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Determination of the active and inactive metabolites of prasugrel in human plasma by liquid chromatography/tandem mass spectrometry
Abstract
Two fast and sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS)-based bioanalytical assays were developed and validated to quantify the active and three inactive metabolites of prasugrel. Prasugrel is a novel thienopyridine prodrug that is metabolized to the pharmacologically active metabolite in addition to three inactive metabolites, which directly relate to the formation and elimination of the active metabolite. After extraction and separation, the analytes were detected and quantified using a triple quadrupole mass spectrometer using positive electrospray ionization. The validated concentration range for the inactive metabolites assay was from 1 to 500 ng/mL for each of the three analytes. Additionally, a 5x dilution factor was validated. The interday accuracy ranged from -10.5% to 12.5% and the precision ranged from 2.4% to 6.6% for all three analytes. All results showed accuracy and precision within +/-20% at the lower limit of quantification and +/-15% at other levels. The validated concentration range for the active metabolite assay was from 0.5 to 250 ng/mL. Additionally, a 10x dilution factor was validated. The interbatch accuracy ranged from -7.00% to 5.98%, while the precision ranged from 0.98% to 3.39%. Derivatization of the active metabolite in blood with 2-bromo-3'-methoxyacetophenone immediately after collection was essential to ensure the stability of the metabolite during sample processing and storage. These methods have been applied to determine the concentrations of the active and inactive metabolites of prasugrel in human plasma.
... 22,25 In addition, the prodrug forms only 15% of the clopidogrel metabolite, with 85% deesterised into an inactive carboxylic acid. 28 Prasugrel has also been shown to be associated with lower drug resistance than clopidogrel. Brandt et al demonstrated that 42% of clopidogrel-treated patients were associated with <20% platelet noninhibition at 4 hours after administration compared with 0% of prasugrel-treated patients. ...
... 25 This may be related to the fact that prasugrel and ticagrelor metabolism have been shown not to be affected by cytochrome P450 polymorphisms. 28,29 It is interesting to speculate that prasugrel 30 and ticagrelor, 21,31 which have anti-inflammatory and antiapoptotic activity, may protect against reperfusion injury, which is known to contribute to CMR-derived infarct size, MVO, and intramyocardial hemorrhage. 32 ...
Background
Third‐generation P2Y12 antagonists (prasugrel and ticagrelor) are recommended in guidelines on ST‐segment elevation myocardial infarction. Mechanisms translating their more potent antiplatelet activity into improved clinical outcomes versus the second‐generation P2Y12 antagonist clopidogrel are unclear. The aim of this post hoc analysis of the Complete Versus Lesion‐Only PRImary PCI Trial‐CMR (CvLPRIT‐CMR) substudy was to assess whether prasugrel and ticagrelor were associated with reduced infarct size compared with clopidogrel in patients undergoing primary percutaneous coronary intervention. Methods and ResultsCvLPRIT‐CMR was a multicenter, prospective, randomized, open‐label, blinded end point trial in 203 ST‐segment elevation myocardial infarction patients with multivessel disease undergoing primary percutaneous coronary intervention with either infarct‐related artery–only or complete revascularization. P2Y12 inhibitors were administered according to local guidelines. The primary end point of infarct size on cardiovascular magnetic resonance was not significantly different between the randomized groups. P2Y12 antagonist administration was not randomized. Patients receiving clopidogrel (n=70) compared with those treated with either prasugrel or ticagrelor (n=133) were older (67.8±12 versus 61.5±10 years, P
... It is metabolized by esterases to a thiolactone (R-95913, inactive), which is then oxidatively metabolized by several cytochrome P450 enzymes to thiol metabolite (R-138727, active). The active metabolite is further metabolized to its S-methyl metabolite (R-106583, inactive) and cysteine conjugate (R-119251, inactive) [1,100,135]. Accordingly, stability of prasugrel metabolites is monitored in the biological fluids rather than the parent compound. ...
... The ratio of the four stereoisomers of R-138727 remained constant when they were exposed to room temperature for By-product [34,117,132] Continued 24 h, subjected to four freeze/thaw cycles, or held during long-term frozen storage for 31 days at À20°C. The stability of the three inactive metabolites (R-95913, R-106583, and R-119251) in human plasma with concentrations of 2.5, 400, and 2000 ng/ml were evaluated by HPLC/MS method [100]. It was found that they are stable in human plasma at room temperature for 3.5 h, when frozen and thawed for three cycles at both À20°C and À70°C, and in plasma extracts with concentration range of 2.5 and 400 ng/ml stored at room temperature for 24 h. ...
A comprehensive profile of prasugrel HCl is reported herein with 158 references. A full description including nomenclature, formulae, elemental analysis, and appearance is included. Methods of preparation for prasugrel HCl, its intermediates, and derivatives are fully discussed. In addition, the physical properties, analytical methods, stability, uses and applications, and pharmacology of prasugrel HCl are also discussed.
© 2015 Elsevier Inc. All rights reserved.
... Blood samples for PK analysis of the Clop-AM were collected into EDTA tubes at 0.5, 1, 2, 3, and 4 h following the first dose of each period and the last dose of period three and processed as described by Farid and colleagues [16]. Clop-AM was measured using validated liquid chromatography methods with tandem mass spectrometric detection, as previously described [16,17]. ...
... Blood samples for PK analysis of the Clop-AM were collected into EDTA tubes at 0.5, 1, 2, 3, and 4 h following the first dose of each period and the last dose of period three and processed as described by Farid and colleagues [16]. Clop-AM was measured using validated liquid chromatography methods with tandem mass spectrometric detection, as previously described [16,17]. Noncompartmental analysis was performed using WinNonlin version 5.3 (Pharsight, Cary, NC). ...
Body weight is a predictor of clopidogrel response. However, no prospective studies have compared pharmacodynamic (PD) and pharmacokinetic (PK) data based on body weight. We compared PD and PK effects of clopidogrel 75 mg in low body weight (LBW, <60 kg) and higher body weight (HBW, ≥60 kg) patients with stable coronary artery disease. LBW (n = 34, 56.4 ± 3.7 kg) and HBW (n = 38, 84.7 ± 14.9 kg) aspirin-treated patients received clopidogrel 75 mg for 10-14 days. The area under the concentration-time curve of active metabolite (Clop-AM) calculated through the last quantifiable concentration up to 4 h postdose, AUC(0-tlast), was calculated by noncompartmental methods. Light transmission aggregometry (LTA) (maximum platelet aggregation and inhibition of platelet aggregation to 20 μM adenosine diphosphate (ADP), and residual platelet aggregation to 5 μM ADP), VerifyNow(®) P2Y12 reaction units (PRU), and vasodilator-associated stimulated phosphoprotein phosphorylation platelet reactivity index (VASP-PRI) were performed. Mean AUC(0-tlast) was lower in HBW than LBW patients: 12.8 versus 17.9 ng h/mL. HBW patients had higher platelet reactivity as measured by LTA (all p ≤ 0.01), PRU (207 ± 68 vs. 152 ± 57, p < 0.001), and VASP-PRI (56 ± 18 vs. 39 ± 17, p < 0.001). More HBW patients exhibited high on-treatment platelet reactivity (HPR) using PRU (35 vs. 9 %) and VASP-PRI (65 vs. 27 %). Body weight correlated with PRU and VASP-PRI (both p < 0.001), and inversely with log transformed AUC(0-tlast) (p < 0.001). In conclusion, HBW patients had lower levels of Clop-AM, and higher platelet reactivity and rates of HPR than LBW subjects, contributing to their suboptimal response to clopidogrel.
... Establishment of an in-vitro dissolution test as a surrogate for animal testing/human studies was explored in this study to support PHCl nanosuspension formulation development. Such a development for nanosuspensions was proposed and investigated for the first time 13 . ...
The objective of this study was to investigate nanosuspensions for enhancing the solubility and dissolution rate of Prasugrel HCl (PHCl) so as to reduce the fluctuations in its oral bioavailability. Prasugrel Hydrochloride nanosuspensions were prepared using evaporative precipitation method. After preparation, various nanosuspensions were characterized for particle size, scanning electron microscope (SEM), zeta potential, drug entrapment efficiency (EE), Fourier transform infrared spectroscopy (FTIR), and differential scanning calorimetry (DSC). Solubility and in-vitro release of the drug from nanoparticles are determined. The formulations were characterized using techniques such as powder x-ray diffractometry, scanning electron microscopy, in-vitro dissolution and in-vivo absorption in rats. The dissolution rate and oral absorption of Prasugrel Hydrochloride in the form of nanosuspensions was significantly higher than that of oral suspension and pure drug. All the techniques investigated in this study can be used to enhance dissolution rate and oral absorption of prasugrel hydrochloride and thus can reduce the fluctuations in its oral bioavailability. Nanosuspensions demonstrated to be better and superior technique when compared to other techniques investigated in enhancing oral bioavailability of Prasugrel Hydrochloride. Prasugrel hydrochloride nanosuspension was successfully developed using and the bioavailability is 4 folds increased than its pure drug.
... Антитромбоцитарная активность ингибиторов P2Y 12 рецепторов У тикагрелора и прасугрела различные механизмы связывания с рецепторами P2Y 12 и различные химические классы препаратов в рамках одной группыингибиторов P2Y 12 , которые могут приводить к значительным различиям с точки зрения биологических и клинических исходов. Прасугрел -тиенопиридин третьего поколения, который необратимо ингибирует рецепторы P2Y 12 тромбоцитов, в то время как тикагрелор представляет собой циклопентил-триазолопиримидин и является обратимым антагонистом этих рецепторов [12,13]. ...
Dual antiplatelet therapy is the most important step in acute coronary syndrome (ACS) treatment. The new generation of inhibitors of P2Y 12 platelet receptors (prasugrel and ticagrelor) provide more pronounced platelet inhibition than clopidogrel. The drugs differ in pharmacodynamics and platelet reactivity tests due to different mechanisms of binding to P2Y 12 receptors. The antiplatelet effect of prasugrel and ticagrelor provides clinical benefit and better prognosis in patients with various forms of ACS. In patients with ST-segment elevation ACS prasugrel and ticagrelor are preferred over clopi-dogrel due to their higher efficacy and better clinical outcomes, and currently have preferential positions in guidelines compared to clopidogrel. The comparison of prasugrel versus ticagrelor (ISAR-REACT 5 trial) demonstrated superiority of prasugrel over ticagrelor in patients with ST-segment elevation ACS, for whom an invasive evaluation is planned and in early invasive treatment non-ST-segment elevation ACS. The choice of a drug for dual antiplatelet therapy in various clinical situations remains controversial. The latest ESC guidelines on non-ST elevation ACS (2020) [1] demonstrate the possible preference for prasugrel in patients with ACS without ST-segment elevation undergoing percutaneous coronary intervention. Current article demonstrates the results of recent clinical studies and the real clinical data regarding antiplatelet therapy in patients with coronary interventions. The indications for the use of P2Y 12 platelet inhibitors in certain groups of patients are outlined. Treatment selection of the most effective and safe drugs in patients with ACS is highlighted according to the updated European guidelines.
... HPLC-UV (77) and LC-MS (78-83) methods were reported for the determination of CCA impurity in plasma samples. A literature survey revealed few analytical methods for PRG alone or with ASA, such as UV spectrophotometry (84,85), HPLC (86)(87)(88)(89), LC-MS (90)(91)(92) and HPTLC (93). There is no published method for the simultaneous determination of all the three drugs (as well as in the presence of CCA impurity). ...
An isocratic reversed-phase high-performance liquid chromatographic method has been developed and validated for the simultaneous determination of aspirin, prasugrel HCl and clopidogrel bisulfate in the presence of clopidogrel-related compound (impurity-A) in focus on counterfeit. This method was used to determine counterfeited antiplatelet drugs in two substandard Indian pharmaceutical products sold on the market in Yemen and two traditional herbal medicines sold on the market in China. Thin layer chromatography and mass spectrometry of counterfeit herbal medicines have additionally been carried out to verify the identification of adulterants. Chromatographic separation was performed on Inertsil ® ODS-3 C18 (4.6 × 250 mm, 5 μm) with isocratic mobile phase elution containing a mixture of acetonitrile: (25 mM) potassium dihydrogen phosphate buffer, pH 2.7 adjusted with 0.1 M o-phosphoric acid (79: 21, v/v), at a flow rate of 1 mL/min and UV detection at 220 nm. Designs of experiment methodology, Plackett–Burman and Box–Behnken designs were used for the screening and optimization of the mobile phase composition. The method validation was also performed in accordance with the International Council on Harmonization (ICH) guidelines. The method developed for routine analysis was found to be sensitive, simple, accurate and highly robust. The results were statistically compared to reference methods using Student’s t-test and variance ratio F-test at P < 0.05.
... Препарат после приема быстро гидролизуется карбоксиэстеразами, а затем превращается в ходе одноступенчатого механизма в активный метаболит, сходный по химической структуре с метаболитом клопидогрела, который далее необратимо связывается с P2Y 12 рецепторами тромбоцитов, препятствуя их взаимодействию с АДФ и, соответственно, подавляя АДФ-индуцированную агрегацию тромбоцитов. От момента приема прасугрела до ингибирования агрегации >50% тромбоцитов проходит порядка 30 мин [24]. ...
The article presents modern approaches to choosing a P2Y12 inhibitor as part of combined antithrombotic therapy in patients with acute coronary syndrome, depending on the indications for interventional treatment and the presence of comorbidities such as atrial fibrillation and diabetes.
... Two P2Y 12 antagonists: cangrelor and prasugrel were utilized to assess whether AR agonists can enhance their antiplatelet effect. Prasugrel is a pro-drug and requires metabolization to its active components, therefore its most abundant stable active metabolite R-138727 was used [28]. P2Y 12 antagonists, as well as AR agonists, were used at IC 50 values [19,21]. ...
We have shown previously that platelet activity can be lowered through the simultaneous inhibition of P2Y12 receptor and activation of adenosine receptors (AR). This work explores this concept by testing the antiplatelet potential of nine AR agonists in combination with P2Y12 receptor antagonists—cangrelor and prasugrel metabolite. A panel of in vitro methods was used to assess platelet viability, P-selectin expression, GPIIb-IIIa activation, fibrinogen binding, calcium ion mobilization, VASP-P level, and cAMP formation, utilizing whole blood or isolated platelets from healthy volunteers. The AR agonists demonstrated anti-platelet effects, but stimulated signaling pathways to varying degrees. AR agonists and P2Y12 antagonists reduced expression of both P-selectin and the activated form of GPIIb-IIIa on platelets; however, the combined systems (AR agonist + P2Y12 antagonist) demonstrated stronger effects. The antiplatelet effects of AR when combined with P2Y12 were more pronounced with regard to exogenous fibrinogen binding and calcium mobilization. The cAMP levels in both resting and ADPactivated platelets were increased by AR agonist treatment, and more so when combined with P2Y12 inhibitor. In conclusion, as AR agonists are fast-acting compounds, the methods detecting early activation events are more suitable for assessing their antiplatelet action. The exogenous fibrinogen binding, calcium mobilisation and cAMP level turned out to be sensitive markers for detecting the inhibition caused by AR agonists alone or in combination with P2Y12 receptor antagonists.
... Prasugrel active metabolites (PAM) will be measured at each time point under the secondary hypothesis that chewed as compared with integer prasugrel will lead to higher PAM bioavailability at 30 min (and other time points, 15 min to 4-6 h). Blood samples for the determination of PAM plasma concentration will be collected into precooled EDTA tubes at the established time points (baseline, 15 min, 30 min, 1 h, 2 h, 3 h and 4-6 h) and will be treated with 25 μl of 500 mM 3′-methoxyphenacyl bromide in acetonitrile within 30 s of collection to derivatize and stabilize the PAM as previously described [14,30]. Centrifugation of the sample will be performed within 30 min (2800 rpm for 15 min at 4°C), and plasma samples prepared from these blood samples will be stored at − 20/− 80°C in polypropylene tubes until they will be shipped to the central laboratory at University Hospital of Verona (LURM) for analysis. ...
Antithrombotic therapy is a critical component of the management of ST-elevation myocardial infarction (STEMI) patients treated with primary percutaneous coronary intervention (PCI). Rapid and profound inhibition of platelet reactivity has been shown to mitigate the ischemic risks and improve myocardial salvage. High residual platelet reactivity (HRPR) has been reported up to 4 or 6 h after loading dose of prasugrel or ticagrelor; therefore, multiple alternative strategies, including crushed or chewed oral tables or intravenous agents, have been investigated to provide a more rapid and sustained inhibition of platelet function and bridge the initial treatment gap. The FABOLUS FASTER is the first investigator-initiated, multicentre, open-label, prospective, randomized study to directly compare the pharmacodynamics effects of cangrelor, tirofiban, chewed or integer prasugrel. This study will add new insights in the management of antiplatelet therapy in patients with STEMI undergoing primary PCI and might be hypothesis-generating for future clinical trials in this field. The trial is registered on clinicaltrials.gov NCT02978040, and EudraCT 2017-001065-24.
... Prasugrel is rapidly metabolized by esterases to diastereomeric forms of 5-(2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl)-5,6,7,7a-tetrahydrothieno [3,2-c]pyridin-2(4H)-one, thiolactone isomers (aka OXTP diastereomers) that are further oxidatively metabolized by cytochrome P450 enzymes, ultimately to its active metabolite (R-138727). [1][2][3] As part of the development process, stress testing studies are typically carried out to discover the intrinsic stability characteristics of the compound, 4 providing an understanding of the degradation pathways available to the molecule under pharmaceutical-relevant conditions and enabling the development of stability-indicating methodologies. ...
Prasugrel hydrochloride is the active ingredient in Effient™, a thienopyridine platelet inhibitor. An extensive study of the degradation chemistry of prasugrel hydrochloride (LY640315 hydrochloride)has been carried out on the drug substance (part I)and on the drug product (part II, future article)using a multidimensional approach including hydrolytic, oxidative, and photolytic stressing, computational chemistry, HPLC analysis, and structure elucidation by various spectroscopic techniques. The major degradation products formed from the drug substance under the various stress conditions have been isolated and structures unambiguously determined, and the pathways leading to these products have been proposed. Fourteen new (not previously disclosed)products were discovered and characterized, in addition to 4 degradation products that had been previously identified in the literature. The pathways indicate that prasugrel is susceptible to hydrolysis, autoxidation (both radical-initiated and single-electron mediated), and peroxide-mediated oxidation; in solution, prasugrel is susceptible to photodegradation.
... Thus research work focused on the development of methods of extraction and determination of new drugs used in cardiology is very important. Several analytical methods have been reported in literature for the quantification of ALS [8][9][10][11][12][13][14], RIV [15][16][17], and PRS [18,19] in biological samples. These methods used either high performance liquid chromatography (HPLC) with ultra-violet (UV) detection, diode-array detection (DAD), or coupled with tandem mass spectrometry (MS/MS). ...
In this study electrochemistry (EC) coupled with electrospray ionization mass spectrometry (ESI-MS) was used to study the metabolic fate of three novel cardiovascular drugs: rivaroxaban (RIV), aliskiren (ALS), and prasugrel (PRS). Mimicry of the oxidative phase I metabolism was achieved in a simple amperometric thin-layer cell equipped with a boron-doped diamond (MD) working electrode. Structures of the electrochemically-generated metabolites were elucidated from MS/MS experiments. Additionally, a sensitive, specific, and rapid ultra-high performance liquid chromatography–tandem mass spectrometer (UHPLC–MS/MS) method has been developed and validated for the selected drugs in human urine samples. Three different sample preparation methods were compared and finally, sample preparation was accomplished through an ultrasound-assisted emulsification microextraction process (USAEME). The drugs were detected using a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source with positive ionization mode (ESI(+)). The results obtained by EC–MS were compared with conventional in vivo studies by analyzing urine samples from patients. Data from in vivo experiments showed good agreement with the data from electrochemical oxidation. Thus, EC–MS is very well-suited for the simulation of the oxidative metabolism of rivaroxaban, aliskiren, and prasugrel as well. Moreover, electrochemical conversion of target compounds appears to be a new in vitro technology for the prediction of potential metabolites.
... Although Clopidogrel is a widely prescribed agent it has some limitation, because of that, researchers developed more effective agents such as the novel Prasugrel, which makes it different is its safety profile and pharmacokinetic properties. Literature revealed very few methods for the estimation of Prasugrel hydrochloride such as LS-MS [5], HPTLC [6], HPLC [7] and UV spectrophotometric method [8]. ...
The construction and performance characteristics of Prasugrel hydrochloride (PRS) selective electrodes were developed. Electrodes were based on the incorporation of PRS with pairing agents, Ammonium Molybdate (MOL) and Nessler reagent (NES). The electrodes displayed a Nernstian response with a mean calibration graphs slopes of 59.21, 27.57 mv.decade-1 for the two electrodes respectively, over linear concentration range 10-2 –10-5 mol L-1 of the drug, with detection limits 0.063, 0.090 µM and quantification limits 0.27, 0.192 µM for electrode 1 and 2 respectively. The safe pH range of the proposed electrodes was (1-4). The influence of possible interfering species such as inorganic cations and pharmacologically related compounds was studied. The electrodes showed a fast response of (20 ± 2 sec, 17 ± 2 sec) for a period of 11 days, 9 days for electrode 1 and 2 respectively without significant change in electrode parameters. The methods are precise as shown by mean recoveries range of (97.7-102.5 %) – (97.3-101 %) with a mean relative standard deviation less than 3.14 and 4.46% for electrode 1 and 2 respectively. The results were compared to those obtained by a reference method. The proposed electrodes were used for the determination of PRS in pure form and pharmaceutical formulation.
... The plasma concentration of the active metabolite of prasugrel, R-138727, was measured by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. [19][20][21] Inertsil ODS-3 (GL Sciences Inc.) was used as the high-pressure liquid chromatography (HPLC) column. The LC mobile phase was methanol with 1% formic acid (54:46), and the methanol and 1% formic acid were mixed using an HPLC pump. ...
We evaluated the pharmacokinetics and pharmacodynamics of prasugrel used in combination with aspirin in healthy Japanese subjects. All subjects received aspirin 100 mg/day. Subsequently, in the single-administration study, 23 subjects also received prasugrel 20 or 30 mg, and in the multiple-administration study, 20 subjects received a loading dose of prasugrel 20 or 30 mg on day 1, followed by a maintenance dose of prasugrel 5 or 7.5 mg/day, respectively, on days 2-5. In both studies, the plasma concentration of the active metabolite of prasugrel, R-138727, reached a maximum 0.5 hours after administration and rapidly decreased within 4 hours. In the single-administration study, the inhibitory effect on adenosine diphosphate-induced platelet aggregation was significantly higher in the prasugrel 20- and 30-mg groups than in the placebo group at all times (1-144 hours) after administration. In the multiple-administration study, a similar antiplatelet effect was found after both the loading dose and the maintenance dose and was maintained for 3-6 days after the last administration. There were study drug-related adverse events; however, all were mild, and none was clinically significant.
... Prasugrel inhibits adenosine diphosphate-induced platelet aggregation more rapidly, more consistently and to a greater extent than do standard and higher doses of clopidogrel in healthy volunteers and in patients with coronary artery disease. Literature survey revealed that only a few analytical methods such as UV, [31][32] HPLC, [33][34] LC-MS, [35][36][37] HPTLC [38] have been reported. According to the information collected from literature there is no reported method for simultaneous determination of ASP and PRA. ...
A simple, reliable, rapid, precise, sensitive and validated RP-HPLC method has been developed to determine Aspirin and Prasugrel in synthetic mixture form. Chromatographic separation achieved isocratically on Luna C18 column (5µm, 150mm × 4.60mm) and acetonitrile: 0.05M ammonium acetate buffer (pH 4.5) in the ratio of 75:25 (v/v) as the mobile phase, at a flow rate of 0.6 ml/min. Detection was carried out at 245 nm. Parameters such as linearity, precision, accuracy, recovery, specificity and ruggedness are studied as reported in the ICH guidelines. The retention times for Aspirin and Prasugrel was found to be 2.25±0.5 and 8.72±0.5 min respectively. Linearity for Aspirin and Prasugrel was in the range of 75-375μg/ml and 10-50μg/ml respectively. The mean recoveries obtained for Aspirin and Prasugrel were 99.58 and 99.48 % respectively and RSD was less than 2. The correlation coefficients for all components are close to 1. Developed method was found to be accurate, precise, selective and rapid for simultaneous estimation of Aspirin and Prasugrel.
... The plasma concentrations of the active metabolites were determined using validated liquid chromatography-tandem mass spectrometry methods. 13,14 The lower limit of detection of both active metabolites is 0.50 ng/mL. ...
Herein, the dosing regimen of prasugrel adjusted for Japanese patients was compared to that of clopidogrel by analyzing the pharmacokinetics and pharmacodynamics in 40 healthy Japanese subjects in a randomized, single blind, crossover study. In period 1, the subjects received either 300 mg clopidogrel or 20 mg prasugrel; after a >2-week interval (period 2), the drug was switched. Blood samples of 36 out of the 40 subjects were collected for analysis of pharmacokinetics, pharmacodynamics, and CYP2C19 genotypes. The plasma concentration of the active metabolite of prasugrel increased rapidly and reached its peak at 30 min post administration, whereas that of the active metabolite of clopidogrel reached its peak at 1 h post administration. The mean AUC and Cmax of the active metabolite of clopidogrel, but not those of prasugrel, were CYP2C19 genotype-dependent. Prasugrel rapidly inhibited platelet aggregation, reaching its maximum effect at 1 h post administration. Clopidogrel, on the other hand, showed a maximum inhibition at 2 h post administration. Platelet aggregation inhibition by clopidogrel was significantly lower in the poor metabolizer subjects than that in the extensive metabolizer subjects was. Overall, prasugrel inhibited platelet aggregation more rapidly and more effectively in healthy Japanese subjects than was observed for clopidogrel. This article is protected by copyright. All rights reserved
... Healthy subjects in six studies (N = 275) involving treatment with clopidogrel (N = 106) and/or prasugrel (N = 169) were included in the pharmacodynamic and pharmacokinetic analyses (Supplemental Table 1) [10][11][12][13][14][15]. Plasma concentrations of clopidogrel and prasugrel active drug metabolite were measured by liquid chromatography with mass spectrometry [16]. The area under the plasma concentration-time curve was analyzed using the log-linear trapezoidal method from time of dose to last measurable concentration (AUC 0-t ). ...
Clopidogrel and prasugrel are antiplatelet therapies commonly used to treat patients with cardiovascular disease. They are both pro-drugs requiring biotransformation into active metabolites. It has been proposed that a genetic variant Q192R (rs662 A>G) in PON1 significantly alters the biotransformation of clopidogrel and affects clinical outcomes; however, this assertion has limited support. The relationship between this variant and clinical outcomes with prasugrel has not been studied. We genotyped PON1 Q192R in 275 healthy subjects treated with clopidogrel or prasugrel and 2922 patients with an ACS undergoing PCI randomized to treatment with clopidogrel or prasugrel in the TRITON-TIMI 38 trial. A meta-analysis was performed including 13 studies and 16,760 clopidogrel-treated patients. Among clopidogrel-treated subjects, there were no associations between Q192R and active drug metabolite levels (P = 0.62) or change in platelet aggregation (P = 0.51). Consistent with these results, in clopidogrel-treated patients in TRITON-TIMI 38, there was no association between Q192R and the rates of CV death, myocardial infarction, or stroke (RR 11.2 %, QR 8.6 %, and QQ 9.3 %; P = 0.66) or stent thrombosis (RR 2.4 %, QR 0.7 %, and QQ 1.6 %, P = 0.30), with patients with the putative at-risk Q variant having numerically lower event rates. Likewise, among prasugrel-treated subjects, there were no associations between Q192R and active drug metabolite levels (P = 0.88), change in platelet aggregation (P = 0.97), or clinical outcomes (P = 0.72). In a meta-analysis, the Q variant was not significantly associated with MACE (QQ vs. RR 1.22, 95 % CI 0.84-1.76) or stent thrombosis (QQ vs. RR OR 1.36, 95 % CI 0.77-2.38). Furthermore, when restricted to the validation studies, the OR (95 % CI) for MACE and stent thrombosis were 0.99 (0.77-1.27) and 1.23 (0.74-2.03), respectively. In the present study, the Q192R genetic variant in PON1 was not associated with the pharmacologic or clinical response to clopidogrel, nor was it associated with the response to prasugrel. The meta-analysis reinforced a lack of a significant association between Q192R and cardiovascular outcomes in clopidogrel-treated patients.
... Prasugrel inhibits adenosine diphosphate-induced platelet aggregation more rapidly, more consistently and to a greater extent than do standard and higher doses of clopidogrel in healthy volunteers and in patients with coronary artery disease. Literature survey revealed that only a few analytical methods such as UV, [31][32] HPLC, [33][34] LC-MS, [35][36][37] HPTLC [38] have been reported. According to the information collected from literature there is no reported method for simultaneous determination of ASP and PRA. ...
A simple, reliable, rapid, precise, sensitive and validated RP-HPLC method has been developed to determine Aspirin and
Prasugrel in synthetic mixture form. Chromatographic separation achieved isocratically on Luna C18 column (5µm, 150mm
× 4.60mm) and acetonitrile: 0.05M ammonium acetate buffer (pH 4.5) in the ratio of 75:25 (v/v) as the mobile phase, at a
flow rate of 0.6 ml/min. Detection was carried out at 245 nm. Parameters such as linearity, precision, accuracy, recovery,
specificity and ruggedness are studied as reported in the ICH guidelines. The retention times for Aspirin and Prasugrel was
found to be 2.25±0.5 and 8.72±0.5 min respectively. Linearity for Aspirin and Prasugrel was in the range of 75-375μg/ml
and 10-50μg/ml respectively. The mean recoveries obtained for Aspirin and Prasugrel were 99.58 and 99.48 % respectively
and RSD was less than 2. The correlation coefficients for all components are close to 1. Developed method was found to be
accurate, precise, selective and rapid for simultaneous estimation of Aspirin and Prasugrel.
... The applied system consisted of a Symbiosis ALIAS chromatographic system (Spark Holland B.V., Emmen, Netherlands) and an AB Sciex detector (QTRAP 5500, AB Sciex, Framingham, MA, USA). A published procedure [32] was modified to perform the analyses. ...
Background:
Morphine decreases the concentrations and effects of clopidogrel, which could lead to treatment failure in myocardial infarction.
Objectives:
To clarify whether more potent P2Y12-inhibitors may provide an effective alternative, we examined drug-drug interactions between morphine and prasugrel.
Methods:
Twelve healthy volunteers received 60 mg prasugrel with placebo or 5 mg morphine intravenously in a randomized, double-blind, placebo-controlled, cross-over trial. Pharmacokinetics were determined by liquid chromatography tandem mass spectrometry, and prasugrel effects were measured by platelet function tests.
Results:
Morphine neither diminished total drug exposure (AUC), which was the primary endpoint, nor significantly delayed drug absorption of prasugrel. However, morphine reduced maximal plasma concentrations (C max) of prasugrel active metabolite by 31 % (p = 0.019). Morphine slightly, but not significantly, delayed the onset of maximal inhibition of platelet plug formation under high shear rates (30 vs. 20 min). Whole blood aggregation was not influenced.
Conclusions:
Although morphine significantly decreases the maximal plasma concentrations of prasugrel active metabolite, it does not diminish its effects on platelets to a clinically relevant degree in healthy volunteers. However, it should be considered that the observed decrease in C max of prasugrel active metabolite caused by morphine co-administration may gain relevance in STEMI patients.
Clinical trial registration:
NCT01369186, EUDRA-CT#: 2010-023761-22.
... It is available with brand names Effient and Prasita. Literature survey revealed very few methods for the estimation of Prasugrel hydrochloride such as LC-MS 6 , HPTLC 7 , UV-Visible spectrophotometric methods 8 As the analysis is an important component in the formulation development of any drug molecule. It becomes essential to develop a simple, sensitive, accurate, precise, reproducible method for the estimation of drug samples. ...
Prasugrel is an anti-platelet agent of the thieno-pyridine class approved by FDA in July 2009 is a Prodrug with restricted prescribing. A new visible spectrophotometric method has been developed and validated for the estimation of Prasugrel hydrochloride in bulk and pharmaceutical formulations. The method makes use of ion pair complex formation between drug and bromophenol blue (BPB). Bromophenol blue and 0.1 H2SO4 were used for the Spectrophotometric analysis of Prasugrel HCl in its pure and its dosage form. The absorbance of the complex formed, by Bromophenol blue and the drug was found maximum on wavelength 421 nm and for 0.1 H2SO4 at the wavelength 223 nm against the corresponding blank. Both the methods were found to be simple, precise, and rapid for detection of Prasugrel hydrochloride and can be conveniently adopted for routine analysis of Prasugrel hydrochloride.
... It is of note that the amount of non-inhibited platelets which needed to be added to overcome prasugrel's effects in this study implied that there were no active metabolite left. Indeed, prasugrel's active metabolite is not stable in vitro and has a very short half-life [26]; even if some blood samples were collected shortly after the first MD, prasugrel's active metabolite was no longer effective in P-PRP when we prepared the mixture with C-PRP. Thus, the results obtained in the prasugrel group reflect the effect of platelet supplementation given at least 6 h after the last drug intake. ...
Managing bleeding in patients receiving P2Y12 inhibitors is challenging. Few data are available regarding the efficacy of platelet transfusion in patients treated with prasugrel or ticagrelor. The aim of this study was to evaluate the minimal amount of platelet supplementation (in terms of ratio of non-inhibited platelets to inhibited platelets) necessary to restore platelet reactivity in platelet-rich plasma (PRP) of patients treated with aspirin and a prasugrel or ticagrelor loading dose for an acute coronary syndrome. PRP samples from patients were mixed ex vivo with increasing proportions of pooled PRP from healthy volunteers. Platelet reactivity was challenged with adenosine diphosphate (ADP), arachidonic acid, collagen or thrombin receptor activating peptide using light transmission aggregometry. The primary endpoint was the proportion of patient samples recovering an ADP-induced maximal aggregation (ADP-Aggmax) value above 40%. In patients treated with prasugrel (n = 32), ADP-Aggmax increased progressively with supplements of pooled PRP, with an average increase of 7.9% (95% CI [7.1; 8.8], p < 0.001) per each 20% increase in the ratio of non-inhibited platelets to inhibited platelets. A ratio of 60% was associated with 90% of patients reaching the primary endpoint. In patients treated with ticagrelor (n = 15), ADP-Aggmax did not significantly increase with any level of supplements. In conclusions, ex vivo addition of non-inhibited platelets significantly improved ADP-Aggmax in patients treated with prasugrel with a dose-dependent effect. There was no evidence of such a reversal in patients treated with ticagrelor. These results suggest that platelet transfusion may be more effective in blunting bleeding in patients treated with prasugrel, than those treated with ticagrelor.
... Blood samples for R-95913 analysis were collected in heparinized tubes, and samples for R-138727 were collected in EDTA tubes; 500 mM of 2-bromo-3'methoxyacetophenone (M815, Tokyo Chemical Industry Co, Ltd, Tokyo, Japan) was then added and mixed with acetonitrile (J.T. Baker Chemical Co, Phillipsburg, New Jersey) to produce R-138727MP. 16 All blood samples for PK analysis were drawn via an indwelling intravenous angiocatheter; the first 1 mL of blood was discarded when obtained from the catheter. Plasma was extracted within 30 minutes after collection by centrifugation at 1500g for 15 minutes at 41C and immediately transferred to 1.5-mL tubes (Eppendorf, Hamburg, Germany), which were frozen at -701C. ...
A combination of clopidogrel and aspirin is the standard treatment for patients with acute coronary syndrome and those undergoing percutaneous coronary intervention. Two novel antiplatelet agents, ticagrelor and prasugrel, have been shown to rapidly and more effectively inhibit the P2Y12 receptor compared with clopidogrel. The aim of this study was to evaluate and compare the pharmacokinetics (PK) and pharmacodynamics (PD) of ticagrelor and prasugrel in healthy male Korean volunteers.
Two separate studies were conducted. One study was performed by using a single-sequence, open-label, crossover design in 12 volunteers who received a single oral dose of ticagrelor (180 mg) and then a single oral dose of prasugrel (60 mg for 4 volunteers and 30 mg for 8 volunteers) with a 7-day washout period. The other study was a randomized, open-label, parallel-group investigation in which 8 volunteers received a single oral dose of prasugrel (10 mg for 4 volunteers and 30 mg for 4 volunteers). In each study, blood samples for PK and platelet aggregation inhibition analysis were serially collected after the administration of each dose. Plasma concentrations of ticagrelor, AR-C124910XX (the active metabolite of ticagrelor), R-95913 (the inactive metabolite of prasugrel), and R-138727 (the active metabolite of prasugrel) were measured by using a validated LC-MS/MS method. PK was analyzed by using a noncompartmental method. Maximal platelet aggregations were assessed with light transmission aggregometry after induction with 20 μmol/L of adenosine diphosphate.
Twenty healthy male Korean volunteers participated in the 2 studies. Plasma concentrations of ticagrelor and AR-C124910XX were obtained from 12 subjects, R-95913 from 20 subjects, and R-138727 from 8 subjects. Both ticagrelor and prasugrel were rapidly absorbed, with the shortest median Tmax of 2.0 and 2.25 hours for ticagrelor and AR-C124910XX, respectively, and a Tmax of 0.5 hour for both R-95913 and R-138727. Strong inhibition of platelet aggregation was shown after administration of both ticagrelor and prasugrel, with slightly stronger and more rapid inhibition with prasugrel in the tested doses. Inhibitory activities of prasugrel lasted longer than those of ticagrelor, reflecting the difference in binding kinetics between the 2 drugs.
Prasugrel 30 and 60 mg exhibited slightly stronger, more rapid, and sustainable platelet inhibitory effects compared with ticagrelor 180 mg. These differing effects should be considered when determining the efficacy and adverse effects of ticagrelor and prasugrel. ClinicalTrials.gov identifier: NCT01876797 and NCT02075268.
Copyright © 2015 Elsevier HS Journals, Inc. All rights reserved.
... Plasma concentrations of R-95913 and R-138727 were determined after derivatizing the sample with 2-bromo-3methoxyacetophenone immediately after blood collection as previously described [18], with minor modifications. Liquid chromatography-tandem mass spectrometry measurements were conducted using a Shimadzu Nexera LC system (Shimadzu, Kyoto, Japan) coupled to a 5500 Qtrap mass spectrometer (AB Sciex, Toronto, ON, Canada) equipped with a TurboIonSpray ionization interface. ...
AimThe P2Y12 inhibitor prasugrel is a prodrug which is activated after its initial hydrolysis partly by CYP3A4. Grapefruit juice, a strong inactivator of intestinal CYP3A4, greatly reduces the activation and anti-platelet effects of clopidogrel. The aim of this study was to investigate the effects of grapefruit juice on prasugrel.Methods
In a randomized crossover study, seven healthy volunteers ingested 200 ml of grapefruit juice or water thrice daily for four days. On day three, they ingested a single 10-mg dose of prasugrel with an additional 200 ml of grapefruit juice or water. Plasma concentrations of prasugrel metabolites and the antiplatelet effect were measured.ResultsGrapefruit juice increased the geometric mean area under the plasma concentration–time curve (AUC0-∞) of prasugrel's primary, inactive metabolite to 164% of control (95% confidence interval 122%, 220%; P=0.008), without a significant effect on its peak plasma concentration (Cmax). The Cmax and AUC0-∞ of the secondary, active metabolite were decreased to 51% (95% confidence interval 32%, 84%; P=0.017) and 74% of control (95% confidence interval 60%, 91%; P=0.014) by grapefruit juice (P <0.05). The average platelet inhibition, assessed with the VerifyNow® method at 0 to 24 h after prasugrel intake, was 5 percentage points (95% confidence interval 1, 10 percentage points) lower in the grapefruit juice phase than in the water phase (P=0.034).Conclusion
Grapefruit juice reduces the bioactivation of prasugrel, but this has only a limited effect on the antiplatelet effect of prasugrel.
... Venous blood samples were collected 0.5, 1, 1.5, 2, and 4 hours after each single-dose administration in Part A and after the first dose of each dosing period in Part B. Samples were processed to plasma and analyzed by LC/MS/MS using previously described methods. 25 Pharmacokinetics of Pras-AM were assessed using these blood samples. Results of noncompartmental analyses of concentration-time profiles were used to determine relationships between dose level and Pras-AM exposure expressed as area under the concentration-time curve through the sampling time of the last quantifiable Pras-AM concentration (AUC[0Àt last ]) and maximum observed concentration (C max ) through 4 hours postdose. ...
This phase 2 study was designed to characterize the relationship among prasugrel dose, prasugrel's active metabolite (Pras-AM), and platelet inhibition while evaluating safety in children with sickle cell disease. It was open-label, multicenter, adaptive design, dose ranging, and conducted in 2 parts. Part A: Patients received escalating single doses leading to corresponding increases in Pras-AM exposure and VerifyNow®P2Y12 (VN) platelet inhibition and decreases in VNP2Y12 reaction units and vasodilator-stimulated phosphoprotein platelet reactivity index. Part B: Patients were assigned daily doses (0.06, 0.08, and 0.12 mg/kg) based on VN pharmacodynamic measurements at the start of 2 dosing periods, each 14±4 days. Platelet inhibition was significantly higher at 0.12 mg/kg (56.3%±7.4%; least squares mean±SE) compared with 0.06 mg/kg (33.8%±7.4%) or 0.08 mg/kg (37.9%±5.6%). Patients receiving 0.12 mg/kg achieved ≥30% platelet inhibition; only 1 patient receiving 0.06 mg/kg exceeded 60% platelet inhibition. High interpatient variability in response to prasugrel and the small range of exposures precluded rigorous characterization of the relationship among dose, Pras-AM, and platelet inhibition.
No hemorrhagic events occurred in Part A; 3 occurred in Part B, all mild and self-limited.
Most children with sickle cell disease may achieve clinically relevant platelet inhibition with titration of daily-dose prasugrel.
... 2-bromo-3′-methoxyacetophenone (MPB) derivatization of the thiol-containing active metabolite R-138727 of prasugrel in blood was performed immediately after sample collection to ensure the stability of the metabolites during sample processing and storage. The MPB-derivatized R-138727 showed a stable transition m/z 498→348 from the cleavage of the carbon-sulfur bond [136]. ...
Liquid chromatography-mass spectrometry (LC-MS) is one of the most prominent analytical techniques, due to its inherent selectivity and sensitivity. In LC-MS, chemical derivatizations are frequently used to enhance the MS ionization efficiency and selectivity, to facilitate structure elucidation, and to improve the chromatographic separation. In this review, we present an overview of derivatization-based LC-MS analysis. We summarize the reaction mechanisms of representative derivatization reagents and the selection strategy to guide and to stimulate future studies. Furthermore, we emphasize applications of derivatization in peptide and protein analysis, metabolite analysis, environmental analysis, pharmaceutical analysis, food-safety evaluation and MS imaging.
... S-(59-adenosyl)-L-methionine chloride (SAM), glucose-6-phosphate dehydrogenase from baker's yeast (G-6-PDH), D-glucose 6-phosphate disodium salt hydrate (G-6-P), b-nicotinamide adenine dinucleotide phosphate sodium salt (NADP), L-glutathione reduced (GSH), and DCMB were also purchased from Sigma-Aldrich Corporation. The derivatizing reagent for the assay of the pharmacologically active metabolites of prasugrel by LC-MS/MS (Farid et al., 2007b) m-methoxyphenacyl bromide (MPBr) and m-anisic acid were obtained from Tokyo Chemical Industry (Tokyo, Japan). All other reagents were commercially available and of guaranteed reagent grade. ...
Prasugrel, a thienopyridine anti-platelet drug, is converted in animals and humans to the pharmacologically active metabolite (R-138727, (2Z)-{1-[(1RS)-2-Cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-sulfanylpiperidin-3-ylidene}ethanoic acid) that has 2 chiral centers, occurring as a mixture of 4 isomers. The RS- and RR-isomers are more active than the SS- and SR-isomers (RS > RR > SR = SS). The pharmacologically active metabolite is further metabolized to an S-methylated metabolite that is the major identified inactive metabolite in humans . In rat, dog and human liver microsomes supplemented with S-adenosyl methione, the SS- and SR-isomers of the active metabolite were extensively S-methylated, while the RS- and RR-isomers were not. Addition of 2,3-dichloromethyl benzylamine (50 μM) completely inhibited the S-methylation reaction, indicating that the microsomal and cytosolic thiol methyltransferase but not the cytosolic thiopurine S-methyltransferase is involved in the methylation. The hepatic intrinsic clearance values for methylation of the RS-, RR-, SS- and SR-isomers (mL/min/kg) were 0, 0, 40.4 and 37.6, respectively, in rat liver microsomes, 0, 0, 11.6 and 2.5, respectively, in dog liver microsomes, and 0, 0, 17.3 and 17.7, respectively, in human liver microsomes, indicating that the RS- and RR-isomers are not methylated in vitro and that the methylation of SS- and SR-isomers is high with rat > human > dog. This finding in vitro agreed well with the in vivo observation in rats and dogs, where the S-methylated SS- and SR-isomers were the major metabolites in the plasma while negligible amounts of S-methylated RS- and RR-isomers were detected after intravenous administration of the pharmacologically active metabolites.
... Blood samples for plasma PK analysis were collected at 0.5, 1, 2, 3, and 4 hours (h) following the first dose of prasugrel and clopidogrel, and concentrations of the active metabolites of each were assayed as described previously (22,23). The primary parameter for analysis was the area under the concentration-time curve calculated through the last quantifiable concentration up to 4 h post dose, AUC (0-tlast) . ...
We compared results obtained with the Nanosphere Verigene® System, a novel point-of-care (POC) genetic test capable of analysing 11 CYP2C19 variants within 3 hours, to an established, validated genotyping method (Affymetrix™ DMET+; reference assay) for identifying extensive and reduced metabolisers of clopidogrel. Based on genotyping, patients (N=82) with stable coronary artery disease on clopidogrel 75 mg daily were defined as extensive metabolisers (*1/*1, *1/*17, *17/*17), reduced metabolisers (*1/*2, *1/*8, *2/*2, *2/*3), or of indeterminate metaboliser status (*2/*17). Pharmacokinetic exposure to clopidogrel's active metabolite and pharmacodynamic measures with protein reaction units (PRU) (VerifyNow® P2Y12 assay) and VASP PRI (PRI) were also assessed. There was a 99.9% overall concordance of marker-level data between the Nanosphere Verigene and DMET+ systems in identifying the CYP2C19 variants and 100% agreement in classifying the patients as extensive (n=59) or reduced metabolisers (n=15). Extensive metabolisers had significantly higher active metabolite exposure than reduced metabolisers (LS means 12.6 ng*h/ml vs 7.7 ng*h/ml; p<0.001). Extensive metabolisers also had lower PRU (LS means 158 vs 212; p=0.003) and VASP PRI (LS means 48% vs 63%, p=0.01) compared to reduced metabolisers. Rates of high on-treatment platelet reactivity were higher in reduced metabolisers compared to extensive metabolisers (VASP PRI ≥50%: 79% vs 47%; PRU ≥ 235: 33% vs 16%). The Nanosphere Verigene CBS system identified 11 CYP2C19 alleles in less than 3 hours with a high degree of accuracy when compared to a conventional method, and was further validated against pharmacokinetic and pharmacodynamic phenotypes.
Aims
Quantitative determination of prasugrel (PG) and it’s all possible process-related impurities.
Background
To the best of our knowledge very few analytical methods are available in the literature for monitoring of process related impurities and degradation products of PG in bulk drug substance/active pharmaceutical ingredient (API).
Objective
Separation of Prasugrel and it’s all possible process-related impurities viz., desacetyl prasugrel- tautomeric forms, intermediates including desacetyl impurity existing in its keto-enol form and positional tautomer impurities with degradation products.
Method
A simple and robust HPLC-UV method having Zorbax XDB C18 column (15 cm x 4.6 mm) 3.5µm particle size column.
Result
Prasugrel and its process related impurities were separated as well as analyzed in pharmaceutical samples biological matrices.
Conclusion
RP-LC method was developed for quantitative determination of PG and related substantial impurities was found to be highly specific, sensitive and precise. The major oxidative degradant was identified as PG desacetyl IMPs (keto-enol and positional tautomer) and hydroxyl IMP.
• Razuprotafib, a sulphamic acid-containing phosphatase inhibitor, is shown in vivo to undergo enzymatic oxidation and methylation to form a major metabolite in monkey and human excreta with an m/z– value of 633.
• LC-MS/MS analysis of samples derived from incubations of razuprotafib with human liver microsomes and recombinant CYP2C8 enzyme has elucidated the metabolic pathway for formation of the thiol precursor to the S-methyl metabolite MS633 (m/z– 633).
• Under in vitro conditions, the major pathway of razuprotafib metabolism involves extensive oxidation of the thiophene and phenyl rings.
• A single oxidation takes place at one of the phenyl groups. Multiple oxidations occur at the thiophene moiety: initial oxidation results in the formation of a thiolactone followed by a second oxidation giving rise to an S-oxide of the thiolactone, which is further metabolised to the ring-opened form and ultimate formation of a thiol (m/z– 619).
• An additional mono-oxidation pathway involves epoxidation of the thiophene followed by hydrolysis to a diol.
• The thiol and diol metabolites are trapped by the addition of a nucleophilic trapping agent, 3-methoxyphenacyl bromide (MPB), giving adducts with m/z– 767.
• The thiol is a likely precursor to the major in vivo razuprotafib metabolite, MS633.
Background: Standard administration of newer oral P2Y 12 inhibitors, including prasugrel or ticagrelor, provides suboptimal early inhibition of platelet aggregation (IPA) in ST-segment elevation myocardial infarction (STEMI) patients undergoing primary percutaneous coronary intervention (PCI). We sought to investigate the effects of cangrelor, tirofiban and prasugrel, administered as chewed or integral loading dose, on IPA in patients undergoing primary PCI.
Methods: The FABOLUS-FASTER is an investigator-initiated, multicenter, open-label, randomized study. A total of 122 P2Y 12 -naïve STEMI patients were randomly allocated (1:1:1) to cangrelor (n=40), tirofiban (n=40), both administered as bolus and 2h infusion followed by 60 mg of prasugrel, or 60 mg loading dose of prasugrel (n=42). The latter group underwent an immediate 1:1 sub-randomization to chewed (n=21), or integral (n=21) tablets administration. The trial was powered to test 3 hypotheses (non-inferiority of cangrelor compared with tirofiban using a non-inferiority margin of 9%, superiority of both tirofiban and cangrelor compared with chewed prasugrel and superiority of chewed prasugrel as compared with integral prasugrel; each with alpha of 0.016) for the primary endpoint that was 30-minute IPA at light transmittance aggregometry (LTA) in response to 20 μmol/L adenosine diphosphate (ADP).
Results: At 30 min, cangrelor did not satisfy non-inferiority compared with tirofiban, which yielded superior IPA over cangrelor (IPA: 95.0±8.9 vs. 34.1±22.5; P<0.001); cangrelor or tirofiban were both superior to chewed prasugrel (IPA: 10.5±11.0, P<0.001 for both comparisons), which did not provide higher IPA over integral prasugrel (6.3±11.4; P=0.47), despite yielding higher prasugrel's active metabolite concentration (ng/ml; 62.3±82.6 vs 17.1±43.5; P=0.016).
Conclusions: Cangrelor provided inferior IPA compared with tirofiban; both treatments yielded greater IPA compared with chewed prasugrel which led to higher active metabolite concentration but not greater IPA compared with integral prasugrel.
Clinical Trial Registration: clinicaltrials.gov NCT02978040, and EudraCT 2017-001065-24
LC–MS based bioanalysis has recently progressed to become the method of choice for analyzing various types of compounds in complex matrices. However, the analysis can be challenging due to various factors, including inherent low concentrations in biological samples, undesirable ionization efficiency or chromatographic behavior, and instability in complex matrices. Chemical derivatization could be a valuable means to improve chromatographic behaviors, MS detection, and lability of the molecule of interest. By introducing exogenous functional groups via chemical derivatization, unfavorable physicochemical properties for LC‐MS bioanalysis of the analytes will be improved to strengthen the LC–MS performance. Although the development and optimization of chemical derivatization employed bioanalysis may be time‐consuming, it has been acknowledged as an indispensable technique to extend the application of LC–MS analysis to a wider range of analytes. The past decades has witnessed numerous successful applications of derivatization integrated LC–MS bioanalysis for multiple compounds such as drug metabolites, proteins, biomarkers, residues and toxins in various matrices. This chapter introduced the derivatization strategies in sample preparation for quantitative LC–MS bioanalysis, and provided applications highlighting the impact of chemical derivatization to enhance the LC–MS performance.
Vorapaxar is a first-in-class antagonist of the protease-activated receptor-1, the primary thrombin receptor on human platelets, which mediates the downstream effects of thrombin in hemostasis and thrombosis. Prasugrel is a platelet inhibitor that acts as a P2Y12 receptor antagonist through an active metabolite, R-138727. This study investigated the interaction of these 2 platelet antagonists when coadministered. This was a randomized, open-label, multiple-dose study in 54 healthy volunteers consisting of a fixed-sequence crossover and a parallel group design. In sequence 1, 36 subjects received prasugrel 60 mg on day 1 and then prasugrel 10 mg once daily on days 2 to 7, followed by vorapaxar 40 mg and prasugrel 10 mg on day 8 and then vorapaxar 2.5 mg and prasugrel 10 mg orally once daily on days 9 to 28. In sequence 2, 18 subjects received vorapaxar 40 mg on day 1 and then vorapaxar 2.5 mg once daily on days 2 to 21. The geometric mean ratios (90% confidence intervals) for AUCτ and Cmax of coadministration/monotherapy for vorapaxar (0.93 ng·h/mL[0.85–1.02 ng·h/mL] and 0.95 ng/mL [0.86–1.05 ng/mL]) and R-138727 (0.91 ng·h/mL [0.85– 0.99 ng·h/mL] and 1.02 ng/mL [0.89–1.17 ng/mL]) were within prespecified bounds, demonstrating the absence of a pharmacokinetic interaction between vorapaxar and prasugrel. There was no specific safety or tolerability risk associated with multiple-dose coadministration of vorapaxar and prasugrel. In conclusion, in this study in healthy volunteers, there was no pharmacokinetic drug–drug interaction between vorapaxar and prasugrel. Multiple-dose coadministration of the 2 drugs was generally well tolerated.
This chapter focuses on the development of prasugrel (4), whose 2-OCOCH3 group plays an essential role in modulating the in vivo antiplatelet activity of thienopyridine (TP). It deals with the history, synthesis, mode of action, and structure-activity relationships of prasugrel (4). Prasugrel (4) is used to reduce thrombotic cardiovascular events, including stent thrombosis, in patients with acute coronary syndrome (ACS), and to prevent thrombosis after percutaneous coronary intervention. The third-generation antiplatelet prasugrel (4) is the acetate ester prodrug of 2-hydroxy TP. It was designed and synthesized to overcome the resistance (non- or poor responsiveness) to the clopidogrel (3), which is mainly caused by CYP2C19 loss-of-function polymorphism. Prasugrel (4) produces rapid and long-lasting inhibition of platelet function, has a greater potency than clopidogrel (3), and shows additive effects when used in combination with aspirin to prevent blood clots after angioplasty or coronary bypass graft.
Recently, there has been an increasing use of the cyclopropyl ring in drug development to transition drug candidates from the preclinical to clinical stage. Important features of the cyclopropane ring are, the 1) coplanarity of the three carbon atoms, 2) relatively shorter (1.51 Å) C-C bonds, 3) enhanced pi-character of C-C bonds, and 4) C-H bonds are shorter and stronger than those in alkanes. The present review will focus on the contributions that a cyclopropyl ring makes to the properties of drugs containing it. Consequently, the cyclopropyl ring addressess multiple roadblocks that can occur during drug discovery such as: a) enhancing potency, b) reducing off-target effects, c) increasing metabolic stability, d) increasing brain permeability, e) decreasing plasma clearance, f) contributing to an entropically more favorable binding to the receptor, g) conformational restriction of peptides/peptidomimetics to prevent proteolytic hydrolysis, and h) altering drug pKa to reduce its P-glycoprotein efflux ratio.
Bioequivalence studies are performed to obtain marketing authorization for generic medicinal products. Assessment of bioequivalence is usually performed after a single dose administration on the basis of the maximum plasma concentration (Cmax) and area under the plasma concentration vs. time curve from time zero until the last measurable concentration (AUC(0-t)). The European Medicines Agency (EMA) recommends conducting bioequivalence studies based on the parent compound (active pharmaceutical ingredient). However, it is not possible for all medicines and the problem is especially important in case of prodrugs. If determination of the parent compound is not applicable EMA recommends choosing active metabolite as the analyte. The draft EMA guideline on bioequivalence of medicinal products containing prasugrel questions this general rule.
A cost effective, sensitive, simple, and rapid high-performance liquid chromatography-tandem mass spectrometry method was developed and validated for the quantification of the prasugrel metabolite in human plasma. Following solid phase extraction, the analyte (prasugrel active metabolite; R-138727) and internal standard (emtricitabine) were separated using a mobile phase in an isocratic elution mode on a reverse phase C18 column and were analyzed by an LC-MS/MS in the multiple reaction monitoring mode using the respective [M+H](+) ions, m/z 498.3-206.0 for R-138727 and m/z 248.2-130.1 for the internal standard. The assay exhibited a linear dynamic range of 0.2-120ng/mL. The lower limit of quantification was 0.2ng/mL with a relative standard deviation of 5.0%. This LC-MS/MS method was validated with intra-batch and inter-batch precision and accuracy. Results for precision and accuracy are in range of 3.9-9.6% and 95.2-102.2% respectively. This validated method is simple and repeatable to use in bioequivalence/pharmacokinetic studies.
Background:
Platelet inhibitory effects induced by oral P2Y12 receptor antagonists are delayed in patients with ST-segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PPCI), which may be attributed to impaired absorption affecting drug pharmacokinetics (PK) and pharmacodynamics (PD). Crushing tablets has been suggested to lead to more favorable PK/PD profiles. To date, no studies have investigated the PK/PD effects of crushing prasugrel.
Objectives:
This study sought to determine whether crushing prasugrel is associated with more favorable drug bioavailability and platelet inhibitory effects compared with whole tablets in STEMI patients undergoing PPCI.
Methods:
Our prospective, randomized, open-label study assessed STEMI patients undergoing PPCI (n = 52) who were treated with a prasugrel 60-mg loading dose (LD) either as whole or crushed tablets. PK/PD analyses were performed at 7 time points. PD effects were measured as P2Y12 reaction units and platelet reactivity index, and PK by plasma levels of prasugrel's active metabolite.
Results:
Compared with whole tablets, crushed prasugrel led to reduced P2Y12 reaction units by 30 min post-LD, which persisted at 1, 2 (164 vs. 95; least square mean difference = 68; 95% confidence interval: 10 to 126; primary endpoint), and 4 h post-LD. Significant differences were no longer present at 6 h post-LD. Parallel findings were shown with platelet reactivity index. Accordingly, high on-treatment platelet reactivity rates were reduced with crushed prasugrel. PK analyses showed a >3-fold faster absorption with crushed compared with whole prasugrel.
Conclusions:
In STEMI patients undergoing PPCI, crushed prasugrel leads to faster drug absorption, and consequently, more prompt and potent antiplatelet effects compared with whole tablet ingestion. (Pharmacological Effects of Crushing Prasugrel in STEMI Patients; NCT02212028).
Prasugrel, being a potent platelet aggregation inhibitor, is used widely around the world to reduce cardiovascular risks in patients with stroke, myocardial infarction, and atherosclerosis. The aim of this review firstly to focus on a comprehensive update of chromatography determination of Prasugrel and its metabolites in human plasma, and in pharmaceutical preparations. It has been described using TLC, HPLC/MS, RP-HPLC, and UV methods. Secondly to localize the chromatographic conditions for separation and quantification. This review provides detailed information on separation conditions for Prasugrel alone, with Aspirin, and in the presence of its related compounds.
List of Abbreviations Introduction Thienopyridines Direct-Acting P2Y12 Antagonists Summary References
Accurate and specific analysis of target molecules in complex biological matrices remains a significant challenge, especially when ultra-trace detection limits are required. Liquid chromatography with mass spectrometry is often the method of choice for bioanalysis. Conventional sample preparation and clean-up methods prior to analysis of biological fluids such as liquid-liquid extraction, solid phase extraction, or protein precipitation are time-consuming, tedious, and can negatively affect target recovery and detection sensitivity. An alternative or complementary strategy is the use of an off-line or on-line in situ derivatization technique. In situ derivatization can be incorporated to directly derivatize target analytes in their native biological matrices, without any prior sample clean-up methods, to substitute or even enhance the extraction and pre-concentration efficiency of these traditional sample preparation methods. Designed appropriately, it can reduce the number of sample preparation steps necessary prior to analysis. Moreover, in situ derivatization can be used to enhance the performance of the developed liquid chromatography with mass spectrometry based bioanalysis methods regarding stability, chromatographic separation, selectivity, and ionization efficiency. This review presents an overview of the commonly used in situ derivatization techniques coupled to liquid chromatography with mass spectrometry based bioanalysis to guide and to stimulate future research. This article is protected by copyright. All rights reserved.
A new, simple, sensitive, precise and accurate High Performance Thin Layer Chromatographic (HPTLC) method for determination of Prasugrel in their tablet dosage form has been developed, validated. Chromatographic separation was achieved on aluminum plates precoated with silica gel 60 F254 as stationary phase and toluene-methanol-acetic acid (8:2:0.04, v/v/v) as mobile phase. Densitometric measurement of their spots was achieved at 220 nm over the concentration ranges of 0.4-1.4 μg spot-1 with mean recoveries of 100.61 ± 1.04. Limit of detection and Limit of quantification for Prasugrel were found to be 0.08 μg spot-1 and 0.26 μg spot-1 respectively. The proposed method was successfully applied for analysis of Prasugrel in commercial tablets and applied to the content uniformity testing of tablets.
A stability-indicating RP-LC method for the determination of prasugrel in tablets was developed and validated. Stress testing of prasugrel was carried out in accordance with ICH guidelines, where the drug was submitted to acidic and basic hydrolysis, oxidative, thermal and photolytic conditions. Prasugrel was unstable under all the conditions and the degradations products were analyzed by HPLC-UV. Furthermore, two main degradation products found under alkaline and thermal conditions were investigated by LC-MS. Based on the fragmentation patterns, two products resulted from hydrolysis of the acetate ester moiety of prasugrel were observed. Due the chemical equilibrium, tautomerism occurs between the ketone and alcohol functions justifying the similar molecular weight and fragment pattern obtained in degradation products analysis. Successful separation was achieved on a RP-18 octadecyl silane column using acetonitrile and triethylamine 0.5% mixture (50:50, v/v) as the mobile phase at 25 °C. The flow rate was 1.0 mL/min and the detector wavelength was 263 nm. The method proposed in this work was successfully applied to quality control of prasugrel and contribute to stability assessment of pharmaceutical products containing this drug.
Current study develops and validates a dissolution test for Prasugrel hydrochloride 10 mg in coated tablets. After sink condition, filters and drug stability were evaluated, the discriminatory dissolution conditions were achieved with a USP apparatus 1 (basket) at 50 rpm stirring speed and 900 mL of 0.01 M HCl as dissolution medium. The UV spectrometric method at 220 nm was performed and validated for the determination of Prasugrel. The parameters specificity, linearity, accuracy, precision and robustness were evaluated according to international protocols. UV method and dissolution test proposed in current analysis may be applied for quality control of coated tablets containing Prasugrel since there is no official monograph for this drug. © 2014, Eduem - Editora da Universidade Estadual de Maringa. All rights reserved.
Prasugrel was subjected to forced degradation studies under conditions of hydrolysis (acid, base and neutral), photolysis, oxidation and thermal stress. The drug showed lability in hydrolytic as well as oxidative conditions, resulting in a total of four degradation products. In order to characterize the degradation products, initially mass fragmentation pathway of the drug was established with the help of mass spectrometry/time-of-flight, multiple stage mass spectrometry and hydrogen/deuterium exchange data. The degradation products were then separated on a C18 column using a stability-indicating volatile buffer method, which was later extended to liquid chromatography with mass spectrometry studies. The latter highlighted that three degradation products had the same molecular mass, while one was different. To characterize all, their mass fragmentation pathways were established first, in the same manner as the drug. Subsequently, liquid chromatography with NMR spectroscopy data were collected. Proton and correlation liquid chromatography with NMR spectroscopy studies highlighted existence of diastereomeric behavior in one pair of degradation products. Lastly, toxicity prediction by computer-assisted technology (TOPKAT) and deductive estimation of risk from existing knowledge (DEREK) software were employed to assess the in silico toxicity of the characterized degradation products. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
The anti-thrombotics of the tetrahydrothienopyridine series, clopidogrel and prasugrel, are prodrugs that must be metabolized in two steps to become pharmacologically active. The first step is the formation of a thiolactone metabolite. The second step is a further oxidation with formation of a thiolactone sulfoxide whose hydrolytic opening leads to a sulfenic acid that is eventually reduced into the corresponding active cis thiol. Very few data were available on the formation of the isomer of the active cis thiol having a trans configuration of the double bond, the most striking result in that regard being that both cis and trans thiols were formed upon metabolism of clopidogrel by human liver microsomes in the presence of glutathione (GSH), whereas only the cis thiol was detected in the sera of patients treated with this drug. This article shows that trans thiols are also formed upon microsomal metabolism of prasugrel or its thiolactone metabolite in the presence of GSH, and that metabolites having the trans configuration of the double bond are only formed when microsomal incubations are done in the presence of thiols, such as GSH, N-acetyl-cysteine and mercaptoethanol. Intermediate formation of thioesters resulting from reaction of GSH with the thiolactone sulfoxide metabolite appears to be responsible for trans thiol formation. Addition of human liver cytosol to the microsomal incubations led to a dramatic decrease of the formation of the trans thiol metabolites. These data suggest that cytosolic esterases would accelerate the hydrolytic opening of thiolactone sulfoxide intermediates and disfavour the formation of thioesters resulting from reaction of these intermediates with GSH that is responsible for trans isomer formation. This would explain why trans thiols have not been detected in the sera of patients treated with clopidogrel.
Introduction
Patients treated with clopidogrel who have higher body size exhibit greater platelet reactivity than patients with lower body size. In a retrospective analysis of the FEATHER trial, we examined the relationship between platelet response to thienopyridines clopidogrel 75 mg (Clop-75), prasugrel 5 mg (Pras-5), and prasugrel 10 mg (Pras-10) using 3 body size indices: body weight (BW), body mass index (BMI), and body surface area (BSA). Relationships are assessed as continuous variables and as 4 incremental body size groups.
Materials and Methods
Aspirin-treated patients with stable coronary artery disease (N = 72) and a BW range of 45-134 kg received Clop-75, Pras-5, and Pras-10 in a 3-period, blinded, cross-over study. Platelet assays included maximum platelet aggregation (MPA) to 20 μM ADP by light transmission aggregometry, VerifyNow-P2Y12 reaction units (PRU), and vasodilator-associated stimulated phosphoprotein (VASP) phosphorylation platelet reactivity index (PRI). Exposure to active metabolites (AMs) was also assessed.
Results
Body size was a determinant of AM exposure and residual platelet reactivity regardless of type and dose of thienopyridine. BW and BSA demonstrated marginally stronger correlations with platelet reactivity; VASP-PRI demonstrated a stronger correlation with the body size than the other tests. Correlation coefficients ranged from a high of 0.64 (BW vs. PRI on Pras-5) to a low of 0.34 (BMI vs. MPA on Pras-10), but all were statistically significant (p < 0.01).
Conclusions
Using a comprehensive selection of body size indices, AM exposures, platelet function tests, and thienopyridine doses, we demonstrated a consistent inverse relationship between body size and response to clopidogrel and prasugrel.
A sensitive and selective ultra-high performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) method was developed for the fast, simultaneous quantification of three novel cardiac drugs (aliskiren, prasugrel and rivaroxaban) in human urine. Sample preparation was performed with microextraction with packed sorbent (MEPS), which is a miniaturization of solid phase extraction. The optimal conditions for MEPS extraction were obtained using C8 sorbent, small sample volumes and a short time period (about 3min for the entire sample preparation step). Chromatographic separation of the selected compounds was achieved in less than 1.5min on a Zorbax Rapid Resolution High Definition SB-C18 column using isocratic elution with 0.1% formic acid and acetonitrile (70:30, v/v) at a flow rate of 0.8mLmin(-1). The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring via an electrospray ionization source with positive ionization mode. The method was fully validated according to the latest recommendations of international guidelines. The lower limit of quantification was 5.0pgmL(-1) for aliskiren and rivaroxaban and 0.5pgmL(-1) for prasugrel. The intra- and inter-day precision was within 7.12% and the accuracy ranged from -7.54% to 4.17%. The mean extraction recoveries of the MEPSC8 methodology were found to be 98.3% for aliskiren, 100.3% for rivaroxaban and 99.9% for prasugrel. This MEPSC8-UHPLC-MS/MS method offers a fast, simple and precise way to determine selected novel cardiac drugs in human urine that could be applied to therapeutic drug monitoring and pharmacokinetic studies.
Human platelets express 2 G protein-coupled nucleotide receptors: the platelet adenosine diphosphate (ADP) receptor coupled to stimulation of phospholipase C (P2Y(1)) via heterotrimeric guanosine 5-triphosphate (GTP)-binding protein G(q), and the platelet ADP receptor coupled to inhibition of adenylyl cyclase (P2Y(12)) via heterotrimeric GTP-binding protein G(i). Although these 2 receptors are encoded on the same chromosome and have similar pharmacologic profiles, they have different reactivities toward thiol reagents. The thiol agent p-chloromercuribenzene sulfonic acid (pCMBS) and the active metabolites from antiplatelet drugs, clopidogrel and CS-747, inactivate the P2Y(12) receptor and are predicted to interact with the extracellular cysteine residues on the P2Y(12) receptor. In this study we identified the reactive cysteine residues on the human P2Y(12) receptor by site-directed mutagenesis using pCMBS as the thiol reagent. Cys97Ser and Cys175Ser mutants of the P2Y(12) receptor did not express when transfected into Chinese hamster ovary (CHO-K1) cells, indicating the essential nature of a disulfide bridge between these residues. The Cys17Ser, Cys270Ser, and Cys17Ser/Cys270Ser double mutants had similar median effective concentration (EC(50)) values for ADP and 2-methylthio-ADP (2-MeSADP) when compared with the wild-type P2Y(12). Similarly, the median inhibitory concentration (IC(50)) values for BzATP (2',3'-O-(4- benzoylbenzoyl) adenosine 5'-triphosphate), an antagonist of the P2Y(12) receptor, also did not differ dramatically among these mutants and the wild-type P2Y(12) receptor. pCMBS inactivated the wild-type P2Y(12) receptor in a concentration-dependent manner, whereas it had no effect on the P2Y(1) receptor. Finally, pCMBS partially affected the G(i) coupling of Cys17Ser or Cys270Ser receptor mutants, but had no effect on Cys17Ser/Cys270Ser P2Y(12) receptor-mediated inhibition of adenylyl cyclase. These results indicate that, unlike the P2Y(1) receptor, which has 2 essential disulfide bridges linking its extracellular domains, the P2Y(12) receptor has 2 free cysteines in its extracellular domains (Cys17 and Cys270), both of which are targets of thiol reagents. We speculate that the active metabolites of clopidogrel and CS-747 form disulfide bridges with both Cys17 and Cys270 in the P2Y(12) receptor, and thereby inactivate the receptor.
The biotransformation of prasugrel to R-138727 (2-[1-2-cyclopropyl-1-(2-fluorophenyl)-2-oxoethyl]-4-mercapto-3-piperidinylidene]acetic acid) involves rapid deesterification to R-95913 (2-[2-oxo-6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl]-1-cyclopropyl-2-(2-fluorophenyl)ethanone) followed by cytochrome P450 (P450)-mediated formation of R-138727, the metabolite responsible for platelet aggregation. For identification of the P450s responsible for the formation of the active metabolite, the current studies were conducted with R-95913 as the substrate. Incubations required supplementation with reduced glutathione. Hyperbolic kinetics (K(m) 21-30 microM), consistent with a single enzyme predominating, were observed after incubations with human liver microsomes. Correlation analyses revealed a strong relationship between R-138727 formation and CYP3A-mediated midazolam 1'-hydroxylation (r(2) = 0.98; p < 0.001) in a bank of characterized human liver microsomal samples. The human lymphoblast-expressed enzymes capable of forming R-138727, in rank order of rates, were CYP3A4>CYP2B6>CYP2C19 approximately CYP2C9>CYP2D6. A monoclonal antibody to CYP2B6 and the CYP3A inhibitor ketoconazole substantially inhibited R-138727 formation, whereas inhibitors of CYP2C9 (sulfaphenazole) and CYP2C19 (omeprazole) did not. Scaling of in vitro intrinsic clearance values from expressed enzymes to the whole liver using a relative abundance approach indicated that either CYP3A4 alone or CYP3A4 and CYP2B6 are the major contributors to R-138727 formation. R-95913 and R-138727 were also examined for their ability to inhibit metabolism mediated by five P450s. R-138727 did not inhibit the P450s tested. In vitro, R-95913 inhibited CYP2C9, CYP2C19, CYP2D6, and CYP3A, with K(i) values ranging from 7.2 microM to 82 microM, but did not inhibit CYP1A2. These K(i) values exceed circulating concentrations in humans by 3.8- to 43-fold. Therefore, neither R-95913 nor R-138727 is expected to substantially inhibit the P450-mediated metabolism of coadministered drugs.
A method was optimalised for the quantitative determination of ceftiofur and its active metabolite desfuroylceftiofur in horse plasma and synovial fluid.The principle of the method was that bound desfuroylceftiofur is first released by dithioerythritol, a reducing agent, followed by the derivatization of the free sulfhydryl group with iodoacetamide. The stable derivative—desfuroylceftiofuracetamide—was then further purified using an Oasis HLB solid-phase extraction column. Chromatography was performed using a PLRP-S polymeric column (100 Å, dp: 5 μm, mm i.d.), with a mixture of 0.1% trifluoro acetic acid in water and acetonitrile as the mobile phase. Gradient elution was performed. The flow-rate was 0.4 ml/min and the UV detector was set at a wavelength of 266 nm. The method was validated in plasma and synovial fluid (linearity, precision, trueness, LOQ, LOD, specificity, susceptibility to interferences). Calibration graphs were prepared over a concentration range of 0–20 μg/ml and good linearity was achieved (r≥0.99, g≤10%). A limit of quantification of 0.5 μg/ml was obtained for ceftiofur in both matrices. Limits of detection were 0.36 and 0.27 μg/ml for ceftiofur in plasma and synovial fluid, respectively. The results of the within-run and between-run precision and the trueness fell within the ranges specified.The main advantage of our method, compared to previously reported methods, was that the sample preparation procedure was less time consuming, resulting in a higher sample throughput (up to 40 samples a day). In addition, the analysis cost was reduced due to the consumption of a lower amount of solvents and reagents and of only one solid-phase extraction column per sample. The method was successfully applied during a pharmacokinetic study in horses after the administration of ceftiofur sodium via regional intravenous perfusion and systemic intravenous injection.
The determination of thiol-containing compounds in biological fluids is important in biochemistry and clinical chemistry. In this paper, derivatization reagents for thiols are reviewed with respect to their reactivity, selectivity, spectroscopic characteristics and their applicability especially to high-performance liquid chromatography. Derivatization used in ultraviolet and electrochemical detection. The derivatization reagents contain a functional group, e.g. an N-substituted maleimide, active halogen or aziridine, which react with the thiol group. Derivatization for use in flow injection analysis, thin-layer chromatography or gas chromatography-mass spectrometry is also described.
Many reagents suitable for the derivatization of neurotransmitters are selective for the amino function. Others, however, are selective for the carboxyl-, thiol- and hydroxyl function, and recently, reagents selective for more than one function have been produced. Interest persists in the established reagents, with their well understood behaviour which assists automation of analysis as much as new technology. Workers appear reluctant to tackle the optimization of many novel reagents. Chiral reagents may become important if d-amino acids are shown to be significant from a physiological point of view. Solid-phase reagents offer better regulated chemistry and combined derivatization/solid-phase extraction, which make them an exciting prospect.
A high-performance liquid chromatographic method for the sensitive determination of 2,2'-[(2-aminoethyl)imino]diethanol bis(butylcarbamate) (I) and its metabolites in human serum has been developed. The method was based on a pre-column derivatization with o-phthalaldehyde. The derivatives were stabilized at least for 24 h at 4 degrees C by using N-acetyl-L-cysteine as a thiol and by eliminating the excess o-phthalaldehyde in the reaction mixture by solvent extraction and the addition of an ammonium salt after the reaction. The recoveries and reproducibilities in human serum spiked with I and its two metabolites were satisfactory, and the responses were linear over a wide range of analyte concentrations. The detection limits of I and its metabolites, II and III, in serum were 0.5, 4 and 2 ng/ml, respectively, at a signal-to-noise ratio of 5. The method was satisfactorily applied to the clinical study of I.
As it is extremely unstable in blood, the thiol compound BMS186716 was stabilized by the addition of methyl acrylate (MA) to blood samples. The blood samples were then kept in ice for 10-15 min for completion of the Michael addition reaction to occur between the thiol group of BMS186716 and MA, after which the plasma was separated by centrifugation under refrigeration. For sample analysis, the standard and quality control samples were prepared by spiking blank plasma with the BMS186716-MA adduct. After addition of the internal standard, BMS 188035-MA, each sample was acidified with HCI and then extracted with methyl tert.-butyl ether. Each reconstituted extract was injected into a high-performance liquid chromatography-positive ion electrospray ionization mass spectrometric system. The electrospray condition was chosen to enhance the [M+NH4]+ signal at the expense of the [M+H]+ signal. Monitoring the [M+NH4]+ signal, a lower limit of quantitation of 2.5 ng/ml was achieved, with 0.5 ml plasma. We have thus shown that a sulfhydryl compound (BMS186716) in blood can successfully be stabilized by reacting it with MA and that the adduct produced is adequately stable in blood and plasma to allow the development of a rugged quantitative bioanalytical method.
During method development in support of non-clinical studies in animal models, BMS-186716 was found to be extremely unstable in blood and plasma. Stabilization of the compound was achieved by reacting the compound with methyl acrylate (MA) in blood, from which the plasma was then prepared. While the resulting BMS-186716-MA adduct was found to be stable in dog plasma, and hence it was used as the basis for the method developed for analysis of dog plasma samples, the BMS-186716-MA adduct was found to be unstable in rat plasma as it was readily hydrolyzed to BMS-186716-acrylic acid (AA) by native esterases found in rat plasma. Although the finding of the instability of BMS-186716-MA in rat plasma was not the result of prospective planning, we were able to successfully develop a quantitative bioanalytical method using BMS-186716-AA as the analyte instead of the originally planned BMS-186716-MA analyte. The standard and quality-control (QC) samples were prepared by spiking blank plasma with BMS-186716-MA, and then allowing them to stand at room temperature for 1 h to convert BMS-186716-MA to BMS-186716-AA. After adding the internal standard BMS-188035-AA, each sample was acidified with HCl and then extracted with methyl tert.-butyl ether. The reconstituted extract was injected into a HPLC-electrospray ionization mass spectrometric system for detection by positive ion electrospray ionization. A lower limit of quantitation (LLQ) of 5 ng/ml was achieved, using 0.1 ml plasma and a standard curve range of 5-5000 ng/ml.
Omapatrilat, the most clinically advanced member of a new class of cardiovascular agents, vasopeptidase inhibitors, is under development at Bristol-Myers Squibb Pharmaceutical Research Institute for the treatment of hypertension and heart failure. An electrospray LC/MS/MS method has been developed and validated for the simultaneous determination of omapatrilat and its four metabolites in human plasma. Since omapatrilat and two of the metabolites are sulfhydryl-containing compounds, methyl acrylate was used to stabilize these compounds in human blood and plasma samples. Methyl acrylate reacted instantly with the sulfhydryl group to form a derivative that was stable in blood and plasma. Extraction of the analytes from plasma samples was achieved by semiautomated liquid-liquid extraction, where a robotic liquid handler performed the liquid-transferring steps. The mass spectrometer was operated in the negative ion selected-reaction-monitoring mode. The calibration curve ranges were 0.5-250 ng/mL for omapatrilat and one metabolite and 2.0-250 ng/mL for the other three metabolites.
In order to improve the sensitivity and stability of human blood samples containing WR-1065 (i.e., active metabolite of the cytoprotective agent amifostine), a high-performance liquid chromatographic method was developed and validated using fluorescent derivatization with ThioGlo3. Using a sample volume of only 100 microl, the method was specific, sensitive (limit of quantitation=10 nM in deproteinized blood or 20 nM in whole blood), accurate (error < or = 3.2%) and reproducible (CV < or = 8.7%). In addition, the stability of WR-1065 in deproteinized and derivatized blood samples was assured for at least four weeks at -20 degrees C. This method should be particularly valuable in translating the kinetic-dynamic relationship of WR-1065 in preclinical models to that in cancer patients.
Membrane-bound P2-receptors mediate the actions of extracellular nucleotides in cell-to-cell signalling. P2X-receptors are ligand-gated ion channels, whereas P2Y-receptors belong to the superfamily of G-protein-coupled receptors (GPCRs). So far, the P2Y family is composed out of 8 human subtypes that have been cloned and functionally defined; species orthologues have been found in many vertebrates. P2Y1-, P2Y2-, P2Y4-, P2Y6-, and P2Y11-receptors all couple to stimulation of phospholipase C. The P2Y11-receptor mediates in addition a stimulation of adenylate cyclase. In contrast, activation of the P2Y12-, P2Y13-, and P2Y14-receptors causes an inhibition of adenylate cyclase activity. The expression of P2Y1-receptors is widespread. The receptor is involved in blood platelet aggregation, vasodilatation and neuromodulation. It is activated by ADP and ADP analogues including 2-methylthio-ADP (2-MeSADP). 2'-Deoxy-N6-methyladenosine-3',5'-bisphosphate (MRS2179) and 2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate (MRS2279) are potent and selective antagonists. P2Y2 transcripts are abundantly distributed. One important example for its functional role is the control of chloride ion fluxes in airway epithelia. The P2Y2-receptor is activated by UTP and ATP and blocked by suramin. The P2Y2-agonist diquafosol is used for the treatment of the dry eye disease. P2Y4-receptors are expressed in the placenta and in epithelia. The human P2Y4-receptor has a strong preference for UTP as agonist, whereas the rat P2Y4-receptor is activated about equally by UTP and ATP. The P2Y4-receptor is not blocked by suramin. The P2Y6-receptor has a widespread distribution including heart, blood vessels, and brain. The receptor prefers UDP as agonist and is selectively blocked by 1,2-di-(4-isothiocyanatophenyl)ethane (MRS2567). The P2Y11-receptor may play a role in the differentiation of immunocytes. The human P2Y11-receptor is activated by ATP as naturally occurring agonist and it is blocked by suramin and reactive blue 2 (RB2). The P2Y12-receptor plays a crucial role in platelet aggregation as well as in inhibition of neuronal cells. It is activated by ADP and very potently by 2-methylthio-ADP. Nucleotide antagonists including N6-(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-beta,gamma-dichloromethylene-ATP (=cangrelor; AR-C69931MX), the nucleoside analogue AZD6140, as well as active metabolites of the thienopyridine compounds clopidogrel and prasugrel block the receptor. These P2Y12-antagonists are used in pharmacotherapy to inhibit platelet aggregation. The P2Y13-receptor is expressed in immunocytes and neuronal cells and is again activated by ADP and 2-methylthio-ADP. The 2-chloro-5-nitro pyridoxal-phosphate analogue 6-(2'-chloro-5'-nitro-azophenyl)-pyridoxal-alpha5-phosphate (MRS2211) is a selective antagonist. mRNA encoding for the human P2Y14-receptor is found in many tissues. However, a physiological role of the receptor has not yet been established. UDP-glucose and related analogues act as agonists; antagonists are not known. Finally, UDP has been reported to act on receptors for cysteinyl leukotrienes as an additional agonist--indicating a dual agonist specificity of these receptors.
A highly sensitive and simple HPLC method with fluorescence detection for the determination of phentermine (Phen), fenfluramine (Fen) and norfenfluramine (Norf, the active metabolite of Fen) in rat brain and blood microdialysates has been developed. The brain and blood microdialysates were directly subjected to derivatization with 4-(4,5-diphenyl-1H-imidazol-2-yl) benzoyl chloride (DIB-Cl) in the presence of carbonate buffer (0.1 M, pH 9.0) at room temperature. The chromatographic conditions consisted of an ODS column and mobile phase composition of acetonitrile and water (65:35, v/v) with flow rate set at 1.0 ml/min. The detection was performed at excitation and emission wavelengths of 325 and 430 nm, respectively. Under these conditions, the DIB-derivatives of Phen, Fen and Norf were well separated and showed good linearities in the studied ranges (5-2000 nM for Phen and 10-2000 nM for Norf and Fen) with correlation coefficients greater than 0.999. The obtained detection limits were less than 23 fmol on column (for the three compounds) in both brain and blood microdialysates at a signal-to-noise ratio of 3 (S/N=3). The intra- and the inter-assay precisions were lower than 10%. The method coupled with microdialysis was applied for a pharmacokinetic drug-drug interaction study of Phen and Fen following individual and combined intraperitoneal administration to rats. In addition, since the role of protein binding in drug interactions can be quite involved, the method was applied for the determination of total and free Phen and Fen in rat plasma and ultrafiltrate, respectively. The results showed that Fen and/or Norf significantly altered the pharmacokinetic parameters of Phen in both blood and brain but did not alter its protein binding. On the other hand, there was no significant difference in the pharmacokinetics of Fen when administered with Phen.