Clopidogrel ameliorates high-fat diet-induced hepatic steatosis in mice through activation of the AMPK signaling pathway and beyond.

Introduction: Metabolic dysfunction-associated steatotic liver disease (MASLD) frequently confers an increased risk of vascular thrombosis; however, the marketed antiplatelet drugs are investigated for the prevention and treatment of MASLD in patients with these coexisting diseases.

Methods: To determine whether clopidogrel could ameliorate high-fat diet (HFD)-induced hepatic steatosis in mice and how it works, mice were fed on normal diet or HFD alone or in combination with or without clopidogrel for 14 weeks, and primary mouse hepatocytes were treated with palmitate/oleate alone or in combination with the compounds examined for 24 h. Body weight, liver weight, insulin resistance, triglyceride and total cholesterol content in serum and liver, histological morphology, transcriptomic analysis of mouse liver, and multiple key MASLD-associated genes and proteins were measured, respectively.

Sex differences in the metabolic activation of and platelet response to vicagrel in mice: Androgen as a key player.

As a biological variable, sex influences the metabolism of and/or response to certain drugs. Vicagrel is being developed as an investigational new drug in China; however, it is unknown whether sex could affect its metabolic activation and platelet responsiveness. This study aimed to determine whether such differences could exist, and to elucidate the mechanisms involved.

The metabolic activation of and platelet response to vicagrel vary with P-glycoprotein deficiency, rather than P-glycoprotein inhibition, in mice.

This study aimed to determine changes in the hydrolysis of vicagrel, a substrate drug of arylacetamide deacetylase (Aadac) and carboxylesterase 2 (Ces2), in P-glycoprotein (P-gp)-deficient or P-gp-inhibited mice and to elucidate the mechanisms involved.Male wild-type (WT) and P-gp knock-out (KO) mice were used to investigate the systemic exposure of vicagrel thiol active metabolite H4 and platelet response to vicagrel, and the mRNA and protein expression levels of intestinal Aadac and Ces2. Moreover, WT mice were administered vicagrel alone or in combination with elacridar (a potent P-gp inhibitor) to determine drug-drug interactions.

The Complement System and C4b-Binding Protein: A Focus on the Promise of C4BPα as a Biomarker to Predict Clopidogrel Resistance.

The complement system plays a dual role in the body, either as a first-line defense barrier when balanced between activation and inhibition or as a potential driver of complement-associated injury or diseases when unbalanced or over-activated. C4b-binding protein (C4BP) was the first circulating complement regulatory protein identified and it functions as an important complement inhibitor. C4BP can suppress the over-activation of complement components and prevent the complement system from attacking the host cells through the binding of complement cleavage products C4b and C3b, working in concert as a cofactor for factor I in the degradation of C4b and C3b, and consequently preventing or reducing the assembly of C3 convertase and C5 convertase, respectively.

Enhanced metabolic activation of and platelet response to clopidogrel in T cell-deficient mice through induction of Cyp2c and Cyp3a and inhibition of Ces1.

Background: T cells and platelets reciprocally coordinate mutual functions through crosstalk or interaction. However, it is not known whether metabolic activation of and platelet response to clopidogrel could be changed if T cells were deficient or impaired in some cases and, if any, how it would work.

Objectives: The objective of this study was to dissect the potential changes in platelet responses to and metabolic activation of clopidogrel in the case of T cell deficiency and to elucidate their mechanisms involved.

Vicagrel is hydrolyzed by Raf kinase inhibitor protein in human intestine.

As an analog of clopidogrel and prasugrel, vicagrel is completely hydrolyzed to intermediate thiolactone metabolite 2-oxo-clopidogrel (also the precursor of active thiol metabolite H4) in human intestine, predominantly by AADAC and CES2; however, other unknown vicagrel hydrolases remain to be identified. In this study, recombinant human Raf kinase inhibitor protein (rhRKIP) and pooled human intestinal S9 (HIS9) fractions and microsome (HIM) preparations were used as the different enzyme sources; prasugrel as a probe drug for RKIP (a positive control), vicagrel as a substrate drug of interest, and the rate of the formation of thiolactone metabolites 2-oxo-clopidogrel and R95913 as metrics of hydrolase activity examined, respectively. In addition, an IC value of inhibition of rhRKIP-catalyzed vicagrel hydrolysis by locostatin was measured, and five classical esterase inhibitors with distinct esterase selectivity were used to dissect the involvement of multiple hydrolases in vicagrel hydrolysis.

Is platelet responsiveness to clopidogrel attenuated in overweight or obese patients and why? A reverse translational study in mice.

Background And Purpose: Overweight or obese patients exhibit poorer platelet responses to clopidogrel. However, the mechanisms behind this phenotype remain to be elucidated. Here, we sought to discover whether and why obesity could affect the metabolic activation of and/or platelet response to clopidogrel in obese patients and high-fat diet-induced obese mice.

Short-term standard alcohol consumption enhances platelet response to clopidogrel through inhibition of Nrf2/Ces1 pathway and induction of Cyp2c in mice.

Aims: Drinking alcohol is prevalent worldwide; however, it is unknown whether alcohol could affect the antiplatelet effects of clopidogrel in patients when taking both concomitantly. This study was designed to investigate the influence of short-term standard alcohol consumption on the metabolic activation of and platelet response to clopidogrel in mice as well as the mechanisms involved.

Main Methods: Male C57BL/6J mice were administered with normal saline (vehicle control) or alcohol at 2 g/kg/day for 7 days, and then gavaged with vehicle control or a single dose of clopidogrel at 10 mg/kg.

P-glycoprotein deficiency enhances metabolic activation of and platelet response to clopidogrel through marked up-regulation of Cyp3a11 in mice: Direct evidence for the interplay between P-glycoprotein and Cyp3a.

Variability in P-glycoprotein (P-gp) efflux transporting activity was supposed to be involved in altered intestinal absorption and bioavailability of clopidogrel in patients; however, reliable evidence is still lacking. In this study, we sought to determine whether P-gp could play an important role in the metabolic activation of and platelet response to clopidogrel in mice. Abcb1a/1b knock-out (KO) and wild-type (WT) mice were used to evaluate differences in the intracellular accumulation of clopidogrel in the intestine, liver, and brain tissues and in systemic exposure of clopidogrel and its main metabolites as well as the mechanisms involved.

Multidrug Resistance-Associated Protein 3 Is Responsible for the Efflux Transport of Curcumin Glucuronide from Hepatocytes to the Blood.

Curcumin, a major polyphenol present in turmeric, is predominantly converted to curcumin--glucuronide (COG) in enterocytes and hepatocytes via glucuronidation. COG is a principal metabolite of curcumin in plasma and feces. It appears that the efflux transport of the glucuronide conjugates of many compounds is mediated largely by multidrug resistance-associated protein (MRP) 3, the gene product of the ATP-binding cassette, subfamily C, member 3.

Development and validation of a UPLC-MS/MS method for simultaneous determination of vicagrel and its major metabolites in rat or human plasma: An optimized novel strategy for the stabilization of vicagrel.

Vicagrel is a promising novel antiplatelet drug. However, the quantification of vicagrel in plasma is currently unavailable since it is liable to be hydrolyzed in plasma by esterases. In this study, an optimized strategy was developed and validated to stabilize vicagrel, 2-oxo-clopidogrel (thiolactone metabolite), and H4 (active thiol metabolite) before quantification of the analytes, such as addition of citric acid (for plasma acidification) and NaF (a non-specific esterase inhibitor) to inhibit esterase activity, immediate addition of a thiol-alkylating reagent MPB into blood samples to derivatize H4 for the formation of stable H4 derivative (i.

Vicagrel enhances aspirin-induced inhibition of both platelet aggregation and thrombus formation in rodents due to its decreased metabolic inactivation.

Both aspirin and vicagrel are effective antiplatelet drugs, with the potential for concomitant use as another dual-antiplatelet therapy for the prevention of recurrent thrombotic or ischemic events. Because they both are the substrates of carboxylesterase 2 (CES2), aspirin attenuated the metabolic activation of and platelet response to vicagrel in mice treated with the two drugs concomitantly. In this study, we sought to clarify whether vicagrel could affect platelet responses to aspirin and their underlying mechanisms.

Enhanced responsiveness of platelets to vicagrel in IL-10-deficient mice through STAT3-dependent up-regulation of the hydrolase arylacetamide deacetylase in the intestine.

Background And Purpose: Vicagrel is a novel promising antiplatelet drug designed for overcoming clopidogrel resistance. There is limited evidence indicating that exogenous IL-10 suppresses CYP3A4 activity in healthy subjects and that IL-10 knockout (KO) mice exhibit increased clopidogrel bioactivation compared with wild-type (WT) mice. In this study, we sought to determine whether IL-10 could play an important role in the metabolism of and platelet response to vicagrel in mice.

Aspirin Attenuates the Bioactivation of and Platelet Response to Vicagrel in Mice.

Vicagrel, a novel acetate analogue of clopidogrel, exerts more potent antiplatelet effect than clopidogrel in rodents. Relevant evidence indicated that aspirin and vicagrel are the drug substrate for carboxylesterase 2. Accordingly, it is deduced that concomitant use of aspirin could attenuate the bioactivation of and platelet response to vicagrel.

Human UGT2B7 is the major isoform responsible for the glucuronidation of clopidogrel carboxylate.

Clopidogrel is predominantly hydrolyzed to clopidogrel carboxylic acid (CCA) by carboxylesterase 1, and subsequently CCA is glucuronidated to clopidogrel acyl glucuronide (CAG) by uridine diphosphate-glucuronosyltransferases (UGTs); however, the UGT isoenzymes glucuronidating CCA remain unidentified to date. In this study, the glucuronidation of CCA was screened with pooled human liver microsomes (HLMs) and 7 human recombinant UGT (rUGT) isoforms. Results indicated that rUGT2B7 exhibited the highest catalytical activity for the CCA glucuronidation as measured with a mean V value of 120.

Mrp3 Transports Clopidogrel Acyl Glucuronide from the Hepatocytes into Blood.

Clopidogrel acyl glucuronide (CLP-G) is a major phase II metabolite of clopidogrel generated in the liver for further excretion into urine; however, it is unclear whether CLP-G transports from hepatocytes into blood. Because multidrug resistance-associated protein 3 (MRP3) is predominantly expressed in the sinusoidal side of hepatocytes and preferentially transports glucuronide conjugates of drug metabolites from hepatocytes into bloodstream, we hypothesized that MRP3 could be such an efflux transporter for CLP-G. In this study, we compared the liver-to-plasma ratios of clopidogrel and its metabolites (including CLP-G) between (ATP-binding cassette, subfamily C, member 3) knockout (KO) and wild-type (WT) mice.

Overcoming Clopidogrel Resistance: Three Promising Novel Antiplatelet Drugs Developed in China.

Clopidogrel is one of the most frequently prescribed drugs worldwide; however, the presence of clopidogrel resistance and high susceptibility to genetic variations and drug interactions are facilitating the development of other antiplatelet drugs. To overcome clopidogrel resistance, several promising clopidogrel analogues have been developed in China, such as vicagrel (and its deuterated analogues), PLD-301, and W1. These novel chemical analogues are all characterized by much faster and more efficient bioconversion to clopidogrel thiolactone (or 2-oxo-clopidogrel, the precursor of clopidogrel active metabolite) in the intestine than clopidogrel itself through bypassing the first-step P450-mediated oxidation of clopidogrel in the liver.

Enhanced Platelet Response to Clopidogrel in Abcc3-deficient Mice Due to Its Increased Bioactivation.

Resistance of the patient to clopidogrel (an inactive prodrug) has been recently reported to be associated with increased messenger RNA expression of ABCC3 that encodes MRP3 (multidrug resistance-associated protein 3). However, there is no evidence showing the effects of MRP3 on altered platelet responses to clopidogrel and their underlying mechanisms. To further clarify whether the presence or absence of Mrp3 could affect the formation of and response to clopidogrel active metabolite (CAM) in Abcc3 knockout (KO) versus wild-type (WT) mice, we determined pharmacokinetic profiles of clopidogrel and CAM and measured inhibition of adenosine diphosphate-induced platelet aggregation by clopidogrel after administration of a single oral dose of clopidogrel to KO and WT mice, respectively.

[Research on Resistant Starch Content of Rice Grain Based on NIR Spectroscopy Model].

A new method based on near-infrared reflectance spectroscopy (NIRS) analysis was explored to determine the content of rice-resistant starch instead of common chemical method which took long time was high-cost. First of all, we collected 62 spectral data which have big differences in terms of resistant starch content of rice, and then the spectral data and detected chemical values are imported chemometrics software. After that a near-infrared spectroscopy calibration model for rice-resistant starch content was constructed with partial least squares (PLS) method.

Polysaccharide from Angelica sinensis protects chondrocytes from H2O2-induced apoptosis through its antioxidant effects in vitro.

This study aimed to explore the protective effects of Angelica sinensis polysaccharide (ASP) on rat chondrocyte injury induced by hydrogen peroxide (H2O2). Rat chondrocytes were cultured and treated with different concentrations of ASP alone or in combination with H2O2, and they were measured with cell viability, apoptosis, release of inflammatory cytokines, such as interleukin-1beta (IL-1β) and tumor necrosis factor-alpha (TNF-α), activity of superoxide dismutase (SOD), and catalase (CAT), and levels of malondialdehyde (MDA) production, respectively. In addition, quantitative real-time reverse transcription polymerase chain reaction was used to estimate the relative expression levels of osteoarthritis (OA)-associated genes, such as collagen type II (Col2a1), aggrecan, SOX9, matrix metalloproteinase (MMP)-1, -3, and -9, as well as tissue inhibitor of matrix metalloproteinase (TIMP)-1, respectively.

CRISPR/Cas: A Faster and More Efficient Gene Editing System.

Gene editing technology has been at its mature stage with the successful development of TALENs and CRISPR/Cas enzymes. The genetically modified endonucleases of ZFNs, TALENs, and CRISPR/Cas are widely used in the development of genetically modified cells or organisms. Among the enzymes that possess gene editing ability, CRISPR/Cas is the latest member with high efficiency in gene editing and simplicity in cloning.

The molecular mechanism underlying the induction of hepatic MRP3 expression and function by omeprazole.

Previous work has indicated that there is increased protein expression of multidrug resistance-associated protein 3 (MRP3) in the liver samples of patients treated with omeprazole compared with those who were not. However, evidence is still lacking to show the mechanisms underlying that induction. This study aimed to assess changes in the fold-induction of MRP3 mRNA and protein expression over controls in omeprazole-treated HepG2 cells after transient transfection of human MRP3 siRNA, or after pretreatment with actinomycin D (Act-D).

IL-10 deficiency increases renal ischemia-reperfusion injury.

Background: Renal ischemia-reperfusion (IR) injury is a frequent cause of acute kidney injury, which results in high morbidity and mortality. Inflammation is an important factor that is involved in kidney repair after renal IR injury. IL-10 is a potent anti-inflammatory cytokine that inhibits inflammatory pathways, but the role of IL-10 in repairing renal IR injury is not known.

CES1A -816C as a genetic marker to predict greater platelet clopidogrel response in patients with percutaneous coronary intervention.

This study was designed to determine whether CES1A -816A/C polymorphism could be associated with altered clopidogrel response. Recruited patients were pretreated with 300 mg clopidogrel loading dose before undergoing percutaneous coronary intervention for stenting and genotyped with CYP2C19 *2, *3, or *17, and CES1A -816A/C, respectively. Adenosine diphosphate-induced maximum platelet aggregation (MPA) was determined on day 3 after initiation of daily clopidogrel maintenance doses.