Liv Endresen

Resistance against cis-dichlorodiammineplatinum in cultured cells with a high content of metallothionein

Resistance against the antitumor drug cis-dichlorodiammineplatinum (cis-DDP) was tested in cultured cells: one human epithelial line derived from normal skin (HE), one mouse fibroblast line derived from L cells (Cl lD), as well as Cd-resistant substrains of both cell lines (designated HE100 and Cl lD100, respectively). The substrains are resistant to 100 μmol Cd/liter and contain large amounts of cytoplasmic Cd-binding proteins which, in the present study, were confirmed by spectrophotometry and amino acid analysis as being metallothioneins (MT). Counting of cells at certain intervals during a growth period with cis-DDP treatment (0.5–60 μmol/liter) showed a resistance against the drug in both MT-containing strains (HE100 and Cl lD100), e.g., after 4 days treatment with 5 μmol/liter the number of HE cells was 8% of controls, whereas 32% of the HE100 cells survived (p < 0.001, t test). The resistance in the MT-containing strain was confirmed by colony formation techniques applicable to Cl lD and Cl lD100 cells both in soft agar and on a plastic substratum. There was a significant difference in survival (p < 0.005, F test) over a dose range of 3–60 μmol/liter. Gel filtration showed that 70% of the Pt in cytosols from cis-DDP-treated resistant cells appeared in the MT fraction. Less than 5% was recovered in the corresponding fractions from nonresistant cells. Determination of the cell cycle phase distribution did not reveal any differences between the corresponding cell strains, nor were glutathione levels increased. A three- to fourfold increase of total sulfhydryl content in the resistant strains was accounted for by MT. This investigation makes it probable that endogeneously synthesized MT contributes to the resistance against cis-DDP.

Kinetics of mercaptopurine and thioguanine nucleotides in renal transplant recipients during azathioprine treatment.

Summary The purpose of this study was to examine the pharmacokinetics of mercaptopurine (6-MP) and thioguanine nucleotides (6-TGN) during azathioprine treatment. Plasma profiles and urinary excretion of 6-MP and 6-TGN concentrations in red blood cells (RBCs) were measured repeatedly during the first 3 weeks following transplantation in 10 adults, who had received kidney grafts from living related donors. Mean maximal 6-MP plasma concentration (Cmax) was 340 nmol/L (SD = 290), mean time to Cmax (7max) was 2 h (SD = 1.8), and mean area under the plasma concentration-time curve (AUC) was 930 nmol/L/h (SD = 770). The mean fraction of azathioprine dose excreted as 6-MP in urine was 1.32% (SD = 1.11). Up to eightfold variability of Cmax and AUC was observed from day to day within each patient. The correlation between 6-MP AUC and amount excreted in the urine was weak (r = 0.37, 95% CI from 0.02 to 0.64). In this group of patients the observed 6-TGN levels in RBCs were low; maxima during the observation period ranged from undetectable to 250 pmol/8 x 108 RBCs. In individual patients, 6-TGN levels were relatively stable throughout the dosing interval (“within-dose-interval-CV” < 19%), even when sharp and high 6-MP peaks in plasma were observed.

Pharmacokinetics of oral 6-mercaptopurine: relationship between plasma levels and urine excretion of parent drug.

Plasma levels and cumulative urine excretion of 6-mercaptopurine (6-MP) were measured using a specific and sensitive high-performance liquid chromatographic assay in seven children with acute lymphoblastic leukemia (ALL) as well as in one healthy volunteer. The dose of 6-MP varied in the range of 25–75 mg/m2 of body surface area and was administered with a standard breakfast. A 4− to 11-fold variation between individuals was found in the pharmacokinetic parameters: peak concentration, time to reach peak, area under the plasma concentration-time curve (AUC), and fraction of dose excreted in the urine. Three repeated determinations in one individual revealed that AUC also varied more than sixfold following an overnight fast. In three individuals, the reducing agents glutathione (10 mg/kg) and ascorbic acid (15 mg/kg) were coadministered with 6-MP to evaluate their possible role in the protection of 6-MP from oxidation and degradation in the intestinal lumen. No consistent effect was observed, however, on the AUCs of either of these agents. A clear relationship was found between AUCs and the 24-h urinary excretion of unchanged drug (r = 0.9381), indicating that determinations of 6-MP in the urine may replace the painful procedure of repeated blood sampling. Further studies are necessary to determine the factors contributing to the unpredictable plasma levels following oral doses of 6-MP and to determine the value of pharmacokinetic monitoring in ALL patients.

Stability of cadmium resistance and metallothionein levels in vitro and in vivo.

Cultured cells can be adapted to large concentrations of the toxic cadmium ion, apparently by induction of synthesis of the Cd-binding protein metallothionein (MT). One human epithelial line derived from normal skin (HE100) and one murine fibroblast line, derived from L cells (C1 1D100), were used to study the stability of Cd resistance, the MT levels following omission of Cd, and the inducibility of MT synthesis in cells on reexposure to Cd. In the murine cells there was no significant loss of resistance during a 4-week period either after cultivation in vitro or after growing the cells in nude mice. In the human cells a decrease (50%) in resistance was noted the first week after Cd omission. After removing Cd from the cells, a rapid decrease in MT content was demonstrated. After 3 weeks of cultivation only trace amounts were left in both cell lines. However, approximately 60% (HE100) and 80% (C1 1D100) of the previous levels were demonstrated after reexposure to maximum tolerable doses for 24 hr. The data indicate that the degree of stability of Cd resistance is dependent on the capacity in cells for an immediate de novo synthesis of large amounts of MT on reexposure to Cd. The animal experiments demonstrate that Cd resistance is maintained even after growing the cells in vivo.

Protein mapping of two metallothionein-rich cell strains and their parent lines, using high-resolution two-dimensional electrophoresis☆

A high-resolution two-dimensional gel electrophoresis (2-D) technique was used to characterize one human and one murine cadmium-resistant substrain and their parental wild-type lines. The substrains are cultured on 100 microM cadmium and contain high levels of the cysteine-rich protein metallothionein (MT). All four cell lines were labeled with [35S]methionine during growth. A remarkable consistency was found in the protein maps of the resistant strains compared to those obtained from their corresponding wild-type lines. Thus, in the maps from the human substrain only two spots were detected which were not found in the parent cells. In the murine substrain, two spots were more abundant and two diminished compared to the parent cells. No distinct spots corresponding to authentic MT were detected in any of the autoradiographs from the cadmium-resistant cells. The reason for this was found to be failure of the protein to focus in the first dimension. Purified [35S]cystine-labeled MT appeared as a diffuse labeling over the entire gel, and subsequently as wide horizontal bands in the second dimension. These bands were also clearly visible in the protein maps when MT-rich cells had been labeled with [35S]cysteine. This study shows that the standardized 2-D gel system used in many laboratories cannot be used to screen cell populations for MT.

The influence of partial hepatectomy on the pharmacokinetics of preoperatively injected 4'-epidoxorubicin in rats

SummaryPreoperative administration of 4′-epidoxorubicin (Epi-A) has been suggested as adjuvant therapy in patients undergoing liver resection for hepatocarcinoma. To assess the influence of partial hepatectomy on the pharmacokinetics of Epi-A, an experimental study in rats was undertaken in which 5 mg/kg Epi-A was given i.v. 10 min prior to a 2/3 hepatic resection or sham operation. Epi-A levels in liver tissue and plasma were determined using a sensitive and specific HPLC method. A marked uptake of Epi-A in liver tissue was found at 10 min after injection. The partially hepatectomized rats showed a 2-fold increase in AUC between 4 and 72 h as compared with the shamoperated controls. The terminal half-life from 24 to 72 h was not significantly changed by the partial hepatectomy. The plasma binding of Epi-A was measured at 4 h post-surgery. The fraction of unbound Epi-A was 0.16 in partially hepatectomized animals and 0.20 in sham-operated rats. The results indicate that when Epi-A is given prior to liver resection, a dose reduction might be necessary to avoid increased side effects due to the rise in AUC.

Pharmacology and General Therapeutic Principles of Methotrexate

Methotrexate (MTX) is one of the most potent and widely used drugs in cancer chemotherapy. Synthesized in 1949 by Seeger and co-workers, it soon proved to be strikingly effective in childhood leukemia (Farber et al., 1956). The immunosuppressive potential of MTX was also recognized in the 1950s; this aspect received minimal attention, however, in the following years. During the 1970s, its primary use in nonneoplastic disease was for the treatment of psoriasis, based on the assumption that the rapidly proliferating epidermal cells in psoriatic lesions are more sensitive to the effects of MTX than normal epidermal cells.