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THE PATIENT A 24-year-old male patient, known to have tuberous sclerosis complex, developed renal epithelioid angiomyolipomas with hepatic metastasis. Everolimus treatment failed, this lead to a switch to temsirolimus treatment without more efficiency that appeared later on was due to drug-drug interaction which was dealt with in an effective manner

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.

Simple Summary More and more relapsing or refractory pediatric cancers are described to present hypoxic features linked to a worse outcome. Therefore, the aim of our phase I study RAPIRI was the targeting of the central node mTor/HIF-1α with rapamycin plus irinotecan and determine the appropriated dose of this combination. As expected, the tolerance was optimal across all dose levels and no maximum tolerated dose of both drugs was reached. The pharmacokinetics (PK) helped us to refine the doses to use in the future phase II trial and the importance of PK follow-up in such combination. We also confirmed in almost half of the interpretable patients for tumor response a non-progressive disease. All those observations additionally to the ancillary’s studies provide strong evidence to propose a next trial focusing on brain tumors and sarcomas and using biweekly 125 mg/m2 irinotecan dose with a PK follow-up and a rapamycin dose of 1.5 mg/m2/day, reaching a blood concentration above 10 µg/L. Abstract 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)-1α 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 µg/L.

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

Background: Hydroxyurea (HU) is a FDA- and EMA-approved drug that earned an important place in the treatment of patients with severe sickle cell anemia (SCA) by showing its efficacy in many studies. This medication is still underused due to fears of physicians and families and must be optimized. Methods: We analyzed our population and identified HU pharmacokinetic (PK) parameters in order to adapt treatment in the future. Working with a pediatric population, we searched for the most indicative sampling time to reduce the number of samples needed. Results: Nine children treated by HU for severe SCA were included for this PK study. HU quantification was made using a validated gas chromatography/mass spectrometry (GC/MS) method. Biological parameters (of effectiveness and compliance) and clinical data were collected. None of the nine children reached the therapeutic target defined by Dong et al. as an area under the curve (AUC) = 115 h.mg/L, four patients were suspected to be non-compliant. Only two patients had an HbF over 20%. The 2 h sample was predictive of the medication exposure (r2 = 0.887). Conclusions: It is urgent to be more efficient in the treatment of SCA, and pharmacokinetics can be an important asset in SCA patients.

International audience; The impact of vitamin D on sensory function, including pain processing, has been receiving increasing attention. Indeed, vitamin D deficiency is associated with various chronic pain conditions, and several lines of evidence indicate that vitamin D supplementation may trigger pain relief. However, the underlying mechanisms of action remain poorly understood. We used inflammatory and non-inflammatory rat models of chronic pain to evaluate the benefits of vitamin D3 (cholecalciferol) on pain symptoms. We found that cholecalciferol supplementation improved mechanical nociceptive thresholds in monoarthritic animals and reduced mechanical hyperalgesia and cold allodynia in a model of mononeuropathy. Transcriptomic analysis of cerebrum, dorsal root ganglia and spinal cord tissues indicate that cholecalciferol supplementation induces a massive gene dysregulation which, in the cerebrum, is associated with opioid signaling (23 genes), nociception (14), and allodynia (8), and, in the dorsal root ganglia, with axonal guidance (37 genes), and nociception (17). Among the identified cerebral dysregulated nociception-, allodynia-and opioid-associated genes, 21 can be associated with vitamin D metabolism. However, it appears that their expression is modulated by intermediate regulators such as diverse protein kinases and not, as expected, by the vitamin D receptor. Overall, several genes-Oxt, Pdyn, Penk, Pomc, Pth, Tac1, Tgfb1-encoding for peptides/hormones stand out as top candidates to explain the therapeutic benefit of vitamin D3 supplementation. Further studies are now warranted to detail the precise mechanisms of action but also the most favourable doses and time windows for pain relief.

Introduction Serotonin (5-HT) plays a major role in cardiovascular regulations. The 5-HT4 Receptor (5-HT4R) elicits positive cardiac inotropic responses and contributes to atrial arrhythmias and ventricular hypertrophy. Nevertheless, headache is a common side effect of prucalopride, a non-arrhythmogenic 5-HT4R agonist. 5-HT released by platelets has been suggested to modulate Blood-Brain-Barrier (BBB) permeability. We hypothesized that 5-HT, through the stimulation of 5-HT4Rs, could affect BBB permeability. Objective This work aims to characterize the functional role of 5-HT4R at the BBB interface. Method Studies have been performed on human cerebral microvascular endothelial cells hCMEC/D3. The BBB permeability was assessed by TransEndothelial Electrical Resistance (TEER). The hCMEC/D3 were stimulated with prucalopride 10 μM in absence or presence of the 5-HT4R antagonist GR113808 1 μM. Additionally, protein expression of occludin was evaluated. Finally, the intracellular pathway involved in this regulation was investigated. Results A 96-hour treatment with prucalopride decreased the TEER (47.0%, n = 6, P 0.05; GR113808 + prucalopride: 130%, P > 0.05), a pathway described to affect occludin expression. Conclusion Our findings support the hypothesis that 5-HT4R plays a role in the regulation of BBB permeability. Studies are ongoing to determine the entire signalling pathway. The contribution of platelets and 5-HT4R in the pathological change in BBB permeability is suggested.

Dihydropyridine calcium-channel blockers are a known substrate for the cytochrome P450 isoform 3A4. Rifampicin, an antitubercular agent, is one of the most potent inducers of hepatic and intestinal CYP3A4 thus increasing dihydropyridine metabolism. We report a case of a 67-year-old hypertensive female treated with a 4-drug antihypertensive regimen including a dihydropyridine (nicardipine 50mg bid), who was admitted for septic arthritis of the knee requiring antibiotic treatment with teicoplanin 400mg od and rifampicin 600mg bid. Six days after rifampicin initiation, she presented with Posterior Reversible Encephalopathy Syndrome due to uncontrolled hypertension. We hypothesized that disequilibrium of previously controlled hypertension was partially due to nicardipine ineffectiveness. Plasma nicardipine concentration was assessed through high performance liquid chromatography 5 hours after co-administration of the two drugs and proved undetectable. This article is protected by copyright. All rights reserved.

Background: Hypoxic environment is a pejorative prognostic factor in pediatric solid tumors (ST). It is favoring neoangiogenesis, tumor progression and resistance to treatments through the activation of mTor and HIF-1α pathways. Those hypoxic markers can be targeted by rapamycin and irinotecan, respectively. Therefore, we designed a dose-escalation phase I trial combining both drugs to inhibit tumor hypoxic mechanisms present in several pediatric refractory/relapsing ST. Procedure: Patients were enrolled in this 3+3 escalation trial with ten dose levels. The primary endpoint was to determine the maximum tolerated dose (MTD) of rapamycin when combined with irinotecan. Rapamycin was administered orally once daily in a 28-day cycle (1 to 2.5 mg/m2/day) and associated biweekly with intravenous irinotecan (125 to 240 mg/m2/dose). Toxicities, pharmacokinetics (PK), efficacy analyses and pharmacodynamics (UGT1A1 status, cytokines/chemokines assessment, MRI markers and proteins' expression) were evaluated. Results: 42 patients were treated and had mild to moderate reversible toxicities. No MTD was present up to the tenth level. The PK were highly variable. Nevertheless, the usually targeted concentrations of rapamycin over time were reached at 1.5 mg/m2/day dose. When looking to the 2 partial responses (PR) and 19 stable diseases (SD) in 31 assessable patients, the mean irinotecan AUC was 363 min.mg/L and the mean D8 rapamycin AUC was 9.3 min.mg/L. No significant relationship was observed between dose escalation and response, but progressive tumors' subgroup (PD) had slightly lower mean irinotecan AUC (321 min.mg/L) and D8 rapamycin AUC (6.5 min.mg/L). The two main tumor groups were brain tumors (BTs) and sarcomas. A high proportion of those patients experienced tumor responses (3/4 ependymomas, 2/3 medulloblastomas, 1 rhabdoid tumor, 2/3 high-grade gliomas and 5/6 osteosarcomas). In all 17 assessable patients for immunohistochemistry, the two targeted proteins, mTor and HIF-1α, were expressed, but the resistance to this double strategy was significantly linked to the HIF-2α protein hyperexpression. In the BTs, lower ADC values in MRI were also a prerequisite for tumor responsiveness. Cytokine analyses, distinguished specific proinflammatory and angiogenic signatures during first course of treatment: PR patients had an increase of TNF, associated to a stable PIGF and a decrease of VEGF-C; the SD subgroup had a significant increase of VEGF-D and a stable or increase of sFLT1 and PD group had a constant increase of IL8 and VEGF-D. Conclusion: Despite no MTD, we can propose a next phase II trial with rapamycin dose of 1.5 mg/m2/day associated with 125 mg/m2 irinotecan in STs not expressing HIF-2α protein. A precise PK follow-up will be used to reach an irinotecan AUC up to 360 min.mg/L and a D8 rapamycin blood concentration of 10 µg/L. The cytokine evaluation will help us to predict during first cycle the potential tumor response. Citation Format: Sarah Jannier, Veronique Kemmel, Consuelo Sebastia, Agathe Chammas, Amelia-Naomi Sabo, Erwan Pencreach, Françoise Farace, Benoit Lhermitte, Birgit Geoerger, Isabelle Aerts, Didier Frappaz, Pierre Leblond, Nicolas Andre, Yves Perel, Nadège Corradini, Anne Isabelle Bertozzi, Florence Vincent, Natacha Entz-Werle. Phase I study with rapamycin combined with irinotecan, the SFCE RAPIRI I trial, a new way to target tumor hypoxia in pediatric refractory cancers [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT136.

The impact of vitamin D on sensory function, including pain processing, has been receiving increasing attention. Indeed, vitamin D deficiency is associated with various chronic pain conditions, and several lines of evidence indicate that vitamin D supplementation may trigger pain relief. However, the underlying mechanisms of action remain poorly understood. We used inflammatory and non-inflammatory rat models of chronic pain to evaluate the benefits of vitamin D

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.

Clinical observations suggested that gamma-hydroxybutyrate (GHB) protects nerve cells against death but the direct proofs are missing. Here, we combined several approaches to investigate GHB capacity to protect human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced death. To increase the patho-physiological relevancy of our study, we used native SH-SY5Y cells and SH-SY5Y cells stably transfected with the wild-type amyloid-precursor-protein (APPwt) or control-vector-pCEP4. Trypan Blue exclusion and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide) assays combined with pharmacological analyses showed that H2O2 reduced native and genetically modified cell viability and APPwt-transfected cells were the most vulnerable. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 staining assessed by flow cytometry revealed a basally elevated apoptotic signal in APPwt-transfected cells. Reverse-transcription, real-time quantitative polymerase chain reaction (qPCR) and Western blotting showed that mRNA and protein basal ratios of apoptotic modulators Bax/Bcl-2 were also high in APPwt-transfected cells. GHB efficiently and dose-dependently rescued native and genetically modified cells from H2O2-induced death. Interestingly, GHB, which strongly decreased elevated basal levels of TUNEL-staining, activated caspase 3-labeling and Bax/Bcl-2 in APPwt-transfected cells, also counteracted H2O2-evoked increased apoptotic markers in native and genetically modified SH-SY5Y cells. Since GHB did not promote cell proliferation, anti-apoptotic action through the down-regulation of Bax/Bcl-2 ratios and/or caspase 3 activity appears as a critical mechanism involved in GHB-induced protection of SH-SY5Y cells against APPwt-overexpression- or H2O2-evoked death. Altogether, these results, providing multi-parametric evidence for the existence of neuroprotective action of GHB, also open interesting perspectives for the development of GHB analog-based strategies against neurodegeneration or nerve cell death.

Patients with schizophrenia have often been described as insensitive to nociceptive signals, but objective evidence is sparse. We address this question by combining subjective behavioral and objective neurochemical and neurophysiological measures. The present study involved 21 stabilized and mildly symptomatic patients with schizophrenia and 21 control subjects. We applied electrical stimulations below the pain threshold and assessed sensations of pain and unpleasantness with rating scales, and Somatosensory Evoked Potentials (SEPs/EEG). We also measured attention, two neurochemical stress indices (ACTH/cortisol), and subjective VEPs/EEG responses to visual emotional stimuli. Our results revealed that, subjectively, patients’ evaluations do not differ from controls. However, the amplitude of EEG evoked potentials was greater in patients than controls as early as 50 ms after electrical stimulations and beyond one second after visual processing of emotional pictures. Such responses could not be linked to the stress induced by the stimulations, since stress hormone levels were stable. Nor was there a difference between patients and controls in respect of attention performance and tactile sensitivity. Taken together, all indices measured in patients in our study were either heightened or equivalent relative to healthy volunteers.

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.

The neurosteroid allopregnanolone (AP) modulates neuroendocrine/neurobiological processes, including hypothalamic-­pituitary-­adrenocortical activities, pain, anxiety, neurogenesis and neuroprotection. These observations raised the hope of developing AP-­based therapies against neuroendocrine and/or neurodegenerative disorders. However, the pleiotropic actions of AP, particularly its cell-­proliferation-­promoting effects, hamper the development of selective/targeted therapies. For example, although AP-­induced neurogenesis may serve to compensate neuronal loss in degenerative brains, AP-­evoked cell-­proliferation is contraindicated for steroid-­sensitive cancer patients. To foster progress, we synthesised 4 novel AP analogues of neurosteroids (ANS) designated BR053 (12-o­ xo-e­ pi-­AP), BR297 (O-­allyl-­epi-­AP), BR351 (O-­allyl-­AP) and BR338 (12-­oxo-­AP). First, because AP is well-­known as allosteric modulator of GABAA receptors (GABAA-­R), we used the electrophysiological patch-­clamp technique to determine the structure-­activity relationship of our ANS on GABAA-­activated current in NCB20 cells expressing functional GABAA-­R. We found that the addition of 12-o­ xo-g­ roup did not significantly change the respective positive or negative allosteric effects of 3α-­AP or 3β-(­epi)-­AP analogues. Importantly, substitution of the 3α-h­ ydroxyl-­ group by 3α-O­ -­allyl highly modified the ANS activities. Unlike AP, BR351 induced a long-l­asting desensitisation/inhibition of GABAA-­R. Interestingly, replacement of the 3β-­hydroxyl by 3β-O­ -­allyl (BR297) completely reversed the activity from negative to positive allosteric action. In a second step, we compared the actions of AP and ANS on SH-­SY5Y neuronal cell viability/proliferation using MTT-­reduction assays. Different dose-r­ esponse curves were demonstrated for AP and the ANS. By contrast to AP, BR297 was totally devoid of cell-­proliferative effect. Finally, we compared AP and ANS abilities to protect against oxidative stress-­induced neuronal death pivotally involved in neurodegenerative diseases. Both BR351 and BR297 had notable advantages over AP in protecting SH-­SY5Y cells against oxidative stress-­induced death. Thus, BR297 appears to be a potent neuroprotective compound devoid of cell-­proliferative activity. Altogether, our results suggest promising perspectives for the development of neurosteroid-­based selective and effective strategies against neuroendocrine and/or neurodegenerative disorders.

*INSERM U-575 and Department of Biochemistry, Faculty of Medicine, Strasbourg, France CEA/Saclay, Department d’Inge´nierie et d’Etudes des prote´ines, Gif Sur Yvette, FranceThe kynurenine pathway is the principal metabolic route forcatabolism of tryptophan. Several intermediates of the path-way are active at central neurotransmitter sites, for example,the NMDA receptor agonist quinolinic acid and the gluta-mate receptor antagonist kynurenic acid. The latter is mainlysynthesized in rat brain astrocytes by kynurenine aminotrans-ferase and is released into the immediate vicinity of gluta-matergic synapses to possibly reduce the neurotoxic effectsof excitatory amino acid (Guidetti et al. 1995, 2007; Ceresoliet al. 1997). These findings raised the possibility that thekynurenine pathway and the metabolites of tryptophan couldbe involved in various neuroregulatory and neuropatholo-gical phenomenon in the CNS (Stone and Darlington 2002).Xanthurenic acid (Xa) is one metabolite of this kynureninepathway and could be considered as the parent compound ofkynurenic acid. Indoleamine 2,3 dioxygenase which ispresent in the brain transforms tryptophan into kynureninethat is either transaminated into kynurenic acid by kynure-nine aminotransferase or hydroxylated into 3-hydroxykynur-enine by kynurenine 3-hydroxylase. Transamination of3-hydroxykynurenine leads to xanthurenic acid (Fig. 1).The latter pathway is generally considered to be part of adetoxification process that reduces the concentration of 3-hydroxykynurenine because spontaneous oxidation of thissubstance induces free radical generation and apoptosis, bothin vitro and in vivo (Okuda et al. 1996; Wei et al. 2000).However, a transaminase specific for 3-hydroxykynureninehas not been identified in man. In mammals, it is generallythought that 3-hydroxykynurenine is transformed to Xa bythe same kynurenine transaminase isoenzymes that catalyzethe transamination of kynurenine to kynurenic acid (Urenjakand Obrenovitch 2000).

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.

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. 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. 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. 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.

Two parent clones of a gamma-hydroxybutyrate (GHB) receptor, C12K32 and GHBh1, were isolated from a human frontal cortex cDNA library. The two clones differ by a deleted cytosine in C12K32. CHO cells transfected with either C12K32 or GHBh1 responded positively to submicromolar GHB stimulation. However, unlike C12K32, GHBh1 desensitizes rapidly on application of low concentrations of GHB. GHB receptor properties were then studied on C12K32 expressed in CHO cells. C12K32 bound GHB with a Kd of 114 nM and has no affinity for GABA or glutamate. GHB and NCS-382 displaced [3H]GHB with an IC50 of 53 +/- 8 and 120 +/- 18 nM, respectively. In patch-clamp experiments, GHB induced a dose-dependent response with an EC50 of 130 nM. This response was antagonized by NCS-382, was not reproduced by GABA, and was sensitive to the addition of GTP-gamma-S in the recording pipette. CHO cells transfected with C12K32 exhibited GTPgamma-35S binding with an EC50 of 462 nM for GHB and an IC50 of 2.9 microM for NCS-382. The present data led to the conclusion that both C12K32 and GHBh1 are two closely related isoforms of a human GHB receptor, GHBh1, that is described in the databank as the GPCR 172A.

Le γ-hydroxybutyrate (GHB), synthétisé dans les années 1960 pour ses propriétés GABAergiques (GABA, acide γ-amino-butyrique), est une substance qui pénètre facilement et rapidement dans le cerveau. Il induit un sommeil proche du sommeil physiologique, avec un réveil de bonne qualité, et possède des indications en anesthésiologie et dans le traitement des troubles narcoleptiques et de l’addiction à l’alcool. Ses propriétés sédatives, anxiolytiques et euphorisantes ont détourné ce composé de ses indications en thérapeutique, pour une utilisation à des fins récréatives et une consommation illicite. Le GHB à doses pharmacologiques interagit avec les récepteurs GABAB cérébraux et avec une famille de récepteurs spécifiques, exprimés principalement par le système nerveux central. Cette dernière famille représente l’un des constituants essentiels d’un système GHB endogène qui aurait comme rôle principal de contrôler l’activité de certaines synapses GABA du système nerveux central., γ-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.

gamma-Hydroxybutyrate (GHB) is an endogenous metabolite of gamma-aminobutyric acid (GABA), which is synthesized in the neuronal compartment of the central nervous system. This substance possesses several properties that support its role as a neurotransmitter/neuromodulator in brain. In particular, it is synthesized by a specific pathway that transforms GABA into succinic semialdehyde via GABA-T activity; then succinic semialdehyde is converted into GHB by a specific succinic semialdehyde reductase (SSR). The last enzyme is considered as a marker for neurons that synthesize GHB. This compound binds in brain to receptors whose distribution, ontogenesis, kinetics, and pharmacology are specific. Endogenous GHB, but also GHB exogenously administered to rats, participate in the regulation of dopaminergic activity of the nigrostriatal pathway. To investigate the distribution of GHB neurons in this pathway and the anatomic relationships between dopaminergic and GHB neurons, immunocytochemical identification of dopamine, GABA, and GHB neurons was carried out in the substantia nigra and striatum of the rat. The following markers for these neurons were used: anti-tyrosine hydroxylase (TH) antibodies for dopamine neurons, anti-glutamate decarboxylase (GAD) antibodies for GABA neurons, and anti-succinic semialdehyde reductase (SSR) antibodies for GHB neurons. GABA neurons were studied because GAD and SSR co-exist frequently in the same neuron, and GABA alone also exerts its own regulatory effects on dopaminergic neurons. This study reveals the co-existence of GAD/SSR and GAD/SSR/TH in numerous neurons of the substantia nigra. However, some neurons appear to be only GAD or SSR positive. In the striatum, TH-positive terminals surround many GHB neurons. GAD innervation is abundant in close contact with unlabeled neurons in the caudate-putamen, whereas distinct SSR-positive punctuates are also present. The existence of SSR-reactive synapses and neurons was confirmed in the striatum at the electron microscopic level. On the basis of these results, a clear anatomo-functional relationship between GHB and dopamine networks cannot be defined; however, we propose the modulation by GHB of striatal intrinsic neurons that could then interfere with the presynaptic control of dopaminergic activity.

Previous results have shown that stimulation of the gamma-hydroxybutyrate (GHB) receptor modulates Ca2+ channel permeability in cell cultures. In order to confirm this result, we investigated the consequence of GHB receptor stimulation on nitric oxide synthase (NOS) activity in rat brain cortical punches rich in GHB receptors. The stimulation of these receptors by increasing amounts of GHB induced a progressive decrease in NOS activity. However, for GHB doses above 10 microM, this reduction was progressively lost, either after receptor desensitization or after stimulation of an additional class of GHB receptor having lower affinity. The effect of GHB was reproduced by the GHB receptor agonist NCS-356 and blocked by the GHB receptor antagonist NCS-382. The GHB-induced effect on Ca2+ movement was additive to those produced by veratrine, indicating that GHB modulates a specific Ca2+ conductance, which explains the modification in NOS activity and the increase in cyclic guanosine monophosphate levels previously reported.

Introduction L’infiltration intra-articulaire (LIA) d’anesthesiques locaux est une alternative au bloc peri-nerveux femoral (BF) pour l’analgesie postoperatoire lors d’une arthroplastie totale de genou (TKA). La LIA preserve le verrou musculaire quadricipital, permettant une reprise d’appui precoce. Le profil pharmacocinetique de la ropivacaine administree par LIA prolongee n’est pas connu. L’objectif de cette etude est de comparer la pharmacocinetique de la ropivacaine administree soit par LIA continue soit par BF continu, apres TKA. Patients et methodes Apres avis du CPP et consentement informe ecrit, 24 patients operes d’une TKA ont ete randomises en 2 groupes. Dans le groupe LIA la ropivacaine (400 mg ; 2 mg.mL−1) etait administree par infiltration intra-articulaire peroperatoire puis par catheter intra-articulaire. Dans le groupe BF, l’injection de 150 mg de ropivacaine 7,5 mg.mL−1 etait poursuivie par catheter peri-nerveux. L’infiltration de ropivacaine 2 mg.mL−1 etait poursuivie pendant 48 h a un debit de 20 mg/h dans les 2 groupes. Les concentrations plasmatiques de ropivacaine (chromatographie couplee a un detecteur UV–Visible) ont ete mesurees pendant 72 heures afin de determiner le pic de concentration (Cmax), son temps de survenue (Tmax) et l’aire sous la courbe. Les signes cliniques de toxicite de la ropivacaine ont ete recherches quotidiennement. Resultats Treize patients ont beneficie d’une LIA et 11 d’un BF. L’evolution de la concentration plasmatique de ropivacaine est presentee sur la Fig. 1 . Dans les deux groupes LIA et BF la Tmax est observee 48 heures apres le debut de l’administration. Dans le groupe LIA la Cmax egale 3,9 ± 4,1 mg/L, l’aire sous la courbe etant de 152 ± 141,6 mg.h/L alors que dans le groupe BF la Cmax egale 2,9 ± 2,0 mg/L et l’aire sous la courbe moyenne est de 112 ± 79,7 mg.h/L. Cinq patients ont des concentrations de ropivacaine superieures a 4 mg/L, 3 dans le groupe BF et 2 dans le groupe LIA. Deux patients (1 dans le groupe LIA, 1 dans le groupe BF) ont presente des signes de toxicite (gout metallique dans la bouche ou bourdonnements d’oreille). Discussion L’administration de ropivacaine continue durant 48 heures postoperatoires par LIA ou BF entraine un pic de concentration systemique en fin d’administration, a la 48e heure. Ces resultats suggerent un maintien de la surveillance clinique des signes de toxicite d’une duree au moins equivalente a la perfusion. L’absence de resorption systemique de ropivacaine dans le groupe LIA durant la premiere heure est liee a la presence du garrot chirurgical. Une LIA de ropivacaine de 48 heures ne semble pas induire de profil de resorption ni de symptomatologie clinique menacants en comparaison avec l’administration perinerveuse femorale mais la posologie minimale efficace reste a determiner afin d’optimiser la securite de cette modalite d’analgesie postoperatoire.

Clonal neurohybridoma NCB-20 cells express a valproate-insensitive succinic semialdehyde reductase activity that transforms succinic semialdehyde into γ -hydroxybutyrate. This activity (1.14±0.16 nmol/min/mg protein) was similar to the lowest activity existing in adult rat brain. [ 3 H] γ -Hydroxybutyrate labels a homogeneous population of sites on NCB-20 cell membranes ( K d =250±44.4 nM, B max =180±16.2 fmol/mg protein) that apparently represents specific γ -hydroxybutyrate binding sites characterized previously on brain cell membranes. Finally, an Na + -dependent uptake of [ 3 H] γ -hydroxybutyrate was expressed in NCB-20 cells with a K m of 35+21.1 μ M and a V max of 80±14.2 pmol/min/mg protein. A three-day treatment with 1 mM dibutyryl-cyclic-AMP induced a three-fold increase in the cellular succinic semialdehyde reductase activity. In parallel, a K + -evoked release of [ 3 H] γ -hydroxybutyrate occurred. This release was Ca 2+ dependent and was not present in undifferentiated cells. Cyclic-AMP treatment induced a decrease of [ 3 H] γ -hydroxybutyrate binding sites, which could be due to spontaneous γ -hydroxybutyrate release. Patch-clamp experiments carried out on differentiated NCB-20 cells revealed the presence of Ca 2+ conductances which were partially inhibited by 50 μ M γ -hydroxybutyrate. This γ -hydroxybutyrate-induced effect was blocked by the γ -hydroxybutyrate receptor antagonist NCS-382, but not by the GABA B antagonist CGP-55 845. These results demonstrate the presence of an active γ -hydroxybutyratergic system in NCB-20 cells which possesses the ability to release γ -hydroxybutyrate. These cells express specific γ -hydroxybutyrate receptors which modulate Ca 2+ currents independently of GABA B receptors.

Five days of gamma-hydroxybutyrate (GHB) administration (3 x 500 mg kg(-1) day(-1) i.p.) to rats resulted in a significant decrease in the density of GHB receptors measured in the whole rat brain without modification of their corresponding affinity. Similar administration of (-)-sulpiride (2 X 100 mg kg(-1) day(-1) i.p. for 5 days) induces an up-regulation of GHB receptors without change in their dissociation constants (Kd). Haloperidol (2 X 2 mg day(-1) i.p. for 5 days) showed no effect. Administered chronically via osmotic minipumps directly into the lateral ventricles, (-)-sulpiride (60 microg day(-1) for 7 days) and GHB (600 microg day(-1) for 7 days) up-regulated and down-regulated rat brain GHB receptors, respectively. Finally, in a mouse hybridoma cell line (NCB-20 cells) expressing GHB receptors, the treatment of these cells with 1 mM GHB, 100 microM (-)-sulpiride or 1 mM GABA decreases, increases and induces no change, respectively, in the density of GHB receptors after 3 days of treatments. These results indicate that chronic GHB treatment modifies the expression of its receptor and that sulpiride also induces plastic changes in GHB receptors perhaps via antagonistic properties.

Xanthurenic acid (XA) is a metabolite of the tryptophan oxidation pathway through kynurenine and 3-hydroxykynurenine. XA was until now considered as a detoxification compound and dead-end product reducing accumulation of reactive radical species. Apart from a specific role for XA in the signaling cascade resulting in gamete maturation in mosquitoes, nothing was known about its functions in other species including mammals. Based upon XA distribution, transport, accumulation and release in the rat brain, we have recently suggested that XA may potentially be involved in neurotransmission/neuromodulation, assuming that neurons presumably express specific XA receptors. Recently, it has been shown that XA could act as a positive allosteric ligand for class II metabotropic glutamate receptors. This finding reinforces the proposed signaling role of XA in brain. Our present results provide several lines of evidence in favor of the existence of specific receptors for XA in the brain. First, binding experiments combined with autoradiography and time-course analysis led to the characterization of XA binding sites in the rat brain. Second, specific kinetic and pharmacological properties exhibited by these binding sites are in favor of G-protein-coupled receptors (GPCR). Finally, in patch-clamp and calcium imaging experiments using NCB-20 cells that do not express glutamate-induced calcium signals, XA elicited specific responses involving activation of cationic channels and increases in intracellular Ca(2+) concentration. Altogether, these results suggest that XA, acting through a GPCR-induced cationic channel modulatory mechanism, may exert excitatory functions in various brain neuronal pathways. journal article research support, non-u.s. gov't 2012 2012 11 06 imported

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.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.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 children9 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 children9 and ≥9 kg, respectively.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.

Background-: Mice deficient for the stable tubule only peptide (STOP) display altered dopaminergic neurotransmission associated with severe behavioural defects including disorganized locomotor activity. Endogenous morphine, which is present in nervous tissues and synthesized from dopamine, may contribute to these behavioral alterations since it is thought to play a role in normal and pathological neurotransmission. Results-: In this study, we showed that STOP null brain structures, including cortex, hippocampus, cerebellum and spinal cord, contain high endogenous morphine amounts. The presence of elevated levels of morphine was associated with the presence of a higher density of mu opioid receptor with a higher affinity for morphine in STOP null brains. Interestingly, STOP null mice exhibited significantly lower nociceptive thresholds to thermal and mechanical stimulations. They also had abnormal behavioural responses to the administration of exogenous morphine and naloxone. Low dose of morphine (1 mg/kg, i.p.) produced a significant mechanical antinociception in STOP null mice whereas it has no effect on wild-type mice. High concentration of naloxone (1 mg/kg) was pronociceptive for both mice strain, a lower concentration (0.1 mg/kg) was found to increase the mean mechanical nociceptive threshold only in the case of STOP null mice. Conclusions-: Together, our data show that STOP null mice displayed elevated levels of endogenous morphine, as well as an increase of morphine receptor affinity and density in brain. This was correlated with hypernociception and impaired pharmacological sensitivity to mu opioid receptor ligands.

Delayed elimination of methotrexate associated with serious side-effects has been attributed to the co-administration of benzimidazole proton pump inhibitors.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.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.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.

γ-Hydroxybutyrate (GHB) is a natural brain neuromodulator that has its own enzymatic machinery for synthesis and degradation, release, and transport systems and several receptors that belong to the G protein-coupled receptor (GPCR) family. Targeting of this system with exogenous GHB is used in therapy to induce sleep and anesthesia and to reduce alcohol withdrawal syndrome. GHB is also popular as a recreational drug for its anxiolytic and mild euphoric effects. However, in both cases, GHB must be administered at high doses in order to maintain GHB concentrations in brain of ∼800–1,000 μM. These high concentrations are thought to be necessary for interactions with low-affinity sites on GABABreceptor, but the molecular targets and cellular mechanisms modulated by GHB remain poorly characterized. Therefore, to provide new insights into the elucidation of GHB mechanisms of action and open new tracks for future investigations, we explored changes of GHB-induced transcriptomes in rat hippocampus and prefrontal cortex by using DNA microarray studies. We demonstrate that a single acute anesthetic dose of 1 g/kg GHB alters a large number of genes, 121 in hippocampus and 53 in prefrontal cortex; 16 genes were modified simultaneously in both brain regions. In terms of molecular functions, the majority of modified genes coded for proteins or nucleotide binding sites. In terms of Gene Ontology (GO) functional categories, the largest groups were involved in metabolic processing for hippocampal genes and in biological regulation for prefrontal cortex genes. The majority of genes modified in both structures were implicated in cell communication processes. Western blot and immunohistochemical studies carried out on eight selected proteins confirmed the microarray findings.

The NCB-20 neurohybridoma cells differentiated with dibutyryl-cyclic-AMP represent an interesting model to study several components of the gamma-hydroxybutyrate (GHB) system in brain. In particular, an active Na(+)-dependent uptake and a depolarization-evoked release of GHB is expressed by these cells, together with high affinity specific binding sites for this substance. However, only little is known about cellular mechanisms following GHB receptor(s) stimulation in these neurons. Electrophysiological data indicate that GHB can differently affect Ca(2+) currents. L-type calcium channels were typically inhibited by GHB when NCB-20 cells were depolarized. In contrast, when NCB-20 cells were at resting potential, GHB induced a specific Ca(2+) entry through T-type calcium channels. In this study, we investigated the effect induced on cytosolic free Ca(2+) level and cAMP production by GHB receptor(s) stimulated with micromolar concentrations of GHB or structural analogues of GHB. Ca(2+) movements studied by cellular imaging were dose-dependently increased but disappeared for GHB concentrations >25 microM. In addition, nanomolar doses of GHB inhibited forskolin-stimulated adenylate cyclase. This effect was also rapidly desensitized at higher GHB concentrations. Acting as an antagonist, NCS-382 decreased GHB receptor(s) mediated cAMP and calcium signals. The agonist NCS-356 mimicked GHB effects which were not affected by the GABA(B) receptor antagonist CGP-55-845. Our results reveal the occurrence of Ca(2+)-dependent adenylate cyclase inhibition in NCB-20 neurons after GHB receptor(s) stimulation by GHB concentrations

International audience; 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.

Gamma-hydroxybutyrate (GHB) is a substance derived from the metabolism of GABA and is heterogeneously distributed in various regions of the brain. This compound possesses a neuromodulatory role on several types of synapses, particularly those using GABA as a neurotransmitter. At physiological concentrations, this effect of GHB is mediated via specific receptors that induce neuronal hyperpolarization and bind radioactive GHB with a specific distribution, ontogenesis, kinetics, and pharmacology. A membrane protein that possesses six to seven transmembrane domains and which binds and is activated by micromolar amounts of GHB was recently cloned from rat brain hippocampus. In order to study the regional and cellular distribution of this receptor in rat brain, we selected several specific peptides belonging to the extracellular domains of the receptor to be used as specific immunogens to raise polyclonal antibodies in the rabbit. Among the antisera obtained, one of them gave particularly good results in terms of specificity and reactivity at high dilution. Immunohistochemical analyses, both at the confocal and electron microscopic level, showed receptor protein distribution closely resembling the distribution of GHB high-affinity binding sites, except for cerebellum, where GHB receptor(s) of lower affinity exist(s). In all regions studied the GHB receptor-like protein labeling appears to be distributed specifically in neurons and not in glial cells. At the cellular level the antibody specifically labels dendrites, and no immunoreactivity was detected in presynaptic endings or in axons. Accordingly, electron microscopy reveals strong labeling of postsynaptic densities and of neuronal cytosol. J. Comp. Neurol. 498:508–524, 2006. © 2006 Wiley-Liss, Inc.

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.

Gamma-Hydroxybutyrate is derived from GABA in brain and plays specific functional roles in the CNS. It is thought to exert a tonic inhibitory control on dopamine and GABA release in certain brain areas, through specific gamma-hydroxybutyrate receptors. Apart from modifying certain calcium currents, the specific transduction mechanism induced by stimulation of gamma-hydroxybutyrate receptors remains largely unknown. We investigated the possible contribution of K(+) channels to the hyperpolarization phenomena generally induced by gamma-hydroxybutyrate in brain, by monitoring (86)Rb(+) movements in a neuronal cell line (NCB-20 cells), which expresses gamma-hydroxybutyrate receptors. Physiological concentrations of gamma-hydroxybutyrate (5-25 microM) induce a slow efflux of (86)Rb(+), which peaks at 5-15 min and returns to baseline levels 20 min later after constant stimulation. This effect can be reproduced by the gamma-hydroxybutyrate receptor agonist NCS-356 and blocked by the gamma-hydroxybutyrate receptor antagonist 6,7,8,9-tetrahydro-5-[H]-benzocycloheptene-5-ol-4-ylidene. The GABA(B) receptor antagonist CGP 55845 has no effect on gamma-hydroxybutyrate-induced (86)Rb(+) efflux. The pharmacology of this gamma-hydroxybutyrate-dependent efflux of (86)Rb(+) is in favor of the involvement of tetraethylammonium and charybdotoxin insensitive, apamin sensitive Ca(2+) activated K(+) channels, identifying them as small conductance calcium activated channels. We demonstrated a gamma-hydroxybutyrate dose-dependent entry of calcium ions into NCB-20 neuroblastoma cells at resting potential. Electrophysiological data showed that this Ca(2+) entry corresponded mainly to a left-hand shift of the current/voltage relation of the T-type calcium channel. This process must at least partially trigger small conductance calcium activated channel activation leading to gamma-hydroxybutyrate-induced hyperpolarization.

Gamma-hydroxybutyric acid was synthesized 35 years ago to obtain a GABAergic substance that penetrates the brain freely. Since then, gamma-hydroxybutyric acid has been used in human beings for its sedative and anesthetic properties when administered at high doses, and most of the studies on gamma-hydroxybutyric acid have focused on its pharmacological effects. However, gamma-hydroxybutyric acid is also an endogenous substance, which is synthesized and released in the brain by specific neuronal pathways, implicated in the control of the GABAergic, dopaminergic, and opioid systems. This control is mediated by specific gamma-hydroxybutyric acid receptors with a unique distribution in brain and a specific ontogenesis and pharmacology. Stimulation of these receptors induces specific cellular responses. Taken together, these results suggest that gamma-hydroxybutyric acid possesses most of the properties required of a neurotransmitter/neuromodulator in the brain.

Abstract 1811 Background: Busulfan (Bu) is a standard component of HSCT regimens and shows a narrow therapeutic range. In very young children, Bu exhibits large pharmacokinetic (PK) variability. Intravenous (IV) Bu clearance was demonstrated to be non-linearly related to body weight (BW) and thus BW is used to optimally define the IV Bu dosage in children [Vassal G. et al., CCP 2006]. Search for additional relevant covariates is still a topical question to further explain the variability in young children (Pts) and infants. Objective: to screen for the effect of heterogeneous patient characteristics on IV Bu PK disposition, and to evaluate the consistency of weight-based dosing strategy (as recommended in the European SmPC) in a large cohort of children, especially in patients with rare diseases and a BW less than 9 kg. Patients and Method: The prospective observational study included 115 patients (pts), mostly infants (median age: 13 months [0.3-148], median BW: 9 kg [3.5-58]). Main diagnoses were immunodeficiency (n=71, mainly SCID and lymphohistiocytosis) and inherited metabolic disorders (n=15). Additional data (90 pts, mostly malignant diseases) from two prospective clinical trials previously described [Nguyen L. et al., BMT 2004] [Vassal G. et al., CCP 2006] were pooled with the observational dataset. Altogether, 205 children aged from 10 days to 15 years (median of 30 months) and weighed from 3.5 to 62.5 kg (median of 11.0 kg) were analysed. IV Bu (Busilvex®) was given over 2 h q6h × 16 doses. For most of the pts (88%), the starting doses were given according to the European SmPC: 1.0 mg/kg, 1.2 mg/kg, 1.1 mg/kg, 0.95 mg/kg and 0.8 mg/kg for pts 23–34 kg, and >34 kg BW, respectively. PK was assessed on plasma samples from the 1st, 9th and 13th dose (536 PK datasets). PK analysis was performed using Non Linear Mixed Effect modelling with the NONMEM program. Results: A one-compartment PK model suitably fitted the concentrations vs time data. BW remained the main determinant of IV Bu PK disposition. Increase of IV Bu clearance with child's growth was observed to be faster in younger than in older children. For children < 9 kg, a doubling in BW is associated with a 2.4-fold increase in Bu clearance while a 1.7-fold increase in Bu clearance is observed in pts ≥ 9 kg for the same BW growth. To take into account this difference, the relationships between Clearance (CL) and BW was modelled using a different allometric exponent for children with a BW of < 9 kg and ≥ 9 kg. The predictive equations were: CL (L/h) = 2.18 × (BW/9)1.25 and CL(L/h) = 2.18 × (BW/9)0.76 in pts < 9 kg and ≥ 9 kg, respectively. The inter-individual variability decreased from 64% (covariate-free model) to 23% for Bu CL. No further significant impact was identified even when considering large fluctuations of the following covariates: creatinine clearance (up to 11 × Upper Normal Limit), bilirubin (up to 4 × UNL), ferritine (up to 43 × UNL), hepatic transaminases (up to 39 × UNL), alkaline phosphatases (up to 4 × UNL), gGT (up to 86 × UNL), total protein (0.5 to 1.4 × UNL) and lactate deshydrogenase (up to 3 × UNL). No PK difference was observed between patients under Benzodiazepine and those under Phenytoin prophylaxis, neither when comparing different groups of diagnoses. Similar performances for targeting an AUC range of 900 – 1500 μM.min were achieved when comparing Bu dosing either adjusted according to the model predictive equation or with the use of the European SmPC recommendations. The success rates were about 60% and 80% in pts < 9 kg and ≥ 9 kg respectively. Conclusion: BW is confirmed to be the only relevant predictor to optimally define the IV Bu dosing in children. The population PK model has established a higher allometric exponent for younger children (< 9kg) in order to take into account the faster maturation of clearance in this weight group. This model is consistent with the dosing strategy recommended in the EMA registration. Disclosures: Daher Abdi: Pierre Fabre Médicament: Employment. Pétain:Pierre Fabre Médicament: Employment. Nguyen:Pierre Fabre Médicament: Employment.

Background: Busulfan (Bu) is the corner stone of hematopoietic stem-cell transplantation (HSCT) regimens with a narrow therapeutic window (TW). Graft rejection or toxicities are reported according to plasmatic exposure. In very young children, Bu exhibits large pharmacokinetic (PK) variability. Bu clearance was demonstrated to be non-linearly related to body weight (BW) and thus BW is used to optimally define the Bu dosage in children [Vassal et al., CCP 2006]. Search for additional data to confirm the TW and to describe clinical outcomes is still a topical question to better understand Pk/Pd relationship and to safely use Bu in young children and infants. Objective: To study the Pk/Pd relationship of Bu in pediatric recipients of bone marrow transplantation (BMT) receiving Bu-based conditioning regimens (CR). To correlate early toxicities (mainly hepatic veno-occlusive disease (VOD), non infectious pulmonary disease (niPD) and outcome i.e. overall survival (OS) at last follow up with type of CR, the underlying diseases, age at transplantation and Pk of busulfan Patients and Method: This multicenter prospective observational study included 307 pts transplanted between 2006 and 2013 from 14 French Pediatric BMT units; median age at transplantation was 18.4 months [1.3-289], with a median BW of 11.3 kg [3.4-82]. 100 pts were younger than 1 year, 71 pts < 9 kg and 171 pts < 16 kg. Patients were mostly affected by non-malignant diseases (primary immune deficiency (n=143), inherited metabolic disorders (n=50), hemoglobinopathies (n=20) while 78 patients suffered from malignant disease. 257 patients received allogenic HSCT (genoidentical donor n=79, matched unrelated donor n=33, mismatch unrelated donor n=18, haploidentical intra-familial donor n=59, other mismatched intra familial donor n=8 or unrelated cord blood n=51) and 41 autologous BMT. All patients received Bu-based conditioning regimen in combination with cyclophosphamide (BuCy n=119), fludarabine (BuFlu n=88), melphalan (BuMel n=36) or thiotepa (BuTTP n=3). 40 patients received BuFlu associated with a second alkylating agent (Mel, TTP or Cy), 12 patients received BuCy associated with a third agent (Mel or VP16). Serotherapy was given in 70% of the patients. Starting doses of Bu were given according to the European SmPC or EBMT-ESID recommendations. The median posology was 1 mg/kg 4x/day for 4 days [0.6-1.3 mg/kg]. PK was assessed on plasma samples with area under the curve (AUC) evaluation from the 1st(D1), 9th (D9) and 13th (D13) dose in 150 patients, D1 and D9 in 40 patients, D1 and D13 in 46 patients while 62 patients were monitored on D1 only (684 PK datasets). PK analysis was performed using Non linear Mixed Effect Modelling. A one-compartment PK model suitably fitted the concentrations vs time data. Median follow up is 27 months (0.33 to 96 months post BMT). Results: At D1, 67% of patients were within the therapeutic window (TW) [900-1500 µM.min] and 66% of patients reached the cumulative TW [14400-24000 µM.min]. Incidence of VOD and niPD were respectively 35% and 14%. Both toxicities correlated with type of CR and age 900 µM.min. OS was 69.1% at last follow up and was not significantly associated with CR and age at transplantation. Conclusion: In this large series of pediatric BMT recipients, we found that toxicities as VOD and niPD correlated with type of CR and age at transplantation in univariate analysis. Combination of 2 alkylating agents with Bu or BuFlu and transplantation < 1 year of age were associated with the highest incidence of toxicites. The TW targeting performance was improved in this cohort compared to previous reported data (Paci et al. TDM 2012). Specific TW for each group of pathologies will be proposed. Disclosures No relevant conflicts of interest to declare.