ABSTRACT
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.Compared with WT mice, P-gp KO mice exhibited significant increases in the systemic exposure of H4, the protein expression levels of intestinal Aadac and Ces2, and inhibition of ADP-induced platelet aggregation by vicagrel. Further, the H4 exposure was positively correlated with intestinal Aadac protein expression levels but did not vary with short-term inhibition of P-gp efflux activity by elacridar.P-gp-deficient mice, rather than elacridar-treated mice, exhibited significant upregulation of intestinal Aadac and Ces2 and thus, enhanced metabolic activation of and platelet response to vicagrel, suggesting that the metabolic activation of vicagrel may vary with P-gp deficiency, not P-gp inhibition, in mice.
ABSTRACT
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. C4BP, particularly C4BP α-chain (C4BPα), exerts its unique inhibitory effects on complement activation and opsonization, systemic inflammation, and platelet activation and aggregation. It has long been acknowledged that crosstalk or interplay exists between the complement system and platelets. Our unpublished preliminary data suggest that circulating C4BPα exerts its antiplatelet effects through inhibition of both complement activity levels and complement-induced platelet reactivity. Plasma C4BPα levels appear to be significantly higher in patients sensitive to, rather than resistant to, clopidogrel, and we suggest that a plasma C4BPα measurement could be used to predict clopidogrel resistance in the clinical settings.
Subject(s)
Complement C4b-Binding Protein , Complement System Proteins , Humans , Biomarkers , Clopidogrel , Complement C3-C5 Convertases/metabolism , Complement C4b-Binding Protein/metabolismABSTRACT
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. METHODS: BALB/c athymic nude mice or euthymic mice (controls) pretreated with cyclosporine A (CsA), thymosin α1 (Tα1), or their combination were used to investigate the changes in ADP-induced platelet activation and aggregation, systemic exposure of clopidogrel and its metabolites, and mRNA/protein expression and activity levels of clopidogrel-metabolizing enzymes in the liver, respectively. RESULTS: Nude mice exhibited significantly enhanced antiplatelet effects of clopidogrel due to increased formation of clopidogrel active metabolite in the liver, where the enzyme activity levels of Cyp2c and Cyp3a were significantly elevated compared with control mice. Furthermore, the effects of CsA pretreatment on the metabolism of clopidogrel in euthymic mice were identical to those seen in athymic mice. As expected, concomitant use of Tα1 reversed all the observed effects of CsA on clopidogrel metabolism and relevant metabolic enzymes. CONCLUSIONS: T cell deficiency or suppression enhances the antiplatelet effects of clopidogrel due to the boosted metabolic activation of clopidogrel in the liver through a dramatic induction of Cyp2c and Cyp3a in mice, suggesting that the metabolism of substrate drugs of Cyp2c and Cyp3a may be enhanced by T cell impairment.
Subject(s)
Platelet Aggregation Inhibitors , Ticlopidine , Animals , Mice , Activation, Metabolic , Blood Platelets/metabolism , Clopidogrel/pharmacology , Cytochrome P-450 CYP3A/metabolism , Mice, Nude , Platelet Aggregation , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation Inhibitors/therapeutic use , T-Lymphocytes/metabolism , Ticlopidine/pharmacologyABSTRACT
BACKGROUND: Trimethylamine N-oxide (TMAO), a gut microbe-generated metabolite, elicits thrombotic events by enhancing platelet reactivity; however, no studies have reported the effects of TMAO on the metabolism of and response to clopidogrel. OBJECTIVES: To determine whether choline and TMAO could significantly impair metabolic activation of and platelet response to clopidogrel in choline- or TMAO-fed mice and the mechanisms involved. METHODS: Male mice were fed with vehicle control (Ctrl), TMAO, choline alone or in combination with 3,3-dimethyl-1-butanol, N-acetyl-L-cysteine, or ML385 for 14 days and then treated with Ctrl or a single oral dose of clopidogrel. Plasma TMAO, protein levels of clopidogrel-metabolizing enzymes in the liver, plasma concentrations of clopidogrel and its metabolites, and adenosine diphosphate-induced platelet aggregation and activation were measured. In addition, HepG2 cells were treated with Ctrl or TMAO alone or in combination with N-acetyl-L-cysteine, ML385, or apocynin, and CES1, reactive oxygen species (ROS), and Nrf2 protein levels were measured, respectively. RESULTS: TMAO significantly increased Ces1 protein expression and activity and clopidogrel hydrolysis in the liver as well as intracellular ROS and CES1 levels and Nrf2 nucleus translocation in HepG2 cells but decreased the formation of clopidogrel active metabolite and impaired platelet response to clopidogrel. Furthermore, concomitant use of 3,3-dimethyl-1-butanol, N-acetyl-L-cysteine, or ML385 effectively reversed choline- or TMAO-induced impairment of inhibition of platelet aggregation by clopidogrel in mice, respectively. CONCLUSIONS: Choline and TMAO impair the metabolic activation of and platelet response to clopidogrel through the activation of the NOX-dependent ROS/Nrf2/CES1 pathway, suggesting novel strategies for overcoming clopidogrel resistance from bench to bedside.
Subject(s)
Choline , NF-E2-Related Factor 2 , Male , Animals , Mice , Choline/metabolism , Clopidogrel , NF-E2-Related Factor 2/metabolism , Reactive Oxygen Species , Activation, Metabolic , Acetylcysteine/pharmacology , Acetylcysteine/metabolismABSTRACT
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 IC50 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. The results showed that rhRKIP hydrolyzed vicagrel in vitro, with the values of Km , Vmax , and CLint measured as 20.04 ± 1.99 µM, 434.60 ± 12.46 nM/min/mg protein, and 21.69 ± 0.28 ml/min/mg protein, respectively, and that an IC50 value of locostatin was estimated as 1.24 ± 0.04 mM for rhRKIP. In addition to locostatin, eserine and vinblastine strongly suppressed vicagrel hydrolysis in HIM. It is concluded that RKIP can catalyze the hydrolysis of vicagrel in the human intestine, and that vicagrel can be hydrolyzed by multiple hydrolases, such as RKIP, AADAC, and CES2, concomitantly.
Subject(s)
Hydrolases , Phosphatidylethanolamine Binding Protein , Humans , Prasugrel Hydrochloride/metabolism , Phosphatidylethanolamine Binding Protein/metabolism , Clopidogrel , Hydrolases/metabolism , Esterases/metabolism , IntestinesABSTRACT
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. EXPERIMENTAL APPROACH: A post hoc stratified analysis of an observational clinical study was performed to investigate changes in residual platelet reactivity with increasing body weight in patients taking clopidogrel. Furthermore, high-fat diet-induced obese mice were used to reveal alterations in systemic exposure of clopidogrel thiol active metabolite H4, ADP-induced platelet activation and aggregation, the expression of genes involved in the metabolic activation of clopidogrel, count of circulating reticulated and mature platelets, and proliferation profiles of megakaryocytes in bone marrow. The relevant genes and potential signalling pathways were predicted and enriched according to the GEO datasets available from obese patients. KEY RESULTS: Obese patients exhibited significantly attenuated antiplatelet effects of clopidogrel. In diet-induced obese mice, systemic exposure of clopidogrel active metabolite H4 was reduced but that of its hydrolytic metabolite was increased due to down-regulation of certain P450s but up-regulation of carboxylesterase-1 in the liver. Moreover, enhanced proliferation of megakaryocytes and elevated platelet count also contributed. CONCLUSION AND IMPLICATIONS: Obesity attenuated metabolic activation of clopidogrel and increased counts of circulating reticulated and mature platelets, leading to impaired platelet responsiveness to the drug in mice, suggesting that clopidogrel dosage may need to be adjusted adequately in overweight or obese patients.
Subject(s)
Blood Platelets , Ticlopidine , Animals , Clopidogrel/metabolism , Clopidogrel/pharmacology , Humans , Mice , Obesity/drug therapy , Obesity/metabolism , Overweight/metabolism , Platelet Aggregation , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation Inhibitors/therapeutic use , Ticlopidine/pharmacologyABSTRACT
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. Inhibition of ADP-induced platelet aggregation and activation by clopidogrel, plasma concentrations of clopidogrel and its active metabolite H4, and changes in mRNA and protein expression of genes related to clopidogrel metabolism and its regulation were measured in mice pretreated with or without alcohol. KEY FINDINGS: Compared with vehicle control, alcohol pretreatment significantly reduced hydrolysis of clopidogrel as a result of significant down-regulation of Nrf2-mediated Ces1 expression (responsible for the formation of clopidogrel carboxylate), increased metabolic activation of clopidogrel due to significant up-regulation of Cyp2c (for the formation of active thiol metabolite H4), and consequently enhanced inhibition of ADP-induced platelet aggregation and activation by clopidogrel. SIGNIFICANCE: Short-term standard alcohol consumption would significantly enhance suppression of ADP-induced platelet aggregation and activation by clopidogrel through significant inhibition of Nrf2/Ces1 signaling pathway and induction of Cyp2c, suggesting that alcohol may interact with drugs that are predominantly metabolized by CES1 or CYP2C in patient care, including clopidogrel.
Subject(s)
Alcohol Drinking/adverse effects , Carboxylic Ester Hydrolases/antagonists & inhibitors , Clopidogrel/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Gene Expression Regulation/drug effects , NF-E2-Related Factor 2/antagonists & inhibitors , Platelet Aggregation/drug effects , Animals , Drug Synergism , Male , Mice , Mice, Inbred C57BL , Platelet Aggregation Inhibitors/pharmacologyABSTRACT
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. Results indicated that, compared with WT mice, KO mice exhibited an 84% increase in systemic exposure of clopidogrel active thiol metabolite H4 and a 14.5% rise of suppression of ADP-induced platelet integrin αIIbß3 activation, paralleled by a 41% decrease in systemic exposure of clopidogrel due to enhanced systemic clearance. Furthermore, KO mice displayed a 45% increase in Cyp3a11 but a 23% decrease in Ces1 at their protein levels compared with WT mice. Concurrently, intracellular clopidogrel concentrations in the tissues examined did not differ significantly between KO and WT mice. We conclude that although P-gp does not transport clopidogrel and its major metabolites in mice, P-gp-deficient mice exhibit elevated formation of the active metabolite H4 and enhanced antiplatelet effect of clopidogrel through up-regulation of Cyp3a11 and down-regulation of Ces1, suggesting that P-gp activity may correlate inversely with the formation of H4 and antiplatelet efficacy of clopidogrel in clinical settings due to P-gp and CYP3A4 interplay.
Subject(s)
ATP Binding Cassette Transporter, Subfamily B, Member 1/deficiency , Blood Platelets/metabolism , Clopidogrel/pharmacology , Cytochrome P-450 CYP3A/biosynthesis , Membrane Proteins/biosynthesis , Platelet Aggregation Inhibitors/pharmacology , Up-Regulation/drug effects , Animals , Blood Platelets/drug effects , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Up-Regulation/physiologyABSTRACT
Curcumin, a major polyphenol present in turmeric, is predominantly converted to curcumin-O-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. However, it is currently unknown whether this was the case with COG. In this study, Mrp3 knockout (KO) and wild-type (WT) mice were used to evaluate the pharmacokinetics profiles of COG, the liver-to-plasma ratio of COG, and the COG-to-curcumin ratio in plasma, respectively. The ATP-dependent uptake of COG into recombinant human MRP3 inside-out membrane vesicles was measured for further identification, with estradiol-17ß-d-glucuronide used in parallel as the positive control. Results showed that plasma COG concentrations were extremely low in KO mice compared with WT mice, that the liver-to-plasma ratios of COG were 8-fold greater in KO mice than in WT mice, and that the ATP-dependent uptake of COG at 1 or 10 µM was 5.0- and 3.1-fold greater in the presence of ATP than in the presence of AMP, respectively. No significant differences in the Abcc2 and Abcg2 mRNA expression levels were seen between Mrp3 KO and WT mice. We conclude that Mrp3 is identified to be the main efflux transporter responsible for the transport of COG from hepatocytes into the blood. SIGNIFICANCE STATEMENT: This study was designed to determine whether multidrug resistance-associated protein (Mrp) 3 could be responsible for the efflux transport of curcumin-O-glucuronide (COG), a major metabolite of curcumin present in plasma and feces, from hepatocytes into the blood using Mrp3 knockout mice. In this study, COG was identified as a typical Mrp3 substrate. Results suggest that herb-drug interactions would occur in patients concomitantly taking curcumin and either an MRP3 substrate/inhibitor or a drug that is predominantly glucuronidated by UDP-glucuronosyltransferases.
Subject(s)
Curcumin/analogs & derivatives , Glucuronides/pharmacokinetics , Hepatocytes/metabolism , Multidrug Resistance-Associated Proteins/metabolism , Administration, Oral , Animals , Curcumin/administration & dosage , Curcumin/pharmacokinetics , Drug Evaluation, Preclinical , Estradiol/administration & dosage , Estradiol/analogs & derivatives , Estradiol/pharmacokinetics , Glucuronides/administration & dosage , Male , Mice , Mice, Knockout , Multidrug Resistance-Associated Protein 2 , Multidrug Resistance-Associated Proteins/geneticsABSTRACT
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.e., MP-H4), use of the anticoagulant K2EDTA to minimize the conversion of 2-oxo-clopidogrel to H-endo, and keeping the analytes at 4⯰C or on wet ice to minimize degradation of the analytes when processed and analyzed. The stability was measured as percent of each analyte remained in plasma samples after their storage for 4â¯h at 4⯰C or in blood samples after 1â¯h at 4⯰C. The results indicated that stability of vicagrel was increased significantly in stabilized plasma or blood samples compared with non-stabilized controls for rats and humans, respectively, and that the stability of 2-oxo-clopidogrel was increased to a certain extent. In contrast, MP-H4 formed was stable in plasma immediately after thorough mixture of MPB with blood. We conclude that the above strategy is useful for improving the stability of vicagrel, 2-oxo-clopidogrel, and H4 in rat or human plasma, and that vicagrel and its two major metabolites can be quantified accurately and simultaneously.
Subject(s)
Chromatography, High Pressure Liquid/methods , Phenylacetates/analysis , Platelet Aggregation Inhibitors/analysis , Tandem Mass Spectrometry/methods , Thiophenes/analysis , Animals , Female , Humans , Male , Phenylacetates/pharmacokinetics , Platelet Aggregation Inhibitors/pharmacokinetics , Rats , Rats, Sprague-Dawley , Specimen Handling , Temperature , Thiophenes/pharmacokinetics , Time FactorsABSTRACT
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. Plasma levels of aspirin and salicylic acid were determined by liquid chromatography-tandem mass spectrometry, inhibition of arachidonic acid (AA)-induced whole-blood platelet aggregation by aspirin was assessed with an aggregometer, and their antithrombotic effects were evaluated by arteriovenous shunt thrombosis model. The results showed that concomitant use of vicagrel (5, 10, or 20 mg/kg) led to an average of 55% and 77% increases in systemic exposure of aspirin (Cmax and AUC0-t) and 2.8-fold increase in suppression of AA-induced platelet aggregation in mice when compared with use of aspirin alone. In the rat thrombus formation model, vicagrel (1 mg/kg) enhanced inhibition of thrombosis formation by aspirin (5 mg/kg), but not vice versa. We conclude that vicagrel increases platelet responses to aspirin and also enhances inhibition of thrombus formation of aspirin due to decreased CES2-catalyzed aspirin inactivation in rodents, and that an integrated net effect on thrombus formation in vivo is superior to inhibition of AA- or ADP-induced platelet aggregation ex vivo by either of the two drugs if taken concomitantly.
Subject(s)
Aspirin/pharmacology , Phenylacetates/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Thiophenes/pharmacology , Thrombosis/drug therapy , Animals , Aspirin/administration & dosage , Aspirin/metabolism , Blood Platelets/drug effects , Blood Platelets/metabolism , Dose-Response Relationship, Drug , Drug Interactions , Drug Therapy, Combination , Inactivation, Metabolic , Male , Mice, Inbred C57BL , Phenylacetates/administration & dosage , Phenylacetates/metabolism , Platelet Aggregation Inhibitors/administration & dosage , Platelet Aggregation Inhibitors/metabolism , Rats, Sprague-Dawley , Thiophenes/administration & dosage , Thiophenes/metabolism , Thrombosis/metabolismABSTRACT
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. EXPERIMENTAL APPROACH: IL-10 KO and WT mice were administered vicagrel, then their plasma H4 (active metabolite of vicagrel) concentrations were determined by LC-MS/MS, and inhibition of ADP-induced whole-blood platelet aggregation by vicagrel was assessed with an aggregometer. The mRNA and protein levels of several relevant genes between IL-10 KO and WT mice were measured by qRT-PCR and Western blots, respectively. Intestinal Aadac protein levels were measured in IL-10 WT mice injected i.p. with vehicle control, Stattic, or BAY 11-7082. KEY RESULTS: Compared with WT mice, IL-10 KO mice exhibited significantly increased plasma levels of H4 and enhanced platelet responses to vicagrel, as well as significantly higher mRNA and protein levels of arylacetamide deacetylase (Aadac) in the intestine. In WT mice, STAT3, not NF-κB, mediated Aadac expression in the intestine. CONCLUSIONS AND IMPLICATIONS: IL-10 suppresses metabolic activation of vicagrel through down-regulation of Aadac in mouse intestine in a STAT3-dependent manner and, consequently, attenuates platelet responses to vicagrel, suggesting that the antiplatelet effect of vicagrel may be modulated by changes in plasma IL-10 levels in relevant clinical settings.
Subject(s)
Carboxylic Ester Hydrolases/metabolism , Interleukin-10/metabolism , Intestinal Mucosa/metabolism , Phenylacetates/pharmacology , Platelet Aggregation Inhibitors/pharmacology , STAT3 Transcription Factor/metabolism , Thiophenes/pharmacology , Animals , Interleukin-10/genetics , Male , Mice, Inbred C57BL , Mice, Knockout , Phenylacetates/pharmacokinetics , Platelet Aggregation/drug effects , Platelet Aggregation Inhibitors/pharmacokinetics , Thiophenes/pharmacokinetics , Up-RegulationABSTRACT
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. To clarify whether there could be such an important drug-drug interaction, the differences in both the formation of vicagrel active metabolite H4 and the inhibition of adenosine diphosphate-induced platelet aggregation by vicagrel were measured and compared between mice treated with vicagrel alone or in combination with aspirin. The plasma H4 concentration was determined by liquid chromatography-tandem mass spectrometry, and the inhibition of platelet aggregation by vicagrel was assessed by whole-blood platelet aggregation. Compared with vicagrel (2.5 mg·kg) alone, concurrent use of aspirin (5, 10, or 20 mg·kg) significantly decreased systemic exposure of H4, an average of 38% and 41% decrease in Cmax and AUC0-∞ in mice when in combination with aspirin at 10 mg·kg, respectively. Furthermore, concomitant use of aspirin (10 mg·kg) and vicagrel (2.5 mg·kg) resulted in an average of 66% reduction in the inhibition of adenosine diphosphate-induced platelet aggregation by vicagrel. We conclude that aspirin significantly attenuates the formation of vicagrel active metabolite H4 and platelet response to vicagrel in mice, and that such an important drug-drug interaction would appear in clinical settings if vicagrel is taken with aspirin concomitantly when marketed in the future.
Subject(s)
Aspirin/pharmacology , Blood Platelets/drug effects , Phenylacetates/pharmacology , Platelet Aggregation Inhibitors/pharmacology , Platelet Aggregation/drug effects , Thiophenes/pharmacology , Activation, Metabolic , Animals , Aspirin/metabolism , Blood Platelets/metabolism , Carboxylesterase , Carboxylic Ester Hydrolases/metabolism , Chromatography, Liquid , Drug Interactions , Male , Mice, Inbred C57BL , Phenylacetates/blood , Phenylacetates/pharmacokinetics , Platelet Aggregation Inhibitors/blood , Platelet Aggregation Inhibitors/pharmacokinetics , Platelet Function Tests , Tandem Mass Spectrometry , Thiophenes/blood , Thiophenes/pharmacokineticsABSTRACT
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 Abcc3 (ATP-binding cassette, subfamily C, member 3) knockout (KO) and wild-type (WT) mice. We also evaluated the ATP-dependent uptake of clopidogrel and CLP-G as well as estradiol-17ß-d-glucuronide into human recombinant MRP3 inside-out membrane vesicles in the presence or absence of ATP. The results indicated that the liver-to-plasma ratio of CLP-G was 11-fold higher in KO mice than in WT mice, and that uptake of CLP-G (1 or 10 µM each) into the membrane vesicles was 11.8- and 3.8-fold higher in the presence of ATP than in the presence of AMP, respectively. We conclude that Mrp3 transports CLP-G from the hepatocytes into blood in an ATP-dependent manner.
ABSTRACT
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 Vmax value of 120.9 pmol/min/mg protein, 3- to 12-fold higher than that of the other rUGT isoforms tested. According to relative activity factor approach, the relative contribution of rUGT2B7 to CCA glucuronidation was estimated to be 58.6%, with the minor contributions (3%) from rUGT1A9. Moreover, the glucuronidation of CCA followed Michaelis-Menten kinetics with a mean Km value of 372.9 µM and 296.4 µM for pooled HLMs and rUGT2B7, respectively, showing similar affinity for both. The formation of CAG was significantly inhibited by azidothymidine and gemfibrozil (well-characterized UGT2B7 substrates) in a concentration-dependent manner, or by fluconazole (a typical UGT2B7-selective inhibitor) in a time-dependent manner, for both HLMs and rUGT2B7, respectively. In addition, CCA inhibited azidothymidine glucuronidation (catalyzed almost exclusively by UGT2B7) by HLMs and rUGT2B7 in a concentration-dependent manner, indicating that CCA is a substrate of UGT2B7. These results reveal that UGT2B7 is the major enzyme catalyzing clopidogrel glucuronidation in the human liver, and that there is the potential for drug-drug interactions between clopidogrel and the other substrate drugs of UGT2B7.
Subject(s)
Glucuronides/metabolism , Glucuronosyltransferase/metabolism , Ticlopidine/analogs & derivatives , Clopidogrel , Drug Interactions , Fluconazole/pharmacology , Gemfibrozil/pharmacology , Glucuronosyltransferase/antagonists & inhibitors , Humans , Isoenzymes/metabolism , Kinetics , Microsomes, Liver/metabolism , Recombinant Proteins/metabolism , Ticlopidine/metabolism , Zidovudine/pharmacologyABSTRACT
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. Of them, metabolic conversion of vicagrel and PLD-301 to 2-oxo-clopidogrel is catalyzed by intestinal carboxylesterase 2 and alkaline phosphatase, respectively. In this review article, we summarized all evidence on highly efficient bioconversion to their shared precursor of clopidogrel active metabolite and the mechanisms underlying such a pronounced improvement. These drugs in the pipeline would be promising antiplatelet drugs that could be superior to clopidogrel in future patient care.
Subject(s)
Drug Discovery/trends , Drug Resistance/drug effects , Platelet Aggregation Inhibitors/chemistry , Ticlopidine/analogs & derivatives , Acute Coronary Syndrome/blood , Acute Coronary Syndrome/drug therapy , Acute Coronary Syndrome/epidemiology , China/epidemiology , Clopidogrel , Drug Discovery/methods , Drug Resistance/physiology , Humans , Phenylacetates/administration & dosage , Phenylacetates/chemistry , Platelet Aggregation Inhibitors/administration & dosage , Prodrugs/administration & dosage , Prodrugs/chemistry , Thiophenes/administration & dosage , Thiophenes/chemistry , Ticlopidine/administration & dosage , Ticlopidine/chemistryABSTRACT
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. Results indicated that Abcc3 KO mice exhibited increased formation of CAM and greater systemic exposure to clopidogrel and enhanced inhibition of adenosine diphosphate-induced platelet aggregation ex vivo by clopidogrel when compared with well-matched WT mice. We conclude that Abcc3 KO mice have enhanced platelet response to clopidogrel due to increased formation of CAM.
Subject(s)
Blood Platelets/drug effects , Multidrug Resistance-Associated Proteins/deficiency , Platelet Activation/drug effects , Platelet Aggregation Inhibitors/pharmacology , Ticlopidine/analogs & derivatives , Animals , Blood Platelets/metabolism , Clopidogrel , Dose-Response Relationship, Drug , Male , Mice , Mice, Knockout , Platelet Activation/physiology , Platelet Aggregation Inhibitors/metabolism , Ticlopidine/metabolism , Ticlopidine/pharmacologyABSTRACT
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. Results are as follows: In respect of internal cross validation, the coefficient of determination (R2) of untreated, pretreatment with MSC+1thD, pretreatment with 1thD+SNV were 0.920 2, 0.967 0 and 0.976 7 respectively. Root mean square error of prediction (RMSEP) were 1.533 7, 1.011 2 and 0.837 1 respectively. In respect of external validation, the coefficient of determination (R2) of untreated, pretreatment with MSC+ 1thD, pretreatment with 1thD+SNV were 0.805, 0.976 and 0.992 respectively. The average absolute error was 1.456, 0.818, 0.515 respectively. There was no significant difference between chemical and predicted values (Turkey multiple comparison), so we think near infrared spectrum analysis is more feasible than chemical measurement. Among the different pretreatment, the first derivation and standard normal variate (1thD+SNV) have higher coefficient of determination (R2) and lower error value whether in internal validation and external validation. In other words, the calibration model has higher precision and less error by pretreatment with 1thD+SNV.
Subject(s)
Oryza/chemistry , Seeds/chemistry , Spectroscopy, Near-Infrared , Starch/chemistry , Calibration , Edible Grain/chemistry , Least-Squares Analysis , Models, TheoreticalABSTRACT
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-1ß) 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. Results indicated that ASP protected chondrocytes from H2O2-induced oxidative stress and subsequent cell injury through its antioxidant, antiapoptotic and anti-inflammatory effects in vitro. Our study suggests that ASP could become a therapeutic supplementation for the treatment of OA.
Subject(s)
Angelica sinensis/chemistry , Antioxidants/pharmacology , Apoptosis/drug effects , Chondrocytes/cytology , Chondrocytes/drug effects , Hydrogen Peroxide/pharmacology , Polysaccharides/pharmacology , Animals , Antioxidants/isolation & purification , Biomarkers/metabolism , Cell Survival/drug effects , Chondrocytes/metabolism , Cytoprotection/drug effects , Down-Regulation/drug effects , Male , Polysaccharides/isolation & purification , Rats , Up-Regulation/drug effectsABSTRACT
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. This review discusses the discovery of CRISPR, the development of the CRISPR/Cas system, and its applications as a new gene editing system.