A liquid chromatography/tandem mass spectrometry (LC/MS/MS) method was developed and validated to determine the concentrations of adefovir [9-(2-phosphonylmethoxyethyl)adenine, PMEA] in human plasma. After one-step protein precipitation of plasma samples by methanol, adefovir was analyzed by LC/MS/MS using positive electrospray ionization. Chromatography was performed on a C18 column. The extraction recoveries of adefovir were found to be 85.1-89.3%. Adefovir was stable under routine laboratory conditions. A minimal matrix effect resulting in a slight ionization enhancement of adefovir (<10.9%) was observed, which did not markedly affect the behavior of the calibrations curves and accuracy and precision data. The method had a chromatographic run time of 7.8min and a linear calibration curve over the concentration range 1.5-90ng/mL for adefovir. The lower limit of quantification of the method was 1.5ng/mL. The intra-and inter-day precision was less than 8.4%. These results indicated that this LC/MS/MS method has high selectivity and efficiency, and acceptable accuracy, precision and sensitivity. The validated LC/MS/MS method has been successfully used in a pharmacokinetic study in healthy volunteers treated with oral adefovir dipivoxil at 10 and 20mg.

In vitro and in vivo studies have indicated that the induction or inhibition of cytochrome P450 (CYP) is one of the major mechanisms for some clinically important pharmacokinetic herb-drug interactions. An attempt was made to simulate the effects of herbal preparation with single or multiple CYP-inhibiting constituents on the area of the plasma concentration-time curve (AUC) of coadministered drug that was either a low clearance drug by intravenous (i.v.) injection or a high clearance drug by oral route. Our simulation studies indicated that the expected increase (Rc) in the AUC of the coadministered drug by inhibiting herbal constituent(s) was dependent on the route of administration. For low clearance drug by i.v. injection, Rc was generally determined by inhibition constant (Ki), unbound inhibitor concentration ([I]), hepatic fraction (fh), number of inhibitory herbal constituents (n) and metabolic pathway fraction in hepatic metabolism (fm), while Rc for a high clearance drug by oral route, Rc was determined by Ki, [I], n and fm. By varying these parameters, Rc changed accordingly. It appeared likely to predict a herb-drug metabolic interaction, if the inhibiting herbal constituents could be quantitatively determined. However, many herb- and drug-related factors may cause difficulties with the prediction, and thus in vivo animal and human studies are always necessary. Copyright

Purpose. The multidrug resistance associated protein (MRP) 4 is a member of the adenosine riphosphate (ATP)-binding cassette transporter family. Camptothecins (CPTs) have shown substantial anticancer activity against a broad spectrum of tumors by inhibiting DNA topoisomerase I, but tumor resistance is one of the major reasons for therapeutic failure. Pglycoprotein, breast cancer resistance protein, MRP1, and MRP2 have been implicated in resistance to various CPTs including CPT-11 (irinotecan), SN-38 (the active metabolite of CPT-11), and topotecan. In this study, we explored the resistance profiles and intracellular accumulation of a panel of CPTs including CPT, CPT-11, SN-38, rubitecan, and 10-hydroxy-CPT (10-OH-CPT) in HepG2 cells with stably overexpressed human MRP4. Other anticancer agents such as paclitaxel, cyclophosphamide, and carboplatin were also included. Methods. HepG2 cells were transfected with an empty vehicle plasmid (V/HepG2) or human MRP4 (MRP4/HepG2). The resistance profiles of test drugs in exponentially growing V/HepG2 and MRP4/HepG2 cells were examined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazonium bromide (MTT) assay with 4 or 48 h exposure time of the test drug in the absence or presence of various MRP4 inhibitors. The accumulation of CPT-11, SN-38, and paclitaxel by V/HepG2 and MRP4/HepG2 cells was determined by validated high-performance liquid chromatography methods. Results. Based on the resistance folds from the MTT assay with 48 h exposure time of the test drug, MRP4 conferred resistance to CPTs tested in the order 10-OH-CPT (14.21) >SN-38 carboxylate (9.70) >rubitecan (9.06) >SN-38 lactone (8.91) >CPT lactone (7.33) >CPT-11 lactone (5.64) >CPT carboxylate (4.30) >CPT-11 carboxylate (2.68). Overall, overexpression of MRP4 increased the IC50 values 1.78-to 14.21-fold for various CPTs in lactone or carboxylate form. The resistance of MRP4 to various CPTs tested was significantly reversed in the presence of DL-buthionine-(S,R)-sulfoximine (BSO, a gglutamylcysteine synthetase inhibitor), MK571, celecoxib, or diclofenac (all MRP4 inhibitors). In addition, the accumulation of CPT-11 and SN-38 over 120 min in MRP4/HepG2 cells was significantly reduced compared to V/HepG2 cells, whereas the addition of celecoxib, MK571, or BSO significantly increased their accumulation in MRP4/HepG2 cells. There was no significant difference in the intracellular accumulation of paclitaxel in V/HepG2 and MRP4/HepG2 cells, indicating that Pglycoprotein was not involved in the observed resistance to CPTs in this study. MRP4 also conferred resistance to cyclophosphamide and this was partially reversed by BSO. However, MRP4 did not increase resistance to paclitaxel, carboplatin, etoposide (VP-16), 5-fluorouracil, and cyclosporine. Conclusions. Human MRP4 rendered significant resistance to cyclophosphamide, CPT, CPT-11, SN-38, rubitecan, and 10-OH-CPT. CPT-11 and SN-38 are substrates for MRP4. Further studies are needed to explore the role of MRP4 in resistance, toxicity, and pharmacokinetics of CPTs and cyclophosphamide.

Background: The glutathione S-transferase (GST) superfamily comprises multiple isozymes with compelling evidence of functional polymorphisms in various ethnic groups. All these mutations, in particular those in class A, k and u GST, are likely to contribute to interindividual differences in responses to xenobiotics including response to chemotherapy and associated with altered disease. The frequency of common GST mutations in Uygur Chinese is unknown. We investigated the common mutations of GSTM1, GSTT1, and GSTP1 in Uygur (N =154) Chinese and compare with Han Chinese (N =196). Method: GSTM1 and GSTT1 polymorphisms were analyzed by multiplexed PCR, and GSTP1 polymorphism was detected by PCR-based restriction fragment length polymorphism (RFLP) analysis. Results: GSTM1 null genotype was found in 53.2% Uygur Chinese, which was close to that in Han Chinese (56.1%) ( P=0.592). A significantly lower frequency ( P <0.05) of GSTT1 null genotype in Uygur Chinese (26.6%) was observed compared with Han Chinese (50.0%). Uygur Chinese exhibited a GSTP1 genotype distribution of 51.3% I/I, 40.2% I/V and 8.4% V/V, which was different from that in Han Chinese (60.7% I/I, 35.2% I/V and 4.1% V/V). Conclusions: There is marked ethnic difference in the frequency of common GSTT1 and GSTP1 mutation, but not GSTM1 mutation, between Uygur and Han Chinese. D 2005 Elsevier B.V. All rights reserved.