In-vitro and in-vivo metabolism of different aspirin formulations studied by a validated liquid chromatography tandem mass spectrometry method.

Low-dose aspirin (ASA) is used to prevent cardiovascular events. The most commonly used formulation is enteric-coated ASA (EC-ASA) that may be absorbed more slowly and less efficiently in some patients. To uncover these "non-responders" patients, the availability of proper analytical methods is pivotal in order to study the pharmacodynamics, the pharmacokinetics and the metabolic fate of ASA. We validated a high-throughput, isocratic reversed-phase, negative MRM, LC-MS/MS method useful for measuring circulating ASA and salicylic acid (SA) in blood and plasma. ASA-d4 and SA-d4 were used as internal standards. The method was applied to evaluate: (a) the "in vitro" ASA degradation by esterases in whole blood and plasma, as a function of time and concentration; (b) the "in vivo" kinetics of ASA and SA after 7 days of oral administration of EC-ASA or plain-ASA (100 mg) in healthy volunteers (three men and three women, 37-63 years). Parameters of esterases activity were Vmax 6.5 ± 1.9 and Km 147.5 ± 64.4 in plasma, and Vmax 108.1 ± 20.8 and Km 803.2 ± 170.7 in whole blood. After oral administration of the two formulations, tmax varied between 3 and 6 h for EC-ASA and between 0.5 and 1.0 h for plain-ASA. Higher between-subjects variability was seen after EC-ASA, and one subject had a delayed absorption over eight hours. Plasma AUC was 725.5 (89.8-1222) for EC-ASA, and 823.1(624-1196) ng h/mL (median, 25-75% CI) for plain ASA. After the weekly treatment, serum levels of TxB2 were very low (< 10 ng/mL at 24 h from the drug intake) in all the studied subjects, regardless of the formulation or the tmax. This method proved to be suitable for studies on aspirin responsiveness.

Patients with Essential Thrombocythemia may be Poor Responders to Enteric-Coated Aspirin, but not to Plain Aspirin.

Essential thrombocythemia (ET) patients are treated with aspirin (acetylsalicylic acid [ASA]) to prevent thrombosis. Previous studies showed that serum thromboxane (Tx) B2 was high 24 hours after enteric-coated (EC)-ASA in ET patients, due to increased number of noninhibited reticulated platelets (RPs), consequent to high platelet turnover, and that ASA should be given twice a day to ET patients. We studied ET patients (n = 17) and healthy subjects (n = 10) on 100 mg EC-ASA once daily; experiments were repeated after 14-day treatment with 100 mg plain-ASA once daily. Serum TxB2, plasma ASA, and salicylic acid (SA) were measured before the morning dose and up to 8 hours thereafter. Blood activity of ASA-deacethylating esterases, in vitro inhibition of collagen-induced TxB2 production by ASA (10-1,000 µM), and number of RP were measured. TxB2 inhibition by ASA in vitro and esterases activities were normal in all subjects. EC-ASA elicited highly variable responses; 6 ET patients were poor responders, as their serum TxB2 was high after EC-ASA; their plasma levels of ASA and SA were low/undetectable. In contrast to EC-ASA, plain ASA decreased serum TxB2 and increased plasma ASA and SA in all subjects. Serum TxB2 was high in ET patients at 24 hours and significantly correlated with RP count (but not RP percentage) and platelet count. Plain ASA should be used in ET patients to inhibit platelets efficiently. The identification of ET patients who might benefit from twice a day ASA could simply be based on their platelet count: since their platelet turnover is not increased, ET patients with normalized platelet count should not need twice a day ASA treatment.

Determination of the serine palmitoyl transferase inhibitor myriocin by electrospray and Q-trap mass spectrometry.

Myriocin is a potent inhibitor of serine-palmitoyl-transferase, the first and rate-determining enzyme in the sphingolipids biosynthetic pathway. This study developed, validated and applied a LC-MS/MS method to measure myriocin in minute specimens of animal tissue. The chemical analog 14-OH-myriocin was used as the internal standard. The two molecules were extracted from the tissue homogenate by solid-phase extraction, separated by gradient reversed-phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection was accomplished by multiple reaction monitoring, employing the most representative transitions, 400@104 and 402@104 for myriocin and 14-OH-myriocin, respectively. The typical limit of detection and lower limit of quantitation of the optimized method were 0.9 pmol/mL (~0.016 pmol injected) and 2.3 pmol/mL, respectively, and the method was linear up to 250 pmol/mL range (r2 = 0.9996). The intra- and between-day repeatability afforded a coefficient of variation ≤7.0%. Applications included quantification of myriocin in mouse lungs after 24 h from administration of ~4 nmol by intra-tracheal delivery. Measured levels ranged from 4.11 (median; 2.3-7.4 IQR, n = 4) to 11.7 (median; 7.6-22.7 interquartile range (IQR), n = 6) pmol/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (less than the limit of detection) to 0.35 pmol/retina.

Left Ventricular Outflow Tract Obstruction in Hypertrophic Cardiomyopathy Patients Without Severe Septal Hypertrophy: Implications of Mitral Valve and Papillary Muscle Abnormalities Assessed Using Cardiac Magnetic Resonance and Echocardiography.

BACKGROUND: In patients with hypertrophic cardiomyopathy and left ventricular outflow tract (LVOT) obstruction, but without basal septal hypertrophy, we sought to identify mitral valve (MV) and papillary muscle (PM) abnormalities that predisposed to LVOT obstruction, using echo and cardiac magnetic resonance. METHODS AND RESULTS: We studied 121 patients with hypertrophic cardiomyopathy hypertrophic cardiomyopathy (age, 49±17 years; 60% men; 57% on ß-blockers) with a basal septal thickness of ≤1.8 cm who underwent echocardiography (rest+stress) and cine cardiac magnetic resonance. Echo measurements included maximal LVOT gradient (rest/provocable), MV leaflet length (parasternal long, 4 and 3-chamber views), and abnormal chordal attachment to mid/base of anterior MV. Cine cardiac magnetic resonance measurements included basal septal thickness, number/area of PM heads, and bifid PM mobility (in systole and diastole). Mean basal septal thickness, LVOT gradient, and LV ejection fraction were 1.5±0.3 cm, 72±54 mm Hg, and 61±6%, respectively. The number of anterolateral and posteromedial PM heads was 2.7±0.7 and 2.6±0.7, respectively. Anterolateral and posteromedial PM areas were 19.9±7 cm(2) and 17.1±6 cm(2), respectively. PM mobility was 11±6°. On multivariable analysis, predictors of maximal LVOT gradient were basal septal thickness, bifid PM mobility, anterior mitral leaflet length, and abnormal chordal attachment to base of anterior mitral leaflet. Forty-five patients underwent surgery to relieve LVOT obstruction, of which 52% needed an additional nonmyectomy (MV repair/replacement or PM reorientation) approach. CONCLUSIONS: In hypertrophic cardiomyopathy patients without significant LV hypertrophy, in addition to basal septal thickness, anterior MV length, abnormal chordal attachment, and bifid PM mobility are associated with LVOT obstruction. In such patients, additional procedures on MV and PM (±myectomy) could be considered.

The interaction of bacteria with engineered nanostructured polymeric materials: a review.

Bacterial infections are a leading cause of morbidity and mortality worldwide. In spite of great advances in biomaterials research and development, a significant proportion of medical devices undergo bacterial colonization and become the target of an implant-related infection. We present a review of the two major classes of antibacterial nanostructured materials: polymeric nanocomposites and surface-engineered materials. The paper describes antibacterial effects due to the induced material properties, along with the principles of bacterial adhesion and the biofilm formation process. Methods for antimicrobial modifications of polymers using a nanocomposite approach as well as surface modification procedures are surveyed and discussed, followed by a concise examination of techniques used in estimating bacteria/material interactions. Finally, we present an outline of future sceneries and perspectives on antibacterial applications of nanostructured materials to resist or counteract implant infections.