H.W. van den Berg

Can severe vincristine neurotoxicity be prevented

SummaryNeurotoxicity in vincristine treatment has generally been considered a consequence of the cumulative dose of the drug, and liver dysfunction has been recognised as an indication to reduce the dosage. We demonstrate that neurotoxicity is also related to individual doses and that even when there is no other evidence of liver dysfunction, a raised level of serum alkaline phosphatase may predict severe neurotoxicity.Exposure to vincristine following IV injection of the drug was studied in 27 subjects by measuring the area under the vincristine plasma concentration time curve (AUC0–∞). A statistically significant relationship was found between the AUC0–∞ and the degree of neurotoxicity. The AUC0–∞ was related both to dose and to elevation of serum alkaline phosphatase, suggesting that elimination of the drug is impaired when serum alkaline phosphatase is raised. Among patients with elevated serum alkaline phosphatase, a small reduction in the dose of the drug resulted in lower vincristine plasma AUC0–∞ and less neurotoxicity.

Recombinant human interferon alpha increases oestrogen receptor expression in human breast cancer cells (ZR-75-1) and sensitizes them to the anti-proliferative effects of tamoxifen.

Exposure of ZR-75-1 human breast cancer cells for 48 h to human recombinant interferon alpha (IFN alpha) resulted in increased expression of oestrogen receptors as measured in a whole cell binding assay. This effect was inversely proportional to dose being significant following treatment with 10-100 IU IFN ml-1 and was only observed at a low initial cell plating density. The extent of the increase in oestrogen receptor levels ranged from 1.2- to 7.2-fold following treatment with 10 IU IFN ml-1. No increase in progesterone receptor expression was observed under the same experimental conditions. Concentrations of IFN which increased oestrogen receptor levels had no effect on cell proliferation. IFN (500 IU ml-1) inhibited cell proliferation and the combination of this treatment with tamoxifen (2 microM) had a greater anti-proliferative effect than either drug alone although there was no evidence of synergism. However, a 5-day pretreatment of cells with IFN (10 IU ml-1) markedly sensitised them to the growth-inhibiting effect of a subsequent 6-day exposure to tamoxifen.

Nonlinear pharmacokinetics for the elimination of 5-fluorouracil after intravenous administration in cancer patients

SummaryPlasma concentrations of 5-fluorouracil (FU) and its primary catabolite, 5′, 6′-dihydro-5-fluorouracil (DHFU) were measured using gas-liquid chromatography after single-dose therapy with 7.2–14.4 mg/kg. Because of the limited sensitivity of the assay for drug levels in plasma, the urinary excretion of FU and metabolites was investigated using an ion-specific electrode after either a single bolus (7.0–9.6 mg/kg) or multiple-dose therapy (6.4–7.4 mg/kg/day). Half-life values for the elimination of FU from plasma (mean, 123.5 min) were greater in each patient than for the catabolite (mean, 109.2 min). Values of the area under the curve for FU profiles varied between patients (mean±SE, 12.7±1.9 μg·h/ml) by comparison with the relatively constant values for curves of DHFU concentrations (mean±SE, 2.8±0.15 μg·h/ml). In pharmacokinetic profiles of urinary excretion a transient phase of convex shape was apparent after 80%–98% of single doses of FU was excreted. Half-lives for the elimination of FU in urine were 2.6–5.9 h, which increased to 18–44 h on multiple dosing. The results demonstrate saturation in the elimination of FU after therapeutic doses, and are consistent with the proposal that reduction of FU to DHFU provides the rate-limiting step.

The pharmacokinetics of vincristine in man

SummaryA radioimmunoassay has been used to investigate the pharmacokinetics of vincristine in 39 cancer patients who received between 0.4 and 1.54 mg vincristine/m2 as part of standard treatment protocols. There was wide interindividual variation in both the terminal elimination half-life of vincristine (t1/2β) and the associated volume of distribution (Vd), resulting in an 11-fold range of dose-corrected area under the plasma concentration versus time curve values (AUC0–∞). Elevated vincristine AUC0–∞ values were observed in those patients with raised serum alkaline phosphatase at the time of vincristine estimation. The t1/2β was significantly longer in these patients than in those with serum alkaline phosphatase within normal limits, suggesting that biochemical evidence of cholestasis is associated with reduced clearance of vincristine. Evidence is also presented to suggest that the clearance of vincristine is dose-dependent within the therapeutic dose range. We observed a disproportionate rise in vincristine plasma concentration at doses exceeding 1 mg/m2, due primarily to a lengthening of mean t1/2β compared with that observed for patients receiving 1 mg vincristine/m2 or less.

Changes in epidermal growth factor receptor expression and response to ligand associated with acquired tamoxifen resistance or oestrogen independence in the ZR-75-1 human breast cancer cell line.

We have examined the expression of receptors for epidermal growth factor (EGFR) by the ZR-75-1 human breast cancer cell line and tamoxifen resistant (ZR-75-9al 8 microM) and oestrogen independent/tamoxifen sensitive (ZR-PR-LT) variants. The parent line expressed a single class of high affinity binding sites (4,340 +/- 460 receptors/cell; Kd 0.23 +/- 0.04 nM). ZR-75-9al 8 microM cells, routinely maintained in medium containing 8 microM tamoxifen, were negative for oestrogen receptor (ER) and progesterone receptor (PGR) and expressed a markedly increased number of EGFR (14,723 +/- 2116 receptors/cell). Receptor affinity was unchanged. Time dependent reversal of the tamoxifen resistant phenotype was accompanied by a return to ER and PGR positivity and a fall in EGFR numbers to parent cell levels. In contrast ZR-PR-LT cells had a greatly reduced EGFR content (803 +/- 161 receptors/cell) accompanying elevated PGR numbers. Pre-treatment of these cells with suramin or mild acid stripping failed to expose receptors which may have been occupied by endogenously produced ligand. Increased proliferation of ZR-75-1 cells treated with EGFR (0.01-10 ng ml-1) was only observed in serum-free medium lacking insulin and oestradiol. Under these conditions untreated cells failed to proliferate. Both variant lines continued to proliferate in serum free medium in the absence or presence of insulin and oestradiol but failed to respond to exogenous EGF.

Initial high anti-emetic efficacy of granisetron with dexamethasone is not maintained over repeated cycles

We have reported previously that the anti-emetic efficacy of single agent 5HT3 antagonists is not maintained when analysed with the measurement of cumulative probabilities. Presently, the most effective anti-emetic regimen is a combination of a 5HT3 antagonist plus dexamethasone. We, therefore, assessed the sustainment of efficacy of such a combination in 125 patients, scheduled to receive cisplatin > or = 70 mg m(-2) either alone or in combination with other cytotoxic drugs. Anti-emetic therapy was initiated with 10 mg of dexamethasone and 3 mg of granisetron intravenously, before cisplatin. On days 1-6, patients received 8 mg of dexamethasone and 1 mg of granisetron twice daily by oral administration. Protection was assessed during all cycles and calculated based on cumulative probability analyses using the method of Kaplan-Meier and a model for transitional probabilities. Irrespective of the type of analysis used, the anti-emetic efficacy of granisetron/dexamethasone decreased over cycles. The initial complete acute emesis protection rate of 66% decreased to 30% according to the method of Kaplan-Meier and to 39% using the model for transitional probabilities. For delayed emesis, the initial complete protection rate of 52% decreased to 21% (Kaplan-Meier) and to 43% (transitional probabilities). In addition, we observed that protection failure in the delayed emesis period adversely influenced the acute emesis protection in the next cycle. We conclude that the anti-emetic efficacy of a 5HT3 antagonist plus dexamethasone is not maintained over multiple cycles of highly emetogenic chemotherapy, and that the acute emesis protection is adversely influenced by protection failure in the delayed emesis phase.

The anti-proliferative effects of tyrosine kinase inhibitors towards tamoxifen-sensitive and tamoxifen-resistant human breast cancer cell lines in relation to the expression of epidermal growth factor receptors (EGF-R) and the inhibition of EGF-R tyrosine kinase.

We have investigated the anti-proliferative effect of the tyrosine kinase inhibitors genistein, lavendustin A and 2,5-dihydromethylcinnimate (2,5-MeC) (a stable erbstatin analogue) against the epidermoid A431 cell line, the ZR-75-1 human breast cancer cell line and tamoxifen-resistant (ZR-75-9a1) and oestrogen-independent (ZR-PR-LT) variants. Increased EGF-R expression by ZR-75-9a1 was associated with decreased sensitivity to genistein and 2,5-MeC whilst decreased EGF-R expression (ZR-PR-LT) cells was associated with increased sensitivity to lavendustin A and 2,5-MeC. Anti-proliferative IC50 concentrations of 2,5-MeC, but not genistein or lavendustin A, inhibited EGFR-R associated tyrosine kinase activity of ZR-75-1 cells and variants by 20-50%.

Expression of receptors for epidermal growth factor and insulin-like growth factor I by ZR-75-1 human breast cancer cell variants is inversely related: the effect of steroid hormones on insulin-like growth factor I receptor expression

We have investigated the expression of insulin-like growth factor I receptors (IGFR) by the ZR-75-1 human breast cancer cell line and tamoxifen-resistant (ZR-75-9a1) and oestrogen-independent (ZR-PR-LT) variants. ZR-75-1 cells expressed 6633+/-953 receptors per cell,(K(d) 0.24+/-0.06 nM). IGFR expression was reduced in ZR-75-9a1 cells (1180+/-614 receptors per cell, K(d) 0.13+/-0.05) and increased in the ZR-PR-LT cell line (18 430+/-3210 receptors per cell, K(d) 0.24+/-17). A comparison of these data with previously published findings for epidermal growth factor receptor (EGFR) expression by these cell lines revealed that IGFR and EGFR expression are inversely related in the variant lines whereas ZR-75-1 cells express similar numbers of both receptors. Since the changes in IGFR expression observed are associated with changes in steroid hormone receptor status, we also investigated the effects of oestradiol, the synthetic progestin ORG 2058 and dexamethasone on IGFR expression. Oestradiol increased IGFR expression only in the ZR-75-1 cell line. Low concentrations of ORG 2058 increased IGFR levels in the two cell lines positive for progesterone receptor (ZR-75-1 and ZR-PR-LT). High concentrations of ORG 2058 increased IGFR expression in all cell lines, as did dexamethasone. These data suggest that EGFR and IGFR expression may be linked in breast cancer, and that EGFR/IGFR ratios in breast cancer may be a more sensitive prognostic indicator than EGFR expression alone. Regardless of basal IGFR expression by the cell studied, ORG 2058 increased IGFR expression, possibly via both the progesterone and glucocorticoid receptors.

A novel method for measuring incorporation of radioactive precursors into nucleic acids and proteins of cells in monolayer culture

Abstract A straightforward and accurate method for assaying DNA, RNA, and protein synthesis in small numbers of cells in monolayer culture is described which is particularly useful when large numbers of different treatment conditions are to be compared.

Mechanistic and anti-proliferative studies of two novel, biologically active bis-benzimidazoles

We have previously synthesised a number of novel head-to-head bis-benzimidazole derivatives that are structurally related to the fluorochrome, Hoechst 33258, and which possess strong affinity for A:T sites in the minor groove of duplex DNA. Initial studies revealed these compounds to exhibit potent antiproliferative activity against a range of ovarian cell lines and to inhibit transcription in an in vitro setting. In this study, we have examined their cellular behaviour in detail and have shown that two of these compounds (ABA13 and ABA833) potently inhibit the proliferation of a range of human tumour cell lines, and show some specificity towards breast carcinoma cell lines. In most of the cell lines investigated, ABA833 was the more potent of the two compounds. Flow cytometric analysis revealed that ABA13 and ABA833 (50-500 nM) induced an S phase block and increased the pre-G1 population in MCF-7 and MDA 468 human breast cancer cells. An increase in the pre-G1 population of RKO colon carcinoma cells was seen only at 500 nM with ABA833, reflecting the reduced sensitivity of this cell line to the bis-benzimidazoles in comparison to the breast cancer cell lines. Mechanistic studies revealed that neither ABA13 or ABA833 act as topoisomerase I (topo I) or topoisomerase II (topo II) poisons in plasmid or kinetoplast DNA (kDNA) relaxation assays, but both compounds do inhibit the catalytic activity of these enzymes. Drug uptake studies showed that reduced sensitivity of MCF-7adr and RKO cells compared with MCF-7 to both ABA13 and ABA833 correlated with a markedly reduced intracellular drug accumulation.