Solid self nano-emulsifying system for the enhancement of dissolution and bioavailability of Prasugrel HCl: in vitro and in vivo studies.

Prasugrel Hydrochloride (PHCl) is an antiplatelet drug. It is a class II drug with variable bioavailability. The objective of this work was to enhance the solubility and hence the bioavailability and efficacy of PHCl. A Self Nano-Emulsifying Drug Delivery System (SNEDDS) was prepared using Kolliphor El, Maisine 35-1, and Transcutol P as surfactant, oil, and co-surfactant, respectively in a ratio 10:72:18 v/v%. The SNEDDS was converted into solid by adsorption onto Neusilin. In vitro release of the drug from SNEDDS in (pH = 4) at 37 °C and 75 rpm for 45 min was studied. The results were compared to those from the unprocessed PHCl and Lexar® (the commercial drug). In-vivo studies (platelet Aggregation and bleeding time) were conducted using rats as animal models. It was found that the particle size of the SNEDDS ranged between 80 and 155 nm and EE% was in the range of 90.2% ± 0.4. The release from SNEDDS was about 84% compared to around 25% from unprocessed PHCl and 65% from Lexar® after 15 min. The platelet aggregation of the formula was lower than the PHCl, and Lexar® indicating higher bioavailability. In conclusion, SNEDDS with high EE% was prepared and was successful in enhancing the solubility, dissolution rate, and the bioavailability.

A Validated LC-MS/MS Method for the Simultaneous Determination of Ticagrelor, Its Two Metabolites and Major Constituents of Tea Polyphenols in Rat Plasma and Its Application in a Pharmacokinetic Study.

Ticagrelor is recommended for management of patients with acute coronary syndromes. Green tea is one of the most popular beverages in China and around the world. Their concomitant use is unavoidable. In this study, a selective and sensitive liquid chromatography-tandem mass spectrometry method for the simultaneous determination of plasma concentrations of ticagrelor, its two metabolites and four major constituents of tea polyphenols (TPs) in rats was developed for co-administration study of ticagrelor and TPs. Diazepam was used as internal standard (IS). Plasma samples were extracted employing a liquid-liquid extraction technique. Chromatographic separation was carried out on a Kinetex C18 column (2.1 × 75 mm, 2.6 µm) by gradient elution using 0.1% formic acid in water, acetonitrile and methanol. Seven analytes and IS were detected by a mass spectrometer with both positive and negative ionization by multiple reaction monitoring mode. The method was fully validated to be reliable and reproducible in accordance with food and drug administration (FDA) guidelines on bioanalytical method validation. The method was then successfully applied for pharmacokinetic study of ticagrelor, its two metabolites and four major constituents of TPs in rat plasma after oral administration of ticagrelor and tea polyphenol extracts.

Effects of ticagrelor on the pharmacokinetics of rivaroxaban in rats.

CONTEXT: Rivaroxaban and ticagrelor are two common drugs for the treatment of atrial fibrillation and acute coronary syndrome. However, the drug-drug interaction between them is still unknown. OBJECTIVE: To investigate the effects of ticagrelor on the pharmacokinetics of rivaroxaban in rats both in vivo and in vitro. MATERIALS AND METHODS: A sensitive and reliable UPLC-MS/MS method was developed for the determination of rivaroxaban in rat plasma. Ten Sprague-Dawley rats were randomly divided into ticagrelor pre-treated group (10 mg/kg/day for 14 days) and control group. The pharmacokinetics of orally administered rivaroxaban (10 mg/kg, single dose) with or without ticagrelor pre-treatment was investigated with developed UPLC-MS/MS method. Additionally, Sprague-Dawley rat liver microsomes were also used to investigate the drug-drug interaction between these two drugs in vitro. RESULTS: The C max (221.34 ± 53.33 vs. 691.18 ± 238.31 ng/mL) and the AUC(0-t) (1060.97 ± 291.21 vs. 3483.03 ± 753.83 µg·h/L) of rivaroxaban increased significantly (p < 0.05) with ticagrelor pre-treatment. The MRT(0-∞) of rivaroxaban increased from 4.41 ± 0.79 to 5.97 ± 1.11 h, while the intrinsic clearance decreased from 9.93 ± 2.55 to 2.89 ± 0.63 L/h/kg (both p < 0.05) after pre-treated with ticagrelor. Enzyme kinetic study indicated that ticagrelor decreased rivaroxaban metabolic clearance with the IC50 value of 14.04 µmol/L. CONCLUSIONS: Our in vivo and in vitro results demonstrated that there is a drug-drug interaction between ticagrelor and rivaroxaban in rats. Further studies need to be carried out to verify whether similar interactions truly apply in humans and whether these interactions have clinical significance.

Population Pharmacokinetic/Pharmacodynamic Modeling of Glenzocimab (ACT017) a Glycoprotein VI Inhibitor of Collagen-Induced Platelet Aggregation.

Glenzocimab (ACT017) is a humanized monoclonal antigen-binding fragment (Fab) directed against the human platelet glycoprotein VI, a key receptor for collagen and fibrin that plays a major role in thrombus growth and stability. Glenzocimab is being developed as an antiplatelet agent to treat the acute phase of ischemic stroke. During a phase I study in healthy volunteers, the population pharmacokinetics (PK) and pharmacodynamics (PD) of glenzocimab were modeled using Monolix software. The PK/PD model thus described glenzocimab plasma concentrations and its effects on ex vivo collagen-induced platelet aggregation. Glenzocimab was found to have dose-proportional, 2-compartmental PK with a central distribution volume of 4.1 L, and first and second half-lives of 0.84 and 9.6 hours. Interindividual variability in clearance in healthy volunteers was mainly explained by its dependence on body weight. The glenzocimab effect was described using an immediate effect model with a dose-dependent half maximal inhibitory concentration: Larger doses resulted in a stronger effect at the same glenzocimab plasma concentration. The mechanism of the overproportional concentration effect at higher doses remained unexplained. PK/PD simulations predicted that 1000-mg glenzocimab given as a 6-hour infusion reduced platelet aggregation to 20% in 100% of subjects at 6 hours and in 60% of subjects at 12 hours after dosing. Simulations revealed a limited impact of creatinine clearance on exposure, suggesting that no dose adjustments were required with respect to renal function. Future studies in patients with ischemic stroke are now needed to establish the relationship between ex vivo platelet aggregation and the clinical effect.

Pharmacokinetics and Bioequivalence of Clopidogrel Hydrogen Sulfate Tablets in Fed and Fasted Conditions: An Open-Label, Randomized, Semireplicated Crossover Study in Healthy Chinese Volunteers.

Clopidogrel is an antiplatelet drug with high intraindividual variability in systemic exposure and efficacy. It has been used for treating atherosclerosis and acute coronary syndrome and in preventing stent restenosis and thrombotic complications after stent implantation in clinical practice for nearly 20 years. In this study we aimed to evaluate the bioequivalence of 2 clopidogrel hydrogen sulfate formulations (75-mg tablets) under fed (n = 66) and fasted (n = 66) conditions by using the reference-scaled average bioequivalence method. An open-label, randomized, 3-sequence and 3-period crossover (3×3), semireplicated study was designed and conducted. Clopidogrel concentration of plasma samples was measured by high-precision liquid chromatography and tandem mass spectrometry. The pharmacokinetic parameters were derived by a noncompartmental model. In the fed condition the geometric least-squares mean ratios of peak concentration (Cmax ) and area under the concentration-time curve (AUC0-t ) were, respectively, 103.38% and 98.97%, and the corresponding 90%CIs were 95.68% to 111.70% and 94.67% to 103.47%. In the fasted condition the geometric least squares mean ratios of Cmax and AUC0-t were, respectively, 106.53% and 105.77%, and the corresponding 90%CIs were 97.62% to 116.25% and 96.96% to 115.38%. According to the criteria for bioequivalence (80.00% to 125.00%), the test formulations of clopidogrel and Plavix were determined to be bioequivalent.

Safety and Pharmacokinetics of DS-1040 Drug-Drug Interactions With Aspirin, Clopidogrel, and Enoxaparin.

DS-1040, a novel low-molecular-weight inhibitor of activated thrombin-activatable fibrinolysis inhibitor, is under development for the treatment of thromboembolic diseases including venous thromboembolism and acute ischemic stroke. Here we describe the results of 3 studies that evaluated the safety and tolerability of DS-1040 along with the effect on DS-1040 pharmacokinetic (PK) parameters, when dosed alone or when coadministered with aspirin (NCT02071004), clopidogrel (NCT02560688), or enoxaparin in healthy subjects. Concomitant administration of single-dose DS-1040 with multiple-dose aspirin, multiple-dose clopidogrel, or single-dose enoxaparin, consistent with clinically relevant dose regimens, was safe and well tolerated with no serious treatment-emergent adverse events (TEAEs), TEAEs leading to discontinuation, bleeding-related TEAEs, and no significant changes in coagulation parameters. DS-1040 did not prolong bleeding time when administered concomitantly with aspirin or clopidogrel. In the aspirin study, DS-1040 PK was evaluated following the concomitant administration with multiple-dose aspirin, where the plasma DS-1040 exposure (peak plasma concentration [Cmax ] and area under the concentration-time curve [AUCinf ]) was to be similar to the data previously published in the first-in-human study of DS-1040 in healthy subjects. The PK parameters of DS-1040 coadministered with clopidogrel were similar to those of DS-1040 alone, with small increases in geometric means for Cmax (7%) and AUClast (9%). When coadministered with enoxaparin, the PK parameters of DS-1040 were not affected (1.1% and 1.5% decreases in geometric means for Cmax and AUClast , respectively). Therefore, concomitant administration of DS-1040 and clopidogrel or enoxaparin did not demonstrate PK drug-drug interactions.

A comparison of multifunctional donors of carbon monoxide: Their anticoagulant, antioxidant, anti-aggregatory and cytotoxicity activities in an in vitro model.

The study examines the effect of two water-soluble carbon monoxide (CO) donors, CORM-3 and CORM-A1, on selected parameters of oxidative stress and hemostasis in human plasma and blood platelets in vitro. It also compares their activity with that of the lipid-soluble CORM-2. The oxidation of amino acid residues in plasma proteins was evaluated by measuring the amounts of thiol and carbonyl groups. Plasma lipid peroxidation was measured as thiobarbituric acid reactive substance (TBARS) concentration. In addition, three haemostatic parameters of plasma were studied, viz. activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT), and one haemostatic parameter of platelets (platelet aggregation). Treatment with CORM-3 and CORM-A1 (all concentrations from 0.1 to 100 µM) decreased thiol group oxidation induced by H2O2/Fe. Incubation with CORM-3 and CORM-A1 also influenced plasma coagulation activity, e.g. CORM-3 and CORM-A1 significantly prolonged TT at the two highest tested concentrations (50 and 100 µM). Only CORM-2 at the highest tested concentration (100 µM) and CORM-3 (50 and 100 µM) reduced platelet aggregation induced by ADP. None of the tested CORMs caused platelet damage. The treatment of various diseases associated with oxidative stress, including cardiovascular diseases, may be enhanced by the administration of CO donors CORM-2 and CORM-3, these being modulators of oxidative stress and hemostasis.

Freeze-Dried Clopidogrel Loaded Lyotropic Liquid Crystal: Box-Behnken Optimization, In-Vitro and In-Vivo Evaluation.

BACKGROUND: Clopidogrel (CLP) suffers from extensive first pass metabolism results in a negative impact on its oral systemic bioavailability. Cubosomes are Lyotropic Liquid Crystalline (LLC) nano-systems comprising monoolein, a steric stabilizer and an aqueous system, it considered a promising carrier for different pharmaceutical compounds. Box-Behnken Design (BBD) is an efficient tool for process analysis and optimization skipping forceful treatment combinations. OBJECTIVE: The study was designed to develop freeze-dried clopidogrel loaded LLC (cubosomes) for enhancement of its oral bioavailability. METHODS: A 33 BBD was adopted, the studied independent factors were glyceryl monooleate (GMO lipid phase), Pluronic F127 (PL F127steric stabilizer) and polyvinyl alcohol powder (stabilizer). Particle Size (PS), Polydispersity Index (PDI) and Zeta Potential (ZP) were set as independent response variables. Seventeen formulae were prepared in accordance with the bottom up approach and in-vitro evaluated regarding PS, PDI and ZP. Statistical analysis and optimization were achieved using design expert software®, then the optimum suggested formula was prepared, in-vitro revaluated, freeze-dried with 3% mannitol (cryoprotectant), solid state characterized and finally packed in hard gelatin capsule for comparative in-vitro release and in-vivo evaluation to Plavix®. RESULTS: Results of statistical analysis of each individual response revealed a quadratic model for PS and PDI where a linear model for ZP. The optimum suggested formula with desirability factor equal 0.990 consisting of (200 mg GMO, 78.15 mg PL F127 and 2% PVA). LC/MS/MS study confirmed significant higher Cmax, AUC0-24h and AUC0-∞ than that of Plavix®. CONCLUSION: The results confirm the capability of developed carrier to overcome the low oral bioavailability.

Impact of low-dose prasugrel on platelet reactivity and cardiac dysfunction in acute coronary syndrome patients requiring primary drug-eluting stent implantation: A randomized comparative study.

OBJECTIVE: The aim of this study was to compare how prasugrel and clopidogrel affect platelet aggregation reactivity, cardiac enzyme release, cardiac remodeling, and the formation of in-stent thrombi after primary percutaneous coronary intervention (PCI). BACKGROUND: The advantages of using prasugrel over clopidogrel in cardiac injury following acute coronary syndrome (ACS) remain unclear. METHODS: A total of 78 ACS patients were randomly allocated into clopidogrel (300 mg loading/75 mg maintenance) or prasugrel (20 mg loading/3.75 mg maintenance) treatment groups, followed by undergoing primary PCI. Platelet reactivity and cardiac enzymes were measured before and after primary PCI. Moreover, cardiac function was measured by ultrasound echocardiography and coronary angioscopic observation was after primary PCI up to 8 months later. RESULTS: Antiplatelet reactivity in the prasugrel treatment group reached optimal levels (P2Y12 reaction units [PRU] less than 262) immediately after the administration and was maintained even at 8 months, independently of the CYP2C19 genotype. Prasugrel treatment significantly suppressed creatine kinase elevation compared to clopidogrel treatment (median value 404 IU/L to 726 IU/L vs. 189 IU/L to 1,736 IU/L, p = 0.018 for maximum values) and reduced left ventricular mass (217.2-168.8 g in prasugrel, p = 0.045; 196.9-176.4 g in clopidogrel, p = 0.061). There were no significant differences in the incidence of in-stent attached thrombi between the two groups. CONCLUSIONS: Compared to clopidogrel, prasugrel produced a stable platelet aggregation inhibitory effect in patients with ACS regardless of CYP2C19 genotype, reduced cardiac enzyme release, and prevented cardiac remodeling after ACS.

Active Micropump-Mixer for Rapid Antiplatelet Drug Screening in Whole Blood.

There is a need for scalable automated lab-on-chip systems incorporating precise hemodynamic control that can be applied to high-content screening of new more efficacious antiplatelet therapies. This paper reports on the development and characterization of a novel active micropump-mixer microfluidic to address this need. Using a novel reciprocating elastomeric micropump design, we take advantage of the flexible structural and actuation properties of this framework to manage the hemodynamics for on-chip platelet thrombosis assay on type 1 fibrillar collagen, using whole blood. By characterizing and harnessing the complex three-dimensional hemodynamics of the micropump operation in conjunction with a microvalve controlled reagent injection system we demonstrate that this prototype can act as a real-time assay of antiplatelet drug pharmacokinetics. In a proof-of-concept preclinical application, we utilize this system to investigate the way in which rapid dosing of human whole blood with isoform selective inhibitors of phosphatidylinositol 3-kinase dose dependently modulate platelet thrombus dynamics. This modular system exhibits utility as an automated multiplexable assay system with applications to high-content chemical library screening of new antiplatelet therapies.

Pharmacokinetics and bioequivalence of low-dose clopidogrel in healthy Chinese volunteers under fasted and fed conditions.

Clopidogrel is an antiplatelet drug whose performance at a low dose (25 mg) have not been evaluated in Chinese patients, who may be subject to different effects from Caucasians. We carried out this evaluation and compared a generic (Taijia) and a reference drug (Plavix®). We evaluated Taijia and Plavix® in 128 subjects, with 64 in a fasted state and 64 receiving a high-fat diet, and computed Cmax, AUC0-∞, and AUC0-t. Reference-scaled average bioequivalence (RSABE) methods and average bioequivalence (ABE) methods were used, and adverse events were assessed. Average maximum plasma concentrations (Cmax) of clopidogrel were significantly greater after 25 mg dose under fed conditions compared to fasted. Reference-scaled Cmax, AUC0-t, and AUC0-∞ were higher than the 0.294 cutoff during fasted, meeting RSABE criteria. Under fed conditions, SWR for AUC0-t and AUC0-∞ were lower than 0.294, permitting use of ABE. The 90% confidence intervals for AUC0-t and AUC0-∞ indicated bioequivalence. Pharmacokinetic parameters differed between fasted and fed states. The generic product was bioequivalent to the reference drug, and was safe and well tolerated. This suggests that they can be used interchangeably in a clinical setting.

Population Pharmacokinetics/Pharmacodynamics of Ticagrelor in Children with Sickle Cell Disease.

BACKGROUND AND OBJECTIVE: Ticagrelor, a reversible P2Y12 platelet inhibitor, is under investigation as a sickle cell disease (SCD) therapy in children. HESTIA1 (NCT02214121) was the first ticagrelor study generating pharmacokinetic (PK), pharmacodynamic (PD, P2Y12 reactivity units [PRU]), and safety data in 45 pediatric SCD patients. Population PK and PK/PD relationships for ticagrelor were quantified using a PK approach. METHODS: An adult population PK model was refined to describe ticagrelor and AR-C124910XX (active metabolite) plasma concentration and time data over a wide range of single/repeated ticagrelor doses (0.125-2.25 mg/kg). Population PK/PD modeling was used to describe the time course and extent of platelet inhibition. Demographic covariate relationships were investigated. RESULTS: The final population PK model adequately described ticagrelor and AR-C124910XX plasma concentrations over time. An allometric body weight relationship between ticagrelor and AR-C124910XX clearances and volumes of distribution was used. Significant covariates for ticagrelor were sex (relative bioavailability) and cholecystectomy (central volume of distribution). Estimated oral clearances (35 kg patient; median bodyweight) were 22.8 L/h (ticagrelor) and 9.97 L/h (AR-C124910XX). The final population PK/PD model well-described the time course and extent of platelet inhibition. Estimated baseline PRU was 283, maximum PRU effect was fixed at 1, and the ticagrelor concentration for half-maximum PRU effect was 233 nmol/L. CONCLUSIONS: These analyses offer the first quantitative characterization of the dose-exposure-response relationship for ticagrelor in pediatric SCD patients. This model-based approach may be used to inform dose selection and design of subsequent studies that aim to define ticagrelor safety and efficacy in pediatric SCD patients.

Clopidogrel Increases Dasabuvir Exposure With or Without Ritonavir, and Ritonavir Inhibits the Bioactivation of Clopidogrel.

Dasabuvir is mainly metabolized by cytochrome P450 (CYP) 2C8 and is predominantly used in a regimen containing ritonavir. Ritonavir and clopidogrel are inhibitors of CYP3A4 and CYP2C8, respectively. In a randomized, crossover study in 12 healthy subjects, we examined the impact of clinical doses of ritonavir (for 5 days), clopidogrel (for 3 days), and their combination on dasabuvir pharmacokinetics, and the effect of ritonavir on clopidogrel. Clopidogrel, but not ritonavir, increased the geometric mean AUC0-∞ of dasabuvir 4.7-fold; range 2.0-10.1-fold (P = 8·10-7 ), compared with placebo. Clopidogrel and ritonavir combination increased dasabuvir AUC0-∞ 3.9-fold; range 2.1-7.9-fold (P = 2·10-6 ), compared with ritonavir alone. Ritonavir decreased the AUC0-4h of clopidogrel active metabolite by 51% (P = 0.0001), and average platelet inhibition from 51% without ritonavir to 31% with ritonavir (P = 0.0007). In conclusion, clopidogrel markedly elevates dasabuvir concentrations, and patients receiving ritonavir are at risk for diminished clopidogrel response.

Clinical Pharmacology of the Reversible and Potent P2Y12 Receptor Antagonist ACT-246475 After Single Subcutaneous Administration in Healthy Male Subjects.

ACT-246475 is a selective and reversible P2Y12 receptor antagonist inducing inhibition of platelet aggregation (IPA). A randomized, double-blind, placebo-controlled, parallel-design study was performed to investigate the safety, tolerability, pharmacokinetics, and pharmacodynamics of escalating single subcutaneous doses of ACT-246475 (1, 2, 4, 8, 16, or 32 mg) in healthy male subjects (N = 8 per dose, 3:1 active:placebo ratio). Pharmacodynamic effects were assessed based on maximum platelet aggregation and P2Y12 reaction units using light transmission aggregometry and VerifyNow® assays, respectively. ACT-246475 was safe and well tolerated up to 32 mg based on adverse event data and absence of clinically relevant changes in hematology, biochemistry, vital signs, and electrocardiogram variables. Median time to reach maximum plasma concentration was 0.5-0.75 hours, and geometric mean terminal half-life ranged from 1.3 to 9.2 hours across the tested dose range. Exposure to ACT-246475 was dose proportional across all dose groups. The maximal %IPA was reached within 30 minutes after subcutaneous administration of ACT-246475. A dose-dependent duration and extent of effect were observed based on area under the effect curve and maximum effect data. Similar results were observed for maximum platelet aggregation and P2Y12 reaction units. The %IPA was ≥85% at doses ≥2 mg. This level of %IPA was extended to at least 12 hours in the 32-mg dose group. The safety and pharmacokinetic/pharmacokinetic profile with quick onset and adequate duration of IPA support further investigation in patients with coronary artery disease.

Study on bioequivalence of beraprost in healthy volunteers by liquid chromatography with tandem mass spectrometry.

Beraprost sodium is an oral prostacyclin analog that was first approved in 1992 (Japan) for the treatment of peripheral vascular disorders. It is administered orally as a tablet available in strength 20 µg. In this paper, we described a liquid chromatography tandem mass spectrometry method that was developed for the quantification of beraprost in human plasma with high sensitivity at picogram per milliliter concentration. The method had been validated in terms of selectivity, sensitivity, accuracy and precision, matrix effect, linearity, recovery and carry-over according to the Guideline on Bioanalytical Validation from the European Medicines Agency. The standard calibration curve for beraprost was 9.5-1419 pg/mL. This method has been applied successfully to a bioequivalence study with 60 µg of beraprost (three tablets) in 29 healthy volunteers. The results showed that the two formulations of beraprost are bioequivalent.

The association between clopidogrel and 2-oxo-clopidogrel plasma levels and the long-term clinical outcome after acute myocardial infarction
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BACKGROUND AND OBJECTIVES: A significant number of ischemic events occur after acute myocardial infarction (MI), even when adhering to dual antiplatelet therapy including aspirin and clopidogrel. The aim of our study was to investigate the association between the concentration of the prodrug clopidogrel and its intermediary metabolite 2-oxo-clopidogrel plasma as well as demographic and clinical factors, and the long-term clinical outcome in patients with their first acute MI, ST-elevation myocardial infarction (STEMI) or non-ST-elevation myocardial infarction NSTEMI, treated with percutaneous coronary intervention (PCI). MATERIALS AND METHODS: This study included 172 consecutive patients with their first acute MI, 88 STEMI, and 84 NSTEMI, treated with PCI. On the third day of hospitalization, blood samples were collected from each patient to measure the concentration of clopidogrel and its metabolite 2-oxo-clopidogrel using the UHPLC-DAD-MS method. The following clinical outcomes were registered during the 28-month follow-up: mortality from cardiovascular causes, nonfatal MI, nonfatal stroke, and hospitalization for urgent myocardial revascularization or heart failure. RESULTS: Lower dose-adjusted clopidogrel concentrations (p < 0.05) were measured in NSTEMI patients with a composite of the hard clinical endpoint events of cardiovascular mortality, non-fatal MI, or a nonfatal stroke. During the follow-up, there was a 3.4 times higher risk of hard clinical endpoint events (p < 0.05) for each unit decrement of the dose-adjusted clopidogrel plasma concentration. Lower dose-adjusted concentrations of clopidogrel in these patients were associated with lower left ventricular ejection fraction (p < 0.001), and fentanyl (p < 0.001) and pantoprazole administration (p < 0.01) during the acute phase of MI. CONCLUSION: In patients with acute MI treated with PCI, lower dose-adjusted clopidogrel and dose-adjusted 2-oxo-clopidogrel plasma concentrations were associated with an increased risk of ischemic events.
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Hemorrhagic Complications of External Ventriculostomy in the Aspirin and P2Y12 Response Assay Era.

OBJECTIVE: Hemorrhagic complications reported from external ventricular drain (EVD) placement range from 10% to 44%. There remains limited literature investigating the incidence, risk factors, and mechanisms to prevent its occurrence, especially in the setting of antiplatelet agent use. We investigated EVD-related hemorrhagic complications after the implementation of VerifyNow platelet inhibition assays at our institution. METHODS: Medical records from 445 patients requiring EVD placement during a 2-year period during which our institution used the assays were reviewed. In total 345 patients were included, and 208 of them underwent assay testing. Indications for EVD included complications of cerebrovascular disease (n = 215), traumatic brain injury (n = 74), primary hydrocephalus (n = 23), and tumor (n = 33). Hemorrhage was defined as any new area of hyperdensity adjacent to or immediately along the catheter trajectory on computed tomography. RESULTS: There was no significant decrease in catheter-induced hemorrhage (CIH) between patients who underwent the VerifyNow assay and those who did not. Platelet transfusion did not significantly decrease the risk of CIH. CIH occurred in 17.7% of patients, significantly decreased when compared with our previously published incidence of 33% before platelet inhibition assay use (P < 0.05). Patients with cerebrovascular disease complications exhibited a significant decrease in CIH, 20% versus 39%, before assay use (P < 0.01). CONCLUSIONS: The incidence of hemorrhage is lower in our new cohort when compared with that of our previously published cohort. Despite the overall decreased rate of hemorrhage, there was no significant difference in hemorrhage rates between patients who did or did not undergo the assay. Platelet transfusion did not decrease the incidence of hemorrhage in patients with inhibited platelet function.

Pharmacokinetics and pharmacokinetic/pharmacodynamic relationship of vicagrel, a novel thienopyridine P2Y12 inhibitor, compared with clopidogrel in healthy Chinese subjects following single oral dosing.

BACKGROUND AND OBJECTIVES: Vicagrel, a novel thienopyridine antiplatelet agent, is an analogue of clopidogrel in development for the treatment of acute coronary syndromes. This study investigated the pharmacokinetic properties of vicagrel after single oral dosing with a direct comparison with clopidogrel in healthy Chinese subjects in the first two phase I clinical studies. The relationship between the exposure to the active metabolite and the platelet reactivity was also assessed for vicagrel. METHODS: Study A was a single-ascending-dose study of vicagrel (5-75 mg) compared with clopidogrel (75 mg) in 67 healthy volunteers. Study B was a randomized, two-period, crossover, loading-dose study of vicagrel 20 mg compared with clopidogrel 300 mg in 12 healthy subjects. Plasma concentrations of three common metabolites of vicagrel and clopidogrel, the active thiol metabolite H4, the inactive thiol metabolite H3, and the S-methylated form of H3 (SM3, the major metabolite of vicagrel), were determined using a validated UHPLC-MS/MS method. The relationship between the AUC0-t of active H4 and the P2Y12 reaction units at 4 h after administration of vicagrel was investigated. Blood concentrations of vicagrel were determined after a single oral administration of vicagrel 25 mg to two healthy Chinese subjects. RESULTS: In the single-ascending-dose study, vicagrel was metabolized rapidly with the median tmax for the three metabolites, namely, H4, H3, and SM3, ranging from 0.25-1.75 h. The pharmacokinetics of the three metabolites for vicagrel were linear across the dose range of 5-75 mg, with the mean Cmax and AUCs for H4 and H3 increasing in an approximately 1:1 dose-proportional manner and for SM3 increasing in a <1:1 dose-proportional manner. The median tmax for active H4 in the vicagrel 5 mg group was slightly shorter than that in the clopidogrel 75 mg group (0.50 versus 0.75 h). The mean AUC0-t for H4 in the vicagrel 5 mg group was similar to that in the clopidogrel 75 mg group (11.7 versus 11.8 ng∙h/mL). The AUC0-t of active H4 was apparently associated with the P2Y12 reaction units at 4 h for vicagrel. In the loading-dose study, for active H4, the median tmax was slightly shorter (0.50 versus 0.75 h) and the mean AUC0-t was 29% higher in the vicagrel 20 mg group than those in the clopidogrel 300 mg group. After a single oral administration of vicagrel 25 mg to 2 subjects, vicagrel was detected in blood but in very low concentrations. CONCLUSIONS: Vicagrel was rapidly and extensively metabolized, and the levels of the parent drug in circulation were very low. The pharmacokinetics of the three metabolites of vicagrel were linear and predictable across the dose range of 5-75 mg. The AUC of active H4 was apparently associated with the P2Y12 reaction units for vicagrel. For active H4, vicagrel 5 mg produced similar exposure (AUC) with more rapid appearance compared with clopidogrel 75 mg, and vicagrel 20 mg produced even slightly higher exposure (AUC) with more rapid appearance compared with clopidogrel 300 mg in humans. TRIAL REGISTRATION: CTR20150346, CTR20160379.

Spectrofluorimetric determination of eptifibatide in human plasma and dosage form.

A spectrofluorimetric method for the determination of eptifibatide is presented based on its native fluorescence. The type of solvent and the wavelength of maximum excitation and emission were carefully selected to optimize the experimental conditions. Under the specified experimental conditions, the linearities obtained between the emission intensity and the corresponding concentrations of eptifibatide were in the range 0.1-2.5 µg/ml for the calibration curve constructed for direct determination of eptifibatide in dosage form and 0.05-2.2 µg/ml for the calibration curve constructed in spiked human plasma with a good correlation coefficient (r > 0.99). The lower limit of quantification for the calibration curve constructed in human plasma was 0.05 µg/ml. Recovery results for eptifibatide in spiked plasma samples and in dosage form, represented as mean ± % RSD, were 95.17 ± 1.94 and 100.29 ± 1.33 respectively. The suggested procedures were validated according to the International Conference on Harmonization (ICH) guidelines for the direct determination of eptifibatide in its pure form and dosage form and United States Food and Drug Administration (US FDA) Guidance for Industry, Bioanalytical Method Validation for the assay of eptifibatide in human plasma.

Development of an LC-MS/MS method for simultaneous determination of ticagrelor and its active metabolite during concomitant treatment with atorvastatin.

A combination of antiplatelet drugs with high-intensity statin therapy is a standard in patients with coronary events. Concomitant treatment with ticagrelor, a moderate CYP3A4 inhibitor, and CYP3A4-metabolized statins such as atorvastatin, might lead to an increased risk of muscle-related adverse events. Therefore, investigation of concentrations of these compounds in clinical samples is necessary. For this purpose, an LC-MS/MS method was developed for simultaneous determination of ticagrelor and its active metabolite (AR-C124910XX), as well as 2-hydroxyatorvastatin, which is the main metabolite of atorvastatin. Protein precipitation was used for sample preparation and afterwards the analytes were separated on a Kinetex XB-C18 column with an isocratic elution (water and acetonitrile with 0.1% formic acid, 57:43, v/v). Detection was performed on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization. The method was fully validated according to the EMA's recommendations. Determination was possible within ranges: 1.25-2000 ng/mL for ticagrelor, 1.25-1000 ng/mL for its AR-C124910XX, 1.25-50 ng/mL for atorvastatin and 1.14-45.73 for 2-hydroxyatorvastatin. Within and between-run accuracy, expressed as a relative error, was within 0.05-10.56% for all analytes, while within and between-run precision, expressed as coefficient of variation, was within 0.61-9.91%. Ticagrelor, atorvastatin and their main metabolites were found to be stable in acetonitrile stock solutions, and in plasma samples stored for 24 h at room temperature, 1 month at -25 °C, after 3 cycles of freezing and thawing, and in processed samples stored as a dry residue for 24 h at 4 °C and for 24 h in autosampler at room temperature. This simple and rapid method allowed simultaneous determination of the analytes for the first time. The procedure was applied for the pharmacokinetic study of ticagrelor, its active metabolite AR-C124910XX, and 2-hydroxyatorvastatin in patients simultaneously treated with ticagrelor and atorvastatin.