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International audience; Background and objective: Daptomycin has been recommended in the treatment of bone and joint infection. Previous work showed that the approved dosage of daptomycin may be insufficient to achieve optimal exposure in patients with bone and joint infection. However, those studies assumed that bone exposure was similar to steady-state daptomycin-free plasma concentrations. We sought to establish a physiologically based pharmacokinetic (PBPK) model of daptomycin to describe the dynamics of daptomycin disposition in bone and skin tissue.Methods: a PBPK model of daptomycin was built using PK-Sim®. Daptomycin concentrations in plasma and bone were obtained from three previously published studies. Physicochemical drug characteristics, mass balance, anthropometrics, and experimental data were used to build and refine the PBPK model. Internal validation of the PBPK model was performed using the usual diagnostic plots. The final PBPK model was then used to run simulations with doses of 6, 8, 10, and 12 mg/kg/24 h. Pharmacokinetic profiles were simulated in 1.000 subjects and the probabilities of target attainment for the area under the concentration-time curve over the bacterial minimum inhibitory concentration were computed in blood, skin, and bone compartments.Results: the final model showed a good fit of all datasets with an absolute average fold error between 0.5 and 2 for all pharmacokinetic quantities in blood, skin and bone tissues. Results of dosing simulations showed that doses ≥10 mg/kg should be used in the case of bacteremia caused by Staphylococcus aureus with a minimum inhibitory concentration >0.5 mg/L or Enterococcus faecalis with a minimum inhibitory concentration >1 mg/L, while doses ≥12 mg/kg should be used in the case of bone and joint infection or complicated skin infection. When considering a lower minimum inhibitory concentration, doses of 6-8 mg/kg would likely achieve a sufficient success rate. However, in the case of infections caused by E. faecalis with a minimum inhibitory concentration >2 mg/L, a higher dosage and combination therapy would be necessary to maximize efficacy.Conclusions: we developed the first daptomycin PBPK/pharmacodynamic model for bone and joint infection, which confirmed that a higher daptomycin dosage is needed to optimize exposure in bone tissue. However, such higher dosages raise safety concerns. In this setting, therapeutic drug monitoring and model-informed precision dosing appear necessary to ensure the right exposure on an individual basis.

Purpose We hypothesized that a protocol of standardized fixed dose using prolonged infusion during the early phase of sepsis may avoid insufficient β-lactam concentrations. Methods In this single center prospective study, patients with sepsis and vasopressors were enrolled if they were treated by either piperacillin-tazobactam, meropenem or cefepime. Βeta-lactams were administered at fixed dose by prolonged infusion. Targeted plasma concentrations for piperacillin, meropenem and cefepime were above 80 mg/L, 8 mg/L and 38 mg/L respectively. Three blood samples were collected per patient over the first 48 h of treatment. Primary endpoint was target concentration achievement during the 48 first hours, defined as all plasma concentrations above the targeted threshold. Results Among the 89 patients completing the three samples, target concentrations were achieved for 61 (69%). Target concentrations were achieved in 20 (53%), 32 (89%), and 9 (60%) of the patients treated with piperacillin, meropenem and cefepime, respectively. By multivariate analysis, lower APACHE 2 score, higher baseline MDRD creatinine clearance, and piperacillin use were independently associated with insufficient β-lactam concentrations. Conclusion Despite a fixed dose antibiotic administration protocol with prolonged infusion insufficient β-lactam concentration was frequent at the early phase of sepsis, especially in less severe patients, without renal failure, and treated with piperacillin. In septic patients with vasopressors, piperacillin dosing higher than 16 g may be needed to achieve the recommended target concentration. Trial registration: NCT02820987

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Direct Oral Anticoagulants (DOACs) available for the treatment and prevention of thromboembolic diseases include dabigatran, a direct thrombin (IIa) inhibitor, and apixaban, edoxaban and rivaroxaban, which are direct inhibitors of Stuart factor (Xa). DOACs have a different pharmacokinetic and pharmacodynamics profiles, with less probable drug-drug interactions than vitamin K antagonists. They do not require systematic therapeutic monitoring except in specific clinical situations such as emergency procedures or drug non-compliance. Furthermore, anticoagulant effects of DOACs could be impacted by renal impairment, drug-drug interactions, food interactions, or pharmacogenetic variability. In this context, we developed a method for simultaneous determination of dabigatran, rivaroxaban and apixaban in human plasma using high performance liquid chromatography coupled with a mass spectrometry assay and applied it to 26 patient samples. Our method presents a total run time of 5 min and extends from 25 to 1000 μg/L for apixaban and dabigatran; and from 5 to 1000 μg/L for rivaroxaban. Intra- and inter-assay accuracy were between −22.3 and 25.4%; and − 23.7 and 3.8%, respectively. Precision at low and high concentrations were below 17.5%. Frozen samples were stable up to 3 months. No significant cross-contamination was observed. In conclusion, our assay can be used during clinical studies and in daily routine practice for the management of specific clinical situations at reasonable cost.

Ticagrelor is an antiplatelet agent that inhibits platelet activation via P2Y12 antagonism. There are several studies showing that P2Y12 needs lipid rafts to be activated, but there are few data about how ticagrelor impacts lipid raft organization. Therefore, we aimed to investigate how ticagrelor could impact the distribution of cholesterol and consequently alter the organization of lipid rafts on platelet plasma membranes. We identified cholesterol-enriched raft fractions in platelet membranes by quantification of their cholesterol levels. Modifications in cholesterol and protein profiles (Flotillin 1, Flotillin 2, CD36, P2Y1, and P2Y12) were studied in platelets stimulated by ADP, treated by ticagrelor, or both. In ADP-stimulated and ticagrelor-treated groups, we found a decreased level of cholesterol in raft fractions of platelet plasma membrane compared to the control group. In addition, the peak of cholesterol in different experimental groups changed its localization on membrane fractions. In the control group, it was situated on fraction 2, while in ADP-stimulated platelets, it was located in fractions 3 to 5, and in fraction 4 in ticagrelor-treated group. The proteins studied also showed changes in their level of expression and localization in fractions of plasma membrane. Cholesterol levels of plasma membranes have a direct role in the organization of platelet membranes and could be modified by stimulation or drug treatment. Since ticagrelor and ADP both changed lipid composition and protein profile, investigating the lipid and protein composition of platelet membranes is of considerable importance as a focus for further research in anti-platelet management.

Raltitrexed is a thymidylate synthase inhibitor that can be administered safely to patients with cardiovascular disease or dihydropyrimidine dehydrogenase deficiency, as opposed to 5FU. The recommended dose of 3mg/m2 every 3 weeks often leads to toxicity. Interestingly, the 2mg/m2 every 2 weeks dose appears to be less toxic. A pharmacokinetic trial was then performed by our team to investigate such phenomenon. However, there are currently, two main methods for RTX measurement described in the literature: a radioimmunoassay (RIA) and chromatographic-based methods with either UV or mass spectrometry detections. The RIA methods: display a low limit of quantification (below 1μg/L), but also a low extent of linearity for the calibration curve. The chromatographic-based methods: include high level of calibrators, but have poor sensitivity (>2μg/mL). If a high sensitivity is essential to satisfactorily describe the elimination of RTX, high concentrations in the calibration curve are also needed to avoid bias linked to the dilutions of the samples. A new LC-MS/MS method was then developed that allows to simultaneously measure very low (0.1μg/L) and very high (3000μg/L) concentrations in the same run. Moreover, the extraction steps are very simple and fast with mainly a precipitation and a filtration steps. This method was validated following the EMA recommendations. In view of the extent of the calibration curve, the carry-over effect was more deeply investigated. With this method, it was possible to measure RTX in samples taken 3 weeks after the administration. Taken together, this method allows to simply and quickly measure RTX in plasma of patients.

Lipid-rafts are defined as membrane microdomains enriched in cholesterol and glycosphingolipids within platelet plasma membrane. Lipid raft-mediated clot retraction requires factor XIII and other interacting proteins. The aim of this study was to investigate the proteins that interact with factor XIII in raft and non-raft domains of activated and non-activated platelet plasma membrane. By lipidomics analysis, we identified cholesterol- and sphingomyelin-enriched areas as lipid rafts. Platelets were activated by thrombin. Proteomics analysis provided an overview of the pathways in which proteins of rafts and non-rafts participated in the interaction network of FXIII-A1, a catalytic subunit of FXIII. "Platelet activation" was the principal pathway among KEGG pathways for proteins of rafts, both before and after activation. Network analysis showed four types of interactions (activation, binding, reaction, and catalysis) in raft and non-raft domains in interactive network of FXIII-A1. FXIII-A1 interactions with other proteins in raft domains and their role in homeostasis highlight the specialization of the raft domain in clot retraction via the Factor XIII protein network.

It is well known that the standard doses of a given drug may not have equivalent effects in all patients. To date, the management of depression remains mainly empirical and often poorly evaluated. The development of a personalized medicine in psychiatry may reduce treatment failure, intolerance or resistance, and hence the burden and costs of mood depressive disorders. The Geneva Cocktail Phenotypic approach presents several advantages including the “in vivo” measure of different cytochromes and transporter P-gp activities, their simultaneous determination in a single test, avoiding the influence of variability over time on phenotyping results, the administration of low dose substrates, a limited sampling strategy with an analytical method developed on DBS analysis. The goal of this project is to explore the relationship between the activity of drug-metabolizing enzymes (DME), assessed by a phenotypic approach, and the concentrations of Venlafaxine (VLX) + O-demethyl-venlafaxine (ODV), the efficacy and tolerance of VLX. This study is a multicentre prospective non-randomized open trial. Eligible patients present a major depressive episode, MADRS over or equal to 20, treatment with VLX regardless of the dose during at least 4 weeks. The Phenotype Visit includes VLX and ODV concentration measurement. Following the oral absorption of low doses of omeprazole, midazolam, dextromethorphan, and fexofenadine, drug metabolizing enzymes activity is assessed by specific metabolite/probe concentration ratios from a sample taken 2 h after cocktail administration for CYP2C19, CYP3A4, CYP2D6; and by the determination of the limited area under the curve from the capillary blood samples taken 2–3 and 6 h after cocktail administration for CYP2C19 and P-gp. Two follow-up visits will take place between 25 and 40 days and 50–70 days after inclusion. They include assessment of efficacy, tolerance and observance. Eleven french centres are involved in recruitment, expected to be completed within approximately 2 years with 205 patients. Metabolic ratios are determined in Geneva, Switzerland. By showing an association between drug metabolism and VLX concentrations, efficacy and tolerance, there is a hope that testing drug metabolism pathways with a phenotypical approach would help physicians in selecting and dosing antidepressants. The MARVEL study will provide an important contribution to increasing the knowledge of VLX variability and in optimizing the use of methods of personalized therapy in psychiatric settings. ClinicalTrials.gov NCT02590185 (10/27/2015). This study is currently recruiting participants.

Everolimus has demonstrated its efficacy in metastatic renal cell carcinoma (mRCC). Preliminary studies have shown high variability of everolimus blood concentrations (EBC). In other settings, its activity was correlated with EBC. We therefore decided to monitor EBC in patients treated with mRCC to assess its influence on oncologic outcomes. Our study analyzed first 3 months’ trough EBC levels in 42 patients treated in 4 French oncologic centers between March 2010 and August 2013. Patients presented a histologically confirmed diagnosis of mRCC and have failed prior anti-angiogenic (AA) therapies. Median follow-up was 25.9 months. A total of 113 EBC were analyzed. The median trough concentration was 14.1 μg/L (range 2.6–91.5). Fourteen patients (67 %) versus 8 (38 %) patients with median EBC above or below 14.1 μg/L were free from progression at 6 months (p = 0.06). Median progression-free survival was 13.3 versus 3.9 months (HR 0.66 95 % CI 0.33–1.31; p = 0.23), and the median overall survival was 26.2 versus 9.9 months (HR 0.62 95 % CI 0.28–1.37; p = 0.24), for patients above or below the median value of trough concentrations, respectively. Impact of drug exposure for AA tyrosine kinase inhibitors activity has been demonstrated in mRCC setting. Interpatients EBC variability was confirmed in the present study, and the results suggest a relationship between initial EBC within the first 3 months and the drug activity. It underlines the need to prospectively include EBC monitoring in future clinical trials to determine the need of its implementation in routine use.

International audience; The fox tapeworm Echinococcus multilocularis is the causative agent of alveolar echinococcosis, a severe zoonotic disease that may be fatal if untreated. A broad spectrum of mammalian species may be accidentally infected even in captivity. In April 2011, liver lesions due to E. multilocularis were observed during the necropsy of a captive-born nutria (Myocastor coypus) in a French wildlife park, leading to initiation of a study to survey the parasite's presence in the park. A comparable environmental contamination with fox's feces infected by E. multilocularis was reported inside (17.8%) and outside (20.6%) the park. E. multilocularis worms were found in the intestines of three of the five roaming foxes shot in the park. Coprological analyses of potential definitive hosts in captivity (fox, lynx, wildcat, genet, wolf, bear and raccoon) revealed infection in one Eurasian wolf. Voles trapped inside the park also had a high prevalence of 5.3%. After diagnosis of alveolar echinococcosis in a Lemur catta during necropsy, four other cases in L. catta were detected by a combination of ultrasound and serology. These animals were treated twice daily with albendazole. The systematic massive metacestode development and numerous protoscoleces in L. catta confirmed their particular sensitivity to E. multilocularis infection. The autochthonous origin of the infection in all the captive animals infected was genetically confirmed by EmsB microsatellite analysis. Preventive measures were implemented to avoid the presence of roaming foxes, contact with potential definitive hosts and contaminated food sources for potential intermediate hosts.

Lipid rafts play a pivotal role in physiological functions of platelets. Their isolation using nonionic mild detergents is considered as the gold standard method, but there is no consensual detergent for lipid raft studies. We aimed to investigate which detergent is the most suitable for lipid raft isolation from platelet membrane, based on lipidomics and proteomics analysis. Platelets were obtained from healthy donors. Twelve sucrose fractions were extracted by three different detergents, namely Brij 35, Lubrol WX, and Triton X100, at 0.05% and 1%. After lipidomics analysis and determination of fractions enriched in cholesterol (Ch) and sphingomyelin (SM), proteomics analysis was performed. Lipid rafts were mainly observed in 1-4 fractions, and non-rafts were distributed on 5-12 fractions. Considering the concentration of Ch and SM, Lubrol WX 1% and Triton X100 1% were more suitable detergents as they were able to isolate lipid raft fractions that were more enriched than non-raft fractions. By proteomics analysis, overall, 822 proteins were identified in platelet membrane. Lipid raft fractions isolated with Lubrol WX 0.05% and Triton X100 1% contained mainly plasma membrane proteins. However, only Lubrol WX 0.05 and 1% and Triton X100 1% were able to extract non-denaturing proteins with more than 10 transmembrane domains. Our results suggest that Triton X100 1% is the most suitable detergent for global lipid and protein studies on platelet plasma membrane. However, the detergent should be adapted if investigation of an association between specific proteins and lipid rafts is planned.

Prolonged fractionated oral administration of etoposide may present a theoretical advantage over intravenous administration of the bolus. This phase I trial was carried out to determine the recommended duration of oral etoposide in combination with a fixed dose of carboplatin. Nineteen patients with varied solid tumors, who were not candidates for standard chemotherapy, were administered an escalating duration (6, 9 or 12 consecutive days) of oral etoposide (a 25 mg capsule three times daily) combined with carboplatin AUC5 administered on day 1, by a 30 min intravenous infusion, to define the maximum tolerated dose on the basis of the acute toxicities that were reported. Etoposide was started on day 2; the cycles repeated every 28 days until disease progression or toxicity. Pharmacokinetics was carried out during the two first cycles. The maximum tolerated dose was determined to be the 12-day treatment level, with two cases of grade 4 neutropenia, grade 3 anemia and thrombocytopenia. As no severe toxicity occurred with the 9-day treatment level and in an attempt to explore an optimal combination, a new 10-day treatment plan was studied in three patients. As one patient presented dose-limiting toxicity at that level, five additional patients were included to establish the recommended regimen. Nonhematological toxicities among all patients were moderate, consisting of grade 2 nausea and asthenia. No treatment-related death occurred. Objective responses were observed in four patients and stabilization in three patients. Pharmacokinetics highlighted no interaction between etoposide and carboplatin. Fractionated oral etoposide (3×25 mg/day) for 10 days in combination with carboplatin AUC 5 presents acceptable toxicity and efficacy. The main toxicity remains hematological.

Background: Factor XIII plays a pivotal role in platelet aggregation. Factor XIII depends on lipid rafts to assemble the proteins required for clot retraction. Several proteins in this assembly remain unidentified. The aim of this study was to investigate which proteins localized in platelet lipid rafts are related to Factor XIII. Methods: Lipid rafts of platelets were isolated by using Lubrol WX 0.05% (w/v) and sucrose gradient. After ultracentrifugation, sucrose fractions were sampled. Lipidomic analysis was performed to identify the platelet lipid raft fractions enriched in cholesterol (Ch) and sphingomyelin (SM) and less so in phosphatidylcholine (PC). These fractions were selected for further proteomic analyses. The network of associated proteins was drawn by STRING database. Results: Sucrose fractions containing platelet lipid rafts were identified (Figure 1A). After proteomics analysis, an interactive network of proteins around FXIIIA was observed (Figure 1B). For these 19 proteins, their STRING score (range 0.576 to 0.900) and the type of their interaction with FXIIIA (activation, binding, catalysis and reaction) are summarized in Table 1. Conclusion: We identified 19 associated proteins with Factor XIII in human platelet lipid rafts. This could provide further insights into clinical targets of pathways through this molecule.

Background: The presence of CD34+ and CD146+ cells in the peripheral blood in response to myocardial ischemia has been advocated as a marker of an organism’s regenerative capacity by late and early progenitor cells respectively. The aim of this study was to evaluate the relationship between circulating CD34+ and CD146+ levels and the change in infarct size, as assessed by magnetic resonance imaging (MRI) in patients (pts) with acute coronary syndrome (ACS including ST and non ST elevation myocardial infarction). Methods: Patients Results: In total, 34 patients aged 57±10 years were included. Mean ejection fraction was 62±11%. Log-transformed number of CD 34+ and CD146+ were significantly correlated with the relative change in infarct size between day 5 and 6 month follow-up (p=0.01, r=-0;41 and p=0.001, r=-0.52 respectively) (Figure) Conclusion: The highest CD34+ and CD146+ cell levels were associated with the greatest relative reduction in infarct size 6 months after ACS. These results support the hypothesis that these cells could be considered as a marker of regenerative capacity in patients with acute coronary syndromes.

To determine the relationship between the number of CD14(+) cells, myocardial infarct (MI) size and left ventricular (LV) volumes in ST segment elevation MI (STEMI) and non-ST segment elevation MI (NSTEMI) patients.A total of 62 patients with STEMI (n=34) or NSTEMI (n=28) were enrolled. The number of CD14(+) cells was assessed at admission. Infarct size, left ventricular ejection fraction (LVEF) and LV volumes were measured using magnetic resonance imaging five days after MI and six months after MI.In STEMI patients, the number of CD14(+) cells was positively and significantly correlated with infarct size at day 5 (r=0.40; P=0.016) and after six months (r=0.34; P=0.047), negatively correlated with LVEF at day 5 (r=-0.50; P=0.002) and after six months (r=-0.46; P=0.005) and positively correlated with end-diastolic (r=0.38; P=0.02) and end-systolic (r=0.49; P=0.002) volumes after six months. In NSTEMI patients, no significant correlation was found between the number of CD14(+) cells and infarct size, LVEF or LV volumes at day 5 or after six months.The number of CD14(+) cells at admission was associated with infarct size and LV remodelling in STEMI patients with large infarct size, whereas in NSTEMI patients, no relationship was observed between numbers of CD14(+) cells and LV remodelling.

International audience; BACKGROUND: Intraperitoneal (IP) perioperative chemotherapy with cisplatin is an interesting option in ovarian cancer treatment. A combination of cisplatin with IP epinephrine (already shown to improve IP and decrease systemic platinum (Pt) exposure) was evaluated using a population pharmacokinetic analysis. METHODS: Data from 55 patients treated with cisplatin-based IP perioperative chemotherapy with (n=26) or without (n=29) epinephrine were analysed using NONMEM. RESULTS: Epinephrine halves clearance between peritoneum and serum (IPCL) and increases the Pt central volume of distribution, IP exposure and penetration in tissue. IPCL has a better predictive value than any other parameter with respect to renal toxicity. CONCLUSION: This confirms that IPCL could be useful in assessing renal toxicity. As IPCL is also linked to tissue penetration and IP exposure, it may be proposed as biomarker. In addition to a Bayesian estimation, we propose a single-sample calculation-way to assess it. Prospective studies are needed to validate IPCL as a biomarker in this context.

International audience; The anticancer drug capecitabine and its metabolites [including the active metabolite 5-fluorouracil (5-FU)] display high pharmacokinetic inter-patient variability. Such variability, which may lead to treatment failure or toxicity, could need drug concentration measurement to individualize dosing regimen. However, usual assay methods are often long and fastidious. A simultaneous and cost-effective method was thus developed for the determination of the concentrations of these compounds in human plasma. Compounds were extracted via a classic liquid-liquid extraction. Chromatographic analysis was performed on a C18 reverse phase column with detection by atmosphere pressure chemical ionization LC-MS/MS. Our method allows a good chromatographic separation of the compounds and was fully validated following Food and Drug Administration (FDA) recommendations (good selectivity, no carry-over, linearity of the calibration curves without weighting, deviations from nominal concentrations of standard samples lower than 15%, intra- and inter-assay precision and accuracy lower than 15%). Recovery and stability were also acceptable following the FDA guidelines. A matrix effect impairing the determination of 5-FU was avoided by using a stable isotopic derivative of 5-FU as internal standard. Interestingly, this method allows detection of TetraHydroUridine, an inhibitor of ex vivo degradation of metabolites, which is essential for the stability, the adequate conditioning of blood samples and for good laboratory practice, essential in routine determination. This method seems usable to routinely determine concentrations of capecitabine and its metabolites in blood and may be helpful in further studies aiming at performing therapeutic drug monitoring.

International audience

International audience; Ovarian cancer is the leading cause of gynecological cancer-related death in Western countries. The present treatment standards for ovarian cancer are based on the association of debulking surgery with platinum-based chemotherapy. Another strategy that could be further investigated is intraperitoneal chemotherapy (IP). We previously described that the 2-h administration of intraoperative IP cisplatin did not reach satisfactory concentrations. In the present study, we present the results of a pharmacokinetic analysis performed after two consecutive 1-h IP 30 mg/l cisplatin administrations. Twenty-seven patients with advanced epithelial cancer classified FIGO stage IIIC were included in the study. Blood and IP samples were taken over a 24-h period, during and after IP treatment. Both total and ultrafiltered (Uf) platinum (Pt) concentration levels were analyzed. Biological and clinical toxicities were also recorded. With this strategy, IP Pt concentrations stayed above the target concentration (10 mg/l) for a satisfactory length of time. The serum Pt concentrations were higher than those observed with the "one-bath" protocol and they induced the occurrence of recoverable renal toxicities (3 grade 1, 7 grade 2 and 4 grade 3). The best predictive parameter for renal failure was the total Pt 24-h Area Under the Curve (AUC) with a threshold value of 25 mg h/l RR = 0.31 (95% CI 0.13 - 0.49, P < 0.01). Administration of an increased amount of cisplatin is feasible and a satisfactory level of IP Pt concentrations is obtained. However, this improvement is associated with an increase in serum Pt levels and resulting renal toxicities. An attractive solution would be to decrease Pt transfer from peritoneum to bloodstream. A phase 1 study using intraoperative IP epinephrine in order to decrease this transfer is presently being carried out.

923 EMERGENCE OF A NEW OPPORTUNISTIC INFECTION IN EUROPE: HEPATIC ALVEOLAR ECHINOCOCCOSIS. A FIFTY-CASE REPORT A. Chauchet, F. Grenouillet, J. Knapp, C. Richou, E. Delabrousse, C. Dentan, S. Capelle, V. Di Martino, E. Deconinck, O. Blagosklonov, D.A. Vuitton, S. Bresson-Hadni, FrancEchino Network. WHO-Collaborating Centre for Prevention and Treatment of Human Echinococcosis and French National Reference Centre on Alveolar Echinococcosis, Franche-Comte University and University Hospital, Department of Hematology, Besancon University Hospital, UMR 6249 Chrono-Environnement, Besancon, Department of Rheumatology, University Hospital Grenoble, Grenoble, EA 3186, Franche Comte University, EA 3181, Franche-Comte University, Besancon, France E-mail: dr.bresson.hadni@wanadoo.fr