Estimating arterial blood gases with the EABC® System (“Earlobe Arterialized Blood Collector”) in critically ill patients. A validation study.

The Earlobe Arterialized Blood Collector® is a minimally invasive system able to perform arterialized capillary blood gas analysis from the earlobe (EL). A prospective validation study was performed in 55 critical ill patients. Sampling failure rate was high (53.6%). Risk factors were age > 65 years, diabetes, vasoactive drug therapy and noradrenaline (NA) doses above 0.22 μg / kg / min. Multivariate analysis showed age > 65 years was the only factor independently associated with failure. Concordance analysis with arterial blood gases and Bland-Altman agreement evaluation were insufficient for validating the new system for all gasometrical variables.

Imatinib plasma concentrations variability in oncologic patients

Imatinib (Glivec®) has transformed the treatment and short-term prognosis of chronic myeloid leukemia (CML) and gastrointestinal stromal tumor (GIST). However, the treatment must be taken indefinitely, it is not devoid of inconvenience and toxicity. Moreover, resistance or escape from disease control occurs in a significant number of patients. Imatinib is a substrate of the cytochromes P450 CYP3A4/5 and of the multidrug transporter P-glycoprotein (product of the MDR1 gene). Considering the large inter-individual differences in the expression and function of those systems, the disposition and clinical activity of imatinib can be expected to vary widely among patients, calling for dosage individualization. The aim of this exploratory study was to determine the average pharmacokinetic parameters characterizing the disposition of imatinib in the target population, to assess their inter-individual variability, and to identify influential factors affecting them. A total of 321 plasma concentrations, taken at various sampling times after the latest dose, were measured in 59 patients receiving Glivec at diverse regimens, using a validated HPLC-UV method developed for this study. The results were analyzed by non-linear mixed effect modeling (NONMEM). A one-compartment model with first-order absorption appeared appropriate to describe the data, with an average apparent clearance of 12.4 l/h, a distribution volume of 268 l and an absorption constant of 0.47 h-1. The clearance was affected by body weight, age and sex. No influences of interacting drugs were found. DNA samples were used for pharmacogenetic explorations. At present, only the MDR1 polymorphism has been assessed and seems to affect the pharmacokinetic parameters of imatinib. Large inter-individual variability remained unexplained by the demographic covariates considered, both on clearance (40 %) and distribution volume (71 %). Together with intra-patient variability (34 %), this translates into an 8-fold width of the 90 %-prediction interval of plasma concentrations expected under a fixed dosing regimen. This is a strong argument to further investigate the possible usefulness of a therapeutic drug monitoring program for imatinib. It may help to individualize the dosing regimen before overt disease progression or observation of treatment toxicity, thus improving both the long-term therapeutic effectiveness and tolerability of this drug.

Genetic dissection of azole resistance mechanisms in Candida albicans and their validation in a mouse model of disseminated infection.

Principal mechanisms of resistance to azole antifungals include the upregulation of multidrug transporters and the modification of the target enzyme, a cytochrome P450 (Erg11) involved in the 14alpha-demethylation of ergosterol. These mechanisms are often combined in azole-resistant Candida albicans isolates recovered from patients. However, the precise contributions of individual mechanisms to C. albicans resistance to specific azoles have been difficult to establish because of the technical difficulties in the genetic manipulation of this diploid species. Recent advances have made genetic manipulations easier, and we therefore undertook the genetic dissection of resistance mechanisms in an azole-resistant clinical isolate. This isolate (DSY296) upregulates the multidrug transporter genes CDR1 and CDR2 and has acquired a G464S substitution in both ERG11 alleles. In DSY296, inactivation of TAC1, a transcription factor containing a gain-of-function mutation, followed by sequential replacement of ERG11 mutant alleles with wild-type alleles, restored azole susceptibility to the levels measured for a parent azole-susceptible isolate (DSY294). These sequential genetic manipulations not only demonstrated that these two resistance mechanisms were those responsible for the development of resistance in DSY296 but also indicated that the quantitative level of resistance as measured in vitro by MIC determinations was a function of the number of genetic resistance mechanisms operating in any strain. The engineered strains were also tested for their responses to fluconazole treatment in a novel 3-day model of invasive C. albicans infection of mice. Fifty percent effective doses (ED(50)s) of fluconazole were highest for DSY296 and decreased proportionally with the sequential removal of each resistance mechanism. However, while the fold differences in ED(50) were proportional to the fold differences in MICs, their magnitude was lower than that measured in vitro and depended on the specific resistance mechanism operating.

Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells.

In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process.

Intestinal gluconeogenesis is a key factor for early metabolic changes after gastric bypass but not after gastric lap-band in mice.

Unlike the adjustable gastric banding procedure (AGB), Roux-en-Y gastric bypass surgery (RYGBP) in humans has an intriguing effect: a rapid and substantial control of type 2 diabetes mellitus (T2DM). We performed gastric lap-band (GLB) and entero-gastro anastomosis (EGA) procedures in C57Bl6 mice that were fed a high-fat diet. The EGA procedure specifically reduced food intake and increased insulin sensitivity as measured by endogenous glucose production. Intestinal gluconeogenesis increased after the EGA procedure, but not after gastric banding. All EGA effects were abolished in GLUT-2 knockout mice and in mice with portal vein denervation. We thus provide mechanistic evidence that the beneficial effects of the EGA procedure on food intake and glucose homeostasis involve intestinal gluconeogenesis and its detection via a GLUT-2 and hepatoportal sensor pathway.

Correlation between vasoconstrictor roles and mRNA expression of alpha1-adrenoceptor subtypes in blood vessels of genetically engineered mice.

We examined the contribution of each alpha(1)-adrenoceptor (AR) subtype in noradrenaline (NAd)-evoked contraction in the thoracic aortas and mesenteric arteries of mice. Compared with the concentration-response curves (CRCs) for NAd in the thoracic aortas of wild-type (WT) mice, the CRCs of mutant mice showed a significantly lower sensitivity. The pD(2) value in rank order is as follows: WT mice (8.21) > alpha(1B)-adrenoceptor knockout (alpha(1B)-KO) (7.77) > alpha(1D)-AR knockout (alpha(1D)-KO) (6.44) > alpha(1B)- and alpha(1D)-AR double knockout (alpha(1BD)-KO) (5.15). In the mesenteric artery, CRCs for NAd did not differ significantly between either WT (6.52) and alpha(1B)-KO mice (7.12) or alpha(1D)-KO (6.19) and alpha(1BD)-KO (6.29) mice. However, the CRC maximum responses to NAd in alpha(1D)- and alpha(1BD)-KO mice were significantly lower than those in WT and alpha(1B)-KO mice. Except in the thoracic aortas of alpha(1BD)-KO mice, the competitive antagonist prazosin inhibited the contraction response to NAd with high affinity. However, prazosin produced shallow Schild slopes in the vessels of mice lacking the alpha(1D)-AR gene. In the thoracic aorta, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.25 and 8.46, respectively, and in alpha(1B)-KO mice they were 8.49 and 9.13, respectively. In the mesenteric artery, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.34 and 7.47, respectively, and in alpha(1B)-KO mice they were 8.11 and 7.82, respectively. These pharmacological findings were in fairly good agreement with findings from comparison of CRCs, with the exception of the mesenteric arteries of WT and alpha(1B)-KO mice, which showed low affinities to BMY7378. We performed a quantitative analysis of the mRNA expression of each alpha(1)-AR subtype in these vessels in order to examine the correlation between mRNA expression level and the predominance of each alpha(1)-AR subtype in mediating vascular contraction. The rank order of each alpha(1)-AR subtype in terms of its vasoconstrictor role was in fairly good agreement with the level of expression of mRNA of each subtype, that is, alpha(1D)-AR > alpha(1B)-AR > alpha(1A)-AR in the thoracic aorta and alpha(1D)-AR > alpha(1A)-AR > alpha(1B)-AR in the mesenteric artery. No dramatic compensatory change of alpha(1)-AR subtype in mutant mice was observed in pharmacological or quantitative mRNA expression analysis.

Review of therapeutic drug monitoring of anticancer drugs part two - Targeted therapies.

Most of oral targeted therapies are tyrosine kinase inhibitors (TKIs). Oral administration generates a complex step in the pharmacokinetics (PK) of these drugs. Inter-individual PK variability is often large and variability observed in response is influenced not only by the genetic heterogeneity of drug targets, but also by the pharmacogenetic background of the patient (e.g. cytochome P450 and ABC transporter polymorphisms), patient characteristics such as adherence to treatment and environmental factors (drug-drug interactions). Retrospective studies have shown that targeted drug exposure, reflected in the area under the plasma concentration-time curve (AUC) correlates with treatment response (efficacy/toxicity) in various cancers. Nevertheless levels of evidence for therapeutic drug monitoring (TDM) are however heterogeneous among these agents and TDM is still uncommon for the majority of them. Evidence for imatinib currently exists, others are emerging for compounds including nilotinib, dasatinib, erlotinib, sunitinib, sorafenib and mammalian target of rapamycin (mTOR) inhibitors. Applications for TDM during oral targeted therapies may best be reserved for particular situations including lack of therapeutic response, severe or unexpected toxicities, anticipated drug-drug interactions and/or concerns over adherence treatment. Interpatient PK variability observed with monoclonal antibodies (mAbs) is comparable or slightly lower to that observed with TKIs. There are still few data with these agents in favour of TDM approaches, even if data showed encouraging results with rituximab, cetuximab and bevacizumab. At this time, TDM of mAbs is not yet supported by scientific evidence. Considerable effort should be made for targeted therapies to better define concentration-effect relationships and to perform comparative randomised trials of classic dosing versus pharmacokinetically-guided adaptive dosing.

Genetics of cadmium hyperaccumulation variance in Thlaspi caerulescens natural populations from Switzerland

SUMMARY Heavy metal presence in the environment is a serious concern since some of them can be toxic to plants, animals and humans once accumulated along the food chain. Cadmium (Cd) is one of the most toxic heavy metal. It is naturally present in soils at various levels and its concentration can be increased by human activities. Several plants however have naturally developed strategies allowing them to grow on heavy metal enriched soils. One of them consists in the accumulation and sequestration of heavy metals in the above-ground biomass. Some plants present in addition an extreme strategy by which they accumulate a limited number of heavy metals in their shoots in amounts 100 times superior to those expected for a non-accumulating plant in the same conditions. Understanding the genetic basis of the hyperaccumulation trait - particularly for Cd - remains an important challenge which may lead to biotechnological applications in the soil phytoremediation. In this thesis, Thlaspi caerulescens J. & C. Presl (Brassicaceae) was used as a model plant to study the Cd hyperaccumulation trait, owing to its physiological and genetic characteristics. Twenty-four wild populations were sampled in different regions of Switzerland. They were characterized for environmental and soil parameters as well as intrinsic characteristics of plants (i.e. metal concentrations in shoots). They were as well genetically characterized by AFLPs, plastid DNA polymorphism and genes markers (CAPS and microsatellites) mainly developed in this thesis. Some of the investigated genes were putatively linked to the Cd hyperaccumulation trait. Since the study of the Cd hyperaccumulation in the field is important as it allows the identification of patterns of selection, the present work offered a methodology to define the Cd hyperaccumulation capacity of populations from different habitats permitting thus their comparison in the field. We showed that Cd, Zn, Fe and Cu accumulations were linked and that populations with higher Cd hyperaccumulation capacity had higher shoot and reproductive fitness. Using our genetic data, statistical methods (Beaumont & Nichols's procedure, partial Mantel tests) were applied to identify genomic signatures of natural selection related to the Cd hyperaccumulation capacity. A significant genetic difference between populations related to their Cd hyperaccumulation capacity was revealed based on somè specific markers (AFLP and candidate genes). Polymorphism at the gene encoding IRTl (Iron-transporter also participating to the transport of Zn) was suggested as explaining part of the variation in Cd hyperaccumulation capacity of populations supporting previous physiological investigations. RÉSUMÉ La présence de métaux lourds dans l'environnement est un phénomène préoccupant. En effet, certains métaux lourds - comme le cadmium (Cd) -sont toxiques pour les plantes, les animaux et enfin, accumulés le long de la chaîne alimentaire, pour les hommes. Le Cd est naturellement présent dans le sol et sa concentration peut être accrue par différentes activités humaines. Certaines plantes ont cependant développé des stratégies leur permettant de pousser sur des sols contaminés en métaux lourds. Parmi elles, certaines accumulent et séquestrent les métaux lourds dans leurs parties aériennes. D`autres présentent une stratégie encore plus extrême. Elles accumulent un nombre limité de métaux lourds en quantités 100 fois supérieures à celles attendues pour des espèces non-accumulatrices sous de mêmes conditions. La compréhension des bases génétiques de l'hyperaccumulation -particulièrement celle du Cd - représente un défi important avec des applications concrètes en biotechnologies, tout particulièrement dans le but appliqué de la phytoremediation des sols contaminés. Dans cette thèse, Thlaspi caerulescens J. & C. Presl (Brassicaceae) a été utilisé comme modèle pour l'étude de l'hyperaccumulation du Cd de par ses caractéristiques physiologiques et génétiques. Vingt-quatre populations naturelles ont été échantillonnées en Suisse et pour chacune d'elles les paramètres environnementaux, pédologique et les caractéristiques intrinsèques aux plantes (concentrations en métaux lourds) ont été déterminés. Les populations ont été caractérisées génétiquement par des AFLP, des marqueurs chloroplastiques et des marqueurs de gènes spécifiques, particulièrement ceux potentiellement liés à l'hyperaccumulation du Cd (CAPS et microsatellites). La plupart ont été développés au cours de cette thèse. L'étude de l'hyperaccumulation du Cd en conditions naturelles est importante car elle permet d'identifier la marque, éventuelle de sélection naturelle. Ce travail offre ainsi une méthodologie pour définir et comparer la capacité des populations à hyperaccumuler le Cd dans différents habitats. Nous avons montré que les accumulations du Cd, Zn, Fe et Cu sont liées et que les populations ayant une grande capacité d'hyperaccumuler le Cd ont également une meilleure fitness végétative et reproductive. Des méthodes statistiques (l'approche de Beaumont & Nichols, tests de Martel partiels) ont été utilisées sur les données génétiques pour identifier la signature génomique de la sélection naturelle liée à la capacité d'hyperaccumuler le Cd. Une différenciation génétique des populations liée à leur capacité d'hyperaccumuler le Cd a été mise en évidence sur certains marqueurs spécifiques. En accord avec les études physiologiques connues, le polymorphisme au gène codant IRT1 (un transporteur de Fe impliqué dans le transport du Zn) pourrait expliquer une partie de la variance de la capacité des populations à hyperaccumuler le Cd.

Population pharmacokinetics of imatinib in CML and GIST patients under long-term treatment

Imatinib (Glivec®) has transformed the treatment and short-term prognosis of chronic myeloid leukaemia (CML) and gastro-intestinal stromal tumour (GIST). However, the treatment must be taken indefinitely, it is not devoid of inconvenience and toxicity. Moreover, resistance or escape from disease control occur in a significant number of patients. Imatinib is a substrate of the cytochromes P450 CYP3A4/5 and of the multidrug transporter P glycoprotein (product of the MDR1 gene). Considering the large inter-individual differences in the expression and function of those systems, the disposition and clinical activity of imatinib can be expected to vary widely among patients, calling for dosage individualisation. The aim of this exploratory study was to determine the average pharmacokinetic parameters characterizing the disposition of imatinib in the target population, to assess their inter-individual variability, and to identify influential factors affecting them. A total of 321 plasma concentrations, taken at various sampling times after latest dose, were measured in 59 patients receiving Glivec® at diverse regimens, using a validated chromatographic method (HPLC-UV) developed for this study. The results were analysed by non-linear mixed effect modelling (NONMEM). A one- compartment model with first-order absorption appeared appropriate to describe the data, with an average apparent clearance of 12.4 l/h, a distribution volume of 268 l and an absorption constant of 0.47 h-1. The clearance was affected by body weight, age and sex. No influences of interacting drugs were found. DNA samples were used for pharmacogenetic explorations. The MDR1 polymorphism 3435C>T appears to affect the disposition of imatinib. Large inter-individual variability remained unexplained by the demographic covariates considered, both on clearance (40%) and distribution volume (71%). Together with intra-patient variability (34%), this translates into an 8-fold width of the 90%-prediction interval of plasma concentrations expected under a fixed dosing regimen ! This is a strong argument to further investigate the possible usefulness of a therapeutic drug monitoring programme for imatinib. It may help to individualise the dosing regimen before overt disease progression or observation of treatment toxicity, thus improving both the long-term therapeutic effectiveness and tolerability of this drug.

Neurochemical profile of the mouse hypothalamus using in vivo 1H MRS at 14.1T.

The hypothalamus plays an essential role in the central nervous system of mammals by among others regulating glucose homeostasis, food intake, temperature, and to some extent blood pressure. Assessments of hypothalamic metabolism using, e.g. (1)H MRS in mouse models can provide important insights into its function. To date, direct in vivo (1)H MRS measurements of hypothalamus have not been reported. Here, we report that in vivo single voxel measurements of mouse hypothalamus are feasible using (1)H MRS at 14.1T. Localized (1)H MR spectra from hypothalamus were obtained unilaterally (2-2.2 microL, VOI) and bilaterally (4-4.4 microL) with a quality comparable to that of hippocampus (3-3.5 microL). Using LCModel, a neurochemical profile consisting of 21 metabolites was quantified for both hypothalamus and hippocampus with most of the Cramér-Rao lower bounds within 20%. Relative to the hippocampus, the hypothalamus was characterized by high gamma-aminobutryric acid and myo-inositol, and low taurine concentrations. When studying transgenic mice with no glucose transporter isoform 8 expressed, small metabolic changes were observed, yet glucose homeostasis was well maintained. We conclude that a specific neurochemical profile of mouse hypothalamus can be measured by (1)H MRS which will allow identifying and following metabolic alterations longitudinally in the hypothalamus of genetic modified models.

Use of new biochemical plasmatic markers, plasma free and total metanephrines, for the diagnosis and follow-up of neuroblastoma in children with clinical correlation

Background: Neuroblastoma is a paediatrictumour derived from the neural crest. Biochemical diagnosis and follow up rely on quantitation of urinary catecholamines (dopamine and noradrenaline) and their metabolites vanillylmandelic acid (VMA) and homovanillic acid (HVA) (gold-standard). When combined, these analyses have a sensitivity of 95%. However, they are clearly limited by inaccuracy of urine collection in young children and normalisation of catecholamine concentrations by creatininuria. Recent development in biochemical diagnosis of pheochromocytoma, another neural crest tumour found in adults, shows that plasmatic measurement of methoxylated catecholamines called metanephrines are more sensitive and specific than other biomarkers. Moreover, a study to determine the reference intervals for metanephrines in a pediatric population has recently been completed. The aim of this work is to describe the role of metanephrines monitoring in the follow up of neuroblastoma. Method: This retrospective study included patients with neuroblastoma in whom the following parameters were determined: plasma free and total metanephrines, plasma catecholamines, 24h urinary catecholamines and metanephrines in absolute value and corrected by creatinine, VMA and HVA at the diagnosis and during treatment at the University Hospital of Lausanne (Switzerland). Eleven patients aged between the first day of life and 7 years old were followed between 2005 and 2012. Clinical outcome and biochemical concentrations of the analytes were correlated. Results: At diagnosis, plasma free and total normetanephrines and methoxytyramine have a sensitivity of 100% compared to 85% for the actual gold standard. Metanephrine remain below the upper reference limit as expected since these tumours do not produce adrenaline. The relationship between biochemical markers and clinical outcome is illustrated graphically. Plasma or urinary normetanephrine and methoxytyramine correlate better with the history of the patient than VMA and HVA, as evaluated by ordinal logistic regression. Concentrations of analytes in urine show a better correlation with clinical events when the results are corrected by creatininuria. Conclusion: Normetanephrine and methoxytyramine reflect disease history in neuroblastoma patients and could play a significant role in the follow up of this type of tumour. Formal studies in a sufficient number of patients are needed to confirm this preliminary observation.

Evidence from glut2-null mice that glucose is a critical physiological regulator of feeding.

A role for glucose in the control of feeding has been proposed, but its precise physiological importance is unknown. Here, we evaluated feeding behavior in glut2-null mice, which express a transgenic glucose transporter in their beta-cells to rescue insulin secretion (ripglut1;glut2-/- mice). We showed that in the absence of GLUT2, daily food intake was increased and feeding initiation and termination following a fasting period were abnormal. This was accompanied by suppressed regulation of hypothalamic orexigenic and anorexigenic neuropeptides expression during the fast-to-refed transition. In these conditions, however, there was normal regulation of the circulating levels of insulin, leptin, or glucose but a loss of regulation of plasma ghrelin concentrations. To evaluate whether the abnormal feeding behavior was due to suppressed glucose sensing, we evaluated feeding in response to intraperitoneal or intracerebroventricular glucose or 2-deoxy-D-glucose injections. We showed that in GLUT2-null mice, feeding was no longer inhibited by glucose or activated by 2-deoxy-D-glucose injections and the regulation of hypothalamic neuropeptide expression by intracerebroventricular glucose administration was lost. Together, these data demonstrate that absence of GLUT2 suppressed the function of central glucose sensors, which control feeding probably by regulating the hypothalamic melanocortin pathway. Furthermore, inactivation of these glucose sensors causes overeating.

A missing sugar prevents glucose entry: a new twist on insulin secretion.

The signaling pathway that regulates glucose-stimulated insulin secretion depends on glucose metabolism, which is itself controlled by glucokinase. In a recent issue of Cell, show that altering N-glycosylation of the GLUT2 glucose transporter prevents its anchoring and retention at the cell surface; this impairs glucose uptake and insulin secretion.

Creatine, central nervous system and creatine deficiency syndromes

It was long thought that most of brain creatine was of peripheral origin. However, recentworks have demonstrated that creatine crosses blood-brain barrier only with poor efficiency, and thatCNS must ensure parts of its creatine needs by its own creatine synthesis pathway, thank to the brainexpression of AGAT and GAMT (creatine synthesis) and SLC6A8 (creatine transporter). This newunderstanding of creatine metabolism and transport in CNS allows a better comprehension of creatinedeficiency syndromes, which are due to deficiencies in AGAT, GAMT and SLC6A8 and mainly affectthe brain of patients who show severe neurodevelopmental delay and present neurological symptomsin early infancy.