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To investigate the role of thrombin in regulating apoptosis, we have used CCl39 cells, a fibroblast cell line in which thrombin-induced cell proliferation has been extensively studied. Withdrawal of serum from CCl39 cells resulted in a rapid apoptotic response that was completely prevented by the inclusion of thrombin. The protective effect of thrombin was reversed by pertussis toxin, suggesting that cell-survival signalling pathways are activated via a Gi or Go heterotrimeric GTPase. Serum-withdrawal-induced death required de novo gene expression and was preceded by the rapid de novo expression of the pro-apoptotic ‘BH3-only’ protein Bim (Bcl-2-interacting mediator of cell death). Thrombin strongly inhibited the up-regulation of both Bim protein and Bim mRNA. The ability of thrombin to repress Bim expression, and to protect cells from apoptosis, was reversed by U0126, a MEK1/2 [MAPK (mitogen-activated protein kinase) or ERK (extracellular-signal-regulated kinase) 1/2] inhibitor, or LY294002, a phosphoinositide 3′-kinase (PI3K) inhibitor, suggesting that both the Raf→MEK→ERK1/2 and PI3K pathways co-operate to repress Bim and promote cell survival. A PAR1p (protease-activated receptor 1 agonist peptide) was also able to protect cells from serum-withdrawal-induced apoptosis, suggesting that thrombin acts via PAR1 to prevent apoptosis.

The pharmacokinetics and pharmacodynamics of prolonged oral etoposide chemotherapy were investigated in 15 women with metastatic breast cancer who received oral etoposide 100mg as a single daily dose for up to 15 days. There was considerable interpatient variability in the day 1 pharmacokinetic parameters: area under the plasma concentration time curve (AUC) (0-24h) 1.950.87mg/ml per min (meanSD), apparent oral clearance 60.921.7ml/min per 1.73m², peak plasma concentration 5.6 2.5g/ml, time to peak concentration 7335min and half-life 22083min. However, intrapatient variability in systemic exposure to etoposide was much less with repeated doses. The intrapatient coefficient of variation (CV) of AUC for day 8 relative to day 1 was 20% and for day 15 relative to day 1 was 15%, compared to the day 1 interpatient CV of 45%. Neutropenia was the principal toxicity. Day 1 pharmacokinetic parameters were related to the percentage decrease in absolute neutrophil count using the sigmoidal E_max equation. A good fit was found between day 1 AUC and neutrophil toxicity (R²=0.77). All patients who had a day 1 AUC >2.0mg/ml per min had WHO grade III or IV neutropenia. The predictive performance of the models for neutrophil toxicity was better for AUC (percentage mean predictive error 5%, percentage root mean square error 18.1%) than apparent oral clearance, peak plasma concentration, or daily dose (mg/m²). A limited sampling strategy was developed to predict AUC using a linear regression model incorporating a patient effect. Data sets were divided into training and test sets. The AUC could be estimated using a model utilizing plasma etoposide concentration at only two time points, 4h and 6h after oral dosing (R²=98.9%). The equation AUC_pr=0.376+ 0.631C_4h+0.336C_6h was validated on the test set with a relative mean predictive error of 0.88% and relative root mean square error of 6.4%. These results suggest monitoring of AUC to predict subsequent myelosuppression as a strategy for future trials with oral etoposide.

Resistance to cancer therapeutics targeting the second kinase in a three-kinase cascade involves amplification of the upstream kinase, not the inhibited kinase.