Clinical phase I and pharmacokinetic study of S 16020, a new olivacine derivative: report on three infusion schedules.

S 16020, a new 9-OH olivacine derivative, is a novel topoisomerase II inhibitor with activity in cell lines presenting the classical multidrug resistance phenotype. This report summarizes, in addition to pharmacokinetic data, the whole phase I clinical experience of S 16020 using three different infusion schedules. Asthenia and skin toxicity were the main side effects. In an attempt to understand the skin toxicity mechanism, experiments in animals were performed, the results of which are reported. S 16020 showed rapid tumor necrotizing activity in some patients, with soft tissue metastases of epidermoïd tumors and pain at the tumor site. To document the side effects of S 16020 and tumor site reactions (pain, edema, inflammatory signs), inflammatory parameters and some cytokines were measured. In our patients there was no hemolysis and no detection of anti-S 16020 antibodies, confirming the absence of immunogenicity of the compound. Based on the overall data of the three infusion schedules of S 16020, the dose of 100 mg/m(2) over 3 h every 3 weeks was selected for phase II studies.

Comparison of in vitro exemestane activity versus other antiaromatase agents.

Anastrozole, letrozole, and exemestane are the most selective and potent oral antiaromatase agents currently available. However, in vitro and in vivo studies comparing these agents are lacking. Anastrozole and letrozole are reversible, competitive nonsteroidal type II inhibitors, whereas exemestane is an irreversible steroidal type I inactivator. The study was conducted to determine the impact of this characteristic on in vitro residual aromatase activity and protein levels after incubation of JEG-3 cells with aminoglutethimide (a type II inhibitor), anastrozole, exemestane, or letrozole. Aromatase activity was measured after various incubation times with each antiaromatase agent at a concentration 10 times higher than IC50 (concentration giving 50% inhibition). Only exemestane induced a residual inhibition of aromatase activity after its removal, without any change in the aromatase protein level. Aromatase activity increased after preincubation of JEG-3 cells with either aminoglutethimide or anastrozole without any change in the aromatase protein level. The aromatase protein level increased rapidly when cells were incubated with letrozole and aromatase activity inhibition disappeared immediately after removal of the drug. The breakthrough effects in aromatase activity or protein levels observed after treatment with reversible inhibitors may be a factor in therapeutic failure with these agents. These results suggest a possible advantage for exemestane because it is the only clinically available oral irreversible aromatase inactivator.

Time-schedule dependency of S 16020, a new topoisomerase II inhibitor.

S 16020 is a new olivacine derivative which has been shown to intercalate into DNA and to stabilize the cleavable complex formed by DNA and purified topoisomerase II. The aim of the present study was to estimate the impact of time exposure on the in vitro activity of S 16020. This was done on seven cancer cell lines of human origin (head and neck, kidney, and ovary). Doxorubicin was used as a reference drug. The cytotoxic activity of S 16020 remained stable during at least 3 h. A loss of activity of about 30% was apparent after 6 or 24 h preincubation. This relative loss of activity reached about 50% after 72 h preincubation. Considering all tested cell lines, the average IC50 decrease was 89+/-8% for S 16020 with incubation times between 1 and 72 h. An exposure index (El) was calculated to evaluate the effect of time on the cytotoxic efficacy. The reference time was 1 h exposure. The El values were corrected to take into account the loss of drug activity. For the majority of cell lines EI values were greater than 1 for both drugs, particularly after a 6 h exposure time. This means that, in this case as compared to the shorter exposure (1 h), increasing time has a relative detrimental effect on drug efficacy. For the two cancer cell lines of ovarian origin, El values remained close to 1 for both drugs whatever the total exposure time. This means that, in this case, time and concentration have symmetrical effects on cell survival. The pharmacological information provided by the present study may be useful in designing future clinical trials on this potentially interesting new topoisomerase II inhibitor. As a consequence of these data, 1 and 3 h drug administration schedules are currently tested during phase I trials.

Bisbenzylisoquinolines as modulators of chloroquine resistance in Plasmodium falciparum and multidrug resistance in tumor cells.

Ten naturally occurring bisbenzylisoquinolines (BBIQ) and two dihydro derivatives belonging to five BBIQ subgroups were evaluated in vitro for their ability to inhibit Plasmodium falciparum growth and, in drug combination, to reverse the resistance to chloroquine of strain FcB1. The same alkaloids were also assessed in vitro for their potentiating activity against vinblastine with the multidrug-resistant clone CCRF-CEM/VLB, established from lymphoblastic acute leukemia. Three of the BBIQ tested had 50% inhibitory concentrations of less than 1 microM. The most potent antimalarial agent was cocsoline (50% inhibitory concentration, 0.22 microM). Regarding the chloroquine-potentiating effect, fangchinoline exhibited the highest biological activity whereas the remaining compounds displayed either antagonistic or slight synergistic effects. Against the multidrug-resistant cancer cell line, fangchinoline was also by far the most active compound. Although there were clear differences between the activities of tested alkaloids, no relevant structure-activity relationship could be established. Nevertheless, fangchinoline appears to be a new biochemical tool able to help in the comprehension of the mechanism of both chloroquine resistance in P. falciparum and multidrug resistance in tumor cells.

In vitro activity of S 9788 on a multidrug-resistant leukemic cell line and on normal hematopoietic cells-reversal of multidrug resistance by sera from phase I-treated patients.

The triazinoaminopiperidine derivative S 9788 is a new multidrug-resistance modulator that is currently being evaluated in phase I clinical trials. In this study, the reversal effect of S 9788 in comparison with verapamil was shown in vitro in human T-leukemic CCRF-CEM/VLB cells expressing the multidrug-resistance (MDR) phenotype. S 9788 increased in a dose-dependent manner the cytotoxic activity of doxorubicin or vinblastine, with complete reversal of resistance occurring at 2 microM for a concomitant continuous exposure (96 h) to the cytotoxic drugs. At respective concentrations equivalent to the IC10 value (the concentration inhibiting 10% of cell growth), S 9788 was 44 times more potent than verapamil in CCRF-CEM/VLB cells. S 9788 at 2 microM did not enhance the in vitro toxicity of doxorubicin or vinblastine in the human normal bone-marrow erythroid (BFU-E) and myeloid (CFU-GM) progenitors. The effect of exposure duration and concentrations on the synergistic action of modulator and cytotoxic agent closely depended on the cytotoxic agent studied. Post-incubations with S 9788 alone after a 1-h coadministration with vinblastine and S 9788 dramatically increased the reversal effect (4-41 times) in proportion to both the duration of postincubation and the concentration of S 9788. In contrast, for doxorubicin resistance, post-incubation with S 9788 alone induced a maximal 2-fold increase in the reversal effect that was not proportional to the post-incubation duration. In patients treated with S 9788 as a 30-min intravenous infusion during phase I trials, a good correlation was found between the serum levels of S 9788 and the ability to reverse MDR in CCRF-CEM/VLB cells. The reversal effect was dose-dependent and was effective beginning at a plasma concentration of 0.25 microM. These data form a basis for the design of phase II trials using a combination of a loading dose of S 9788 given before vinblastine or doxorubicin administration followed by a maintenance infusion of S 9788 alone for a period of 2-24 h.

Inactivation of the p53 gene expression by a splice donor site mutation in a human T-cell leukemia cell line.

An abnormally sized 3.5 kb p53 transcript was detected in the KE-37R human leukemic T-cell line in which no p53 protein could be detected by immunoprecipitation. S1 nuclease protection experiments and sequencing analysis indicated conservation of the entire intron 4 (755 bp) in the 3.5 kb transcript and the presence of a G to A substitution in the last exonic nucleotide of the splice donor site. These data support the notion that p53 gene inactivation by point mutations in splice junctions also exists in hemopoietic neoplasia.

c-myc gene expression in a leukemic T-cell line bearing a t(8;14) (q24;q11) translocation.

We have examined the expression of the c-myc protooncogene in human T-cell leukemic KE-37R cells carrying a t(8;14) (q24;q11) translocation. The breakpoint on chromosome 8 is located at 2.2 kb downstream of c-myc exon 3 and the 3' part of the TcR-alpha gene (14q11) has been juxtaposed to c-myc. Our results showed that the steady-state levels of c-myc RNA transcripts were increased and the P1/P2 ratio of c-myc promoter utilization did not change, indicating that preferential utilization of P2 was maintained in the rearranged gene. High levels of electrophoretically normal p64 and 67 c-myc proteins were detected and both products kept their instability. In addition, transcription from promoter P0 was not detectable. Our results suggest that the activation of the gene is likely to result from its juxtaposition to the enhancer element of the TcR-alpha gene located downstream of the Ca region which stimulates constitutive synthesis of normal c-myc transcripts from the rearranged allele.

[Lack of expression of the protein p53 gene in a human T-cell leukemia line]. / Absence d'expression du gène de la protéine p53 dans une lignée humaine issue d'une leucémie de type T.

Expression of the gene encoding the nuclear phosphoprotein p53 (a proto-oncogene classified in the same functional family as c-myc and E1a adenovirus gene) was examined in a human T-cell leukemia (KE-37R cell line). No p53 (or a modified product) could be detected by immunoprecipitation with monoclonal antibodies P Ab 421 and P Ab 122 in KE-37R cell extracts, and no p53-specific RNA was characterized by Northern blot analysis. Southern blot using a murine p53 cDNA clone as a probe, did not reveal any gross rearrangement in the structure of the gene. However, this molecular probe was not suited for investigating the 5' end of the gene which contains the promoter and the non coding exon 1. It is interesting to notice that in KE-37R cells, c-myc has been activated by a t(8; 14) (q24; q11) translocation, suggesting that the c-myc product might substitute to some functions normally requiring p53.