CN113274504A - Application of compound acting on choline or TMAO related target in preparation of drugs for preventing and/or reversing clopidogrel resistance - Google Patents
Application of compound acting on choline or TMAO related target in preparation of drugs for preventing and/or reversing clopidogrel resistance Download PDFInfo
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Abstract
The invention provides application of a compound acting on a choline or trimethylamine oxide (TMAO) related target in preparation of a medicament for preventing and/or reversing clopidogrel resistance, wherein the compound comprises one or more of an NOX inhibitor, an ROS scavenger, an Nrf2 inhibitor and an intestinal microorganism choline TMA lyase inhibitor. The invention systematically explores the influence of choline or TMAO on the metabolism activation and antiplatelet effect of clopidogrel and various intervention mechanisms thereof, and provides a brand-new treatment target and prevention strategy for effectively overcoming clopidogrel resistance associated with choline or TMAO in clinic.
Description
Technical Field
The invention belongs to the technical field of drug research and development, and particularly relates to application of a compound acting on a choline or trimethylamine oxide (TMAO) related target in preparation of a drug for preventing and/or reversing clopidogrel resistance.
Background
Clopidogrel (clopidogrel) is one of the antiplatelet therapy "primary drugs" selected by the World Health Organization (WHO) (Patel a et al, Circ cardio Qual outsomes 2015; 8: 447-51). Since the approval of FDA in the united states for marketing in 1997, clopidogrel is one of the most commonly used drugs for preventing thrombosis or reoccurrence of cardiovascular and cerebrovascular embolism or ischemic events in intravascular stents by clinicians in various countries, and "dual antiplatelet therapy" combined with aspirin is a widely accepted antiplatelet therapy regimen "gold standard". Clopidogrel itself is not pharmacologically active as a prodrug (produgs). After being absorbed into blood orally, about 85 percent of clopidogrel in blood is hydrolyzed and inactivated by liver carboxylesterase CES1, and the residual unhydrolyzed drug needs to be catalyzed by a series of P450 oxidases in liver to generate an active metabolite H4, and then the active metabolite H4 is combined with ADP receptor P2y12 on platelet membrane by H4, so that platelet activation and aggregation are induced by ADP in blood to play an antiplatelet role. Obviously, the induction or inhibition of the expression level of liver carboxylesterase CES1 protein and the activity of the hydrolase thereof are important for the influence of liver hydrolytic metabolism (inactivation) of clopidogrel and antiplatelet effect thereof.
"clopidogrel resistance" refers to a phenomenon in which, after taking standard dose of clopidogrel, a patient fails to achieve an expected antiplatelet effect for various reasons by weakening the metabolic activation of the drug in the liver and/or the reactivity of platelets to the drug, resulting in poor or ineffective clinical efficacy of clopidogrel to the patient. It is estimated that about 10% to 45% of patients taking drugs may have clopidogrel resistance, seriously affecting the drug treatment effect and prognosis of the patients. In clinical practice, patients diagnosed with clopidogrel resistance are at risk of reoccurrence of cardiovascular and cerebrovascular thrombosis or ischemic events, and physicians often switch to other more antiplatelet agents (e.g., ticagrelor). However, the same class of drugs with stronger antiplatelet effect is often associated with an increased risk of bleeding. Therefore, further and systematically researching the generation mechanism of clopidogrel resistance can provide a new target for drug therapy of clopidogrel resistance. Therefore, the screened new drug capable of effectively preventing clopidogrel resistance is one of the clinical treatment strategies for overcoming clopidogrel resistance.
Disclosure of Invention
In view of the above, the present invention aims to provide an application of a drug or a compound acting on a choline or TMAO-related target in the preparation of a drug for preventing and/or reversing clopidogrel resistance; the invention systematically explores the influence of choline or TMAO on the metabolic activation and antiplatelet effect of clopidogrel and various intervention mechanisms thereof, and provides a brand-new treatment target and prevention strategy for effectively overcoming clopidogrel resistance clinically.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides application of a medicine or compound acting on a choline or TMAO related target in preparation of a medicine for preventing and/or reversing clopidogrel resistance, wherein the medicine or compound acting on the choline or TMAO related target comprises one or more of an NOX inhibitor, an ROS scavenger, an Nrf2 inhibitor and an intestinal microorganism choline TMA lyase inhibitor.
Preferably, the NOX inhibitor comprises APO (vanillone).
Preferably, the ROS scavenger comprises NAC (N-acetylcysteine).
Preferably, the antioxidant transcription factor Nrf2 inhibitor comprises ML 385.
Preferably, the gut microbial choline Trimethylamine (TMA) lyase inhibitor comprises DMB (3, 3-dimethyl-1-butanol).
Preferably, the drug or compound acting on the choline or TMAO related target comprises 2-4 of APO, NAC, ML385 and DMB.
Preferably, the medicament for preventing and/or reversing clopidogrel resistance is an oral preparation.
The invention systematically explores the influence of the dietary choline or TMAO on the metabolic activation and antiplatelet effect of clopidogrel and various intervention mechanisms thereof, and provides a brand-new treatment target and prevention strategy for effectively overcoming clopidogrel resistance clinically. The invention discloses a causal relationship and an action mechanism between induction of liver carboxylesterase Ces1 protein expression and enhancement of hydrolase activity and clopidogrel resistance due to the fact that the level of TMAO in blood is remarkably increased after a mouse takes excessive food choline, discloses that a series of drugs or compounds can effectively prevent and/or reverse clopidogrel resistance caused by the feeding of high-choline food or TMAO by effectively reducing the generation of TMAO, inhibiting the activity of NADPH Oxidase (NOX), eliminating active oxygen free Radicals (ROS), inhibiting the activity of an antioxidant transcription factor Nrf2 and the like, and discloses that the compounds or analogues of the compounds with the same structure have clinical application value of preventing and/or treating clopidogrel resistance mainly mediated by the TMAO.
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Fig. 1 shows the platelet aggregation rates of the different treatment groups. The results show that choline or TMAO can significantly weaken the inhibition effect of clopidogrel on mouse platelet aggregation. Wherein A shows: the ingestion of choline or TMAO can significantly enhance the platelet aggregation of mice whole blood induced by ADP and weaken the inhibition of clopidogrel to platelet aggregation induced by ADP; b shows that: DMB can remarkably reverse inhibition of choline on anti-platelet effect of clopidogrel. Data are expressed as mean ± standard deviation; n is 6; p < 0.05; p < 0.01; p < 0.001. In the figure, Ctrl: (solvent) control; TMAO: trimethylamine oxide; choline: choline; CLP: clopidogrel; DMB: 3, 3-dimethyl-1-butanol.
FIG. 2 is the effect of dietary choline or TMAO on the pharmacokinetic process of clopidogrel and its parameters in mice; the kit comprises a substrate, a CLP-C-CLP-C-CLP-C-CLP-C-CLP-C-Choline-C-Choline-C-CLP-C-CLP-C-CLP-C-CLP-C-. The results show that the ingestion of choline or TMAO significantly enhances the hepatic metabolism of clopidogrel (a), increases the production (C) of the Ces1 enzymatic hydrolysate CLP-C, and significantly reduces the production (B) of the clopidogrel active metabolite H4; DMB is administered at the same time, and can significantly reverse the enhancing effect of choline on clopidogrel metabolism (D), the increase of production of Ces1 hydrolysate CLP-C (F), and the reduction of production of clopidogrel active metabolite H4 (E). Data are expressed as mean ± standard deviation; n is 6. CLP-C: clopidogrel carboxylate (a hydrolysate of the Ces1 enzyme); h4: clopidogrel active metabolite; other English abbreviations are illustrated in the figure of FIG. 1.
FIG. 3 is a graph showing the effect of dietary choline or TMAO on the expression level of drug metabolizing enzyme protein in mouse liver. Wherein, A and B are protein expression level of mouse liver carboxylesterase Ces1 detected after choline or TMAO is ingested, A is Westernblot (WB) detection result, and B is relative concentration of enzyme protein expression. The results show that choline or TMAO intake can significantly induce the protein expression level of mouse liver carboxylesterase Ces 1. C and D are protein expression level of carboxylesterase Ces1 in mouse liver after DMB administration, C is WB detection result, and D is relative concentration of enzyme protein expression. The results show that the induction of the expression level of the mouse liver Ces1 enzyme protein by choline can be remarkably inhibited or reversed after the DMB is simultaneously administered. Data are expressed as mean ± standard deviation; n is 6. P < 0.05. Ces 1: (mouse) carboxylesterase 1; cyp is: (mouse) cytochrome P450 enzyme line; gapdh: WB internal controls (for cytoplasmic protein analysis); other English abbreviations are illustrated in the figure of FIG. 1.
FIG. 4 is a graph showing the induction of TMAO on the level of Reactive Oxygen Species (ROS) production and antioxidant transcription factor (Nrf2) protein expression in HepG2 cells. Wherein A is the relative concentration of intracellular ROS after the TMAO with different concentrations is used for treating the cells, and shows that the TMAO concentration dependently induces the intracellular ROS generation; b and C are the expression levels of related proteins in cells after the cells are treated by TMAO with different concentrations, B is a WB detection result, and C is the relative concentration of protein expression, which shows that the concentration of TMAO dependently promotes Nrf2 protein to enter nucleus. Data are expressed as mean ± standard deviation; n is 3; p < 0.05. Lamin B: WB internal controls (for nucleoprotein analysis); keap 1: physiological inhibitory protein of intracytoplasmic Nrf 2.
Fig. 5 shows that NAC (ROS scavenger, tool) significantly reverses TMAO-induced increases in intracellular ROS production by scavenging intracellular ROS (a) and nuclear entry increases of Nrf2 with upregulation of hydrolase Ces1 protein expression (B and C). Wherein, A is the relative concentration of ROS in cells of different treatment groups, B and C are the protein expression levels in cells of different treatment groups, B is a WB detection result, and C is the relative concentration of protein expression. Data are expressed as mean ± standard deviation; n is 3; p < 0.05; p < 0.01; p < 0.001. NAC: n-acetylcysteine; other English abbreviations are shown in the figure description of FIG. 4.
Fig. 6 shows that ML385(Nrf2 inhibitor, tool drug) significantly reverses TMAO-induced nuclear entry of Nrf2 and upregulation of hydrolase Ces1 protein expression by inhibiting Nrf2 activity. Wherein, A is a WB detection result of protein expression levels of different treatment groups (Ctrl, TMAO + ML385), and B is a relative concentration of protein expression of different treatment groups (Ctrl, TMAO + ML 385). Data are expressed as mean ± standard deviation; n is 3; p < 0.05; p < 0.01; other English abbreviations are shown in the figure description of FIG. 4.
Fig. 7 shows that co-administration of APO (NOX inhibitor, tool) significantly inhibited TMAO-induced ROS production in hepatocytes (a), and inhibited TMAO-induced upregulation of Ces1 protein (B and C). Wherein, A is the relative concentration of ROS in cells of different treatment groups (Ctrl, TMAO + APO), B is the WB detection result of Ces1 enzyme protein expression in cells of different treatment groups (Ctrl, TMAO + APO), and C is the relative concentration of Ces1 enzyme protein expression in cells of different treatment groups (Ctrl, TMAO + APO). Data are expressed as mean ± standard deviation; n is 3; p < 0.05; APO: vanillyl ketone.
Fig. 8 is a graph showing that simultaneous administration of NAC or ML385 can significantly reverse TMAO-attenuated clopidogrel antiplatelet effects. Data are expressed as mean ± standard deviation; n is 6; p < 0.05; p < 0.01; p < 0.001.
Detailed Description
In the present invention, the clopidogrel resistance specifically refers to: after taking standard dose of clopidogrel, patients fail to achieve the expected antiplatelet effect for various reasons by weakening the metabolic activation of the drug in the liver and/or the reactivity of platelets to the drug, eventually resulting in a phenomenon that clopidogrel has poor or ineffective clinical efficacy to patients.
The causes of clopidogrel resistance are complex and various, genetic factors (such as CYP2C19 gene polymorphism) and non-genetic factors are involved, and the degree of clopidogrel resistance also varies from person to person. Choline (choline) components in some foods (such as red meat, eggs, milk, cheese, seafood, deep-sea fish and the like) are metabolized by intestinal microorganism choline Trimethylamine (TMA) lyase to generate TMA, and then TMA is catalyzed, oxidized and metabolized by FMO3 enzyme in the liver to generate TMAO, thereby causing the TMAO in blood to be remarkably increased. Thus, a person who often eats a high choline diet may cause platelet hyperreactivity due to a significant increase in TMAO in blood.
Based on the application, the invention provides the application of the medicine or the compound acting on the choline or TMAO related target in preparing the medicine for preventing and/or reversing clopidogrel resistance. In the invention, the medicine or compound acting on the choline or TMAO related target comprises one or more of a NOX inhibitor, a ROS scavenger, a Nrf2 inhibitor and a gut microorganism choline TMA lyase inhibitor.
In the present invention, the NOX inhibitor reverses TMAO-induced upregulation of Ces1 enzyme protein expression in hepatocytes by inhibiting NOX enzyme activity, reducing TMAO-induced ROS production in hepatocytes. In the practice of the present invention, the NOX inhibitor includes, but is not limited to, Apocynin (APO), which is exemplified by APO.
In the present invention, the ROS scavenger is capable of significantly scavenging ROS, reducing intracellular ROS levels, reversing TMAO-induced nuclear entry increase of Nrf2, and upregulating expression of Ces1 enzyme protein. In the present invention, the ROS scavenger includes, but is not limited to, N-acetylcysteine (NAC), which is exemplified herein by NAC.
In the invention, the Nrf2 inhibitor remarkably reverses TMAO-induced Nrf2 nuclearity increase and Ces1 enzyme protein expression up-regulation by inhibiting Nrf2 nuclearity. In the present invention, the Nrf2 inhibitor includes but is not limited to ML385, and the present invention is exemplified by ML 385.
In the invention, the intestinal microorganism choline TMA lyase inhibitor can reduce the generation of intestinal TMA by inhibiting the microorganism choline TMA lyase, remarkably inhibit the induction effect of choline on the expression of liver hydrolase Ces1, reverse the influence of choline on the liver metabolism of clopidogrel, lead to the remarkable reduction of the generation of hydrolysate CLP-C and the remarkable increase of the generation of active metabolite H4, and further remarkably reverse the inhibition of choline on the antiplatelet effect of clopidogrel. In the present invention, the intestinal microorganism choline TMA lyase inhibitor includes, but is not limited to, DMB (3, 3-dimethyl-1-butanol), and the present invention is illustrated by way of example of DMB.
In the invention, the combined application of the medicament for preventing and/or reversing clopidogrel resistance and clopidogrel can remarkably enhance the attenuated clopidogrel antiplatelet effect, thereby preventing and/or reversing clopidogrel resistance. In the invention, the compound acting on the choline or TMAO related target comprises 2-4 of APO, NAC, ML385 and DMB.
The research of the invention shows that TMAO can significantly increase the generation of ROS in liver cells, promote Nrf2 to enter nucleus, up-regulate Ces1 enzyme protein expression level, induce Ces1 hydrolase activity, increase the hydrolysis (inactivation) of clopidogrel, reduce the metabolic activation of clopidogrel and cause clopidogrel resistance. Simultaneous administration of NAC (ROS scavenger) significantly reduced intracellular ROS levels, reversed TMAO-induced nuclear increase of Nrf2, and upregulation of Ces1 enzyme protein expression; co-administration of ML385(Nrf2 inhibitor) significantly reversed TMAO-induced nuclear increase of Nrf2 and upregulation of Ces1 enzyme protein expression; and APO (NOX inhibitor) is simultaneously administered, so that the generation of ROS in hepatocytes induced by TMAO can be obviously inhibited, and the expression up-regulation of Ces1 enzyme protein induced by TMAO is reversed. Through the verification of in vivo research of mice, NAC or ML385 given at the same time can obviously reverse the attenuation of the platelet effect of clopidogrel induced by TMAO.
Based on the new findings, the invention provides a plurality of new treatment targets (NOX activity, ROS generation amount, Nrf2 nuclear invasion, liver Ces1 hydrolase activity and the like) related to clopidogrel resistance caused by TMAO, provides scientific basis and direction for research and development of related new drugs, and also provides a plurality of brand new treatment strategies for effectively preventing and/or reversing clopidogrel resistance caused by TMAO for clinicians.
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
Animal experiments
1. Male, 6-8 week old C57BL/6J mice (weight 18 + -1 g) were purchased from Nanjing university animal model center, and fed water and plain food as required for routine animal experiments.
After obtaining animal experiment ethics audit application batch, mice were randomly grouped according to different study objectives: saline (solvent) control group (Ctrl), TMAO group, choline group (choline), choline + DMB group, gavage daily for 14 days. The specific dosage for intragastric administration was as follows: TMAO (6 mg/day), choline (50 mg/day), DMB (1.2. mu. mol/day), reference is made to the relevant literature (Wang Z et al, Cell 2015; 163: 1585-95; ZhuW et al, Cell 2016; 165: 111-24). TMAO concentration in mouse plasma was measured after 14 days of each gavage with physiological saline (solvent control, Ctrl), TMAO, choline + DMB and quantitatively analyzed by the LC-MS/MS method described in the literature (Chen ML et al, mBio 2016; 7: e 02210-15). The results show that after the continuous gavage of choline or TMAO for 14 days, the concentration of TMAO in the plasma of the mice is respectively 89.0 +/-13.0 mu M and 94.5 +/-13.9 mu M, which indicates that the related mouse model is successfully established.
2. Mice were randomly divided into a solvent control (Ctrl) group, a TMAO group, and a choline group, and were administered with physiological saline, TMAO, or choline for 14 days, respectively, and then administered with clopidogrel (10mg/kg) alone or an equivalent volume of physiological saline (solvent control, Ctrl) by gavage, respectively, and then whole blood platelet aggregation induced by ADP (adenosine diphosphate) and inhibition of the same by ADP (adenosine diphosphate) under various experimental conditions, pharmacokinetic characteristics of blood concentrations of clopidogrel and its hydrolysate CLP-C and active metabolite H4, and protein expression levels of drug metabolizing enzymes related to clopidogrel metabolism in liver tissues of mice were examined by the method described in the reference (Tai T et al, J Cardiovasc Pharmacol 2016; 68: 433-40; Ji JZ et al, Biochem Pharmacol 2021; 183: 114313). The results are shown in FIG. 1A, FIG. 2A to C and FIG. 3A to B, respectively.
The results show that TMAO or cholinergic activity significantly increased the ADP-induced whole blood platelet aggregation rate in mice compared to normal saline (solvent control) (see a in fig. 1). In addition, the combination of choline or TMAO significantly increased the ADP-induced whole blood platelet aggregation rate in mice compared to the clopidogrel group (see a in fig. 1). The result shows that choline or TMAO can significantly induce platelet aggregation induced by ADP and weaken the antiplatelet effect of clopidogrel. The main reasons for this are that choline or TMAO can significantly induce protein expression of mouse liver carboxylesterase Ces1 (see a-B in fig. 3), and enhanced activity of Ces1 hydrolase results in that a large amount of clopidogrel in blood is hydrolyzed to form clopidogrel carboxylate (CLP-C) (see C in fig. 2), and systemic exposure (AUC) of clopidogrel parent drug is significantly reduced (see a in fig. 2). Since the systemic exposure amount of the clopidogrel parent drug (raw material compound) used to activate the metabolism to produce its active metabolite H4 is significantly reduced (see a in fig. 2), the production amount (or systemic exposure amount) of the clopidogrel active metabolite H4 in the liver is also significantly reduced accordingly (see B in fig. 2). The results of a series of researches reveal that choline or TMAO significantly weakens the antiplatelet effect of clopidogrel by inducing the protein expression and the hydrolase activity of liver hydrolase Ces1, enhancing the hydrolysis of clopidogrel and reducing the generation amount of an active metabolite H4.
3. In order to confirm whether the choline food is metabolized by intestinal microorganisms to generate TMA and then is further oxidized to generate TMAO to play the role of the choline food, mice are randomly divided into 4 groups of Ctrl group, choline group, DMB group and choline and DMB group, and after single dose of clopidogrel (10mg/kg) or equal volume of normal saline (solvent control, Ctrl) is respectively administered by intragastric gavage, ADP-induced platelet aggregation in whole blood and the degree of inhibition of clopidogrel against ADP-induced platelet aggregation, the blood levels of clopidogrel and its hydrolysate CLP-C and active metabolite H4 and its pharmacokinetic profile, and the protein expression levels of drug metabolizing enzymes involved in clopidogrel metabolism in mouse liver tissues were examined under various experimental conditions as described in the references (Tai T et al, JCardiovasc Pharmacol 2016; 68: 433-40; Ji JZ et al, Biochem Pharmacol 2021; 183: 114313). The results are shown in FIG. 1B, FIG. 2D to F and FIG. 3C to D, respectively.
The results showed that the combination of DMB significantly inhibited the induction of the expression of liver hydrolase Ces1 by choline (see C-D in fig. 3) compared to the choline group, thus significantly reversed the influence of choline on clopidogrel liver metabolism, resulting in a decrease in the production of the hydrolysis product CLP-C (see F in fig. 2), a decrease in clopidogrel hydrolysis (see D in fig. 2), an increase in the production of the active metabolite H4 (see E in fig. 2), and significantly reversed the inhibition of the antiplatelet effect by choline (see B in fig. 1). The results of this series of studies revealed that choline-induced clopidogrel resistance can be effectively reversed with DMB.
4. The curative effect of the experimental drug treatment is examined and evaluated:
mice were randomly divided into 4 groups of solvent control (Ctrl) group, TMAO + NAC group, TMAO + ML385 group. The specific doses administered were as follows: TMAO (6 mg/day), NAC (150 mg/kg/day), ML385(30 mg/kg/day), reference is made to the relevant literature (Deng S et al, Neurotheropeuterics 2020; Xie C et al, OxydMed CellLongev 2018; 1874985), the solvent control is an equal volume of physiological saline, and the administration is continued for 14 days each by gavage. The extent of inhibition of ADP-induced platelet aggregation in whole blood and clopidogrel against ADP-induced platelet aggregation under various experimental conditions was determined as described in the literature (Tai T et al, JCardiovasc Pharmacol 2016; 68: 433-40; Ji JZ et al, Biochem Pharmacol 2021; 183: 114313). The result is shown in fig. 8, TMAO can significantly increase the ADP-induced mouse whole blood platelet aggregation rate, and significantly inhibit the anti-platelet effect of clopidogrel; the combination of NAC or ML385 respectively can remarkably reverse the inhibition of the clopidogrel antiplatelet effect by TMAO.
Example 2
Second, cell experiment
1. Selecting HepG2 cells as a human hepatocyte model, and detecting whether the influence of different doses of TMAO (0, 50, 100 and 200 mu M) on the generation of ROS in cells and the expression of Keap1/Nrf2 protein presents dose dependence or not; whether TMAO (100. mu.M) -induced intracellular ROS production, Nrf2 nuclear entry and increased expression levels of the Ces1 protein can be significantly inhibited by using NAC (1mM) as an ROS scavenger; the combination of Nrf2 inhibitor ML385 (10. mu.M) can significantly inhibit TMAO (100. mu.M) induced upregulation of intracellular Nrf2 nuclear import and Ces1 protein expression. According to the conventional cell culture method, when the fusion degree of the cultured HepG2 cells is 75-85%, TMAO and/or other compounds to be detected are respectively added according to experimental groups, and the cells are cultured for 24 h. The relative intracellular concentration of ROS was measured using ROS kit (DCFH-DA active oxygen fluorescence probe method), and the relative expression level of each protein was measured using WB technique (binding corresponding primary and secondary antibodies). The results are shown in FIGS. 4, 5 and 6, respectively. The results show that TMAO can significantly induce ROS production in hepatocytes (see a in fig. 4), promote Nrf2 nucleation (see B-C in fig. 4), and exhibit dose-dependence (see fig. 4). Combination with NAC significantly scavenges intracellular ROS in hepatocytes (see a in fig. 5), significantly reducing TMAO-induced nuclear entry of Nrf2 and upregulation of Ces1 protein expression (see B-C in fig. 5). TMAO-induced nuclear entry of Nrf2 and upregulation of Ces1 protein expression were significantly reduced with ML385 (see fig. 6). The results of a series of researches reveal that as known ROS scavengers (NCA) and Nrf2 inhibitors (ML385), NCA and ML385 respectively eliminate ROS and inhibit the action mechanism of Nrf2 activity to further remarkably reduce TMAO-induced nucleation of Nrf2 and the expression up-regulation of Ces1 protein, so as to reverse the attenuation of the platelet anti-platelet effect of TMAO-induced clopidogrel, and provide scientific basis for NCA and ML385 to effectively prevent and treat TMAO-mediated clopidogrel resistance (as shown in figure 8).
2. HepG2 cells were selected as a human hepatocyte model, and whether the co-administration of APO (100. mu.M) could significantly inhibit TMAO (100. mu.M) -induced ROS production in hepatocytes and TMAO (100. mu.M) -induced upregulation of the expression level of Ces1 protein was detected. According to a conventional cell culture method, when the fusion degree of the cultured HepG2 cells is 75-85%, respectively adding a culture medium (solvent control), TMAO and TMAO + APO according to experimental groups, and culturing the cells for 24 h. The relative expression level of each protein was determined using WB techniques (binding corresponding primary and secondary antibodies). As a result, as shown in fig. 7, TMAO significantly induced ROS production in hepatocytes, while APO administration significantly inhibited TMAO-induced ROS production in hepatocytes (see a in fig. 7). TMAO can remarkably induce the up-regulation of the expression level of Ces1 protein in liver cells, and APO can remarkably inhibit the induction of Ces1 hydrolase in liver cells by TMAO (see B-C in figure 7). Thus, APO significantly reduced TMAO-induced ROS production in hepatocytes by inhibiting NOX enzymatic activity in hepatocytes (see a in fig. 7).
The above studies showed that NAC significantly scavenges intracellular ROS in hepatocytes (see a in fig. 5), significantly reducing TMAO-induced nuclear entry of Nrf2 and upregulation of Ces1 protein (see B-C in fig. 5). APO significantly inhibited TMAO-induced upregulation of Ces1 protein expression, and should also be by significantly inhibiting TMAO-induced, ROS-promoted Nrf2 nuclear entry. Obviously, the final cellular biochemical effects of the isoqu of APO, NAC and ML385 are all significant inhibition of induction of protein expression and activity of Ces1 enzyme in hepatocytes by TMAO.
The embodiment can know that the invention provides the application of the medicine or the compound acting on the related target of choline or TMAO in the preparation of the medicine for preventing and/or reversing clopidogrel resistance, provides scientific basis and direction for the research and development of related new medicines, and provides a plurality of brand new treatment strategies for effectively preventing and/or reversing clopidogrel resistance caused by TMAO for clinicians.
The foregoing is only a preferred embodiment of the present invention. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention.
Claims (8)
1. The application of the compound acting on the choline or TMAO related target in preparing the medicine for preventing and/or reversing clopidogrel resistance is characterized in that the medicine or the compound acting on the choline or TMAO related target comprises one or more of a NOX inhibitor, a ROS scavenger, a Nrf2 inhibitor and an intestinal microorganism choline TMA lyase inhibitor.
2. Use according to claim 1, wherein the NOX inhibitor comprises vanillyl ketone.
3. The use of claim 1, wherein said ROS scavenger comprises N-acetylcysteine.
4. The use of claim 1, wherein the Nrf2 inhibitor comprises ML 385.
5. The use of claim 1, wherein the gut microbial choline TMA lyase inhibitor comprises 3, 3-dimethyl-1-butanol.
6. The use of any one of claims 1-5, wherein the compound acting on a choline-or TMAO-related target comprises 2-4 of vanillone, N-acetylcysteine, ML385 and 3, 3-dimethyl-1-butanol.
7. The use according to claim 6, wherein the medicament for preventing and/or reversing clopidogrel resistance is an oral formulation.
8. The application of the drug or compound acting on the choline or TMAO related target point in combination with clopidogrel in preparing the drug for resisting platelet aggregation.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116407526A (en) * | 2022-03-11 | 2023-07-11 | 复旦大学附属肿瘤医院 | Breast cancer therapeutic drug, auxiliary therapeutic drug, anti-tumor immune activator and application of trimethylamine oxide and precursor product choline thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101984971A (en) * | 2010-11-08 | 2011-03-16 | 北京阜康仁生物制药科技有限公司 | Drug composition containing clopidogrel and nicotinic acid compounds |
| CN107880094A (en) * | 2016-09-30 | 2018-04-06 | 中国科学院大连化学物理研究所 | Black bearberry acids neutrality cholesterol lipolytic enzymes inhibitor and its application |
| WO2018236899A1 (en) * | 2017-06-19 | 2018-12-27 | The Cleveland Clinic Foundation | Treating disease and promoting weight loss by inhibiting the tma/fmo3/tmao pathway |
| CN110468196A (en) * | 2019-08-20 | 2019-11-19 | 南京市第一医院 | It is a kind of predict clopidogrel Resistant biomarker application and its kit |
| CN112791116A (en) * | 2019-11-13 | 2021-05-14 | 上海交通大学医学院附属新华医院 | Application of musk heart-through dropping pill in preparing antiplatelet medicine for treating gene mutation clopidogrel resistance |
-
2021
- 2021-06-30 CN CN202110733817.XA patent/CN113274504B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101984971A (en) * | 2010-11-08 | 2011-03-16 | 北京阜康仁生物制药科技有限公司 | Drug composition containing clopidogrel and nicotinic acid compounds |
| CN107880094A (en) * | 2016-09-30 | 2018-04-06 | 中国科学院大连化学物理研究所 | Black bearberry acids neutrality cholesterol lipolytic enzymes inhibitor and its application |
| WO2018236899A1 (en) * | 2017-06-19 | 2018-12-27 | The Cleveland Clinic Foundation | Treating disease and promoting weight loss by inhibiting the tma/fmo3/tmao pathway |
| CN110468196A (en) * | 2019-08-20 | 2019-11-19 | 南京市第一医院 | It is a kind of predict clopidogrel Resistant biomarker application and its kit |
| CN112791116A (en) * | 2019-11-13 | 2021-05-14 | 上海交通大学医学院附属新华医院 | Application of musk heart-through dropping pill in preparing antiplatelet medicine for treating gene mutation clopidogrel resistance |
Non-Patent Citations (5)
| Title |
|---|
| RUISONG MA ET AL: "TMAO: a potential mediator of clopidogrel resistance", 《SCIENTIFIC REPORTS》 * |
| ZHAOJIAN ZHANG: "A Review of Danshen Combined with Clopidogrel in the Treatment of Coronary Heart Disease", 《EVIDENCE-BASED COMPLEMENTARY AND ALTERNATIVE MEDICINE》 * |
| 丁柏宇等: "冠心病人群氯吡格雷抵抗与基因多态性的研究进展", 《医学综述》 * |
| 刘帅兵等: "氯吡格雷的体外相互作用研究", 《中国血液流变学杂志》 * |
| 李雷等: "氯吡格雷抵抗在缺血性脑血管病中的研究进展", 《中国临床神经科学》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116407526A (en) * | 2022-03-11 | 2023-07-11 | 复旦大学附属肿瘤医院 | Breast cancer therapeutic drug, auxiliary therapeutic drug, anti-tumor immune activator and application of trimethylamine oxide and precursor product choline thereof |
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