Egorin M J, Snyder S W, Pan S S, Daly C
Division of Developmental Therapeutics, University of Maryland Cancer Center, Baltimore 21201.
Semin Oncol. 1990 Feb;17(1 Suppl 3):7-17.
Because the transport and accumulation of N,N',N''-triethylenethiophosphoramide (thiotepa) by cells has not been characterized, these processes were investigated with [14C]thiotepa and cultured L1210 or freshly obtained human or avian RBCs. The octanol: phosphate-buffered saline (PBS) partition coefficient of thiotepa was 2.4 +/- 0.1 (n = 8). With this value, the permeability coefficient (Ps) for thiotepa was estimated to be between 2.8 x 10(-4) and 1.81 x 10(-3) cm/sec, and the half-life of accumulation of thiotepa by L1210 cells was estimated to be 0.063 to 0.40 seconds. Thiotepa accumulation by cells was measured after incubation of cells with [14C]thiotepa and subsequent harvesting of cells by centrifugation through silicone fluid. Thiotepa accumulation by L1210 cells was biphasic. The initial phase was rapid and essentially complete by 10 seconds. The amount of cell-associated 14C increased linearly with increasing extracellular concentrations of thiotepa or with increasing size of the cell pellet. The absolute amount of cell-associated 14C was consistent with that expected if the [14C]thiotepa had been evenly distributed in the incubation medium and a volume equal to that of the cell pellet had been sampled and counted. This rapid phase of thiotepa accumulation was not slowed when cells were incubated on ice. The second phase of [14C]thiotepa accumulation occurred at a rate much slower than that of the initial phase. This slower phase of drug accumulation was linear for at least 5 hours. The rate of 14C accumulation increased progressively over a range of extracellular thiotepa concentrations from 5 to 100 nmol/mL and could not be saturated under acceptable tissue culture conditions. The slower rate of 14C accumulation was ablated by incubating cells on ice and was reduced by 30% to 50% in the presence of 1mM of sodium azide or 2,4-dinitrophenol. The slow rate of accumulation of 14C reflected summation of a relatively stable or constant amount of exchangeable 14C and an amount of nonexchangeable 14C that increased linearly from almost undetectable levels at the start of the experiment to amounts equal to 64 +/- 11% of total cellular radioactivity after 5 hours. The initial association of [14C]thiotepa with both human and avian RBCs was also very rapid. Avian RBCs also exhibited a slow rate of 14C accumulation that was linear for at least 5 hours but that was 15% to 20% that of L1210 cells.(ABSTRACT TRUNCATED AT 400 WORDS)
由于细胞对氮芥气（噻替派）的转运和积累尚未明确，因此利用[14C]噻替派以及培养的L1210细胞或新鲜获取的人或禽红细胞对这些过程进行了研究。噻替派在辛醇：磷酸盐缓冲盐水（PBS）中的分配系数为2.4±0.1（n = 8）。根据该值，噻替派的渗透系数（Ps）估计在2.8×10(-4)至1.81×10(-3) cm/秒之间，L1210细胞积累噻替派的半衰期估计为0.063至0.40秒。在用[14C]噻替派孵育细胞并随后通过硅油离心收获细胞后，测量细胞对噻替派的积累情况。L1210细胞对噻替派的积累呈双相性。初始阶段迅速，在10秒时基本完成。与细胞结合的14C量随细胞外噻替派浓度的增加或细胞沉淀大小的增加而呈线性增加。细胞结合的14C绝对量与预期一致，即如果[14C]噻替派在孵育培养基中均匀分布，并且对等于细胞沉淀体积的部分进行取样和计数的话。当细胞在冰上孵育时，噻替派积累的这个快速阶段并未减慢。[14C]噻替派积累的第二阶段发生速率比初始阶段慢得多。药物积累的这个较慢阶段至少在5小时内呈线性。在细胞外噻替派浓度从5至100 nmol/mL的范围内，14C积累速率逐渐增加，并且在可接受的组织培养条件下无法达到饱和。14C积累的较慢速率在细胞在冰上孵育时被消除，并且在存在1mM叠氮化钠或2,4 - 二硝基苯酚的情况下降低30%至50%。14C积累的缓慢速率反映了相对稳定或恒定数量的可交换14C与不可交换14C数量的总和，不可交换14C的数量从实验开始时几乎不可检测的水平线性增加，直至5小时后达到总细胞放射性的64±11%。[14C]噻替派与人及禽红细胞的初始结合也非常迅速。禽红细胞也表现出14C积累的缓慢速率，该速率至少在5小时内呈线性，但仅为L1210细胞的15%至20%。（摘要截断于400字）
