Review Role of Thymidine in Biochemical Modulation: a Review Peter J

[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 intracellular 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 reducÃase 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 deoxyguanosine 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 inridÂ¡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 reducÃase(36), dThd efficiently at low doses and are consistent with the earlier reversal studies show that low levels (Â«3/Â¿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). This suggests tional Cancer Institute in 1978. that tubular secretion of dThd occurs; at millimolar levels, the data are consistent with saturation of tubular secretion and decreased tubular reabsorption. Thymine, on the other hand, Pharmacology appears to undergo tubular reabsorption (47). These data sug The pharmacology of dThd has been studied in animals and gest that the use of dThd in patients with impaired renal humans (41-51). From the antitumor activity studies discussed function would require dose and schedule modification based above, it is clear that the most important determinant for on blood level monitoring during the course of treatment. cytotoxicity is prolonged exposure of cells to millimolar levels CSF penetration of dThd occurs in the monkey and in of dThd, some 4 log orders higher than normal plasma levels. humans. A steady state CSF level one-tenth that of plasma was Following i.v. administration, dThd exhibits dose-dependent found in the monkey (43), while the ratio for man was 0.29 pharmacokinetics (Table 1). Daily doses of dThd 5*8g/m2 are (47). CSF levels of thymine were equivalent to plasma levels at cleared rapidly from the plasma of rodents and humans by first steady state (47). The ability of dThd to cross the blood-brain order kinetics, with a half-life of about 10 min (41-43, 45, 49). barrier may explain some of its clinical toxicity. At these doses, peak plasma dThd levels are 1-10 /Â¿M(45,51) and clearance, which has been calculated to be of the order of 24 liters/min (43), is predominantly nonrenal. Renal clearance Phase I-II Studies accounts for only 2% of the administered dose (45). The greater part is metabolized, in the peripheral circulation and in the Phase I studies of dThd, with doses escalating to toxicity, liver, via dThd phosphorylase to thymine. At low doses (3 g/ were begun in 1978 and are summarized in Table 2. Doses m ') plasma thymine levels in humans are less than those of ranging from 34 to 230 g/m2 were administered by continuous dThd, and they decay with a half-life of 32 min (49). Similar infusion for 2 to 29 days with moderate toxicity, which affected pharmacokinetic behavior was observed in the dog (44). At principally bone marrow (anemia, leukopenia, thrombocyto- higher doses in both species, however (7.5 g/m2 in humans), penia), the central nervous system (somnolence, headache, vis sustained high levels of thymine are observed; the terminal half- ual disturbances), and gastrointestinal tract (nausea, vomiting, life of thymine increases to 308 min and provides evidence of a diarrhea) (48, 43). In several patients with leukemia, the cyto- saturable metabolic process (44, 49). reduction observed prompted extension of the duration of in The pharmacokinetic behavior of dThd at high doses is fusion. Toxicity was not increased in patients receiving 75 g/ considerably different. Doses on the order of 75 g/m2/day yield nr/ilay as the duration of infusion was expanded to 14 days steady state plasma levels in the millimolar range. This .Vorder (48). Hence, dThd is well tolerated as a single agent at levels

Table 1 Pharmacokinetics oflhymidine in animals and humans half- SpeciesRatDog life (min)KD."8 drug (41) HPLC 1.8 mol/kg/min 2-80 ml/min/kg Covey and Straw (44) Human Radioimmunoassay 8g/m2/day75 8-10 45 Ensminger and Frei (45) Human Radiolabeled drug 20-25 Rubini et al. (46) Human HPLC g/m2/day 100 45.5 95-266 ml/min/m2 Zaharko et al. (47) Human HPLC 75 g/m2/day 80 Kufe et al. (48) Human' HPLC 3-45 g/m2/day 7-98CL7-67 Woodcock et al. (49) Human*MethodRadiolabeledHPLCDose0.396-1 23 g/m2Terminal 10-325 liters/kg/dayRef.Beltzrta/.Au et al. (50) " 'n... volume of distribution at steady state; CL, plasma clearance; HPLC, high pressure liquid chromatography. * Studies conducted with concurrent 5-fluorouracil administration. 3912 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. ROLE OF THYMIDINE IN BIOCHEMICAL MODULATION Table 2 Phase l-ll studies of single-agent thymidine Dose No. of Study Schedule patients Toxicity Comment Chiuten et al. (53) 75 d x 5-10Â° 12 Hematological Minor responses in 1 melanoma, 2 Gastrointestinal ALL, 1 ANLL Central nervous system

Kufe et al. (48) 75 d x 2-14 11 Hematological Peripheral cytoreduction in 5 patients Gastrointestinal ( I also marrow) with leukemia Central nervous system

Kufe et al. (54) 75 d x 4-7 3 Hematological Mycosis fungoides patients: 2 with Gastrointestinal partial clearing of plaques and 1 Central nervous system with minimal response

Leyva et al. (55) 34-75 d x 3-5 10 Hematological Gastrointestinal Central nervous system

Blumenreich et al. (56) 90-240 d x 14-29 6 Hematological Cytoreduction in 5 patients with leu Gastrointestinal kemia or lymphoma including 1 Central nervous system complete remission in ALL Hepatic ' d x, daily for following range of days; ALL, acute lymphocytic leukemia. which in preclinical models both in vitro and in vivo produce FUra incorporation into RNA. The most striking effect is on modulating effects. the pharmacokinetic behavior of FUra. When dThd is admin Investigators at Memorial Sloan-Kettering performed a istered shortly before or concurrently with FUra in humans, Phase II study in leukemia. At the upper dose levels (90-240 the half-life of FUra is increased from a mean of 6-38 min to g/m2) a single patient with acute lymphocytic leukemia achieved 76-190 min (49, 51) and plasma clearance reduced from 389 a complete remission (56). The high doses required make to 56 liters/kg/day (50). Steady state levels of FUra (7.5 mg/ treatment with d I lui as a single agent impractical because of kg/day by infusion) in the presence of 1 to 10 /Â¿MdThdincreased the inordinate fluid loads involved. From this and the Phase I from 0.38 to 1.30 /Â¿M(50).Studies in dogs showed equivalent studies, a total of 29 patients with acute leukemia (19 of whom changes in plasma elimination and demonstrated that at higher had ANLL) were treated with doses of dThd in excess of 40 g/ levels, FUra elimination was nonlinear, confirming the predic m2 with but a single complete remission (48, 53-56). These tions of Collins et al. (75). The fall in plasma clearance could results, albeit in a heavily pretreated population, led to the be totally accounted for by inhibition of FUra catabolism (44). abandonment of studies with dThd as a single agent, and the This supports the data of Woodcock et al. (49) which indicate focus shifted to its use as a modulator of the activity of other that dThd administration suppresses almost completely the active cytotoxic drugs. production of labeled CO2 from [2-'4C]-5-fluorouracil. The increased half-life of FUra is most closely related to Thymidine as a Modulator of 5-Fluorouracil levels of thymine, which is believed to inhibit competitively dihydrouracil dehydrogenase (44, 50). The K, for thymine as The mechanisms by which FUra exerts its cytotoxic effects derived from plasma levels in the dog was 17.4 Â±7.81 JÂ¿M(44). include: (a) incorporation into RNA, which leads to impaired Since available data indicate that the human and dog enzymes processing, particularly of preribosomal RNA (57-60); (A) in are not substantially different for this interaction, dThd doses of the order of 8 g/m2/day may suffice to produce a near- hibition of TS, following metabolism to FdUMP (61, 62); and (c) incorporation of FUra residues into DNA (63, 64) which maximal pharmacokinetic interaction. may contribute to cytotoxicity by the loss of DNA integrity Other factors may be important, however, to a true enhance which follows their excision (65, 66). The relative importance ment of the therapeutic index of FUra. The metabolism of of each of these mechanisms is unknown and may differ from dThd to thymine by dThd phosphorylase generates deoxyribose one cell line to another (67). 1-phosphate which in turn may be utilized by the same enzyme Umeda and Heidelberger (68) found that dThd reverses to convert FUra to FdUrd (67), elevated levels of which have FUra-induced growth inhibition of Novikoff hepatoma cells, been described by Au et al. (50). Elevation of intracellular levels which was interpreted as supporting a primary role for TS of FdUrd have been demonstrated; however, conversion of inhibition. Subsequent studies confirmed that in some cell lines FdUrd to FdUMP is inhibited by high levels of dTTP (76, 77), in vitro and some tissues in vivo TS inhibition correlates with which also inhibits ribonucleotide reducÃase,the other major FUra-induced cytotoxicity (67, 69, 70). Others have found that pathway to FdUMP (67). It is assumed, but not established in FUra is incorporated into RNA, that RNA processing is thereby vivo, that the blockage of FdUMP generation enhances FUTP disrupted (58, 71, 72), and that the extent of incorporation levels and thereby RNA incorporation. Sommadossi et al. (78) relates to cytotoxicity (60). Martin et al. (4, 73), using dThd to were unable to demonstrate an increase in FUra metabolites or increase RNA incorporation of FUra, found marked enhance in FUra (RNA) in hepatocytes exposed to high levels of thy ment of antitumor activity against two murine solid tumors and mine. Attempts to relate enhanced RNA incorporation to confirmed that this was associated with augmented incorpora plasma levels of dThd have not been successful. Thus, it cannot tion of FUra into nRNA. These observations were confirmed be conclusively stated what the target level of dThd should be in additional preclinical tumor models (5, 50, 74). Hence, for Phase II studies, but on pharmacokinetic grounds, a dose incorporation of FUra into RNA is an important parameter to of 8 g/m2/day should be optimal. follow in trials of FUra modulation by dThd. Several mecha Using doses in this range in a regimen of simultaneous FUra nisms may be invoked to explain the enhancement by dThd of and dThd infusion over 5 days Au et al. (50) defined a regimen 3913 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. ROLE OF THYMIDINE IN BIOCHEMICAL MODULATION with tolerable toxicity. Similar dose-limiting toxicity was noted previously untreated colorectal cancer patients on this study, in another trial in which both drugs were administered by rapid there were 2 partial responses. None of the 4 patients who had infusion, each as a single dose per course (49). Myelosuppres- been treated previously with FUra responded. Sternberg et al. sion and central nervous system toxicity were prominent, while (85) on a somewhat similar schedule observed only 1 partial mucositis was minimal. This increase in hematological over remission in a FUra-naive patient among 22 Ã©valuablepatients gastrointestinal toxicity suggests a selective effect of dThd on (6 of whom had no prior FUra). It remains possible that the 5- FUra metabolism and supports similar conclusions from mu day regimen of FUra/dThd might be more advantageous and a rine studies (79). The reason(s) for this selective change in full Phase II trial of this regimen is warranted. toxicity is unknown; it does not seem to relate solely to elevated Further modulation of FUra by the addition of PALA, an or prolonged plasma FUra levels, since the biochemical effects inhibitor of de novo pyrimidine biosynthesis, has been at in the mouse were seen only in tumor, and not in normal gut tempted in a number of clinical studies (Table 4). The predili or bone marrow cells (79). However, an effect on RNA incor ical basis for the combination of PALA/dThd/FUra derives poration of FUra has not been examined in tumor or in normal from the in vivo studies of Martin et al. (4) who demonstrated tissue in humans, and for this reason one cannot define an that this combination is superior to dThd/FUra in the CD8F1 optimal schedule to maximize the synergistic interaction of model. The biochemical rationale is that by reducing uridine FUra and dThd on biochemical grounds. nucleotide pools with PALA, the ratio of FUTP to UTP will It is noteworthy that clinical responses were observed in be increased, favoring increased RNA incorporation of the several of the Phase I studies (Table 3) and that some respond fraudulent nucleotide by RNA polymerase. ing patients had received extensive prior chemotherapy with Phase I studies have shown that PALA, 250-2000 mg/m2, drugs which included FUra (49, 51, 80-85). In a study by followed 24 h later by dThd, 30-45 g, given with or immediately Woodcock et al. (49), there were two partial responses (1 preceding FUra allows a total FUra dose of 300 mg/m2 to be colorectal cancer, 1 ovarian cancer) among 17 good risk patients given (88). When the dose of PALA is increased to 4 g/m2, the who had received prior FUra therapy. There were also 2 partial tolerable dose of FUra falls to 200 mg/m2 (89). However, with responses (1 colorectal cancer, 1 non-Hodgkin's lymphoma) of low dose PALA in the former studies, central nervous system 5 good risk patients who had not received prior FUra. Not one toxicity, probably related to dThd, made the regimen unsuitable of the 15 poor risk patients (Karnofsky performance, ss60) had for Phase II testing. Responses have been observed in advanced an objective response on this study (49). The majority of studies colorectal cancer using high (4 g/m2) doses of PALA, with in Table 3 have doses and schedules of FUra which are lower dThd and FUra (89); other studies have not confirmed these and less frequent than those conventionally used. Such sched results (87). It is not clear that this regimen is truly modulating ules have failed to show superiority to FUra in a randomized the action of FUra; a Phase I study to maximize the dose of trial (84). Using a 5-day continuous infusion schedule, Vogel et FUra in the modulatory combination should refine this regimen al. (81) conducted a Phase I trial with 12 patients. Among 8 prior to further Phase II testing.

Table 3 Phase I-ll studies of combination thymidine and 5-fluorouracil scheduleStudyKirkwoodeia/. Dose and of patients71212ToxicityHematological (51)Ohniiina mg/tnVday only3course 5-FUra 51i.v. bolus d x Gastrointestinal CentralsystemHematological nervous

(80)Vogelet al. g/m2/dayCI or 2.5 h i.v.d 00-400 mg/m2/day schedules2 I8x 5; d I, 3; d i.v. bolus d x 5; d 3, Gastrointestinal 5;di5-20 SkinHematological

Â«ai (81)dThdSg/mVdayCI'dxS8g/m'/day CI d x 5.55-FUra277-525mg/kg/day CI d x partial remissions in colon 5No. GastrointestinalCommentsFirst

Woodcock et al. (49) 15 g 30 min i.v. d x l 7.5-10 mg/kg d x 1-2 43 Hematological 4 partial remissions (2 colon, 1 ovar Gastrointestinal ian, 1 NHL); 10 minor responses. Central nervous system Other schedules also used for pharmacokinetic study.

Presant et al. (82) 9 g/m2 30 min i.v. d x 1 225-325 mg/m2 d x l 27 Hematological 3 partial remissions (2 colon, 1 Gastrointestinal breast) Central nervous system

Lynch et al. (83) 6-8 g/m2/h x 3 h 1/wk 100-200 mg/m2 i.v. Central nervous system push 1/wk Hematological Gastrointestinal

18 g/m2/h x 1.5 h 1/wk 100-175 mg/m2 i.v. I/ 29 Central nervous system wk Gastrointestinal

Buroker et al. (84) 45 g total 90 min i.v. d 1 300 mg/m2 i.v. d 1 67 Hematological Colorectal patients: 6 objective re Gastrointestinal sponses in 34 patients with meas Central nervous system urable disease

Sternberg et al. (85) 405 mg/kg as W min load- 7.5 mg/kg/day x 5 CI 27 Hematological 1 partial remission ing dose then 216 mg/kg/ Central nervous system day x 6 O Gastrointestinal * CI, continuous infusion; NHL, non-Hodgkin's lymphoma; d x 5, daily for 5 days (other schedules similarly treated). 3914 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. ROLE OF THYMIDINE IN BIOCHEMICAL MODULATION Table 4 Clinical trials with thymidine, PALA, and 5-fluorouracil scheduleStudyChiuten Dose and of patients27ToxicityGastrointestinal el al. (86)dThd30 g i.v. d yPALA1 g/m2 i.v. d 15-FUra150-300 mg/m2 i.v. sequential d2No. Central nervous system No responses HematologicalCommentsdThd/5-FUra

Chiuten et al. (87) 30 g i.v. d 2 1 g/m2 i.v. d l 150-300 mg/m2 i.v. 28 Gastrointestinal dThd/5-FUra simultaneous d2 Central nervous system l complete remission colon Hematological Phase II study

Casper et al. (88) 45 g i.v. d 2 l / 0.25-2 g/m2 i.v. dl 100-150 mg/m2 d 2 15 Central nervous system wk 1/wk 1/wk Hematological Gastrointestinal

O'Connell et al. (89) 15 g i.v. d 2 4.0 g/m2 i.v. 200 mg/m2 i.v. d 2 37 Hematological 1 complete and 9 partial colon Central nervous system remissions; 18 stable disease Gastrointestinal â€¢d,day.

It is generally accepted that FUra is most effective when mice and ara-C toxicity in normal rats, while simultaneous administered on schedules of frequent administration (90). For infusion of the agents was only modestly effective (39, 115). this reason attempts to modulate its action are probably best Bolus administration of dThd had minimal effects on circulat conducted on one of those schedules. Phase II studies of dThd/ ing deoxycytidine levels, while after 24 h infusion they were FUra in colon cancer or other tumors on repeated dose sched reduced to 20% of control (39). The selective advantage from ules might be followed by trials of regimens incorporating the combination was abolished in mice when the dose of dThd PALA in that setting in an attempt to modulate FUra maxi was increased from 5 to 10 g/kg/day, or when the agents were mally. administered concurrently (115). This implies that there is a dThd concentration range within which one modulates ara-C Thymidine as a Modulator of ara-C selectively in tumor cells and above which one increases toxicity to both tumor and normal tissues, negating any advantage from Two major biochemical mechanisms have been proposed to the combination. This concentration range has not been defined explain the cytotoxicity of ara-C: inhibition of DNA polymer- for human tumors; its importance is manifest. ase; and incorporation into DNA. Circulating ara-C is phos- Five clinical trials of combined dThd and ara-C have been phorylated intracellularly by deoxycytidine kinase to ara-CTP conducted (Table 5). In the study by Van Echo et al. (117), (91, 92), which inhibits DNA polymerase (93, 94) and is incor dThd administration preceded that of ara-C by 2 h; thereafter porated into DNA (94, 95). The importance of high levels of the drugs were administered concurrently to marrow aplasia. ara-CTP is demonstrated by studies in vitro (96-98) and in vivo dThd doses from 8 to 40 g/m2/day were used. In addition to (99-102) which relate formation and retention of ara-CTP to cytotoxicity and antileukemic effect. However, ara-CTP is not Table 5 Clinical studies of thymidine as a modulator of ara-C a potent competitive inhibitor of DNA polymerase (K-,approx PatientsStudyVan imately equal to the Km of dCTP, the natural substrate) (103), andscheduledThd, and inhibition of DNA synthesis occurs at levels of ara-CTP al.(116)VanEcho et 8g/m2/day;ara-C, some 100-fold lower than those of dCTP (104). Incorporation 100-250mg/m2/day of ara-C residues into DNA, on the other hand, also correlates un with ara-C cytotoxicity (105-107) and may be a a more impor hy-poplasiadThd,til marrow tant determinant then DNA polymerase inhibition (108, 109). Hence, it seems likely that the formation of ara-CTP is a al.(117)BlumenreichEcho et 8g/m2/day;ara-C, 250mg/nr'/il necessary, but not always sufficient, criterion for cytotoxicity. simultane Competition for the formation and incorporation of ara-CTP ously tomarrowaplasiadThd, comes from the natural substrate (110-112). Efforts to increase the efficacy of ara-C have therefore included measures to reduce etal. 75g/m2/day, (118)Zittoun g/m2/day;then 30 intracellular dCTP levels. The effects of dThd administration ara-C,200 in this regard have been discussed above. Plagemann et al. (111) mg/m2/dayuntil showed that pretreatment of rat hepatoma cells with 1 HIM marrowaplasiadThd, dThd increased ara-C incorporation into DNA over 2-fold. Harris et al. (113) increased the sensitivity to ara-C of four cell etal.(119)Frame! 30g/m2/day, lines; concentrations as low as 10 ^M dThd were effective in day 1;ara-C, 200mg/m2/day, certain of the lines, and the effect was observed only in those 3dThd, days 2, lines which exhibited a reduction in dCTP pools. These results were confirmed in L1210 leukemia cells by Grant et al. (114), al. (120)Dose 75g/m2/days, -2;ara-C,days 1 who showed maximal reduction of dCTP pools at 0.1 HIM 25-625mg/m2/day,days dThd, at which level incorporation of ara-C into DNA was increased 5-fold. Confirmation of these results in vivo empha 3-4DiseaseANLLALLCML-BANLLALLCML-BAMLCMLAULCML-BANLLALLMyelosclerosisAMLAMMLNHLCMLALLEvaluate14331551411141111101315CR"300501200310000000PR0002000000000!0000 sized the importance of dose, sequence, and schedule of the * CR, complete response; PR, partial response; ALL, acute lymphocytic leu kemia; CML-B, chronic myeloid leukemia blast crisis; AUL, acute undifferen- agents (39, 115). Pretreatment with dThd for 24 h prior to ara- tiated leukemia; AMML, acute monomyelocytic leukemia; NHL, non-Hodgkin's C administration maximized antitumor activity in tumorous lymphoma. 3915 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. ROLE OF THYMIDINE IN BIOCHEMICAL MODULATION myelotoxicity, mucositis, dermatitis, and hepatitis were prom Borsa and Whitmore (128) showed that administration of dThd inent. However, neither time to aplasia nor the incidence of alone could protect against MTX-induced cytotoxicity. Tatter- these toxicities appeared to be related to the dose of dThd, and sail et al. (129) demonstrated that while depletion of dTTP all doses were tolerable. pools was a uniform consequence of MTX administration, some In the population of ANLL patients on these trials, almost cell lines required the addition of a purine as well as dThd to all of whom had relapsed on or failed induction with ara-C- prevent cytotoxicity. Although subsequent studies by several containing regimens, 11 of 55 achieved complete remission. investigators failed to define the biochemical basis for such The incidence of complete remission was not related to the differences, further testing of MTX and dThd in combination dose of dThd. was stimulated by evidence of an enhanced therapeutic index Less severe toxicity was observed in the study of Blumenreich with the combined use of MTX and dThd against LI210 e/ al. (118), in which dThd administration preceded that of ara- leukemia in mice (130). C by 5-8 days. Two of six patients with ANLL achieved a While simultaneous administration of MTX and dThd by complete remission. Flash-labeling of bone marrow cells with continuous infusion in this species allowed a 7.5-fold increase ara-C and deoxycytidine did not demonstrate conclusively that in the total MTX dose, the therapeutic advantage could be enhancement of ara-C incorporation occurred. In addition, the negated by giving too high a dose of dThd, or both dThd and dose of dThd in this study (75 g/m2) yielded millimolar levels, inosine together (131). Further, the protection afforded by dThd higher than those which yielded maximal synergy /';/ vitro and was greater in tumor-bearing than in non-tumor-bearing mice //; vivo. Zittoun et al. (119) used a less intensive regimen and (132), and in bone marrow than in gut mucosa cells (132,133). observed complete remissions in 3 of 14 patients with chronic These studies and the detailed clinical-biochemical correlations myelocytic leukemia and 1 of 11 with ANLL. by Howell et al. (34, 134, 135) provided indirect evidence that These results do not prove that the combination is superior dThd protection of target cell populations is based on: (a) their to ara-C alone. Complete remissions in those patients who had ability to salvage preformed purines; and/or (/>)shunting of the failed ara-C-containing regimens suggest that the modulation remaining supply of reduced folates to maintain purine synthe of ara-C by dThd merits further study in the treatment of sis, because of end product allosteric inhibition by dTTP of the human leukemia. A study with clinical and biochemical end enzymes of de novo pyrimidine biosynthesis. They also empha points is needed to define the optimal doses of both dThd and sized the dependence of a therapeutic advantage on the dose ara-C for maximal ara-CTP pool expansion and/or (ara-C) and schedule of dThd used. DNA formation. The effect of one on the pharmacokinetics of The initial clinical studies showed that doses of MTX some the other has not been described; optimal tailoring of a regimen 2 orders of magnitude greater than those conventionally used for use in humans requires such information. Finally, descrip were well tolerated with concurrent administration of dThd at tion of the selectivity of the modulation for leukemic cells may a dose of 8 g/m2 (126). This dose was based on the in vitro levels of 10~6 to IO"5 M needed to achieve optimal rescue; it be crucial to its ultimate therapeutic role. The activity demon strated by high-dose ara-C in leukemic patients refractory to was clear that continued administration of dThd for 48-72 h ara-C suggests that resistance may be overcome by increased after the MTX dose was required for full protection from intracellular ara-C levels (121). However, while leukemia blast toxicity (136,137). Subsequent studies showed that 2 g/m2/day cells exposed to increasing extracellular ara-C levels generate dThd sufficed to protect from over 3 g/m2/day MTX by 72 h ara-CTP levels almost proportional to the increase, the degree infusion (138), while 1 g/m2/day dThd was effective following of incorporation of ara-C residues into DNA does not keep a single bolus dose of MTX at 3 g/m2 (139). The observation pace, probably as a result of DNA polymerase inhibition (122). that dThd prolonged the plasma half-life of MTX (138), and Thus it appears that there may be an "optimal" concentration possibly its retention in longer-acting polyglutamylated forms, of ara-CTP for maximal selectivity for leukemic cells. Wide provides a possible explanation for this dose effect. However, interpatient variability in ara-C pharmacokinetics and phar- although these and other MTX/dThd combinations were estab macodynamics has been demonstrated (123). A further poten lished as being safe for human administration (140), none has tial role of dThd may be in reducing the pharmacodynamic been tested rigorously in the clinic to demonstrate if an advan variability by producing uniformly low levels of dCTP, to allow tage exists over the use of MTX as a single agent. Sporadic development of a regimen suitable for testing in comparative responses occurred in a variety of tumors in several of the trials studies. (131-138), but there is no general agreement on the optimal Finally it should be noted that in this instance also, the dose and schedule for combined administration of the agents. addition of PALA to dThd/ara-C yields enhanced efficacy in A recent study in small numbers of head and neck cancer preclinical models (124). Practical development of this regimen patients using two different schedules of dThd rescue yielded requires the determination of the parameters already discussed low response rates for both regimens (141). Again, the concern for dThd/ara-C and more extensive Phase I study. is raised that the current schedules may rescue both host and tumor. Two concepts are receiving attention in the clinic at this time: I li) inuline and Methotrexate (a) the possibility that early and brief administration of dThd MTX inhibits dihydrofolate reducÃase,which results in de may protect the cells of the gastrointestinal tract during their pletion of the reduced folate cofactors required for de novo period of MTX exposure (142); and (b) the selection of specific pyrimidine and purine biosynthesis (125). Reversal of MTX tumors with defined biochemical abnormalities, e.g., hypoxan- cytotoxicity may be achieved by passing the block in reduced tliincignali ine phophoribosyltransferase deficiency (143), which folate synthesis [with 5-formyltetrahydrofolate, folinic acid] or would prevent the tumor cells being rescued by dThd alone. by providing the end products of the reactions inhibited [dThd Trials to explore these ideas are in progress. and inosine (or hypoxanthine)]. However, the most widely used application of dThd at this While early in vitro studies suggested that MTX cytotoxicity time is for the protection of a small group of patients who was largely a consequence of its antipurine effect (126, 127), manifest MTX toxicity, especially renal failure, following high- 3916 Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1987 American Association for Cancer Research. ROLE OF THYMIDINE IN BIOCHEMICAL MODULATION

dose MTX despite adequate i.v. folinic acid rescue. Formal 11 Meuth, M. The genetic consequences of nucleotide precursor pool imbalance in mammalian cells. MutÃ¢t.Res., Â¡26:107-112, 1984. evaluation of this type of rescue has not been conducted but 12. Snyder, R. D. The role of deoxynucleoside triphosphate pools in the sporadic case reports (data on file at the National Cancer inhibition of DNA-excision repair and replication in human cells by hy Institute) suggest that catastrophic generalized MTX toxicity droxyurea. MutÃ¢t.Res., 131: 163-172, 1984. may be averted by the administration of dThd, 8 g/m2/day, 13. Snyder, R. D. Inhibitors of ribonucleotide reducÃasealter DNA repair in human fibroblasts through specific depletion of purine deoxynucleotide until MTX levels have fallen below 5 x 10~8M. triphosphates. Cell Biol. Toxicol., /: 49-57, 1984. In summary, the optimal dose and schedule of MTX/dThd !4 Bjursell, ( >..and Reichard, P. 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