Cooper Todd, Ayres Mary, Nowak Billie, Gandhi Varsha
Department of Experimental Therapeutics, University of Texas M. D. Anderson Cancer Center, 1515 Holcombe Blvd, Houston, TX, 77030, USA.
Department of Pediatrics, University of Texas M. D. Anderson Cancer Center, Houston, TX, USA.
Cancer Chemother Pharmacol. 2005 Apr;55(4):361-368. doi: 10.1007/s00280-004-0906-y. Epub 2004 Oct 16.
Clofarabine has proven to be effective in the treatment of adult and pediatric acute myelogenous leukemia (AML). To investigate if clofarabine could be used with success in biochemical modulation strategies, we investigated the biochemical modulation of cytarabine triphosphate (ara-CTP) by clofarabine in a myeloid leukemia cell line and the effect of this combination on cytotoxicity.
K562 cells were incubated with clofarabine and ara-C either sequentially or simultaneously to evaluate the combination effect on their phosphorylated metabolites. Clonogenic assays were used to determine the cytotoxicity of each agent alone and in combination. Deoxynucleotide analysis was performed to assess the effect of clofarabine on dNTPs.
Clofarabine added either simultaneously or in sequence increased ara-CTP accumulation. The maximal modulation of ara-CTP accumulation occurred with 1 microM clofarabine. This level was achieved at the maximum tolerated dose for adult and pediatric patients with AML. With 10 microM ara-C alone, 86 microM ara-CTP had accumulated after 3 h. The optimal sequence for the drug combination, i.e., clofarabine followed 4 h later by ara-C, resulted in 248 microM ara-CTP at 3 h. Clofarabine accumulated maximally in the monophosphate form. Preincubation with ara-C did not affect the triphosphate form, but it lowered clofarabine monophosphate. Clofarabine resulted in the intracellular decrease of dATP and dGTP levels. Clonogenic assays revealed that the combination of clofarabine and ara-C produced synergistic killing of myeloid leukemia cells.
These findings demonstrate that combination of clofarabine followed by ara-C results in a biochemical modulation of ara-CTP and synergistic cell kill. These studies provide a compelling rationale for clinical trials using this combination regimen for adult and pediatric patients with AML.
氯法拉滨已被证明在治疗成人和儿童急性髓性白血病（AML）方面有效。为了研究氯法拉滨是否能成功用于生化调节策略，我们研究了氯法拉滨对髓系白血病细胞系中三磷酸阿糖胞苷（ara-CTP）的生化调节作用以及该联合用药对细胞毒性的影响。
将K562细胞与氯法拉滨和阿糖胞苷（ara-C）依次或同时孵育，以评估联合用药对其磷酸化代谢产物的影响。采用克隆形成试验来确定每种药物单独及联合使用时的细胞毒性。进行脱氧核苷酸分析以评估氯法拉滨对脱氧核苷酸三磷酸（dNTPs）的影响。
同时或依次添加氯法拉滨均可增加ara-CTP的积累。ara-CTP积累的最大调节作用出现在1 μM氯法拉滨时。这一水平在成人和儿童AML患者的最大耐受剂量时达到。单独使用10 μM ara-C时，3小时后积累了86 μM ara-CTP。药物联合的最佳顺序，即氯法拉滨4小时后再用ara-C，在3小时时产生了248 μM ara-CTP积累。氯法拉滨以单磷酸形式最大程度地积累。预先用ara-C孵育不影响三磷酸形式，但降低了氯法拉滨单磷酸。氯法拉滨导致细胞内dATP和dGTP水平降低。克隆形成试验表明，氯法拉滨和ara-C联合用药对髓系白血病细胞产生协同杀伤作用。
这些发现表明，氯法拉滨后用ara-C联合用药可对ara-CTP进行生化调节并产生协同细胞杀伤作用。这些研究为使用该联合方案治疗成人和儿童AML患者的临床试验提供了令人信服的理论依据。
