Simultaneous determination of human plasma levels of citalopram, paroxetine, sertraline, and their metabolites by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry method is presented which allows the simultaneous determination of the plasma concentrations of the selective serotonin reuptake inhibitors citalopram, paroxetine, sertraline, and their pharmacologically active N-demethylated metabolites (desmethylcitalopram, didesmethylcitalopram, and desmethylsertraline) after derivatization with the reagent N-methyl-bis(trifluoroacetamide). No interferences from endogenous compounds are observed following the extraction of plasma samples from six different human subjects. The standard curves are linear over a working range of 10-500 ng/mL for citalopram, 10-300 ng/mL for desmethylcitalopram, 5-60 ng/mL for didesmethylcitalopram, 20-400 ng/mL for sertraline and desmethylsertraline, and 10-200 ng/mL for paroxetine. Recoveries measured at three concentrations range from 81 to 118% for the tertiary amines (citalopram and the internal standard methylmaprotiline), 73 to 95% for the secondary amines (desmethylcitalopram, paroxetine and sertraline), and 39 to 66% for the primary amines (didesmethylcitalopram and desmethylsertraline). Intra- and interday coefficients of variation determined at three concentrations range from 3 to 11% for citalopram and its metabolites, 4 to 15% for paroxetine, and 5 to 13% for sertraline and desmethylsertraline. The limits of quantitation of the method are 2 ng/mL for citalopram and paroxetine, 1 ng/mL for sertraline, and 0.5 ng/mL for desmethylcitalopram, didesmethylcitalopram, and desmethylsertraline. No interferences are noted from 20 other psychotropic drugs. This sensitive and specific method can be used for single-dose pharmacokinetics. It is also useful for therapeutic drug monitoring of these three drugs and could possibly be adapted for the quantitation of the two other selective serotonin reuptake inhibitors on the market, namely fluoxetine and fluvoxamine.

Potent synergism of the combination of fluconazole and cyclosporine in Candida albicans.

Several types of drugs currently used in clinical practice were screened in vitro for their potentiation of the antifungal effect of the fungistatic agent fluconazole (FLC) on Candida albicans. These drugs included inhibitors of multidrug efflux transporters, antimicrobial agents, antifungal agents, and membrane-active compounds with no antimicrobial activity, such as antiarrhythmic agents, proton pump inhibitors, and platelet aggregation inhibitors. Among the drugs tested in an agar disk diffusion assay, cyclosporine (Cy), which had no intrinsic antifungal activity, showed a potent antifungal effect in combination with FLC. In a checkerboard microtiter plate format, however, it was observed that the MIC of FLC, as classically defined by the NCCLS recommendations, was unchanged when FLC and Cy were combined. Nevertheless, if a different reading endpoint corresponding to the minimal fungicidal concentration needed to decrease viable counts by at least 3 logs in comparison to the growth control was chosen, the combination was synergistic (fractional inhibitory concentration index of <1). This endpoint fitted to the definition of MIC-0 (optically clear wells) and reflected the absence of the trailing effect, which is the result of a residual growth at FLC concentrations greater than the MIC. The MIC-0 values of FLC and Cy tested alone in C. albicans were >32 and >10 microg/ml, respectively, and decreased to 0.5 and 0.625 microg/ml when the two drugs were combined. The combination of 0.625 microg of Cy per ml with supra-MICs of FLC resulted in a potent antifungal effect in time-kill curve experiments. This effect was fungicidal or fungistatic, depending on the C. albicans strain used. Since the Cy concentration effective in vitro is achievable in vivo, the combination of this agent with FLC represents an attractive perspective for the development of new management strategies for candidiasis.

High-throughput hydrophilic interaction chromatography coupled to tandem mass spectrometry for the optimized quantification of the anti-Gram-negatives antibiotic colistin A/B and its pro-drug colistimethate.

Colistin is a last resort's antibacterial treatment in critically ill patients with multi-drug resistant Gram-negative infections. As appropriate colistin exposure is the key for maximizing efficacy while minimizing toxicity, individualized dosing optimization guided by therapeutic drug monitoring is a top clinical priority. Objective of the present work was to develop a rapid and robust HPLC-MS/MS assay for quantification of colistin plasma concentrations. This novel methodology validated according to international standards simultaneously quantifies the microbiologically active compounds colistin A and B, plus the pro-drug colistin methanesulfonate (colistimethate, CMS). 96-well micro-Elution SPE on Oasis Hydrophilic-Lipophilic-Balanced (HLB) followed by direct analysis by Hydrophilic Interaction Liquid Chromatography (HILIC) with Ethylene Bridged Hybrid - BEH - Amide phase column coupled to tandem mass spectrometry allows a high-throughput with no significant matrix effect. The technique is highly sensitive (limit of quantification 0.014 and 0.006μg/mL for colistin A and B), precise (intra-/inter-assay CV 0.6-8.4%) and accurate (intra-/inter-assay deviation from nominal concentrations -4.4 to +6.3%) over the clinically relevant analytical range 0.05-20μg/mL. Colistin A and B in plasma and whole blood samples are reliably quantified over 48h at room temperature and at +4°C (<6% deviation from nominal values) and after three freeze-thaw cycles. Colistimethate acidic hydrolysis (1M H2SO4) to colistin A and B in plasma was completed in vitro after 15min of sonication while the pro-drug hydrolyzed spontaneously in plasma ex vivo after 4h at room temperature: this information is of utmost importance for interpretation of analytical results. Quantification is precise and accurate when using serum, citrated or EDTA plasma as biological matrix, while use of heparin plasma is not appropriate. This new analytical technique providing optimized quantification in real-life conditions of the microbiologically active compounds colistin A and B offers a highly efficient tool for routine therapeutic drug monitoring aimed at individualizing drug dosing against life-threatening infections.

Changes in the transcriptional profile of transporters in the intestine along the anterior-posterior and crypt-villus axes.

BACKGROUND: The purpose of this work was to characterize the expression of drug and nutrient carriers along the anterior-posterior and crypt-villus axes of the intestinal epithelium and to study the validity of utilizing whole gut tissue rather than purified epithelial cells to examine regional variations in gene expression. RESULTS: We have characterized the mRNA expression profiles of 76 % of all currently known transporters along the anterior-posterior axis of the gut. This is the first study to describe the expression profiles of the majority of all known transporters in the intestine. The expression profiles of transporters, as defined according to the Gene Ontology consortium, were measured in whole tissue of the murine duodenum, jejunum, ileum and colon using high-density microarrays. For nine transporters (Abca1, Abcc1, Abcc3, Abcg8, Slc10a2, Slc28a2, Slc2a1, Slc34a2 and Slc5a8), the mRNA profiles were further measured by RT-PCR in laser micro-dissected crypt and villus epithelial cells corresponding to the aforementioned intestinal regions. With respect to differentially regulated transporters, the colon had a distinct expression profile from small intestinal segments. The majority (59 % for p cutoff < or = 0.05) of transporter mRNA levels were constant across the intestinal sections studied. For the transporter subclass "carrier activity", which contains the majority of known carriers for biologically active compounds, a significant change (p < or = 0.05) along the anterior-posterior axis was observed. CONCLUSION: All nine transporters examined in laser-dissected material demonstrated good replication of the region-specific profiles revealed by microarray. Furthermore, we suggest that the distribution characteristics of Slc5a8 along the intestinal tract render it a suitable candidate carrier for monocarboxylate drugs in the posterior portion of the intestine. Our findings also predict that there is a significant difference in the absorption of carrier-mediated compounds in the different intestinal segments. The most pronounced differences can be expected between the adjoining segments ileum and colon, but the differences between the other adjoining segments are not negligible. Finally, for the examined genes, profiles measured in whole intestinal tissue extracts are representative of epithelial cell-only gene expression.

Plasma and urine profiles of Delta9-tetrahydrocannabinol and its metabolites 11-hydroxy-Delta9-tetrahydrocannabinol and 11-nor-9-carboxy-Delta9-tetrahydrocannabinol after cannabis smoking by male volunteers to estimate recent consumption by athletes.

Since 2004, cannabis has been prohibited by the World Anti-Doping Agency for all sports competitions. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In doping urine analysis, the target analyte is 11-nor-9-carboxy-Delta(9)-tetrahydrocannabinol (THC-COOH), the cutoff being 15 ng/mL. However, the wide urinary detection window of the long-term metabolite of Delta(9)-tetrahydrocannabinol (THC) does not allow a conclusion to be drawn regarding the time of consumption or the impact on the physical performance. The purpose of the present study on light cannabis smokers was to evaluate target analytes with shorter urinary excretion times. Twelve male volunteers smoked a cannabis cigarette standardized to 70 mg THC per cigarette. Plasma and urine were collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-Delta(9)-tetrahydrocannabinol (THC-OH), and THC-COOH were determined after hydrolysis followed by solid-phase extraction and gas chromatography/mass spectrometry. The limits of quantitation were 0.1-1.0 ng/mL. Eight puffs delivered a mean THC dose of 45 mg. Plasma levels of total THC, THC-OH, and THC-COOH were measured in the ranges 0.2-59.1, 0.1-3.9, and 0.4-16.4 ng/mL, respectively. Peak concentrations were observed at 5, 5-20, and 20-180 min. Urine levels were measured in the ranges 0.1-1.3, 0.1-14.4, and 0.5-38.2 ng/mL, peaking at 2, 2, and 6-24 h, respectively. The times of the last detectable levels were 2-8, 6-96, and 48-120 h. Besides high to very high THC-COOH levels (245 +/- 1,111 ng/mL), THC (3 +/- 8 ng/mL) and THC-OH (51 +/- 246 ng/mL) were found in 65 and 98% of cannabis-positive athletes' urine samples, respectively. In conclusion, in addition to THC-COOH, the pharmacologically active THC and THC-OH should be used as target analytes for doping urine analysis. In the case of light cannabis use, this may allow the estimation of more recent consumption, probably influencing performance during competitions. However, it is not possible to discriminate the intention of cannabis use, i.e., for recreational or doping purposes. Additionally, pharmacokinetic data of female volunteers are needed to interpret cannabis-positive doping cases of female athletes.

Smoked cannabis and doping control: looking for the wrong target analyte?

Introduction: Since 2004, cannabis is prohibited by the World Anti-Doping Agency (WADA) for all sports in competition. In the years since then, about half of all positive doping cases in Switzerland have been related to cannabis consumption. In most cases, the athletes plausibly claim to have consumed cannabis several days or even weeks before competition and only for recreational purposes not related to competition. In doping analysis, the target analyte in urine samples is 11-nor-delta-9-tetrahydrocannabinol- 9-carboxylic acid (THC-COOH), the reporting threshold for laboratories is 15 ng/mL. However, the wide detection window of this long-term THC metabolite in urine does not allow a conclusion concerning the time of consumption or the impact on the physical performance. Aim: The purpose of the present pharmacokinetic study on volunteers was to evaluate target analytes with shorter urinary excretion time. Subsequently, urines from athletes tested positive for cannabis should be reanalyzed including these analytes. Methods: In an one-session clinical trial (approved by IRB, Swissmedic, and Federal Office of Public Health), 12 healthy, male volunteers (age 26 ± 3 yrs, BMI 24 ± 2 kg/m2) with cannabis experience (> once/month) smoked a Cannabis cigarette standardized to 70 mg THC/cigarette (Bedrobinol® 7%, Dutch Office for Medicinal Cannabis) following a paced-puffing procedure. Plasma and urine was collected up to 8 h and 11 days, respectively. Total THC, 11-hydroxy-THC (THC-OH), and THC-COOH were determined after enzymatic hydrolyzation followed by SPE and GC/MS-SIM. The limit of quantitation (LOQ) for all analytes was 0.1 ng/mL. Visual analog scales (VAS) and vital functions were used for monitoring psychological and somatic side-effects at every timepoint of specimen collection (up to 480 min). Results: Eight puffs delivered a mean THC dose of 45 mg. Mean plasma levels of total THC, THC-OH and THC-COOH were measured in the range of 0.1-20.9, 0.1-1.8, and 1.8-7.5 ng/mL, respectively. Peak concentrations were observed at 5, 10, and 90 min. Mean urine levels were measured in the range of 0.1-0.7, 0.10-6.2, and 0.1-13.4 ng/mL, respectively. The detection windows were 2-8, 2-96, and 2-120 h. No or only mild effects were observed, such as dry mouth, sedation, and tachycardia. Besides high to very high THC-COOH levels (0-978 ng/mL), THC (0.1-24 ng/mL) and THC-OH (1-234 ng/mL) were found in 90 and 96% of the cannabis-positive urines from athletes. Conclusion: Instead of or in addition to THC-COOH, the pharmacologically active THC and THC-OH should be the target analytes for doping urine analysis. This would allow the estimation of more recent Cannabis consumption, probably influencing performance during competition. Keywords: cannabis, doping, clinical trial, plasma and urine levels, athlete's samples

Mechanisms of antifungal resistance in the opportunistic fungus Aspergillus fumigatus

ABSTRACT Aspergillus fumigatus is one of the most prevalent airbone fungal pathogen and can cause severe fatal invasive aspergillosis in immunocompromised patients. Several antifungal agents are available to treat these infections but with limited success. These agents include polyenes (amphotericin B), echinocandins (caspofungin) and azoles, which constitute the most important class with itraconazole (ITC) and voriconazole as major active compounds. Azole-derived antifungal agents target the ergosterol biosynthesis pathway via the inhibition of the lanosterol 14α-demethylase (cyp51/ERG1 1), a cytochrome P450 responsible for the conversion of lanosterol to ergosterol, which is the main component of cell membrane in fungi. A. fumigatus is also found in the environment as a contaminant of rotting plant or present in composting of organic waste. Among antifungal agents used in the environment for crop protection, the class of azoles is also widely used with propiconazole or prochloraz as examples. However, other agents such as dicarboximide (iprodione), phenylamide (benalaxyl) or strobilurin (azoxystrobin) are also used. Emergence of clinical azole-resistant isolates has been described in several European countries. However the incidence of antifungal resistance has not been yet reported in details in Switzerland. In this study, the status of antifungal resistance was investigated on A. fumigatus isolates collected from Swiss hospitals and from different environmental sites and. tested for their susceptibility to several currently used antifungal agents. The data showed a low incidence of resistance for all tested agents among clinical and environmental isolates. Only two azole-resistant environmental isolates were detected and none among the clinical tested isolates. In general, A. fumigatus was susceptible to all antifungals tested in our study, except to azoxystrobin which was the less active agent against all isolates. Since mechanisms of antifungal resistance have been poorly investigated until now in A. fumigatus, this work was aimed 1) to identify A. fumigatus genes involved in antifungal resistance and 2) to test their involvement in the development of resistance in sampled isolates. Therefore, this work proposed to isolate A. fumigatus genes conferring resistance to a drug-hypersusceptible Saccharomyces cerevisiae strain due to a lack of multidrug transporter genes. Several genes were recovered including three distinct efflux transporters (atrF, atrH and mdrA) and a bZip transcription factor, yapA. The inactivation of each transporter in A. fumigatus indicated that the transporters were involved in the basal level of azole susceptibility. The inactivation of YapA led to a hypersusceptibility to H2O2, thus confirming the involvement of this gene in the oxidative stress response of A. fumigatus. The involvement of the abovementioned transporters genes and of other transporters genes identified by genome analysis in azole resistance was tested by probing their expression in some ITC-resistant isolates. Even if upregulation of some transporters genes was observed in some investigated isolates, the correlation between azole resistance and expression levels of all these transporters genes could not be clearly established for all tested isolates. Given these results, the present work addressed 1) alteration in the expression of cyp51A encoding for the azole target enzyme, and 2) mutation(s) in the cyp51A sequence as potential mechanisms of azote resistance in A. . However, overexpression of cyp51A in the investigated isolates was not linked with azote resistance. Since it was reported that mutation(s) in cyp51A were participating in azote resistance in A. fumigatus, a functional complementation of cyp51A cDNAs from ITC-resistant A. fumigatus strains in S. cerevisiae ergl 1 Δ mutant strain was attempted. Expression in S. cerevisiae allowed the testing of these cDNAs with regards to their functionality and involvement in resistance to specific azote compounds. We could demonstrate that Cyp51A protein with a G54E or M220K mutations conferred resistance to specific azoles in S. cerevisiae, therefore suggesting that these mutations were important for the development of azote resistance in A. fumigatus. In conclusion, this work showed a correlation between ITC resistance and mechanisms involving overexpression of transporters and cyp51A mutations in A. fumigatus isolates. However, azole resistance of some isolates has not been solved and thus it will be necessary to approach the study of resistance mechanisms in this fungal species using alternative methodologies. RESUME Aspergillus fumigatus est un champignon opportuniste répandu et est la cause d'aspergilloses invasives le plus souvent fatales chez des patients immunodéprimés. Plusieurs antifongiques sont disponibles afin de traiter ces infections, cependant avec un succès limité. Ces agents incluent les polyènes (amphotericin B), les échinocandines (caspofungin) et les azoles, qui représentent la plus importante classe d'antifongiques avec l'itraconazole (ITC) et le voriconazole comme principaux agents actifs. Les dérivés azolés ciblent la voie de biosynthèse de l'ergostérol via l'inhibition de la lanostérol 14α-demethylase (cyp51/ERG11), un cytochrome P450 impliqué dans la conversion du lanostérol en ergostérol, qui est un composant important de la membrane chez les champignons. A. fumigatus est également répandu dans l'environnement. Parmi les antifongiques employés en agriculture afin de protéger les cultures, les azoles sont aussi largement utilisés. Cependant, d'autres agents tels que les dicarboximides (iprodione), les phenylamides (benalaxyl) et les strobilurines (azoxystrobin) peuvent être également utilisés. L'émergence de souches cliniques résistantes aux azoles a été décrite dans différents pays européens. Cependant, l'incidence d'une telle résistance aux azoles n'a pas encore été reportée en détails en Suisse. Dans ce travail, l'émergence de la résistance aux antifongiques a été étudiée par analyse de souches d'A. fumigatus provenant de milieux hospitaliers en Suisse et de différents sites et leur susceptibilité testée envers plusieurs antifongiques couramment utilisés. Les données obtenues ont montré une faible incidence de la résistance parmi les souches cliniques et environnementales pour les agents testés. Seulement deux souches environnementales résistantes aux azoles ont été détectées et aucune parmi les souches cliniques. Les mécanismes de résistance aux antifongiques ayant été très peu étudiés jusqu'à présent chez A. fumigatus , ce travail a eu aussi pour but 1) d'identifier les gènes d' A. fumigatus impliqués dans la résistance aux antifongiques et 2) de tester leur implication dans la résistance de certaines souches. Ainsi, il a été proposé d'isoler les gènes d' A. fumigatus pouvant conférer une résistance aux antifongiques à une souche de Saccharomyces cerevisiae hypersensible aux antifongiques. Trois transporteurs à efflux (atrF, atrH et mdrA) et un facteur de transcription appartenant à la famille des bZip (YapA) ont ainsi été isolés. L'inactivation, dans une souche d'A. fumigatus, de chacun des ces transporteurs a permis de mettre en évidence leur implication dans la susceptibilité d'A. fumigatus aux antifongiques. L'inactivation de YapA a engendré une hypersusceptibilité à l' H2O2, confirmant ainsi le rôle de ce gène dans la réponse au stress oxydatif chez A . fumigatus. La participation dans la résistance aux antifongiques des gènes codant pour des transporteurs ainsi que d'autres gènes identifiés par analyse du génome a été déterminée en testant leur niveau d'expression dans des souches résistantes à l'ITC. Bien qu'une surexpression de transporteurs ait été observée dans certaines souches, une corrélation entre la résistance à l'ITC et les niveaux d'expression de ces transporteurs n'a pu être clairement établie. Ce présent travail s'est donc porté sur l'étude de 2 autres mécanismes potentiellement impliqués dans la résistance aux azoles : 1) la surexpression de cyp51A codant pour l'enzyme cible et 2) des mutations dans cyp51A. Cependant, la surexpression de cyp51A dans les souches étudiées n'a pas été constatée. L'effet des mutations de cyp51A dans la résistance aux azoles a été testée par complémentation fonctionnelle d'une souche S. cerevisiae déletée dans son gène ERG11. L'expression de ces gènes chez S. cerevisiae a permis de démontrer que les protéines Cyp51Ap contenant une mutation G54E ou M220K pouvaient conférer une résistance spécifique à certains azoles, ainsi suggérant que ces mutations pourraient être importantes dans le développement d'une résistance aux azoles chez A. fumigatus. En conclusion, ce travail a permis de mettre en évidence, dans des souches d'A. fumigatus , une corrélation entre leur résistance à l' ITC et les mécanismes impliquant une surexpression de transporteurs et des mutations dans cyp51A. Cependant, ces mécanismes n'ont pu expliquer la résistance aux azoles de certaines souches et c'est pourquoi de nouvelles approches doivent être envisagées afin d'étudier ces mécanismes.

Cheminformatic studies of molecular properties and their influence on bioactivity in the context of drug discovery

Molecular shape has long been known to be an important property for the process of molecular recognition. Previous studies postulated the existence of a drug-like shape space that could be used to artificially bias the composition of screening libraries, with the aim to increase the chance of success in Hit Identification. In this work, it was analysed to which extend this assumption holds true. Normalized Principal Moments of Inertia Ratios (NPRs) have been used to describe the molecular shape of small molecules. It was investigated, whether active molecules of diverse targets are located in preferred subspaces of the NPR shape space. Results illustrated a significantly stronger clustering than could be expected by chance, with parts of the space unlikely to be occupied by active compounds. Furthermore, a strong enrichment of elongated, rather flat shapes could be observed, while globular compounds were highly underrepresented. This was confirmed for a wide range of small molecule datasets from different origins. Active compounds exhibited a high overlap in their shape distributions across different targets, making a purely shape­ based discrimination very difficult. An additional perspective was provided by comparing the shapes of protein binding pockets with those of their respective ligands. Although more globular than their ligands, it was observed that binding sites shapes exhibited a similarly skewed distribution in shape space: spherical shapes were highly underrepresented. This was different for unoccupied binding pockets of smaller size. These were on the contrary identified to possess a more globular shape. The relation between shape complementarity and exhibited bioactivity was analysed; a moderate correlation between bioactivity and parameters including pocket coverage, distance in shape space, and others could be identified, which reflects the importance of shape complementarity. However, this also suggests that other aspects are of relevance for molecular recognition. A subsequent analysis assessed if and how shape and volume information retrieved from pocket or respective reference ligands could be used as a pre-filter in a virtual screening approach. ln Lead Optimization compounds need to get optimized with respect to a variety of pararneters. Here, the availability of past success stories is very valuable, as they can guide medicinal chemists during their analogue synthesis plans. However, although of tremendous interest for the public domain, so far only large corporations had the ability to mine historical knowledge in their proprietary databases. With the aim to provide such information, the SwissBioisostere database was developed and released during this thesis. This database contains information on 21,293,355 performed substructural exchanges, corresponding to 5,586,462 unique replacements that have been measured in 35,039 assays against 1,948 molecular targets representing 30 target classes, and on their impact on bioactivity . A user-friendly interface was developed that provides facile access to these data and is accessible at http//www.swissbioisostere.ch. The ChEMBL database was used as primary data source of bioactivity information. Matched molecular pairs have been identified in the extracted and cleaned data. Success-based scores were developed and integrated into the database to allow re-ranking of proposed replacements by their past outcomes. It was analysed to which degree these scores correlate with chemical similarity of the underlying fragments. An unexpectedly weak relationship was detected and further investigated. Use cases of this database were envisioned, and functionalities implemented accordingly: replacement outcomes are aggregatable at the assay level, and it was shawn that an aggregation at the target or target class level could also be performed, but should be accompanied by a careful case-by-case assessment. It was furthermore observed that replacement success depends on the activity of the starting compound A within a matched molecular pair A-B. With increasing potency the probability to lose bioactivity through any substructural exchange was significantly higher than in low affine binders. A potential existence of a publication bias could be refuted. Furthermore, often performed medicinal chemistry strategies for structure-activity-relationship exploration were analysed using the acquired data. Finally, data originating from pharmaceutical companies were compared with those reported in the literature. It could be seen that industrial medicinal chemistry can access replacement information not available in the public domain. In contrast, a large amount of often-performed replacements within companies could also be identified in literature data. Preferences for particular replacements differed between these two sources. The value of combining different endpoints in an evaluation of molecular replacements was investigated. The performed studies highlighted furthermore that there seem to exist no universal substructural replacement that always retains bioactivity irrespective of the biological environment. A generalization of bioisosteric replacements seems therefore not possible. - La forme tridimensionnelle des molécules a depuis longtemps été reconnue comme une propriété importante pour le processus de reconnaissance moléculaire. Des études antérieures ont postulé que les médicaments occupent préférentiellement un sous-ensemble de l'espace des formes des molécules. Ce sous-ensemble pourrait être utilisé pour biaiser la composition de chimiothèques à cribler, dans le but d'augmenter les chances d'identifier des Hits. L'analyse et la validation de cette assertion fait l'objet de cette première partie. Les Ratios de Moments Principaux d'Inertie Normalisés (RPN) ont été utilisés pour décrire la forme tridimensionnelle de petites molécules de type médicament. Il a été étudié si les molécules actives sur des cibles différentes se co-localisaient dans des sous-espaces privilégiés de l'espace des formes. Les résultats montrent des regroupements de molécules incompatibles avec une répartition aléatoire, avec certaines parties de l'espace peu susceptibles d'être occupées par des composés actifs. Par ailleurs, un fort enrichissement en formes allongées et plutôt plates a pu être observé, tandis que les composés globulaires étaient fortement sous-représentés. Cela a été confirmé pour un large ensemble de compilations de molécules d'origines différentes. Les distributions de forme des molécules actives sur des cibles différentes se recoupent largement, rendant une discrimination fondée uniquement sur la forme très difficile. Une perspective supplémentaire a été ajoutée par la comparaison des formes des ligands avec celles de leurs sites de liaison (poches) dans leurs protéines respectives. Bien que plus globulaires que leurs ligands, il a été observé que les formes des poches présentent une distribution dans l'espace des formes avec le même type d'asymétrie que celle observée pour les ligands: les formes sphériques sont fortement sous­ représentées. Un résultat différent a été obtenu pour les poches de plus petite taille et cristallisées sans ligand: elles possédaient une forme plus globulaire. La relation entre complémentarité de forme et bioactivité a été également analysée; une corrélation modérée entre bioactivité et des paramètres tels que remplissage de poche, distance dans l'espace des formes, ainsi que d'autres, a pu être identifiée. Ceci reflète l'importance de la complémentarité des formes, mais aussi l'implication d'autres facteurs. Une analyse ultérieure a évalué si et comment la forme et le volume d'une poche ou de ses ligands de référence pouvaient être utilisés comme un pré-filtre dans une approche de criblage virtuel. Durant l'optimisation d'un Lead, de nombreux paramètres doivent être optimisés simultanément. Dans ce contexte, la disponibilité d'exemples d'optimisations réussies est précieuse, car ils peuvent orienter les chimistes médicinaux dans leurs plans de synthèse par analogie. Cependant, bien que d'un extrême intérêt pour les chercheurs dans le domaine public, seules les grandes sociétés pharmaceutiques avaient jusqu'à présent la capacité d'exploiter de telles connaissances au sein de leurs bases de données internes. Dans le but de remédier à cette limitation, la base de données SwissBioisostere a été élaborée et publiée dans le domaine public au cours de cette thèse. Cette base de données contient des informations sur 21 293 355 échanges sous-structuraux observés, correspondant à 5 586 462 remplacements uniques mesurés dans 35 039 tests contre 1948 cibles représentant 30 familles, ainsi que sur leur impact sur la bioactivité. Une interface a été développée pour permettre un accès facile à ces données, accessible à http:/ /www.swissbioisostere.ch. La base de données ChEMBL a été utilisée comme source de données de bioactivité. Une version modifiée de l'algorithme de Hussain et Rea a été implémentée pour identifier les Matched Molecular Pairs (MMP) dans les données préparées au préalable. Des scores de succès ont été développés et intégrés dans la base de données pour permettre un reclassement des remplacements proposés selon leurs résultats précédemment observés. La corrélation entre ces scores et la similarité chimique des fragments correspondants a été étudiée. Une corrélation plus faible qu'attendue a été détectée et analysée. Différents cas d'utilisation de cette base de données ont été envisagés, et les fonctionnalités correspondantes implémentées: l'agrégation des résultats de remplacement est effectuée au niveau de chaque test, et il a été montré qu'elle pourrait également être effectuée au niveau de la cible ou de la classe de cible, sous réserve d'une analyse au cas par cas. Il a en outre été constaté que le succès d'un remplacement dépend de l'activité du composé A au sein d'une paire A-B. Il a été montré que la probabilité de perdre la bioactivité à la suite d'un remplacement moléculaire quelconque est plus importante au sein des molécules les plus actives que chez les molécules de plus faible activité. L'existence potentielle d'un biais lié au processus de publication par articles a pu être réfutée. En outre, les stratégies fréquentes de chimie médicinale pour l'exploration des relations structure-activité ont été analysées à l'aide des données acquises. Enfin, les données provenant des compagnies pharmaceutiques ont été comparées à celles reportées dans la littérature. Il a pu être constaté que les chimistes médicinaux dans l'industrie peuvent accéder à des remplacements qui ne sont pas disponibles dans le domaine public. Par contre, un grand nombre de remplacements fréquemment observés dans les données de l'industrie ont également pu être identifiés dans les données de la littérature. Les préférences pour certains remplacements particuliers diffèrent entre ces deux sources. L'intérêt d'évaluer les remplacements moléculaires simultanément selon plusieurs paramètres (bioactivité et stabilité métabolique par ex.) a aussi été étudié. Les études réalisées ont souligné qu'il semble n'exister aucun remplacement sous-structural universel qui conserve toujours la bioactivité quel que soit le contexte biologique. Une généralisation des remplacements bioisostériques ne semble donc pas possible.

Identification and characterization of novel classes of macrophage migration inhibitory factor (MIF) inhibitors with distinct mechanisms of action.

Macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, is considered an attractive therapeutic target in multiple inflammatory and autoimmune disorders. In addition to its known biologic activities, MIF can also function as a tautomerase. Several small molecules have been reported to be effective inhibitors of MIF tautomerase activity in vitro. Herein we employed a robust activity-based assay to identify different classes of novel inhibitors of the catalytic and biological activities of MIF. Several novel chemical classes of inhibitors of the catalytic activity of MIF with IC(50) values in the range of 0.2-15.5 microm were identified and validated. The interaction site and mechanism of action of these inhibitors were defined using structure-activity studies and a battery of biochemical and biophysical methods. MIF inhibitors emerging from these studies could be divided into three categories based on their mechanism of action: 1) molecules that covalently modify the catalytic site at the N-terminal proline residue, Pro(1); 2) a novel class of catalytic site inhibitors; and finally 3) molecules that disrupt the trimeric structure of MIF. Importantly, all inhibitors demonstrated total inhibition of MIF-mediated glucocorticoid overriding and AKT phosphorylation, whereas ebselen, a trimer-disrupting inhibitor, additionally acted as a potent hyperagonist in MIF-mediated chemotactic migration. The identification of biologically active compounds with known toxicity, pharmacokinetic properties, and biological activities in vivo should accelerate the development of clinically relevant MIF inhibitors. Furthermore, the diversity of chemical structures and mechanisms of action of our inhibitors makes them ideal mechanistic probes for elucidating the structure-function relationships of MIF and to further determine the role of the oligomerization state and catalytic activity of MIF in regulating the function(s) of MIF in health and disease.

Free and total plasma levels of lopinavir during pregnancy, at delivery and postpartum: implications for dosage adjustments in pregnant women.

BACKGROUND: Physiological changes associated with pregnancy may alter antiretroviral plasma concentrations and might jeopardize prevention of mother-to-child HIV transmission. Lopinavir is one of the protease inhibitors more frequently prescribed during pregnancy in Europe. We described the free and total pharmacokinetics of lopinavir in HIV-infected pregnant and non-pregnant women, and evaluated whether significant alterations in its disposition and protein binding warrant systematic dosage adjustment. METHODS: Plasma samples were collected at first, second and third trimester of pregnancy, at delivery, in umbilical cord and postpartum. Lopinavir free and total plasma concentrations were measured by HPLC-MS/MS. Bayesian calculations were used to extrapolate total concentrations to trough (Cmin). RESULTS: A total of 42 HIV-positive pregnant women and 37 non-pregnant women on lopinavir/ritonavir were included in the study. Compared to postpartum and control values, total lopinavir Cmin was decreased moderately (31-39%) during pregnancy, and free Cmin minimally, showing significant alteration only at delivery (-35%). However, total and free Cmin remained in all patients above the target concentrations for wild-type virus of 1,000 ng/ml, and above the unbound IC50(WT) of 0.64-0.77 ng/ml of lopinavir, respectively. Lopinavir free fractions remained higher during pregnancy compared to postpartum and controls, and were influenced by α-1-acid-glycoprotein and albumin decrease. Free cord-to-mother ratio (0.43) was 2.7-fold higher than total cord-to-mother ratio (0.16), suggesting higher fetal exposure. CONCLUSIONS: The moderate decrease of total lopinavir concentrations during pregnancy is not associated with proportional decrease in free concentrations. Both reach a nadir at delivery, albeit not to an extent that would put treatment-naive women at risk of insufficient exposure to the free, pharmacologically active concentrations of lopinavir. No dosage adjustment is therefore needed during pregnancy as it is unlikely to further enhance treatment efficacy but could potentially increase the risk of maternal and fetal toxicity. Nonetheless, in case of viral resistance in treatment-experienced pregnant women, loss of virological control or questionable adherence, it is justified to consider lopinavir dosage adjustment based on total plasma concentration measurement.

STRATEGIC-1: A multiple-lines, randomized, open-label GERCOR phase III study in patients with unresectable wild-type RAS metastatic colorectal cancer.

BACKGROUND: The management of unresectable metastatic colorectal cancer (mCRC) is a comprehensive treatment strategy involving several lines of therapy, maintenance, salvage surgery, and treatment-free intervals. Besides chemotherapy (fluoropyrimidine, oxaliplatin, irinotecan), molecular-targeted agents such as anti-angiogenic agents (bevacizumab, aflibercept, regorafenib) and anti-epidermal growth factor receptor agents (cetuximab, panitumumab) have become available. Ultimately, given the increasing cost of new active compounds, new strategy trials are needed to define the optimal use and the best sequencing of these agents. Such new clinical trials require alternative endpoints that can capture the effect of several treatment lines and be measured earlier than overall survival to help shorten the duration and reduce the size and cost of trials. METHODS/DESIGN: STRATEGIC-1 is an international, open-label, randomized, multicenter phase III trial designed to determine an optimally personalized treatment sequence of the available treatment modalities in patients with unresectable RAS wild-type mCRC. Two standard treatment strategies are compared: first-line FOLFIRI-cetuximab, followed by oxaliplatin-based second-line chemotherapy with bevacizumab (Arm A) vs. first-line OPTIMOX-bevacizumab, followed by irinotecan-based second-line chemotherapy with bevacizumab, and by an anti-epidermal growth factor receptor monoclonal antibody with or without irinotecan as third-line treatment (Arm B). The primary endpoint is duration of disease control. A total of 500 patients will be randomized in a 1:1 ratio to one of the two treatment strategies. DISCUSSION: The STRATEGIC-1 trial is designed to give global information on the therapeutic sequences in patients with unresectable RAS wild-type mCRC that in turn is likely to have a significant impact on the management of this patient population. The trial is open for inclusion since August 2013. TRIAL REGISTRATION: STRATEGIC-1 is registered at Clinicaltrials.gov: NCT01910610, 23 July, 2013. STRATEGIC-1 is registered at EudraCT-No.: 2013-001928-19, 25 April, 2013.

A quantitative analysis of antagonism and inverse agonism at wild-type and constitutively active hamster alpha1B-adrenoceptors.

In order to characterize inverse agonism at alpha1B-adrenoceptors, we have compared the concentration-response relationships of several quinazoline and non-quinazoline alpha1-adrenoceptor antagonists at cloned hamster wild-type (WT) alpha1B-adrenoceptors and a constitutively active mutant (CAM) thereof upon stable expression in Rat-1 fibroblasts. Receptor activation or inhibition thereof was assessed as [3H]inositol phosphate (IP) accumulation. Quinazoline (alfuzosin, doxazosin, prazosin, terazosin) and non-quinazoline alpha1-adrenoceptor antagonists (BE 2254, SB 216,469, tamsulosin) concentration-dependently inhibited phenylephrine-stimulated IP formation at both WT and CAM with Ki values similar to those previously found in radioligand binding studies. At CAM in the absence of phenylephrine, the quinazolines produced concentration-dependent inhibition of basal IP formation; the maximum inhibition was approximately 55%, and the corresponding EC50 values were slightly smaller than the Ki values. In contrast, BE 2254 produced much less inhibition of basal IP formation, SB 216,469 was close to being a neutral antagonist, and tamsulosin even weakly stimulated IP formation. The inhibitory effects of the quinazolines and BE 2254 as well as the stimulatory effect of tamsulosin were equally blocked by SB 216,469 at CAM. At WT in the absence of phenylephrine, tamsulosin did not cause significant stimulation and none of the other compounds caused significant inhibition of basal IP formation. We conclude that alpha1-adrenoceptor antagonsits with a quinazoline structure exhibit greater efficacy as inverse agonists than those without.

Pi*-pi* bonding interactions generated by halogen oxidation of zirconium(IV) redox-active ligand complexes.

The new complex, [Zr(pda)2]n (1, pda2- = N,N'-bis(neo-pentyl)-ortho-phenylenediamide, n = 1 or 2), prepared by the reaction of 2 equiv of pdaLi2 with ZrCl4, reacts rapidly with halogen oxidants to afford the new product ZrX2(disq)2 (3, X = Cl, Br, I; disq- = N,N'-bis(neo-pentyl)-ortho-diiminosemiquinonate) in which each redox-active ligand has been oxidized by one electron. The oxidation products 3a-c have been structurally characterized and display an unusual parallel stacked arrangement of the disq- ligands in the solid state, with a separation of approximately 3 A. Density functional calculations show a bonding-type interaction between the SOMOs of the disq- ligands to form a unique HOMO while the antibonding linear combination forms a unique LUMO. This orbital configuration leads to a closed-shell-singlet ground-state electron configuration (S = 0). Temperature-dependent magnetism measurements indicate a low-lying triplet excited state at approximately 750 cm-1. In solution, 3a-c show strong disq--based absorption bands that are invariant across the halide series. Taken together these spectroscopic measurements provide experimental values for the one- and two-electron energies that characterize the pi-stacked bonding interaction between the two disq- ligands.

Novel Macrocyclic Amidinoureas: Potent Non-Azole Antifungals Active against Wild-Type and Resistant Candida Species.

Novel macrocyclic amidinourea derivatives 11, 18, and 25 were synthesized and evaluated as antifungal agents against wild-type and fluconazole resistant Candida species. Macrocyclic compounds 11 and 18 were synthesized through a convergent approach using as a key step a ring-closing metathesis macrocyclization reaction, whereas compounds 25 were obtained by our previously reported synthetic pathway. All the macrocyclic amidinoureas showed antifungal activity toward different Candida species higher or comparable to fluconazole and resulted highly active against fluconazole resistant Candida strains showing in many cases minimum inhibitory concentration values lower than voriconazole.

EADock: docking of small molecules into protein active sites with a multiobjective evolutionary optimization.

In recent years, protein-ligand docking has become a powerful tool for drug development. Although several approaches suitable for high throughput screening are available, there is a need for methods able to identify binding modes with high accuracy. This accuracy is essential to reliably compute the binding free energy of the ligand. Such methods are needed when the binding mode of lead compounds is not determined experimentally but is needed for structure-based lead optimization. We present here a new docking software, called EADock, that aims at this goal. It uses an hybrid evolutionary algorithm with two fitness functions, in combination with a sophisticated management of the diversity. EADock is interfaced with the CHARMM package for energy calculations and coordinate handling. A validation was carried out on 37 crystallized protein-ligand complexes featuring 11 different proteins. The search space was defined as a sphere of 15 A around the center of mass of the ligand position in the crystal structure, and on the contrary to other benchmarks, our algorithm was fed with optimized ligand positions up to 10 A root mean square deviation (RMSD) from the crystal structure, excluding the latter. This validation illustrates the efficiency of our sampling strategy, as correct binding modes, defined by a RMSD to the crystal structure lower than 2 A, were identified and ranked first for 68% of the complexes. The success rate increases to 78% when considering the five best ranked clusters, and 92% when all clusters present in the last generation are taken into account. Most failures could be explained by the presence of crystal contacts in the experimental structure. Finally, the ability of EADock to accurately predict binding modes on a real application was illustrated by the successful docking of the RGD cyclic pentapeptide on the alphaVbeta3 integrin, starting far away from the binding pocket.