f Voriconazole (VRC) plasma trough concentrations (C min ) are highly variable, and this could affect treatment efficacy and safety in patients undergoing allogeneic hematopoietic stem cell transplantation (AHSCT). We aimed to describe the intra-and interindividual variation of VRC C min throughout the course of VRC therapy and to identify the determinants of this variation. Clinical data, medications, and VRC C min (n ‫؍‬ 308) of 33 AHSCT patients were retrospectively collected. Cytochrome P450 (CYP450) genotypes of CYP2C19, CYP3A4, and CYP3A5 patients were retrospectively determined before allografting, and a combined genetic score was calculated for each patient. The higher the genetic score, the faster the metabolism of the patient. The VRC C min inter-and intraindividual coefficients of variation were 84% and 68%, respectively. The VRC dose (D) was correlated to VRC C min (r ‫؍‬ 0.412, P < 0.0001) only for oral administration. The administration route and the genetic score significantly affected the initial VRC C min . Considering oral therapy, patients with a genetic score of <2 had higher initial VRC C min /D than patients with a genetic score of >2 (P ‫؍‬ 0.009). Subsequent VRC C min remained influenced by the genetic score (P ‫؍‬ 0.004) but were also affected by pump proton inhibitor comedication (P < 0.0001). The high variability of VRC C min in AHSCT patients is partially explained by the route of administration, treatment with pump proton inhibitors, and the combined genetic score. This study suggests the interest in combined genetic score determination to individualize a priori the VRC dose and underlines the need for longitudinal therapeutic drug monitoring to adapt subsequent doses to maintain the VRC C min within the therapeutic range.

Microtubules are centrally involved in cell division, being the principal components of mitotic spindle. Tubulin, the constituent of microtubules, can be cyclically modified on its A-subunit by enzymatic removal of the COOH-terminal tyrosine residue by an ill-defined tubulin carboxypeptidase (TCP) and its readdition by tubulin tyrosine ligase (TTL). We and others have previously shown that suppression of TTL and resulting accumulation of detyrosinated tubulin are frequent in human cancers of poor prognosis. Explanations for the involvement of TTL and detyrosinated tubulin in tumor progression arise from the recent discovery that tubulin detyrosination leads to CAP-Gly protein mislocalization, which correlates with defects in spindle positioning during mitosis. Impaired control of spindle positioning is one factor favoring tumor invasiveness. Thus, TCP could be a target for developing novel therapeutic strategies against advanced stages of cancers. Inhibitors of TCP, by reversing abnormal detyrosinated tubulin accumulation in tumor cells, could impair tumor progression. TCP has never been isolated and this has hampered search of specific inhibitors. In this article, we describe a cell-based assay of TCP activity and its use to screen a library of natural extracts for their inhibitory potency. This led to the isolation of two sesquiterpene lactones. We subsequently found that parthenolide, a structurally related compound, can efficiently inhibit TCP. This inhibitory activity is a new specific property of parthenolide independent of its action on the nuclear factor-KB pathway. Parthenolide is also known for its anticancer properties. Thus, TCP inhibition could be one of the underlying mechanisms of these anticancer properties.

Hypoxia is an important selective force in the clonal evolution of tumours. Through HIF-1 and other transcription factors combined with tumour-specific genetic alterations, hypoxia is a dominant factor in the angiogenic phenotype. Cellular adaptation to hypoxia is an important requirement of tumour progression independent of angiogenesis. The adaptive changes, insofar as they alter hypoxiainduced apoptosis, are likely to determine responsiveness to antiangiogenic strategies. To investigate this adaptation of tumour cells to hypoxia, we recreated in vitro the in vivo situation of chronic intermittent exposure to low-oxygen levels. The colon carcinoma cell lines HT29 and HCT116 were subjected to 40 episodes of sublethal hypoxia (4 h) three times a week. The resulting two hypoxiaconditioned cell lines have been maintained in culture for more than 2 years. In both cell lines changes in doubling times occurred: in HT29 an increase, and in HCT116 a decrease. Cell survival in response to hypoxia and to DNA damage differed strikingly in the two cell lines. The HT29 hypoxia-conditioned cells were more resistant than the parental line to a 24 h hypoxic challenge, while those from HCT116 surprisingly were more sensitive. Sensitivity to cisplatin in vitro was also significantly different for the hypoxiaconditioned compared with the parental lines, suggesting a change in pathways leading to apoptosis following DNA damage signaling. The growth of both conditioned cell lines in vivo as xenografts in immunodeficient (SCID) mice was more rapid than their parental lines, and was accompanied in each by evidence of enhanced vascular proliferation as a consequence of the hypoxia-conditioning. Thus the changes in apoptotic susceptibility were independent of altered angiogenesis. The derivation of these lines provides a model for events within hypoxic regions of colon cancers, and for the acquisition of resistance and sensitivity characteristics that may have therapeutic implications for the use of antiangiogenesis drugs.

How pharmacogenetics modulates the inhibitory effects of inflammation on voriconazole trough concentration (Cmin) remains unknown. In 29 recipients of allogeneic hematopoietic stem cell transplantation retrospectively studied, both a genetic score (which aggregated CYP2C19 and CYP3A genotypes) and inflammation significantly influenced voriconazole Cmin (n = 260). A trend toward (p = 0.03) a greater impact of inflammation in patients with the highest genetic score (corresponding to ultra-rapid metabolizers) was observed. Further researches are needed to confirm these data.

Voriconazole (VRC) overdoses are frequent and expose patients at high risk of adverse effects. This case–control study performed in hematological patients who benefited from VRC therapeutic drug monitoring from January 2012 to December 2015 aimed to identify risk factors of VRC overdose. Pharmacogenetic, biological, and demographic parameters at the time of VRC trough concentration (Cmin) were retrospectively collected from medical records. Cases (VRC overdose: defined by a VRC Cmin ≥ 4 mg/L; n = 31) were compared to controls (no VRC overdose: defined by VRC Cmin < 4 mg/L; n = 31) using nonparametric or chi‐square tests followed by multivariable analysis. VRC overdoses were significantly associated with high CRP and bilirubin levels, intravenous administration, and age in univariable analysis. In contrast, the proportion of CYP genotypes (CYP2C19, CYP3A4, or CYP3A5, considered alone or combined in a combined genetic score) were not significantly different between patients who experienced a VRC overdose and those who did not. In multivariable analysis, the class of CRP level (defined by median CRP levels of 96 mg/L) was the sole independent risk factor of VRC overdose (P < 0.01). Patients with CRP levels > 96 mg/L) had a 27‐fold (IC 95%: [6–106]) higher risk of VRC overdose than patients with CRP levels ≤ 96 mg/L. This study demonstrates that inflammatory status, assessed by CRP levels, is the main risk factor of VRC overdose in French hematological patients, whereas pharmacogenetic determinants do not appear to be involved.

Background. Tacrolimus trough concentrations (mean/variability), as well as concentration-to-dose ratio (C/D ratio), affect kidney allograft outcomes. We investigated the link between the C/D ratio and death-censored kidney graft survival (DCGS). Methods. We performed a retrospective study on 1029 kidney transplant patients (2004–2016) with the following criteria: tacrolimus-based immunosuppression, >1-year graft survival, no initial use of everolimus, and available anti–human leukocyte antigen antibody data. We analyzed the impact of the time-varying C/D ratio on DCGS. Fast metabolizers were defined by a C/D ratio < 1.05. We also investigated the effect of an early (mo 3 to mo 6 post transplantation) C/D ratio below 1.05. Cox survival analyses were performed, adjusting for potential confounders (tacrolimus trough, variability of tacrolimus trough, de novo donor-specific antibody development, cytochrome P450 3A5 genotype, pregraft sensitization, mo 3 glomerular filtration rate). Results. Time-varying C/D ratio was significantly associated with DCGS (hazard ratio [HR], 2.35; P < 0.001) in a univariate model, on the full analysis set comprising 1029 patients. In the multivariate time-varying model, based on 666 patients with available cytochrome P450 3A5 genotypes, the effect of the C/D ratio remained significant (HR, 2.26; P = 0.015); even when glomerular filtration rate at month 3 < 30 mL/min/1.73 m2 (HR, 2.61; P = 0.011), de novo donor-specific antibody development (HR, 4.09; P < 0.001) and continued steroid prescription (HR=2.08, P = 0.014) were taken into account (other covariates, including tacrolimus trough concentrations, were nonsignificant). In the same multivariate model, the effect of early C/D ratio (median at mo 3 and mo 6) remained significantly associated with DCGS (HR, 2.25; P = 0.041). Conclusions. C/D ratio is an independent and early predictor of DCGS. Identification of fast metabolizers could be a strategy to improve graft survival, for example, by optimizing tacrolimus formulation. Mechanistic studies to understand the C/D ratio effect are required.

The authors describe a cell-based assay for anti-microtubule compounds suitable for automation. This assay allows the identification, in a single screening campaign, of both microtubule-destabilizing and microtubule-stabilizing agents. Its rationale is based on the substrate properties of the tubulin-modifying enzymes involved in the tubulin tyrosination cycle. This cycle involves the removal of the C-terminal tyrosine of the tubulin α-subunit by an ill-defined tubulin carboxypeptidase and its readdition by tubulin tyrosine ligase. Because of the substrate properties of these enzymes, dynamic microtubules, sensitive to depolymerizing drugs, are composed of tyrosinated tubulin, whereas nondynamic, stabilized microtubules are composed of detyrosinated tubulin. Thus depolymerization or stabilization of the microtubule network can easily be detected with doubleimmunofluorescence staining using antibodies specific to tyrosinated and detyrosinated tubulin. The authors have scaled this assay to the 96-well plate format and adapted its process for an automated handling, including a readout using a microplate reader. They describe the different steps of this adaptation. This assay was validated using known compounds. This new cellbased assay represents an alternative to both global cytotoxicity assays and in vitro tubulin assembly assays commonly used for the detection of microtubule poisons. (Journal of Biomolecular Screening 2006:377-389)

Fluoropyrimidines (FU) are still the most prescribed anticancer drugs for the treatment of solid cancers. However, fluoropyrimidines cause severe toxicities in 10 to 40% of patients and toxic deaths in 0.2 to 0.8% of patients, resulting in a real public health problem. The main origin of FU-related toxicities is a deficiency of dihydropyrimidine dehydrogenase (DPD), the rate-limiting enzyme of 5-FU catabolism. DPD deficiency may be identified through pharmacogenetics testing including phenotyping (direct or indirect measurement of enzyme activity) or genotyping (detection of inactivating polymorphisms on the DPYD gene). Approximately 3 to 15% of patients exhibit a partial deficiency and 0.1 to 0.5% a complete DPD deficiency. Currently, there is no regulatory obligation for DPD deficiency screening in patients scheduled to receive a fluoropyrimidine-based chemotherapy. Based on the levels of evidence from the literature data and considering current French practices, the Group of Clinical Pharmacology in Oncology (GPCO)-UNICANCER and the French Network of Pharmacogenetics (RNPGx) recommend the following: (1) to screen DPD deficiency before initiating any chemotherapy containing 5-FU or capecitabine; (2) to perform DPD phenotyping by measuring plasma uracil (U) concentrations (possibly associated with dihydrouracil/U ratio), and DPYD genotyping (variants *2A, *13, p.D949V, HapB3); (3) to reduce the initial FU dose (first cycle) according to DPD status, if needed, and further, to consider increasing the dose at subsequent cycles according to treatment tolerance. In France, 17 public laboratories currently undertake routine screening of DPD deficiency.