Quantification of urinary excretion of ethosuximide enantiomers and their major metabolites in the rat.

A chiral gas chromatographic assay has been developed for quantitative analysis of ethosuximide and its major metabolites in rat urine. The extraction procedure was found to be precise and reproducible. Recovery was in the range of 94-98%, intraday CV(%) was 0.92% for (S)-ethosuximide (50 mug/ml) and 0.51% for (R)-ethosuximide (50 mug/ml). Interday CV(%) was 1.12% for (S)-ethosuximide and 0.72% for (R)-ethosuximide. The limit of detection was determined to be around 0.01 mug/ml for each enantiomer. Following administration of rac-ethosuximide by i.v., i.p. and oral routes, unchanged ethosuximide was detected in urine up to 72h after drug administration. The appearance of all detected metabolites occurred Within 24h of drug administration. Significantly more (S)-ethosuximide was excreted unchanged than (R)-ethosuximide with all three routes studied. A substantial amount of the drug was eliminated as the 2-(1-hydroxyethyl)-2-methylsuccinimide (2 pairs of diastereoisomers). Much less drug was eliminated as the 2-ethyl-3-hydroxy-2-methylsuccinimide with only one diastereoisomer observed. Examination of the one pair of diastereoisomers of 2-(1-hydroxyethyl)-2-methylsuccinimide that was resolved showed preferential excretion of one isomer. Comparison of both pairs of diastereoisomers showed that one pair was formed in preference to the other with a ratio of approximately 0.8:1. It is concluded that stereoselective metabolism of ethosuximide occurs. Copyright (C) 2001 John Wiley & Sons, Ltd. Author Keywords: chiral pharmacokinetics; ethosuximide enantiomers; metabolism; rat; urinary excretion; gas chromatography

Inositol 1,4,5-trisphosphate receptors modulate Ca2+ sparks and Ca2+ store content in vas deferens myocytes.

Spontaneous Ca2+ sparks were observed in fluo 4-loaded myocytes from guinea pig vas deferens with line-scan confocal imaging. They were abolished by ryanodine (100 microM), but the inositol 1,4,5-trisphosphate (IP3) receptor (IP3R) blockers 2-aminoethoxydiphenyl borate (2-APB; 100 microM) and intracellular heparin (5 mg/ml) increased spark frequency, rise time, duration, and spread. Very prolonged Ca2+ release events were also observed in approximately 20% of cells treated with IP3R blockers but not under control conditions. 2-APB and heparin abolished norepinephrine (10 microM; 0 Ca2+)-evoked Ca2+ transients but increased caffeine (10 mM; 0 Ca2+) transients in fura 2-loaded myocytes. Transients evoked by ionomycin (25 microM; 0 Ca2+) were also enhanced by 2-APB. Ca2+ sparks and transients evoked by norepinephrine and caffeine were abolished by thimerosal (100 microM), which sensitizes the IP3R to IP3. In cells voltage clamped at -40 mV, spontaneous transient outward currents (STOCs) were increased in frequency, amplitude, and duration in the presence of 2-APB. These data are consistent with a model in which the Ca2+ store content in smooth muscle is limited by tonic release of Ca2+ via an IP3-dependent pathway. Blockade of IP3Rs elevates sarcoplasmic reticulum store content, promoting Ca2+ sparks and STOC activity.

Pilot-scale evaluation of the application of low pH-inducible polyphosphate accumulation to the biological removal of phosphate from wastewaters.

To investigate the possible biotechnological application of the phenomenon of low pH-inducible phosphate uptake and polyphosphate accumulation, previously reported using pure microbial cultures and under laboratory conditions, a 2000 L activated sludge pilot plant was constructed at a municipal sewage treatment works. When operated as a single-stage reactor this removed more than 60% of influent phosphate from primary settled sewage at a pH of 6.0, as opposed to approximately 30% at the typical operational pH for the works of 7.0-7.3-yet without any deleterious effect on other treatment parameters. At these pH values the phosphorus content of the sludge was, respectively, 4.2% and 2.0%. At pH 6.0 some 33.9% of sludge microbial cells were observed to contain polyphosphate inclusions; the corresponding value at pH 7.0 was 18.7%. Such a process may serve as a prototype for the development of alternative biological and chemical options for phosphate removal from wastewaters.

The Role of Thymidylate Synthase Induction in Modulating p53-regulated Gene Expression in Response to 5-Fluorouracil and Antifolates

Thymidylate synthase (TS) is a critical target for chemotherapeutic agents such as 5-fluorouracil (5-FU) and antifolates such as tomudex (TDX),multitargeted antifolate, and ZD9331. Using the MCF-7 breast cancer line, we have developed p53 wild-type (M7TS90) and null (M7TS90-E6) isogenic lines with inducible TS expression (approximately 6-fold induction compared with control after 48 h). In the M7TS90 line, inducible TS expression resulted in a moderate approximately 3-fold increase in 5-FU IC-50(72 h) dose and a dramatic >20-fold increase in the IC-50(72 h) doses of TDX, multitargeted antifolate, and ZD9331. S-phase cell cycle arrest and apoptosis induced by the antifolates were abrogated by TS induction. In contrast, cell cycle arrest and apoptosis induced by 5-FU was unaffected by TS expression levels. Inactivation of p53 significantly increased resistance to 5-FU and the antifolates with IC-50(72 h) doses for 5-FU and TDX of >100 and >10 microM, respectively, in the M7TS90-E6 cell line. Furthermore, p53 inactivation completely abrogated the cell cycle arrest and apoptosis induced by 5-FU. The antifolates induced S-phase arrest in the p53 null cell line; however, the induction of apoptosis by these agents was significantly reduced compared with p53 wild-type cells. Both inducible TS expression and the addition of exogenous thymidine (10 microM) blocked p53 and p21 induction by the antifolates but not by 5-FU in the M7TS90 cell line. Similarly, inducible TS expression and exogenous thymidine abrogated antifolate but not 5-FU-mediated up-regulation of Fas/CD95 in M7TS90 cells. Our results indicate that in M7TS90 cells, inducible TS expression modulates p53 and p53 target gene expression in response to TS-targeted antifolate therapies but not to 5-FU.

Three-stage contingent screening for down syndrome

Objective To demonstrate the potential value of three-stage sequential screening for Down syndrome. Methods Protocols were considered in which maternal serum pregnancy associated plasma protein-A (PAPP-A) and free -human chorionic gonadotropin (hCG) measurements were taken on all women in the first trimester. Those women with very low Down syndrome risks were screened negative at that stage and nuchal translucency (NT) was measured on the remainder and the risk reassessed. Those with very low risk were then screened negative and those with very high risk were offered early diagnostic testing. Those with intermediate risks received second-trimester maternal serum -fetoprotein, free -hCG, unconjugated estriol and inhibin-A. Risk was then reassessed and those with high risk were offered diagnosis. Detection rates and false-positive rates were estimated by multivariate Gaussian modelling using Monte-Carlo simulation. Results The modelling suggests that, with full adherence to a three-stage policy, overall detection rates of nearly 90% and false-positive rates below 2.0% can be achieved. Approximately two-thirds of pregnancies are screened on the basis of first-trimester biochemistry alone, five out of six women complete their screening in the first trimester, and the first-trimester detection rate is over 60%. Conclusion Three-stage contingent sequential screening is potentially highly effective for Down syndrome screening. The acceptability of this protocol and its performance in practice, should be tested in prospective studies. Copyright © 2006 John Wiley & Sons, Ltd.