Targeting Inhibition of Notch1 Signaling Pathway on the Study of Human Gastric Cancer Stem Cells with Chemosensitization.

Background: Gastric cancer is the second most frequent cause of cancer death worldwide, although much geographical variation in incidence exists. Prevention and personalized treatment are regarded as the best options to reduce gastric cancer mortality rates (Hartgrink et al., 2009). Numerous studies have suggested that Notch1 and its ligands are overexpressed in gastric cancer, and its knockdown can inhibit the proliferation and survival of gastric cancer cells. Objective: To investigate the effect of Notch1 on the stemness and drug sensitivity of human gastric cancer SGC-7901 cells. Methods: Highly expressed Notch1 intracellular domain (NICD1) and Notch1-shRNA lentiviral expression vector were used to infect human gastric cancer SGC-7901 cells cultured in vitro, and western blot and immunofluorescence staining were used to identify highly expressed NICD and Notch1 silenced cells. The percentage of CD133+ cells was analyzed by flow cytometry, the expression of nestin and CFAP by immunofluorescence staining, the formation rate of tumor cell spheres and the tumorigenicity of SCID mice in vivo, and the regulation of cell stemness by Notch1. The sensitivity of each group of cells to the chemotherapeutic drugs teniposide (VM-26) and carmustine (BCNU) was also detected by the MTT method. Results: The stemness phenotype of tumor cells with the increased NICD expression was enhanced, such as an increased proportion of CD133+ cells, enhanced nestin expression, decreased GFAP expression, increased tumor cell sphere formation rate and tumorigenic rate of SCID mice implantation, and decreased sensitivity to VM-26 and BCNU. In contrast, the stemness phenotype of tumor cells with downregulated Notch1 gene expression was significantly suppressed, while the sensitivity to VM-26 and BCNU was increased. Conclusion: High Notch1 expression increased the stemness of SGC-7901 cells and decreased the sensitivity of SGC-7901 cells to chemotherapeutic drugs.

Kinetics and regulation of cytochrome P450-mediated etoposide metabolism.

Etoposide is a DNA topoisomerase II inhibitor widely used in the treatment of a variety of malignancies that is also associated with therapy-related leukemia. The cytochrome P450 (P450)-derived catechol and quinone metabolites of etoposide may be important in the damage to the MLL (mixed lineage leukemia) gene and other genes resulting in leukemia-associated chromosomal translocations. Kinetic analysis of catechol formation by recombinant P450s was determined using liquid chromatography/selected reaction monitoring/mass spectrometry. CYP3A4 was found to play a major role in etoposide metabolism (K(m) = 77.7 +/- 27.8 microM; V(max) = 314 +/- 84 pmol of catechol/min/nmol of P450). However, CYP3A5 (K(m) = 13. 9 +/- 3.1 microM; V(max) = 19.4 +/- 0.4 pmol of catechol/min/nmol of P450) may be involved in etoposide metabolism at therapeutic concentrations of free drug. Other P450s do not appear to be involved in etoposide catechol formation. Real-time polymerase chain reaction and Western blot analysis revealed significantly increased CYP3A4 mRNA and protein levels in hepatocytes treated with 10 microM rifampicin compared with untreated cells, but only modest effects of rifampicin on CYP3A5 induction. Etoposide (40, 5, 1, and 0.25 microM) caused a slight increase in CYP3A4 mRNA in three of five batches of hepatocytes but did not result in proportionately increased CYP3A4 protein levels. At high concentrations, etoposide induced only a modest increase in CYP3A5 mRNA and protein levels in four of five batches of hepatocytes. Alternatively, coadministration of other drugs with etoposide may account for the increase in etoposide catechol formation during therapy with etoposide.

In vitro drug resistance profiles of adult versus childhood acute lymphoblastic leukaemia.

The difference in the current cure rates between adult and childhood acute lymphoblastic leukaemia (ALL) may be caused by differences in drug resistance. Earlier studies showed that in vitro cellular drug resistance is a strong independent adverse risk factor in childhood ALL. Knowledge about cellular drug resistance in adult ALL is still limited. The present study compared the in vitro drug resistance profiles of 23 adult ALL patients with that of 395 childhood ALL patients. The lymphoblasts were tested by the MTT assay. The group of adult ALL samples was significantly more resistant to cytosine arabinoside, L-asparaginase, daunorubicin, dexamethasone and prednisolone. The resistance ratio (RR) was highest for prednisolone (31.7-fold) followed by dexamethasone (6.9-fold), L-asparaginase (6. 1-fold), cytosine arabinoside (2.9-fold), daunorubicin (2.5-fold) and vincristine (2.2-fold). Lymphoblasts from adult patients were not more resistant to mercaptopurine, thioguanine, 4-HOO-ifosfamide, mitoxantrone and teniposide. There were no significant differences in drug resistance between adult T-cell (T-) ALL (n = 11) and adult common/pre-B-cell (B-) ALL (n = 10). Additionally, adult T-ALL did not differ from childhood T-ALL (n = 69). There were significant differences between adult common/pre-B-ALL and childhood common/pre-B-ALL (n = 310) for prednisolone (RR = 302, P = 0.008), dexamethasone (RR = 20.9, P = 0.017) and daunorubicin (RR = 2.7, P = 0.009). Lymphoblasts from adults proved to be relatively resistant to drugs commonly used in therapy. This might contribute to the difference in outcome between children and adults with ALL.

[A high performance liquid chromatographic method for the determination of teniposide in brain tissue using electro-chemical detection].

A reversed-phase HPLC method for the determination of teniposide in brain tissue is described. Teniposide can be separated on a Hypersil ODS column with a mobile phase of methyl alcohol-water-acetic acid (56:41:3, V/V) and flow rate of 0.8 mL/min (10 MPa). Column temperature was 35 degrees C and operating potentials for electrochemical detection was 0.70 V. To 50 mg brain tissue were 1 mL 50% ammonium sulfate and 4 mL ethyl acetate. The sample was vortexed for 5 min and ultrasonically agitated for 15 min, then centrifuged at 3000 r/min for 15 min. The upper (organic) layer was collected and the lower layer reextracted with 4 mL of ethyl acetate by vigorously vortexing, then centrifuged at 3000 r/min for 15 min. The organic layer was combined with the previously collected ethyl acetate layer. This organic extract was dried under a gentle nitrogen stream, and the residue was reconstituted with 1 mL internal standard of guaifenesinum before HPLC analysis. The linear range was 0.1-10.0 mg/L, and the detection limit 0.1 mg/L. Intra-day and inter-day RSD for assaying brain tissue sample were 0.87% and 1.41%, respectively. The average recovery was 92.87% with coefficient of variation of 2.35%.

Deletion and duplication sequences induced in CHO cells by teniposide (VM-26), a topoisomerase II targeting drug, can be explained by the processing of DNA nicks produced by the drug-topoisomerase interaction.

Frameshift mutations induced by acridines in bacteriophage T4 have been shown to be due to the ability of these mutagens to cause DNA cleavage by the type II topoisomerase of T4 and the subsequent processing of the 3' ends at DNA nicks by DNA polymerase or its associated 3' exonuclease followed by ligation of the processed end to the original 5' end. An analysis of the ability of nick-processing models is presented here to test the ability of nick processing to account for the DNA sequences of duplications and deletions induced in the aprt gene of CHO cells by teniposide (VM-26) [Han et al. (1993) J. Mol. Biol., 229, 52]. Although teniposide is not an acridine, it induces topoisomerase II-mediated DNA cutting in aprt sequences in vitro and mutagenesis in vivo. Although the previous study noted a correlation between mutation sites and nearby DNA discontinuities induced by the enzyme in vitro, neither the nick-processing model responsible for T4 mutations, nor double-strand break models alone were able to account for most of the mutant sequences. Thus, no single model explained the correlation between teniposide-induced DNA cleavage and mutagenic specificity. This report describes an expanded analysis of the ways that nick-processing models might be related to mutagenesis and demonstrates that a modified nick-processing model provides a biochemical rationale for the mutant specificities. The successful nick-processing model proposes that either 3' ends at nicks are elongated by DNA polymerase and/or that 5' ends of nicks are subject to nuclease activity; 3'-nuclease activity is not implicated. The mutagenesis model for nick-processing of teniposide-induced nicks in CHO cells when compared to the mechanism of nick-processing in bacteriophage T4 at acridine-induced nicks provides a framework for considering whether the differences may be due to cell-specific modes of DNA processing and/or due to the precise characteristics of topoisomerase-DNA intermediates created by teniposide or acridine that lead to mutagenesis.

O-demethylation of epipodophyllotoxins is catalyzed by human cytochrome P450 3A4.

We previously demonstrated that O-demethylation of the pendant dimethoxyphenol ring of epipodophyllotoxins to produce their respective catechol metabolites is catalyzed by cytochrome(s) P450 in human liver microsomes. Our objective was to identify the specific human cytochrome(s) P450 responsible for catechol formation. Using a panel of prototypical substrates and inhibitors for specific cytochromes P450, we identified substrates for CYP3A4 (midazolam, erythromycin, cyclosporin, and dexamethasone) as inhibitors of catechol formation from both etoposide and teniposide. Dexamethasone inhibition was competitive, with Ki values of 60 and 45 microM for etoposide and teniposide, respectively. In 58 human livers, the correlation coefficients for teniposide catechol formation versus 1'- and 4-hydroxymidazolam formation were 80% and 85%, respectively; for etoposide catechol formation versus 1'- and 4-hydroxymidazolam formation r2 was 83% and 79%, respectively. Teniposide and etoposide catechol formation rates were also significantly correlated with immunodetectable CYP3A (r2 = 49% and 51%, respectively) and not with immunodetectable CYP1A2, 2E1, or 2C8. Finally, cDNAs for human CYP3A4, 3A5, 2A6, 2B6, 2C8, and 2C9 were functionally expressed in HepG2 cells, using a vaccinia viral vector. Teniposide and etoposide catechol formation was catalyzed primarily by 3A4 (15.4 and 40.9 pmol/pmol/hr, respectively) and to a lesser degree by 3A5 (1.94 and 11.3 pmol/pmol/hr, respectively), whereas there was no detectable O-demethylation of epipodophyllotoxins by 2A6, 2B6, 2C8, 2C9, or the control virus alone. Moreover, the relative activities of midazolam hydroxylation, compared with O-demethylation of epipodophyllotoxins, were similar for heterologously expressed 3A4 and for human liver microsomes. We conclude that catechol formation from teniposide and etoposide is primarily mediated by human CYP3A4, making these reactions susceptible to inhibition by prototypical 3A substrates and inhibitors.

Multidrug resistance-associated protein gene overexpression and reduced drug sensitivity of topoisomerase II in a human breast carcinoma MCF7 cell line selected for etoposide resistance.

A human breast cancer cell line (MCF7/WT) was selected for resistance to etoposide (VP-16) by stepwise exposure to 2-fold increasing concentrations of this agent. The resulting cell line (MCF7/VP) was 28-, 21-, and 9-fold resistant to VP-16, VM-26, and doxorubicin, respectively. MCF7/VP cells also exhibited low-level cross-resistance to 4'-(9-acridinylamino)-methanesulfon-m-anisidide, mitoxantrone, and vincristine and no cross-resistance to genistein and camptothecin. Furthermore, these cells were collaterally sensitive to the alkylating agents melphalan and chlorambucil. DNA topoisomerase II levels were similar in both wild-type MCF7/WT and drug-resistant MCF7/VP cells. In contrast, topoisomerase II from MCF7/VP cells appeared to be 7-fold less sensitive to drug-induced cleavable complex formation in whole cells and 3-fold less sensitive in nuclear extracts than topoisomerase II from MCF7/WT cells. Although this suggested that the resistant cells may contain a qualitatively altered topoisomerase II, no mutations were detected in either the ATP-binding nor the putative breakage/resealing regions of either DNA topoisomerase II alpha or II beta. In addition, the steady-state intracellular VP-16 concentration was reduced by 2-fold in the resistant cells, in the absence of detectable mdr1/P-gp expression and without any change in drug efflux. In contrast, expression of the gene encoding the MRP was increased at least 10-fold in resistant MCF7/VP cells as compared to sensitive MCF7/WT cells. These results suggest that resistance to epipodophyllotoxins in MCF7/VP cells is multifactorial, involving a reduction in intracellular drug concentration, possibly as a consequence of MRP overexpression, and an altered DNA topoisomerase II drug sensitivity.

Function of the hydrophilic carboxyl terminus of type II DNA topoisomerase from Drosophila melanogaster. I. In vitro studies.

The function of the hydrophilic carboxyl-terminal region of Drosophila DNA topoisomerase II was examined by constructing a series of deletion mutants at the 3'-end of the Drosophila Top2 cDNA. The truncated enzymes were then expressed in Saccharomyces cerevisiae. Deletion of up to 240 out of 1447 total amino acids had no apparent effect on the enzyme's ability to catalyze topisomerization reactions. When 273, or more, amino acids were deleted, the enzyme was no longer active. Examples were found where deletion of less than 240 amino acids inactivated the enzyme. Based on the hydrodynamic properties determined for one of these mutants, the lack of activity was most likely due to misfolding of the polypeptides. The active mutants have similar hydrodynamic properties and heat inactivation profiles as the intact enzyme, suggesting that they are dimeric and stably folded. The carboxyl-terminal 240 amino acids also were not required for interaction with the drug VM26. The only difference noted between the shortest, active mutant and the full-length enzyme was a decrease in the stability of the interaction of the truncated enzyme with DNA as evidenced by a decrease in the ionic strength at which catalysis was optimal and at which the transition between a processive and distributive mode of supercoil relaxation occurred.

Topoisomerase II as a target of VM-26 and 4'-(9-acridinylamino)methanesulfon-m-aniside in atypical multidrug resistant human small cell lung carcinoma cells.

The Adriamycin-resistant small cell lung carcinoma cell line, GLC4/ADR, showed large differences in cross-resistance to drugs such as Adriamycin, etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA), and mitoxantrone, which stimulate the formation of topoisomerase (Topo) II-DNA complexes. GLC4/ADR cells demonstrated a reduced Topo II activity and no detectable levels of the P-glycoprotein compared to the parental GLC4 cells (S. De Jong et al., Cancer Res., 50: 304-309, 1990). In the present study, the resistance to VM-26 (59.5-fold) and to m-AMSA (4-fold) of GLC4/ADR after a 1-h incubation was further analyzed. Using the K(+)-sodium dodecyl sulfate precipitation assay, a reduction in VM-26- and m-AMSA-induced cleavable complex formation was found in GLC4/ADR cells compared to GLC4 cells that was related to the degree of resistance to each drug. Cellular accumulation of the VM-26 analogues VP-16 was 3- to 8-fold less and the accumulation of m-AMSA 1- to 2-fold less in GLC4/ADR cells than in the parental cells. Following the removal of VM-26, the cleavable complexes in GLC4/ADR cells disappeared at least 2-fold faster than in GLC4 cells, while the efflux of VP-16 was also enhanced in the resistant cells. On the contrary, no differences in cleavable complex disappearance or drug efflux between these cell lines were observed with m-AMSA. Efflux of both drugs, however, occurred at a much higher rate than cleavable complex disappearance. Using isolated nuclei, a reduction in cleavable complexes in GLC4/ADR was still observed with VM-26 as well as m-AMSA compared to GLC4. The resistant nuclei and nuclear extracts showed a 3-fold decrease in M(r) 170,000 Topo II by immunoblotting. No differences in cleavable complex formation were found between nuclear extracts of both cell lines, when the Topo II activities were equalized. These findings suggest that the cross-resistance to m-AMSA is due to a decreased amount of Topo II and decreased drug accumulation, while in addition to these mechanisms an increased rate of cleavable complex disappearance is involved in the cross-resistance to VM-26 of the GLC4/ADR cell line.

Human cytochrome P450 metabolism of teniposide and etoposide.

Although teniposide (VM26) and etoposide (VP16) are eliminated mostly by nonrenal mechanisms, their cytochrome P450 metabolism in humans has not been reported. Our objective was to determine the affinity and capacity of P450 O-demethylation of VM26 and VP16 in a variety of human livers. Formation of catechols of VM26 and VP16 was detected in 24 and 26 of 26 liver microsomal preparations, respectively, with wide variability in maximum catechol formation rates from VM26 (41-fold) and VP16 (39-fold range), even among normal livers. Maximal activity measurements at 500 microM substrate were lower for VM26 catechol formation (mean = 1.5 nmol/mg/hr) than for VP16 catechol (mean = 3.2 nmol/mg/hr) in 26 livers (P less than .001). Maximal activities for VP16 and VM26 O-demethylation, and ethoxycoumarin O-deethylation were significantly higher in normal than in diseased livers. No differences were found in activities related to age, sex or race of the liver donor. In all five livers tested over a range of substrate concentrations, Km (19.7, 23.2, 43.5, 30.1 and 22.0 microM) and Vmax values (1.0, 1.2, 4.4, 8.2 and 4.0 nmol/mg/hr) for VM26 were lower compared to values for VP16 (Km = 60.2, 115.1, 87.3, 42.1 and 81.9 microM; Vmax = 1.4, 3.3, 10.3, 27.5 and 10.1 nmol/mg/hr). Despite higher VP16 catechol formation, VM26 underwent greater overall reduced nicotinamide adenine dinucleotide phosphate-dependent metabolism than VP16, consistent with greater nonrenal clearance of VM26 in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)

Variability in teniposide plasma protein binding is correlated with serum albumin concentrations.

Teniposide is a widely used anticancer drug that is extensively bound to plasma proteins (greater than 95%). We evaluated the drug's plasma protein binding in nine patients with acute lymphocytic leukemia who were in their first complete remission, and in a second group of nine patients at the time of relapse and subsequently after achieving another complete remission. Plasma protein binding was assessed by equilibrium dialysis, with direct high-performance liquid chromatographic measurement of total and free teniposide. The mean unbound fraction was 0.44% (0.21-0.88%) in the plasma of patients in first remission. It was significantly higher in patients at the time of relapse (mean = 0.86%; range 0.68-1.08%) and after achieving another complete remission (mean = 1.25%; range 0.51-2.11%). Serum albumin values were significantly lower at the time of relapse (mean = 4.6 vs 4.0 mg/dl; p less than 0.014), and decreased further during intensive postremission therapy containing L-asparaginase (mean = 3.2; p less than 0.05). For all 18 patients, a significant negative correlation (r2 = 0.667; p less than 0.001) was found between serum albumin and unbound teniposide, with low albumin being associated with higher unbound fraction. Such patients have higher systemic exposure to unbound (presumably active) teniposide at any given total plasma concentration of the agent.

Effects of the lipophilic anticancer drug teniposide (VM-26) on membrane transport.

The epipodophyllotoxin glucopyranosides have previously been shown to interact with membrane lipids and to alter the activity of several lipid-embedded membrane proteins. To determine if these agents are acting as general membrane perturbants, we have further examined their effects on membrane processes in Ehrlich ascites tumor cells. [3H]VM-26 and [3H]VP-16 were taken up rapidly and concentrated within the cells in proportion to their lipophilicity. Neither agent was found to have any significant effect on the influx of L-[3H]leucine or alpha-[3H]aminoisobutyric acid. Likewise, these drugs had no significant effects on the hexose transporter. The nucleoside transporter, which is structurally and functionally similar to the hexose transporter, was dramatically affected, however. VM-26 was a non-competitive inhibitor of equilibrium-exchange influx of cytosine arabinoside in Ehrlich cells with a Ki of 15 microM. Equilibrium-exchange influx increased with temperature in control cells (Q10 = 2) but not in VM-26-treated cells; thus, VM-26 was a more potent inhibitor at higher temperatures. VM-26 also significantly reduced zero-trans influx in Ehrlich, P388, L5178Y, and ML-1 cells, and these effects were immediate in onset. VM-26 inhibited high-affinity binding of the nucleoside transport inhibitor nitrobenzylmercaptopurine riboside (NBMPR), but VM-26 enhanced non-specific NBMPR binding to Ehrlich cells. The apparent specificity of the epipodophyllotoxins for the nucleoside transporter is discussed.

Isolation of two Chinese hamster ovary cell mutants hypersensitive to topoisomerase II inhibitors and cross-resistant to peroxides.

We have isolated two Chinese hamster ovary cell lines, designated ADR-4 and ADR-5, which exhibit hypersensitivity to intercalating agents and epipodophyllotoxins. These drugs are thought to exert their cytotoxicity via an interaction with the enzyme topoisomerase II. However, there is no apparent change in the level or catalytic activity of topoisomerase II in the mutant cells. Drug sensitivity does not appear to be due to increased drug transport because accumulation of radiolabeled actinomycin D is similar in mutant and wild-type cells. Both mutant cell lines show enhanced resistance to hydrogen peroxide and to organic peroxides. ADR-4 cells show a degree of temperature sensitivity. ADR-5 cells show mild sensitivity to UV irradiation. Neither cell line shows significant sensitivity to mono- or bifunctional alkylating agents, ionizing radiation, or bleomycin. Cell fusion studies indicate that the phenotype of each mutant cell line is recessive and that the mutants represent two different genetic complementation groups. These studies also indicate that ADR-4 and ADR-5 Adriamycin-sensitive mutant, ADR-1. These results indicate that sensitivity to topoisomerase II inhibitors can result from abnormalities in several genes. These drug-sensitive mutants may be useful for studying the mechanisms of cell killing by topoisomerase II inhibitors, free radicals, and heat.

Structure-bioactivation relationship of a series of podophyllotoxin derivatives.

With the aim of elucidating the structural requirements for O-demethylation of the antitumor agent VP-16-213 by cytochrome P-450, the binding of a series of podophyllotoxin derivatives to rat liver microsomal cytochrome P-450 was studied. The examined podophyllotoxin derivatives were: VP-16-213, VM-26, podophyllotoxin, 4'-demethylepipodophyllotoxin (the aglycone of VP-16-213 and VM-26) and 3,5-dimethoxy-4-hydroxytoluene (a model compound for the E-ring of VP-16-213). The binding to phenobarbital (Pb)-induced microsomes was more extensive than that to 3-methylcholanthrene (3-MC)-induced microsomes. Experiments on the binding to cytochrome P-450 in Pb-induced microsomes led to the following findings: (a) the presence of the polycyclic skeleton is necessary for binding; (b) the presence of the sugar moiety gives a further extension of binding, and changes in the sugar moiety affect binding; (c) binding increases on elevation of hydrophobicity; (d) the E-ring itself does not bind. For binding to cytochrome P-450 in 3-MC-induced microsomes conclusions (a) and (d) appeared to hold true. For the O-demethylation of the podophyllotoxin derivatives containing the dimethoxyphenol ring by Pb- and 3-MC-induced microsomes, the following order was observed: VM-26 greater than VP-16-213 greater than aglycone much greater than E-ring. A similar sequence was observed for the cytotoxicity against Chinese hamster ovary cells.

Etoposide and teniposide. Bioanalysis, metabolism and clinical pharmacokinetics.

Etoposide (VP 16-213) and teniposide (VM 26) are semisynthetic epipodophyllotoxin derivatives active against a variety of tumours. The clinical efficacy has led to an increasing interest in these compounds. This review presents information on the mechanism of action, biochemical pharmacology, bioanalysis, metabolism and pharmacokinetics of etoposide and teniposide.

Clinical pharmacodynamics of continuous infusion teniposide: systemic exposure as a determinant of response in a phase I trial.

Teniposide (VM-26) is an effective anticancer drug usually administered as a short infusion in doses of 150 to 165 mg/m2. The objectives of the trial reported here were to evaluate clinical responses and assess pharmacokinetic parameters as a determinant of outcome when VM-26 was administered as a 72-hour continuous infusion (CI) with doses escalated from 300 to 750 mg/m2 per course. Twenty-eight patients with recurrent leukemia, lymphoma, or neuroblastoma received 53 courses of CI VM-26 and 16 had measurable responses. There were two partial remissions and one stable disease among seven neuroblastoma patients and 13 of 21 leukemia/lymphoma patients had oncolytic responses (greater than or equal to 75% decrease in circulating blasts). Toxicity included moderate to severe mucositis and myelosuppression. Pharmacokinetic parameters determined during 35 courses administered to 23 patients were highly variable. Clearance (CI) ranged between 3.7 and 43.8 ml/min/m2, resulting in VM-26 plasma concentrations from 2.8 to 30.6 mg/L across all dose levels. The interpatient pharmacokinetic variability reflected in CI and VM-26 steady state concentrations (Css), obscured any dose-response relationship. However, when pharmacokinetic parameters for responding and nonresponding patients were compared, statistically significant relationships were observed. For responders, the mean Css was 15.2 mg/L and mean CI was 12.1 mL/min/m2; for nonresponders, mean Css was 6.2 mg/L (P less than .01) and mean CI was 21.3 mL/min/m2 (P less than .05). Thus, patients with higher CI and lower Css were less likely to respond. Clinical responses occurred in ten of ten patients with Css greater than 12 mg/L, and only five of 13 patients with Css less than 12 mg/L (P less than .01). In this study, interpatient pharmacokinetic variability yielded a four- to sixfold difference in intensity of systemic exposure (Css) within the same dose level, which was an important determinant of clinical response. These data indicate that achieving a VM-26 target concentration for individual patients could ensure an increased intensity of systemic exposure in patients with a high CI and improve the likelihood of effective therapy.

Pharmacokinetics of high-dose teniposide.

This paper describes the pharmacokinetics of teniposide (VM-26) after being administered iv in high doses to eight cancer patients (maximum dose, 1.0 g/m2). VM-26 levels in plasma, urine, saliva, duodenal fluid, and cerebrospinal fluid were determined using high-performance liquid chromatography in combination with electrochemical detection. The plasma concentration-time curve of VM-26 showed a triphasic decay with a slow third phase in five patients, whereas in two patients the plasma concentration decay was biphasic. The plasma pharmacokinetics of VM-26 proved to be linear and could be fitted to a three-compartment model (five patients) and to a two-compartment model (two). The steady-state volume of distribution varied from 13.2 to 24.7 L/m2. The total-body clearance ranged from 5.84 to 10.18 ml/minute/m2. Low concentrations of VM-26 were found in saliva, duodenal fluid, cerebrospinal fluid, and urine. Excretion of unchanged VM-26 into the urine varied from 8.8% to 13.9% of the administered dose. No glucuronide of VM-26 could be detected in plasma or other biological fluid.

Uptake and binding of teniposide (VM26) in Krebs II ascites cells.

With [3H] VM26 as marker, the uptake and binding of teniposide have been made in cells of Krebs II ascitic tumors. The intracellular accumulation of drug displayed a passive diffusion and a saturation kinetics with an apparent Michaelis-Menten constant of 37.54 10(-6) M and a flux of 13.4 nM/min/mg of protein. VM26 was rapidly taken and an equilibrium was established with the extracellular drug in about 30 min. The steady-state accumulation was diminished by Na+ and Ca2+ absence and VP16-213, whereas, K+ and Mg2+ have no effect. Energy dependence of the system was characterized by a Q10 of 1.75 +/- 0.2 and the uptake was reduced by ouabain and iodoacetamide, when 2-4-dinitrophenol and glucose absence were without appreciable change. The study of the efflux showed that about 87% of the uptaken drug was removed, the residual amount being probably irreversibly bound. The intracellular accumulation of the drug was associated with various cell organelles, however, only the nuclear fraction demonstrated a high affinity binding.

The clinical pharmacology of etoposide and teniposide.

Etoposide and teniposide are semisynthetic derivatives of podophyllotoxin and are increasingly used in cancer medicine. Teniposide is more highly protein-bound than etoposide, and its uptake and binding to cells is also greater. Etoposide and teniposide are phase-specific cytotoxic drugs acting in the late S and early G2 phases of the cell cycle. They appear to act by causing breaks in DNA via an interaction with DNA topoisomerase II or by the formation of free radicals. Teniposide is more potent as regards the production of DNA damage and cytotoxicity. Most studies show a biexponential decay following intravenous administration of etoposide and teniposide. The terminal elimination half-life of etoposide is less than that of teniposide, and the plasma and renal clearances of etoposide are greater. The peak plasma concentrations of drug and the area under the concentration versus time curve are linearly related to the intravenous dose of both drugs. Considerable interpatient variability of pharmacokinetic parameters exists following intravenous etoposide and teniposide. Various metabolites of etoposide and teniposide have been identified but their detection and quantitation are disputed. Approximately 30 to 70% of a dose of etoposide is accounted for by excretion, whereas the figure appears to be only 5 to 20% for teniposide. The bioavailability of oral etoposide is about 50% but its absorption is not linear with increasing dose within the range in clinical use. There is considerable inter- and intrapatient variability in the pharmacokinetics of oral etoposide. There is no evidence of accumulation of etoposide and teniposide after multiple consecutive doses by the intravenous or oral routes. The exact roles of the liver and kidney in metabolism and excretion of etoposide and teniposide are uncertain. Etoposide has been shown to be a highly schedule-dependent drug in clinical studies. This together with the phase-specific action of etoposide and teniposide and their increasingly widespread use in cancer medicine make the clinical pharmacology of these drugs of great clinical importance.

Atypical multiple drug resistance in a human leukemic cell line selected for resistance to teniposide (VM-26).

Resistance to the cytotoxic effects of many natural product drugs after exposure to a single agent is a common observation. The classes of drugs included in the "classic" multiple drug resistance phenotype are Vinca alkaloids, anthracyclines, epipodophyllotoxins, and antibiotics. We report here the characterization of a human leukemic cell line (CEM/VM-1) with "atypical" multiple drug resistance: despite resistance and cross-resistance to etoposide, anthracyclines, mitoxantrone, and 4'-[(9-acridinyl)amino]methanesulphon-m-anisidide (mAMSA), these cells retain sensitivity to the Vinca alkaloids. Further, even though this cell line is approximately equal to 40-fold cross-resistant to the cytotoxic effect of etoposide (VP-16), it is similar to drug-sensitive CEM cells in the cellular pharmacology of [3H]VP-16 as determined by zero time binding, initial influx rate, steady state drug concentration, and unidirectional efflux. Our studies suggest that the resistance of CEM/VM-1 cells to epipodophyllotoxins is due to an altered interaction between drug and its cellular target(s) by a mechanism unrelated to the decreased cellular concentration of drug associated with the "classic" multiple drug resistance phenotype.