A pig model of chronic hepatitis E displaying persistent viremia and a downregulation of innate immune responses in the liver.

BACKGROUND: Hepatitis E virus (HEV) is a zoonotic virus transmitted by pig meat and responsible for chronic hepatitis E in immunocompromised patients. It has proved challenging to reproduce this disease in its natural reservoir. We therefore aimed to develop a pig model of chronic hepatitis E to improve the characterization of this disease. METHODS: Ten pigs were treated with a tacrolimus-based regimen and intravenously inoculated with HEV. Tacrolimus trough concentration, HEV viremia, viral diversity, innate immune responses, liver histology, clinical disease and biochemical markers were monitored for 11 weeks post-infection (p.i.). RESULTS: HEV viremia persisted for 11 weeks p.i. HEV RNA was detected in the liver, small intestine, and colon at necropsy. Histological analysis revealed liver inflammation and fibrosis. Several mutations selected in the HEV genome were associated with compartmentalization in the feces and intestinal tissues, consistent with the hypothesis of extrahepatic replication in the digestive tract. Antiviral responses were characterized by a downregulation of IFN pathways in the liver, despite an upregulation of RIG-I and ISGs in the blood and liver. CONCLUSIONS: We developed a pig model of chronic hepatitis E that reproduced the major hallmarks of this disease. This model revealed a compartmentalization of HEV genomes in the digestive tract and a downregulation of innate immune responses in the liver. These original features highlight the relevance of our model for studies of the pathogenesis of chronic hepatitis E and for validating future treatments.

Adjunction of a fish oil emulsion to cytarabine and daunorubicin induction chemotherapy in high-risk AML.

The treatment of acute myeloid leukemia (AML) with unfavorable cytogenetics treatment remains a challenge. We previously established that ex vivo exposure of AML blasts to eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), or fish oil emulsion (FO) induces Nrf2 pathway activation, metabolic switch, and cell death. The FILO group launched a pilot clinical study to evaluate the feasibility, safety, and efficacy of the adjunction of a commercial FO emulsion to 3 + 7 in untreated AML with unfavorable cytogenetics. The primary objective was complete response (CR). Thirty patients were included. FO administration raised the plasma levels of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids (p < 0.001). The pharmacokinetics of cytarabine and daunorubicin were unaffected. A historical comparison to the LAM2001 trial (Lioure et al. Blood 2012) found a higher frequency of grade 3 serious adverse events, with no drug-related unexpected toxicity. The CR rate was 77%, and the partial response (PR) 10%, not significantly superior to that of the previous study (CR 72%, PR 1%). RT-qPCR analysis of Nrf2 target genes and antioxidant enzymes did not show a significant in vivo response. Overall, FO emulsion adjunction to 3 + 7 is feasible. An improvement in CR was not shown in this cohort of high-risk patients. The present data does not support the use of FO in adjunction with 3 + 7 in high-risk AML patients.ClinicalTrials.gov identifier: NCT01999413.

High silver concentrations in biological samples following different exposures: Two case reports.

BACKGROUND: Silver is used in various industrial applications, but also in confectioneries and for therapeutic use due to its antibiotic properties. Its toxicity is not well documented and most often only in the context of professional exposure. AIM: Here we report two cases of high silver concentrations in biological samples in two women: the first patient presented grey marks around her cuticles, probably due to her consumption of silvered sweets and the second patient presented agranulocytosis and thrombocytopenia occurring within 24 h after the topical application of a cream containing sulfadiazine and silver to burns over a large surface area. METHODS: Silver concentrations were determined in blood and urine samples and sweets using inductively coupled plasma- mass spectrometry (ICP-MS). RESULTS: The silver concentrations were elevated compared to population reference values and confirmed the hypotheses for the patients: the significant consumption of sweets coated with silver nanoparticles and the topical application of a cream containing silver to burns over a large area. DISCUSSION-CONCLUSION: After initial questioning by the dermatologist, Patient 1 explained that she consumed more than 30 bags of the sweets per year. She decreased her consumption of the sweets and the control performed one year later showed a plasma silver concentration of 1.5 µg/L. For Patient 2, the absorption of silver through burns over a large area appeared relatively significant, whereas it is very low through undamaged skin. The haematological cells counts returned to normal levels quickly and no other major effects were highlighted. To apply these findings to a larger population, further investigation to determine sulfadiazine and silver concentrations in plasma and urine have been initiated in a cohort of patients with burns over a large area.

Intra-tracheal amikacin spray delivery in healthy mechanically ventilated piglets.

BACKGROUND: Nebulization during mechanical ventilation is impeded by large extra-pulmonary drug deposition and long administration durations which currently limit implementation of inhaled antibiotic therapy. Direct intra-tracheal delivery using a sprayer represents an appealing alternative investigated in small animal models, but large animal data are lacking. METHODS: Amikacin was administered through intravenous infusion (20 mg/kg), nebulization (60 mg/kg) and direct intra-tracheal spray (30 mg/kg) to 10 intubated piglets, in a randomized cross-over design. Amikacin concentrations were measured in the serum and pulmonary parenchyma. Anatomic deposition was investigated using immuno-histochemistry. RESULTS: Spray delivery resulted in higher amikacin outputs than nebulization and infusion. Pulmonary inhaled delivery techniques yielded much higher lung concentrations and much lower serum concentrations than intravenous infusion. However, unlike nebulization and infusion, intra-tracheal spray delivery was associated with more than 100- and 1000-fold variability in lung concentrations between and within animals. Amikacin specific immuno-histochemistry showed consistent bronchial and alveolar drug deposition with all modalities. CONCLUSION: Nebulization remains the most reliable and simple technique to deliver inhaled amikacin uniformly to the lung during mechanical ventilation. Further development of tracheal sprays is required to take advantage of potential benefits related to high drug output and low extra-pulmonary deposition in large animals.

A successful compartmental approach for the treatment of breast cancer brain metastases.

BACKGROUND: Brain metastases are challenging daily practice in oncology and remain a compartmental problem since most anti-cancer drugs do not cross the blood-brain barrier at relevant pharmacological concentrations. METHODS: In a young woman with HER2-overexpressing breast cancer resistant to standard treatments, at the time of brain metastases progression, a ventricular reservoir was implanted for intrathecal drug injections and detailed pharmacokinetic studies. RESULTS: A first association of intrathecal trastuzumab with intravenous cisplatin was offered to the patient. For trastuzumab, the mean cerebrospinal fluid trough concentration of 53.4 mg/L reached relevant levels, enabling the stabilization of the metastases. Adding intravenous cisplatin was not beneficial, since the cerebrospinal fluid exposure was almost undetectable under 0.08 mg/L. We then offered the patient an intrathecal combination of trastuzumab and methotrexate, because of their in vitro synergic cytotoxicity. The cerebrospinal fluid peak of methotrexate was 1037 µmol/L at 2 h, and the concentrations remained above the theoretical therapeutic concentration. After 2 months of this drug combination, we obtained an excellent response on the brain metastases. CONCLUSION: Our preliminary study supports the interest of a compartmental approach through a direct administration of drugs into the cerebrospinal fluid for the treatment of breast cancer brain metastases.

Enhanced Amikacin Diffusion With Ultrasound and Microbubbles in a Mechanically Ventilated Condensed Lung Rabbit Model.

The poor diffusion of intravenous antibiotics in lung tissue makes nosocomial pneumonia challenging to treat, notably in critical patients under mechanical ventilation. The combination of ultrasound and microbubbles (USMB) is an emerging method for non-invasive and targeted enhancement of uptake of various drugs in several organs. This study aims to evaluate if USMB may increase amikacin concentration in condensed lung tissues in a mechanically ventilated rabbit model. When applied 60 or 160 min after the beginning of an intravenous amikacin infusion, USMB increased amikacin concentration in the condensed lung tissue by 1.33 (p = 0.025) or 1.56-fold (p = 0.028) respectively. When applied 70 min after the beginning of an intravenous amikacin infusion, USMB increased amikacin concentration in the muscle tissue by 2.52 (p = 0.025). In conclusion, this study demonstrates that USMB is a promising method for the targeted delivery of amikacin in mechanically ventilated condensed lung, thus opening new therapeutic fields against lung infections.

T lymphocyte immunophenotyping in bronchoalveolar lavage on AQUIOS CL® cytometer.

Bronchoalveolar lavage (BAL) is one of the tests for orientation in the diagnosis of diffuse parenchymal lung disease. Lymphocyte subpopulation study by flow cytometry in the BAL is part of the criteria for the diagnosis of sarcoidosis. We investigated the feasibility of T lymphocyte immunophenotyping in BAL on a new automaton: AQUIOS CL®, a closed access cytometer with fully automated process designed for blood samples. Repeatability and contamination studies showed acceptable results (variation coefficients <5% and contamination indices <1%). We also compared BAL immunophenotyping results performed on AQUIOS CL® with those performed with an open access cytometer: Navios®. The correlation coefficient r is close to 1 on the studied parameters. The number of unacceptable measurement deviations is <5% and does not affect the biological interpretation of the results indicating a good correlation between the two automata. These results show that immunophenotyping in BAL is feasible on AQUIOS CL®.

A novel way to manage trastuzumab cardiotoxicity.

PURPOSE: Trastuzumab is the most widely prescribed anti-HER2 humanized monoclonal antibody. Cardiac toxicity is the only limiting toxicity of trastuzumab and it is of particular concern in patients with complete response, since the drug needs to be stopped, with a risk of disease relapse. To date, no pharmacological data on trastuzumab cardiotoxicity in patients have been made available. Here, we provide proof of concept, demonstrating that it was possible to prevent trastuzumab-induced cardiotoxicity by modifying the drug administration schedule. METHODS: In this paper, we report the case of a patient with metastatic breast cancer responding to trastuzumab, who developed severe cardiac toxicity twice using a 3-weekly regimen. Considering preclinical pharmacological data on trastuzumab cardiotoxicity, we hypothesized that a weekly schedule of trastuzumab with lower peaks of serum concentration could be safe while remaining efficient. With the patient's consent, we started a weekly combination of carboplatin (AUC2) and trastuzumab (2 mg/kg) with close monitoring of trastuzumab concentrations. RESULTS: We successfully controlled the disease for an additional 6 months with relevant trough concentrations of trastuzumab of around 50 mg/L. Another important aspect is that, with this weekly schedule, we observed no cardiac toxicity, and the left ventricular ejection fraction remained stabilized, at over 50%. CONCLUSIONS: Trastuzumab is the most widely prescribed anti-HER2 monoclonal antibody for the treatment of HER2 metastatic breast cancer, and it is the only drug that has been approved for the treatment of localized HER2 breast cancer, 1-year treatment being required after surgery. In case of cardiac toxicity, particularly in women over 60 years of age, a weekly regimen with lower peaks of concentration could be an alternative to the standard 3-weekly regimen.

Immunoassays for Measuring Serum Concentrations of Monoclonal Antibodies and Anti-biopharmaceutical Antibodies in Patients.

Monoclonal antibodies (mAbs) may be used as biopharmaceuticals to treat various diseases, ranging from oncology to inflammatory and cardiovascular affections. Trustworthy analytical methods are necessary to study their pharmacokinetics, both during their development and in post-marketing studies. Because biopharmaceuticals are macromolecules, ligand-binding assays (both immunoassays and bioassays) are methods of choice to measure their concentrations. Immunoassays are based on the capture of biopharmaceuticals by their target, which may be a circulating or membrane antigen or by an antibody recognizing their structure. Bioassays measure the activity of the biopharmaceutical in a specific in vitro test. A number of techniques have been reported, but their limits of detection and quantification vary widely. Anti-drug antibodies (ADA) against biopharmaceuticals are often formed and sometimes interfere with clinical efficacy. Accurate and reliable detection of ADA is therefore necessary. Binding of ADA is dependent on affinity and avidity, which makes quantification challenging. In this review, we discuss the benefits and limitations of each method to determine mAb levels and carefully compare ADA assays.

Vancomycin during the Last Hour of the Hemodialysis Session: A Pharmacokinetic Analysis.

BACKGROUND: Vancomycin is usually administered after the dialysis sessions to patients undergoing hemodialysis. Administration of vancomycin during (as opposed to after) sessions would save time, and would be more acceptable to patients and staff, but may lead to vancomycin underexposure. The aim of this study was to propose a new dosing regimen of vancomycin taking into account the dialysis-related losses of vancomycin when administered during dialysis. METHODS: In this monocentric prospective study, vancomycin was infused to dialyzed patients during the last hour of the dialysis session at increased doses. Monitoring of vancomycin was performed using repeated blood samples by pharmacokinetics modeling. Patients were treated according to our protocol and guidelines. RESULTS: Twenty patients were included. Vancomycin protocol was efficient: 17 of 20 (85%) patients were cured, 1 needed additional vancomycin treatment, and 2 died (sepsis n = 1, multiple organ failure n = 1). Median pre-dialysis concentration was adequate (16.2 [10.2-24.4] µg/mL), and there was no emergence of resistant bacteria. Infusion of vancomycin during dialysis therefore decreased the exposure to vancomycin by 25% compared to infusion of vancomycin after dialysis. For a typical patient, the dose of vancomycin to be administered during dialysis would be 1.4 g. CONCLUSION: Administration of vancomycin during the last hour of dialysis session is safe, efficacious with regards to infection control, achieved recommended vancomycin concentrations despite the use of high-flux membranes, and improved patients' quality of life.

Development and validation of an enzyme-linked immunosorbent assay to measure adalimumab concentration.

BACKGROUND: Adalimumab is a therapeutic antibody used for treating inflammatory diseases. To understand interindividual PK variability, there is a need to develop and validate an assay to measure serum adalimumab concentrations. METHODS: An ELISA was developed on microtiter plates coated with TNF-α. Seven nonzero adalimumab standards ranging from 0.05 to 50 mg/l and three quality controls (0.2, 2.5 and 7 mg/l) were tested for their intra and interday precision on six occasions. RESULTS: The LOD, LLOQ and ULOQ of the assay were 0.022, 0.073 and 9 mg/l, respectively. CONCLUSION: This method is accurate, reproducible and may be useful for PK studies and for therapeutic drug monitoring of adalimumab.

Aerosol Route to Administer Teicoplanin in Mechanical Ventilation: In Vitro Study, Lung Deposition and Pharmacokinetic Analyses in Pigs.

BACKROUND: Glycopeptides given intravenously achieve low airway concentrations. Nebulization of teicoplanin may be an efficient way of delivering a high concentration of this antibiotic to the lung. This multistep study assessed the feasibility of teicoplanin nebulization during mechanical ventilation by evaluating: the stability of its antibiotic effect; epithelial tolerance; lung deposition and systemic absorption in ventilated pigs. METHODS: Nebulized and non-nebulized teicoplanin activity was tested on Staphylococcus aureus cultures. The cytotoxic effect of teicoplanin on human respiratory epithelial cells was assessed by measuring lactate dehydrogenase activity released, cell viability, and transepithelial electrical resistance. Volume median diameter of particles of nebulized teicoplanin was measured by laser diffraction during mechanical ventilation. The deposited mass of teicoplanin nebulized with a vibrating mesh nebulizer in ventilated piglets was assessed by scintigraphy. Blood pharmacokinetics of teicoplanin administered either intravenously or by nebulization was compared. RESULTS: No decrease of antibiotic activity was observed after nebulization. In vitro cytotoxicity of teicoplanin was only observed with 1000 times the dose recommended for intravenous administration. Volume median diameter of particles was 2.5±0.1 µm. Of the initial nebulizer charge of teicoplanin, 24±7% was present in the lungs of ventilated pigs after the nebulization. Amount absorbed in blood was low (3.4%±0.9%) after nebulization, and blood stream elimination half-life value was 25.4 h. CONCLUSIONS: Teicoplanin was administered efficiently by nebulization during mechanical ventilation, without any effect on its pharmacological properties or any cytotoxicity. The pharmacokinetic parameters are promising in view of its time-dependent killing process. All the results of our multi-step study highlighted the potential of teicoplanin to be nebulized during mechanical ventilation.

Lung and serum teicoplanin concentration after aerosol and intravenous administration in a rat model.

BACKGROUND: Ventilator-associated pneumonia (VAP) caused by methicillin-resistant Staphylococcus aureus is a major cause of mortality in mechanically ventilated patients. Nebulization of teicoplanin is an alternative way of administration that may provide higher lung tissue concentrations than intravenous (IV) delivery. The aim of this study was to compare the administration of teicoplanin via aerosol with the IV route by measuring the lung and the serum teicoplanin concentrations in a rat model. METHODS: Eighty healthy male Sprague-Dawley rats were anesthetized and received a single dose of teicoplanin by the IV or aerosol route. After sacrifice, lung and blood samples were collected and teicoplanin concentrations were measured with fluorescence polarization. A noncompartmental approach was used. The area under the concentration curve/minimal inhibition concentration ratio (AUC/MIC), AUC, absorbed fraction, mean residence time (MRT), and mean absorption time (MAT) of teicoplanin were calculated. RESULTS: Mean±SD lung tissue concentrations of teicoplanin in the aerosol group were significantly higher than those in the IV group (p<0.0001). The mean lung tissue concentration achieved at 15 min was 1.94±1.33 mg/g in the aerosol group and 0.04±0.01 mg/g in the IV group. The mean AUClung was 67.4 mg hr(-1)g(-1) after aerosol and 0.8 mg hr(-1)g(-1) after the IV route. In the aerosol group, AUCserum/MIC ratio was 605/2, and in the IV route, AUCserum/MIC ratio was 682/2. MAT was longer after aerosol than after the IV route (0.91 hr versus 0.06 hr), and MRT was longer after aerosol than after IV bolus administration (6.52 hr versus 5.61 hr). CONCLUSION: Teicoplanin concentrations in the lung tissue of the rat model were significantly higher by the aerosol route than by the IV route. The AUClung after nebulization was 84 times higher than delivery by the IV route, and the AUClung/MIC ratio after nebulization met the recommended target to eradicate Staphylococcus aureus. Administration of teicoplanin by the aerosol route could represent one of the new therapeutic weapons of the treatment of the VAP.

An automated method for measurement of methoxetamine in human plasma by use of turbulent flow on-line extraction coupled with liquid chromatography and mass spectrometric detection.

Methoxetamine is a new ketamine derivative designer drug which has recently become available via the Internet marketed as "legal ketamine". It is a new dissociative recreational drug, acting as an NMDA receptor antagonist and dopamine reuptake inhibitor. The objective of this study was to develop on-line automated sample preparation using a TurboFlow device coupled with liquid chromatography with ion-trap mass spectrometric detection for measurement of methoxetamine in human plasma. Samples (100 µL) were vortex mixed with internal standard solution (ketamine-d4 in acetonitrile). After centrifugation, 20 µL of the supernatant was injected on to a 50 mm × 0.5-mm C18XL Turboflow column. The retained analytes were then back-flushed on to a 50 mm × 3-mm (3 µm) Hypersil Gold analytical column for chromatographic separation, then eluted with a formate buffer-acetonitrile gradient. Methoxetamine and the IS were ionized by electrospray in positive mode. Parent [M + H](+) ions were m/z 248.1 for methoxetamine and m/z 242.0 for the IS. The most intense product ions from methoxetamine (m/z 203.0) and the IS (m/z 224.0) were used for quantification. The assay was accurate (96.8-108.8% range) and precise (intra and inter-day coefficients of variation <8.8%) over the range of 2.0 (lower limit of quantification) to 1000.0 ng mL(-1) (upper limit of quantification). No matrix effect was observed. This method has been successfully applied to determination of plasma concentrations of methoxetamine in the first French hospitalization case report after acute intoxication; the plasma concentration was 136 ng mL(-1).

Isotretinoin exposure during pregnancy: assessment of spontaneous reports in France.

BACKGROUND: In three previous studies, we have shown that pregnant women were still being exposed to isotretinoin and that compliance with recommendations was incomplete. The relaxation of these recommendations (summary of product characteristics 2004), combined with the release of generic brands, encouraged us to carry out a fourth study. OBJECTIVE: To assess isotretinoin exposure during pregnancy following the application of less stringent recommendations and the marketing of generic isotretinoin brands. METHODS: All cases of isotretinoin exposure during pregnancy, between 1 January 2003 and 31 December 2006, spontaneously reported to pharmacovigilance centres, the Teratogenic Agent Information Centre, and pharmaceutical companies in France were assessed. Cases were classified for analysis into the following groups: 'conception <1 month after isotretinoin discontinuation', 'conception during isotretinoin treatment' and 'patient already pregnant when isotretinoin was started'. The rate of spontaneously reported isotretinoin exposure during pregnancy was estimated by dividing the number of isotretinoin-exposed pregnancies by the number of women of childbearing age treated with isotretinoin. RESULTS: Over 4 years, 147 cases of isotretinoin exposure during the teratogenic risk period were spontaneously reported, i.e. 'conception <1 month after isotretinoin discontinuation' (23%), 'conception during isotretinoin treatment' (61%), and 'patient already pregnant when isotretinoin was started' (16%). Nineteen percent of the patients did not use any form of contraception. In 23% of the patients, the method of contraception used did not comply with recommendations, while in 86% of the cases, isotretinoin was prescribed by a dermatologist. Among the 44 pregnancies with available data on fetuses or neonates, there were two (4.5%) malformations compatible with the time of exposure and with isotretinoin embryopathy. The rate of spontaneously reported isotretinoin exposure during pregnancy has increased by approximately 30%, from 0.32 (95% CI 0.26, 0.38) to 0.41 (95% CI 0.34, 0.49) per 1000 women of childbearing age treated since 1999-2002. CONCLUSIONS: We suggest that recommendations be tightened, with specific information regarding the most effective contraceptive method combined with compulsory monthly pregnancy testing during treatment. The French Drug Agency has informed the European Medicines Agency of the need for measures aimed at improving compliance.

Molecular lipophilicity determination of a huperzine series by HPLC: comparison of C18 and IAM stationary phases.

Two hydrophobic parameters (logkw-C18 and logkw-IAM, respectively) of a huperzine A series were extrapolated by high performance liquid chromatography (HPLC) using both C18 and immobilised artificial membrane (IAM) columns. A mathematical correlation between C18 and IAM hydrophobic parameters was completed, suggesting a similar behaviour on both columns. This behaviour was principally led by hydrophobic forces. The theoretical lipophilicity (logP) of each compound was computed using Pallas software and compared to experimental values, showing a similar lipophilic behaviour. Finally, the huperzine logkw-IAM and logkw-C18 values were correlated with the relative bound percentage of huperzine in human serum albumin, confirming that hydrophobic forces are predominant in the huperzine-HSA binding mechanism.

Huperzine A--human serum albumin association: chromatographic and thermodynamic approach.

The synthesis of six new huperzine analogues was reported. Each product presents an amidification of the free amine on huperzine A. The synthesis strategy of these new huperzine A derivatives is based on a condensation with an acyl anhydride. The binding on HSA of two molecule series (huperzine and benzodiazepine, respectively) was investigated with high performance liquid affinity chromatography (HPLAC) using an HSA column. A thermodynamic approach showed that binding huperzine A on HSA involved hydrophobic and Van der Waals interactions. A comparative thermodynamic study with benzodiazepine molecules was carried out to determine the potential binding site of huperzine derivatives on HSA.