Ferrocenyl Nucleoside Analogs, a New Class of Cytostatic Drugs, Overcome Multiple Drug Resistance in Acute Lymphoplastic Leukemia Ex Vivo.

In spite of the relatively good prognosis for de novo acute lymphoplastic leukemia (ALL), the survival rate for relapsed ALL is only 40%. The therapy of relapsed ALL and secondary leukemias with the common cytostatic drugs often show a poor response. Therefore new substances are required to lead multiple drug resistant leukemia cells into cell death. Nucleoside analogs play a very important role in anti-leukemic therapy. Here we present a new, own-synthesized class of Ferrocenyl Nucleosides, a type of organometallic sandwich compounds, where two cyclopentadenyl rings bind to a central Fe²⁺ ion. We could show anti-leukemic and anti-cancer activity in different leukemia and tumor cell lines and primary cells from children with primary ALL and AML. The apoptosis induction was efficient in low micro molar concentrations (LC₅₀ = 10–20μM). The DNA fragmentation has been extraordinary high (LC₉₀ = 30–50μM). Ex vivo experiments showed that some Ferrocenyl Nucleoside Analogs, like PJ184, are able to overcome resistance against anthracyclines like Doxorubicin (p<0,001), Daunorubicin (p<0,001) and Idarubicin (p<0,001) or alkaloids like Vincristin (p<0,001). Furthermore Ferrocenyl Cytosin Analog (PJ184) mediated apoptosis induction is significant higher than apoptosis induced by the clinical used nucleoside analogs like Fludarabin (p<0,001), Cytarabine (p<0,001) and Cladribine (p<0,001; all p-values by ANOVA test). These results have been achieved by testing primary lymphoblasts of seven primary ALL patients after incubation for 60h with LC₅₀ determined in leukemic cell lines. The testing of one AML revealed similar results. Apoptosis induction has been investigated by flowcytometric measurement of DNA-fragmentation, mitochondrial membrane potential reduction and phosphatedylserin-staining on cell membrane surface. The cell death by necrosis could be excluded by a lactatdehydrogenase-release assay. In order to characterize the Ferrocenyl Nucleoside apoptosis pathway we used cellular model systems with deficiencies in different molecular parts of the apoptosis cascade. Apoptosis reveals to be independent of CD95 receptor and the pro-apoptotic factor SMAC, but shows a dependency of the apoptosis inhibitor Bcl-2. We also investigated a loss of mitochondrial membrane potential, which indicates the involvement of the mitochondrial apoptosis machinery. Taken together, these new class of Ferrocenyl Nucleoside Analogs revealed very promising in vitro and ex vivo results and may lead to a new approach in antileukemic therapy. In vivo experiments will be performed soon.