Phase II clinical trial of fludarabine in chronic lymphocytic leukemia on a weekly low-dose schedule.

The major complication during therapy of chronic lymphocytic leukemia (CLL) with the purine nucleotide analogue fludarabine is infection, which is also the main cause of morbidity and mortality in the disease. As the incidence of infectious episodes during therapy correlated with severity of neutropenia, stage of disease, and response to therapy, an effort was made to reduce therapy-related myelosuppression and improve response by altering the conventional therapy regimen. The protocol which yielded a response rate of 57% in previously treated patients with CLL consisted of five consecutive daily doses of 25-30 mg/m2 fludarabine given every three to four weeks. Based on observations from intracellular pharmacology studies it was hypothesized that repetitive single weekly doses of fludarabine would allow normal bone marrow cells to recover while maintaining cytotoxic levels in the leukemic cells. The cumulative four-week dose of the once-weekly regimen was approximately 80% of the original protocol. Eleven out of 46 evaluable patients (24%) responded to the therapy. Seven patients (15%) achieved a complete remission, and four (9%) a partial remission. While myelosuppression was reduced by about 30% compared with the original protocol, the incidence of febrile episodes was increased by 17%. Pretreatment serum IgG levels below the normal range correlated significantly with a high incidence of infectious episodes and with a short median survival time. These observations suggest that in addition to myelosuppressive therapy, disease related depressed immune function causes morbidity and mortality due to infections. The results further show that changes in the scheduling of the therapy regimen, associated with a slightly lower dose, resulted in reduced efficacy as measured by the response rate.

Cellular pharmacology of fludarabine triphosphate in chronic lymphocytic leukemia cells during fludarabine therapy.

The pharmacology of fludarabine triphosphate (F-ara-ATP) in leukemic lymphocytes was studied during a phase II trial of fludarabine in 24 patients with chronic lymphocytic leukemia (CLL). Fludarabine was given as a 30-min i.v. infusion at a dose of 25 or 30 mg/m2 daily for 5 days. The concentrations of F-ara-ATP, the active metabolite of fludarabine, were determined in leukemic lymphocytes at intervals up to 24 hr after the first infusion. A median peak concentration of 19 microM (range, 6-52 microM) was generally reached 4 hr after the beginning of the infusion. No significant relationship was observed between clinical response and the median peak level of F-ara-ATP or the retention of F-ara-ATP in leukemic lymphocytes. In vitro incubation of CLL cells with the parent nucleoside of fludarabine, arabinosyl-2-fluoroadenine (F-ara-A), indicated that F-ara-ATP accumulated in a linear fashion in response to the product of the F-ara-A concentration times the duration of incubation. Exposing cells longer with lower F-ara-A concentrations or shorter with higher F-ara-A concentrations resulted in similar intracellular levels of F-ara-ATP as long as the products of fludarabine concentration and time of exposure were equal. These results and the fact that the fludarabine dose rate currently administered is well tolerated suggest that it may be the optimal dose rate for F-ara-ATP accumulation in CLL cells.

Fludarabine infusion potentiates arabinosylcytosine metabolism in lymphocytes of patients with chronic lymphocytic leukemia.

Our previous work has shown that incubation of K562 cells or lymphocytes from patients with advanced chronic lymphocytic leukemia (CLL) with arabinosyl-2-fluoroadenine (F-ara-A) potentiates the rate of arabinosylcytosine 5'-triphosphate (ara-CTP) synthesis during subsequent treatment with arabinosylcytosine (ara-C). To test the biochemical modulation of ara-CTP in a clinical setting, we designed a protocol to administer fludarabine (Fludara, F-ara-AMP) and ara-C in a pharmacologically directed sequence for patients with CLL refractory to conventional fludarabine therapy. ara-C was infused in seven patients with progressive CLL at a dose rate that maximizes ara-CTP accumulation (0.5 g/m2 during 2 h). Fludarabine (30 mg/m2 during 30 min) was infused 20 h later, followed by a second, identical dose of ara-C at 24 h, when the concentration of F-ara-A 5'-triphosphate (F-ara-ATP) was maximal in CLL cells. Comparison of ara-CTP pharmacokinetics in circulating CLL cells demonstrated that the ara-CTP area under the curve increased by a median of 1.5-fold (range, 1.1- to 1.7-fold) after fludarabine infusion. Plasma pharmacokinetics indicated that neither the median steady-state ara-C concentrations nor the levels of its deamination product arabinosyluracil were significantly affected by fludarabine infusion. The median rate of ara-CTP elimination was slightly faster after fludarabine treatment (t1/2, 6.7 versus 5.8 h), suggesting that catabolism of ara-CTP was not responsible for the increased ara-CTP area under the curve. The rate of ara-CTP accumulation by CLL cells after fludarabine infusion, however, was increased by a median of 1.3-fold in seven of the eight patients (range, 1.2- to 1.8-fold); the peak occurred within 1 h of the end of the infusion. In vitro incubation of leukemic lymphocytes with F-ara-A before ara-C also showed a median 1.3-fold increase in the rate of ara-CTP accumulation. Thus, infusion of fludarabine before ara-C augments ara-CTP metabolism in leukemic lymphocytes. This knowledge should be considered in the design of combination chemotherapy.

Inhibition of fludarabine metabolism by arabinosylcytosine during therapy.

The active 5'-triphosphate of arabinosyl-2-fluoroadenine (F-ara-ATP) increases the anabolism of arabinosylcytosine (ara-C), whereas ara-C 5'-triphosphate inhibits the phosphorylation of arabinosyl-2-fluoroadenine (F-ara-A) in human leukemia cells in vitro. These interactions have a potential impact on drug scheduling. Clinical trials of relapsed leukemia in which fludarabine (F-ara-A 5'-monophosphate) and ara-C were given in sequence provided the opportunity to evaluate the effects of ara-C infusion on two sequelae: the pharmacokinetics of F-ara-A in plasma and that of F-ara-ATP in leukemia cells. First, F-ara-A pharmacokinetics were altered by ara-C infusion. This was visualized as a transient increase in F-ara-A plasma levels during the ara-C infusion that was given 4 h after fludarabine. The perturbation in F-ara-A plasma levels was dependent on the dose ara-C. Second, peak F-ara-ATP concentrations were lower in leukemia cells of patients who received ara-C in addition to fludarabine as compared with those who received fludarabine alone. The terminal half-life of F-ara-A in plasma and the half-life of intracellular F-ara-ATP were reduced after the ara-C infusion in a concentration-dependent manner. Studies using purified deoxycytidine kinase support the conclusion that the increase in plasma levels of F-ara-A is in part the result of an effective competition by ara-C for phosphorylation by this enzyme, leading to a perturbation of the pharmacokinetics of intracellular F-ara-ATP.

A sensitive fluorescence assay for quantitation of fludarabine and metabolites in biological fluids.

Rapid and quantitative dephosphorylation of the new anticancer nucleotide analogue fludarabine phosphate to its nucleoside 9-beta-D-arabinofuranosyl-2-fluoroadenine (F-ara-A) renders this metabolite the target for pharmacologic investigations. At clinically effective doses of fludarabine phosphate (18-30 mg/m2 per day) comprehensive pharmacokinetic analysis of F-ara-A has been limited by the sensitivity of UV based HPLC assays. To address this problem we developed a sensitive test based on the condensation of F-ara-A with chloroacetaldehyde to form the fluorescent derivative, arabinosyl-1,N6-etheno-isoguanine. Combined with a solid-phase extraction step prior to derivatization and separation of the reaction products by reverse-phase HPLC, this assay had a quantitation limit of 2 pmol F-ara-A per ml plasma. Slightly modified, the system was also applicable to urine specimens, with a quantitation limit of 1 nmol F-ara-A per ml urine.

Investigations on metabolism and carcinogenicity of 1,1,2-trichloroethane.

Two groups of male and female Sprague-Dawley rats (50 animals/group per sex) were treated with either 15.37 or 46.77 mumole of 1,1,2-TCE in DMSO/rat for 2 years. The animals were treated once a week by s.c. injection of test compound in the skin of neck. Two groups of controls received either DMSO or no treatment at all. The incidence of benign mesenchymal and epithelial tumors was not significant when compared with either DMSO-treated or untreated controls. The animals treated with 46.77 mumole 1,1,2-TCE significantly developed sarcomas when compared with the untreated controls. In a further experiment, either 40 mumole or 160 mumole 1,1,2-TCE was injected into male Wistar rats and the metabolites, TdGA and HEMA, were determined in 24-h urine samples. Comparative studies were carried out giving equimolar amounts of chloroethanol and 2-chloroacetaldehyde diethyl acetal. Analysis of the metabolites showed that no detectable HEMA was excreted in urine after treatment of rats with 1,1,2-TCE or chloroethanol. TdGA was excreted in urine much more among chloroacetaldehyde-treated animals than among 1,1,2-TCE- or chloroethanol-treated rats.

Investigations on metabolism, genotoxic effects and carcinogenicity of 2,2'-dichlorodiethylether.

Either 40 mumole or 160 mumole 2,2'-DDE was injected into male Wistar rats and the metabolites, TdGA and HEMA, were determined in the 24-h urine specimens. Comparative investigations were carried out giving equimolar amounts of chloroethanol and 2-chloroacetaldehyde diethyl acetal. In a further step, inhalation experiments were performed to determine urinary excretion of the two metabolites after an 8-h exposure of male Wistar rats to 10, 50, 100, and 500 ppm 2,2'-DDE and to 50, 200, und 1,000 ppm vinyl chloride. A long-term study was conducted to investigate the possible carcinogenicity of 2,2'-DDE in male and female Sprague-Dawley rats following s.c. injections of 4.36 mumole and 13.1 mumole 2,2'-DDE in DMSO per week. The evaluation of tumor development in treated groups and controls were based on macroscopic inspection and histological examinations of the suspect organs and tissues. Analysis of the metabolites showed that HEMA excretion was much lower than the excretion of TdGA following the uptake of 2,2'-DDE, 2-chloroethanol and 2-chloroacetaldehyde diethyl acetal. Contrary to these, vinyl chloride uptake resulted in a higher urinary excretion of HEMA than TdGA. There was no appreciable increase in the number of tumors detected in 2,2'-DDE-treated animals when compared with untreated or DMSO-treated groups. Since irradiation of 2,2'-DDE with UV did not elevate mutagenic activity of the compound against Salmonella typhimurium TA100, the high mutagenicity of the compound found in a desiccator cannot be due to the liberation of mutagenic compounds produced under the influence of UV light.

The influence of 18 environmentally relevant polycyclic aromatic hydrocarbons and Clophen A50, as liver monooxygenase inducers, on the mutagenic activity of benz[a]anthracene in the Ames test.

In the presence of various liver S9 preparations induced by different polycyclic aromatic hydrocarbons, the weak carcinogen, benz[a]anthracene, exhibited mutagenic activities comparable to those of benzo[a]pyrene in the Ames test. A series of positive correlations were found between mutagenic activity and the hydroxylation products of benz[a]anthracene at the 3,4-, 5,6- and 8,9-positions, of which 5,6-position was the most pronounced one (p less than 0.01). Our results suggest that the observed mutagenic activity is due mainly to the generation of K-region epoxides.

Expression of the plant sulphite reductase in cell suspension cultures from Catharanthus roseus L.

Sulphur-heterotrophic growth exhibited a dual response to the expression of sulphate-assimilating enzymes. The level of ATP-sulphurylase (EC 2.7.7.4) appeared "repressed" while sulphite reductase (EC 1.8.7.1) and O-acetyl-L-serine sulphhydrylase (EC 4.2.99.8) were "derepressed" and coordinated in their occurrence. The capability of the cells to reduce adenylylphosphosulphate or 3'-phospho adenylylphosphosulphate to cysteine coincided with the activity of sulphite reductase. The expression of these reducing steps lacked correlation with the regulation of ATP-sulphurylase.

On the role of S: Sulfotransferases in assimilatory sulfate reduction by plant cell suspension cultures.

Cell suspension cultures of Catharanthus roseus (L.) G. Don were grown under S-auxotrophic (1.8 mM sulfate) and under S-heterotrophic (0.5 and 1.0 mM cysteine or methionine) conditions. The development of activity of the thiol sulfotransferases was followed during the complete growth period. Under auxotrophic growth, an NADPH-dependent S: sulfotransferase and a GSH-dependent S: sulfotransferase developed identically, whereas under heterotrophic growth, differences in the amount of enzymes and in the time course of their development occurred. The NADPH-dependent sulfotransferase appeared "repressed" by the S-amino acids but the GSH-dependent enzyme was "derepressed." In that phenomenon, the development of the GSH sulfotransferase paralleled the development of the ATP-sulfurylase (EC 2774) activity of the cells.