Change in plasma concentration of clozapine and norclozapine following a switch of oral formulation.

BACKGROUND: Clozapine formulation has been shown to affect plasma concentrations of clozapine and norclozapine. Changes in formulation might result in toxicity or treatment failure. METHODS: We identified, from electronic records, patients who switched from clozapine tablets to oral liquid or vice versa and compared plasma concentrations before and after the switch. RESULTS: We identified 13 patients with 85 blood samples who changed formulation of clozapine. Overall mean standardized clozapine plasma level was 0.67 mg/l daily dose on liquid and 0.87 mg/l daily dose on tablets (p = 0.035). CONCLUSION: Use of clozapine liquid results in lower plasma clozapine concentration than the same dose of tablets.

The infant mouse as a in vivo model for the detection and study of DNA damage-induced changes in the liver.

The present work describes the use of the infant (4-wk-old) mouse as an animal model for the study of DNA damage-induced G(1) checkpoint response, changes in p53 protein levels, and multiple gene expression changes after DNA damage has been induced in the liver. Hepatocytes in the infant B6C3F1 mouse had a proliferation index that was 27 times greater than that of the 12-wk-old B6C3F1 mouse (57.4 vs. 2.1%, respectively). Eight hours after infant mice were exposed to the DNA damaging agents bleomycin (100 mg/kg, i.p.) or 10 Gy of whole body gamma irradiation, the G(1)/S ratio significantly increased from 21 (control) to 66 and 75, respectively, because of the induction of the G(1)/S checkpoint response. One hour after whole body irradiation of infant mice the levels of the p53 protein, phosphoserine 18-p53 and phosphoserine 23-p53 increased dramatically and tended to peak at 1 h in the liver, whereas the p21(WAF1) protein increased more slowly and tended to peak at 2 h after irradiation. The mRNA expression of the p53-response genes p21, murine double minute clone 2 (mdm2), and cyclin G was increased at 2 h after irradiation but was decreased by 8 h postirradiation, relative to the 2-h time-point. The expression of insulin-like growth factor binding protein-1 (IGFBP-1) and growth-regulated oncogene 1 (GRO1) increased at 2 and 8 h postirradiation. This work characterizes various parameters in the infant mouse, thus validating the use of this model to study in vivo DNA damage-induced cell-cycle-related changes.

Development of a high-throughput viral-free assay for the measurement of CCR5-mediated HIV/cell fusion.

M-tropic HIV strains gain access to their host cell via interaction of the viral envelope protein gp120 with the CCR5 coreceptor and CD4 located on the host cell. Inhibition of this event has been shown to reduce viral fusion and entry into cells in vitro. In the present study we describe the development of a novel cell/cell fusion assay that both mimics the viral/cell fusion process and allows quantification of this event. The assay has been characterized both biochemically, using selective antibodies, and pharmacologically, using selective CCR5 antagonists, and has been shown to be selective for examining the interaction of viral gp120 with hCCR5/hCD4. In addition, compound pIC50 data obtained from this cell/cell fusion assay correlates well (r2 = 0.7274) with data obtained from an HIV-1 replication assay. Furthermore, this assay has the added ability to simultaneously determine compound toxicity, thus allowing rapid determination of active, non-toxic compounds. In conclusion, the cell/cell fusion assay developed has been demonstrated to be a suitable surrogate assay that can be used to assess the effects of compounds on gp120/CCR5/CD4 mediated viral fusion into host cells.

Application of cDNA microarray technology to in vitro toxicology and the selection of genes for a real-time RT-PCR-based screen for oxidative stress in Hep-G2 cells.

Large-scale analysis of gene expression using cDNA microarrays promises the rapid detection of the mode of toxicity for drugs and other chemicals. cDNA microarrays were used to examine chemically induced alterations of gene expression in HepG2 cells exposed to a diverse group of toxicants at an equitoxic exposure concentration. The treatments were ouabain (43 microM), lauryl sulfate (260 microM), dimethylsulfoxide (1.28 M), cycloheximide (62.5 microM), tolbutamide (12.8 mM), sodium fluoride (3 mM), diethyl maleate (1.25 mM), buthionine sulfoximine (30 mM), potassium bromate (2.5 mM), sodium selenite (30 microM), alloxan (130 mM), adriamycin (40 microM), hydrogen peroxide (4 mM), and heat stress (45 degrees C x 30 minutes). Patterns of gene expression were correlated with morphologic and biochemical indicators of toxicity. Gene expression responses were characteristically different for each treatment. Patterns of expression were consistent with cell cycle arrest, DNA damage, diminished protein synthesis, and oxidative stress. Based upon these results, we concluded that gene expression changes provide a useful indicator of oxidative stress, as assessed by the GSH:GSSG ratio. Under the conditions of this cell culture test system, oxidative stress upregulated 5 genes, HMOX1, p21(waf1/cip1), GCLM, GR, TXNR1 while downregulating CYP1A1 and TOPO2A. Primers and probes for these genes were incorporated into the design of a 7-gene plate for RT-PCR. The plate design permitted statistical analysis and allowed clear discrimination between chemicals inducing oxidative vs nonoxidative stress. A simple oxidative stress score (0-1), based on the responses by the 7 genes (including p-value) on the RT-PCR plate, was correlated with the GSH:GSSG ratio using linear regression and ranking (Pearson product) procedures. These analyses yielded correlation coefficients of 0.74 and 0.87, respectively, for the treatments tested (when 1 outlier was excluded), indicating a good correlation between the biochemical and transcriptional measures of oxidative stress. We conclude that it is essential to measure the mechanism of interest directly in the test system being used when assessing gene expression as a tool for toxicology. Tables 1-15, referenced in this paper, are not printed in this issue of Toxicologic Pathology. They are available as downloadable text files at http://taylorandfrancis.metapress.com/openurl.asp?genre=journal&issn=0192-6233. To access them, click on the issue link for 30(4), then select this article. A download option appears at the bottom of this abstract. In order to access the full article online, you must either have an individual subscription or a member subscription accessed through www.toxpath.org.

Transcriptional and physiological responses of HepG2 cells exposed to diethyl maleate: time course analysis.

Expression levels of 767 genes were measured in HepG2 cells at eight time points (0, 0.5, 1, 6, 12, 16, 20, and 24 h) following exposure to the oxidizing agent, diethyl maleate (DEM). DEM treatment caused an immediate and sustained loss of intracellular GSH, with a concomitant increase in GSSG. From 6-12 h after exposure, there was a substantial increase in the percentage of cells undergoing S phase arrest and apoptosis. Expression profiles of approximately 90% of the genes fell into one of five clusters generated using hierarchical-clustering software, indicating the well-ordered nature of the stress response. The directional movement and timing of induction for many genes matched closely the known physiological role of the proteins they encode. Inhibitors of the cell cycle (CDKN1, CDKN4D, ATM) were induced, whereas cyclins [proliferating cell nuclear antigen (PCNA), cyclin A, cyclin D1, cyclin K] were downregulated during the period from 6--20 h. Likewise, pro-apoptotic genes such as the caspases (CASP9, CASP3, CASP2) and apoptotic protease activating factor (APAF) were induced during the same period. Results of this study indicate that there is a good correlation between time-dependent physiological, biochemical, and gene expression data.