[Assessment of the use by hospital pharmacists of the opinions issued by the Haute Autorité de santé and the economic analysis tools proposed to them]. / Évaluation de l'utilisation par les pharmaciens hospitaliers des avis émis par la Haute Autorité de santé et des outils économiques qui leur sont proposés.

BACKGROUND: In French hospitals, the supply and management of drugs are missions of pharmacists. The aim of this study is to assess the use of efficacy and economics opinions of the Haute Autorite de santé (HAS) in hospital referencing of drugs in France in 2017. METHODS: A questionnaire for hospital pharmacists was developed to establish their knowledge and their uses of Transparency Commission and Economic and Public Health Evaluation Committee opinions. This survey was distributed by the ADIPH association in July 2017. This questionnaire included 35 questions. RESULTS: Despite the health professional are more and more interesting by economics analysis, only 30 % of hospitals pharmacists of this panel declared to use HAS economic opinions (versus 80% for Transparency Commission opinions). Among these pharmacists, 86% used this report in the hospital referencing of drugs. CONCLUSION: Through this analysis, some prospects for improvement can be seen in the health professional formation, cost-effectiveness report publication and the use of cost-effectiveness analysis. This study is an overview of the use of opinions provided by the HAS. This thesis established a basis to reflection on the use of economics reports and models in the hospital decisions.

[Use of a Delphi survey to assess the hospital economic impact of innovative products: The example of idarucizumab a dabigatran-specific reversal agent]. / Utilisation du questionnaire Delphi pour anticiper l'impact économique des innovations thérapeutiques à l'hôpital : application à l'idarucizumab, le nouvel agent de réversion spécifique au dabigatran.

OBJECTIVES: The economic impact of therapeutic innovations on the hospital patient management cannot be easily estimated. The objective of this study is to illustrate the use of a Delphi survey as a support tool to identify the changes following the use of idarucizumab in dabigatran-treated patients with uncontrolled/life-threatening bleeding or who required emergency surgery/urgent procedures. METHODS: The Delphi questionnaires have been administrated to 8 emergency physicians or anesthetists from 6 different hospital centers. Following the answers, an economic valorization has been carried out on every parameter on which a consensus was reached (at least 4 answers showing an identical trend). A mean management cost for each etiology with and without the use of idarucizumab has thus been identified. RESULTS: For gastro-intestinal and other life-threatening bleedings (excepted intracranial bleedings), the total management cost of the hospital stay was respectively 6058 € (-35%) and 6219 € (-34%) following the use of the reversal agent. The hospital management cost for intracranial bleeding is slightly increasing to 9790 € (+3%). The cost of a stay for emergency surgery decreases to 6962€ (-2%). CONCLUSIONS: This study shows a positive economic impact following the use of the dabigatran-specific reversal agent for patients with uncontrolled/life-threatening bleeding excepted in the case of intracranial bleeding. Moreover, it points out that a Delphi survey is an easy way to predict the hospital economic impact of a therapeutic innovation when no other evaluation is possible.

Organizational and budget impact model (OBIM) of same™ a new autotransfusion medical device.

Objectives: This study aimed to assess the budget and organizational impact of progressively replacing the intraoperative cell salvage centrifugation-based systems currently installed in French hospitals with the same™ system, a new autotransfusion medical device.Methods: An Organizational and Budget Impact Model (OBIM) was developed based on two methodological guidelines issued by the French Health Authority (Haute Autorité de Santé, HAS). The OBIM was also developed based on a pragmatic literature review, hospital data and hospital pharmacists' expertise.Results: Considering an average hospital cohort of 600 cardio-thoracic surgery patients, with 57% experiencing mild hemorrhages, 23% moderate hemorrhages, and 20% massive hemorrhages, and a same™ market share of 33% in Year 1, the implementation of same™ resulted in significant savings. With an average allogeneic transfusion of 4.19 packed red blood cells (RBC) and 0.62 platelet concentrate per patient based on National Hemovigilance Report of the ANSM (French National Agency for the Safety of Medicines and Health Products) and when using same™ system a reduction of 45% of RBC transfusion associated with a reduction of 60% and 90% of platelet use for moderate and massive hemorrhages respectively, the first year annual saving amounted to €44,601 and the cumulated saving over 5 years to €535,206.Discussion: This model structure was developed based on overall hospitals' needs and acknowledged guidelines, with inputs based on French literatures and hospital data, so findings were specifics to a context. Among the inputs, the number of annual same™ procedure is not based on device capability but rather on hospital capability with the number of operating rooms used for cardio-thoracic surgery equipped with the device.Conclusions: The results of this OBIM demonstrated the economic and organizational benefits of same™ for hospitals. This benefit results mainly of a reduction in the use of allogeneic blood products (RBCs, platelets).

Trypanosoma cruzi: in vitro activity of Epoxy-alpha-Lap, a derivative of alpha-lapachone, on trypomastigote and amastigote forms.

Chagas disease is an endemic parasitic infection caused by Trypanosomacruzi that affects 18-20 million people in Central and South America. Recently we described the Epoxy-alpha-Lap, an oxyran derivative of alpha-lapachone, which presents a low toxicity profile and a high inhibitory activity against T.cruzi epimastigotes forms, the non-infective form of this parasite. In this work we described the trypanocidal effects of Epoxy-alpha-Lap on extracellular (trypomastigote) and intracellular (amastigote) infective forms of two T. cruzi strains (Y and Colombian) known by their different infective profile. Our results showed that Epoxy-alpha-Lap is lethal to trypomastigote Y and Colombian strains (97% and 84%, respectively). Interestingly, Epoxy-alpha-Lap also showed a trypanocidal effect in human macrophage infected with T. cruzi Y (85.6%) and Colombian (71.9%) strains amastigote forms. Similar effects were observed on T. cruzi amastigote infected Vero cells (96.4% and 95.0%, respectively). Our results pointed Epoxy-alpha-Lap as a potential candidate for Chagas disease chemotherapy since it presents trypanocidal activity on all T. cruzi forms with low) toxicity profile.

Synthesis, structure-activity relationship and trypanocidal activity of pyrazole-imidazoline and new pyrazole-tetrahydropyrimidine hybrids as promising chemotherapeutic agents for Chagas disease.

Drug therapy for Chagas disease remains a major challenge as potential candidate drugs have failed clinical trials. Currently available drugs have limited efficacy and induce serious side effects. Thus, the discovery of new drugs is urgently needed in the fight against Chagas' disease. Here, we synthesized and evaluated the biological effect of pyrazole-imidazoline (1a-i) and pyrazole-tetrahydropyrimidine (2a-i) derivatives against relevant clinical forms of Trypanosoma cruzi. The structure-activity relationship (SAR), drug-target search, physicochemical and ADMET properties of the major active compounds in vitro were also assessed in silico. Pyrazole derivatives showed no toxicity in Vero cells and also no cardiotoxicity. Phenotypic screening revealed two dichlorinated pyrazole-imidazoline derivatives (1c and 1d) with trypanocidal activity higher than that of benznidazole (Bz) against trypomastigotes; these were also the most potent compounds against intracellular amastigotes. Replacement of imidazoline with tetrahydropyrimidine in the pyrazole compounds completely abolished the trypanocidal activity of series 2(a-i) derivatives. The physicochemical and ADMET properties of the compounds predicted good permeability, good oral bioavailability, no toxicity and mutagenicity of 1c and 1d. Pyrazole nucleus had high frequency hits for cruzipain in drug-target search and structure activity relationship (SAR) analysis of pyrazole-imidazoline derivatives revealed enhanced activity when chlorine atom was inserted in meta-positions of the benzene ring. Additionally, we found evidence that both compounds (1c and 1d) have the potential to interact non-covalently with the active site of cruzipain and also inhibit the cysteine proteinase activity of T. cruzi. Collectively, the data presented here reveal pyrazole derivatives with promise for further optimization in the therapy of Chagas disease.

Potency evaluation of antivenoms in Brazil: the national control laboratory experience between 2000 and 2006.

Envenoming from snakebites is an important public health issue in Brazil. In 2005, 28,597 cases were notified (15 cases/100,000 inhabitants), 87.5% due to Bothrops and 9.2% to Crotalus genus. Antivenoms available in Brazil are liquid preparations containing purified equine Fab'2. Since 1987, the National Institute for Quality Control in Health (INCQS/FIOCRUZ) has been testing all lots prior to batch release. Between 2000 and 2006, 619 lots of antivenoms were tested, comprising 2,513,690 ampoules. The potency assay was performed only for bothropic and crotalic antivenoms (485 lots corresponding to 1,866,726 ampoules) due to the unavailability of the other reference venoms. This paper aims to report the last 7-year activities of INCQS on the quality control, batch release and potency evaluation of antivenoms.

Efficacy of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinone derivatives against different Trypanosoma cruzi discrete type units: Identification of a promising hit compound.

The limited efficacy of benznidazole (Bz) indicated by failures of current Phase II clinical trials emphasizes the urgent need to identify new drugs with improved safety and efficacy for treatment of Chagas disease (CD). Herein, we analyzed the efficacy of a series of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones against different Trypanosoma cruzi discrete type units (DTUs) of relevant clinical forms of CD. Cytotoxic and trypanocidal effect of naphthoquinone derivatives were assessed in mammalian cells, trypomastigotes and intracellular amastigotes using, luminescent assays (CellTiter-Glo and T. cruzi Dm28c-luciferase) and/or counting with a light microscope. Reactive oxygen species (ROS) production and intracellular targets of promising compounds were assessed with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) probe and ultrastructural analysis, respectively. ADMET properties were analyzed by in silico modeling. Most of the compounds showed low cytotoxic effect. Only two compounds (Compounds 2 and 11) had IC50 values lower than Bz, showing higher susceptibility of bloodstream trypomastigotes. Compound 2 exhibited greater efficacy against trypomastigotes from different T. cruzi DTUs, even better than Bz against Brazil and CL strains. Ultrastructural analysis revealed changes in intracellular compartments, suggesting autophagy as one possible mechanism of action. Oxidative stress, induced by Compound 2, resulted in elevated level of ROS, leading to parasite death. Compound 2 was also effective against intracellular amastigotes, showing high selectivity index. ADMET analysis predicted good oral bioavailability, reduced drug metabolism and no carcinogenic potential for Compound 2. The data highlight Compound 2 as a hit compound and stimulate further structural and pharmacological optimization to potentiate its trypanocidal activity and selectivity.

Transgenic mice overexpressing protein kinase C epsilon in their epidermis exhibit reduced papilloma burden but enhanced carcinoma formation after tumor promotion.

To determine the role that protein kinase C epsilon (PKCepsilon) may play in skin growth, differentiation, and tumor promotion, transgenic mice were generated that overexpressed an epitope-tagged protein kinase C epsilon (T7-PKCepsilon) in their epidermis using the human keratin 14 promoter. Three independent mouse lines that overexpressed the T7-PKCepsilon in their epidermis were produced. The three independent lines 206, 224, and 215 exhibited a 3-, 6-, and 18-fold elevation, respectively, in the level of PKCepsilon immunoreactive protein. Line 215 exhibited a 19-fold greater phosphatidylserine and 12-O-tetradecanoylphorbol-13-acetate (TPA) stimulated kinase activity than line 224. Line 206 exhibited a low basal T7-PKCepsilon activity, which failed to be stimulated by phosphatidylserine and TPA. All of the line 215 transgenic mice (F0 to the F2 generation) displayed phenotypic changes in the skin. The phenotypic changes progressed gradually, starting around 4-5 months of age, with mild dryness of the tail accompanied by hair loss and inflammation at the base of the tail. Hyperproliferation and ulceration of the affected regions were observed around 7-8 months of age. The hyperproliferative epidermis from the affected regions exhibited an expansion of the suprabasal epidermal cells. Inflammation and/or ulceration were also observed in the dorsal skin, the ears, and around the eyes. The line 215 mice, which expressed the highest level of PKCepsilon, were evaluated for sensitivity to mouse skin tumor promotion by TPA. Tumors were elicited by the initiation (7,12-dimethylbenz[a]anthracene, 100 nmol)-promotion (TPA, 5 nmol/twice weekly) protocol. The papilloma burden was reduced by 95-96% for male and female T7-PKCepsilon mice compared to wild-type controls. However, carcinomas developed rapidly in the T7-PKCepsilon mice treated with 7, 12-dimethylbenz[a]anthracene and TPA. These carcinomas appeared to form independently of prior papilloma development. These results demonstrate that PKCepsilon is an important regulator of skin tumor development.

Detrimental effect of nitric oxide on Trypanosoma cruzi and Leishmania major like cells.

The production of nitric oxide (NO) by activated macrophages has been reported to be a non-specific immune-effect mechanism against several parasites. In this work we investigate whether the NO has a detrimental effect on the intracellular parasites of the genus Leishmania and as well as Trypanosoma cruzi. This was assessed by co-cultivating infective Leishmania promastigotes and T. cruzi trypomastigotes and non-infective T. cruzi epimastigotes forms of the parasites in the presence of the NO releasing molecule, S-nitroso-N-acetyl-DL-penicillamine (SNAP). We demonstrate that the NO has the ability to inhibits the growth of all parasites in a concentration dependent manner. In addition, by analysing purified protein and cell homogenates of L. major (promastigotes) and T. cruzi (epimastigotes and trypomastigotes) we demonstrated that the NO may regulate the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity of both parasites.

Expression of B7-1 costimulatory molecules in patients with multibacillary leprosy and reactional states.

BACKGROUND: The expression of B7 as a costimulatory molecule on the surface of antigen-presenting cells such as macrophages and on dendritic cells characterizes the efficiency of the cell-mediated immune response. AIMS: Our purpose was to evaluate B7-1 expression in peripheral blood mononuclear cells (PBMCs) immediately after cell isolation ('spontaneous' B7 expression), and in inflammatory cells from cutaneous lesions of patients with multibacillary leprosy (MB-L) without and during the reactional states of erythema nodosum leprosum (ENL) or reversal reaction (RR). METHODS: Peripheral blood samples and skin biopsies of eight patients without (MB-L) and with reactional episodes (ENL and RR) were studied using antibodies against B7-1, CD1b, DR and CD14 in flow-cytometry and immunohistochemistry experiments. RESULTS: The flow-cytometry studies (mean +/- SD% of fluorescent cells) revealed significant B7-1 expression on PBMCs isolated from patients with ENL (8.0 +/- 0.6%) and RR (15.0 +/- 1.4%) compared with that observed for patients with MB-L (0.4 +/- 0.2%). Similar results were observed for cutaneous lesions of these patients by immunohistochemical assays. One patient studied before and during ENL revealed weak B7 expression before the reactional episode (0.3% of cells) compared with the marked level of B7-expressing cells detected during ENL (8.5% fluorescent cells). Interestingly, an even higher B7 expression (15% of cells) was observed in patients with RR. CONCLUSIONS: Our results strongly suggest that B7 expression precedes reactional episodes in MB-L, which could be related to the acquisition of effective immunity to Mycobacterium leprae during reactional episodes in leprosy. We propose B7 expression as a marker of CMI response in reactional episodes in leprosy.

Leishmania amazonensis: early proteinase activities during promastigote-amastigote differentiation in vitro.

Leishmania proteinase activity is known as parasite differentiation marker, and has been considered relevant for leishmanial survival and virulence. These properties suggest that Leishmania proteinases can be promising targets for development of anti-leishmania drugs. Here, we analyze the activities of four proteinases during the early phase of the Leishmania amazonensis promastigotes differentiation into amastigotes induced by heat shock. We have examined activities of cysteine-, metallo-, serine-, and aspartic-proteinase by hydrolysis of specific chromogenic substrates at pH 5.0 and at the optimal pH for each enzyme. Our results show that metallo-, serine-, and aspartic-proteinases activities were down-regulated during the shock-induced transformation of promastigotes into amastigotes. In contrast, cysteine-proteinase activity increased concomitantly with the promastigote differentiation. Immunocytochemical localization using two anti-cysteine-proteinase monospecific rabbit antibodies detected the enzyme in several cell compartments of both parasite stages. Our results show different proteinase activity modulation and expression during the early phases of the shock-induced parasite transformation.

Localization of lectin-binding sites on the surface of Trypanosoma cruzi grown in chemically defined conditions.

The transformation of Trypanosoma cruzi epimastigotes to mammal-infective metacyclic trypomastigotes (metacyclogenesis) can be performed in vitro under chemically defined conditions (TAU 3AAG medium). During this process, changes in the nature of cell surface sugar composition and sugar distribution was evaluated using FITC and gold-labeled lectins and observed by flow cytometry and transmission electron microscopy. The pattern of labeling with the lectins from Triticum vulgaris (WGA), Arachis hypogaea (PNA), Limax flavus (LFA), Canavalia ensiformis (Con-A), and Ricinus communis (RCA-I) significantly changed during the metacyclogenic process. The results obtained are discussed in relation to the role played by T. cruzi cell surface carbohydrate residues on the process of parasite-host cell interaction.

Purification and Partial characterization of Trypanosoma cruzi triosephosphate isomerase.

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on phenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isoelectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytochemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins.

Purification and partial characterization of Trypanosoma cruzi triosephosphate isomerase

The enzyme triosephosphate isomerase (TPI, EC 5.3.1.1) was purified from extracts of epimastigote forms of Trypanosoma cruzi. The purification steps included: hydrophobic interaction chromatography on plenyl-Sepharose, CM-Sepharose, and high performance liquid gel filtration chromatography. The CM-Sepharose material contained two bands (27 and 25 kDa) with similar isolectric points (pI 9.3-9.5) which could be separated by gel filtration in high performance liquid chromatography. Polyclonal antibodies raised against the porcine TPI detected one single polypeptide on western blot with a molecular weight (27 kDa) identical to that purified from T. cruzi. These antibodies also recognized only one band of identical molecular weight in western blots of several other trypanosomatids (Blastocrithidia culicis, Crithidia desouzai, Phytomonas serpens, Herpertomonas samuelpessoai). The presence of only one enzymatic form of TPI in T. cruzi epimastigotes was confirmed by agarose gel activity assay and its localization was established by immunocytichemical analysis. The T. cruzi purified TPI (as well as other trypanosomatid' TPIs) is a dimeric protein, composed of two identical subunits with an approximate mw of 27,000 and it is resolved on two dimensional gel electrophoresis with a pI of 9.3. Sequence analysis of the N-terminal portion of the 27 kDa protein revealed a high homology to Leishmania mexicana and T. brucei proteins.