Publications by authors named "Luigi Silvestro"

16 Publications

  • Page 1 of 1

Silencing the Cytoskeleton Protein Iba1 (Ionized Calcium Binding Adapter Protein 1) Interferes with BV2 Microglia Functioning.

Cell Mol Neurobiol 2020 Aug 16;40(6):1011-1027. Epub 2020 Jan 16.

Department of Anatomy, Animal Physiology and Biophysics, Faculty of Biology, University of Bucharest, Splaiul Independentei 91-95, Sector 5, 050095, Bucharest, Romania.

Iba1 (ionized calcium binding adapter protein 1) is a cytoskeleton protein specific only for microglia and macrophages, where it acts as an actin-cross linking protein. Although frequently regarded as a marker of activation, its involvement in cell migration, membrane ruffling, phagocytosis or in microglia remodeling during immunological surveillance of the brain suggest that Iba1 is not a simple cytoskeleton protein, but a signaling molecule involved in specific signaling pathways. In this study we investigated if Iba1 could also represent a drug target, and tested the hypothesis that its specific silencing with customized Iba1-siRNA can modulate microglia functioning. The results showed that Iba1-silenced BV2 microglia migrate less due to reduced proliferation and cell adhesion, while their phagocytic activity and P2x7 functioning was significantly increased. Our data are the proof of concept that Iba1 protein is a new microglia target, which opens a new therapeutic avenue for modulating microglia behavior.
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http://dx.doi.org/10.1007/s10571-020-00790-wDOI Listing
August 2020

The 10th GCC Closed Forum: rejected data, GCP in bioanalysis, extract stability, BAV, processed batch acceptance, matrix stability, critical reagents, ELN and data integrity and counteracting fraud.

Bioanalysis 2017 Apr 24;9(7):505-516. Epub 2017 Mar 24.

WuXi Apptec, Plainsboro, NJ, USA.

The 10th Global CRO Council (GCC) Closed Forum was held in Orlando, FL, USA on 18 April 2016. In attendance were decision makers from international CRO member companies offering bioanalytical services. The objective of this meeting was for GCC members to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues discussed at this closed forum included reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, biomarker assay validation, processed batch acceptance criteria, electronic laboratory notebooks and data integrity, Health Canada's Notice regarding replicates in matrix stability evaluations, critical reagents and regulatory approaches to counteract fraud. In order to obtain the pharma perspectives on some of these topics, the first joint CRO-Pharma Scientific Interchange Meeting was held on 12 November 2016, in Denver, Colorado, USA. The five topics discussed at this Interchange meeting were reporting data from failed method validation runs, GCP for clinical sample bioanalysis, extracted sample stability, processed batch acceptance criteria and electronic laboratory notebooks and data integrity. The conclusions from the discussions of these topics at both meetings are included in this report.
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http://dx.doi.org/10.4155/bio-2017-5000DOI Listing
April 2017

Synthesis and Structural Investigation of New Bio-Relevant Complexes of Lanthanides with 5-Hydroxyflavone: DNA Binding and Protein Interaction Studies.

Molecules 2016 Dec 16;21(12). Epub 2016 Dec 16.

Department of General and Inorganic Chemistry, Faculty of Pharmacy, "Carol Davila" University of Medicine and Pharmacy, 6 Traian Vuia Str., 020956 Bucharest, Romania.

In the present work, we attempted to develop new metal coordination complexes of the natural flavonoid 5-hydroxyflavone with Sm(III), Eu(III), Gd(III), Tb(III). The resultant hydroxo complexes have been characterized by a variety of spectroscopic techniques, including fluorescence, FT-IR, UV-Vis, EPR and mass spectral studies. The general chemical formula of the complexes is [Ln(CH₉O₃)₃(OH)₂(H₂O)]·H₂O, where Ln is the lanthanide cation and x = 0 for Sm(III), x = 1 for Eu(III), Gd(III), Tb(III) and = 0 for Sm(III), Gd(III), Tb(III), = 1 for Eu(III), respectively. The proposed structures of the complexes were optimized by DFT calculations. Theoretical calculations and experimental determinations sustain the proposed structures of the hydroxo complexes, with two molecules of 5-hydroxyflavone acting as monoanionic bidentate chelate ligands. The interaction of the complexes with calf thymus DNA has been explored by fluorescence titration and UV-Vis absorption binding studies, and revealed that the synthesized complexes interact with DNA with binding constants (K) ~ 10⁴. Human serum albumin (HSA) and transferrin (Tf) binding studies have also been performed by fluorescence titration techniques (fluorescence quenching studies, synchronous fluorescence spectra). The apparent association constants (K) and thermodynamic parameters have been calculated from the fluorescence quenching experiment at 299 K, 308 K, and 318 K. The quenching curves indicate that the complexes bind to HSA with smaller affinity than the ligand, but to Tf with higher binding affinities than the ligand.
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http://dx.doi.org/10.3390/molecules21121737DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6273368PMC
December 2016

Evaluation of Clopidogrel Conjugation Metabolism: PK Studies in Man and Mice of Clopidogrel Acyl Glucuronide.

Drug Metab Dispos 2016 09 11;44(9):1490-7. Epub 2016 Jul 11.

Department of Biopharmacy, Faculty of Pharmacy, University of Medicine and Pharmacy "Carol Davila," Bucharest, Romania (S.N.S., C.M.); 3S-Pharmacological Consultation and Research GmbH, Harpstedt, Germany (S.N.S, L.S., S.R.S.); Pharma Serv International SRL, Bucharest, Romania (M.S.); Clinical Hospital of the Ministry of Health of the Moldavian Republic, Chisinau, The Moldavian Republic (L.R.); Pharmacology Department, National Institute for Chemical Pharmaceutical Research and Development (ICCF), Bucharest, Romania (Y.R.).

The existence of a glucuronide conjugate of the major circulating clopidogrel metabolites, called clopidogrel acyl glucuronide (CAG), is already known. However, information regarding its pharmacokinetics (PK), metabolism, and clearance are modest. We investigated in vivo the potential CAG trans-esterification to clopidogrel (reaction occurring in vitro in particular conditions) by administering the metabolite to mice. Experiments were then carried out on men, clopidogrel administered alone or followed by activated charcoal intake (intestinal reabsorption blockade). Study objectives included: PK comparison of CAG, clopidogrel carboxylic acid (CCA), and clopidogrel in plasma, determination of their elimination patterns in urine and feces, and tracking of charcoal-induced changes in PK and/or urinary excretion that would indicate relevant enterohepatic recycling of CAG. In mice, CAG was rapidly hydrolyzed to CCA after oral administration, whereas by intravenous route metabolic conversion to CCA was delayed. No levels of clopidogrel were detected in mice plasma, excluding any potential trans-esterification or other form of back-conversion in vivo. PK experiments in man showed that CAG is hydrolyzed in the gastrointestinal tract (very low concentrations in feces), but there is no evidence of enterohepatic recirculation. Quantitation of the three moieties in stool samples accounted for only 1.2% of an administered dose, suggesting that other yet unknown metabolites/degradation products formed through metabolic processes and/or the activity of local microflora are mainly excreted by this route. In man CAG was confirmed as one of the major terminal metabolites of clopidogrel, with a PK behavior similar to CCA.
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http://dx.doi.org/10.1124/dmd.116.071092DOI Listing
September 2016

9th GCC closed forum: CAPA in regulated bioanalysis; method robustness, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, regulatory audit experiences and electronic laboratory notebooks.

Bioanalysis 2016 Mar 26;8(6):487-95. Epub 2016 Feb 26.

WuXi/XBL, 107 Morgan Lane, Plainsboro, NJ, USA.

The 9th GCCClosed Forum was held just prior to the 2015 Workshop on Recent Issues in Bioanalysis (WRIB) in Miami, FL, USA on 13 April 2015. In attendance were 58 senior-level participants, from eight countries, representing 38 CRO companies offering bioanalytical services. The objective of this meeting was for CRO bioanalytical representatives to meet and discuss scientific and regulatory issues specific to bioanalysis. The issues selected at this year's closed forum include CAPA, biosimilars, preclinical method validation, endogenous biomarkers, whole blood stability, and ELNs. A summary of the industry's best practices and the conclusions from the discussion of these topics is included in this meeting report.
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http://dx.doi.org/10.4155/bio.16.16DOI Listing
March 2016

Bioavailability and safety study of resveratrol 500 mg tablets in healthy male and female volunteers.

Exp Ther Med 2016 Jan 25;11(1):164-170. Epub 2015 Nov 25.

Research and Development Department, Medochemie Ltd., Limassol 3011, Cyprus.

Over the past few decades, -resveratrol has received widespread attention as a preventive agent for numerous diseases. Several studies have demonstrated that it has significant biological and pharmacological properties. -resveratrol has been reported to possess anti-oxidant, anti-inflammatory, anticarcinogenic, antidiabetic, anti-aging, cardioprotective and neuroprotective properties, which can be relevant in chronic diseases and longevity in humans. The aim of the present study was to investigate the rate and extend of absorption, and also the safety of resveratrol following a single 500 mg oral dose. This was an open label, single dose, one period, bioavailability study in 15 healthy volunteers under fasting conditions. Blood samples were collected at predefined time points up to 24 h after resveratrol administration, and plasma concentrations of resveratrol and its conjugated (glucuronated and sulphated) metabolites were determined using a validated high performance liquid chromatography/tandem mass spectrometry method. Pharmacokinetic parameters, including Cmax, AUC0-t, AUC-inf, Tmax, T1/ and MRT, were determined from plasma concentration-time profiles and found to be in good agreement with previously reported data. Cmax and AUC-inf were lower for resveratrol when compared with the values for its glucuronated and sulphated metabolites. Cmax for resveratrol, glucuronated resveratrol and sulphated resveratrol were 71.2±42.4 ng/ml, 4,083.9±1,704.4 ng/ml and 1,516.0±639.0 ng/ml, respectively, while the AUC0-inf values were 179.1±79.1 ng/ml, 39,732.4±16,145.6 ng/ml and 14,441.7±7,593.2 ng/ml, respectively. No adverse reactions associated with resveratrol were reported during the study. The plasma concentrations of resveratrol (free and conjugated) were in agreement with those mentioned in the literature, and were adequate to promote the pharmacological activities of resveratrol. In conclusion, resveratrol 500 mg tablets were well-tolerated by all participants of the study.
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http://dx.doi.org/10.3892/etm.2015.2895DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4726856PMC
January 2016

Influence of sample handling conditions on drug partitioning in blood: a major problem in PK studies?

Bioanalysis 2015 Dec 30;7(23):2973-6. Epub 2015 Nov 30.

3S-Pharmacological Consultation & Research GmbH, Koenigsbergerstrasse 1, 27243 Harpstedt, Germany.

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http://dx.doi.org/10.4155/bio.15.209DOI Listing
December 2015

An update on solid phase-supported liquid extraction.

Bioanalysis 2015 17;7(17):2177-86. Epub 2015 Sep 17.

3S-Pharmacological Consultation & Research GmbH, Koenigsberger Strasse 1 27243 Harpstedt, Germany.

Solid phase-supported liquid extraction (SLE) is a technique almost 40 years old being rediscovered in the last few years due to its simplicity, optimal for automation and giving very clean extracts with minimal matrix effects when analyzed by techniques like HPLC-MS/MS, GC-MS/MS, CE-MS/MS. In the next paragraphs the evolution of SLE, according to literature, will be presented first, followed by some considerations on the SLE material now available and a typical protocol of work. To conclude, considerations based on the author's practical experiences with SLE will be done, as well as few remarks on potential future areas of SLE development.
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http://dx.doi.org/10.4155/bio.15.144DOI Listing
July 2016

Pharmacokinetic study for the establishment of bioequivalence of two inhalation treatments containing budesonide plus formoterol.

J Pharm Pharmacol 2014 Dec 11;66(12):1677-85. Epub 2014 Aug 11.

ELPEN Pharmaceutical Co Inc., Pikermi, Greece.

Objectives: The aim of this study was to compare lung deposition and assess the bioequivalence of two formulations containing budesonide and formoterol and being delivered via Elpenhaler and Turbuhaler, respectively. A pharmacokinetic (PK) study was conducted.

Methods: An open, randomized, two-sequence, two-period, crossover, single-dose study in 100 asthmatic patients under fasting conditions was performed. Wash out period was 6 days. Equivalence in lung deposition was assessed after a single inhalation of each treatment with concomitant oral administration of activated charcoal (40 g) to prevent gastrointestinal absorption of the drugs. Several PK parameters were estimated, the area under the drug concentration in plasma versus time curve (AUC0-t ) and the maximum drug concentration in plasma (Cmax ) being the primary response variables. Equivalent lung deposition was concluded if the 90% confidence interval (CI) for the Elpenhaler/Turbuhaler geometric mean ratio of AUC0-t and Cmax , for both drug substances fell within the regulatory limits (0.80-1.25).

Key Findings: Acceptance criteria were met. Equivalent lung deposition can be concluded. No statistically significant differences between treatments in the incidence of adverse events were found.

Conclusions: The formulations are bioequivalent regarding both rate and extent of absorption. The treatments were also well tolerated by the participating subjects.
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http://dx.doi.org/10.1111/jphp.12303DOI Listing
December 2014

Confirmation of diosmetin 3-O-glucuronide as major metabolite of diosmin in humans, using micro-liquid-chromatography-mass spectrometry and ion mobility mass spectrometry.

Anal Bioanal Chem 2013 Oct 16;405(25):8295-310. Epub 2013 Aug 16.

3S-Pharmacological Consultation and Research GmbH, Koenigsbergerstrasse 1, 27243, Harpstedt, Germany,

Diosmin is a flavonoid often administered in the treatment of chronic venous insufficiency, hemorrhoids, and related affections. Diosmin is rapidly hydrolized in the intestine to its aglicone, diosmetin, which is further metabolized to conjugates. In this study, the development and validations of three new methods for the determination of diosmetin, free and after enzymatic deconjugation, and of its potential glucuronide metabolites, diosmetin-3-O-glucuronide, diosmetin-7-O-glucuronide, and diosmetin-3,7-O-glucuronide from human plasma and urine are presented. First, the quantification of diosmetin, free and after deconjugation, was carried out by high-performance liquid chromatography coupled with tandem mass spectrometry, on an Ascentis RP-Amide column (150 × 2.1 mm, 5 μm), in reversed-phase conditions, after enzymatic digestion. Then glucuronide metabolites from plasma were separated by micro-liquid chromatography coupled with tandem mass spectrometry on a HALO C18 (50 × 0.3 mm, 2.7 μm, 90 Å) column, after solid-phase extraction. Finally, glucuronides from urine were measured using a Discovery HSF5 (100 × 2.1 mm, 5 μm) column, after simple dilution with mobile phase. The methods were validated by assessing linearity, accuracy, precision, low limit of quantification, selectivity, extraction recovery, stability, and matrix effects; results in agreement with regulatory (Food and Drug Administration and European Medicines Agency) guidelines acceptance criteria were obtained in all cases. The methods were applied to a pharmacokinetic study with diosmin (450 mg orally administered tablets). The mean C max of diosmetin in plasma was 6,049.3 ± 5,548.6 pg/mL. A very good correlation between measured diosmetin and glucuronide metabolites concentrations was obtained. Diosmetin-3-O-glucuronide was identified as a major circulating metabolite of diosmetin in plasma and in urine, and this finding was confirmed by supplementary experiments with differential ion-mobility mass spectrometry.
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http://dx.doi.org/10.1007/s00216-013-7237-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3777223PMC
October 2013

Development of a sensitive method for simultaneous determination of fluticasone propionate and salmeterol in plasma samples by liquid chromatography-tandem mass spectrometry.

Biomed Chromatogr 2012 May;26(5):627-35

3S-Pharmacological Consultation and Research GmbH, Koenigsbergerstrasse 1, 27243 Harpstedt, Germany.

A new method for the fast simultaneous quantification of fluticasone propionate and salmeterol from plasma samples by liquid chromatography-tandem mass spectrometry, with adequate sensitivity for pharmacokinetic applications, was developed and validated. The chromatographic separation and mass-spectrometric parameters were optimized for the retention and detection of the two compounds, despite quite different structures and properties. Two columns connected in series were used, cation-exchange (Zorbax 300-SCX, 5 cm x 2.1 mm, 5 μm) and octadecyl (Discovery HSC₁₈, 10 cm x 2.1 mm, 5 μm). The mass-spectrometric interface was operated in negative electrospray ionization mode; high sensitivity and lesser matrix effects were obtained, permitting smaller consumption of plasma. The sample preparation was based on supported liquid-liquid extraction in 96-well format plates that provided clean samples with a simplified procedure that was suitable for automation. The method was validated according to regulatory guidelines, by assessing lower limits of quantification, selectivity, linearity, accuracy, precision, extraction recoveries and matrix effects. A comparison with two other methods for the separate determination of fluticasone propionate and salmeterol in plasma samples, previously developed by our group, is presented. The statistical evaluation of the results obtained with the three methods on a set of unknown samples from treated patients demonstrated good correlation (R² 0.987 for fluticasone propionate and 0.967 for salmeterol).
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http://dx.doi.org/10.1002/bmc.1708DOI Listing
May 2012

Development and validation of an HPLC-MS/MS method to quantify clopidogrel acyl glucuronide, clopidogrel acid metabolite, and clopidogrel in plasma samples avoiding analyte back-conversion.

Anal Bioanal Chem 2011 Aug 9;401(3):1023-34. Epub 2011 Jun 9.

3S-Pharmacological Consultation & Research GmbH, Koenigsbergerstrasse 1, 27243 Harpstedt, Germany.

A new sensitive and fast quantitative analytical method for the simultaneous determination of clopidogrel, its main metabolite clopidogrel carboxylic acid, and the newly described acyl glucuronide metabolite, in human plasma samples, is presented. The analytical procedures (plasma storage, handling, and extract storage in the autosampler) were optimized in order to avoid back-conversion; a known drawback in measurements of clopidogrel. Clopidogrel acyl glucuronide was confirmed as a major source of back-conversion to the parent drug in the presence of methanol, and thorough stability experiments were carried out to find the most appropriate conditions for an accurate analysis of clopidogrel and the two metabolites. The method was validated by assessing selectivity, sensitivity, linearity, accuracy, and precision for all three analytes, in accordance to Food and Drug Administration guidelines. Spiked quality controls in plasma as well as incurred samples were used to verify back-conversion in the selected conditions, with results meeting European Medicines Agency acceptance criteria (concentrations within 80-120% of the first reading). The method was then applied to a pharmacokinetic study, and for the first time, a pharmacokinetic curve of clopidogrel acyl glucuronide in human plasma is presented. The concentrations ranged up to 1,048.684 ng/mL, with a mean of 470.268 ng/mL, while clopidogrel had a mean C(max) of 1.348 ng/mL; these orders of magnitude show how much the back-conversion of this metabolite may influence clopidogrel quantification if it is not properly controlled.
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http://dx.doi.org/10.1007/s00216-011-5147-4DOI Listing
August 2011

Development and validation of an HPLC-MS/MS method to determine clopidogrel in human plasma. Use of incurred samples to test back-conversion.

J Chromatogr B Analyt Technol Biomed Life Sci 2010 Nov 1;878(30):3134-42. Epub 2010 Oct 1.

3S-Pharmacological Consultation & Research GmbH, Koenigsbergerstrasse 1, 27243 Harpstedt, Germany.

Quantitative methods using LC-MS/MS allow achievement of adequate sensitivity for pharmacokinetic studies with clopidogrel; three such methods, with LLOQs as low as 5 pg/mL, were developed and fully validated according to the well established FDA 2001 guidelines. The chromatographic separations were performed on reversed phase columns Ascentis RP-Amide (15 cm x 2.1 mm, 5 μm), Ascentis Express C8 (10 cm x 2.1 mm, 2.7 μm) and Ascentis Express RP Amide (10 cm x 2.1 mm, 2.7 μm), respectively. Positive electrospray ionization in MRM mode was employed for the detection and a deuterated analogue (d3-clopidogrel) was used as internal standard. Linearity, precision, extraction recovery, matrix effects and stability tests on blank plasma spiked with clopidogrel and stored in different conditions met the acceptance criteria. During the analysis of the real samples from the first pharmacokinetic study, a significant increase (>100%) of the measured clopidogrel concentrations in the extracts kept in the autosampler at 10 °C was observed. Investigations led to the conclusion that most probably a back-conversion of one or more of the clopidogrel metabolites is occurring. The next methods were optimized in order to minimize this back-conversion. After a series of experiments, the adjustment of the sample preparation (e.g. processing at low temperature and introducing a clean-up step on Supelco HybridSPE-Precipitation cartridges) has proven to be the most effective in order to improve the stability of the extracts. Incurred samples of real subjects were successfully used in the validation of the last two analytical methods to evaluate the back-conversion, while tests using only the known metabolites could not detect this important problem.
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http://dx.doi.org/10.1016/j.jchromb.2010.09.022DOI Listing
November 2010

High-throughput HPLC-MS/MS method to determine ibandronate in human plasma for pharmacokinetic applications.

J Chromatogr B Analyt Technol Biomed Life Sci 2009 Oct 12;877(27):3159-68. Epub 2009 Aug 12.

Pharma Serv Int'l SRL, 52 Sabinelor Str., 5th District, 050853 Bucharest, Romania.

A sensitive high-throughput liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the quantification of ibandronate in human plasma. In a previous study, we have analyzed alendronate in urine samples of subjects treated at therapeutic dosages, using a derivatization approach; a similar derivatization was adapted and improved to determine ibandronate in plasma. The bisphosphonate was isolated from the biological matrix by liquid-liquid extraction, and derivatized with trimethylsilyldiazomethane prior to separation on a reversed-phase column (Supelco Discovery HSC18) and detection on a quadrupole-linear ion trap mass spectrometer (API 4000 QTrap). Various parameters of extraction and derivatization were optimized in order to get adequate recovery, high derivatization yield and minimal ion suppression; a deuterated analogue, d3-ibandronate, was used as internal standard. The transitions 376.1-->114.2 and 379.1-->61.0 were acquired to monitor ibandronate and d3-ibandronate derivatives, respectively. A multiplexing LC system made possible the overlapping of two chromatographic runs, thus the interval between injections being reduced to only 2min, a very short analysis time for compounds of this class. The method was fully validated over the quantification range 0.2-175.0ng/ml, allowing an appropriate evaluation of the plasma concentrations of ibandronate, expected at therapeutic dosage, as proved by application to a pharmacokinetic study. A good linearity over the selected range (r>0.99), accuracy and precision within +/-15% of the target values and a recovery over 50% were obtained.
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http://dx.doi.org/10.1016/j.jchromb.2009.08.007DOI Listing
October 2009

Development and application of a high-performance liquid chromatography-mass spectrometry method to determine alendronate in human urine.

J Chromatogr A 2007 Aug 19;1160(1-2):21-33. Epub 2007 Apr 19.

Pharma Serv International SRL, 52 Sabinelor Street, 5th District, 050853 Bucharest, Romania.

Despite the high potential offered by electrospray ionization on highly polar compounds like biphosphonates, few applications have been developed. High-performance liquid chromatography (HPLC) separation methods suitable for such molecules cannot be used in tandem with mass spectrometry (MS) due to high non-volatile salt content; at the same time the sample preparation, in biological fluids, is also a challenging problem. In the past ion-pair chromatography was mainly used in the case of HPLC-MS of biphosphonates, but no application to quantitative pharmacokinetic (PK) studies has been presented. In this study, after preliminary tests with ion-pair chromatography showing a poor sensitivity, a combined derivatization of the amino group and the biphosphonate has been developed and tested in a PK study. Using this analytical approach we were able to fully validate the quantitation of alendronate in the range of 6.667-4860.0 ng/ml in urine (sample volume 2.0 ml); each analytical run was 5.0 min long. The sensitivity achieved permitted a correct evaluation of the alendronate urinary excretion over the full period of urine collection. Sample preparation despite its complexity permitted to process and analyze up to 200 samples in a working day.
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http://dx.doi.org/10.1016/j.chroma.2007.04.020DOI Listing
August 2007

Intra-plaque production of platelet-activating factor correlates with neoangiogenesis in human carotid atherosclerotic lesions.

Int J Mol Med 2003 Sep;12(3):327-34

Dipartimento di Fisiopatologia Clinica, Universitá degli Studi di Torino, Torino, Italy.

Platelet-activating factor (PAF) is a phospholipid mediator synthesized by activated inflammatory and endothelial cells. Recently PAF has been shown to contribute to neoangiogenesis in several experimental models. Here we evaluated the presence of PAF and its potential role in neovascularization within human atherosclerotic plaques. The amount of PAF extracted from 18 carotid plaques (266.65+/-40.07 pg/100 mg dry tissue; mean +/- SE) was significantly higher than that extracted from 18 normal arterial specimens (6 from carotid artery and 12 from aorta) (4.72+/-2.31 pg/100 mg dry tissue; mean +/- SE). The levels of PAF significantly correlated with the infiltration of CD68-positive monocytes and the extent of neovascularization, detected as von Willebrand Factor-positive cells. The amount of PAF also correlated with the area occupied by TNF-alpha-expressing cells. The absence of enhanced level of PAF in the circulation of atherosclerotic patients suggests a local production of this mediator within the plaque. The lipid extracts of atherosclerotic plaques containing high levels of PAF-bioactivity, but not those of control arteries, were angiogenic in a murine Matrigel model. WEB 2170, a specific PAF receptor antagonist, significantly prevented angiogenesis induced by the lipid extracts of atherosclerotic plaques. Our results indicate a local production of PAF within the atherosclerotic plaques and suggest that it may contribute to intra-plaque neoangiogenesis.
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September 2003