Hans Erik Rugstad

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.

Cadmium resistance and content of cadmium-binding protein in two enzyme-deficient mutants of mouse fibroblasts (L-cells)

Abstract The toxic Cd 2+ ion accumulates in mammalian organisms, the main storage organs are apparently the liver and the kidney. In these organs Cd 2+ is bound to low molecular weight proteins (thioneins) as metallothionein. We describe here the development of resistance to otherwise lethal concentrations of Cd 2+ by two non-epithelial cell lines, both derived from mouse fibroblasts (L-cells). One of the cell lines (clone ID) is deficient in thymidine kinase and resistant to 5-bromodeoxyuridine, the other (A9) deficient in hypoxanthine-guanine phosphoribosyl transferase and resistant to 8-azaguanine. After stepwise increase in Cd 2+ concentration, clone 1D cells had apparently normal growth rate in the presence of 100 micromolar Cd 2+ after 6 months of Cd treatment. The A9 cells were apparently more sensitive to Cd 2+ , after about one years Cd treatment they had apparently normal growth in the presence of 100 micromolar Cd 2+ . This concentration of Cd 2+ would kill cells of both cell lines not previously exposed to Cd. In the resistant A9 cells about 40 per cent of the cadmium were bound to a cadmiumbinding protein (Cd-BP) of molecular weight of about 12,000, most probably metallothionein, in the resistant clone 1D cells the corresponding figure was 60 per cent. The non-resistant cell lines had apparently no metallothionein. We have thus found that also non-epithelial cells can synthesize low molecular weight Cd-BP and that there apparently is a good correlation between cadmium resistance and content of Cd-BP.

Monitored high-dose azathioprine treatment reduces acute rejection episodes after renal transplantation

BACKGROUND Azathioprine (AZA) is widely used in organ transplantation. Common practice is to adjust dose according to body weight only, despite documented pharmacokinetic variability. The purpose of this study was to investigate whether high-dose AZA treatment monitored by 6-thioguanine nucleotides (6-TGN) levels reduces the incidence of rejection episodes in renal transplantation without a corresponding increase in myelotoxicity. METHODS Patients receiving cyclosporine, steroids, and AZA were randomized into either the low-dose AZA group (3 mg/kg on day 0, then 2 mg/kg/day the first week and 1 mg/kg/day thereafter) or the high-dose AZA group. In the latter, AZA was started at 5 mg/kg/day and then adjusted to keep 6-TGN concentrations (measured twice weekly) between 100 and 200 pmol/8 x 10(8) RBCs. RESULTS A total of 360 transplant recipients were included in the final analysis. The cumulative incidence of first rejection episodes was reduced by 21%, from 62.8% in the low-dose group to 49.4% in the high-dose group (difference: 13.3%; 95% confidence interval: 3.2-23.5). Similar results were found in subgroups according to HLA-DR match. The 6-TGN concentration was significantly higher in the high-dose AZA group during the first month, and the reduction in rejection episodes was achieved in the same period. A larger proportion of patients in the high-dose group had nadir white blood cell count below 2.0 x 10(9) leukocytes/L (13.3% vs. 4.4%; difference: 8.9%; confidence interval: 3.1-14.7). CONCLUSIONS High-dose AZA therapy in a triple-drug regimen, monitored by 6-TGN, will keep myelotoxicity within acceptable limits with the benefit of a reduction in acute rejection episodes.

Pharmacokinetics of diltiazem and its metabolites in relation to CYP2D6 genotype

Recently, it was shown in vitro that the polymorphic enzyme cytochrome P450 (CYP) 2D6 mediates O‐demethylation of diltiazem. The aim of this study was to compare the pharmacokinetics of diltiazem and its major metabolites in healthy human volunteers representing different CYP2D6 genotypes.

Patterns of Azathioprine Metabolites in Neutrophils, Lymphocytes, Reticulocytes, and Erythrocytes: Relevance to Toxicity and Monitoring in Recipients of Renal Allografts

Monitoring of azathioprine (AZA) therapy by the measurement of 6-thioguanine nucleotides (6-TGN) concentrations in red blood cells (RBC) may improve safety and ensure optimal immunosuppressive effects of AZA in organ transplantation. The authors explored the rationale for such monitoring by measuring thiopurine metabolites in peripheral blood cell types that are more relevant to the effects and kinetics of AZA and its active metabolites. Neutrophil granulocytes were isolated by density gradient centrifugation, and CD4+ lymphocytes and reticulocytes by using specific immunomagnetic beads. In neutrophils, 6-TGN concentrations had median measurements 31 times higher than in RBCs. In contrast to the high methylated mercaptopurine (me-MP) concentrations in RBCs, these metabolites were not detected in the neutrophils. Thiopurine metabolite levels were lower than the analytic limit of detection in all the CD4+ samples. The concentrations of 6-TGN and me-MPs were lower in reticulocytes than in RBCs in general, indicating that thiopurine metabolites are taken up by RBCs in the circulation. This studys findings, that 6-TGN concentrations are very high in neutrophils, whereas me-MPs are undetectable, many explain the specific neutropenic adverse effect of AZA. The results also add support to monitoring AZA through measurements of 6-TGN and me-MPs in RBCs.

Clinical Pharmacokinetics of Methyldopa

SummaryAbsorption of methyldopa from the gastrointestinal tract is incomplete and variable; bio-availability after oral administration is about 25% (range 8 to 62%). The average lime to reach maximum plasma concentration (tmax) [chemically determined] is 2 hours, when the maximum plasma concentration of active drug accounts for 50% of the radioactivity, the remainder representing various metabolites.Physicochemical determination of methyldopa shows that bi-phasic elimination occurs after both intravenous and oral administration, the half-life of the α-phase being 0.21 hours (range 0.16 to 0.26 hours) and of the β-phase 1.28 hours (range 1.02 to 1.69 hours) in normal subjects. Methyldopa is less than 15% protein bound, whereas the primary metabolite, which most probably is the O-sulphate, is about 50% protein bound.The apparent volume of distribution in the central compartment is about 0.23L/kg (range 0.19 to 0.32L/kg), and the total volume of distribution (calculated as Vdarea) is about 0.60L/kg (range 0.41 to 0.72L/kg) in healthy volunteers.Acid-labile conjugates are formed after oral administration. These acid-labile conjugates, in particular the O-sulphate, are probably formed in the intestinal cells, since they are detected in very small amounts after intravenous administration. Additionally, there is a rapid formation of partly unidentified metabolites after both intravenous and oral administration. After intravenous administration the quantitatively most prominent metabolites are methyldopamine and the glucuronide of dihydroxyphenylacetone, but traces of 5 or 6 other metabolites have also been found and identified. These metabolites are probably formed in the liver, but the complete metabolic pattern is still unknown.The renal clearance of methyldopa (95ml/min/m2) is more than 50% higher than the endogenous creatinine clearance. Renal excretion of some metabolites is slower.Extrarenal elimination accounts for about 50% of the total body clearance of the drug. Renal excretion is very low in patients with renal failure, resulting in accumulation of both active drug and, in particular, its metabolites. There is a marked accumulation of unidentified metabolites in renal failure patients, which possibly explains the strong and prolonged hypotensive action of methyldopa in these patients.

Possibilities for Therapeutic Drug Monitoring of Azathioprine: 6-thioguanine Nucleotide Concentrations and Thiopurine Methyltransferase Activity in Red Blood Cells

The objectives of this study were to establish monitoring of azathioprine (AZA) treatment in renal allograft recipients by red blood cell (RBC) 6-thioguanine nucleotide (6-TGN) measurements and to characterize the variability of RBC thiopurine methyltransferase (TPMT) activity and the effects on 6-TGN levels and the incidence of rejection episodes. In 82 renal allograft recipients, the effect of standard AZA dosage (3 mg/kg tapered to 1 mg/kg) was compared with higher dosages (3 mg/kg for several days) under 6-TGN monitoring. The authors measured TPMT in these patients and in a group not receiving AZA. The authors did not find an inverse correlation between RBC TPMT activity and 6-TGN concentrations, and baseline TPMT activity did not predict the incidence of rejection episodes The slight increase in RBC TPMT activity after transplant was associated with the use of furosemide rather than AZA; in the five patients receiving furosemide for less than 10 days, TPMT activity declined. The higher AZA dosage in the 6-TGN monitored group was not sufficient to increase RBC 6-TGN to target levels (100 to 200 pmol/8 x 10(8) RBC); median 6-TGN levels were similar in the two groups, as was the incidence of rejection episodes. Based on these findings, the authors suggest that higher dosages be studied in conjunction with 6-TGN monitoring, to explore the possibilities for therapeutic improvements.

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.

Monitoring of azathioprine treatment by determination of 6-thioguanine nucleotide concentrations in erythrocytes.

Thioguanine nucleotides (6-TGN) are intracellular metabolites that may contribute to the antiproliferative effects of AZA. The objectives of our study were to describe the variability of 6-TGN concentrations during AZA therapy and to investigate possible correlations between 6-TGN levels and subsequent myelosuppression. We measured 6-TGN concentrations in RBC of 65 renal transplant recipients from day 0 until 11–64 days after transplantation. High 6-TGN concentrations were observed in relation to elevated S-creatinine. In 15 patients, 6-TGN concentrations above 200 pmol/8×108 RBCs were measured (high 6-TGN group: mean maximal 6-TGN = 552 pmol/8×108 RBCs, SE = 91). In the remaining 50 patients, mean maximal 6-TGN was 82 pmol/8×108 RBCs, SE = 6.1 (low 6-TGN group). In the former group, mean S-creatinine measured on the day of maximal 6-TGN was 466 μmol/L (SE = 62.3), while in the latter it was 190 (SE = 14.7). In the high 6-TGN group, we observed a lower mean nadir neutrophil count than in the low 6-TGN group (3.4 vs. 5.1 × 109 neutrophils/L). The nadir neutrophil count occurred, on the average, 12.7 days after maximal 6-TGN in the high 6-TGN group, with no such delay in the low 6-TGN group.

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.