Pharmacological and clinical studies of new ways to improve cytostatic treatment of acute myelocytic leukemia : in vitro and in vivo studies

Abstract

Treatment failure in refractory or relapsed disease is often caused by resistance towards Ara-C or daunorubicin, the two drugs mainly used in the treatment of acute myeloid leukemia (AML). This thesis comprises studies of resistance mechanisms regarding Ara-C and the newer nucleoside analogues CdA and fludarabine, improvement of treatment outcome after enhancement of tumour AraC susceptibility by concomitant treatment with GM-CSF and the effect of liposomal formulation on leukemic cell uptake of daunorubicin. The main objective was to find ways to improve the cytostatic treatment of AML.

First, different anthracycline resistant cell lines were evaluated for resistance towards Ara-C and possible resistance mechanisms. In a HL 60 doxorubicin resistant cell line, expressing high levels of MDR-1 gene and protein we found cross-resi stance to Ara-C, reduced dCK activity, decreased 5´NT activity and only half the uptake and retention of Ara-C compared to wild type cells indicating higher efflux of the drug in resistant cells, probably via the P-gp. This was confirmed, as the sensitivity to Ara-C was increased in combination with reversing agents CsA and PSC, measured by cytotoxicity, DNA fragmentation and caspase-3-like activity. Likewise, in K 562 cell lines with acquired resistance to daunorubicin and vincristine cross-resistance to Ara-C was found. These cells expressed P-gp but not MRP1-6. Decreased dCK (20 - 45%) activity was confirmed, reductions in dCK activity and protein levels in concordance with a decrease in dCK mRNA.

In contrast to the HL 60 cell line elevated 5'NT activity resulting in several fold increased 5'NT/dCK enzyme ratios in resistant cells was found. Next, leukemic cells from 170 AML patients were evaluated for in vitro-activity and cross-resistance patterns of the purine analogues cladribine and fludarabine, and Ara-C using the ATP-assay. After incubation with clinically relevant concentrations about 25% of the samples were highly sensitive for Ara-C or CdA/fludarabine. CdA was significantly more active than Ara-C (p<0.05) but not more than fludarabine. The cytotoxicity of CdA correlated strongly to fludarabine (r=0.82,p<0.0001) but less toward Ara-C (r=0.49,p=0.002). A significant number of Ara-C resistant cells were sensitive to CdA/fludarabine.

To study if addition of growth factor increases the effect of Ara-C, in a phase III randomised study, patients aged > 64 years with newly diagnosed AML received a 3-day MEA protocol + the addition of prior and concomitant GM-CSF. This regimen induced CR in 64.5%, with no difference between the treatment arms. With GM-CSF remission duration was shorter (6 vs. 13 months, ns) as was the median OS (9 vs. 14 months, ns). The median time to neutrophil recovery was significantly shorter in the GMCSF treatment arm (17 vs. 25 d, p=0.03) as was the number of septic episodes. Liposomal daunorubicin, DaunoXome, was compared to conventional daunorubicin with respect to plasma and intracellular pharmacokinetics.

Following DaunoXome the peak values and plasma AUC were more than 100 times higher than after administration of free daunorubicin (AUC; 176.16 vs. 0.98µM x hour) but the intracellular AUCs were comparable (759.5 vs. 715.03µM x hour). Intracellular concentrations after DaunoXome peaked later and half as high as after daunorubicin. After DaunoXome or daunorubicin plasma clearance was 0.001 or 0.4 µmol/h respectively. The volume of distribution was 5.5 1, for DaunoXome vs. 3640 L for daunorubicin indicating low tissue affinity for the liposomal formulation.

In conclusion cross-resi stance to Ara-C is found in cell lines with acquired resistance towards anthracyclines, expressing high levels of P-gp. This is in part explained by increased 5'NT/dCK enzyme ratios in the resistant cells but the addition of reversing agents restores Ara-C sensitivity, indicating efflux via P-gp as a separate mechanism of resistance. In leukemic cells from patients in vitro sensitivity to CdA or Fludarabine correlates strongly to Ara-C, however as this correlation is not complete, CdA and fludarabine can play a role as alternatives or in combination with Ara-C Addition of GM-CSF to anti leukemic therapy shortens ANC recovery and reduces septicaemia but does not enhance treatment outcome compared to chemotherapy alone. Liposomal daunorubicin, DaunoXome, gives two-log higher plasma but similar intracellular concentrations of daunorubicin and active metabolite daunorubicinol compared to administration of free daunorubicin indicating comparable antileukemic effects.