Patulin is a mycotoxin that is found mainly in apple products and causes symptoms such as bleeding from the digestive tract and diarrhea. Efforts to elucidate the mechanism of its toxicity have focused on protein tyrosine phosphatases (PTPs), which regulate the function of tight junctions (TJs) in colon epithelial cells. Patulin reacts with the conserved cysteine residues in the catalytic domains of PTP isoforms. Treatment of Caco-2 human colon cancer cells, used as a colon epithelial model, with 50 µM patulin decreased the level of density-enhanced phosphatase-1 (DEP-1) protein to 30% of the control level after 6 h. The level of DEP-1 mRNA was also decreased during 24 h after treatment with patulin. Moreover, knockdown of DEP-1 increased the level of phosphorylated claudin-4. Destruction of TJs by patulin treatment was observed by immunostaining with an antibody against zonula occludens (ZO)-1. To better understand the mechanistic basis of the decrease in DEP-1 mRNA levels, we searched for a cis-element upstream of the DEP-1 gene and found an element responsive to the peroxisome proliferator-activated receptor gamma (PPARγ ) protein. Using a PPARγ-specific antibody, we showed a decrease in PPARγ abundance to 42% of the control level within 6 h after treatment with patulin. PPARγ has four cysteine residues that are involved in zinc finger formation. Our data suggest that DEP-1 affects TJ function and that PPARγ might control DEP-1 expression. Therefore, the toxicity of patulin to cellular functions might be attributable to its ability to down-regulate the expression of DEP-1 and PPARγ.

A new liquid chromatographic/mass spectrometric assay has been developed for the determination of DX-8951, a new anti-tumor drug, and its 4-hydroxymethyl metabolite (UM-1) in human plasma and urine. Solid-phase extractions were used for sample preparation. A gradient reverse-phase HPLC separation was developed with mobile phases consisting of trifluoroacetic acid and methanol. The detection was conducted using atmospheric pressure chemical ionization tandem mass spectrometry in the selected reaction monitoring mode. A structural analog, camptothecin (CPT), was used as the internal standard. The assay was validated for the determination of DX-8951 and UM-1 in human plasma and urine. The lower limits of quantitation of DX-8951 and UM-1 were 0.1 ng/mL in plasma and 1 ng/mL in urine. The method showed a satisfactory sensitivity, precision, accuracy, recovery and selectivity.

DX-8951f (Exatecan), a camptothecin derivative, is a promising new antitumor drug currently in phase I clinical trials. The drug exhibits high topoisomerase I inhibitory activity and also high antitumor activity in vitro. It was further found to affect the transport of P-glycoprotein-mediated multidrug resistant cell lines. [1][2][3] DX-8951, the anhydrous free base of DX-8951f, contains a lactone ring in its molecule. This lactone of DX-8951 is reversibly hydrolyzed to hydroxy-acid of DX-8951 in alkaline aqueous solution. In a pharmacological study in vitro, the antitumor activity of lactone was found to be higher than that of hydroxy-acid. Thus, the separate quantitation of these forms is important to evaluate the pharmacokinetics and pharmacodynamics of DX-8951.Other camptothecin derivatives such as irinotecan and topotecan also contain lactone rings. In the assay of irinotecan and topotecan, simultaneous quantitation of their lactone and hydroxy-acid by HPLC were reported. [4][5][6][7][8][9] In this paper, we describe a new sensitive and validated HPLC method for lactone and total drug (lactone plus hydroxy-acid) of DX-8951 in mouse plasma using a fluorometric detector. This method can be used successfully to assess the pharmacokinetic study in male mice treated with a single intravenous administration of either lactone or hydroxy-acid of DX-8951. MATERIALS AND METHODSMaterials DX-8951f, (1S,9S)-1-amino-9-ethyl-5-fluoro-1, 2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H-benzo- (Fig. 1). The anhydrous free base of DX-8951f is referred to as DX-8951. Methanol (MeOH) and acetonitrile (MeCN) were of HPLC-grade (Kanto Chemical Co. Inc., Tokyo, Japan). All other chemicals were of analytical reagent grade and used without purification. Water purified by a Mill-Q system (Millipore, Milford, MA, U.S.A.) was used.Chromatographic Conditions The chromatographic system consisted of a Waters 600E multi-solvent pump system, a reverse-phase Puresil C18 column (4.6 mm i.d.ϫ150 mm), and a Waters 474 scanning fluorescence detector (all from Millipore Co., Milford, MA, U.S.A.) set at an excitation wavelength of 365 nm and an emission wavelength of 445 nm. The column temperature was kept at 30°C in a model RCM-100 column oven (Millipore Co.). To the HPLC system equipped with a Waters 717 autosampler (Millipore Co.), 0.08 ml aliquots of samples were injected. The mobile phase, which had been degassed in an ultrasonic bath before use, was a mixture (18 : 82, v/v) of MeCN-0.05 M potassium dihydrogen phosphate (pH 3 adjusted with phosphoric acid). The flow rate was set at 1 ml/min.Standard Solutions DX-8951 has a lactone ring in its structure which closes and opens reversibly depending on the pH value; it also has a lactone form in acidic pH and a hydroxy-acid form in neutral and basic pH (Fig. 2) Drug Metabolism & Physicochemical Property Research Laboratory, Daiichi Pharmaceutical Co., Ltd., Tokyo 134-8630, Japan. Received September 8, 2000; accepted October 24, 2000 A sensitive high-performance liquid chromatographic (HPLC...

No abstract

Urinary metabolites of DX-8951 ((1S,9S)-1-amino-9-ethyl-5-fluoro- 1,2,3,9,12,15-hexahydro-9-hydroxy-4-methyl-10H,13H- benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione, CAS 171335-80-1, exatecan) in rats and humans were identified. Rats were dosed with the drug, and two major metabolites (UM-1 and UM-2) in the urine were isolated and purified by using ion-exchange column and HPLC. From NMR and mass spectra, they are suggested to be 4-hydroxymethyl metabolite (UM-1) and 3-hydroxy metabolite (UM-2) of the drug. Their chemical structures were confirmed by comparing their NMR spectra with those of chemically synthesized metabolites. Two major metabolites were found in human urine obtained in phase I trial. They were also confirmed to be UM-1 and UM-2 by LC/MS/MS by comparing their mass fragment patterns with those of synthetic metabolites.