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Cellular Transport and Accumulation of Thiotepa in Murine, Human, and Avian Cells

Division of Developmental Therapeutics [M. J. E., S. W. S., S. S. P, C. D.], University of Maryland Cancer Center, and Division of Medical Oncology, Department of Medicine, University of Maryland School of Medicine [M. J. E.], Baltimore, Maryland 21201

Because the transport and accumulation of thiotepa by cells has not been characterized, these processes were investigated with [¹⁴C]thiotepa and cultured L1210 or freshly obtained human or avian RBC. The octanol:phosphate buffered saline partition coefficient of thiotepa was 2.4 ± 0.1 (n = 8). With this value, the permeability coefficient (P_s) for thiotepa was estimated to be between 2.8 x 10^-4 and 1.81 x 10^-3 cm/s and the half-life of accumulation of thiotepa by L1210 cells was estimated to be 0.063–0.40 s. 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 s. 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 h. The rate of ¹⁴C accumulation increased progressively over a range of extracellular thiotepa concentrations between 5 and 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 30–50% in the presence of 1 mM 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 which increased linearly from almost undetectable levels at the start of the experiment to amounts approximately equal to those of exchangeable radioactivity after 5 h. 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 which was linear for at least 5 h but which was 15–20% that of L1210 cells. Human RBCs did not exhibit a slower rate of ¹⁴C accumulation and essentially all of the ¹⁴C associated with human RBCs was exchangeable for the 5 h duration of the experiment. All of the ¹⁴C associated with cells after 10 s or 5 h was accounted for by two radioactive spots on thin layer chromatography. One of these had an R_f value identical to that of thiotepa and the other remained at the origin. There was no radioactivity in the area of the thin-layer chromatography plate corresponding to tepa. The radioactivity at the origin accounted for a greater proportion of the total radioactivity associated with L1210 cells incubated for 5 h with [¹⁴C]thiotepa than it did in L1210 cells incubated with radiolabelled drug for a few seconds.

¹ To whom requests for reprints should be addressed at Division of Developmental Therapeutics, University of Maryland Cancer Center, 655 West Baltimore Street, Baltimore, MD 21201.

Received 2/ 3/89. Revised 6/29/89. Accepted 7/13/89.

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