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Supercritical fluid chromatography (SFC) holds the potential to become an orthogonal method to HPLC/UHPLC in xenobiotic metabolism studies, due to its outstanding capacity to simultaneously separate highly similar (as HPLC) and physicochemically different analytes (problematic using HPLC). Paucity of guideline-conform validation, however, has been a major obstacle to clinical application of SFC, even in cases where biotransformation yields chemically dissimilar metabolites that require more than one HPLC method for comprehensive analysis. Here, a method based on supercritical fluid chromatography coupled to single quadrupole MS detection was developed to simultaneously quantify the divisive analgesic flupirtine and its acidic and basic metabolites, represented by 4-fluorohippuric acid (4-FHA) and the active metabolite D-13223 respectively, using custom-made synthetic internal standards. Experimental data on the fundamental retention mechanisms under supercritical conditions, indicating the importance of halogen and π-π-bonding for specific retention on polysaccharide-based stationary-phases, is discussed. Compared to previous HPLC methods, the novel method offers higher versatility in terms of the target metabolite range (addressing both acidic and basic metabolites within a singular method), faster analysis (7.5 min), and compliance with green chemistry principles. Validation was performed according to EMA criteria on bioanalytical method validation, demonstrating selectivity, carry-over, calibration curve parameters (LLOQ, range, and linearity), within- and between-run accuracy and precision, dilution integrity, matrix effect and stability. For proof-of-concept, the SFC method was applied to clinical samples of human urine obtained after single intravenous (100 mg), single oral (100 mg), and repeated oral administration (400 mg). Flupirtine, D-13223, and 4-FHA could be quantified, shedding light on the extent of oxidative flupirtine metabolism in humans in the context of the unresolved biotoxification that has led to the withdrawal of specific neuronal KV7 openers.

On-line solid-phase supercritical fluid extraction (SFE) and chromatography (SFC) coupled to mass spectrometry (MS) has been evaluated for its usefulness with respect to metabolic profiling and pharmacological investigations of ketamine in humans. The aim of this study was to develop and validate a rapid, highly selective and sensitive SFE–SFC–MS method for the quantification of ketamine and its metabolites in miniature amounts in human urine excluding liquid-liquid extraction (LLE). Several conditions were optimized systematically following the requirements of the European Medicines Agency: selectivity, carry-over, calibration curve parameters (LLOQ, range and linearity), within- and between-run accuracy and precision, dilution integrity, matrix effect, and stability. The method, which required a relatively small volume of human urine (20 μL per sample), was validated for pharmacologically and toxicologically relevant concentrations ranging from 25.0 to 1000 ng/mL (r2 > 0.995). The lower limit of quantification (LLOQ) for all compounds was found to be as low as 0.5 ng. In addition, stability of analytes during removal of water from the urine samples using different conditions (filter paper or ISOLUTE® HM-N) was studied. In conclusion, the method developed in this study can be successfully applied to studies of ketamine metabolites in humans, and may pave the way for routine application of on-line SFE–SFC–MS in clinical investigations.

As to the alignment of "Horizon 2020", ir is a more integrated approach to European science policy than expressed in the proposals previously drafted, and specifically considers: (i) promoting excellence in Science, (ii) establishing a sound industrial leadership and (iii) expressing an ambition to address current and future societal challenges. In this respect, the quest for a knowledge-based economy in Europe should result in proposals for industrial and employment policies that will consolidate the major European advantages in the biomedical, healthcare and pharmaceutical sectors. Horizon 2020 also provides the possibility of adopting a more flexible and simplified management route to drive European research through innovation, research and development. What should be additionally considered? Unmet medical needs, under pressure from demographic changes, await the generation of new medicines and health technologies which will evolve into a driver for a unified European policy. We believe that this should be focused on harnessing pharmaceutical knowledge for clinical use, as part of a response to accommodate patient needs and economic growth based on a robust, scientific approach. The bolder ambition for European research is to unlock key bottlenecks currently undermining European competitiveness. The historical lack of an appropriate business/innovation environment with reduced access to adequate risk finance instruments has severed the path for economic growth and industrial development. These issues are of critical importance and a solution is urgently needed to foster translation from the university to the healthcare sector through the generation and support of start-ups, spin-offs, university-industry consortia, and other platforms, which support translational research. The ultimate goal is implementation of holistic programmes: the 'bench to bedside' paradigm of medicines and other healthcare products. The European Research Council supports the basic biomedical research programmes of long term importance for development of medicines; however, fundamental research initiatives on medicines development will not be competitive in such an environment. In order to strengthen the long term outlook, we must foster innovative research within the university sector, EUFEPS proposes that a fund for such research be set up within Horizon 2020, which would be open for individual research groups and which would include Public-Public Partnerships (complementing already existing Public-Private Partnerships). How do we look for implementation? There is an established research agenda for medicines research that is globally focused, and which incorporates a cooperative model between universities and industry, facilitating integration of complex technologies. Regulatory Science will play an important role in this integration. This agenda uses tools arising from systems approaches (including discovery with systems biology and also systems pharmacology) and has the potential for providing better knowledge management, as well as technological innovation (including manufacturing). It also addresses the drive towards personalised medicines and can, with support from both public and private sectors, foster translation of knowledge to new technologies and from that, to new medicinal products and complex integrated systems. This is a part of a strategy capable of solving unmet medical needs, which would increase the quality of life of patients suffering from chronic and debilitating diseases. The instruments to allow the development of a research agenda should strengthen existing partnerships such as the IMI-JU model; allow for the creation of European-network infrastructures that can bring together existing competences with adequate European coordination, thus promoting advanced training and continuous professional development for the pharmaceutical sciences. This will be the cornerstone of a knowledge management strategy, providing education and training for healthcare professionals and scientists. A key role for EUFEPS is to help the research community to embrace these new holistic policies applied to the spectrum of pharmaceutical, medical and cognate sciences.

(2R,3S,4R,5R)-5-(6-Amino-9H-purin-9-yl)-3-hydroxy-4-[2-(methylamino)benzamido]tetrahydrofuran-2-yl-methoxy[(hydroxy)phosphoryloxy][(hydroxy)phosphoryl]dichloromethylphosphonic acid was synthesized as a chemically and metabolically stable analog of ATP substituted with a fluorescent methylanthranoyl (MANT) residue. The compound is intended for studying the binding site and function of adenylyl cyclases (ACs), which was exemplified by studying its interaction with Bacillus anthracis edema factor (EF) AC exotoxin.

The involvement of purine salvage in the accumulation of current trypanocidal drugs is important for the treatment of African sleeping sickness. The substrate specificity of essential nucleoside transporters is therefore of physiological and pharmacological interest. With the intention to contribute to the knowledge in the field, a series of 16 adenosine derivatives with substituents in N(6)-position were prepared in order to evaluate their potential to inhibit Trypanosoma brucei spp. in vitro. An unmodified ribose moiety was selected to conserve key molecular recognition motifs of the arsenal of integral membrane proteins expressed in large numbers on the protozoan plasma membrane. Two of the new compounds prepared using a polymer-assisted acylation protocol showed antitrypanosomal activities in the single digit micromolar concentration range.

Given the more or less global spread of multidrug-resistant plasmodia, structurally diverse starting points for the development of chemotherapeutic agents for the treatment of malaria are urgently needed. Thus, a series of 20 adenosine derivatives with a large lipophilic substituent in N 6 -position were prepared in order to evaluate their potential to inhibit the chloroquine resistant Plasmodium falciparum strain K1 in vitro. The rationale for synthesis of these structures was the high probability of interactions with multiple adenosine associated targets and the assumption that a large hydrophobic N 6 -(4-phenoxy)benzyl substitution should allow the molecules to diffuse across parasite membranes. Starting from readily available inosine, the new compounds were prepared as single isomers using a polymer-assisted acylation protocol enabling the straightforward isolation of the target compounds in pure form. Heterocyclic ring systems were synthesized on-bead on Kenner’s safety-catch linker prior to acylation of the scaffold in solution. Most of the highly pure compounds displayed anti-plasmodial activity in the low micromolar or even submicromolar concentration range.

The parallel acylation of N-{3-[4-(3-aminopropyl)piperazin-1-yl]propyl}-7-chloroquinolin-4-amine with polymer-bound carboxylic acids opened straightforward access to novel aminoquinolines with activity against Plasmodium falciparum strains in vitro. Using this amino scaffold prepared in solution and polymer-bound carboxylic, we have synthesized a series of 29 new compounds in good to excellent yield and purity. Biological evaluation included determination of the activity against a chloroquine (CQ) sensitive strain and a CQ resistant mutant. Most of the novel structures presented here displayed activity against both strains in the lower nanomolar range, four compounds showed an at least fourfold increase in the ratio of inhibition of CQ resistant to sensitive strains over CQ itself. These results suggest that this derivatization technique is a useful method to speed up structure-activity relationship studies on aminoquinolines toward improved activity versus CQ resistant strains of P. falciparum in vitro.

We have applied a fast and high-yielding method for the parallel amidation of 4-[4-(2-methoxyphenyl)piperazin-1-yl]-butylamine yielding analogs of the partial dopamine receptor agonist BP 897. Using this amino scaffold prepared in solution and polymer-bound carboxylic acid equivalents, we have synthesized a series of high affinity dopamine D(3) receptor ligands. The novel compounds were obtained in good to excellent yield and purity. Biological evaluation included determination of binding affinities at hD(2S) and hD(3) receptor subtypes. From the 22 novel structures presented here, compound 4v showed high affinity (K(i) (hD(3)) 1.6nM) and a 136-fold preference for the D(3) receptor versus that for the D(2) receptor subtype. Our results suggest that this derivatization technique is a useful method to speed up structure-activity relationships studies on dopamine receptor subtype modulators.

In 1971 Kenner et al. introduced the safety-catch principle into solid phase peptide synthesis. Thus two contradicting needs were addressed. On the one hand, sufficient stability of the linker substrate bond to impede hydrolysis or similar side reactions, on the other hand mild chemical conditions allowing for unscathed liberation of the precious products. Over the years this linker type emerged in several different chemical disciplines and nowadays it presents a useful and broadly applicable tool. Recent advancements and applications based on Kenner's safety-catch linker are reviewed.

We have developed a method for the chemoselective introduction of spacer modified biotin labels into unprotected multi-functional amines. A range of novel biotin spacer conjugates attached to a polymer-bound sulfonamide anchor was prepared using established amide bond forming procedures. After chemical transformation of the attachment site by alkylation, the resulting reactive species were utilized as N-selective polymer-supported biotinylation reagents. The labeled compounds, obtained in good to excellent yield and purity, are free of residual biotin and possess a custom tailored distance from the immobilization site being especially suited for the immobilization on streptavidin-functionalized dextran layers of surface plasmon resonance detector chips. In addition, derivatives displaying a phenyl group were synthesized in order to demonstrate the versatility of the procedure for the simultaneous introduction of spacer-modified biotin and a UV-light absorbing moiety. The formation of biotin sulfoxides in the presence of in situ generated peroxides was investigated and is discussed. Our results suggest that this derivatization technique is a useful addition to the existing biotin labeling protocols.

The aminoacyl-tRNA synthetases link tRNAs with their cognate amino acid. In some cases, their fidelity relies on hydrolytic editing that destroys incorrectly activated amino acids or mischarged tRNAs. We present structures of leucyl-tRNA synthetase complexed with analogs of the distinct pre- and posttransfer editing substrates. The editing active site binds the two different substrates using a single amino acid discriminatory pocket while preserving the same mode of adenine recognition. This suggests a similar mechanism of hydrolysis for both editing substrates that depends on a key, completely conserved aspartic acid, which interacts with the alpha-amino group of the noncognate amino acid and positions both substrates for hydrolysis. Our results demonstrate the economy by which a single active site accommodates two distinct substrates in a proofreading process critical to the fidelity of protein synthesis.

We have investigated the in vitro antimalarial activity of a new series of adenosine derivatives. The results show that N6-(1-naphthylmethyl)-5‘-deoxy-5‘-(amido)adenosines as well as N6-(4-phenylbenzyl)-5‘-deoxy-5‘-(amido)adenosines display significant activity against the malaria-causing parasites, with the sterically demanding bisubstituted species reported being active in most cases in the low-micromolar range. The novel compounds with unusual substitution pattern were obtained applying an efficient convergent polymer-assisted solution-phase (cPASP) synthesis protocol. Thus, we were able to prepare a series of substituted derivatives in parallel that would have been difficult to synthesize by standard techniques. The scope and limitations of the synthetic methodology are discussed.

In a joint effort with various laboratories we have been aiming at the structure-based design of glycolysis inhibitors as anti-trypanosomal drugs. 2′-Deoxy-2′-(3-methoxybenzamido)- N 6 -(1-naphtylmethyl)adenosine ( 1a ) was thus revealed as a promising lead structure for the development of selective agents against protozoan parasites. Here we describe the polymer-assisted synthesis of novel amido derivatives of the scaffold 2′-amino-2′-deoxy- N 6 -(1-naphtylmethyl)adenosine ( 5a ) we reported recently. This building block synthesized in solution was treated with an excess of polymer-supported carboxylic acids leading to chemoselective, practically quantitative conversion of the amine to the desired analogous amides. The best compound ( 1h ) from this series was obtained after on-bead nucleophilic substitution of the carboxylic acid equivalent attached to the Kenner safety catch linker and exhibited an improved inhibitory effect on T. b. brucei blood stream forms with an IC 50 of 0.85 μM in vitro

An efficient and simple polymer-assisted approach has been developed to biotinylate multifunctional compounds bearing an amino functionality. Biotin was immobilized on aminomethylated polystyrene via the Kenner safety catch linker, activated and subsequently transferred to the amino function of the target compounds chemoselectively, thus avoiding protecting group operations. This approach holds potential for the introduction of spacer-modified biotin derivatives.

The synthesis of a novel scaffold for the simple and high-yielding polymer-assisted solution phase preparation of arrays of 2′-amido-2′-deoxy-N6-(1-naphthylmethyl)adenosine derivatives 10a–h as analogues of a known inhibitor of trypanosomatid glycosomal glyceraldehyde-3-phosphate dehydrogenase is described.

In a pilot study, alterations of polymorphonuclear neutrophil function during systemic thrombolysis in acute myocardial infarction have been investigated in humans. The following parameters of neutrophil function were measured before and at 15 and 45 minutes after initiation of systemic thrombolysis with a recombinant tissue-type plasminogen activator in 20 patients with acute myocardial infarction: (1) neutrophil adhesion and (2) neutrophil activation. During systemic thrombolysis a significant decrease was observed in neutrophil adhesion (5.5+/-6.4 to 3.2+/-3.3; p0.05), in phagocyting neutrophil activation (39+/-18 to 25+/-14%; p0.05), and in resting neutrophil activation (9+/-7 to 3+/-4%; p0.05). Successful reperfusion coincided with a significantly higher reduction of phagocyting neutrophil activation (40+/-14 to 20+/-12% vs. 39+/-24 to 26+/-19% in unsuccessful reperfusion; p0.05), and of neutrophil adhesion (6.2+/-5.7 to 2.7+/-3.0 vs. 4.1+/-3.8 to 3.5+/-4.0 in unsuccessful reperfusion; p0.05) during thrombolysis. Systemic thrombolysis in acute myocardial infarction is accompanied by a reduction in neutrophil adhesion and activation dependent on thrombolytic success.

A new synthetic strategy for the simple and high yielding preparation of arrays of 2′-amido-2′-deoxyadenosine derivatives ready for biological testing without the need for chromatographic purification is described. Acids are coupled to the Kenner or Ellman safety catch linker, respectively, activated by cyanomethylation and subsequently transferred to the 2′-amino group of 2′-amino-2′-deoxyadenosine.