The transport of a hepatoprotective agent, isopropryl 2-(1-3-dithiethane-2-ylidene)-2[N-(4-methyl-thiazole-2-yl) carbamoyl] acetate (YH439), across Caco-2 cell monolayers.

Isopropryl 2-(1-3-dithiethane-2-ylidene)-2[N-(4-methyl-thiazole-2-yl) carbamoyl] acetate (YH439) is currently under phase II clinical trials by the Yuhan Research Center for use as a hepatoprotective agent. Unfortunately, the oral bioavailbility of YH439, which is sparingly soluble in water (i.e., 0.3 microg/ml or 0.91 microM at room temperature), reportedly, is negligible regardless of the dose administered to rats in the 10-300 mg/kg range. The bioavailability of the compound increased up to 24%, when administered in the form of a micellar solution (700 microg/ml or 2.1 mM for YH439) at a dose of 10 mg/kg, suggesting that its limited solubility is associated with its negligible bioavailability. In order to obtain additional information concerning the bioavailability of YH439, the mechanism(s) involved in gastrointestinal (GI) absorption were investigated in the present study. For this purpose, the transport of YH430 across a Caco-2 cell monolayer was measured in a Transwell. A permeability of 4.07 x 10(-5) cm/s was obtained for the absorptive (i.e., apical to basolateral direction) transport of 0.42 pM YH439, implicating that the in vivo GI absorption is nearly complete. The absorptive transport exhibited a slight concentration-dependency with an intrinsic clearance (CLi) of 0.38 microLL/cm2/sec, which accounted for 28.1% of the total intrinsic clearance (i.e., CLi plus the intrinsic clearance for the linear component) of the transport. Thus, saturation of the absorption process appears to be a minor factor in limiting the bioavailability of the compound. The apparent permeability of YH439 from the basolateral to the apical direction (i.e., efflux, 6.67 x 10(-5) cm/s) was comparable to that for absorptive transport, but, interestingly, a more distinct concentration-dependency was observed for this transport. However, the efflux does not appear to influence the bioavailability of the compound, as evidenced by the sufficiently high permeability in the absorption direction. Rather, a reportedly extensive first-pass hepatic metabolism appears to be a principal factor in limiting the bioavailability. In this respect, reducing the first-pass metabolism by some means would lead to a higher bioavailability of the compound. Thus, elevation of the absorption rate of YH439 becomes a necessity. From a practical point of view, increasing the concentration of YH439 in the GI fluid appears to be a feasible way to increase the absorption rate, because the compound is primarily absorbed via a linear mechanism. In summary, the solubilization of YH439, as previously demonstrated for a micellar solution of the compound, appears to be a practical way to increase the oral bioavailability of YH439.

A computational model for quantitative analysis of cell cycle arrest and its contribution to overall growth inhibition by anticancer agents.

Most anticancer agents induce cell cycle arrest (cytostatic effect) and cell death (cytotoxic effect), resulting in the inhibition of population growth of cancer cells. When asynchronous cells are to be examined, the currently used flow cytometric method can not provide checkpoint-specific and quantitative information on the drug-induced cell cycle arrest. Hence, despite its significance, no good method to analyze in detail the mechanism of cell cycle arrest and its contribution to overall growth inhibition induced by an anticancer agent has yet been established. We describe in this study the development of a discrete time (Markov model)-based computational model for cell cycle progression / arrest with transition probability (TP(i)) as a model parameter. TP(i) was calculated using model equations that include easily measurable parameters such as the fraction of cells in each cell cycle phase and population doubling time. The TP(i) was then used to analyze checkpoint-specific and quantitative changes in cell cycle progression. We also used TP(i) in a Monte-Carlo simulation to predict growth inhibition caused by cell cycle arrest only. Human SCLC cells (SBC-3) exposed to UCN-01 were used to validate the model. The model-predicted growth curves agreed with the observed data for SBC-3 cells not treated or treated at a cytostatic concentration (0.2 mM) of UCN-01, indicating validity of the present model. The changes in TP(i) indicated that UCN-01 reduced the G(1)-to-S transition rate and increased the S-to-G(2) / M and G(2) / M-to-G(1) transition rates of SBC-3 cells in a concentration- and time-dependent manner. When the model-predicted growth curves were compared with the observed data for cells treated at a cytotoxic concentration (2 mM), they suggested that 22% out of 65% and 32% out of 73% of the growth inhibition could be attributed to the cell cycle arrest effect after 48 h and 72 h exposure, respectively. In conclusion, we report here the establishment of a novel method of analysis that can provide checkpoint-specific and quantitative information about cell cycle arrest induced by an anticancer agent and that can be used to assess the contribution of cell cycle arrest effect to the overall growth inhibition.

Computational model of intracellular pharmacokinetics of paclitaxel.

The intracellular pharmacokinetics of paclitaxel is closely related to its pharmacodynamics. Although drug transport across the cell membrane and extracellular and intracellular drug binding have been shown to affect intracellular drug accumulation, their quantitative relationship is unknown. This study was designed to establish a mathematical model for computing the intracellular paclitaxel pharmacokinetics. As a starting point, the model assumes drug transport into and out of cells via passive diffusion. Experimental data on the intracellular pharmacokinetics of [(3)H]paclitaxel were obtained using monolayer cultures of human breast MCF7 tumor cells, which have negligible expression of the mdr1 P-glycoprotein. The results indicate that, in addition to drug binding and microtubule concentration, changes in cell number due to cell growth and drug effects also affected intracellular drug accumulation. A kinetic model was developed to describe several concomitant processes: 1) saturable drug binding to extracellular proteins, 2) saturable and nonsaturable drug binding to intracellular components, 3) time- and concentration-dependent drug depletion from culture medium, 4) cell density-dependent drug accumulation, and 5) time- and drug concentration-dependent enhancement of tubulin concentration. The model was validated by the close prediction (<7% deviation) of the effects of extracellular-to-intracellular concentration gradient and cell density on the kinetics of drug accumulation and efflux. This model was used to predict the effects of changing several parameters (number and binding affinity of intracellular binding sites, free fraction, and concentration of drug in extracellular fluid) on intracellular drug accumulation. In conclusion, the computational model of intracellular paclitaxel pharmacokinetics provides the means to predict drug concentration in cells.

Detailed structural analysis on both human MRP5 and mouse mrp5 transcripts.

The multidrug-resistant phenotype in tumor cells is attributed in part to anti-cancer drug efflux transporters such as the MRP family. The amino-terminal structure of MRP5 has not been refined. To determine the amino-terminal structure of a major transcript of the MRP5 gene, we performed primer extension analysis to determine a major transcriptional start site of this gene and compared the structure of human MRP5 and that of mouse mrp5. We successfully determined the structures of human MRP5 and mouse mrp5. Estimated amino acid sequences are 1437 and 1436 amino acids for human MRP5 and mouse mrp5 respectively, and were highly conserved (94.1%). We further showed that our previously identified SMRP mRNA was a splicing variant of the MRP5 gene, which was expressed in various human tissues, suggesting that a short form of MRP5 protein encoded by the SMRP mRNA may have a physiological role.

Molecular determinants of UCN-01-induced growth inhibition in human lung cancer cells.

UCN-01 (7-hydroxystaurosporine) inhibits the growth of various malignant cell lines in vitro and in vivo. In this study, a human small cell lung carcinoma subline resistant to UCN-01, SBC-3/UCN, was established and characterized. SBC-3/UCN cells showed 8-fold greater resistance to the UCN-01-induced growth-inhibitory effect than the parent cells, SBC-3. No UCN-01-induced G1 accumulation in SBC-3 cells was observed in SBC-3/UCN cells and decreased expression of phosphorylated RB protein was found in SBC-3 cells. Neither basal expression nor induction of p21(Cip1) by UCN-01 treatment was detected in the SBC-3/UCN cell line. An inhibitory effect of UCN-01 on CDK2 activity, which is mediated by p21(Cip1)/CDK2 complex formation upon UCN-01 treatment, was observed in SBC-3 but not in SBC-3/UCN cells. SBC-3/UCN showed higher CDK6 activity than SBC-3 cells. UCN-01 did not inhibit the CDK4 and CDK6 activities in both cells. We screened the cell cycle regulatory molecules associated with G(1)/S progression and found a remarked decrease in interferon regulatory factor 1 (IRF-1), which is known to cooperate with p53 in p21(Cip1) induction. Our results suggest that p21(Cip1) regulation via the IRF-1-associated pathway may represent a major determinant of UCN-01-induced growth inhibition in human lung cancer cells.

Determinants of paclitaxel penetration and accumulation in human solid tumor.

The present study examined the determinants of the penetration and accumulation of [(3)H]paclitaxel (12-12,000 nM) in three-dimensional histocultures of patient tumors and of a human xenograft tumor in mice. The results showed 1) significant and saturable drug accumulation in tumors, 2) extensive drug retention in tumors, and 3) a slower penetration but a more extensive accumulation in the xenograft tumor compared with patient tumors. Drug penetration was not rate-limited by drug diffusion from medium through the matrix supporting the histocultures. The difference in the expression of the mdr1 P-glycoprotein did not fully account for the difference in the drug accumulation in xenograft and patient tumors. Autoradiography and imaging were used to evaluate the spatial relationship between tumor architecture, tumor cell distribution, and drug distribution as a function of time and initial drug concentration in culture medium. The tumor cell density and the kinetics of drug-induced apoptosis were also evaluated. The results indicate that a high tumor cell density is a barrier to paclitaxel penetration and that the apoptotic effect of paclitaxel enhances its penetration in solid tumor. These factors are responsible for the time- and concentration-dependent drug penetration rate, with drug penetration confined to the periphery until apoptosis and reduction of epithelial cell density occurred at 24 h, after which time paclitaxel penetrated the inner parts of the tumor.

Activation-induced apoptosis of peripheral lymphocytes treated with 7-hydroxystaurosporine, UCN-01.

7-hydroxystaurosporine (UCN-01) is a new anticancer agent which exerts an inhibitory effect on cell cycle check points and is currently under phase I clinical trials in US and Japan. Preliminary clinical data indicated that UCN-01 remained in plasma at high concentrations for long periods of time. This unavoidable high plasma drug exposure is likely to lead to hematological toxicities in patients. In the present study, cultured human peripheral blood lymphocytes (PBLs) were used to evaluate the possible hematological toxicities of UCN-01 treatment. UCN-01 induces apoptosis, and the induction of apoptosis-related surface markers were also examined to investigate the involvement of these molecules in UCN-01-induced apoptosis in PBLs. In vitro viability of PBLs was decreased by high dose of UCN-01 (25 microM, 3-day exposure). This effect of UCN-01 was significantly suppressed by the presence of human serum, suggesting that some specific inhibitory factor(s) in human serum may antagonize the lympholytic effect of UCN-01. The percentage of annexin V-positive PI-negative cells increased with exposure to UCN-01 in a time- and dose-dependent manner; by up to 30.3% after exposure to 25 microM UCN-01 for 3 days. At the same time, the expression of both interleukin-2 receptor (IL-2R, CD25) and Fas (CD95), analyzed by flow cytometry, was induced. Con A-stimulated PBLs were more sensitive to UCN-01-induced apoptosis than non-stimulated lymphocytes and UCN-01 increased the sFas-L released into culture medium from con A-stimulated PBLs. Therefore, lymphocyte depletion mediated by activation-induced apoptosis is likely to occur in patients treated with UCN-01 at high doses.

Pharmacodynamics of immediate and delayed effects of paclitaxel: role of slow apoptosis and intracellular drug retention.

The kinetics of the time-dependent antitumor effects of paclitaxel are not fully understood; some literature reports indicate a higher activity by prolonging treatment durations, whereas other reports indicate no enhancement under in vitro conditions. The present study was designed to address this controversy and to determine the mechanism of the higher cytotoxicity associated with longer treatment durations. Six human epithelial cancer cell lines (bladder RT4, breast MCF7, pharynx FaDu, ovarian SKOV3, and prostate PC3 and DU145) were used. To determine whether the higher activity observed for the longer treatment durations is due to a delayed exhibition of drug effects and/or a reflection of cumulative effects that required a continuous drug exposure, cells were treated with paclitaxel for 3-96 h and then either: (a) immediately processed for drug effect measurement; or (b) washed, incubated in drug-free medium, and processed for drug effect measurement at 96 h. The overall drug effect (i.e., combination of cytostatic and apoptotic effects) was determined by the sulforhodamine B assay, which measures the cellular protein. In addition, to determine whether apoptosis occurs with a time delay, apoptosis was measured in cells that were collected immediately after drug treatment for various durations or in cells that were treated with drugs for 3 h but collected at later time points. Apoptosis was determined using agarose gel electrophoresis and by measuring the cytoplasmic DNA-histone complex using ELISA. The contribution of the intracellularly retained drug to the delayed drug effect was studied by characterizing the kinetics of cellular drug uptake and efflux and by examining the effect of removal of the intracellularly retained drug. All six cell lines showed similar results, as follows: (a) paclitaxel produced cytotoxicity that was exhibited immediately after treatment (immediate effect) and after treatment was terminated (delayed effect); (b) the immediate and delayed effects showed different pharmacodynamics. The immediate effect increased with treatment duration and drug concentration. For the delayed effect, all treatments produced the same maximum effect at 96 h, although treatments for < or = 12 h showed higher IC50s than longer treatments, whereas treatments for > or = 24 h showed indistinguishable IC50s; (c) treatment for as brief as 3 h was sufficient to induce apoptosis, which occurred with a lag time of about 24 h, although longer treatments produced a greater extent of apoptosis; (d) The intracellular and extracellular concentrations reached an equilibrium at approximately 5 h, which rules out slow and/or insufficient uptake as the cause of the lower effects at shorter treatment times (i.e., < 24 h); (e) upon removal of drug-containing medium, the amount of drug retained intracellularly was about 10% of the applied dose and was reduced to approximately 0.5% after three successive washes, separated by 3-h equilibration periods; and (f) the delayed effect of the 3-h treatment was largely due to the drug retained intracellularly, whereas the delayed effect of the 24 h treatment was independent of the drug retained intracellularly. In conclusion, in human epithelial cancer cells, paclitaxel-induced cytotoxicity occurred after termination of drug treatment, which was partly due to the slow manifestation of apoptosis and partly due to the significant amount of drug retained intracellularly. Based on these findings and recognizing that some previous studies measured the immediate effect whereas the other studies measured the delayed effect, we propose that the conflicting data in the literature regarding the effect of treatment duration on paclitaxel activity under in vitro conditions are in part due to the different pharmacodynamics of the immediate and delayed drug effects. Furthermore, differences in the delayed effects for treatments of < 24 h and the minimal differences for treatments of > or = 24 h indicate that th

Nonlinear renal excretion of theophylline and its metabolites, 1-methyluric acid and 1,3-dimethyluric acid, in rats.

Plasma pharmacokinetics and renal excretion of theophylline (TP) and its metabolites were investigated in rats. Plasma concentrations of TP declined in a monoexponential manner, while those of 1-methyluric (MU) and 1,3-dimethyluric (DMU) declined in a biexponential manner upon respective i.v. bolus injection of each compound at 6 mg/kg dose. The total body clearances (CLt) of the metabolites were 4-6 fold larger than that of TP, while the distribution volumes of them at steady-state (Vdss) were 40-50% smaller than that of TP. The metabolites showed their plasma peaks in 30 min after i.v. injection of TP indicating very rapid metabolism of TP. Metabolism of TP to DMU was more than fourfold faster than that to MU. Renal excretion of TP and its metabolites was studied in urine flow rate (UFR)-controlled rats. The renal clearance (CLr) of TP was inversely related to plasma TP concentrations, and much smaller than the glomerular filtration rate (GFR) suggesting tubular secretion and profound reabsorption in the renal tubule. The CLr of each metabolite also showed that inverse relationship, but far exceeded GFR suggesting that tubular secretion plays a major role in their elimination. The CLr of the metabolites were reduced to less than GFR by i.p. injection of probenecid (142.7 mg/kg). It supports that the metabolites are secreted in the renal tubule, and suggests that they share a common transport system in their secretion processes with probenecid. On the other hand, the CLr of TP was not affected significantly by the probenecid treatment. Considering the inverse relationship of TP between the CLr and its plasma concentrations, no effect of probenecid on CLr of TP is most likely due to negligible contribution of the secretion to the overall CLr of TP.