[CANCER RESEARCH 47, 3911-3919, August 1, 1987] Review Role of Thymidine in Biochemical Modulation: A Review Peter J. O'Dwyer,1 Susan A. King, Daniel F. Hoth, and Brian 1,e>land-Jones Investigational Drug Branch, Cancer Therapy Evaluation Program, Division of Cancer Treatment, National Cancer Institute, Bethesda, Maryland 20892 {P. O., S. A. K., D. F. H., B. L-J.J, and The Fox Chase Cancer Center, Philadelphia, Pennsylvania 19111 [P. OJ Abstract effects on the pathways of precursor synthesis (9), and the effects of their perturbation on DNA integrity and replication The role of thymidine in the treatment of cancer has been under clinical investigation for nearly a decade. C'Unirai trials have demonstrated that (10-13). Administration of exogenous dThd expands the intra- cellular dTTP pool through the salvage pathway of deoxynu- it lacks antitumor activity in its own right. In this review, the mechanism of action and rationale for the use of thymidine as a biochemical modu cleotide biosynthesis (14). The major effect of elevated dTTP lator of standard agents such as 5-fluorouracil, l-/3-r>arabinofuranosyl- levels is believed to be inhibition of ribonucleotide reducíase cytosine, and methotrexate are summarized. With this background, the (IS). Cytotoxicity has been associated with the consequent clinical trials which have been conducted with thymidine, either alone or depletion of dCTP (16, 17); in most cell lines the cytotoxicity in combination, are described. We suggest a number of further studies of can be reversed by replenishing dCTP pools (18,19). Addition the role of thymidine in the biochemical modulation of antimetabolites. ally, elevated levels of ribonucleotide reductase have been as sociated with resistance to dThd-induced cytotoxicity (20). Introduction However, Akman et al. (21) have shown that dCTP administra tion does not universally reverse the effects of dThd, which The naturally occurring nucleoside thymidine has been used suggests that this effect alone may not explain the cytotoxicity for decades to synchronize cells growing in vitro in S-phase (1). to all cell lines. Observations of enhanced sensitivity to DNA- It was introduced into clinical trials in 1978 based on evidence damaging agents (22, 23) and enhanced mutagenesis (24) fol that prolonged exposure to dThd2was cytotoxic to cells in vitro, lowing elevation of dTTP pools support an additional action and that its administration to nude mice bearing human tumor on preformed DNA or its normal repair. xenografts yielded tumor regressions (2, 3). Numerous preclin- The regulation by dTTP of key enzymes of the pyrimidine- ical studies have shown that dThd can modulate the activity of and purine-synthetic pathways, while not primarily responsible active cytotoxic drugs in a synergistic fashion in vitro and in for cytotoxicity, has important implications for the therapeutic vivo. The major goal of clinical studies with dThd has been to use of dThd in combination regimens with other antimetabo replicate this syngergistic interaction in human cancer. Many lites. Alterations in enzyme activity may also play a role in of these studies have incorporated detailed biochemical analysis resistance to the effects of dThd. High dTTP levelsinhibit dThd of the modulation by dThd of antimetabolites such as FUra, kinase and deoxycytidylate deaminase directly and activate ara-C, and MTX. In most cases, however, it is not clear that deoxycytidine kinase (25-27). The ability to maintain high the design of the regimen used in humans replicates that which levels of dTTP depends on the phosphorylases responsible for yields maximal synergy in animals, and no combination has the degradation of dTTP and dThd; differential sensitivity of been subjected to a controlled clinical trial to demonstrate the malignant T-lymphocytes and normal B-lymphocytes has been efficacyof the modulation. dThd has been the subject of several ascribed to differences in phosphorylase activity (28). Hence, excellent reviews (4-6). In this paper, we review the rationale while the major effects of exogenous dThd appear to be exerted for the development of regimens which incorporate dThd in at the level of ribonucleotide reductase, actions at other sites clinical trials, evaluate them from the perspective of their bio (to be described) may determine its synergistic interaction with chemical and pharmacological end points, and indicate the antimetabolites such as ara-C, S-azacytidine, and FUra. clinical studies needed to define the role of dThd used in this A further effect of dThd which may be of relevance to setting. antimetabolite combination is the cytokinetic synchronization of cells in S phase (29,30). From the studies of Kufe and Major Mechanism of Action (31), using ara-C alone, and Ross et al. (32), using deoxygua- nosine as a modulator of ara-C, it would seem that biochemical Deoxynucleoside triphosphate pools in mammalian cells are rather than cytokinetic effects correlate better with cytotoxicity. less than one-tenth the size of corresponding ribonucleoside However, the impetus for the evaluation of dThd as an antitu- triphosphate pools and are sufficient to supply the DNA repli mor agent was provided by its known cytokinetic actions. Lee cation fork with precursors for only 30 s to 5 min (7). Their et al. (2) first demonstrated that continued exposure of cells to importance in the control of DNA synthesis is evidenced by dThd for periods greater than their doubling time resulted in their tight control relative to the cell cycle (8), their regulatory cell death. They later showed that dThd is cytotoxic to human Received 4/3/86; revised 2/26/87; accepted 4/16/87. tumor xenografts in mice, with apparent selectivity for tumor The costs of publication of this article were defrayed in part by the payment over normal cells (3). Negative results were obtained in LI210, of page charges. This article must therefore be hereby marked advertisement in Lewis lung, colon 26, and colon 38, although dThd doses were accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1To whom requests for reprints should be addressed, at Department of Medical lower than in previous studies (33); however, malignant lym Oncology, Fox Chase Cancer Center, Centra] and Shelmire Avenues, Philadel phocytes appeared to be markedly sensitive (28). These data phia, PA 19111. 2The abbreviations used are; dThd, thymidine; FUra, S-fluorouracil; ara-C, 1- justified initial clinical testing of dThd as an antitumor drug. 0-D-arabinofuranosylcytosine; MTX, methotrexate; CSF, cerebrospinal fluid; Is. Plasma levels of dThd in normal subjects and solid tumor thymidylate synthetase; AMI. acute nonlymphocytic leukemia; I ill Ml'. 5- patients exhibit marked variation over a range from <4 x 10~8 fluorodeoxyuridine 5'-monophosphate; FdUrd, 5-fluorodeoxyuridine; FUTP, 5- to 8.7 x IO"7 M (34). The level required for cytotoxicity is IInon inrid¡notriphosphate; PALA, JV-phosphonacetyl-L-aspartate; ara-CTP, 1-/3- D-arabinofuranosylcytosine triphosphate. several orders of magnitude higher although this varies with 3911 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. ROLE OF THYMIDINE IN BIOCHEMICAL MODULATION the cell line studied, from as low as 6.2 x 10~5 M for LSI78Y of magnitude increase in plasma levels for a single order of lymphoblasts (35) to 1 x 10~2 M for HL-60 (21). In general, magnitude dose increase is accounted for by the prolongation millimolar levels are needed. Such levels expand dTTP pools of the half-life to about 100 min (47-49). Renal clearance at up to 4000% of control (18) but reduce dCTP only to 40 to these levels represents some 50% of total plasma clearance, 60% of control (18, 36). Although this modest depression of which is reduced to 95-266 ml/min/m2 (47, 48). These data dCTP levels has been thought to be insufficient to support a reflect saturation of the metabolic processes which catabolize cytotoxic mechanism involving ribonucleotide reducíase(36), dThd efficiently at low doses and are consistent with the earlier reversal studies show that low levels («3/¿M)ofdeoxycytidine data of Ensminger and Frei (52) in humans which indicated partially reverse dThd-induced growth inhibition without in that hepatic extraction of dThd was saturable at doses between creasing dCTP pools, while higher levels (>8 ^M) restore both 16 and 32 g/m2/day and those of Covey and Straw (44) in the dCTP pools and normal growth in parallel (18). Endogenous dog. However, calculations based on liver plasma flow indicate deoxycytidine may also prevent the expression of dThd cyto- that the liver cannot be the major site of dThd metabolism; the toxicity /// rmi against 11210 cells against which it is active in presence of dThd phosphorylase activity in plasma, kidney, vitro (37). Zaharko and Covey (38) have shown that the effects spleen, and intestinal mucosa suggests that dThd is widely of dThd on deoxycytidine metabolism are dependent on the catabolized in the body (44, 47). The major product of this model used; a selective advantage for tumor over normal tissue process, thymine, is present at plasma levels which are of the effects of dThd could not be demonstrated in the mouse. How order of those of dThd but which decay with a half-life over 3- ever, endogenous deoxycytidine levels, which may be as high as fold longer (47). The renal contribution to thymine clearance 2 x IO"5 M in rodents (39), are usually less than 1 x 10~6 M in is some 13-18% of total body clearance (47). humans (39, 40). Therefore, humans may be more susceptible Renal handling of dThd also varies in a dose-dependent to its cytotoxic effects. manner. At micromolar levels, urinary clearance of dThd ex On the basis of its activity against the human tumor xeno- ceeds creatinine clearance by a factor of 4 (42), while at milli grafts (3), dThd was introduced into clinical trials by the Na molar levels this factor is reduced to 1.65 (47).