Pharmacokinetics and bioavailability of different acetylsalicylic acid formulations assessed by liquid chromatography-tandem mass spectrometry in healthy subjects

Low-dose acetylsalicylic acid (ASA, 100 mg/die) is used in thromboprophylaxis. Enteric-coated formulation (EC-ASA) is commonly used for its lower risk of side effects. Some patients on EC-ASA do not respond appropriately and recent studies showed that poor responsiveness is more frequent with EC-ASA [1]. Aim of this study was to validate a method useful to study the pharmacokinetics (PK) of ASA in healthy subjects treated with two different aspirin formulations

Effect of red wine intake on serum and salivary melatonin levels: A randomized, placebo-controlled clinical trial

Melatonin (MLT) is a recently discovered phytochemical in wine, but its influence on physiological MLT levels is still unknown. This study aimed at evaluating variations, in serum and saliva, of MLT concentrations after the intake of MLT-enriched red wine. Twelve healthy volunteers were recruited to receive 125 mL of red wine naturally lacking of MLT (placebo, PLC), or the same wine enriched with MLT (MLT+). A physiological steady decline of serum MLT was observed from baseline up to 90 min, for both wines. After PLC intake, the decrease was significantly faster than the one occurring after MLT+ wine, which thus delayed the drop down of serum MLT with a plateau at 30\u201360 min. Salivary MLT levels slightly peaked at 45 min after MLT+ wine intake, without statistical significance. Therefore, the intake of a glass of MLT-enriched red wine changed serum levels of the indoleamine, supporting the role of wine MLT in counteracting the physiological decline of the hormone into the bloodstream

Pharmacokinetic of myriocin in rabbit’s eyes

Myriocin (Myr) is a suicide inactivator of ceramide synthesis with a complex lipid multifunctional structure. Its biological activity is exerted at very low doses, and thus highly performing quantitative method are needed [1]. The pharmacological development of Myr to modulate ceramide levels also requires currently unavailable ADME information in healthy and pathological animal models

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 \ufb01rst 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 is used as the internal standard. The two molecules are extracted from the tissue homogenate by solid-phase extraction, separated by gradient reverse-phase liquid chromatography and measured by negative ion electrospray mass spectrometry in the triple quadrupole. Detection is accomplished by Multiple Reaction Monitoring, employing the most representative transitions: 400@104 and 402@104 for Myriocin and 14-OH-Myriocin, respectively. The typical LoD and LLoQ of the optimized method are 0.9 pmoles/mL (approx. 0.016 pmoles injected) and 2.3 pmoles/mL, respectively, and the method is linear up to 250 pmoles/mL range (r2= 0.9996). The intra-and between-day repeatability affords a CV% 64 7.0. Applications included quantification of Myriocin in mouse lungs after 24 hrs from administration of ~4 nmoles by intra-trachea delivery. Measured levels ranged from 4.11 (median; 2.3-7.4 IQR, n=4) to 11.7 (median; 7.6-22.7 IQR, n=6) pmoles/lung depending on the different formulations used. Myriocin was also measured in retinas of mice treated by intravitreal injection and ranged from 0.045 (<LoD) to 0.35 pmoles/retina

Total plasma homocysteine analysis by HPLC with SBD-F precolumn derivatization

Over the last two decades, various studies have shown that moderate and persistent hyperhomocysteinemia is implicated in the development of atherosclerosis, which is responsible for 50% of all mortality and morbidity in Western countries. Considering that the most traditional risk factors for heart disease and stroke, such as plasma lipids, cigarette smoking, hypertension, obesity, and diabetes, only account for 50% of cardiovascular disease (1,2), one can understand the reason why homocysteine (Hcy) measurement is included in the list of tests for investigating the causes of atherosclerosis and thrombosis. One of the major problems encountered in studies on the potential atherogenic role of Hcy was the development of an accurate and simple assay, capable of screening, in a normal population, subjects having a congenital predisposition to occlusive vascular disease. Several approaches have been described in literature for measuring total plasma homocysteine (tHcy), which is defined as the sum of free and protein-bound homocysteine, homocystine, and homocysteine-cysteine mixed disulfide. These procedures involve, after a reduction step, the use of gas-chromatography-mass spectrometry (GC-MS) (3), radioenzymic assay (4), and high-performance liquid chromatography (HPLC)

Evaluation of aspirin responsiveness in healthy subjects and essential thrombocythemia patients

Introduction. Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by an increased number of platelets in the circulating blood, which is associated with an increased risk for both bleeding and thrombotic complications (1). Low-dose acetylsalicylic acid (ASA, 75-100 mg/d), which inhibits the platelet thromboxane A2 (TxA2) biosynthesis, is used for preventing thrombotic complications in ET patients at risk. The pharmacodynamics (PD) effects of ASA are blunted in some ET patients (ET-Non responders, ET-NR) (2). Aim of this study was to identify the causes of poor responsiveness to Aspirin in ET-NR. Methods. ET responders (ET-R, n=10), ET-NR (n=9) and healthy controls (n=11) were enrolled in the study. Using ID-LC-MS-MS for determination of ASA and salicylic acid (SA) we evaluated: 1) the in vitro activity of plasma and blood esterases, that hydrolyze ASA to SA; 2) the in vivo kinetics of ASA and SA at different times after the administration of 100 mg ASA (enteric-coated formulation). Serum TxB2 (stable metabolite of TxA2) was measured by ELISA at different time points after drug intake. In vitro effects of ASA on platelet aggregation (Multiplate) and TxB2 production (ELISA) induced by collagen in whole blood were measured in ET-NR and healthy controls. Results. Esterase activity was similar in the three study groups. Pharmacokinetics of ASA was very variable in all subjects: ASA maximum plasma concentration ranged between 500 and 1400 ng/mL 2-6 hours after intake. SA had similar trend. Half of ET-NR did not show ASA absorption within the 8 hours observation period. In these patients serum TxB2 was persistently high. However, in vitro addition of ASA (100 \ub5M) inhibited TxB2 production at the same rate as in controls, excluding an impaired pharmacodynamics effect (3). Conclusions. Excluding enzymatic or pharmacodynamics effects, causes of inadequate response to ASA in ET patients need some more investigations on gastro-intestinal availability. References 1) Patrono C., Rocca B. and De Stefano V. Platelet activation and inhibition in polycythemia vera and essential thrombocythemia. Blood. 2013;121 (10):1701-1711 2) Cattaneo M. Aspirin and Clopidogrel. Efficacy, Safety, and the Issue of Drug Resistance. Arterioscler Thromb Vasc Biol. 2004; 24:1980-1987. 3) Frelinger AL, Furman MI, Linden MD, Li Y, Fox ML, Barnard MR, Michelson AD. Residual arachidonic acid-induced platelet activation via an adenosine diphosphate-dependent but cyclooxygenase-1- and cyclooxygenase-2-independent pathway: a 700-patient study of aspirin resistance. Circulation. 2006 Jun 27;113(25):2888-96

Determination of vitamin K1 in plasma by solid phase extraction and HPLC with fluorescence detection

We describe a procedure for quantification of vitamin K-1 in human plasma by HPLC. Samples, enriched with a vitamin K derivative as internal standard, were deproteinized, purified on polymeric RP-SPE cartridges and injected into HPLC equipped with a post-column on-line zinc metal reactor and a fluorometric detector. Median level in blood donors (n = 87) was 1.967 nmol/L(0.93-4.01, 5th-95th percentiles), with a significant correlation between plasma levels and age (r = 0.276, p = 0.00958) and a lower (not significant) value in women than in men. This method, easy-to-handle and with a high throughput, can be used to identify covert states of vitamin K intake deficiency in patients thus at risk of alterations in blood clotting or bone mineralization

In-vivo pharmacokinetic of paclitaxel released by devitalized microfragmented fat tissue: potential application in chemotherapy

Introduction. Paclitaxel (PTX) is a major chemotherapy drug used for treatment of different types of solid tumors. It belongs to the class of tubulin-targeting drugs, causing interferences in normal function of microtubules during cell division. Devitalized microfragmented adipose tissue (DMAT) was engineered into a biocompatible scaffold for long-term drug delivery of PTX. Thus, pharmacokinetics and pharmacodynamics properties of DMAT uploaded with PTX (DMAT-PTX) was investigated into a mouse model prior to clinical evaluation in pharmaco-oncology. Methods. DMAT was prepared by devitalization (by freezing/thawing procedure) and disaggregation of lipoaspirate obtained by human donors. PTX was internalized by shaking into DMAT prior administration. For the pharmacokinetic and biodistribution experiments, BALB/cOlaHsd mice were injected, either subcutaneously (s.c., n=12) or intraperitoneally (i.p., n=12) with 10 mg/kg of DMAT-PTX. After 2, 24, 72 and 168 hrs post treatment, blood, livers and kidneys from all mice were collected. From mice treated s.c. also the site of DMAT-PTX injection was removed and stored until use. PTX was extracted from samples, either by solid phase extraction or by monophasic liquid extraction, and then analysed in LC-MS/MS. Results. DMAT-PTX after i.p. injections showed a higher Cmax compared to s.c. delivery (1904 vs 279 ng/mL in plasma at 2 h), although after 24 h the plasma levels were higher in s.c. treated animals (19.1 vs 6.1 ng/mL). Similar results were obtained for kidney and liver. In addition, slow release of PTX from DMAT-PTX specimens was demonstrated also by analysing its kinetic disappearance from the site of injection: PTX ranged from 380 \ub5g/g at 2h to 1.5 \ub5g/g at 7 days. Conclusions. The analysis of PTX at the site of DMAT-PTX injection shows that this new lipophilic delivery system is able to produce a slow and long-lasting release of PTX, suggesting its possible application in counteracting tumour relapsing after surgical resection

Dimethylarginines in complicated type 1 diabetes: roles of insulin, glucose, and oxidative stress

To investigate the roles of insulin, glucose, and oxidative stress on plasma asymmetric and symmetric dimethylarginine (ADMA, SDMA) levels in complicated diabetes, we studied patients with type 1 diabetes (T1D; n = 20), T1D + end-stage renal disease under hemodialysis (T1D + ESRD; n = 12), T1D + ESRD who received kidney transplant (KD; n = 16), and T1D + ESRD who received kidney-pancreas transplant (KP; n = 20) and healthy controls (n = 50). Levels of ADMA, SDMA, and free and total malondialdehyde (MDA) were increased in all patients, with the highest rises for SDMA and free MDA in T1D+ESRD. In KP, the normalized glycemia contributes to the recovery of ADMA, SDMA, and MDA levels toward normal values. From the covariance analyses, both glucose and insulin relate significantly to ADMA in T1D + ESRD (beta = +0.004, beta = -0.038, respectively) and in KP (beta = +0.032, beta = +0.032, respectively). Creatinine clearance and insulin relate to SDMA in all patient groups (beta = -0.006). Our results provide evidence for the effect of kidney-pancreas transplant on the recovery of ADMA, SDMA, and indexes of oxidative stress toward normal values. Only free MDA allows one to discriminate the magnitude of the oxidative status, as increased total MDA could also be attributable to a reduced renal function