Synergistic action of Aplidin and cytosine arabinoside in vitro and in vivo against lymphoblastic leukemia

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Aplidin^R (plitidpesin; dehydrodidemnin B, C₅₇H₈₉N₇O₁₅) is a novel antitumor agent isolated from the Mediterranean tunicate (seasquirt) Aplidium albicans. Aplidin has shown impressive in vitro and in vivo activity against different human cancer cells and has recently entered Phase II clinical trials in a variety of solid tumors following promising toxicity and pharmacological properties seen in Phase I studies. Fatigue and muscular pain were the most prevalent toxicities at 5 mg/m2 iv 3 h every other week or 3.4 mg/m2/wk with little or no bone marrow toxicity. Aplidin inhibits protein synthesis via GTP-dependent elongation factors 1-alpha and ornithine decarboxylase (ODC) activity, induces rapid p53-independent apoptosis in vitro, cell cycle perturbation and alteration of gene expression at early times after treatment. Aplidin inhibits vascular endothelial growth factor (VEGF) secretion and vascular endothelial growth factor-receptor 1 (VEGF-R1/flt-1), preventing autocrine stimulation in the human lymphoid leukemic cell line MOLT-4 cells and in AML blasts. Lack of cross resistance with conventional agents against fresh pediatric and adult AML/ALL blasts except fludarabine and Gemcitabine makes it an attractive therapeutic choice. Characterization of gene expression profile is currently underway in an attempt to generate a molecular fingerprint of sensitivity/resistance to Aplidin that will be validated in phase II clinical studies. Based on in vitro antileukemic effect of Aplidin as well as early results of clinical trials, a systematic study of drug combinations with Aplidin, for use possible in hematologic malignancies was undertaken. Three cell lines viz. K562 (acute myeloid leukemia), CCRF-CEM (acute lymphocytic leukemia), and SKI-DLCL (diffuse large cell lymphoma) were used for combination studies. Cytarabine and Aplidin were found to be synergistic in combination with all 3 cell lines as assessed by the Chou-Talalay combination index analysis. Since cytarabine and Aplidin produced impressive synergistic cell kill in all three cell culture models, the combination was further tested in the CCRF-CEM ALL xenograft model in SCID mice. Aplidin (0.7 mg/Kg) potentiated the antitumoral effect of cytarabine (50mg/Kg) in vivo and resulted in greater than 50% reduction in tumor size as compared to the untreated group. T/C ratios indicated that the effect of the combination was maximal at day 5 but was still maintained on day 8 (T/C on day 3 = 0.614; day 5= 0.403 and day 8= 0.703). Initial Gene expression analysis on CEM-S and SKI-DLCL cells using the U133 GeneChip ( Human Genome, Affymetrix) suggests that Aplidin has multiple cellular targets including downregulating ribosomal 18S and 28S mRNA expression while upregulating TNF related death ligand-1 alpha mRNA, TGF-beta, p21 activated kinase 2 and Caspase 5. Further studies are being carried out to confirm these observations.