Evaluation of the INNOVANCE PFA P2Y test cartridge: sensitivity to P2Y(12) blockade and influence of anticoagulant.

Monitoring of platelet ADP receptor P2Y(12) inhibition may be performed by a variety of platelet function assays. Given the lack of sensitivity of the existing PFA-100® cartridge formulations to detect P2Y(12) inhibition, a new cartridge for the PFA-100 (INNOVANCE® PFA P2Y) has recently been developed. The performance of the new PFA-100 test cartridge was compared with standard collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges, light transmission aggregometry, vasodilator-stimulated phosphoprotein, the VerifyNow® P2Y(12) assay and multiple electrode aggregometry. In this study, 20 normal blood samples anticoagulated with either citrate or hirudin were spiked with two different clinically relevant concentrations (1 and 10 µM final concentration) of the prasugrel active metabolite (R-138727, Lilly/Daiichi Sankyo) for 30 min at 37°C. Comparison of the platelet function tests demonstrated that all tests (except CADP and CEPI) were substantially inhibited by 10 µM R-138727. Intermediate results were typically obtained with 1 µM R-138727 in citrated blood. However, both MEA ADP and ADPHS tests were highly sensitive to 1 µM R-138727 in hirudin anticoagulated blood. Further comparison of citrate or hirudin blood samples (N = 5) revealed that all platelet tests (except CEPI) became more sensitive to 1 µM R-138727 in hirudinized blood. The INNOVANCE PFA P2Y cartridge proved to be sensitive to P2Y(12) inhibition and was comparable to other currently available platelet function tests. The sensitivity of all platelet function tests for detecting in vitro inhibition of P2Y(12) is markedly different depending on the anticoagulant used.

The new INNOVANCE® PFA P2Y cartridge is sensitive to the detection of the P2Y12 receptor inhibition.

Insufficient response on antiplatelet medication has become an intensively discussed issue because of the risk factor of recurrent adverse cardiovascular events. However, the monitoring of antiplatelet therapy requires appropriate, robust and reliable test methods. For the measurement of thienopyridine effects, the manufacturer of the PFA-100® System provides the INNOVANCE® PFA P2Y * cartridge. We tested this cartridge for its capacity to detect the inhibition of the P2Y12 receptor, which is the target for thienopyridine medication (e.g. clopidogrel). We compared the INNOVANCE® PFA P2Y * results with those obtained by the receptor specific flow cytometric vasodilator stimulated phosphoprotein (VASP) assay that expresses the status of the P2Y12 receptor as "platelet reactivity index" (PRI). The in vitro addition of the P2Y12 receptor antagonist cangrelor (AR-C69931MX) to citrated human whole blood resulted in a dose-dependent prolongation of closure times (CTs) of the INNOVANCE® PFA P2Y * cartridge correlating with decreased PRI levels. In volunteers, the intake of a 600 mg clopidogrel loading dose caused an increase of the CTs in all volunteers, although some of these volunteers were identified as "poor responders" by the VASP assay (no significant reduction of PRI levels). In 50 patients with stable coronary artery disease undergoing percutaneous coronary intervention (PCI) and under dual antiplatelet therapy, the new cartridge had a detection rate of 84% (CT 106 s as cut-off) for clopidogrel medication. After dividing the 50 patients into two groups according to their response to clopidogrel INNOVANCE® PFA P2Y * recognized all "responders" (defined by a PRI > 50%) using >106 s as cut-off but the specificity for a "good response" was only 42% because several "poor responders" (defined by a PRI > 50%) also showed CTs above the cut-off. The best correlation (substantial agreement) between the results of INNOVANCE® PFA P2Y * and of the VASP phosphorylation assay was achieved using CT > 200 s and PRI < 55% as cut-offs. Then, the sensitivity of INNOVANCE® PFA P2Y * was 97% and the specificity for a "good response" 65%. In summary, INNOVANCE® PFA P2Y * showed a high sensitivity for the detection of P2Y12 receptor blockade, but had only a limited specificity for a "good response" to clopidogrel. Therefore, this new cartridge is a useful tool to rule out P2Y12 receptor inhibition, if normal or only slightly prolonged CTs are obtained. Its predictive value for risk assessment of thromboembolic events, e.g. after coronary stent implantation, needs to be evaluated in clinical trials.

Assessment of clopidogrel non-response by the PFA-100 system using the new test cartridge INNOVANCE PFA P2Y.

Until now, the PFA-100 system has been considered unsuitable for monitoring clopidogrel efficacy. The authors evaluated platelet function in peripheral arterial occlusive disease (PAOD) patients using a new PFA-100(R) test cartridge (product name: INNOVANCE PFA P2Y*) specifically designed for this purpose. Twenty-two stable PAOD patients on antithrombotic therapy with clopidogrel alone (n = 22) and 18 patients undergoing a peripheral catheter intervention, preliminarily treated with 100 mg/day of aspirin followed by co-administration of clopidogrel (loading dose 300 mg, maintenance dose 75 mg/day), were enrolled in this study. Defining non-responsiveness to clopidogrel as an aggregation response within the reference range (90% central interval), four (18.2%) non-responders using light transmittance aggregometry (LTA) induced by 5 microM adenosine diphosphate (ADP) and six (27.3%) non-responders using LTA induced by 2 microM ADP (LateAggr >72.1% and >42.9%, respectively) were identified. INNOVANCE PFA P2Y* determined six (27.3%) non-responders (CT < 87 s). Agreement between the two aggregometry assays and INNOVANCE PFA P2Y* on the definition of clopidogrel response and non-response exceeded 70%. Only three patients were uniformly identified as clopidogrel non-responders by all three assays. When clopidogrel was co-administered with aspirin, two (11.1%) non-responders to clopidogrel were detected with INNOVANCE PFA P2Y*, whereas ADP-induced LTA found all patients to be responsive. INNOVANCE PFA P2Y* appears to be suitable for monitoring the effect of clopidogrel on platelet function. Its sensitivity in detecting responsiveness or non-responsiveness to clopidogrel is comparable to ADP-induced LTA. Additional prospective studies are needed to clarify the clinical relevance of the test results and classification obtained with INNOVANCE PFA P2Y*.

Anthocyanins and colonic metabolites of dietary polyphenols inhibit platelet function.

Maintenance and achievement of an optimal platelet function by dietary solutions might be considered an interesting target to influence cardiovascular health. Polyphenol-rich foods such as vegetables and fruits have been shown to significantly improve platelet function in ex vivo studies in humans. However, until yet, it still remains unclear if polyphenols itself, their metabolites or a mixture of both are responsible for the beneficial effects observed so far. Our study aims to evaluate the effect of anthocyanins, in vivo metabolites of different polyphenols from colonic origin and a representative mixture of both at physiological concentrations on platelet aggregation and activation function. Some anthocyanins [1 microM], colonic metabolites [10 microM] and a mixture (4 phenolic acid [1 microM], 4 anthocyanin [0.1 microM] showed significant platelet sedating and desensitizing effects. Activation of the platelets (P-selectin expression) was significantly reduced by 10-40% in resting platelets, TRAP-activated and hydrogen peroxide-stressed platelets and epinephrine pre-activated platelets relative to controls. The dose-response curve of the weak agonist TRAP was also significantly altered resulting in a >0.8 microM increase of threshold concentration to induce aggregation. The representative mixture was active despite ten times lower concentrations of the individual components, which showed no activity when tested individually at that concentration, indicating synergism of the different components. Platelet reactivity to strong agonists such as collagen and ADP was not influenced. These results show that anthocyanins and in vivo metabolites of polyphenols have anti-thrombotic properties, suggesting themselves as well as their dietary sources and precursors as potential cardiovascular health promoters.

Colonic metabolism of dietary polyphenols: influence of structure on microbial fermentation products.

The metabolism of chlorogenic acid, naringin, and rutin, representative members of three common families of dietary polyphenols, the hydroxycinnamates, the flavanones, and the flavonols, respectively, was studied in an in vitro mixed culture model of the human colonic microflora. Time- and concentration-dependent degradation of all three compounds was observed, which was associated with the following metabolic events after cleavage of the ester or glycosidic bond: reduction of the aliphatic double bond of the resulting hydroxycinnamate caffeic acid residue; dehydroxylation and ring fission of the heterocyclic C-ring of the resulting deglycosylated flavanone, naringenin, and of the deglycosylated flavonol, quercetin (which differed depending on the substitution). The metabolic events, their sequences, and major phenolic end products, as identified by GC-MS or LC-MS/MS, were elucidated from the structural characteristics of the investigated compounds. The major phenolic end products identified were 3-(3-hydroxyphenyl)-propionic acid for chlorogenic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-phenylpropionic acid for naringin, and 3-hydroxyphenylacetic acid and 3-(3-hydroxyphenyl)-propionic acid for rutin. The degree of degradation of the compounds studied was significantly influenced by the substrate concentration as well as individual variations in the composition of the fecal flora. The results support extensive metabolism of dietary polyphenols in the colon, depending on substrate concentration and residence time, with resultant formation of simple phenolics, which can be considered biomarkers of colonic metabolism if subsequently absorbed. It is also apparent that a relatively small number of phenolic degradation products are formed in the colon from the diverse group of natural polyphenols.

The metabolic fate of dietary polyphenols in humans.

Dietary polyphenols are widely considered to contribute to health benefits in humans. However, little is yet known concerning their bioactive forms in vivo and the mechanisms by which they may contribute toward disease prevention. Although many studies are focusing on the bioavailability of polyphenols through studying their uptake and the excretion of their conjugated forms, few are emphasizing the occurrence of metabolites in vivo formed via degradation by the enzymes of colonic bacteria and subsequent absorption. The purpose of this research was to investigate the relationship between biomarkers of the colonic biotransformation of ingested dietary polyphenols and the absorbed conjugated polyphenols. The results show that the majority of the in vivo forms derive from cleavage products of the action of colonic bacterial enzymes and subsequent metabolism in the liver. Those include the glucuronides of 3-hydroxyphenylacetic, homovanillic, vanillic and isoferulic acid as well as 3-(3-methoxy-4-hydroxyphenyl)-propionic, 3-(3-hydroxyphenyl)-propionic acid, and 3-hydroxyhippuric acid. In contrast, intact conjugated polyphenols themselves, such as the glucuronides of quercetin, naringenin and ferulic, p-coumaric, and sinapic acid were detected at much lower levels. The results suggest that consideration should be given to the cleavage products as having a putative role as physiologically relevant bioactive components in vivo.

Uptake and metabolism of epicatechin and its access to the brain after oral ingestion.

Epicatechin is a flavan-3-ol that is commonly present in green teas, red wine, cocoa products, and many fruits, such as apples. There is considerable interest in the bioavailability of epicatechin after oral ingestion. In vivo studies have shown that low levels of epicatechin are absorbed and found in the circulation as glucuronides, methylated and sulfated forms. Recent research has demonstrated protective effects of epicatechin and one of its in vivo metabolites, 3'-O-methyl epicatechin, against neuronal cell death induced by oxidative stress. Thus, we are interested in the ability of ingested epicatechin to cross the blood brain barrier and target the brain. Rats were administered 100 mg/kg body weight/d epicatechin orally for 1, 5, and 10 d. Plasma and brain extracts were analyzed by HPLC with photodiode array detection and LC-MS/MS. This study reports the presence of the epicatechin glucuronide and 3'-O-methyl epicatechin glucuronide formed after oral ingestion in the rat brain tissue.

The metabolism of dietary polyphenols and the relevance to circulating levels of conjugated metabolites.

Berry extracts rich in anthocyanins have been linked to protective effects including the modulation of age-related neurological dysfunction and the improvement of the resistance of red blood cells against oxidative stress in vitro. In this study the bioavailability, metabolism and elimination of polyphenols from blackcurrant juice, rich in anthocyanins, flavonols, and hydroxycinnamates, were investigated. The four major native anthocyanidin glycosides of blackcurrant juice, delphinidin-3-glucoside, delphinidin-3-rutinoside, cyanidin-3-glucoside and cyanidin-3-rutinoside, were detected and identified in low amounts by HPLC and LC-MS in plasma and urine post-ingestion. Elimination of the anthocyanins was fast (maximum excretion after 1 h) and plasma levels (0-128.6 nmol/l) and total urinary exretion (0.07-1.35 mg; 0.007-0.133% of the dose ingested) were low. Most significantly, of the hydroxycinnamates, conjugated and free ferulic, isoferulic, p-coumaric, sinapic and vanillic acids were identified in plasma and urine, using GC-MS techniques. Quercetin and kaempferol (as glucuronides) and the proposed colonic metabolite of quercetin, 3-hydroxyphenylacetic acid, were detectable in a minority of subjects. Increased daily urinary hippuric, 4-hydroxyhippuric and 3-hydroxyhippuric acid levels were also observed post-ingestion in all volunteers.