosis of various subtypes of APJ endogenous ligands Elabella and Apelin.

Disclosure of Invention

The invention relates to a medicine, namely cobicistat, which has the functions of inhibiting platelet aggregation and in-vitro thrombosis of New Zealand rabbit induced by platelet inducer ADP;

the invention discovers a new pharmacological function of the comparasin, and the comparasin can effectively inhibit 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 a human blood vessel model;

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

The invention has the beneficial technical effects

The medicament of the invention can be used for treating platelet aggregation and thrombotic diseases effectively.

Description of the drawings:

FIG. 1A shows that cobicistat alone does not induce platelet aggregation in New Zealand rabbits

FIG. 1B shows that Coxstat inhibits ADP-induced platelet aggregation

FIG. 2 is a graph showing that costab inhibits Apelin subtype-induced platelet aggregation

FIG. 3 is a graph showing that cobicistat inhibits ADP-induced thrombosis in artificial blood vessels in vitro

FIG. 4 is a graph showing that cobicistat inhibits Elabela-induced thrombosis in vitro in an artificial blood vessel

FIG. 5 is a graph showing that Coxstat inhibits Apelin-12-induced thrombosis in vitro in an artificial blood vessel

FIG. 6 is a graph showing that Coxstat inhibits Apelin-17-induced thrombosis in vitro in an artificial blood vessel

FIG. 7 is a graph showing that Coxstat inhibits Apelin-36-induced thrombosis in vitro in an artificial blood vessel

Wherein:

in FIG. 2, A is Apelin12, B is Apelin17, C is Apelin36 and D is Elabela;

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

Detailed Description

Preparation example 1

The function of which is tested, as shown in the examples below. The following examples are intended to illustrate the invention, but are not intended to limit the scope of the invention.

Example 1 determination of the Effect of Coxsitabine on platelet aggregation

The platelet aggregation rate was measured by a platelet aggregation clotting factor analyzer (model LG-PABER-I, peking Shidi scientific instruments). The instrument adopts a photoelectric turbidimetry method to test platelet aggregation: measurements were performed using Platelet Poor Plasma (PPP) as a substrate, using Platelet Rich Plasma (PRP). Under the stirring of magnetic beads, an inducer is added into PRP, so that platelet aggregation occurs, and the transmittance of PRP is increased or turbidity is reduced. The change of the light turbidity is converted into the change of the electric signal, so that the aggregation rate of the blood platelets is calculated.

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

1. Determination of the Effect of Coxstat at different concentrations on platelet aggregation

Collecting blood from middle artery of New Zealand rabbit, anticoagulating with sodium citrate, centrifuging at 800r/min for 10min, collecting supernatant to obtain PRP, centrifuging at 3000r/min for 10min, and collecting supernatant to obtain PPP. And accurately adding 300 mu l of PPP into the test cup, putting the PPP into a test channel, pressing a PPP key to measure the substrate, and taking out the PPP. In another test cup, 300. Mu.l of PRP was added precisely, 1 test bead was added to the test cup using a bead filler after 1min of pre-warming at 37 ℃, and after starting the test, different concentrations (0.001, 0.01, 0.1, 1. Mu. Mol/ml) of Costutzer were added within three seconds, respectively, for 5min, and the maximum aggregation rate of platelets was recorded (see FIG. 1A).

2. Determination of the inhibition of the procoagulant ADP-induced platelet aggregation by Cobicistat

And (3) collecting PPP and PRP, adding a coagulant ADP into the PRP, incubating for 5min, placing into a test area with a regulated baseline, adding test beads, adding the comparastat to test for 5min within three seconds after the test is started, recording the maximum aggregation rate of platelets, and observing a test result to detect the platelet aggregation effect of the comparastat on the ADP-promoted New Zealand rabbit. Thus, the platelet aggregation-inhibiting effect of Coxseed was judged (see FIG. 1B).

3. Determination of the platelet aggregation induced by Coxstatin at different concentrations on various subtypes of Apelin in New Zealand rabbits

Collecting PPP and PRP, respectively adding APJ receptor endogenous ligands ELABELA, apelin-12, apelin-17 and Apelin-36 into PRP, incubating for 5min, placing into a test area with a regulated baseline, adding test beads, adding Coxstar for testing for 5min within three seconds after starting testing, recording the maximum aggregation rate of platelets, and observing the test result to detect the platelet aggregation promotion effect of Coxstar on ELABELA, apelin-12, apelin-17 and Apelin-36. Thus, the platelet aggregation-inhibiting effect of Coxseed was judged (see FIG. 2).

Example 2 measurement of the Effect of Coxsitabine on in vitro thrombosis

An in vitro thrombus model was established using a thrombus detector (LMK-12, zhengzhou Mingtiao scientific Co., ltd.) using the chandler method: blood is injected into the external rotating ring to simulate the flowing state of the blood in vivo so as to form thrombus.

1. Determination of the Effect of Cobicistat in inhibiting coagulant ADP-induced thrombosis in vitro in an artificial blood vessel

Taking blood from middle artery of rabbit ear in New Zealand (without adding any anticoagulant, taking 1ml venous blood directly, and paying attention to remove bubbles in a syringe, treating the blood with different concentrations (0.001, 0.01, 0.1 and 1 mu mol/ml) of comparatives and 10 mu mol/ml of coagulant ADP, filling the blood into a circular ring, slowly injecting 1m1 blood into the tube along one end wall of the plastic ring, connecting the blood into the circular ring at any time, placing the circular ring in a corresponding rotary disc, placing the circular ring at a medium rotating speed of 20 +/-2 rpm, circulating the circular ring for 10min at 37 ℃, taking out the thrombus, measuring and recording the wet weight and length of the thrombus, placing the thrombus after being weighed out the wet weight into an oven at 60 +/-1 ℃ for drying, weighing the dried thrombus again after 30min, and recording the dry weight of the thrombus (see figure 3).

2. Determining the influence of different concentrations of Coxstat on the in vitro thrombosis of artificial blood vessels induced by various subtypes of Apelin

An in vitro thrombus model is constructed by adopting a chandler method. The thrombotic disease recording assay results were taken as described in1 by adding different concentrations (0.001, 0.01, 0.1, 1. Mu. Mol/ml) of comparasin together with 1.0. Mu. Mol/ml ELABELA (see FIG. 4), 1.0. Mu. Mol/ml Apelin-12 (see FIG. 5), 10-4. Mu. Mol/ml Apelin-17 (see FIG. 6) and 0.01. Mu. Mol/ml Apelin-36 (see FIG. 7), respectively, to 1ml of blood of New Zealand rabbits according to the test method in 1. And the effect of colchicine on the procoagulant ADP-induced thrombosis in rabbits was observed (see FIG. 5).

In conclusion, the medicine of the invention can be compared with other medicines and has ideal platelet aggregation resisting effect, so that a basis is provided for resisting the side effect of HIV treatment, and the medicine can be effectively used for treating platelet aggregation and thrombotic diseases.

Claims

1. Use of an anti-HIV drug cobicistat for the preparation of an antithrombotic drug characterized by being useful for the treatment of thrombotic disorders.

2. The use of the anti-HIV drug, cobicistat, according to claim 1 for the preparation of an antithrombotic drug characterized by being useful in the treatment of thrombotic disorders caused by platelet aggregation.

3. Use of the anti-HIV drug, colbicitrat, according to claim 1, for the preparation of an antithrombotic drug characterized by being useful for inhibiting platelet aggregation induced by the platelet inducer ADP.

4. The use of the anti-HIV drug, cobicistat, according to claim 1 for the preparation of an antithrombotic drug characterized by inhibiting platelet aggregation induced by the APJ endogenous ligands Elabela, apelin-12, apelin-13 and Apelin-36.

5. Use of the anti-HIV drug, cobicistat, according to claim 1, for the preparation of an antithrombotic drug characterized by being useful in the treatment of thrombosis.

6. Use of the anti-HIV drug, cobicistat, according to claim 1 for the preparation of an antithrombotic drug characterized by being useful for inhibiting platelet inducer ADP-induced in vitro thrombosis.

7. The use of the anti-HIV drug, cobicistat, according to claim 1, for the preparation of an antithrombotic drug characterized by being useful for inhibiting in vitro thrombosis induced by the APJ endogenous ligands Elabela, apelin-12, apelin-13 and Apelin-36.

8. Use of the anti-HIV agent cobicistat according to any one of claims 1-7 for the preparation of an antithrombotic agent characterized by being useful in the treatment of thrombotic disorders caused by platelet aggregation; or preventing platelet aggregation in the body.