Síntesis d'anàlegs de BEL i de substrat com a nous potencials inhibidors de la fosfolipasa independent de calci(grup VIA)

[cat] En els últims anys s'ha demostrat que els lípids no són només components estructurals de la membrana plasmàtica sinó que a més a més aquests desenvolupen diferents funcions cel·lulars. Concretament s'ha observat que els productes de degradació de les fosfolipases són importants en els processos d'inflamació, entre d'altres; i sembla ser que la fosfolipasa A2 independent de calci podria estar relacionada amb la esquizofrènia. La síntesis de nous potencials inhibidors d'aquesta proteïna per estudiar el metabolisme d'aquests lípids ha estat l'objectiu principal en el transcurs d'aquesta tesi doctoral. Per a dur a terme aquest treball, s'han sintetitzat diferents anàlegs a l'únic inhibidor selectiu d'aquest enzim (BEL), introduint diferents modificacions per intentar obtenir un inhibidor reversible igual de selectiu. Una d'aquestes modificacions va ser la introducció de la funcionalitat trifluorocetona, comú en diferents inhibidors de lipases, en l'esquelet de BEL. Desafortunadament, cap dels compostos sintetitzats sota aquest disseny no va presentar les propietats inhibitòries desitjades. Es va dissenyar una segona família de compostos introduint grups ariloxi- en la estructura de BEL; en aquest cas tampoc es van obtenir bons inhibidors, en canvi, dos d'aquests compostos van augmentar la activitat d'aquest enzim selectivament respecte a la fosfolipasa A2 dependent de calci (cPLA2). Una altra aproximació per dissenyar nous inhibidors va consistir en la síntesi d'anàlegs de substrat. Aquests compostos presentaven un grup n-hexadecilmercaptà a la posició sn-1 i el grup fosforilcolina a la posició sn-3 de l'esquelet de glicerol, amb diferents funcionalitat (amida, carbamat, sulfonamida...) i cadenes laterals diverses a la posició sn-2. Entre els diferents productes sintetitzats, alguns d'ells van presentar nivells d'inhibició destacables, no obstant quan es va estudiar la selectivitat sobre cPLA2 es va observar que també inhibien aquest enzim. Per tal de poder conèixer millor la estructura del centre actiu de l'enzim en estudi i poder dissenyar millors inhibidors es van clonar i expressar en bacteri dos fragments del centre catalític del enzim. Desafortunadament, aquests fragments s'expressaven en petites quantitats i s'agregaven en cossos d'inclusió i els diferents experiments que es van realitzar per resoldre aquests problemes no van conduir a resultats satisfactoris. Durant el transcurs de la tesi, es van preparar, també, diferents anàlegs de ceramida. Els assaigs preliminars d'aquests compostos en diferents enzims del metabolisme d'esfingolípids van mostrar que s'havia dissenyat i sintetitzat una nova família de compostos que obre una línia de recerca prometedora en el camp dels esfingolípids.[eng] In last decades, researchers have demonstrated that lipids are not only structural compounds of cellular membranes. These compounds have several functions in cell metabolism, specifically, phospholipids are cleaved by phospholipases, and their metabolites are secondary messengers in inflammation pathways. Moreover calcium-independent phospholipase A2 (iPLA2) might be linked to schizophrenia. Synthesis of new inhibitors of this enzyme has been the main aim of this work. In our group we have designed different analogues of the only inhibitor of this enzyme (BEL). We introduced several modifications and functional groups, like trifluoroketones, in BEL's structure. Unfortunately, any of these compounds could not inhibit the activity of the enzyme. Some compounds that have an ariloxigroup in alpha position of ketone increase activity of iPLA2 and not produce the same effect in cPLA2. Then, we have synthesized analogues of the natural substrate of phospholipases with several modifications; thioether group in sn-1 position, phosphorilcholine group in sn-3 position and different functional groups (amide, carbamate, sulfonamide.) in sn-2 position. Some of these compounds show inhibition of iPLA2, moreover, they also inhibit cPLA2. To know more information about catalytic mechanism of this phospholipase and design new inhibitors we have cloned and expressed two fragments of catalytic site of the enzyme. However these proteins are not soluble and its levels of expression were very small, and we could not do structural studies like X-ray crystallization. Furthermore, we have designed and synthesized analogues of ceramide. Biological assays of these compounds in several enzymes of sphingolipid metabolism proved that this family opens a new research line in sphingolipid field

Bioanalytical techniques in discrimination between therapeutic and abusive use of drugs in sport

The discrimination between therapeutic and abusive use of drugs in sports is performed using threshold concentrations or reporting levels, and the detection of the substances in a sample is only reported as an adverse analytical finding when the concentration exceeds the threshold or the reporting level. In this paper, the strategies of discrimination and the analytical methods used for the main groups of substances where the distinction is needed (β-2 agonists, ephedrines, glucocorticoids and morphine) will be reviewed. Nowadays, LC-MS is the method of choice for the analysis of these substances and, in most of the cases, a simple dilution of the urine sample is performed before the chromatographic analysis.The financial support received from Consell Català de l’Esport and DIUE (2014 651 SGR 692) (Generalitat de Catalunya, Spain) and the World Antidoping Agency 652 (grants 13D22RV and 14D06RV) is acknowledge

Detection of stanozolol O- and N-sulfate metabolites and their evaluation as additional markers in doping control

Stanozolol (STAN) is one of the most frequently detected anabolic androgenic steroids in sports drug testing. STAN misuse is commonly detected by monitoring metabolites excreted conjugated with glucuronic acid after enzymatic hydrolysis or using direct detection by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It is well known that some of the previously described metabolites are the result of the formation of sulfate conjugates in C17, which are converted to their 17-epimers in urine. Therefore, sulfation is an important phase II metabolic pathway of STAN that has not been comprehensively studied. The aim of this work was to evaluate the sulfate fraction of STAN metabolism by LC-MS/MS to establish potential long-term metabolites valuable for doping control purposes. STAN was administered to six healthy male volunteers involving oral or intramuscular administration and urine samples were collected up to 31 days after administration. Sulfation of the phase I metabolites commercially available as standards was performed in order to obtain MS data useful to develop analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) to detect potential sulfate metabolites. Eleven sulfate metabolites (M-I to M-XI) were detected and characterized by LC-MS/MS. This paper provides valuable data on the ionization and fragmentation of O-sulfates and N-sulfates. For STAN, results showed that sulfates do not improve the retrospectivity of the detection compared to the previously described long-term metabolite (epistanozolol-N-glucuronide). However, sulfate metabolites could be additional markers for the detection of STAN misuse.The financial support received from the World Anti-Doping AgencyWADA (12A21RV), Ministerio de Economía y Competividad(Gob ierno de España) (Project number DEP2012-35612) andGeneralitat de Catalunya (Consell Català de l’Esport, DIUE 2014SGR 692) is gratefully acknowl edged

Surface registration from range image fusion

The registration of full 3-D models is an important task in computer vision. Range finders only reconstruct a partial view of the object. Many authors have proposed several techniques to register 3D surfaces from multiple views in which there are basically two aspects to consider. First, poor registration in which some sort of correspondences are established. Second, accurate registration in order to obtain a better solution. A survey of the most common techniques is presented and includes experimental results of some of the

Detection and characterization of prednisolone metabolites in human urine by LC-MS/MS.

Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. oral), whereas they are allowed using other administration ways. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of prednisolone (PRED) was studied using liquid chromatography coupled to tandem mass spectrometry. A single oral (10 mg) dose of PRED was administered to two healthy male volunteers. Urine samples were collected up to 6 days after administration. Samples were hydrolyzed with β-glucuronidase and subjected to liquid-liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Precursor ion scan methods (m/z 77, 91, 105, 121, 147 and 171) in positive ionization and neutral loss scan methods (76 and 94 Da) in negative ionization modes were applied for the open detection of PRED metabolites. Using these methods, PRED parent compound plus 20 metabolites were detected. PRED and 11 metabolites were characterized by comparison with standards of the compounds (PRED, prednisone, 20β-dihydro-PRED and 20α-dihydro-PRED, 20β-dihydro-prednisone and 20α-dihydro-prednisone, 6β-hydroxy-PRED and 6α-hydroxy-PRED, 20β isomers and 20α isomers of 6β,11β,17α,20,21-pentahydroxypregnan-1,4-diene-3-one, 6α,11β,17α,20β,21-pentahydroxypregnan-1,4-diene-3-one and Δ(6) -PRED). Using mass spectrometric data, feasible structures were proposed for seven of the remaining nine detected metabolites, including several 6-hydroxy-metabolites. Eleven of the characterized metabolites have not been previously described. Maximum excretion rates for PRED metabolites were achieved in first 24 h; however, most of the metabolites were still detectable in the last collected samples (day 6).Grant from Ministerio de Ciencia e Innovación (Spain) (DEP2009-11454), Generalitat de Catalunya (Consell Català de l’Esport and DIUE 2014 SGR 692) and support by ISCIII contrato de formación en investigación Río Hortega (CM12/00085

Detection and characterization of prednisolone metabolites in human urine by LC-MS/MS.

Glucocorticosteroids are prohibited in sports when used by systemic administrations (e.g. oral), whereas they are allowed using other administration ways. Strategies to discriminate between administrations routes have to be developed by doping control laboratories. For this reason, the metabolism of prednisolone (PRED) was studied using liquid chromatography coupled to tandem mass spectrometry. A single oral (10 mg) dose of PRED was administered to two healthy male volunteers. Urine samples were collected up to 6 days after administration. Samples were hydrolyzed with β-glucuronidase and subjected to liquid-liquid extraction with ethyl acetate in alkaline conditions. The extracts were analyzed by liquid chromatography coupled to tandem mass spectrometry. Precursor ion scan methods (m/z 77, 91, 105, 121, 147 and 171) in positive ionization and neutral loss scan methods (76 and 94 Da) in negative ionization modes were applied for the open detection of PRED metabolites. Using these methods, PRED parent compound plus 20 metabolites were detected. PRED and 11 metabolites were characterized by comparison with standards of the compounds (PRED, prednisone, 20β-dihydro-PRED and 20α-dihydro-PRED, 20β-dihydro-prednisone and 20α-dihydro-prednisone, 6β-hydroxy-PRED and 6α-hydroxy-PRED, 20β isomers and 20α isomers of 6β,11β,17α,20,21-pentahydroxypregnan-1,4-diene-3-one, 6α,11β,17α,20β,21-pentahydroxypregnan-1,4-diene-3-one and Δ(6) -PRED). Using mass spectrometric data, feasible structures were proposed for seven of the remaining nine detected metabolites, including several 6-hydroxy-metabolites. Eleven of the characterized metabolites have not been previously described. Maximum excretion rates for PRED metabolites were achieved in first 24 h; however, most of the metabolites were still detectable in the last collected samples (day 6).Grant from Ministerio de Ciencia e Innovación (Spain) (DEP2009-11454), Generalitat de Catalunya (Consell Català de l’Esport and DIUE 2014 SGR 692) and support by ISCIII contrato de formación en investigación Río Hortega (CM12/00085

Budesonide use and misuse in sports: elimination profiles of budesonide and metabolites after intranasal, high-dose inhaled and oral administrations

Budesonide (BUD) is a glucocorticoid (GC) widely used in therapeutics. In sports, the World Anti-doping Agency (WADA) controls the use of GCs, and WADA-accredited laboratories use a reporting level of 30 ng/mL for 6β-hydroxy-budesonide (6βOHBUD) to detect the systemic administration of BUD. In the present work, we examined the urinary excretion profile of 6βOHBUD, BUD, and 16α-hydroxy-prednisolone (16αOHPRED) after intranasal (INT), inhaled (INH) (at high doses) and oral administrations in male and female volunteers. BUD was administered to healthy volunteers using INT route (256 μg/day for three days, n = 4 males and 4 females), INH route (800 μg/day for three days, n = 4 males and 4 females, and 1600 μg/day for three days, n = 4 males) or oral route (3 mg, n = 8 females). Urine samples were collected before and after administration at different time periods, and were analyzed by liquid chromatography-tandem mass spectrometry. 6βOHBUD and BUD concentrations were very low after INT treatment (0.0-7.1 and 0.0-8.1 ng/mL, respectively), and higher after INH treatments (0.0-35.4 and 0.0-48.3 ng/mL, respectively). For 16αOHPRED, elevated concentrations were detected after INT and INH treatments (2.6-66.4 and 3.4-426.5 ng/mL, respectively). Concentrations obtained following oral administration were higher than after therapeutic administrations (2.8-80.6, 1.5-36.1, and 10.4-532.2 ng/mL for 6βOHBUD, BUD, and 16αOHPRED, respectively). After all administrations, concentrations were higher in males than in females. Results demonstrated that 6βOHBUD is the best discriminatory marker and a reporting level of 40 ng/mL was found to be the best criterion to distinguish allowed from forbidden administrations of BUD

Elimination profile of triamcinolone hexacetonide and its metabolites in human urine and plasma after a single intra-articular administration

Triamcinolone hexacetonide (THA) is a synthetic glucocorticoid (GC) used by intra-articular (IA) administration. GCs are prohibited in sports competitions by systemic routes, and they are allowed by other routes considered of local action (IA administration, among others). The aim of the present work was to study the metabolic profile of THA in urine and plasma following IA administration. Eight patients (4 males and 4 females) with knee osteoarthritis received an IA dose of THA (40 mg) in the knee joint. Spot urine and plasma samples were collected before injection and at different time periods up to day 23 and 10 post-administration, respectively. The samples were analysed by liquid chromatography-tandem mass spectrometry. Neither THA nor specific THA metabolites were detected in urine. Triamcinolone acetonide (TA) and 6β-hydroxy-triamcinolone acetonide were the main urinary metabolites. Maximum concentrations wereobtained between 24 and 48 h after administration. Using the reporting level of 30 ng/mL to distinguish allowed from forbidden administrations of GCs, a large number of false adverse analytical findings would be reported up to day 4. On the other hand, TA was detected in all plasma samples collected up to day 10 after administration. THA was also detected in plasma but at lower concentrations. The detection of plasma THA would be an unequivocal proof to demonstrate IA use of THA. A reversible decrease was observed in plasma concentrations of cortisol in some of the patients, indicating a systemic effect of the drug

Detection and characterization of clostebol sulfate metabolites in Caucasian population.

Anabolic androgenic steroids (AAS) are synthetic testosterone derivatives which undergo extensive metabolism in man. Differences in the excretion of phase II metabolites are strongly associated with inter-individual and inter-ethnic variations. Sulfate metabolites have been described as long-term metabolites for some AAS. Clostebol is the 4-chloro derivative of testosterone and the aim of the present study was the evaluation of clostebol sulfate metabolites in Caucasian population by LC-MS/MS technology. Clostebol was orally administered to four healthy Caucasian male volunteers, and excretion study urines were collected up to 31 days. Several analytical strategies (neutral loss scan, precursor ion scan and selected reaction monitoring acquisitions modes) were applied to detect sulfate metabolites in post-administration samples. Sixteen sulfate metabolites were detected, five of them having detectability times above 10 days (S1a, S2a, S3b, S3g and S4b). Interestingly, metabolite S1a could be detected up to the last collected sample of all excretion studies and it was characterized by LC-MS/MS and GC-MS as 4ξ-chloro-5α-androst-3β-ol-17-one 3β-sulfate. Thus, monitoring of S1a improves the detection time of clostebol misuse with respect to the commonly monitored metabolites, excreted in the glucuronide fraction. Importantly, this new metabolite can be incorporated into recently developed LC-MS/MS screening methods base on the direct detection of phase II metabolites.The financial support received from the World Anti-Doping Agency WADA (12A21RV), Ministerio de Economía y Competividad (Gobierno de Espana)(Project number DEP2012-35612) and Generalitat de Catalunya (Consell Català de l’Esport, DIUE 2014 SGR 692) is gratefully acknowledged. Spanish Health National System is also acknowledged for O. J. Pozo contract (MS10/00576)