HLA-A*33:03-Restricted Activation of Ticlopidine-Specific T-Cells from Human Donors.

The HLA class I allele HLA-A*33:03 is a risk factor for ticlopidine-induced liver injury. Herein, we show HLA class I-restricted ticlopidine-specific CD8+ T-cell activation in healthy donors expressing HLA-A*33:03. Cloned CD8+ T-cells proliferated and secreted IFN-γ in the presence of ticlopidine and autologous antigen presenting cells. A reduction of the T-cell response after blocking with HLA-class I and HLA-A*33 antibodies indicates that the interaction between drugs and the HLA allele detected in genetic association studies may be important for T-cell activation.

The mechanism and risk factors of clopidogrel-induced liver injury.

CONTEXT AND OBJECTIVE: Clopidogrel (CLP) is a prodrug which is widely used as a platelet aggregation inhibitor. Hepatotoxicity is rare but a potentially serious adverse reaction that is associated with CLP. Thiophene in CLP (the thienopyridine derivative) is a group that is easily oxidated by cytochrome P450 enzymes (CYP450s) to generate reactive metabolites (RMs), it may be implicated in the mechanism of CLP-induced hepatotoxicity. CYP2C19 and CYP2B6 are important CYP450s involved in the metabolism and activation of CLP, and the aim of this study is to investigate whether the metabolites of CYP2C19 and CYP2B6 are associated with the CLP-induced liver injury. METHOD: Primary rat hepatocytes are applied to evaluate the hepatotoxicity of CLP. Glutathione-depleted mouse model is used to evaluate whether this toxicity of CLP is metabolized by CYP450s. We also used HepG2 cells co-incubated with recombinant CYP2B6 and CYP2C19 enzymes to further assess whether the metabolites of CYP2C19 and CYP2B6 are associated with the CLP-induced hepatocellular toxicity. RESULT: CLP in high dose (100 µM and 300 µM) showed cytotoxicity in primary rat hepatocytes assay. Administration of CLP with l-buthionine-S, R-sulfoxinine (BSO) for seven days enhanced the liver injury of CLP. The level of ALT, AST and TBIL in plasma increased significantly, and the histopathological results showed the obvious liver injury; Pretreatment of 1-aminobenzotriazole, a nonspecific inhibitor of CYP450s, suppressed CLP-induced hepatotoxicity; CLP showed a dose-dependent toxicity in HepG2/CYP2C19 enzyme and HepG2/CYP2B6 enzyme models. CONCLUSION: High activities of CYP2C19 and CYP2B6 are the risk factors for hepatocellular toxicity of CLP.

Aspirin and Clopidogrel Inhibit Aneurysm Healing after HydroCoil Implantation in External Carotid Artery Aneurysm Model.

PURPOSE: To understand whether the use of antiplatelet agents leads to less intra-aneurismal tissue formation following coil implantation in a rat end-pouch external carotid artery (ECA) aneurysm model. METHODS: End-pouch ECA aneurysms were created in adult rats and were then embedded with either platinum or HydroCoils. Rats were treated either with aspirin, clopidogrel, aspirin + clopidogrel, or saline for 2 weeks after coil implantation. At 2 weeks after coil implantation, rats were sacrificed and the aneurysm pouch was removed for histological and immunohistochemical analysis. A blinded single observer calculated the percentage of the organized area and the residual length of elastic lamina within the aneurysm. Student's t-test was used to compare data from image analysis between the different groups. RESULTS: Within the platinum group, the organized tissue area was not affected by antiplatelet administration (aspirin versus saline, P = .83; clopidogrel versus saline, P = .46; aspirin + clopidogrel versus saline, P = .54). For the HydroCoil group, the organized tissue area was significantly reduced (aspirin versus saline, P = .02; clopidogrel versus saline, P = .04; aspirin + clopidogrel versus saline, P = .02) in rats treated with antiplatelet agents; however, no difference (aspirin versus clopidogrel, P = .8; aspirin versus aspirin + clopidogrel, P = .3; clopidogrel versus aspirin + clopidogrel, P = .5) was found among type or combination of antiplatelets administered. HydroCoil-treated aneurysms had a similar number of macrophages compared to the platinum group (P = .3819); however, the HydroCoil group had significant suppression of macrophages in the groups treated with combined antiplatelets (P = .02). CONCLUSION: Following HydroCoil implantation, the area of organized tissue is diminished significantly in a rat end-pouch ECA aneurysm model treated with antiplatelets.

Toxicity studies of a bioactive protein with antithrombotic-thrombolytic activity, DLBS1033.

DLBS1033 is a bioactive protein extract containing Lumbricus rubellus and has been known to have antithrombotic/thrombolytic activity. The present study was aimed to assess the safety aspect of DLBS1033 in a preclinical setting, which included observation on toxic signs after acute and repeated administrations, and the drug's effect on prenatal development and drug interaction. In acute toxicity study, a high dose level (16.2 g/kg) of DLBS1033 was well tolerated. In subchronic toxicity study, after the doses of 270, 540 and 1080 mg/kg of DLBS1033 per day, no mortality was observed and other parameters were all observed to be normal. In prenatal developmental toxicity, no observed adverse effect level (NOAEL) of DLBS1033 was observed at a moderate dose (540 mg/kg). Coadministration of DLBS1033 with clopidogrel or aspirin did not cause gastric lesions, except when all three drugs were coadministrated. Taken together, results of the present study suggested that DLBS1033 is safe for long-term administration, with a caution at a high dose used during pregnancy, and can be used in combination with one of the antiplatelet drugs.

Ticlopidine-induced hepatotoxicity in a GSH-depleted rat model.

We investigated hepatotoxicity induced by ticlopidine (TIC) in glutathione (GSH)-depleted rats by pre-treatment of a well-known GSH synthesis inhibitor, L-buthionine-S,R-sulfoxinine (BSO). Although sole administration of either TIC or BSO showed no signs of hepatotoxicity, combined administration of TIC with BSO induced hepatotoxicity, which was characterized by centrilobular necrosis of the hepatocytes and an elevation of plasma alanine aminotransferase activity. Administration of radio-labeled TIC in combination with BSO resulted in significantly higher covalent binding to rat liver proteins than that observed after sole dosing of radio-labeled TIC. Pre-treatment of 1-aminobenzotriazole, a non-specific inhibitor of P450s, completely suppressed both hepatotoxicity and the increased hepatic covalent binding caused by TIC co-treatment with BSO. The results obtained in this animal model suggest that GSH depletion and covalent binding may be involved in hepatotoxicity induced by TIC. These observations may help to understand the risk factors and the mechanism of hepatotoxicity of TIC in humans.

Cell-based high-throughput screening for the evaluation of reactive metabolite formation potential.

Here, we established a high-throughput in vitro assay system to predict reactive metabolite (RM) formation. First, we performed the glutathione (GSH) consumption assay to monitor GSH levels as an index of RM formation potential using HepaRG cells pretreated with 500 µM D,L-buthionine-(S,R)-sulfoximine (BSO) and then treated with ticlopidine and diclofenac. Both drugs, under GSH-reduced conditions, significantly decreased relative cellular GSH content by 70% and 34%, respectively, compared with that in cells not pretreated with BSO. Next, we examined the correlation between GSH consumption and covalent binding assays; the results showed good correlation (correlation coefficient = 0.818). We then optimized the test compound concentration for evaluating RM formation potential using 76 validation compound sets, and the highest sensitivity (53%) was observed at 100 µM. Finally, using HepG2 cells, PXB-cells, and human primary hepatocytes, we examined the cell types suitable for evaluating RM formation potential. The expression of CYP3A4 was highest in HepaRG cells, suggesting the highest sensitivity (56.4%) of the GSH consumption assay. Moreover, a co-culture model of PXB-cells and HepaRG cells showed high sensitivity (72.7%) with sufficient specificity (85.7%). Thus, the GSH consumption assay can be used to effectively evaluate RM formation potential in the early stages of drug discovery.

Investigation of bioactivation of ticlopidine using linear ion trap/orbitrap mass spectrometry and an improved mass defect filtering technique.

The bioactivation of ticlopidine, a widely used antiplatelet drug, into reactive metabolites and their subsequent covalent binding to cellular macromolecules are thought to be involved in the occurrence of idiosyncratic hepatotoxicity in patients. In the present study, GSH/stable isotope-labeled GSH was used as the trapping agent to investigate the bioactivation pathways of ticlopidine in rat liver microsomes. The samples were analyzed by high-resolution linear ion trap/Orbitrap followed by multiple mass defect filtering (MDF). In total, 17 GSH adducts were detected, and a comprehensive profile for ticlopidine bioactivation has been proposed. The results show that ticlopidine can be directly bioactivated by rat P450s, forming GSH adducts through two major bioactivation pathways, thiophene-S-oxidation and thiophene epoxidation. These adducts were also formed substantially in human liver microsomes. Moreover, ticlopidine can be metabolized via multiple pathways before giving rise to reactive intermediates. The GSH adducts derived from epoxidation of the chlorophenyl moiety of ticlopide and bioactivation of N-dealkylated metabolites are reported here for the first time. The formation of a number of ticlopidine GSH adducts from diversified metabolic pathways mediated by P450s implies a high potential for protein binding and provides a conceivable link between the high reactivity of ticlopidine after bioactivation and the ticlopidine-induced toxicity. Additionally, the current approach has the following advantages as compared to previous high-resolution LC/MS methodologies. First, novel MDF utilized doubly charged ions as filter templates to detect the GSH adducts, mainly doubly charged in the ion source, resulting in broader detection coverage. Second, multiple mass defect filter templates were for the first time applied to reveal different classes of GSH adducts. Finally, a quick check of isotopic doublets and full examination of isotope fingerprints in the accurate mass were introduced to screen out false positives and enhance the identification of low abundant GSH adducts.

Anticoagulant and antiplatelet usage associates with mortality among hemodialysis patients.

Many prescribe anticoagulants and antiplatelet medications to prevent thromboembolic events and access thrombosis in dialysis patients despite limited evidence of their efficacy in this population. This retrospective cohort study examined whether use of warfarin, clopidogrel, and/or aspirin affected survival in 41,425 incident hemodialysis patients during 5 yr of follow-up. The prescription frequencies for warfarin, clopidogrel, and aspirin were 8.3, 10.0, and 30.4%, respectively, during the first 90 d of initiating chronic hemodialysis. Compared with the 24,740 patients receiving none of these medications, Cox proportional hazards analysis suggested that exposure to these medications was associated with increased risk for mortality (warfarin hazard ratio [HR] 1.27 [95% confidence interval (CI) 1.18 to 1.37]; clopidogrel HR 1.24 [95% CI 1.13 to 1.35]; and aspirin HR 1.06 [95% CI 1.01 to 1.11]). The increased mortality associated with warfarin or clopidogrel use remained in stratified analyses. A covariate- and propensity-adjusted time-varying analysis, which accounted for longitudinal changes in prescription, produced similar results. In addition, matching for treatment facility and attending physician revealed similar associations between prescription and mortality. We conclude that warfarin, aspirin, or clopidogrel prescription is associated with higher mortality among hemodialysis patients. Given the possibility of confounding by indication, randomized trials are needed to determine definitively the risk and benefit of these medications.

The FDA-approved drugs ticlopidine, sertaconazole, and dexlansoprazole can cause morphological changes in C. elegans.

Urgent need for treatments limit studies of therapeutic drugs before approval by regulatory agencies. Analyses of drugs after approval can therefore improve our understanding of their mechanism of action and enable better therapies. We screened a library of 1443 Food and Drug Administration (FDA)-approved drugs using a simple assay in the nematode C. elegans and found three compounds that caused morphological changes. While the anticoagulant ticlopidine and the antifungal sertaconazole caused both accumulations that resulted in distinct distortions of pharyngeal anatomy and lethality upon acute exposure, the proton-pump inhibitor dexlansoprazole caused molting defects and required exposure during larval development. Such easily detectable defects in a powerful genetic model system advocate the continued exploration of current medicines using a variety of model organisms to better understand drugs already prescribed to millions of patients.

Clopidogrel versus prasugrel in rabbits. Effects on thrombosis, haemostasis, platelet function and response variability.

The new P2Y(12) antagonist prasugrel produces greater inhibition of ADP-induced platelet aggregation (IPA) and reduction of thrombotic events in patients versus approved doses of clopidogrel, but increases major bleeding. We examined whether IPA level or P2Y(12) receptor occupancy (RO) could be optimized to better balance the efficacy and bleeding effects of these thienopyridines and reduce the response variability in rabbits. Rabbits were given three daily oral doses of clopidogrel (0.3-30 mg/kg/d), prasugrel (0.03-10 mg/kg/d) or vehicle (n = 6-40/group). Electrically-induced carotid artery thrombosis (AT, % thrombus weight reduction), cuticle bleeding time (BT, fold-increase over control), IPA to 20 microM ADP (% inhibition of peak light transmission) and RO (% inhibition of [(33)P]-2MeS-ADP binding to P2Y(1)-blocked platelets) were determined 2-3 hours after the last dose. ED(50) (doses for half-maximal effect, mg/kg/d) of AT, BT, IPA and RO were 1.6, 6.7, 1.9 and 1.4 for clopidogrel vs. 1.2, 1.9, 0.5 and 0.2 for prasugrel. IPA of 30-40% for both compounds produced the optimal balances of efficacy (AT: 50-60%) and BT of about 2-fold with significant RO of 70-80%. IPA of 50-60% achieved higher efficacy (AT: 60-80%), but with increased BT of five- to six-fold and >90% RO. Box-plot suggests no significant difference in the IPA and RO response variability between both compounds. Clopidogrel was 1.3-7 times less potent than prasugrel in rabbits, depending upon which biomarker was studied. The ratio of efficacy: bleeding was most favorable at a moderate IPA of 30% to 40%. Both compounds had similar IPA and RO response variability.