Gene expression predicts differential capecitabine metabolism, impacting on both pharmacokinetics and antitumour activity.

Capecitabine is converted into 5'-deoxy-5-fluorocytidine (5'DFCR), 5'-deoxy-5-fluorouridine (5'DFUR) and 5-fluorouracil (5-FU) by CES1 and 2, CDD, and TP, in both liver and tumour. 5-FU is catabolised by DPD. Gene expression analysis of these enzymes was undertaken in fresh human hepatocytes, mouse liver, colorectal cancer cell lines and xenografts. Cell lines with low CDD expression (<1.5) had 5'DFCR/5'DFUR cytotoxicity ratios>2 and cell lines with TP/DPD<0.6 had 5'DFUR IC50>50 microM (SRB assay). A pharmacokinetic/pharmacodynamic study in nude mice bearing HCT 116 xenografts and treated with capecitabine by oral gavage assessed pharmacokinetic, gene expression and antitumour activity. Low liver CDD correlated with high 5'DFCR plasma concentrations in mice. CDD expression was approximately 100-fold higher in fresh human hepatocytes than mouse liver, explaining the higher plasma 5'DFUR concentrations reported previously in humans. Tumour 5-FU concentration correlated with TP/DPD and with tumour response. These studies identify the potential utility of gene expression analysis and drug monitoring in tumour in patients.

Simultaneous determination of capecitabine and its metabolites by HPLC and mass spectrometry for preclinical and clinical studies.

A reverse-phase high-performance liquid chromatography method with electrospray ionization and detection by mass spectrometry is described for the simultaneous determination of capecitabine, its intermediate metabolites (DFCR, DFUR) and the active metabolite 5-fluorouracil in mouse plasma, liver and human xenograft tumours. The method was also cross-validated in human plasma and human tumour for clinical application. The method has greater sensitivity than previously published methods with an equivalent accuracy and precision. It uses less biological material (plasma, tissue) and should therefore be applicable to biopsies in patients treated with capecitabine.

Aspirin activates the NF-kappaB signalling pathway and induces apoptosis in intestinal neoplasia in two in vivo models of human colorectal cancer.

Substantial evidence indicates that aspirin has antitumour activity against large bowel cancer and modulation of the NF-kappaB (NF-kappaB) signalling pathway has been identified as a key mechanism in this effect. However, studies examining how aspirin affects the NF-kappaB pathway to promote apoptosis have been restricted to in vitro analysis in tissue culture systems and have produced contrasting results. Here, we employed two animal models of human colorectal cancer to determine aspirin effects on the NF-kappaB pathway in colorectal neoplasia in vivo, and the relationship of such effects to the induction of apoptosis. We demonstrate that aspirin induces phosphorylation and degradation of cytoplasmic IkappaBalpha in xenografted HT-29 tumours and in adenomas from APC(Min+/-) mice. Furthermore, we show that this response occurs in a time-dependent manner and is paralleled by nuclear translocation of p65 and caspase activation. Using high performance liquid chromatography analysis, we demonstrate that >0.5 mM salicylate levels are achievable in xenografted tumours after low-dose aspirin (40 mg/kg) treatment and that these levels, which are pharmacologically relevant to humans, are sufficient to stimulate an NF-kappaB and apoptotic response. We demonstrate that activation of the NF-kappaB pathway is associated with increased apoptosis in neoplastic epithelial cells, but found that aspirin has a minimal effect on nuclear p65 and apoptosis in normal intestinal mucosa from APC(Min+/-) mice. These in vivo findings further establish that aspirin induces activation of the NF-kappaB pathway in neoplastic epithelial cells and provide further support that this effect is important for the antitumour activity of the agent. These data have considerable relevance to cancer prevention and therapy.

Liquid chromatography/mass spectrometric bioanalysis of a modified gamma-cyclodextrin (Org 25969) and Rocuronium bromide (Org 9426) in guinea pig plasma and urine: its application to determine the plasma pharmacokinetics of Org 25969.

A sensitive and specific liquid chromatography/mass spectrometry (LC/MS) method has been developed and validated for the quantification of the modified gamma-cyclodextrin Org 25969 and Rocuronium bromide (Roc or Org 9426) in the plasma and urine of guinea pigs. The assay was linear and reproducible over the range 25-10000 ng/mL for both compounds. The lowest limit of quantification (LLOQ) for both compounds in urine was 25 ng/mL. In plasma, the LLOQ was 25 ng/mL for Org 9426 and 50 ng/mL for Org 25969. The inter- and intra-day variation was lower than 20%. The physicochemical properties of both compounds imposed different modes of extraction from plasma. The modified gamma-cyclodextrin was extracted by trifluoroacetic acid (TFA) precipitation while Rocuronium was extracted by acetonitrile precipitation. Both compounds were quantified in urine by direct injection onto the column. The LC/MS analyses of Org 25969 and Org 9426 were performed using two different assay conditions. It was not possible to quantify the complex of cyclodextrin and Roc as it dissociated on the LC column. The use of LC/MS conferred great advantage to the quantification of both Org 25969 and Org 9426, as they were not chromogenic enough to afford the sensitivity and specificity required for the assay.