Flavoured and nicotine-containing e-liquids impair homeostatic properties of an alveolar-capillary cell model.

Most people consider that electronic cigarettes are safer than tobacco and are marketed as quit-smoking products. The e-liquid, which usually contains propylene glycol (PG) and vegetable glycerin (VG) in different ratios, nicotine and a wide variety of flavours, is heated by a coil and the aerosol droplets are primarily delivered to the alveolar area where nicotine and other molecules cross the alveolar-capillary barrier (ACB). However, e-cigarettes effects on the ACB are not yet established. In our study, a well-characterised in vitro model of the ACB was exposed to PG and VG and to five flavoured e-liquids with and without nicotine. The vehicles, due to their hypertonic properties, modulated the ACB integrity by modifying occludin expression. Below a 10% concentration, the vehicles did not trigger oxidative stress or cell death. Different results were observed between flavoured e-liquids: while red fruits and mint-eucalyptus disrupted ACB integrity, triggered oxidative stress and cell death, blond tobacco had no worse effect compared to the vehicles. However, the addition of nicotine in the latter e-liquid increased oxidative stress and cell death compared to the vehicles. Finally, mint-eucalyptus e-liquid increased some inflammation markers. Our results revealed that e-liquids alter ACB homeostasis, depending on flavour and nicotine presence.

Sirolimus Pharmacokinetics Variability Points to the Relevance of Therapeutic Drug Monitoring in Pediatric Oncology.

Sirolimus is widely used in transplantation, where its therapeutic drug monitoring (TDM) is well established. Evidence of a crucial role for sirolimus in the PI3K/AkT/mTor pathway has stimulated interest in its involvement in neoplasia, either as monotherapy or in combination with other antineoplastic agents. However, in cancer, there is no consensus on sirolimus TDM. In the RAPIRI phase I trial, the combination sirolimus + irinotecan was evaluated as a new treatment for refractory pediatric cancers. Blood sampling at first sirolimus intake (D1) and at steady state (D8), followed by LC/MS2 analysis, was used to develop a population pharmacokinetic model (Monolix® software). A mono-compartmental model with first-order absorption and elimination best fit the data. The only covariate retained for the final model was "body surface area" (D1 and D8). The model also demonstrated that 1.5 mg/m2 would be the recommended sirolimus dose for further studies and that steady-state TDM is necessary to adjust the dosing regimen in atypical profiles (36.4% of the population). No correlation was found between sirolimus trough concentrations and efficacy and/or observed toxicities. The study reveals the relevance of sirolimus TDM in pediatric oncology as it is needed in organ transplantation.

SFCE-RAPIRI Phase I Study of Rapamycin Plus Irinotecan: A New Way to Target Intra-Tumor Hypoxia in Pediatric Refractory Cancers.

Hypoxic environment is a prognostic factor linked in pediatric cancers to a worse outcome, favoring tumor progression and resistance to treatments. The activation of mechanistic Target Of Rapamycin (mTor)/hypoxia inducible factor (HIF)-1 pathway can be targeted by rapamycin and irinotecan, respectively. Therefore, we designed a phase I trial associating both drugs in pediatric refractory/relapsing solid tumors. Patients were enrolled according to a 3 + 3 escalation design with ten levels, aiming to determine the MTD (maximum tolerated dose) of rapamycin plus irinotecan. Rapamycin was administered orally once daily in a 28-day cycle (1 to 2.5 mg/m2/day), associating biweekly intravenous irinotecan (125 to 240 mg/m2/dose). Toxicities, pharmacokinetics, efficacy analyses, and pharmacodynamics were evaluated. Forty-two patients, aged from 2 to 18 years, were included. No MTD was reached. Adverse events were mild to moderate. Only rapamycin doses of 1.5 mg/m2/day reached over time clinically active plasma concentrations. Tumor responses and prolonged stable disease were associated with a mean irinotecan area under the curve of more than 400 min.mg/L. Fourteen out of 31 (45.1%) patients had a non-progressive disease at 8 weeks. Most of them were sarcomas and brain tumors. For the phase II trial, we can then propose biweekly 125 mg/m2 irinotecan dose with a pharmacokinetic (PK) follow-up and a rapamycin dose of 1.5 mg/m2/day, reaching a blood concentration above 10 g/L.

New dosing nomogram and population pharmacokinetic model for young and very young children receiving busulfan for hematopoietic stem cell transplantation conditioning.

BACKGROUND: Busulfan (Bu) is the cornerstone of conditioning regimens prior to hematopoietic stem cell transplantation, widely used in both adults and children for the treatment of malignant and nonmalignant diseases. Despite an intravenous formulation, interindividual variability (IIV) remains high and optimal exposure difficult to achieve, especially in neonates and infants. PROCEDURE: To ensure both efficacy and safety, we set up in 2005 an observational study designed for children not fully assessed during the drug registration procedure. From a large cohort of 540 patients, we developed a Bu population pharmacokinetic model based on body weight (BW) and maturation concepts to reduce IIV and optimize exposure. A new dosing nomogram was evaluated to better fit the population pharmacokinetic model. RESULTS: Bu clearance IIV was significantly decreased from 61.3% (covariate-free model) to 28.6% when combining BW and maturation function. Median Bu area under the curve (AUC) was 1179 µmol/L × min compared to 1025 with the EMA dosing nomogram for children <9 kg. The target AUC was reached for each BW strata, significantly increasing the percentages of patients achieving reaching the targeted AUC as compared to FDA schedule. CONCLUSION: This new model made it possible to propose a novel dosing nomogram that better considered children below 16 kg of BW and allowed better initial exposure as compared to existing dosing schedules. This nomogram, which would be easy to use to determine an optimal dosing schedule in daily practice, will need to be validated in clinical routine. Therapeutic drug monitoring remains strongly advisable for small children and those with specific diseases.

Protective effect of 4-Phenylbutyrate against proteolipid protein mutation-induced endoplasmic reticulum stress and oligodendroglial cell death.

Proteolipid protein (PLP) mutation causes oligodendrocyte degeneration and myelin disorders including Pelizaeus-Merzbacher Disease (PMD). As the pathophysiological mechanisms involved in PMD are poorly known, the development of therapies remains difficult. To elucidate the pathogenic pathways, an immortalized oligodendroglial cell line (158JP) expressing PLP mutation has been generated. Previous investigations revealed that 158JP oligodendrocytes exhibit several abnormalities including aberrant PLP insertion into the plasma membrane, cAMP, plasmalogen and cell cycle deficits. However, further clarifications of abnormal PLP-induced oligodendrocyte degeneration are required in order to identify relevant mechanisms to target for efficient protection against oligodendrocyte death. Because PLP overexpression may lead to its accumulation inside the endoplasmic reticulum (ER) and cause ER-stress, we explored whether ER-stress may pivotally determine 158JP cell survival/death. Viability assays, RT-qPCR, western blot and flow cytometry were combined to compare cell survival, ER-stress and apoptotic markers in 158JP and control (158N) oligodendrocytes. We observed a significant decreased viability/survival of 158JP compared to 158N cells. Consistently, ER-stress markers (BiP, caspase-12) increased in 158JP (+30%) compared to the controls. mRNA and protein ratios of apoptotic modulators (Bax/Bcl2) are higher in 158JP oligodendrocytes which are also more vulnerable than 158N cells to tunicamycin-induced ER-stress. Interestingly, 4-Phenylbutyrate (ER-stress inhibitor), which decreased ER-stress and apoptotic markers in 158JP cells, significantly increased their survival. Our results, which show a direct link between the viability and endogenous levels of ER-stress and apoptotic markers in 158JP cells, also suggest that 4-Phenylbutyrate-based strategy may contribute to develop effective strategies against oligodendrocyte dysfunctions/death and myelin disorders.

Skeletal muscle ischemia-reperfusion injury and cyclosporine A in the aging rat.

Old patients exhibit muscle impairments and increased perioperative risk during vascular surgery procedures. Although aging generally impairs protective mechanisms, data are lacking concerning skeletal muscle in elderly. We tested whether cyclosporine A (CsA), which protects skeletal muscle from ischemia-reperfusion (IR) in young rats, might reduce skeletal muscle mitochondrial dysfunction and oxidative stress in aging rats submitted to hindlimb IR. Wistar rats aged 71-73 weeks were randomized to IR (3 h unilateral tourniquet application and 2 h reperfusion) or IR + CsA (10 mg/kg cyclosporine IV before reperfusion). Maximal oxidative capacity (VM ax ), acceptor control ratio (ACR), and relative contribution of the mitochondrial respiratory chain complexes II, III, IV (VS ucc ), and IV (VTMPD /Asc ), together with calcium retention capacity (CRC) a marker of apoptosis, and tissue reactive oxygen species (ROS) production were determined in gastrocnemius muscles from both hindlimbs. Compared to the nonischemic hindlimb, IR significantly reduced mitochondrial coupling, VMax (from 7.34 ± 1.50 to 2.87 ± 1.22 µMO2 /min/g; P < 0.05; -70%), and VS ucc (from 6.14 ± 1.07 to 3.82 ± 0.83 µMO2 /min/g; P < 0.05; -42%) but not VTMPD /Asc . IR also decreased the CRC from 15.58 ± 3.85 to 6.19 ± 0.86 µMCa(2+) /min/g; P < 0.05; -42%). These alterations were not corrected by CsA (-77%, -49%, and -32% after IR for VM ax, VS ucc , and CRC, respectively). Further, CsA significantly increased ROS production in both hindlimbs (P < 0.05; +73%). In old rats, hindlimb IR impairs skeletal muscle mitochondrial function and increases oxidative stress. Cyclosporine A did not show protective effects.

Mitomycin C Pharmacokinetics as Predictor of Severe Neutropenia in Hyperthermic Intraperitoneal Therapy.

BACKGROUND: Cytoreductive surgery (CRS) combined with hyperthermic intraperitoneal chemotherapy (HIPEC) is an approach to overcome peritoneal carcinomatosis from colorectal adenocarcinoma. Mitomycin C (MMC) is frequently used but not devoid of toxicity, of which the most common and feared is neutropenia. Our study explores the clinical and surgical risk factors of neutropenia and a possible link between MMC pharmacokinetics and neutropenia as HIPEC's supervention. METHODS: A total of 45 patients undergoing CRS-HIPEC for peritoneal carcinomatosis of colorectal origin between 2004 and 2010 were followed. For each patient, MMC was measured in plasma at different times during HIPEC and the area under the MMC concentration-time curve (MMC-AUC) was calculated. RESULTS: The incidence of neutropenia was 40 %. No demographic, clinical, or surgical factors increased the risk of neutropenia. However, we found that the occurrence of neutropenia and its gravity increased in direct correlation with an increase in MMC plasma concentration 30 min (T30) and 45 min (T45) after the start of HIPEC. The same correlation was observed between the MMC-AUC and the risk of neutropenia. CONCLUSIONS: Neutropenia is a frequent complication associated with MMC-HIPEC. The results of our study indicate the feasibility and the potential benefit of a protocol including the MMC dosage at T30 after the start of HIPEC. A threshold of 572 µg/L gives a predictive sensitivity of 86 % and a specificity of 80 %. These results must be considered in the management of patients undergoing MMC-HIPEC in order to place high-risk patients under neutropenic monitoring while the other patients can undergo simple hematological monitoring.

Pharmacokinetic behavior and appraisal of intravenous busulfan dosing in infants and older children: the results of a population pharmacokinetic study from a large pediatric cohort undergoing hematopoietic stem-cell transplantation.

BACKGROUND: Intravenous (IV) busulfan (Bu) dosing approved in Europe based on 5 body weight (BW) strata has been validated for targeting Bu exposures in children undergoing hematopoietic stem-cell transplantation and with mostly malignant diseases. The authors conducted an observational study aiming to investigate the behavior and ontogeny of IV Bu pharmacokinetic (PK) disposition, and to reevaluate the consistency of the BW-based dosing in very young children with rare diseases. METHODS: The observational study comprised 115 patients, mostly infants with immunodeficiencies and metabolic inherited disorders and with altered liver function and/or iron overload. Additional data (90 children, mostly malignant diseases) were pooled with the first data set. The overall data (205 children aged from 10 days to 15 years) were analyzed using population PK modeling. RESULTS: The BW remained the main determinant of IV Bu PK, and no further covariate effect was identified. Bu clearance (CL) variability was best described by BW allometric functions. Increase of drug CL with the child's growth was faster in younger children. This pattern is likely related to the maturation of GSTA1 enzymes during infancy and was accounted for in the model by estimating a higher BW allometric exponent in children <9 kg compared with that in children ≥9 kg. IV Bu PK was not modified in children with altered liver function and/or iron overload, and no disease specific difference was observed. Bu dosing either adjusted according to the final model or with the approved EU labeling yields similar targeting performances. For both dosing strategies, the percent of patients achieving the therapeutic area under the curve window (900-1500 µmole·min/L were 60% and 70%-90% in children <9 and ≥9 kg, respectively. CONCLUSIONS: A population PK model accounting for the highest Bu CL in the youngest patients was validated on training and evaluation data sets. The BW-based dosing strategy recommended in Europe proved to be consistent on a large paediatric cohort representative of the population heterogeneity observed in hematopoietic stem-cell transplantation.

Ventricular bigeminy associated with voriconazole, methadone and esomeprazole.

CASE: We report a case of ventricular bigeminy with concomitant administration of methadone, voriconazole and esomeprazole in a Caucasian woman aged 26 with acute lymphoblastic leukaemia. Plasma concentrations of voriconazole and methadone were high, 12.4 mg/l (therapeutic range: 1-4 mg/l) and 1.6 mg/l (therapeutic range: 0.2-0.4 mg/l), respectively. In the absence of esomeprazole, no more episode of cardiac arrhythmia occurred and 7 days after, methadone plasma concentration fell at 0.57 ml/l while voriconazole concentration was at 5.5 mg/l. We speculate that a pharmacokinetic interaction between methadone and voriconazole was amplified by the addition of esomeprazole. This led to the large increase of the plasma concentration of methadone and was potentially responsible for its cardiac toxicity. CONCLUSION: Physicians should be aware of the potential interaction between voriconazole, esomeprazole and methadone leading to arrhythmia. The inhibitory potential of voriconazole is possibly increased by esomeprazole.

Severe intoxication with methotrexate possibly associated with concomitant use of proton pump inhibitors.

BACKGROUND: Delayed elimination of methotrexate associated with serious side-effects has been attributed to the co-administration of benzimidazole proton pump inhibitors. PATIENTS AND METHODS: We have retrospectively analyzed the causes of delayed methotrexate elimination in patients who had received the rescue agent glucarpidase to evaluate the potential implication of benzimidazoles. RESULTS: Between 2002 and 2008, six patients (mean age: 30 years; range: 4-74 years) were treated with glucarpidase. Delayed elimination associated with impaired renal function occured after the first cycle except in 2 patients (2nd and 8th administration of high-dose methotrexate). The possible causes of delayed elimination identified were: insufficient hydration (n=1) and drug-drug interactions (n=5). The potential drug-drug interactions included the co-administration of piperacillin/tazobactam (n=1) and proton pump inhibitors (omeprazole, n=3; esomeprazole, n=2). Impaired elimination of methotrexate was not observed either in the 3 patients who were treated further or during the previous cycles of the 2 pretreated patients in relation to the absence of co-prescription of proton pump inhibitors. CONCLUSION: In line with the recent literature and given the prohibitive cost of glucarpidase, we have advocated the cessation of proton pump inhibitors administration during methotrexate treatment.

Endogenous morphine in SH-SY5Y cells and the mouse cerebellum.

BACKGROUND: Morphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system. METHODOLOGY AND PRINCIPAL FINDINGS: Microscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca(2+)-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10(-10) M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies. CONCLUSIONS/SIGNIFICANCE: The presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a specific function in neuromodulation and/or neurotransmission. Furthermore, its presence in cerebellar basket cell termini suggests that morphine has signaling functions in Purkinje cells that remain to be discovered.

Xanthurenic acid distribution, transport, accumulation and release in the rat brain.

Tryptophan metabolism through the kynurenine pathway leads to several neuroactive compounds, including kynurenic and picolinic acids. Xanthurenic acid (Xa) has been generally considered as a substance with no physiological role but possessing toxic and apoptotic properties. In the present work, we present several findings which support a physiological role for endogenous Xa in synaptic signalling in brain. This substance is present in micromolar amounts in most regions of the rat brain with a heterogeneous distribution. An active vesicular synaptic process inhibited by bafilomycin and nigericin accumulates xanthurenate into pre-synaptic terminals. A neuronal transport, partially dependant on adenosine 5'-triphosphate (ATP), sodium and chloride ions exists in NCB-20 neurons which could participate in the clearance of extracellular xanthurenate. Both transports (neuronal and vesicular) are greatly enhanced by the presence of micromolar amounts of zinc ions. Finally, electrical in vivo stimulation of A10-induced Xa release in the extracellular spaces of the rat prefrontal cortex. This phenomenon is reproduced by veratrine, K+ ions and blocked by EGTA and tetrodotoxin. These results strongly argue for a role for Xa in neurotransmission/neuromodulation in the rat brain, thus providing the existence of specific Xa receptors.

[A mechanism for gamma-hydroxybutyrate (GHB) as a drug and a substance of abuse]. / Mécanismes d'action d'un médicament détourné: le gamma-hydroxybutyrate.

Gamma-hydroxybutyrate (GHB) is mainly known because of its popularity as a drug of abuse among young individuals. However this substance increases slow-wave deep sleep and the secretion of growth hormone and besides its role in anaesthesia, it is used in several therapeutic indications including alcohol withdrawal, control of daytime sleep attacks and cataplexy in narcoleptic patients and is proposed for the treatment of fibromyalgia. GHB is also an endogenous substance present in several organs, including brain where it is synthesized from GABA in cells containing glutamic acid decarboxylase, the marker of GABAergic neurons. GHB is accumulated by the vesicular inhibitory aminoacid transporter (VIAAT) and released by depolarization via a Ca2+ dependent-mechanism. A family of GHB receptors exists in brain which possesses hyperpolarizing properties through Ca2+ and K+ channels. These receptors--one of them has been recently cloned from rat brain hippocampus--are thought to regulate GABAergic activities via a subtle balance between sensitized/desensitized states. Massive absorption of GHB desensitize GHB receptors and this modification, together with a direct stimulation of GABAB receptors by GHB, induce a perturbation in GABA, dopamine and opiate releases in several region of the brain. This adaptation phenomenon is probably responsible for the therapeutic and recreative effects of exogenous GHB.