Search

Resume Introduction L’automatisation de la dispensation individuelle nominative induit des changements de pratique sur l’ensemble du circuit du medicament, c’est pourquoi l’integration d’un automate doit etre anticipee. Une analyse preliminaire des risques a ete realisee avant l’automatisation. Neanmoins, des difficultes organisationnelles intrinseques au sein de la pharmacie a usage interieur (PUI) ont surgi suite a l’automatisation. Objectif L’objectif de notre demarche etait de realiser une analyse de processus et des pratiques pour identifier les causes de ces difficultes puis de mettre en place des mesures correctives ad hoc. Materiels et methodes Une cartographie du processus, une ethnographie organisationnelle et une enquete de satisfaction ont ete realisees. Resultats Suite a l’identification des causes, des mesures correctives ont ete mises en place, telles que : la redaction de nouvelles procedures en fonction des besoins, la designation d’un informaticien referent, la reorganisation de l’espace de travail, du materiel et de la gestion du temps. Depuis la mise en place des mesures correctives nos taux de non-conformite releves etaient de 1,14 % apres 199 jours et de 0,97 % apres 462 jours et sont semblables a ceux retrouves dans la litterature. Discussion/conclusion Il semble que la plupart des difficultes rencontrees soient liees a un changement de pratique majeur, lie a la possibilite d’obtenir rapidement des doses unitaires, et aux conditions de l’environnement de travail. Une nouvelle demarche d’amelioration continue sera initiee pour l’etape d’administration des medicaments.

We report the successful implementation of virtual screening in the discovery of new inhibitors of undecaprenyl pyrophosphate synthase (UppS) from Escherichia coli. UppS is an essential enzyme in the biosynthesis of bacterial cell wall. It catalyzes the condensation of farnesyl pyrophosphate (FPP) with eight consecutive isopentenyl pyrophosphate units (IPP), in which new cis-double bonds are formed, to generate undecaprenyl pyrophosphate. The latter serves as a lipid carrier for peptidoglycan synthesis, thus representing an important target in the antibacterial drug design. A pharmacophore model was designed on a known bisphosphonate BPH-629 and used to prepare an enriched compound library that was further docked into UppS conformational ensemble generated by molecular dynamics experiment. The docking resulted in three anthranilic acid derivatives with promising inhibitory activity against UppS. Compound 2 displayed high inhibitory potency (IC50 = 25 µM) and good antibacterial activity against E. coli BW25113 ΔtolC strain (MIC = 0.5 µg/mL).

Antibiotic resistance represents one of the biggest public health challenges in the last few years. Mur ligases (MurC-MurF) are involved in the synthesis of UDP-N-acetylmuramyl-pentapeptide, the main building block of bacterial peptidoglycan polymer. They are essential for the survival of bacteria and therefore important antibacterial targets. We report herein the synthesis and structure-activity relationships of Mur ligases inhibitors with an azastilbene scaffold. Several compounds showed promising inhibitory potencies against multiple ligases and one compound also possessed moderate antibacterial activity. These results represent a solid ground for further development and optimization of structurally novel antimicrobial agents to combat the rising bacterial resistance.

Summary Intranasal naloxone aims at preventing opioid overdose related deaths in active drug users. In France, it has been available since July 2016 through a temporary approval which requires a hospital-based pharmacy and a nominative registration of each patient. We present the characteristics of the first patients who could receive this prescription in our hospital-based addiction center and how they used naloxone during follow-up. Results favor a larger dispensing of naloxone. Patients’ as well as peers’ and families’ education is needed.

High-dose baclofen is prescribed as a maintenance treatment to reduce alcohol use in patients with alcohol use disorder. Nevertheless, some patients still have massive alcohol intakes and require inpatient alcohol withdrawal. To compare the oral dose of benzodiazepine prescribed to manage alcohol withdrawal symptoms in patients with vs. without steady-state pretreatment with high-dose baclofen. Retrospective chart review study. Prescribed benzodiazepine dose expressed in diazepam-equivalent was compared between groups. Thirty-one patients were assessed in the high-dose maintenance baclofen group and compared to 31 matched patients not receiving baclofen. No statistically significant difference was evident between groups regarding levels of benzodiazepines prescribed. The mean diazepam-equivalent dose during the first 7 days was 294 ± 149 mg in the baclofen group vs. 310 ± 133 mg (t-test = 0.440, P = 0.661) in matched controls. Steady-state high-dose baclofen before an inpatient alcohol cessation hospitalization does not lower the needed benzodiazepine dose in the management of alcohol withdrawal symptoms.

Colicin M is an enzymatic bacteriocin produced by some Escherichia coli strains which provokes cell lysis of competitor strains by hydrolysis of the cell wall peptidoglycan undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) precursor. The overexpression of a gene, cbrA (formerly yidS), was shown to protect E. coli cells from the deleterious effects of this colicin, but the underlying resistance mechanism was not established. We report here that a major structural modification of the undecaprenyl-phosphate carrier lipid and of its derivatives occurred in membranes of CbrA-overexpressing cells, which explains the acquisition of resistance toward this bacteriocin. Indeed, a main fraction of these lipids, including the lipid II peptidoglycan precursor, now displayed a saturated isoprene unit at the α-position, i.e., the unit closest to the colicin M cleavage site. Only unsaturated forms of these lipids were normally detectable in wild-type cells. In vitro and in vivo assays showed that colicin M did not hydrolyze α-saturated lipid II, clearly identifying this substrate modification as the resistance mechanism. These saturated forms of undecaprenyl-phosphate and lipid II remained substrates of the different enzymes participating in peptidoglycan biosynthesis and carrier lipid recycling, allowing this colicin M-resistance mechanism to occur without affecting this essential pathway. IMPORTANCE Overexpression of the chromosomal cbrA gene allows E. coli to resist colicin M (ColM), a bacteriocin specifically hydrolyzing the undecaprenyl-PP-MurNAc(-pentapeptide)-GlcNAc (lipid II) peptidoglycan precursor of targeted cells. This resistance results from a CbrA-dependent modification of the precursor structure, i.e., reduction of the α-isoprenyl bond of C55-carrier lipid moiety that is proximal to ColM cleavage site. This modification, observed here for the first time in eubacteria, annihilates the ColM activity without affecting peptidoglycan biogenesis. These data, which further increase our knowledge of the substrate specificity of this colicin, highlight the capability of E. coli to generate reduced forms of C55-carrier lipid and its derivatives. Whether the function of this modification is only relevant with respect to ColM resistance is now questioned.

Certain Pseudomonas aeruginosa strains produce a homolog of colicin M, namely, PaeM, that specifically inhibits peptidoglycan biosynthesis of susceptible P. aeruginosa strains by hydrolyzing the lipid II intermediate precursor. Two variants of this pyocin were identified whose sequences mainly differed in the N-terminal protein moiety, i.e., the region involved in the binding to the FiuA outer membrane receptor and translocation into the periplasm. The antibacterial activity of these two variants, PaeM1 and PaeM2, was tested against various P. aeruginosa strains comprising reference strains PAO1 and PA14, PaeM-producing strains, and 60 clinical isolates. Seven of these strains, including PAO1, were susceptible to only one variant (2 to PaeM1 and 5 to PaeM2), and 11 were affected by both. The remaining strains, including PA14 and four PaeM1 producers, were resistant to both variants. The differences in the antibacterial spectra of the two PaeM homologs prompted us to investigate the molecular determinants allowing their internalization into P. aeruginosa cells, taking the PAO1 strain that is susceptible to PaeM2 but resistant to PaeM1 as the indicator strain. Heterologous expression of fiuA gene orthologs from different strains into PAO1, site-directed mutagenesis experiments, and construction of PaeM chimeric proteins provided evidence that the cell susceptibility and discrimination differences between the PaeM variants resulted from a polymorphism of both the pyocin and the outer membrane receptor FiuA. Moreover, we found that a third component, TonB1, a protein involved in iron transport in P. aeruginosa, working together with FiuA and the ExbB/ExbD complex, was directly implicated in this discrimination. IMPORTANCE Bacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains of P. aeruginosa that inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility of P. aeruginosa clinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.

International audience; A halophilic organism, SWO25T, was isolated from water sampled in Algeria at the salt lake (sebkha) of Ouargla. The novel strain stained Gram-negative, and cells were pleomorphic with a red pigmentation. Strain SWO25T grew optimally at 35–45 °C, at pH 6.0–8.0 and 0.05–0.25 M MgCl2 concentrations. Cells were extremely halophilic, with optimal growth at 4.3–5.1 M NaCl. The predominant membrane polar lipids were C20C20 glycerol diether derivatives of phosphatidylglycerol, phosphatidylglycerol phosphate, phosphatidylglycerol sulfate, triglycosyl diether and diglycosyl diether. The major respiratory menaquinone component was MK-8. Cells were highly tolerant to the presence of decane and isooctane in the growth medium. Chemotaxonomic properties supported the assignment of strain SWO25T to the genus Haloarcula. The DNA G+C content was 61.1mol%. DNA–DNA hybridization and phylogenetic analyses of the 16S rRNA and rpoB′ genes showed that strain SWO25T is distinct from known Haloarcula species. Based on phenotypic, chemotaxonomic, genotypic and phylogenetic data, we describe a novel species of the genus Haloarcula, for which the name Haloarculasebkhae sp. nov. is proposed. The type strain is SWO25T (=CIP 110583T=JCM 19018T).

International audience; The Mur ligases form a series of consecutive enzymes that participate in the intracellular steps of bacterial peptidoglycan biosynthesis. They therefore represent interesting targets for antibacterial drug discovery. MurC, D, E and F are all ATP-dependent ligases. Accordingly, with the aim being to find multiple inhibitors of these enzymes, we screened a collection of ATP-competitive kinase inhibitors, on Escherichia coli MurC, D and F, and identified five promising scaffolds that inhibited at least two of these ligases. Compounds 1, 2, 4 and 5 are multiple inhibitors of the whole MurC to MurF cascade that act in the micromolar range (IC50, 32-368 mu M). NMR-assisted binding studies and steady-state kinetics studies performed on aza-stilbene derivative 1 showed, surprisingly, that it acts as a competitive inhibitor of MurD activity towards D-glutamic acid, and additionally, that its binding to the D-glutamic acid binding site is independent of the enzyme closure promoted by ATP.

Certain Pseudomonas aeruginosa strains produce a homolog of colicin M, namely, PaeM, that specifically inhibits peptidoglycan biosynthesis of susceptible P. aeruginosa strains by hydrolyzing the lipid II intermediate precursor. Two variants of this pyocin were identified whose sequences mainly differed in the N-terminal protein moiety, i.e., the region involved in the binding to the FiuA outer membrane receptor and translocation into the periplasm. The antibacterial activity of these two variants, PaeM1 and PaeM2, was tested against various P. aeruginosa strains comprising reference strains PAO1 and PA14, PaeM-producing strains, and 60 clinical isolates. Seven of these strains, including PAO1, were susceptible to only one variant (2 to PaeM1 and 5 to PaeM2), and 11 were affected by both. The remaining strains, including PA14 and four PaeM1 producers, were resistant to both variants. The differences in the antibacterial spectra of the two PaeM homologs prompted us to investigate the molecular determinants allowing their internalization into P. aeruginosa cells, taking the PAO1 strain that is susceptible to PaeM2 but resistant to PaeM1 as the indicator strain. Heterologous expression of fiuA gene orthologs from different strains into PAO1, site-directed mutagenesis experiments, and construction of PaeM chimeric proteins provided evidence that the cell susceptibility and discrimination differences between the PaeM variants resulted from a polymorphism of both the pyocin and the outer membrane receptor FiuA. Moreover, we found that a third component, TonB1, a protein involved in iron transport in P. aeruginosa, working together with FiuA and the ExbB/ExbD complex, was directly implicated in this discrimination.IMPORTANCE Bacterial antibiotic resistance constitutes a threat to human health, imposing the need for identification of new targets and development of new strategies to fight multiresistant pathogens. Bacteriocins and other weapons that bacteria have themselves developed to kill competitors are therefore of great interest and a valuable source of inspiration for us. Attention was paid here to two variants of a colicin M homolog (PaeM) produced by certain strains of P. aeruginosa that inhibit the growth of their congeners by blocking cell wall peptidoglycan synthesis. Molecular determinants allowing recognition of these pyocins by the outer membrane receptor FiuA were identified, and a receptor polymorphism affecting the susceptibility of P. aeruginosa clinical strains was highlighted, providing new insights into the potential use of these pyocins as an alternative to antibiotics.

WOS:000450383500008; International audience; Targeted covalent inhibitors have become an integral part of a number of therapeutic protocols and are the subject of intense research. The mechanism of action of these compounds involves the formation of a covalent bond with protein nucleophiles, mostly cysteines. Given the abundance of cysteines in the proteome, the specificity of the covalent inhibitors is of utmost importance and requires careful optimization of the applied warheads. In most of the cysteine targeting covalent inhibitor programs the design strategy involves incorporating Michael acceptors into a ligand that is already known to bind non-covalently. In contrast, we suggest that the reactive warhead itself should be tailored to the reactivity of the specific cysteine being targeted, and we describe a strategy to achieve this goal. Here, we have extended and systematically explored the available organic chemistry toolbox and characterized a large number of warheads representing different chemistries. We demonstrate that in addition to the common Michael addition, there are other nucleophilic addition, addition-elimination, nucleophilic substitution and oxidation reactions suitable for specific covalent protein modification. Importantly, we reveal that warheads for these chemistries impact the reactivity and specificity of covalent fragments at both protein and proteome levels. By integrating surrogate reactivity and selectivity models and subsequent protein assays, we define a road map to help enable new or largely unexplored covalent chemistries for the optimization of cysteine targeting inhibitors. (C) 2018 Elsevier Masson SAS. All rights reserved.

Intranasal naloxone aims at preventing opioid overdose related deaths in active drug users. In France, it has been available since July 2016 through a temporary approval which requires a hospital-based pharmacy and a nominative registration of each patient. We present the characteristics of the first patients who could receive this prescription in our hospital-based addiction center and how they used naloxone during follow-up. Results favor a larger dispensing of naloxone. Patients' as well as peers' and families' education is needed.

In the context of antibacterial drug discovery resurgence, novel therapeutic targets and new compounds with alternative mechanisms of action are of paramount importance. We focused on UDP-N- acetylenolpyruvylglucosamine reductase (i.e. MurB), an underexploited target enzyme that is involved in early steps of bacterial peptidoglycan biosynthesis. On the basis of the recently reported crystal structure of MurB in complex with NADP+ , a pharmacopohore model was generated and used in a virtual screening campaign with combined structure-based and ligand-based approaches. In order to explore chemical space around hit compounds, further similarity search and organic synthesis was employed to obtain several compounds with micromolar IC50 values on MurB. The best inhibitors in the reported series of 5-substituted tetrazol-2-yl acetamides were compounds 13, 26 and 30 with IC50 values of 34, 28 and 25 µM, respectively. None of the reported compounds possessed in vitro antimicrobial activity against S. aureus and E. coli.

Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family—MurC, MurD, MurE and MurF—are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park’s nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC–MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC–MurF enzymes in biochemical inhibition assays and molecules 10–14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC–MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d -Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents.

During the biogenesis of bacterial cell-wall polysaccharides, such as peptidoglycan, cytoplasmic synthesized precursors should be trafficked across the plasma membrane. This essential process requires a dedicated lipid, undecaprenyl-phosphate that is used as a glycan lipid carrier. The sugar is linked to the lipid carrier at the inner face of the membrane and is translocated toward the periplasm, where the glycan moiety is transferred to the growing polymer. Undecaprenyl-phosphate originates from the dephosphorylation of its precursor undecaprenyl-diphosphate, with itself generated by de novo synthesis or by recycling after the final glycan transfer. Undecaprenyl-diphosphate is de novo synthesized by the cytosolic cis-prenyltransferase undecaprenyl-diphosphate synthase, which has been structurally and mechanistically characterized in great detail highlighting the condensation process. In contrast, the next step toward the formation of the lipid carrier, the dephosphorylation step, which has been overlooked for many years, has only started revealing surprising features. In contrast to the previous step, two unrelated families of integral membrane proteins exhibit undecaprenyl-diphosphate phosphatase activity: BacA and members of the phosphatidic acid phosphatase type 2 super-family, raising the question of the significance of this multiplicity. Moreover, these enzymes establish an unexpected link between the synthesis of bacterial cell-wall polymers and other biological processes. In the present review, the current knowledge in the field of the bacterial lipid carrier, its mechanism of action, biogenesis, recycling, regulation, and future perspective works are presented.

MurF ligase is a crucial enzyme that catalyses the ultimate intracellular step of bacterial peptidoglycan biosynthesis, and thus represents an attractive target for antibacterial drug discovery. We designed, synthesized and evaluated a new series of cyanothiophene-based inhibitors of MurF enzymes from Streptococcus pneumoniae and Escherichia coli. The target compounds had increased polarity compared to the first generation of inhibitors, with demonstrated enzyme inhibitory potencies in the low micromolar range. Furthermore, the best inhibitors displayed promising antibacterial activities against selected Gram-positive and Gram-negative strains. These results represent an important step towards the development of new antibacterial agents targeting peptidoglycan biosynthesis.

We present a modern in silico virtual screening workflow towards novel MurB inhibitors. Namely, the field of antibacterial research is in crucial need of novel lead compounds on new or underexplored therapeutic targets such as MurB. We identified three structurally distinct compounds that indicated promising micromolar inhibitory activity on E. coli MurB. All three compounds did not display cytotoxicity on HepG2 cell line, but also did not possess in vitro antimicrobial activity against S. aureus and E. coli. After the second biological evaluation using radio-labelled substrates in a MurA-MurB coupled test, only marginal inhibitory potency could be reproduced on MurB. As no compound in the series was able to significantly decrease the residual activity of the enzyme, we decided to report the complete inactive compound set to be used as decoys.

Introduction La Haute Autorite de Sante promeut la reevaluation de l’antibiotherapie a 72 h (indicateur l’ICATB2, item A11d). Nous avons souhaite connaitre la faisabilite du suivi de cet item, avec un focus sur les cephalosporines de 3e (C3G) et 4e (C4G) generation, qui figurent dans la liste des antibiotiques (ATB) critiques de l’ANSM. Materiels et methode Les identites de tous les patients avec prescription de C3G/C4G ont ete recherchees dans le logiciel d’aide a la prescription medicamenteuse (LAP) (Genois/Phedra®, deploye sur 482 lits de MCO soit 84 % des lits de l‘hopital ; exclus : reanimations, USIC, urgences) sur 14 jours (avril 2018). Deux sources ont ensuite ete auditees : (1) dossier informatise (Middlecare®) : compte-rendus d’hospitalisation (CRH) et avis specialises formalises (contenu saisi en texte libre) ; (2) champ dedie « reevaluation », a saisie facultative, dans le LAP. – Audit « donnees CRH » : les CRH et avis specialises ont ete analyses manuellement a l’aide d’une grille a 13 items. La prescription etait consideree comme reevaluee si le CRH citait explicitement l’ATB, ET mentionnait une poursuite ou une modification. – Audit « donnees LAP » : une requete Business Object® a extrait les donnees du LAP. La prescription etait consideree comme reevaluee a 72 h si le champ « Reevaluation » avait ete renseigne par le medecin OU si l’ATB avait ete arrete dans les 72 h suivant son initiation. Resultats et discussion Soixante-dix-huit dossiers patients ont ete analyses. (1) Audit « CRH » : sur 78 dossiers, la cephalosporine prescrite est mentionnee dans 68 cas (87 %) (ceftriaxone, cefotaxime, ceftazidime, cefepime), la date d’initiation dans 60 cas (77 %), et le site infecte dans 76 cas (98 %). Dans 4 % des dossiers (3/78), la tracabilite de la reevaluation a ete, apres analyse, consideree comme non requise (C3G/C4G en derniere ligne, choix argumente et pertinent). Pour les 75 autres CRH, la reevaluation etait tracee dans 79 % des cas (59/75) : 37 % des patients dans les 72 h, 24 % de reevaluations plus tardives et 39 % non datees. (2) Audit « LAP » : 22 % des prescriptions ont ete considerees comme reevaluees a 72 h (arret de l’ATB). En revanche, le champ « reevaluation » du LAP n’a jamais ete renseigne. Conclusion Un audit de 2 semaines cible sur les C3G/C4G montre que dans un hopital avec dossier patient informatise, la tracabilite de la reevaluation de l’antibiotherapie dans les CRH est possible (retrouvee dans 8 CRH sur 10). Cependant, seul un suivi exhaustif (tous les CRH, tous les antibiotiques sensibles, en continu) permettrait de suivre de facon quantitative l’amelioration des pratiques. Un tel suivi est inenvisageable manuellement. Une evolution rapide des outils informatiques, notamment un champ dedie obligatoire dans les LAP, semble la solution la plus simple a mettre en œuvre. Ces difficultes seront communiquees aux tutelles a l’origine de la redaction de l’indicateur A11d pour le faire evoluer.

Introduction La melatonine est une hormone impliquee dans les rythmes circadiens. Une specialite a base de Melatonine a liberation prolongee (MLP) 2 mg est disponible pour les troubles du sommeil chez les plus de 55 ans. L’objectif etait de realiser un etat des lieux des prescriptions de MLP en SSR geriatrie. Materiels et methode Etude retrospective. Pour les patients ayant beneficies de MLP du 10/02/2017 au 10/08/2018, les informations suivantes ont ete recueillies dans le logiciel prescription et le dossier medical informatise : âge, sexe, taille, poids, IMC, dose journaliere et duree de traitement, prise de benzodiazepine (BZD) a l’entree et a la sortie d’hospitalisation, comorbidites (demence, AVC). Les poursuites du traitement etaient objectivees : en hospitalisation, par la conciliation de sortie realisee systematiquement, et en ambulatoire, par la pharmacie d’officine. Resultats et discussion Sur 18 mois, 31 patients (13 femmes, 18 hommes) d’âge moyen 85,73 ans ont beneficie de MLP. La posologie et la duree moyenne etait de 2,38 mg (min = 2 mg, max = 6 mg) pour 45,22 jours. Aucune influence significative de l’âge, du nombre et du type de comorbidites, de l’IMC, du sexe n’a ete trouvee sur la posologie prescrite. Les comorbidites les plus representees etaient la demence (42 %) et les antecedents d’AVC (34 %). Pour 39 % (n = 12), 32 % (n = 10), 29 % (n = 9) des patients la MLP a ete : arretee la veille de la sortie, au cours du sejour et poursuivi au moment de la sortie. Sur les 9 patients sortis avec de la MLP, 22 % (n = 2) ont recupere de maniere reguliere leur traitement a la pharmacie et 33 % (n = 3) uniquement le premier mois. Soixante-et-onze pourcents (n = 22) des patients avaient une BZD a l’entree et 42 % a la sortie (n = 13). Soixante-six pourcents (n = 6) des patients beneficiaient a la sortie d’une co-prescription de MLP avec une BZD. Dans la litterature les posologies efficaces varient en fonction des comorbidites. Il est retrouve une posologie de 2 mg de MLP pendant 4 semaines pour les patients presentant une demence et de 2 mg de MLP pendant 24 semaines pour les patients aux antecedents d’AVC. La MLP est frequemment arretee la veille de la sortie d’hospitalisation, ainsi qu’au cours du sejour suite a une reponse partielle ou une inefficacite. De plus seul 22 % des patients poursuivent leur traitement par MLP en ville. Cela peut s’expliquer par le cout (environ 30€ non rembourses pour 30 comprimes) mais aussi par un mesusage. En effet la MLP a une action chronobiotique sur le rythme circadien en plus d’une action soporifique. L’action chronobiotique peut provoquer, lors d’une prise trop precoce ou tardive par rapport a l’heure habituelle de coucher, un retard ou une avance de phase du sommeil. Le patient ou le clinicien peuvent alors juger a tort d’une inefficacite ou d’une reponse partielle de la MLP motivant alors l’introduction concomitante de BZD. Conclusion La MLP pourrait presenter un interet pour les personnes âgees atteintes d’insomnie associe a un antecedent d’AVC ou demence. Neanmoins, aucune recommandation de bon usage n’existe encore. Pour le moment, en raison de son cout et de ses modalites d’utilisation, la MLP semble difficile a utiliser correctement.

The synthesis of the peptide stem of bacterial peptidoglycan involves four enzymes, the Mur ligases (MurC, D, E and F). Among them, MurD is responsible for the ATP-dependent addition of d-glutamic acid to UDP-MurNAc-l-Ala, a reaction which involves acyl-phosphate and tetrahedral intermediates. Like most enzymes of peptidoglycan biosynthesis, MurD constitutes an attractive target for the design and synthesis of new antibacterial agents. Escherichia coli MurD has been the first Mur ligase for which the tridimensional (3D) structure was solved. Thereafter, several co-crystal structures with different ligands or inhibitors were released. In the present review, we will deal with work performed on substrate specificity, reaction mechanism and 3D structure of E. coli MurD. Then, a part of the review will be devoted to recent work on MurD orthologs from species other than E. coli and to cellular organization of Mur ligases and in vivo regulation of the MurD activity. Finally, we will review the different classes of MurD inhibitors that have been designed and assayed to date with the hope of obtaining new antibacterial compounds.

Background: MurE controls stereochemical incorporation of lysine or diaminopimelate into peptidoglycan stem peptides. Results: The structure of S. aureus MurE reveals an unexpected lack of specificity for lysine within the active site. Conclusion: Incorporation of lysine is supported by the comparatively high concentration of cytoplasmic lysine, not enzyme specificity. Significance: This study provides new perspectives in targeting Gram-positive peptidoglycan assembly for antimicrobial discovery., Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

International audience; Peptidoglycan is an essential component of the bacterial cell wall, and enzymes involved in its biosynthesis represent validated targets for antibacterial drug discovery. MurF catalyzes the final intracellular peptidoglycan biosynthesis step: the addition of D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc-L-Ala-γ-D-Glu-meso-DAP (or L-Lys). As MurF has no human counterpart, it represents an attractive target for the development of new antibacterial drugs. Using recently published cyanothiophene inhibitors of MurF from Streptococcus pneumoniae as a starting point, we designed and synthesized a series of structurally related derivatives and investigated their inhibition of MurF enzymes from different bacterial species. Systematic structural modifications of the parent compounds resulted in a series of nanomolar inhibitors of MurF from S. pneumoniae and micromolar inhibitors of MurF from Escherichia coli and Staphylococcus aureus. Some of the inhibitors also show antibacterial activity against S. pneumoniae R6. These findings, together with two new co-crystal structures, represent an excellent starting point for further optimization toward effective novel antibacterials.

To improve the appropriate use of long-acting benzodiazepine (la.bzd) prescriptions in the elderly, the Haute Autorite de Sante (HAS) has developed clinical practice indicators (CPI). The alert indicator (AI) evaluates the prevalence of la.bzd prescription among older people. The mastering indicator (MI) corresponds to the prevalence of elderly with a justified, i.e., appropriate, la.bzd prescription among all the elderly with la.bzd prescriptions. The objective of this study was to test the feasibility of routine generation of CPI regarding la.bzd prescriptions among the elderly in the hospital setting. This was a retrospective study. The study was conducted in two university hospitals located in Paris. Eligible cases were patients aged 65 years and older who were hospitalized in acute care units from January to June 2014. The AI calculation was based on information extracted from medical databases from these hospitals. The appropriateness of la.bzd prescription was assessed by a physician and a pharmacist and was based on review of computerized patient records and prescriptions, using an ad hoc algorithm. The MI was then calculated. Variation in the level of indicators was explored according to the characteristics of patients and of their hospitalization using chi2 test. Factors associated with a potentially inappropriate prescription (PIP) of la.bzd were studied using univariate and multivariate logistic regression. Among the 4167 patients included in the study, 362 had la.bzd prescriptions, i.e., the AI was 9%. Prescriptions were found to be appropriate for 83 patients, i.e., the MI was 23% and PIP was 77%. The MI varied between 13 and 31% according to characteristics of patients and of hospitalization. In multivariate analysis, factors associated with PIP were age, number of comorbidities, type of care unit, and concurrent prescription of a neuroleptic or hypnotic. Generation of the AI was routinely possible but only for acute care units with computerized prescriptions, corresponding to 78% of patients. Production of the MI has required medical record review for all patients with a la.bzd prescription and cannot be automated. However, difficulties in generating the MI have identified areas for significant improvement. Moreover, strategies to improve the care of older people with a la.bzd prescription could be targeted using characteristics of patients and of hospitalization associated with PIP. The future deployment of a single electronic medical record in all care departments would make it easier to mine the data and make possible automated production of CPI.

We report on the successful application of ProBiS-CHARMMing web server in the discovery of new inhibitors of MurA, an enzyme that catalyzes the first committed cytoplasmic step of bacterial peptidoglycan synthesis. The available crystal structures of Escherichia coli MurA in the Protein Data Bank have binding sites whose small volume does not permit the docking of drug-like molecules. To prepare the binding site for docking, the ProBiS-CHARMMing web server was used to simulate the induced-fit effect upon ligand binding to MurA, resulting in a larger, more holo-like binding site. The docking of a filtered ZINC compound library to this enlarged binding site was then performed and resulted in three compounds with promising inhibitory potencies against MurA. Compound 1 displayed significant inhibitory potency with IC50 value of 1μM. All three compounds have novel chemical structures, which could be used for further optimization of small-molecule MurA inhibitors.

International audience; Colicins are bacterial toxins produced by some Escherichia coli strains. They exhibit either enzymatic or pore-forming activity towards a very limited number of bacterial species, due to the high specificity of their reception and translocation systems. Yet, we succeeded in making the colicin M homologue from Pectobacterium carotovorum, pectocin M1 (PcaM1), capable of inhibiting E. coli cell growth by bypassing these reception and translocation steps. This goal was achieved through periplasmic expression of this pectocin. Indeed, when appropriately addressed to the periplasm of E. coli, this pectocin could exert its deleterious effects, i.e., the enzymatic degradation of the peptidoglycan lipid II precursor, which resulted in the arrest of the biosynthesis of this essential cell wall polymer, dramatic morphological changes and, ultimately, cell lysis. This result leads to the conclusion that colicin M and its various orthologues constitute powerful antibacterial molecules able to kill any kind of bacterium, once they can reach their lipid II target. They thus have to be seriously considered as promising alternatives to antibiotics.

International audience; Erysipelothrix rhusiopathiae is a Gram-positive bacterium pathogenic to many species of birds and mammals, including humans. The main feature of its peptidoglycan is the presence of l-alanine at position 3 of the peptide stem. In the present work, we cloned the murE gene from E. rhusiopathiae and purified the corresponding protein as His6-tagged form. Enzymatic assays showed that E. rhusiopathiae MurE was indeed an l-alanine-adding enzyme. Surprisingly, it was also able, although to a lesser extent, to add meso-diaminopimelic acid, the amino acid found at position 3 in many Gram-negative bacteria, Bacilli and Mycobacteria. Sequence alignment of MurE enzymes from E. rhusiopathiae and Escherichia coli revealed that the DNPR motif that is characteristic of meso-diaminopimelate-adding enzymes was replaced by HDNR. The role of the latter motif in the interaction with l-alanine and meso-diaminopimelic acid was demonstrated by site-directed mutagenesis experiments and the construction of a homology model. The overexpression of the E. rhusiopathiae murE gene in E. coli resulted in the incorporation of l-alanine at position 3 of the peptide part of peptidoglycan.

The cluster of genes of capsular K5 heparosan is composed of three regions, involved in the synthesis and the exportation of the polysaccharide. The region 2 possesses all the necessary genes involved in the synthesis of heparosan, namely kfiA, encoding alpha-4-N-acetylglucosaminyltransferase, kfiD, encoding β-3-glucuronyl transferase, kfiC, encoding UDP-glucose dehydrogenase (UDP-glucuronic acid synthesis), and kfiB encoding a protein of unknown function. The cloning and expression of kfiADCB into Escherichia coli K-12 were found to be sufficient for the production of heparosan, which accumulates in the cells due to a lack of the exporting system. The concentration of recombinant heparosan reached one gram per liter under fed-batch cultivation. The cytoplasmic localization of heparosan inside the bacteria allowed subsequent enzymatic modifications such as a partial degradation with K5 lyase when expressed intracellularly. Under these conditions, the production of DP 2-10 oligosaccharides occurred intracellularly, at a concentration similar to that of recombinant intracellular heparosan.

International audience; Colicin M (ColM) is the only enzymatic colicin reported to date that inhibits cell wall peptidoglycan biosynthesis. It catalyzes the specific degradation of the lipid intermediates involved in this pathway, thereby provoking lysis of susceptible Escherichia coli cells. A gene encoding a homologue of ColM was detected within the exoU-containing genomic island A carried by certain pathogenic Pseudomonas aeruginosa strains. This bacteriocin (pyocin) that we have named PaeM was crystallized, and its structure with and without an Mg2+ ion bound was solved. In parallel, site-directed mutagenesis of conserved PaeM residues from the C-terminal domain was performed, confirming their essentiality for the protein activity both in vitro (lipid II-degrading activity) and in vivo (cytotoxicity against a susceptible P. aeruginosa strain). Although PaeM is structurally similar to ColM, the conformation of their active sites differs radically; in PaeM, residues essential for enzymatic activity and cytotoxicity converge toward a same pocket, whereas in ColM they are spread along a particularly elongated active site. We have also isolated a minimal domain corresponding to the C-terminal half of the PaeM protein and exhibiting a 70-fold higher enzymatic activity as compared with the full-length protein. This isolated domain of the PaeM bacteriocin was further shown to kill E. coli cells when addressed to the periplasm of these bacteria.

D-Glutamic acid-adding enzyme (MurD ligase) catalyses the addition of D-glutamic acid to UDP-N-acetylmuramoyl-L-alanine, an essential cytoplasmic step in the pathway for bacterial cell-wall peptidoglycan synthesis. As such, it represents an important antibacterial drug-discovery target enzyme. Recently, several series of compounds have been synthesised and found to inhibit MurD from Escherichia coli, the best one having an IC(50) value of 8 μM. In the present work, we have tested 20 of these compounds against the MurD enzymes from Staphylococcus aureus, Streptococcus pneumoniae, Borrelia burgdorferi and Mycobacterium tuberculosis. Most of the E. coli MurD inhibitors appeared less efficient against the four other orthologues. This divergent result can be explained by the differences in amino acid sequences and topologies of the active sites of the MurD ligases studied.

Undecaprenyl phosphate is the essential lipid involved in the transport of hydrophilic motifs across the bacterial membranes during the synthesis of cell wall polymers such as peptidoglycan. A HPLC procedure was developed for the quantification of undecaprenyl phosphate and its two derivatives, undecaprenyl pyrophosphate and undecaprenol. During the exponential growth phase, the pools of undecaprenyl phosphate and undecaprenyl pyrophosphate were ca. 75 and 270 nmol/g of cell dry weight, respectively, in Escherichia coli, and ca. 50 and 150 nmol/g, respectively, in Staphylococcus aureus. Undecaprenol was detected in S. aureus (70 nmol/g), but not in E. coli (

International audience; The biosynthetic pathway of peptidoglycan, an essential component of bacterial cell wall, is a well-recognized target for antibiotic development. Peptidoglycan precursors are synthesized in the bacterial cytosol by various enzymes including the ATP-hydrolyzing Mur ligases, which catalyze the stepwise addition of amino acids to a UDP-MurNAc precursor to yield UDP-MurNAc-pentapeptide. MurD catalyzes the addition of D-glutamic acid to UDP-MurNAc-L-Ala in the presence of ATP; structural and biochemical studies have suggested the binding of the substrates with an ordered kinetic mechanism in which ligand binding inevitably closes the active site. In this work, we challenge this assumption by reporting the crystal structures of intermediate forms of MurD either in the absence of ligands or in the presence of small molecules. A detailed analysis provides insight into the events that lead to the closure of MurD and reveals that minor structural modifications contribute to major overall conformation alterations. These novel insights will be instrumental in the development of new potential antibiotics designed to target the peptidoglycan biosynthetic pathway.

Introduction La scintigraphie aux leucocytes radiomarques a l’exametazime-99mTc (Ceretec®-99mTc) est utilisee dans le cadre des etudes de foyers infectieux. Un controle de viabilite des cellules radiomarquees a ete mis en place en suivant la « methode manuelle par coloration d’exclusion » de la pharmacopee europeenne. Cet essai necessitant l’observation au microscope d’un echantillon radioactif, la question de la radio-exposition du cristallin s’est posee. Une etude a alors ete realisee en vue d’estimer et d’optimiser l’ensemble des doses recues par l’operateur, en particulier lors de l’etape de numeration sous microscope. Materiels et methode La methodologie de l’etude a ete definie avec une Personne competente en radioprotection. Le controle de viabilite a ete decompose en 4 etapes depuis le recueil de la solution mere de leucocytes radiomarques jusque leur observation au microscope. A chaque etape, les activites manipulees ont ete mesurees avec un activimetre CRC15®. Tout au long du processus, les principes fondamentaux de radioprotection (temps, ecran, distance) ont ete appliques. Pour chaque controle (n = 10), une surveillance dosimetrique de l’operateur a ete menee par le port de dosimetres passifs et operationnels (dosimetrie corps entier), et d’une bague thermoluminescente (dosimetrie extremites). Pour le cristallin, en l’absence de dosimetre dedie, une estimation a ete realisee par des mesures a 25 cm avec un radiametre Identifinder®. La prise en compte de la duree de chaque etape (de 30 a 150 s) a permis de passer des debits de doses mesurees (μSv/h) aux doses recues (μSv). Ces mesures estiment egalement la dose corps entier. Resultats et discussion Les activites manipulees ont ete tres basses : de 25 MBq en moyenne pour la solution mere a 0,025 MBq pour la solution observee au microscope. Quant aux mesures de surveillance dosimetrique corps entier et extremites, elles n’ont pas atteint les seuils de detection des appareils et sont donc bien inferieures aux limites reglementaires (20 mSv/an pour le corps entier, 500 mSv/an pour les extremites). L’utilisation d’ecrans adaptes (pot, paravent et valisette plombes) ainsi que l’eloignement de la solution mere restante des la fin de son utilisation a permis de diminuer les doses cristallin (et corps entier) de 12 a 3 nSv (de 0,012 μSv a 0,003 μSv) pour chaque controle. L’exposition restante correspond essentiellement a l’etape d’observation sous microscope qui est la plus longue (2 nSv/150 s). Bien que la Commission Internationale de Protection radiologique (CIPR) propose de diminuer la limite reglementaire de la dose au cristallin de 150 a 20 mSv/an, l’exposition de quelques nSv/controle n’augmente pas le risque de cataracte radio-induite. Conclusion L’etude dosimetrique du controle de viabilite des leucocytes radiomarques a permis de verifier que l’exposition de l’operateur aux rayonnements ionisants est tres inferieure aux limites reglementaires et, en particulier, au niveau du cristallin. Elle a egalement contribue a ameliorer sa radioprotection lors de la realisation de ce controle.

Introduction Notre etablissement est passe de la Depakine Chrono® au valproate de sodium cp LP generique en mars 2016. Notre logiciel de prescription, Phedra [Genois] V6 web (base de donnees Thesorimed), propose au medecin, apres saisie de « DEPAK », toutes les specialites commencant par « DEPAK… » (DEPAKine*, DEPAKote*) et toutes celles contenant le meme principe actif (acide valproique). En octobre 2016, a l’occasion du passage en dispensation nominative du valproate avec analyse pharmaceutique systematique, des erreurs ont ete detectees, notamment prescription de valproate (forme non LP) a la place de la forme LP. L’hypothese causale etait celle de libelles confusionnants dans le referentiel medicament. Nous avons souhaite valider cette hypothese. Materiels et methode Analyse de toutes les prescriptions Phedra de valproate per os (formes seches) sur 4 periodes d’un mois : trois en retrospectif (P1, P2, P3), une (P4) en prospectif apres modification du libelle : « valproate de sodium LP (cp de 500 mg) » en « valproate LP (cp de 500 mg) [=Depakine Chrono] » ; comparaison des prescriptions au compte rendu medical (CR) (logiciel Middlecare). Libelles pour le prescripteur : – P1 (10/2015) : valproate cp [= Depakine*] et Depakine Chrono cp ; – P2 et P3 (04/2016 et 10/2016) : valproate cp [= Depakine*] et valproate LP cp (P2 : analyse a posteriori des dossiers ; P3 : analyse pharmaceutique prospective systematique) ; – P4 (01/2017) : valproate cp [= Depakine*] et valproate LP cp [= Depakine Chrono*] (analyse pharmaceutique prospective systematique). Resultats et discussion Cinquante-quatre prescriptions ont ete analysees. P1 (Depakine Chrono® princeps au livret) : 11 prescriptions sur 12 (92 %) conformes aux CR (prescription discordante : valproate non LP au lieu de Depakine Chrono®). P2 et P3 (changement de marche : Depakine Chrono® remplace par le generique valproate LP*) : 19 prescriptions sur 30 (63 %) conformes aux CR. Sur les 11 prescriptions discordantes : 7 valproate (non LP) au lieu de valproate LP ; 2 valproate LP au lieu de valproate (non LP) ; 2 valproate LP au lieu de Depakote®. P4 (ajout de « = Depakine Chrono* » au libelle du generique valproate LP*) : 11 prescriptions sur 12 (92 %) conformes aux CR (prescription discordante : valproate non LP au lieu de valproate LP). La franche diminution des erreurs de prescription apres ajout de la mention « = Depakine Chrono* » a la fin du libelle du generique conforte notre hypothese d’erreur de nomenclature, et peut s’expliquer par une tendance du prescripteur a selectionner plus volontiers un libelle contenant un nom du princeps. Conclusion Les erreurs dites de nomenclature sont frequentes. Dans les logiciels d’aide a la prescription medicamenteuse, l’affichage du nom du princeps, en plus de celui de la DCI, apparait comme un outil de detrompage utile, y compris pour les generiques, surtout lorsque cohabitent dans un meme hopital des princeps et des generiques pour une meme substance active. Une gestion rigoureuse de la base medicamenteuse est donc primordiale, releve de la responsabilite pharmaceutique et participe a la lutte contre la e-iatrogenie.

Introduction Le « midline » est un catheter (KT) veineux peripherique profond, insere dans la veine basilique ou cephalique, pour une utilisation de courte ou moyenne duree (souvent Materiels et methode Apres recensement des besoins des services et analyse bibliographique, les medecins et pharmaciens ont rencontre les fournisseurs de midlines. Les DM pre-retenus ont ete testes au moyen de fiches d’evaluation, dont les criteres principaux etaient la facilite de pose et l’identification du DM une fois pose. L’Equipe operationnelle d’hygiene (EOH) a aussi ete impliquee afin d’encadrer l’utilisation du DM selectionne. Resultats et discussion Finalement, deux services souhaitent poser ce DM, la RMT et le departement d’anesthesie et reanimation. Leur choix est de le reserver aux patients difficiles a piquer lors de therapeutiques en IV peripherique : les patients a faible capital veineux comme les personnes âgees, les patients obeses, et les patients avec un œdeme generalise classiquement hospitalises en reanimation, les therapeutiques concernees etant des antibiotherapies ou de la rehydratation durant 1 a 4 semaines. Le besoin est estime a 2 patients par mois. Ce DM permet d’eviter l’utilisation d’un KT central avec les risques engendres et les praticiens apprecient la facilite de pose car elle est echoguidee. La bibliographie met en evidence la possible confusion avec le PICCline, avec risque d’administration de produits veinotoxiques sur le midline. Ce risque serait majore par la faible utilisation de ce DM dans les services poseurs. Au total, 3 fournisseurs commercialisent des kits de midlines : 2 sont retenus car leur technique de pose Seldinger est connue des medecins, le 3e presentant une technique nouvelle est ecarte. Les essais ont ete realises de janvier a mars 2017, par 3 medecins sur 5 patients. Les 2 DM ont ete juges equivalents en termes de facilite de pose, d’echogenicite de l’aiguille et du KT, d’ergonomie du kit et d’identification apres la pose. Le DM choisi a l’avantage de disposer d’une etiquette « midline » a rajouter sur le KT une fois pose. Le protocole d’entretien du midline a ete redige en collaboration avec l’EOH. Conclusion Ce travail pluridisciplinaire a permis de selectionner le midline le mieux adapte, en diminuant le risque de confusion avec une voie centrale. Il precise l’emploi de ce DM dans notre etablissement, et pourra servir de base si une extension a d’autres services etait envisagee.

Increasing bacterial resistance to available antibiotics stimulated the discovery of novel efficacious antibacterial agents. The biosynthesis of the bacterial peptidoglycan, where the MurD enzyme is involved in the intracellular phase of the UDP-MurNAc-pentapeptide formation, represents a collection of highly selective targets for novel antibacterial drug design. In our previous computational studies, the C-terminal domain motion of the MurD ligase was investigated using Targeted Molecular Dynamic (TMD) simulation and the Off-Path Simulation (OPS) technique. In this study, we present a drug design strategy using multiple protein structures for the identification of novel MurD ligase inhibitors. Our main focus was the ATP-binding site of the MurD enzyme. In the first stage, three MurD protein conformations were selected based on the obtained OPS/TMD data as the initial criterion. Subsequently, a two-stage virtual screening approach was utilized combining derived structure-based pharmacophores with molecular docking calculations. Selected compounds were then assayed in the established enzyme binding assays, and compound 3 from the aminothiazole class was discovered to act as a dual MurC/MurD inhibitor in the micomolar range. A steady-state kinetic study was performed on the MurD enzyme to provide further information about the mechanistic aspects of its inhibition. In the final stage, all used conformations of the MurD enzyme with compound 3 were simulated in classical molecular dynamics (MD) simulations providing atomistic insights of the experimental results. Overall, the study depicts several challenges that need to be addressed when trying to hit a flexible moving target such as the presently studied bacterial MurD enzyme and show the possibilities of how computational tools can be proficiently used at all stages of the drug discovery process.

Norepinephrine, 5-hydroxytryptophan, octopamine, dihydroxyphenylacetic acid, N-acetyldopamine, dopamine, 5-hydroxyindoleacetic acid, N-acetylserotonin, tyramine, tryptophan and serotonin in larvae (third free stage and parasitic stages) and adult males and females (at defined ages during the intestinal phase) of the parasitic nematode Nippostrongylus brasiliensis were quantified simultaneously by high-performance liquid chromatography with electrochemical detection. Biogenic amine levels depended on the stage, the age and the sex of parasites and on environmental conditions. Their physiological roles in reproductively competent adults of this nematode are discussed in relation to exuviation and egg laying. Parallel fluctuations in free ecdysteroids and norepinephrine were observed in females from the same worm populations.

MurF is an essential bacterial enzyme that is involved in the last intracellular stage of peptidoglycan biosynthesis, and therefore it has the potential to be exploited as a target for the development of new antibacterials. Here, we report on the expression, purification and biochemical characterization of MurF from an important pathogen, Streptococcus pneumoniae. Additionally, ligand-based virtual screening was successfully used and a new hit compound with micromolar inhibitory activities against MurF enzymes from S. pneumoniae and Escherichia coli was identified.

Introduction Depuis 27 ans, la pharmacie realise une DIN manuelle dans le service LS. Pour ameliorer la securisation du circuit du medicament (mdt) et permettre un redeploiement de l’activite, la pharmacie a acquis un automate de DIN : l’Acced S300 (Ecodex ® ). Le but de cette etude est de comparer le temps consacre par les preparateurs en pharmacie hospitaliere (PPH) aux differentes activites de DIN en LS, avant et apres acquisition de l’automate. Materiels & methode Cette etude a ete realisee sur 4 semaines (smn) : 2 pour la DIN manuelle en septembre 2015 et janvier 2016 (1) et 2 pour la DIN automatisee en aout 2016 (2). Chaque PPH s’est chronometre sur les differentes activites de la DIN. Les temps sont donnes pour 1 smn et correspondent a un cumul pour l’ensemble des PPH. Resultats & discussion Pour 7 jours (j) de LS, nous avons prepare en (1) 147 piluliers pour 74 patients et 529 piluliers pour 76 patients en (2). En DIN manuelle, les piluliers etaient prepares 2 fois/smn avec 1 pilulier/patient pour les differents jours alors qu’en DIN automatisee, les piluliers sont faits pour 7 j avec 1 pilulier/j/patient. L’impression des plans de cueillette prend 40 minutes (min) en (1) vs 9 min pour imprimer les ordonnances en (2). La preparation des piluliers occupe 5h15 en (1) vs 5h06 en (2). Ces 5h06 correspondent a une etape de complementation car l’AccedS ne prend en charge que les formes orales seches. Cela comprend la sortie des mdt de la liste de complementation (53 min), la complementation (3h40) et la preparation de la liste verte (mdt non pris en charge pas l’automate, non mis dans les piluliers et en dispensation reglobalisee comme le paracetamol) (34 min). Le temps de production des piluliers par l’automate n’est pas comptabilise car a lieu la nuit. Le double controle des piluliers est fait en aleatoire et prend 39 min en (1) vs 26 min en (2). L’acheminement des chariots vers les services occupe 43 min en (1) vs 21 min en (2) (passage d’une dispensation bi-hebdo a hebdo). Le reapprovisionnement des bacs de cueillette prend 42 min en (1) vs 95 min en (2) pour preparer les mdt de la liste de reassort pour la coupe. Nous gagnons du temps sur la sortie des consommations : 2h09 en (1) vs 17 min en (2) car l’automate facture les mdt qu’il introduit dans les piluliers. Le lancement informatique des prescriptions vers l’automate (40 min), le lancement de l’automate (20 min) et l’entree/sortie des chariots (11 min) sont des etapes specifiques de la DIN automatisee. La coupe (2h55 min) et le retour dans l’automate des doses non utilisees par les services (70 min) sont des etapes confiees aux agents hospitaliers sous la responsabilite des PPH. Au final, le temps de DIN/smn pour les PPH pour le service LS est de 10h07 en (1) vs 9h04 en (2), sans les etapes de coupe ni de retour des doses. Conclusion La DIN automatisee en LS ne nous fait pas gagner de temps car l’automatisation implique des etapes de complementation et manutention importantes. Cependant, l’automatisation participe a l’amelioration de la securisation du circuit du mdt (risque d’erreur humaine diminue, production d’1 pilulier/j/patient, homogeneisation des doses).

Introduction En septembre 2014, l’ANSM a informe les praticiens hospitaliers du retrait d’AMM de la specialite Vistide ® (cidofovir), suite a la diminution de l’incidence des retinites a CMV chez les patients VIH au stade sida, a l’existence d’alternatives therapeutiques dans cette indication, ainsi qu’a des difficultes de fabrication. Depuis, le cidofovir fait l’objet d’une demande d’autorisation temporaire d’utilisation nominative (ATUn). Au sein de notre etablissement, cette ATUn est essentiellement utilisee dans le cadre des papillomatoses oropharyngees recidivantes, dues au virus du papillome humain (ou HPV), avec realisation d’injections en intra-lesionnel. Les papillomatoses sont des affections rares et une des conditions d’acces au traitement par le cidofovir est l’echec de therapeutiques anterieures. L’objectif de cette analyse est de comparer les donnees de la litterature aux pratiques usuelles du service d’ORL. Materiels et methode (1) Analyse des donnees de la litterature depuis la base de donnees PubMed. (2) Recueil de donnees (âge, sexe, type HPV, nombre d’injections et concentrations, antecedents medicaux et chirurgicaux) realise par l’interne en pharmacie avec la participation des medecins du service d’ORL. Analyse retrospective des demandes d’ATUn realisees entre septembre 2014 et septembre 2016. (3) Entretiens avec les 2 praticiens realisant les prescriptions de cidofovir. Resultats et discussion (1) Six articles ont ete retenus, mentionnant des concentrations variant de 2,5 a 75 mg/mL dont les plus utilisees sont : 5 et 7,5 mg/mL. Les effets indesirables les plus frequemment rapportes sont une toxicite renale et/ou hematologique. (2) Sinq patients âges de 19 a 70 ans (2 hommes, 3 femmes, âge moyen 49 ans). Pour 3/5 de ces patients, les papillomes etaient larynges, 1/5 avec une localisation nasale et 1/5 chez qui les papillomes ont ete retrouves dans le nez et le pharynx. Le typage HPV a ete effectue pour 3/5 patients, il a ete retrouve 2 HPV6 et 1 HPV11. Pour un patient, le typage a ete demande mais les resultats n’ont pas encore ete communiques et aucun typage n’a ete demande pour le dernier patient. Concernant les antecedents, des exereses sans injection des papillomes ont ete faites (avec ou sans laser) pour 4/5 patients, et 1/5 a recu un traitement par interferon α-2a. Le protocole utilise consiste en 2 injections espacees de 3 semaines avec de possibles injections ulterieures en cas de recidives. Seulement un patient a necessite une 3 e injection, plus d’un an apres les premieres. (3) Aucun effet indesirable n’a ete rapporte. Conclusion Les donnees emanant de divers articles mettent en evidence la difficulte de proposer un schema therapeutique standardise. Neanmoins, les concentrations utilisees par les praticiens sont celles les plus frequemment retrouvees dans la litterature. Un suivi au long cours de ces patients permettrait de s’assurer de l’absence d’effets indesirables et/ou de recidives des papillomatomes.