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Purine and pyrimidine ddNs¹ play a major role in the treatment of HIV and other viral infections. These compounds must be phosphorylated intracellularly, most commonly to their ddNTPs, to exert their antiviral effects. With few exceptions, however, the ddNTP levels attained in human cells during clinical studies are too low for accurate measurement. As a consequence, studies of intracellular drug levels are usually conducted in model systems (e.g. virus-susceptible human cell lines adapted for rapid growth in tissue culture) and then extrapolated to the clinical situation. In such in vitro systems, radiolabeled drugs can be used, greatly simplifying the chromatographic quantitation of active metabolites, and cell sampling can be conducted under more rigidly controlled conditions than feasible in a clinical study. The parameters of greatest practical interest derived from such in vitro studies are the concentrations attained by ddNTPs in host cells and the duration of ddNTP persistence over time. Such information can be of major importance in establishing rational clinical dosage schedules (1-3).

Anti-HIV ddNs, unlike nucleoside analogs used in cancer chemotherapy, typically are active at levels which, at least over short periods of drug exposure, are not cytostatic or cytotoxic. Because of this absence of toxicity, an unusual situation frequently occurs, i.e. host cell function, including cell replication, continues at normal rates in vitro; consequently, conventional pharmacokinetic parameters (e.g. half-times for intracellular nucleotide accumulation and decay) require correction for the continuous dilution resulting from cell replication proceeding at the same rate as for unperturbed cells. In this short communication, we present a simple general method for determining the corrected t_1/2 values for the decay of ddNTPs and apply it to the adenosine-based agents ddATP and F-ddATP.

Materials and Methods. Chemicals. ddI and F-ddA were provided by the Pharmaceutical Resources Branch, National Cancer Institute. The radiolabeled compounds, [2,3-³H]ddI (30 Ci/mmol) and [5-³H]F-ddA (10 Ci/mmol), were obtained from Moravek Biochemicals (Brea, CA). Radiopurity (97%) was verified by HPLC before use.

Cells. MOLT-4 cells were obtained from the American Type Culture Collection (Rockville, MD) and were grown in RPMI 1640 tissue culture medium supplemented with 10% heat-inactivated (56°C for 30 min) fetal bovine serum, 45 µg/ml gentamycin, and 4 mM L-glutamine at 37°C in a humidified atmosphere of 95% air/5% CO₂. Cells were verified to be in logarithmic growth at the time of use (a 30-hr doubling time at an initial cell density of <1.5 × 10⁶ cells/ml).

Metabolism Studies. Ten-milliliter aliquots of cell suspensions were incubated with a 10 µM concentration of either [³H]ddI or [³H]F-ddA (5 µCi/ml) for 12 hr. Cells were then washed and resuspended in drug-free medium, and aliquots removed over the next 60 hr for determination of intracellular concentrations of ddATP or F-ddATP, using ion-exchange Partisil 10-SAX HPLC chromatography as previously described (4). During the course of the study, cells were maintained in logarithmic growth (i.e. <1.5 × 10⁶ cells/ml) either by diluting the cultures with additional fresh medium or by removing the cells by centrifugation and resuspending the cell pellet in an appropriate volume of fresh medium.

Theoretical. Assuming the simplest case (i.e. cells in exponential growth and a total amount, M, of intracellular ddNTP decaying by a first-order process with a rate constant k), then

(1)

But, typically, the concentration, C, of ddNTP, is the actual measured variable (e.g. pmol/10⁶ cells). This concentration decreases with an experimentally determined rate constant , both because of the loss of total ddNTP and because of ddNTP dilution by the simultaneous increase in total cell number, N. If we assume that the cells are in exponential growth with a rate constant , and note that M = NC, then

(2)

Since dC/dt = C and dN/dt = N, a simple result is obtained that allows correction of measured ddNTP decay for cell growth, i.e.

(3)

(For an alternate derivation of eq. 3, see the Appendix.) Furthermore, if we define t_m, the experimentally measured half-time, as ln2/, and the experimentally determined cell doubling time, t_D, as ln2/, then

(4)

Results and Discussion. Two features of practical interest arise from the formulation developed herein. The first of these is that the correction is greater for agents with slower intracellular clearance rates than for agents with more rapid rates. Thus, for F-ddATP (fig. 1), t_m = 19 hr; t_D for MOLT-4 cells = 30 hr. Consequently, 1/t_1/2 = 1/19  1/30, so that t_1/2 = 52 hr, a 174% increase over the measured value. However, for ddGTP, previously studied in this laboratory under the same conditions and in the same cell line (table 1), t_m = 4.2 hr, giving t_1/2 = 4.9 hr, a difference of only 17% from the experimentally determined value.

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Intracellular levels of ddATP and F-ddATP after incubation with ddI and F-ddA. MOLT-4 cells were treated with ddI or F-ddA (10 µM; 5 µCi/ml) for 12 hr, as described more fully in Materials and Methods. Experiments shown were conducted independently twice, with little variation in results. Results shown are the average of dulplicate values from a single experiment. The measured half-times (i.e. tm values) were calculated from the slopes of the weighted lines generated by linear regression analysis.

View larger version (15K): [in this window] [in a new window]   Fig. 2.   Relationship between measured half-times (tm values) and actual t1/2 values in cell lines with replication times (tD values) ranging from 24 to 60 hr. Curves were generated from the equation 1/t1/2 = 1/tm  1/tD.