Selective Expansion of 5,10-Methylenetetrahydrofolate Pools and Modulation of 5-Fluorouracil Antitumor Activity by Leucovorin in Vivo1

Home , Fluorodeoxyuridylate, Tetrahydrofolic acid

(CANCER RESEARCH 49, 2592-2596, May 15, 1989] Selective Expansion of 5,10-Methylenetetrahydrofolate Pools and Modulation of 5-Fluorouracil Antitumor Activity by Leucovorin in Vivo1

Joel E. Wright,2 Arnon Dreyfuss, Ibrahim El-Magharbel, Dorothy Trites, Steven M. Jones, Sylvia A. Holden, Andre Rosowsky,2 and Emil Frei III

Dana-Farber Cancer Institute, and Departments of Biological Chemistry and Molecular Pharmacology [J. E. W., A. R.J, Pathology fS. A. HJ, and Medicine [A. D., E. F.], Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT (13-15). Recently, all favorable in vivo preclinical results from murine models have been challenged (16), suggesting indirectly Expansion of CH2THF pools in tissues of BÃ•LB/cmice bearing s.c.- that the combination may be more efficacious in the human implanted EMT6 mammary adenocarcinomas was measured after leucovorin administration. Twenty-four mice were treated with leucovorin at than in the mouse. doses of 0, 45, 90, or 180 mg/kg/inject Â¡onx8 injections spaced over 48 Our in vivostudy had three main objectives: to assay CH2THF h. Tumor and bone marrow cytosols were assayed for CHjTHF by pools in EMT6 mammary adenocarcinoma and bone marrow forming ternary complexes with thymidylate synthase and |3H]FdUMP. of BALB/c mice after high-dose leucovorin treatment; to deter Tumor CH2THF pools were expanded significantly at the two higher mine what leucovorin dose is required to provide maximum doses. Marrow levels were not different from controls. Groups of tumor EMT6 tumor levels of CH2THF; and to examine the therapeu bearing mice were treated with saline, leucovorin, 5-fluorouracil or 5- tic effect of combining leucovorin and FU in this refractory fluourouracil plus leucovorin on an optimal dosage schedule. Measured tumor model. We pursued these objectives by first analyzing from the last day of treatment, these tumors grew to 10 mm root-mean- CH2THF tissue levels over a range of leucovorin doses. Subse square diameters in 3.5 Â±1.4, 5.0 Â±1.2, 6.5 Â±1.5, and 9.3 Â±1.2 days, quently we investigated tumor growth delay (17) in response to respectively. Growth rates were significantly different from controls only FU given alone and in combination with leucovorin. in the latter two groups.

MATERIALS AND METHODS INTRODUCTION Enhancement of the cytotoxicity of FU3 by the nontoxic drug (6/f,S>5,10-Methylene-5,6,7,8-tetrahydrofolic Acid. In a nitrogen- filled glove bag, a 50-mg sample of 5,6,7,8-tetrahydrofolic acid (Sigma, leucovorin is an outcome of the cellular metabolism of both St. Louis, MO), in a breakseal vial, was transferred to an amber, septum- drugs used in the combination. The FU is converted enzymati- stoppered 4-ml vial containing 2.0 ml of 100 mM Tris base (pH 10). cally to FdUMP and the leucovorin forms CH2THF. These When the solids were completely dissolved, 25 fil of 40% formalin was metabolites inhibit the target enzyme, thymidylate synthase, by introduced with the aid of a syringe. After 20 min at room temperature, forming a covalent ternary complex, TS-FdUMP-CH2THF (1). a 20-ÃÃlportionof 2-mercaptoethanol was added. The product was In the presence of sufficient FdUMP and CH2THF, or any of purified on a 10-mm ID x 25-cm long Dynamax preparative Cis HPLC its polyglutamylated forms, binding to the second subunit of column (Rainin Instruments, Boston, MA), eluted with 0.1 M ammo thymidylate synthase can give rise to a higher-order complex, nium acetate buffer, pH 7.8, containing 50 mM formaldehyde and a 0 TS-(FdUMP)2-(CH2THF)2 (2), resulting in complete loss of to 10% linear gradient of acetonitrile over 70 min. Fractions containing the major peak and three satellite peaks were collected over dry ice. enzyme activity. The FdUMP binding is reversible with a half- Substances collected with the smaller peaks did not promote [3H]- life of 30 min in the case of Lactobacillus casei enzyme, but in FdUMP binding to L. casei thymidylate synthase, whereas material the presence of excess CH2THF, ternary complex formation is corresponding to the major peak did enhance the complexation. Quan- complete (3). A strategy for elevating intracellular CH2THF in titation of the product by UV spectrophotometry was based upon the conjunction with high-dose methotrexate rescue uses the non- reported 294-nm extinction of 32,000 at pH 7.2 (18). Purity (>97%) toxic compound leucovorin (4), which is efficiently metabolized and concentration were further confirmed using paired-ion HPLC (19). to generate this and other reduced folates. The yield was 80% based upon starting material, tetrahydrofolic acid. Expansion of CH2THF pools by leucovorin has been shown These reaction conditions were also used to synthesize 0.1 mM (Â±)[5,10-I4C]CH2THFfrom [14C]formaldehyde (New England Nuclear, in culture with several murine and human cell lines (5, 6), in vivo with murine leukemia (7) and human colon tumor xeno- Boston, MA). Since the purity achieved in the reaction mixture was grafts (8), and in human patients with advanced colorectal about 88%, estimating from relative HPLC peak areas, this material was used without further Chromatographie separation. The specific cancer (9). Potentiation of FU activity by treatment with leu activity of the resulting radioactive cofactor was 200 cpm/pmol. [6-3H] covorin has also been demonstrated in cell culture (10, 11) FdUMP was obtained from Moravek Biochemicals, Brea, CA, and its while in tumor-bearing mice, results have been less promising purity checked periodically by HPLC. L. casei thymidylate synthase (12). Nonetheless, preclinical development has culminated in a was purchased from Bio-Pure Corp., Boston, MA, and further purified series of studies of the leucovorin-FU combination in humans in the laboratory of Dr. Roy Kisliuk of Tufts-New England Medical Center. Tris-ascorbate buffer (20) was prepared from Tris-base 50 mM, Received 11/28/88; revised 2/14/89; accepted 2/20/89. ethylenediaminetetraacetic acid 1.0 mM, and sucrose 8.5% w/v. It was The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in titrated to pH 7.4 with concentrated HC1 and deaerated with nitrogen. accordance with 18 U.S.C. Section 1734 solely to indicate this fact. This buffer served as the tissue homogenization medium, assay diluent 1This study was supported by Grant CA 19589 from the National Cancer and solvent for CH2THF, [3H]FdUMP, and thymidylate synthase. Institute, DHHS. Tumor Transplants. One group of 26 female BALB/c mice used in 2To whom requests for reprints should be addressed, at Dana-Farber Cancer the leucovorin treatment study received 2 x IO6 EMT6 tumor cells, Institute, 44 Binney Street, Boston, MA 02115. 3The abbreviations used are: FU, 5-fluorouracil; CH2THF, (6A)-5,10-meth- S.C.,in each flank. In the leucovorin-FU combination study, starting ylene-5,6.7,8-tetrahydrofolate; (Â±)-CH2THF, (6A,S)-5,10-methylene-5,6,7,8-tetrahydrofolate; dUMP, 2'-deoxyuridylic acid; FdUMP, 5-fluoro-2'-deoxyuridylic with 52 mice, EMT6 transplants were given to the right flank only. In acid; TS-(FdUMP)â€ž-(CH2THF)â€ž,covalentcomplex formed when one (n = 1) or both studies, mice were examined daily, beginning one week after tumor two (n = 2) moles each of CH2THF and FdUMP bind to thymidylate synthase; implantation. As soon as the tumors were sufficiently developed (2 mm HPLC, high-performance liquid chromatography. x 2 mm), the mice were divided into groups. Two mice in the leucovorin 2592

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1989 American Association for Cancer Research. LEUCOVORIN, FOLATE POOLS, AND FLUOROURACIL ACTIVITY study and four in the combination study were sacrificed at this time The kinetics of dUMP interference were also investigated, using the due to inadequate tumor development. The remaining mice were di standard assay mixture containing 0.75 MMCH2THF and another which vided into four groups with equivalent tumor size distributions. differed only by addition of dUMP to a final concentration of 100 MM. Leucovorin Dose-Response. Three groups of six mice bearing bilateral The CH2THF assay procedure was followed, but the reactions were tumors were given leucovorin at doses of 45, 90, or 180 mg/kg/ quenched by addition of 1% sodium dodecyl sulfate and boiling after injection, spaced over a 48-h course of eight i.p. injections; the control 2, 4, 6, 8, 10, 20, 30, or 45 min and analyzed for [3H]FdUMP binding. group received normal saline. Within l h after the last injection, the Leucovorin-FU Combination. Four groups of 12 mice bearing EMT6 mice were sacrificed by cervical dislocation. Tumors were excised and tumors in the right flank were treated i.p. with saline or 180 mg/kg weighed and bone marrow was harvested from both tibias and femurs leucovorin at 11:00 a.m. daily for 8 days. Two h after the first injection of each animal. Tissues were treated with 0.8% ammonium chloride the mice received either 30 mg/kg FU or saline. This was repeated for 5 min to remove red cells (21), and then were washed with ice-cold every other afternoon for a total of five injections. Following this phosphate-buffered saline and kept at 0-4Â°Cfor subsequent procedures. schedule, the groups were constituted as follows: Group 1 received From each tumor, a 10-20-mg sample was removed and homogenized saline, both in the morning and afternoon. Group 2 was given leu in a Dounce tube (Kontes, Vineland, NJ) with the aid of 0.5 ml of tris- covorin mornings and saline in the afternoon. Group 3 was given saline ascorbate buffer. The tube was washed with another 0.5 ml of buffer in the morning and FU in the afternoon. Group 4 received leucovorin and the homogenate and washings were pooled in a 1.5 ml Eppendorf in the morning and FU in the afternoon. tube. The samples were spun for 15 min in a Fisher model 235B Tumor Growth. Perpendicular, two-dimensional caliper measure microcentrifuge and the supernatant was collected. A 20-M>sample was ments were recorded for each tumor, every third or fourth day, begin immediately assayed for CH2THF content by the method of Priest and ning on the first day of treatment. Mice were weighed weekly. Animals Doig (22). As in their general procedure, limiting quantities of the which appeared moribund were recorded as nonsurvivors and sacrificed. tumor or bone marrow cytosols and several concentrations of the This study adhered to the tumor growth delay assay, both in method CH2THF standard were incubated with excess [3H]FdUMP and L. casei ology and data analysis, as previously applied to the growth pattern of thymidylate synthase for 45 min. The total volume was 100 /tl. After SA S sarcoma in mice (17). The Wilcoxon procedure was used to boiling with 1% sodium dodecyl sulfate to stabilize the complex, an evaluate the significance of pairwise comparisons between controls and aliquot was counted for total radioactivity and an equal volume was each tested group (24). chromatographed on a Sephadex G25-150 spin column to remove uncomplexed radioactivity. The fraction bound at each concentration of standard CH2THF was then plotted. From this calibration curve, RESULTS molar levels of cofactor in the test samples were calculated. These values were expressed as a function of the protein content, determined By modifying a widely used procedure (26) for synthesis of by the Bradford dye-binding method (23). Samples were frozen at (Â±)CH2THF, so that the reaction was carried out at pH 10 -75Â°Cbetween assays. Four repetitions of each assay were performed rather than 8, a substantial improvement in yield and purity on different days. Pairwise comparisons of the mean assay values were was obtained. Preparation of a 1.0-/uManalytical stock solution analyzed and 95% confidence intervals calculated using the Bonferroni from the purified product was also simplified by application of procedure (24). an established paired-ion chromatography method (19). Leu Tumor Resampling. Two representative tumors from each of the four covorin was used as an internal standard in this system. Its groups were resampled in duplicate in order to assess the validity of peak height ratio, relative to (Â±)CH2THF, was 2.0 under the the sampling procedure. Since the range of tumor masses was 0.03-1.8 reported separation conditions. Using the stock (Â±)CH2THF g, it was not practicable to remove two more samples from tumors containing the active 6/?-isomer at 0.50 UM, a standard curve weighing less than 60 mg. Thus these smaller tumors were excluded was obtained with each assay. from the resampling study. From the remainder, two tumors in each of Analysis of dUMP concentrations by conversion to [5-14C]- the four groups were selected according to statistical criteria. Within each group, the assay mean minus its standard deviation was calculated, thymidylate was sensitive to a detection limit of 20 pmol. using the results of the leucovorin dose-response. The tumor selected Tumor and marrow cytosols contained insufficient amounts of to represent the low range in any group was closest to this calculated dUMP for determination by this method. Although dUMP value in CH2THF content. Tumors representing the high end of the concentrations in the cytosols could not be quantitated directly, assay range had levels closest to the group mean plus its standard they were found to lie below the threshold of interference for deviation. However, tumors derived from the same host as the low the CH2THF assay in a series of mixing experiments. Inhibition range sample were also excluded from this selection. Two 10-20 mg of 0.125 MM[3H]FdUMP binding to CH2THF and thymidylate pieces were taken from each of the eight selected tumors and their synthase by dUMP was determined in the presence of added CHiTHF levels assayed (four replicates). These data, combined with nucleotide (0.05-1.0 MM).Using equivalent volumes of tumor the original dose-response results, were used to calculate the coefficient of variation in sampling (24). or marrow extract in place of added dUMP, no inhibition was seen. The inhibition by added dUMP is illustrated in Fig. 1. Interference. Tumor and marrow cytosols and standard dUMP so Use of a lower final concentration, 0.025 MM,of [3H]FdUMP lutions were analyzed for dUMP content by an established method in which the thymidylate synthase catalyzed reaction with [5,10-I4C]- resulted in substantially decreased binding (18-38% lower) CH2THF forms [5-14C]thymidylate (25). Incubations contained 30 ÃŸl when bone marrow cytosols were included in the incubation of L. casei thymidylate synthase, 0.16 units/ml, 40 n\ of 0.1 mM (data not shown). However, tumor cytosols were again not (Â±)[5,10-14C]CH2THFand 20 n\ of cytosol or dUMP stock solutions inhibitory. at 0.5, 1.0, 2.0, 5.0, and 10 MM,in a total assay volume of 175 Mi- Measurement of the time-dependent complexation of [3H]- Inhibition of ternary complex formation by dUMP at final concen FdUMP under normal assay conditions as well as with added trations of 0, 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 MM,or a 60-^1 aliquot 0.1 MMdUMP, shown in Fig. 2, gave invariant initial velocities of cytosol from each tumor or marrow sample was measured. The (1.9 Â±0.1 pmol/min). Interference by dUMP resulted in a 33% samples were supplemented with enough CH2THF to bring the final concentration of 6/Ã®-isomerto0.25 MM.Except for these modifications, lower plateau value for complex formation. the standard assay procedure was followed. [3H]FdUMP binding de Leucovorin dose-response results are given in Table 1. In the creased hyperbolically with increasing dUMP. This provided a more tumors, leucovorin treatment gave a statistically significant (P sensitive means of assessing dUMP interference in the assay. Controls < 0.05) 2.5-fold increase in CH2THF over controls at the two were run without added cytosol. Another series of curves was obtained higher doses. However, when control data, which were more using [3H]FdUMP at 0.025 instead of 0.125 MM. variable, were excluded from the analysis, significant differences 2593

1.2 10

l Ã¶ 0.6 = S

o.o 0.5 1.0 Mean Assay Value (nmol/mg protein) dUMP (uM) Fig. 3. CH2THF content of three different samples from each of eight repre Fig. 1. Equilibrium binding of 0.125 JJM[3H]FdUMP to L. casei thymidylate sentative tumors plotted versus mean value from the same tumor, showing synthase, 23 munits/ml and CH2THF, 0.5 MM,plotted as a function of dUMP variability in sampling technique. concentration. Bars, 2 X SD, N = 4. Table 2 Tumor growth after treatment with FU alone and in combination with leucovorin Days to 10 mm confidence Diameter*ControlsTreatment" RMS interval^2.2-4.9

Leucovorin 5.0 3.9-6.2 0/12 FU 6.5 5.0-8.0 2/12 Leucovorin + FU3.5 9.395% 8.1-10.5Deaths0/12 0/12 " Leucovorin dose, alone or in combination, 180 mg/kg/injection x 8 injec tions; FU dose, 30 mg/kg/injection x 5 injections. Â£1 * From last day of treatment. RMS (root-mean-square). Diameter: geometric 3S â€”¿ o. â€¢¿D mean of two orthogonal measurements. th c Bonferroni 7"test.

Tumor growth delays with FU and FU plus leucovorin were marginal due to the refractory nature of the EMT6 mammary adenocarcinoma, even though the maximum effective dose of FU was used. Composite growth curves confirmed the appli Time (min) cability of the regrowth rate model as the choice of endpoint, Fig. 2. Time-dependent formation of [3H]FdUMP-TS-CH2THF complex. L. casei thymidylate synthase concentration: 23 munits/ml. First-order kinetic curves based upon the effects of treatment on growth rates (17). Tumor generated by nonlinear regression. D, assay mixture with 0.75-0iM CH2THF; â€¢¿ growth endpoints, presented in Table 2, show that high-dose same mixture plus 0.10 nM dUMP. Bars, 2 x SD, N= 4. leucovorin plus FU gives a modest but significant augmentation of response, relative to an optimal course of FU given alone. Table 1vivoLeucovorin Dose-dependent leucovorin modulation of CH2THF pools in Results with leucovorin alone are not significantly different CH2THF from controls. content" (nmol/mg pro dose, tein)Tumor group (mg/kg/ inj x 8inj)0 Marrow1.5 DISCUSSION Controls 0.8 Research into the biochemical determinants of FU cytotox- 45 Low 2.1 0.5 90 Intermediate 3.70.53.7 icity, begun three decades ago (27), has revealed a mosaic of 180 HighIntracellular 1.2 interrelated processes. No individual study has provided a fully " 95% confidence intervals: tumor, Â±1.1;marrow, Â±0.3nmol/mg protein. integrated view of the relative contribution of each metabolic step in killing cells, but the various parts do provide a coherent (P < 0.05) were found between the low and intermediate doses picture. Anabolic transformation of FU, giving rise to riboside (16% increase), between the intermediate and high doses (32%) and deoxyriboside triphosphates, results in RNA and DNA and between the low and high doses (57%). No expansion in misincorporation but is comparatively nonspecific (28). By marrow CH2THF pools was seen at any leucovorin dose, rela administering reduced folate precursors, such as leucovorin, it tive to controls. becomes possible to exploit an earlier event in FU activation, Redetermination of the CH2THF content of samples taken namely its conversion to FdUMP. Binding of this species to from the same tumor is illustrated in Fig. 3. Coefficients of thymidylate synthase, which is greatly enhanced by CH2THF, variation, F, calculated from these data (24), in conjunction blocks the conversion of dUMP to thymidylate. In tumor cells with the replicate assay results, show that variability between lacking alternative salvage pathways, depletion of thymidylate, samples from any tumor (K= 0.31) is less than that from tumor an essential nucleotide, may provide selective killing. Addi to tumor within any group (V= 0.53). This result applies to all tional selectivity may be conferred by differential uptake of the treated and control groups in the dose response study. reduced folates via a postulated oncofetal carrier (29). This 2594

Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1989 American Association for Cancer Research. LEUCOVORIN, FOLATE POOLS. AND FLUOROURACIL ACTIVITY transport system predominates in the uptake of 5-methyltetra- has been shown to result from RNA incorporation (28). As part hydrofolate, leucovorin, and methotrexate in embryonic and of another study presently underway, we have found very low neoplastic cells, but is lacking in other normal tissues. The basal levels of CH2THF in the intestinal mucosa of BALB/c mechanism has previously been used to explain tumor selective female mice, which supports this conclusion and may also help antifolate sensitivity. Since both 5-methyltetrahydrofolate and to explain the gut toxicity of methotrexate in mice. By differ leucovorin are precursors of CH2THF, such transport differ ential expansion of CH2THF pools, we may achieve more ences may also influence the selectivity of fluoropyrimidines selective thymidylate synthase blockade and decreased systemic given in combination with leucovorin. toxicity associated with RNA incorporation. Attempting to gain new insights into the usefulness of leu An effect of FU therapy which tends to be self-defeating is covorin, we have applied a recently developed analytical method the buildup of dUMP, due to its nonutilization during thymi in the measurement of CH2THF in vivo. The increase in dylate synthase blockade. Some authors have reported ex CH2THF levels after administering leucovorin on a multiple- panded pools of dUMP reaching millimolar concentrations injection schedule was then determined. Doses and schedules (25). In the cytosols we have assayed, which did not involve FU were intended to approximate, in mice (30), the steady state treatment, dUMP concentrations in the 20 Â¿tlassay aliquots concentration and duration profile of a clinical protocol for were below the detection limit, 20 pmol, using the [5,10-14C]- continuous intravenous leucovorin administration. High- and CH2THF assay. Since tumor or marrow excision following FU intermediate-dose leucovorin treatments expanded tumor or FU-leucovorin treatment were outside the scope of the CH2THF pools 2.5-fold. This increase is similar to a clinical present study, we did not measure expanded dUMP pools in finding in two patients in whom tumor CH2THF levels rose by such tissues. 1.4 and 2.2 fold after 500 mg/m2 leucovorin was given over 5 Inhibition studies in which dUMP was added as a component days by continuous infusion (9). of the TS-[3H]FdUMP-CH2THF reaction mixture showed that Tumors in mice that did not receive leucovorin contained the presence of even 20 pmol of dUMP was slightly inhibitory. larger amounts of endogenous CH2THF than were present in By the same method, however, the addition of a 60-^1 aliquot marrow. These tumor pools were not significantly expanded at of tumor or marrow cytosol was not inhibitory when the con the lowest dose of leucovorin. The much lower basal levels of centration of [3H]FdUMP in the assay was 0.125 ^M. With a CH2THF in bone marrow compared with tumor in the un smaller excess of [3H)FdUMP, 0.025 UM,cytosols from marrow treated animals is consistent with the relative insensitivity of were inhibitory, but those from tumors were not. These exper the marrow to FU treatment. But the pronounced increase in iments emphasized the importance of using a large excess of tumor/marrow CH2THF mole ratio from 1.9-fold in controls the radioligand to avoid interference in the analysis of to 7.4-fold after the intermediate dose of leucovorin is expected CH2THF. The nature of this effect was further revealed in the to augment the differential effect. When the leucovorin dose kinetic studies, which showed that in the context of the was increased from 90 to 180 mg/kg/injection, no further CH2THF assay, interference is not due to competition between expansion in CH2THF pools was seen and the tumor/marrow dUMP and [3H]FdUMP for thymidylate synthase binding sites, mole ratio dropped to 3.1. Thus the intermediate dose is prob since initial rates of [3H]FdUMP binding under the standard ably optimal in terms of response and selectivity. Previous in assay conditions did not decrease in the presence of 0.10 MM vitro studies with the leucovorin-FU combination in five human dUMP. Instead, we observed a reduction in total [3H]FdUMP and murine cell lines showed progressive augmentation of FU bound at the plateau, consistent with depletion of CH2THF by cytotoxicity when the leucovorin concentration was increased its reaction with dUMP to form thymidylate, resulting in the from 10~8 to 10~6 M (6). Above IO"6 M, no further increase in observed inhibition. activity was seen, consistent with our finding that CH2THF The mixing experiments show that analytical methods can levels do not increase beyond the intermediate leucovorin dose. accurately measure CH2THF pools, as long as interfering We have not, however, measured in vivo concentrations after dUMP pools are not expanded by thymidylate synthase block the various doses, so we cannot determine explicitly what ade. Under the latter conditions, when fluoropyrimidines are leucovorin concentration results in maximum CH2THF pool used, they form nonradioactive FdUMP which competes for expansion in the EMT6 tumor. [3H]FdUMP binding sites, giving erroneously low assay results. When the control treatment is compared with the low dose In some preliminary experiments (data not shown), we were regimen, the difference in CH2THF levels is insignificant by able to overcome this problem, and abrogate dUMP interfer the Bonferroni T test (P > 0.1). In pairwise comparisons be ence at the same time, by the use of 5'-nucleotidase from tween controls and intermediate or high dose leucovorin, how Crotalus adamanteus (Sigma, St. Louis, MO), in the presence ever, significant differences (P < 0.05) are seen in both cases. of excess zinc chloride (31), to dephosphorylate both dUMP A threshold dose lies between 45 and 90 mg/kg/injection x 8 and [3H]FdUMP (32). injections, and the latter dose lies on the tumor response Having shown that CH2THF pools can be modulated by plateau. This plateau is consistent with a recently proposed leucovorin in vivo, in a dose-dependent, saturable fashion, we mechanism whereby fluctuations in CH2THF levels in response next examined the therapeutic consequences of this phenome to folate or antifolate treatment are held within narrow limits, non in the same murine tumor model after treatment with FU while much larger changes in 5-methyltetrahydrofolate and 10- alone or in combination with leucovorin. Protocols were again formyltetrahydrofolate are observed. Thus interconversion of designed to reproduce, insofar as possible, the effect of contin the three metabolites sets an upper limit on CH2THF pool uous i.v. administration of both drugs. Toward this end, the expansion (20). total leucovorin dose, selected from a mouse-to-man equipo- In bone marrow, where basal levels are more variable, con tency table (30), was approximately equivalent to 3900 mg/m2 trols do not differ significantly from any treated group (P > in humans. Fluorouracil was given at the maximum effective 0.1). These cells do not accumulate additional CH2THF after dose in mice, based upon previous experience in our animal high-dose leucovorin treatment. facility. Leucovorin alone had no statistically significant effect The toxicity of fluoropyrimidines to mouse intestinal mucosa on tumor growth (P < 0.18). With FU alone the tumors took 2595

covorin infusion of 5-fluorouracil Cytotoxicity against murine L1210 leuke 6.5 days to reach the 10 mm diameter endpoint from the end mia. Cancer Chemother. Pharmacol., 6: 121-125, 1981. of treatment, longer than controls (P < 0.007). Tumor growth 13. Madajewicz, S., Petrelli, N., Rustum, Y. M., Campbell, J., Herrera, L., to the endpoint after combination treatment required 9.3 days, Mittelman, A., Perry, A., and Creaven, P. Phase I-II trial of high dose calcium leucovorin and 5-fluorouracil in advanced colorectal cancer. Cancer also longer than controls (P < 0.0001). Res., 44:4667-4669, 1984. We conclude that high-dose leucovorin treatment can expand 14. Grem, J. L., Hoth, D. F., Hamilton, J. M., King, S. A., and Leyland-Jones, CH2THF pools in EMT6 tumor cells, but not in marrow. The B. Overview of current status and future direction of clinical trials with 5- effect is dose-dependent and saturable. Leucovorin, given in fluorouracil in combination with folinic acid. Cancer Treat. Rep., 71: 1249- 1264, 1987. combination with FU results in a more pronounced tumor 15. Arbuck, S. G. 5-FU/Leucovorin: biochemical modulation that works? On cology (Basel), ;.â€¢61-71,1987. growth delay than with FU alone. The additional benefit af 16. Martin, D. S., Stolfi, R. L., and Colofiore, J. R. Failure of high-dose forded by the use of leucovorin in this regimen is obtained with leucovorin to improve therapy with the maximum tolerated dose of 5- no increase in host toxicity. fluorouracil: a murine study with clinical relevance? J. Nati. Cancer. Inst., Â«0:496-501,1988. 17. Begg, A. C. Principles and practices of the tumor growth delay. In: R. F. Kaiman (ed.), Rodent Tumor Models, pp. 114-121. New York: Pergamon ACKNOWLEDGMENTS Press, 1987. Myla Hunt and Janet Andersen, Division of Biostatistics, Dana- 18. Blakley, R. L. Spectroscopic studies on the combination of formaldehyde with tetrahydropteroylglutamic acid and other hydropteridines. Biochem. 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Downloaded from cancerres.aacrjournals.org on October 2, 2021. © 1989 American Association for Cancer Research. Selective Expansion of 5,10-Methylenetetrahydrofolate Pools and Modulation of 5-Fluorouracil Antitumor Activity by Leucovorin in Vivo

Joel E. Wright, Arnon Dreyfuss, Ibrahim El-Magharbel, et al.

Cancer Res 1989;49:2592-2596.

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