Dorota Danielak

HPLC-MS/MS method for the simultaneous determination of clopidogrel, its carboxylic acid metabolite and derivatized isomers of thiol metabolite in clinical samples.

A fast and reproducible HPLC-MS/MS method was developed for the simultaneous determination of clopidogrel (CLP), its carboxylic acid derivative (CLPM), derivatized thiol metabolite isomers MP-H3 and the active MP-H4 in incurred human plasma. CLP, CLPM, MP-H3 and MP-H4 isomers together with the internal standard piroxicam were extracted from plasma samples using a simple protein precipitation with acetonitrile. The analytes were separated on HPLC Zorbax Plus C18 column via gradient elution with water and acetonitrile, both containing 0.1% (v/v) formic acid. Detection of the analytes were performed on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. Calibration curves of the analytes prepared in 250μL plasma were found to be linear in ranges: 0.25-5.00ng/mL for CLP, 0.25-50.00ng/mL for MP-H3 and MP-H4 isomers and 50-10,000ng/mL for CLPM. The lower limit of quantitation was 0.25ng/mL for CLP, MP-H3, MP-H4 and 50.00ng/mL for CLPM. Intra- and inter-assay precision, expressed as relative standard deviation, was ≤18.1% for CLP, ≤15.2% for CLPM, ≤10.1% for MP-H3 and ≤19.9% for MP-H4. Intra- and inter-day accuracy of the method, expressed as relative error, was ≤16%. The analytes were stable in samples stored for 6h in autosampler, in plasma samples for 24h at room temperature and for 3 months at -25°C. Resolution of CLP, CLPM and MP-H3 and MP-H4 isomers of thiol metabolite during one analytical run was reported in patient plasma. The HPLC-MS/MS method was applied for pharmacokinetic studies of CLP and its metabolites in patients treated with daily dose of 75mg CLP.

Genetic and non-genetic factors affecting the response to clopidogrel therapy

Introduction: Clopidogrel (CLP) is a second-generation thienopyridine that prevents platelet aggregation by inhibiting the adenosine diphosphate receptor located on the platelet surface. The use of CLP in combination with aspirin has become standard treatment in patients with acute coronary syndromes and stent implantation. Data suggests that a significant percentage of individuals treated with CLP do not receive the expected therapeutic benefit because of a decreased platelet inhibition. The clinical consequences of an inadequate platelet response are cardiovascular complications, which can lead to acute myocardial infarction, stroke and death. The mechanism underlying CLP resistance is multifactorial and includes genetic polymorphisms and non-genetic causes (such as drug–drug interactions, co-morbidities, age). Areas covered: This article reviews the so-far accumulated evidence on the role of genetic polymorphisms and non-genetic factors, as determinants of the antiplatelet response to CLP. Pharmacodynamic and clinical aspects of the CLP nonresponsiveness are also presented. Relevant papers were identified by an extensive PubMed search using appropriate keywords. Expert opinion: Impaired platelet inhibition in CLP poor responders is a real problem, as it leads to serious clinical consequences. Therefore, prediction models that include pharmacogenetic knowledge and non-genetic risk factors of low response to the drug are needed in the individualization of antithrombotic therapy. Alternative antiplatelet strategies that should be considered to overcome this problem include dose modification, adjunctive antiplatelet drug usage, and use of newer agents.

The influence of genetic polymorphism of Cyp2c19 isoenzyme on the pharmacokinetics of clopidogrel and its metabolites in patients with cardiovascular diseases

An extensive investigation on pharmacokinetics of clopidogrel and its metabolites as well as pharmacodynamics of the drug was performed in patients with cardiovascular disease carrying various alleles coding CYP2C19 isoenzyme. The influence of non‐genetic factors on the clopidogrel response was also studied. Plasma concentrations of clopidogrel, its carboxylic metabolite, and diastereoisomers of a thiol metabolite (the inactive H3 and the active H4) following an administration of 75 mg of the drug were determined in three groups of patients divided with respect to their CYP2C19 genotype: ultrametabolizers, extensive metabolizers, and intermediate metabolizers. The mean peak plasma concentration of H4 in intermediate metabolizers was 3.1‐ and 2.8‐fold lower than that of ultrametabolizers (P = 0.055) and extensive metabolizers (P = 0.026), respectively. The mean H4 area under the curve (AUC0–24 h) for intermediate metabolizers were significantly lower than that for ultrametabolizers (P = 0.046). Intermediate metabolizers exhibited a significantly higher platelet aggregation than ultrametabolizers and extensive metabolizers (P = 0.035). A multivariate analysis showed that the effect of CYP2C19*2 allele on an ADP‐induced platelet aggregation was better pronounced in the presence of non‐genetic risk factors (P = 0.008).

Impact of common ABCB1 polymorphism on pharmacokinetics and pharmacodynamics of clopidogrel and its metabolites

The reasons of clopidogrel (CLP) resistance are still unclear. The response to CLP may be influenced by both genetic and non‐genetic factors. Among genetic factors, common polymorphisms in the gene coding glycoprotein‐P (P‐gp, MDR1 and ABCB1) are considered as potential determinants of the efficacy of CLP treatment. The aim of this study was to evaluate the influence of ABCB1 3435C>T genetic polymorphism on the pharmacokinetics and pharmacodynamics of CLP and its metabolites: diastereoisomers of thiol metabolite (the inactive H3 and the active H4) and inactive carboxylic derivative.

Determinants of high on-treatment platelet reactivity and agreement between VerifyNow and Multiplate assays

Abstract Dual antiplatelet therapy with clopidogrel is a regimen used before and after drug-eluting stent (DES) implantation. Point-of-care platelet reactivity assays are easy-to-use methods to determine adequate response to the drug. The aim of this study was a comparison of the two platelet reactivity assays: Multiplate® and VerifyNow® and an identification of factors potentially influencing the results of these tests, including common genetic polymorphisms. The study included 39 patients receiving 75 mg clopidogrel daily before angioplasty with DES implantation. Platelet reactivity was measured with Multiplate and P2Y12 VerifyNow assays. Genetic polymorphisms of CYP2C19*2, ABCB1 3435C > T, and CYP3A4*1G were determined with PCR-RFLP method and CYP2C19*17 was determined by means of an allele-specific PCR. Agreement between Multiplate and VerifyNow assays was poor (Cohen’s κ = 0.056, p = .273). Hematocrit significantly negatively correlated with VerifyNow assayed platelet reactivity (r = −.487, p = .002). Female sex was significantly associated with higher VerifyNow assay results after adjustment to hematocrit (253.2 ± 47.6 PRU vs. 195.9 ± 56.9 PRU, p = .013) and the prevalence of high-on-treatment platelet reactivity (OR: 8.50; 95% CI 1.13–77.60, p = .024). Reactivity measured with Multiplate was lower in women (82.3 vs. 175.6 AU·min, p = .037) and in patients who received calcium channel blockers (74.7 vs. 191.7 AU·min, p = .002). None of the studied polymorphisms significantly influenced platelet aggregation measurements. In conclusion, different aspects modify between-patient variability of the Multiplate and VerifyNow assays and agreement between those two assays was poor.

Ticagrelor in modern cardiology - an up-to-date review of most important aspects of ticagrelor pharmacotherapy

ABSTRACT Introduction: Ticagrelor is a first drug of a new chemical class cyclopentyltriazolopyrimidines. It is an antiplatelet agent with a unique mechanism of action, allowing a direct and reversible competitive inhibition of P2Y12 receptor. According to newest guidelines, it is recommended for prevention of thrombotic events in patients with acute coronary syndromes. Moreover, it is preferred over clopidogrel, an older generation antiplatelet drug, and therefore gains more interest in modern cardiology and vascular medicine. Areas covered: This review is a comprehensive and thorough summary of the most important findings on ticagrelor. Pharmacokinetics, pharmacogenetics, drug-drug interactions, adverse effects, efficacy in specific patient populations and off-label properties of ticagrelor are discussed in this paper. Moreover, the results from pivotal clinical trials are presented. Expert opinion: Introduction of ticagrelor, a first directly-acting and reversible P2Y12 inhibitor, gave some new possibilities as the efficacy of older drugs was often insufficient. Despite some drawbacks, such as a risk of bleeding events or dyspnea, a rapid onset of action, consistency in the antiplatelet effect and reports on pleiotropic properties make this drug a promising candidate for a first-choice antiplatelet agent in patients with acute coronary events.

HPCE AND HPLC METHODS FOR DETERMINATION OF CLOPIDOGREL AND ITS CARBOXYLIC ACID METABOLITE IN BIOLOGICAL SAMPLES: A COMPARATIVE ANALYSIS

A comparison between two analytical techniques (HPCE and HPLC) with UV detection for the determination of clopidogrel (CLP) and its carboxylic acid metabolite (CLPM) in biological fluids was performed. Resolution of the analytes by HPLC method was obtained on a C18 column with the use of a mobile phase consisted of acetonitrile and 0.03 M phosphate buffer at pH 3.0. HPCE analysis was performed in an uncoated capillary filled with an 0.025 M phosphate buffer at pH 2.5. A comparison of the both methods was performed in terms of validation parameters. Statistical analysis, Bland-Altman method, and ATE/LER zone method were also applied for assessing agreement between two methods. It was concluded that the HPLC method produced comparable results to the HPCE method with respect to the analysis of CLPM in plasma samples. Moreover, HPCE offered several advantages such as a lower solvent consumption and a higher selectivity of the analytes in plasma and urine samples. On the other hand, HPLC displayed excellent retention time repeatability in comparison to HPCE method. This study highlights the effective possibilities of application of both methods in the analysis of CLPM in plasma samples obtained from patients undergoing CLP therapy prior to percutaneous coronary intervention.

Relationship between exposure to treosulfan and its monoepoxytransformer – An insight from population pharmacokinetic study in pediatric patients before hematopoietic stem cell transplantation

&NA; Treosulfan (TREO), a structural analog of busulfan, is currently studied as a myeloablative agent in conditioning regimens before hematopoietic stem cell transplantation in pediatric patients. High exposure to TREO (>1650 mg*h/mL) might be related to early toxicity, especially skin toxicity and mucositis. The aim of the present study was to investigate a potential relationship between exposure to TREO and its monoepoxytransformer (S,S‐EBDM), as well as variability of the pharmacokinetics of these entities by means of a population pharmacokinetic approach with a non‐linear mixed‐effects analysis. The study included data from 14 children with malignant and non‐malignant diseases treated with TREO in daily doses 10–14 g/m2. The parent‐metabolite population pharmacokinetic model was developed in NONMEM 7.3 software. Upon the constructed model, an extensive simulation was performed to assess the correlation between exposure to TREO and S,S‐EBDM. It was found that TREO and S,S‐EBDM pharmacokinetics was best described with 2‐compartmental and 1‐compartmental linear models, respectively. The vast majority (>65%) of TREO was transformed to S,S‐EBDM. Overall, a considerable interpatient variability of pharmacokinetic parameters was observed, especially the clearance of S,S‐EBDM. A weak correlation was found between the exposure to TREO and S,S‐EBDM (r = 0.1681, p < 0.0001). Also, patients with an exposure to TREO above 1650 mg*h/mL were most likely to have also a high exposure to S,S‐EBDM (35.38 &mgr;M*h vs. 43.14 &mgr;M*h, p < 0.0001). In summary, a parent‐metabolite population pharmacokinetic model for TREO and S,S‐EBDM was developed for the first time. It was shown that there is a weak correlation between exposure to TREO and S,S‐EBDM. Therefore therapeutic drug monitoring of not only prodrug but also its active epoxide might be needed. Graphical abstract Figure. No Caption available.

Assessment of the Risk of Rhabdomyolysis and Myopathy During Concomitant Treatment with Ticagrelor and Statins

The introduction of ticagrelor, one of the first directly-acting oral antiplatelet drugs, provided new possibilities in the prevention of thrombotic events in patients with acute coronary syndromes (ACS). Current guidelines recommend ticagrelor in dual antiplatelet therapy with aspirin over clopidogrel for prevention of stent thrombosis in patients with ACS. Moreover, in the management of ACS, lipid-lowering treatment with high-intensity statin therapy is advised for secondary prevention of cardiovascular events over the long term. Despite the apparent advantages of combined antiplatelet and lipid-lowering treatments, a possible interaction between statins and ticagrelor may lead to myopathy and rhabdomyolysis. In this review, relevant information was gathered on the ticagrelor-statin interaction that might lead to this life-threatening condition. This review focuses on the most widely used statins—simvastatin, atorvastatin, and rosuvastatin. Possible mechanisms of this interaction are discussed, including CYP3A4 isoenzymes, organic anion transporter polypeptide (OATPs), P-glycoprotein and glucuronidation. PubMed database was searched for relevant case reports and all data gathered from the introduction of ticagrelor to March 2018 are presented and discussed. In summary, co-administration of statins and ticagrelor was found to be relatively safe in routinely prescribed doses. However, caution should be exercised, especially in elder populations.

Development of a Limited Sampling Strategy for the Estimation of Exposure to High-Dose Etoposide After Intravenous Infusion in Pediatric Patients

Background: Etoposide (VP-16), a podophyllotoxin derivative, is used in conditioning regimens before allogeneic hematopoietic stem cell transplantation in children with acute lymphoblastic leukemia. The aim of this study was to develop a limited sampling strategy (LSS) suitable for the prediction of exposure to VP-16 defined as area under time–concentration curve (AUC). Methods: The study included 28 pediatric patients with acute lymphoblastic leukemia, who were administered a 4-hour infusion of 60 mg/kg VP-16. VP-16 concentrations were determined in samples collected 4–124 hours after the beginning of infusion. On obtaining the pharmacokinetic (PK) profiles, a population PK model was developed in NONMEM (ICON Development Solutions, Hanover, MD) with first-order conditional estimation with interaction algorithm. LSSs were chosen by means of a multivariate regression analysis and cross-validated with a leave-one-out approach. Predictive performance of LSSs was assessed by calculating relative prediction error (PE), mean PE, mean absolute PE, and root mean squared PE for model-predicted and observed AUC. Results: VP-16 PKs was best described by a 2-compartment first-order model, and a large variability in the PK parameters was observed. A 3-sample strategy allowed the estimation of VP-16 with highest accuracy and precision (mean relative PE = 0.18%, 95% confidence interval, 1.73%–2.09%; mean absolute relative PE = 3.47%, 95% confidence interval, 2.28%–4.66%; root mean squared PE = 4.43%). The final equation was AUC = 6.85 × C6 h + 3.88 × C12 h + 46.11 × C28 h + 282.0 (adjusted R2 = 0.9540). Conclusions: In conclusion, developed LSS allows accurate and precise estimation of VP-16 AUC and might be useful for therapeutic drug monitoring.