Background and Objectives: Advagraf(R) is a new extended-release once-daily formulation of tacrolimus, a potent immunosuppressant widely used in renal transplantation. The aims of this study were (i) to develop a population pharmacokinetic model for once-daily tacrolimus in adult renal transplant patients; and (ii) to develop a Bayesian estimator able to reliably estimate individual pharmacokinetic parameters and exposure indices. Methods: Full pharmacokinetic profiles obtained from 41 adult renal transplant patients who had been switched from ciclosporin to a single daily dose of the new once-daily tacrolimus formulation for more than 6 months were analysed. Tacrolimus concentrations were measured using validated turbulent flow chromatography-tandem mass spectrometry methods. Population parameters were computed using nonlinear mixed-effect modelling software (NONMEM(R) Version VI). The patients were randomly divided into (i) a model-building test group (n = 29); and (ii) a validation group (n = 12). Population pharmacokinetic analysis was performed to estimate the effects on tacrolimus pharmacokinetics of demographic characteristics (sex, bodyweight, age), drug interaction with prednisolone, laboratory test results (the haematocrit, haemaglobin level and serum creatinine level) and cytochrome P450 (CYP) 3A5 (CYP3A5) genetic polymorphism. The population pharmacokinetic model was further refined by taking into account all of the data from the 41 patients, and the final model was validated using a bootstrap and a visual predictive check. For Bayesian estimation, the best limited-sampling strategy was determined on the basis of the D-optimality criterion and validation performed in the validation group. Results: The trapezoidal area under the whole-blood concentration time curve from 0 to 24 hours (AUC(24)) of tacrolimus varied by up to 50% for the same trough concentration value. The pharmacokinetics of once-daily tacrolimus were well described by a two-compartment model combined with an Erlang distribution to describe the absorption phase. The CYP3A5 genotype was the only covariate retained in the final model. The apparent clearance of tacrolimus was 2-fold higher in expressers (with the CYP3A5*1/*1 and CYP3A5*1/*3 genotypes) than in non-expressers (with the CYP3A5*3/*3 genotype). This factor explained around 25% of the interindividual variability in the apparent clearance. A posteriori Bayesian estimation allowed accurate prediction of the AUC(24) of once-daily tacrolimus, using just three sampling times (0, 1 and 3 hours post-dose) with a nonsignificant mean bias of 0.7% (range 16-20%) and good precision (root mean square error 9%). Conclusions: Population pharmacokinetic analysis of once-daily tacrolimus in renal transplant recipients resulted in identification of the CYP3A5*1/*3 genotype as a significant covariate on the apparent clearance of tacrolimus, and the design of an accurate maximum a posteriori Bayesian estimator based on three blood concentration measurements and this covariate. Such a tool could be helpful for comparing different exposure indices or different target levels. It could contribute to improvement of the efficacy and tolerability of once-daily tacrolimus in some patients.

PiT1/SLC20A1 is a sodium-dependent inorganic phosphate (Pi) transporter expressed by most mammalian cells. Interestingly, PiT1 transcription has been shown to be upregulated by the tumor necrosis factor alpha (TNF), and we have now investigated the possible involvement of PiT1 in TNF-induced apoptosis. We show that PiT1-depleted cells are more sensitive to the pro-apoptotic activity of TNF (i.e. when the anti-apoptotic NFkappaB pathway is inactivated). These observations were made in the human HeLa cancer cell line either transiently or stably depleted in PiT1 by RNA interference and in immortalized mouse embryonic fibroblasts (MEFs) isolated from PiT1 knockout embryos. Depletion of the closely related family member PiT2 had no effect on TNF-induced apoptosis, showing that this effect was specific to PiT1. The increased sensitivity of PiT1-depleted cells was evident regardless of the presence or absence of extracellular Pi, suggesting that a defect in Pi uptake was not involved in the observed phenotype. Importantly, we show that the re-expression of a Pi-uptake mutant of PiT1 in PiT1-/- MEFs delays apoptosis as efficiently as the WT protein, showing that this function of PiT1 is unrelated to its transport activity. Caspase-8 is more activated in PiT1-depleted cells and our data reveal that the sustained activation of the MAPK JNK is upregulated in response to TNF. JNK activity is actually involved in PiT1-depleted cell death as specific JNK inhibitors delay apoptosis.

Understanding the interactions between sleep and the immune system may offer insight into why short sleep duration has been linked to negative health outcomes. We, therefore, investigated the effects of napping and extended recovery sleep after sleep restriction on the immune and inflammatory systems and sleepiness. After a baseline night, healthy young men slept for a 2-hr night followed by either a standard 8-hr recovery night (n=12), a 30-min nap (at 1 p.m.) in addition to an 8-hr recovery night (n=10), or a 10-hr extended recovery night (n=9). A control group slept 3 consecutive 8-hr nights (n=9). Subjects underwent continuous electroencephalogram polysomnography and blood was sampled every day at 7 a.m. Leukocytes, inflammatory and atherogenesis biomarkers (high-sensitivity C-reactive protein, interleukin-8, myeloperoxidase, fibrinogen and apolipoproteins ApoB/ApoA), sleep patterns and sleepiness were investigated. All parameters remained unchanged in the control group. After sleep restriction, leukocyte and -among leukocyte subsets- neutrophil counts were increased, an effect that persisted after the 8-hr recovery sleep, but, in subjects who had a nap or a 10-hr recovery sleep, these values returned nearly to baseline. Inflammatory and atherogenesis biomarkers were unchanged except for higher myeloperoxidase levels after sleep restriction. The increased sleepiness after sleep restriction was reversed better in the nap and extended sleep recovery conditions. Saliva cortisol decreased immediately after the nap. Our results indicate that additional recovery sleep after sleep restriction provided by a midday nap prior to recovery sleep or a sleep extended night can improve alertness and return leukocyte counts to baseline values.

BACKGROUND: Type I and II inosine monophosphate dehydrogenases (IMPDH) are the targets of mycophenolic acid (MPA), a widely used immunosuppressant. The aims of this study were: to check the presence of controversial polymorphisms in the IMPDH II gene; to look for new ones; and to investigate potential associations between the most frequent SNPs in both IMPDH genes and clinical outcome in renal transplant recipients. METHODS: The DNA and clinical data of 456 patients from two clinical trials were collected. We sequenced the IMPDH II gene in 80 patients and we genotyped the 456 patients' DNA for the IMPDH II rs4974081, rs11706052, 787C>T and the IMPDH I rs2278293 and rs2278294 SNPs, all of which were earlier reported to be potentially involved in MPA treatment related outcome. We investigated the associations of biopsy proven acute rejection (BPAR), leucopenia, cytomegalovirus infections and other infections with these IMPDH polymorphisms, as well as with demographic, biological and treatment data using multivariate analysis. RESULTS: Many IMPDH II variant alleles referenced in Genbank were not detected and no new polymorphisms were identified. In the whole group of 456 patients, the IMPDH I rs2278294 SNP was associated with a lower risk of BPAR and a higher risk of leucopenia over the first year post-transplantation. No other IMPDH I or IMPDH II polymorphism was significantly associated with any clinical outcome. Interestingly, calcineurin inhibitor and MPA exposures below the therapeutic range increased the risk of BPAR. Cytomegalovirus infection was the factor most closely linked with leucopenia, whereas tacrolimus was associated with fewer infections than cyclosporine. CONCLUSION: IMPDH II genotyping may not improve MPA treatment outcome over the first year post-transplantation, in contrast to MPA and calcineurine inhibitor therapeutic drug monitoring and IMPDH I genotyping.

The enantiomeric purity determination of a synthetic intermediate of new 3,4-dihydro-2,2-dimethyl-2H-1-benzopyrans, i.e. 4-amino-2,2-dimethyl-6-ethoxycarbonylamino-3,4-dihydro-2H-1-benzopyran, was successfully carried out using an anionic cyclodextrin (CD) derivative combined with a chiral ionic liquid (IL). In order to obtain high resolution and efficiency values, the addition of a chiral IL, i.e. ethylcholine bis(trifluoromethylsulfonyl)imide (EtChol NTf(2)), to the background electrolyte containing heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD (HDMS-beta-CD) was found to be essential. A simultaneous increase in separation selectivity and enantioresolution seems to indicate a synergistic effect of HDMS-beta-CD and EtChol NTf(2). The best enantioseparation of the key intermediate was achieved using a methanolic solution of 0.75M formic acid, 10mM ammonium formate, 1.5mM HDMS-beta-CD and 5mM EtChol NTf(2). Levamisole was selected as internal standard. The optimized conditions allowed the determination of 0.1% of each enantiomer in the presence of its stereoisomer using the method of standard additions. The NACE method was then fully validated with respect to selectivity, response function, trueness, precision, accuracy, linearity and limits of detection and quantification.

AIM: In renal transplant patients given mycophenolate mofetil (MMF), we investigated the relationship between the digestive adverse events and polymorphisms in the UGT genes involved in mycophenolic acid (MPA) intestinal metabolism and biliary excretion of its phase II metabolites. METHODS: Clinical data and DNA from 256 patients transplanted between 1996 and 2006 and given MMF with cyclosporin (CsA, n = 185), tacrolimus (TAC, n = 49) or sirolimus (SIR, n = 22), were retrospectively analysed. The relationships between diarrhoea and polymorphisms in UGT1A8 (2; 518C>G, 3; 830G>A), UGT1A7 (622C>T), UGT1A9 (-275T>A), UGT2B7 (-840G>A) and ABCC2 (-24C>T, 3972C>T) or the co-administered immunosuppressant were investigated using the Cox proportional hazard model. RESULTS: Multivariate analysis showed that patients on TAC or SIR had a 2.8 higher risk of diarrhoea than patients on CsA (HR = 2.809; 95%CI (1.730, 4.545); P < 0.0001) and that non-carriers of the UGT1A8 2 allele (CC518 genotype) had a higher risk of diarrhoea than carriers (C518G and 518GG genotypes) (HR = 1.876; 95%CI (1.109, 3.175); P = 0.0192). When patients were divided according to the immunosuppressive co-treatment, a significant effect of UGT1A8 2 was found in those co-treated with CsA (HR = 2.414; 95%CI (1.089, 5.354); P = 0.0301) but not TAC or SIR (P = 0.4331). CONCLUSION: These results suggest that a possible inhibition of biliary excretion of MPA metabolites by CsA and a decreased intestinal production of these metabolites in UGT1A8 2 carriers may be protective factors against MMF-induced diarrhoea.

Cyclosporin A (CsA) is a substrate for cytochrome P450 3A and the efflux transporter P-glycoprotein (P-gp; ABCB1), both abundantly expressed in the kidney. In a long-term follow-up of a cohort of patients who had received kidney transplants between the years 1990 and 2005, we retrospectively investigated the effect of CYP3A4, CYP3A5, and ABCB1 polymorphisms in kidney graft donors on recipients' renal function and risk of subsequent graft loss. DNA samples from 227 donors and clinical data from the 259 respective recipients were analyzed. Graft loss was significantly associated with the presence of the ABCB1 variant haplotype 1236T/2677T/3435T in the donor (1236T/2677T/3435T vs. other haplotypes: hazard ratio = 9.346; 95% confidence interval (CI) (2.278-38.461); P = 0.0019) and with previous episodes of acute organ rejection (hazard ratio = 3.077; 95% CI (1.213-7.812); P = 0.0178). The variant haplotype was also associated with a greater decrease in renal function (homozygotes for TTT -3.047 ml.min(-1)/year; heterozygotes for TTT -4.435 ml.min(-1)/year; others -2.186 ml.min(-1)/year; P = 0.0240). The study showed that the presence of ABCB1 polymorphisms in donors influences long-term graft outcome adversely with decrease in renal function and graft loss in transplant recipients receiving CsA.

Aims - Low-density lipoprotein (LDL) oxidation is a key step in atherogenesis, promoting the formation of lipid-laden macrophages. Here, we compared the effects of copper-oxidized LDLs (OxLDLs) and of the more physiologically relevant myeloperoxidase-oxidized LDLs (MoxLDLs) in murine RAW264.7 macrophages and in human peripheral blood monocyte-derived macrophages. Results - Both oxidized LDLs, contrary to native LDLs, induced foam cell formation and an intracellular accumulation of reactive oxygen species (ROS). This oxidative stress was responsible for the activation of the NF-E2-related factor 2 (Nrf2) transcription factor, and the subsequent Nrf2-dependent overexpression of the antioxidant genes, Gclm and HO-1, as evidenced by the invalidation of Nrf2 by RNAi. Interestingly, MoxLDLs always induced a stronger response than OxLDLs. These differences could be partly explained by specific ROS-producing mechanisms differing between OxLDLs and MoxLDLs. Whereas both types of oxidized LDLs caused ROS production partly by NADPH oxidase, only MoxLDLs-induced ROS production was dependent on cytosolic PLA2. Conclusion - This study highlights that OxLDLs and MoxLDLs induce an oxidative stress, through distinct ROS-producing mechanisms, which is responsible for the differential activation of the Nrf2 pathway. These data clearly suggests that results obtained until now with copper oxidized-LDLs should be carefully reevaluated taking into consideration physiologically more relevant oxidized LDLs.

In the present study the migration order of the propranolol enantiomers with various native CDs and neutral and charged CD derivatives was examined in capillary electrophoresis (CE). The reversal of the enantiomer migration order was observed due to sulfation of beta-CD on its primary hydroxy groups. The structures of intermolecular selector-select and temporary diastereomeric associates in solution were elucidated based on 1D rotating frame nuclear Overhauser effect spectroscopy (1D ROESY) experiments. Major structural differences were observed between the propranolol complexes with native beta-CD and heptakis(6-O-sulfo)-beta-CD.

A NACE method was developed for the separation of fenbendazole (FBZ), a prochiral drug giving rise to chiral (oxfendazole or OFZ) and nonchiral (FBZ sulphone or FBZSO(2)) metabolites. First, the effect of the nature and the concentration of CD as well as that of the acidic BGE on the enantiomeric separation of OFZ were studied. OFZ enantiomers were completely resolved using a BGE made up of 10 mM ammonium formate and 0.5 M TFA in methanol containing 10 mM heptakis(2,3-di-O-acetyl-6-O-sulfo)-beta-CD and 10 mM heptakis(2,3-di-O-methyl-6-O-sulfo)-beta-CD. Moreover, the NACE method was found to be particularly well suited to the simultaneous determination of FBZ, OFZ enantiomers, and FBZSO(2). Thiabendazole was selected as an internal standard. The CD-NACE potential was then evaluated for in vitro metabolism studies using FBZ as a model case. The OFZ enantiomers and FBZSO(2) could be detected after incubation of FBZ in the phenobarbital-induced male rat liver microsomes systems.

Glioblastoma is one of the most angiogenic human tumours and endothelial proliferation is a hallmark of the disease. A better understanding of glioblastoma vasculature is needed to optimize anti-angiogenic therapy that has shown a high but transient efficacy. We analysed human glioblastoma tissues and found non-endothelial cell-lined blood vessels that were formed by tumour cells (vasculogenic mimicry of the tubular type). We hypothesized that CD133(+) glioblastoma cells presenting stem-cell properties may express pro-vascular molecules allowing them to form blood vessels de novo. We demonstrated in vitro that glioblastoma stem-like cells were capable of vasculogenesis and endothelium-associated genes expression. Moreover, a fraction of these glioblastoma stem-like cells could transdifferentiate into vascular smooth muscle-like cells. We describe here a new mechanism of alternative glioblastoma vascularization and open a new perspective for the antivascular treatment strategy.

Side-population (SP) analysis has been used to identify progenitor cells from normal and malignant tissues as well as revealing tumor cells with increased resistance to radiation and chemotherapy. Despite enhanced chemoresistance, tumor SP cells may still express tumor-associated antigens (TAAs), which may render them susceptible to elimination by the immune system. In this study, we show that both Hodgkin lymphoma (HL) cell lines and primary HL tumor samples contain a distinct SP phenotype. Importantly, while these cells showed increased resistance to gemcitabine, a commonly used drug for the treatment of refractory HL, HL SP cells also expressed higher levels of the TAAs MAGEA4, SSX2, survivin, and NY-ESO-1, which allowed them to be specifically recognized and killed by TAA-specific cytotoxic T lymphocytes. This study suggests that chemoresistant HL SP cells can be targeted by the immune system, providing a rationale for combined chemotherapy and immunotherapy for the treatment of HL.

Separations using CE employing non-aqueous BGE are already as well established as separations in aqueous buffers. The separation mechanisms in achiral CE with non-aqueous BGEs are most likely similar to those in aqueous buffers. However, for the separation of enantiomers involving their interaction with chiral buffer additives, the interaction mechanisms might be very different in aqueous and non-aqueous BGEs. While the hypothesis regarding distinct mechanisms of enantiomer separations in aqueous and non-aqueous BGEs has been mentioned in several papers, no direct proof of this hypothesis has been reported to date. In the present study, the enantiomers of propranolol were resolved using CE in aqueous and non-aqueous methanolic BGEs with two single isomer sulfated derivatives of beta-CD, namely heptakis (2,3-diacetyl-6-sulfo)-beta-CD and heptakis (2,3-dimethyl-6-sulfo)-beta-CD. The enantiomer migration order of propranolol was inverted when an aqueous BGE was replaced with non-aqueous BGE in the case of heptakis (2,3-dimethyl-6-sulfo)-beta-CD but remained the same in the case of heptakis (2,3-diacetyl-6-sulfo)-beta-CD. The possible molecular mechanisms leading to this reversal of enantiomer migration order were studied by using nuclear overhauser effect spectroscopy in both aqueous and non-aqueous BGEs.

The involvement of myeloperoxidase (MPO) in various inflammatory conditions has been the scope of many recent studies. Besides its well studied catalytic activity, the role of its overall structure and glycosylation pattern in biological function is barely known. Here, the N-glycan composition of native dimeric human MPO purified from neutrophils and of monomeric MPO recombinantly expressed in Chinese hamster ovary cells has been investigated. Analyses showed the presence of five N-glycans at positions 323, 355, 391, 483, 729 in both proteins. Site by site analysis demonstrated a well conserved micro- and macro-heterogeneity and more complex-type N-glycans for the recombinant form. Comparison of biological functionality of glycosylated and deglycosylated recombinant MPO suggests that glycosylation is required for optimal enzymatic activity. Data are discussed with regard to biosynthesis and three-dimensional structure of MPO.

OBJECTIVE: To study the neuroimaging features of intracranial solitary fibrous tumors (ISFTs). MATERIALS AND METHODS: Retrospective study of neuroimaging features of 9 consecutive histopathologically proven ISFT cases. Location, size, shape, density, signal intensity and gadolinium uptake were studied at CT and MRI. Data collected from diffusion-weighted imaging (DWI) (3 patients), perfusion imaging and MR spectroscopy (2 patients), and DSA (4 patients) were also analyzed. RESULTS: The tumors most frequently arose from the intracranial meninges (7/9), while the other lesions were intraventricular. Tumor size ranged from 2.5 to 10cm (mean=6.6cm). They presented multilobular shape in 6/9 patients. Most ISFTs were heterogeneous (7/9) with areas of low T2 signal intensity that strongly enhanced after gadolinium administration (6/8). Erosion of the skull was present in about half of the cases (4/9). Components with decreased apparent diffusion coefficient were seen in 2/3 ISFTs on DWI. Spectroscopy revealed elevated peaks of choline and myo-inositol. MR perfusion showed features of hyperperfusion. CONCLUSION: ISFT should be considered in cases of extra-axial, supratentorial, heterogeneous, hypervascular tumor. Areas of low T2 signal intensity that strongly enhance after gadolinium injection are suggestive of this diagnosis. Restricted diffusion and elevated peak of myo-inositol may be additional valuable features.

BACKGROUND.: In the prospective, randomized, multicenter APOMYGRE trial conducted in France, concentration-controlled mycophenolate mofetil (MMF) dosing based on mycophenolic acid (MPA) exposure significantly reduced the treatment failure and acute rejection during the first posttransplantation year compared with fixed-dose MMF. This analysis investigated the cost effectiveness of dose individualization. METHOD.: The study included 65 patients per group (intent-to-treat population). Treatment failure (primary efficacy endpoint) was defined as death, graft loss, acute rejection, or MMF discontinuation because of adverse effects. Data on hospitalizations, drugs prescribed, physicians' fees, laboratory expenses, ambulatory visits, and transportation were retrieved. Costs were calculated from the French National Health System perspective. RESULTS.: The mean (95% confidence interval) total yearly cost per patient was &OV0556;47,477 (&OV0556;43,933; &OV0556;51,020) in the concentration-controlled group and &OV0556;46,783 (&OV0556;44,152; &OV0556;49,414) in the fixed-dose group (P=0.7). The observed incremental cost-effectiveness ratio was &OV0556;3757 per treatment failure (Purchasing Power Parities United States/France: $4129). Hospitalization and drug costs accounted for approximately 50% and 25% of total costs, respectively. The cost for MPA area under the concentration-time curve and dose calculation was &OV0556;452 per patient, less than 1% of the total cost. CONCLUSION.: In the APOMYGRE trial, therapeutic MPA monitoring using a limited sampling strategy reduced the risk of treatment failure and acute rejection in renal allograft recipients during the first 12 months posttransplantation, at neutral cost.

Ribavirin pharmacokinetic and exposure effect trials based on either plasma or serum concentrations have yielded diverging results. This study aimed to compare ribavirin concentrations in serum and plasma and to investigate the influence of blood collection and preanalytical conditions on ribavirin concentration stability. Blood samples from patients with hepatitis virus C and receiving ribavirin were collected in plain (dry) tubes, in tubes containing ethylenediaminetetra-acetic acid or lithium-heparinate, in Type II Serum Separating Tubes with clot activator, or Type II lithium heparinate Plasma Separating Tubes. Different time and temperature conditions were tested before and after blood centrifugation. Ribavirin was determined using liquid chromatography-dual mass spectroscopy. Multiple-way analysis of variance was used for statistical analyses. Ribavirin concentrations showed a higher interlaboratory variability in serum than in plasma. Results were fairly stable over 2 hours in whole blood collected in dry or ethylenediaminetetra-acetic acid tubes and very stable up to 24 hours in serum or plasma kept in gel-containing tubes after immediate centrifugation. When Plasma Separating II gel tubes were kept at +4 degrees C or at ambient temperature for up to 24 hours before centrifugation, ribavirin concentrations decreased by 1% to 8% and 12% to 18%, respectively. These results suggest that blood samples should be collected in gel-containing tubes and centrifuged immediately, after which the tubes can be kept at ambient temperature for the next 24 hours. In case of clinical constraints, Plasma Separating II gel tubes can be kept at +4 degrees C for a maximum of 2 hours before centrifugation with limited impact on the measured concentrations.

The influence of the BGE composition, including the addition of a single-isomer sulfated beta-CD derivative, on the ionization performance of the model compound carvedilol in NACE-ESI-MS was studied using an alternative infusion method. This approach employs voltage-induced infusion of the BGE containing the analyte, and takes into account the effects of variations in EOF and effective analyte mobility on the ESI-MS intensity. First, the optimal composition of the sheath liquid for CE-MS in terms of signal abundance and stability was determined. The BGE ammonium formate, acetate, and camphorsulfonate were found to have similar effects on analyte ionization. Addition of single-isomer sulfated beta-CD derivatives (available as sodium salt) to the BGE revealed that the anionic CD derivatives did not give rise to the same ionization suppression effect. This result can be attributed to differences in the dissociation state of these sodium salts. Finally, it is shown that information about chiral selectivity can also be obtained with the applied infusion method.

The loss of chromosomes 1p-19q is the only prognostic molecular alteration identified in low-grade gliomas (LGGs) to date. Search for loss of heterozygosity (LOH) on chromosomes 1p, 9p, 10q, and 19q was performed in a series of 231 LGGs. Loss of chromosomes 1p-19q was strongly correlated with prolonged progression-free survival (PFS) and overall survival (OS) in univariate and multivariate analyses. LOH on 9p and 10q were associated with shortened PFS (P = .01 and .03, respectively) on univariate analysis. On multivariate analysis, LOH on 9p remained significant for PFS (P = .05), whereas LOH on 10q had a significant effect on OS (P = .02). Search for LOH 9p and 10q appears to be a useful complement to analysis of chromosomes 1p-19q in LGGs.