Blocking catabolism with eniluracil enhances PET studies of 5-[18F]fluorouracil pharmacokinetics.

UNLABELLED: Noninvasive methods for measuring the pharmacokinetics of chemotherapeutic drugs such as 5-fluorouracil (FU) are needed for individualized optimization of treatment regimens. PET imaging of [18F]FU (PET/[18F]FU) is potentially useful in this context, but PET/[18F]FU is severely hampered by low tumor uptake of radiolabel and rapid catabolism of FU in vivo. Pretreatment with eniluracil (5-ethynyluracil) prevents catabolism of FU. Hypothesizing that suppression of catabolism would enhance PET/[18F]FU, we examined the effects of eniluracil on the short-term pharmacokinetics of the radiotracer. METHODS: Anesthetized rats bearing a subcutaneous rat colorectal tumor were given eniluracil or placebo and injected intravenously 1 h later with [18F]FU or [3H]FU. In the 18F studies, dynamic PET image sequences were obtained 0-2 h after injection. Tumors were excised and frozen at 2 h and then analyzed for labeled metabolites by high-performance liquid chromatography. Biodistribution of radiolabel was determined by direct tissue assay. RESULTS: Eniluracil improved tumor visualization in PET images. With eniluracil, tumor standardized uptake values ([activity/g]/[injected activity/g body weight]) increased from 0.72 +/- 0.06 (mean +/- SEM; n = 6) to 1.57 +/- 0.20 (n = 12; P < 0.01), and tumor uptake increased by factors of 2 or more relative to plasma (P < 0.05) and bone, liver, and kidney (P < 0.01). Without eniluracil (n = 5), 57% +/- 4% of recovered radiolabel in tumor at 2 h was on catabolites, with the rest divided among FU (2% +/- 1%), anabolites of FU (38% +/- 7%), and unidentified peaks (4% +/- 2%). With eniluracil (n = 8), catabolites, FU, and anabolites comprised 2% +/- 1%, 41% +/- 5%, and 57% +/- 4%, respectively, of the recovered radiolabel in tumors. CONCLUSION: Eniluracil increased tumor accumulation of 18F relative to host tissues and fundamentally changed the biochemical significance of that accumulation. With catabolism suppressed, tumor radioactivity reflected the therapeutically relevant aspect of FU pharmacokinetics--namely, uptake and anabolic activation of the drug. With this approach, it may be feasible to measure the transport and anabolism of [18F]FU in tumors by kinetic modeling and PET. Such information may be useful in predicting and increasing tumor response to FU.

Validation of fluorouracil metabolite analysis in excised tumor. Lability of anabolites.

Rapid excision and freezing of tissue commonly is assumed to preserve the molecular composition of the tissue just prior to its removal from the host. We examined the lability of radiolabeled 5-fluorouracil (FUra) and its anabolites during excision and freeze-clamping in a rat tumor model. Acid-soluble metabolites were identified by HPLC. Two rats, each bearing multiple, subcutaneously-implanted colon tumors, were treated with eniluracil (an inactivator of dihydropyrimidine dehydrogenase) to prevent catabolism of FUra and then injected intravenously with [(3)H]FUra. After 2 hr, tumors were harvested sequentially and segmented. The tumor pieces were kept at room temperature for various times up to 4 min prior to freezing. These specimens showed a decrease (P < 0.01) in labeled nucleoside triphosphate content of 13 +/- 2%/min and commensurate increases (P < 0.005) in labeled nucleoside monophosphates and nucleosides with increasing time-to-freeze. The amounts of labeled macromolecules, nucleoside diphosphates, and FUra each remained approximately constant. The study indicates that substantial errors may occur in measured tissue concentrations of pyrimidine nucleosides and nucleotides due to lability during tissue excision and freeze-clamping. Such errors can be corrected using data of the type obtained in this study.

Pharmacokinetics of the thymidine analog 2'-fluoro-5-[(14)C]-methyl-1-beta-D-arabinofuranosyluracil ([(14)C]FMAU) in rat prostate tumor cells.

2'-Fluoro-5-[(14)C]-methyl-1-beta-D-arabinofuranosyluracil (FMAU) is an analog of thymidine (TdR) that is resistant to catabolism, is incorporated into DNA, and has been labeled with (11)C for use with positron emission tomography. We compared the uptake and metabolism of [(14)C]FMAU with that of [(3)H]TdR in fast- and slow-growing cell lines of a rat prostate tumor. Although FMAU was incorporated much less rapidly than TdR, FMAU behaved very similarly to TdR with respect to correlation between uptake velocity and cell growth rate, saturability of cellular incorporation, and intracellular metabolite pools. Thus, FMAU warrants further evaluation as an in vivo indicator of tumor cell division.

Deoxyuridylate effects on thymidylate synthase-5-fluorodeoxyuridylate-folate ternary complex formation.

The competitive basis and specificity of deoxyuridylate (dUMP)-mediated decreases in thymidylate synthase-5'-fluorodeoxyuridylate-folate (TS-FdUMP-folate) ternary complex formation at low concentrations of folates were investigated using charcoal isolation of protein-bound [3H]FuUMP ligand. Reaction conditions used 0.02 microM TS (Lactobacillus casei) and 0.10 microM [3H]FdUMP incubated for 10 min at 37 degrees and pH 7.4. Decreases in counts below control (C) values in dUMP-added samples (S) were expressed as C/S ratios. At CH2--H4PteGlu1 or H4PteGlu1 concentrations below 10 microM, highly linear relationships were found to exist between C/S value and dUMP concentrations, expressed as dUMP/FdUMP ratios. For H4PteGlu1, maximal C/S values for dUMP interference occurred at the lowest H4PteGlu1 concentrations, approaching the value of the TS-FdUMP binary complex. The efficiency of ternary complex formation by H4PteGlu1 was 28 +/- 5% of CH2--H4PteGlu1 values at concentrations below 1.0 microM. The protective effect of increasing H4PteGlu1 against dUMP interference resulted in a linear relationship between the logarithm of H4PteGlu1 concentration and the slope of dUMP interference (C/S vs dUMP/FdUMP). In contrast, the results with CH2--H4PteGlu1 were biphasic. At concentrations of CH2--H4PteGlu1 lower than 0.5 microM, C/S values were greater than those for binary complex alone, a result related to CH2--H4PteGlu1 consumption based on [5-3H]dUMP tritium-release studies. At concentrations of CH2--H4PteGlu1 above 1.0 microM, however, dUMP interference was nearly abolished. Kinetic analysis of the data suggests that this effect of the 5,10-methylene moiety may result in part from positive allosteric effects of first site TS-FdUMP-CH2--H4PteGlu1 ternary complex binding on acceleration of second site binding, in addition to slowed rates of dissociation. Other folylmonoglutamates showed relatively poor TS-[3H]FdUMP-folate complex formation: at 500 microM folate, as a percentage of CH2--H4PteGlu1 values, these were 29.6% for dihydrofolate, 7.5% for 5-CH3--H4PteGlu1, 3.0% for CH = H4PteGlu1, 1.6% for folic acid, 1.1% for 5-CHO--H4PteGlu1 (leucovorin) and 0.9% for 10-CHO--H4PteGlu1. Inhibitory effects by dUMP were consistent with binary complex effects alone for these folates. Study of methotrexate, as the monoglutamate and the hexaglutamate, suggested that ternary complexes with dUMP are favored over those with FdUMP at high concentrations of the antifolate. Our results indicate that activation of leucovorin to over 0.5 microM in intracellular CH2--H4PteGlu1 equivalents may be a requirement for achieving complete TS inhibition by FdUMP in the presence of excess conce

Assay and time course of 5-fluorouracil incorporation into RNA of L1210/0 ascites cells in vivo.

A method for determination of levels of incorporation of nonradiolabeled 5-fluorouracil (FUra) into RNA (F-RNA) in tissue samples is shown to be applicable to tissues in vivo. BDF1 mice bearing L1210 ascites cells were injected intraperitoneally with [14C]FUra, 100 mg/kg. The time course of F-RNA levels in L1210 cells was determined by following the radiolabeled drug, and by NaB3H4 labeling of isolated and derivatized nucleoside. RNA ribonucleotides were obtained by KOH hydrolysis of perchloric acid precipitates of cell sonicates. FUMP nucleotides were separated from remaining nucleotides by DEAE-cellulose chromatography. FUMP fractions were treated with alkaline phosphatase, and FUrd was separated from non-FUrd nucleoside contaminants by additional DEAE-cellulose chromatography. FUrd was quantitated by periodate oxidation of ribose and NaB3H4 reduction of the resulting nucleoside dialdehydes. Isolation of tritiated FUrd-trialcohol from remaining tissue contaminants and background radioactivity was done by silica gel thin layer chromatography. Comparison of results obtained by isolation of [14C]FUrd with results of NaB3H4 labeling of the same samples showed parallel results with comparable biological standard deviations, although the tritium method consistently gave slightly lower values. The peak level of F-RNA at 3 hr was 1 base substitution per 174 normal nucleotides. The level of F-RNA after 3 hr declined slowly, so that at 96 hr there still remained 1 FUra base per 597 normal nucleotides. Serial determinations of RNA content showed marked decreases, on the basis of either DNA or protein level, that continued up to 96 hr after FUra administration. These biochemical effects are among the most prolonged reported for FUra, suggesting the possibility that F-RNA represents a storage compartment for release of toxic metabolites and emphasize the need for additional study of RNA effects at long time points. Our method for assay of F-RNA appears to be suitable for study of biopsy specimens of tumors and normal tissues following nonradiolabeled-FUra administration.

In vivo kinetics of thymidylate synthetase inhibition of 5-fluorouracil-sensitive and -resistant murine colon adenocarcinomas.

The predictive utility of several biochemical parameters of 5-fluorouracil (5-FUra) action was evaluated in four murine colonic adenocarcinomas: 5-FUra-sensitive Tumor 38 and 5-FUra-resistant Tumors 07/A, 51 and 06/A. Thymidylate synthetase (TS) was determined by a tritiated 5-fluoro-2'-deoxyuridylate (FdUMP)-binding assay. Bolus 5-FUra (80 mg/kg, i.p.) administrated caused in all tumors a rapid decrease in free TS levels. Only Tumor 38, however, showed inhibition of TS to undetectable (less than 0.05 pmol/g) levels, which lasted up to 6 hr after treatment; correction for dissociation of endogenous TS: FdUMP:folate ternary complex during the TS assay was required. Total TS (free enzyme plus ternary complex) was determined with experimental conditions that achieved quantitative recovery of free TS from ternary complex. By 48 hr after 5-FUra, Tumor 38 showed a decrease in total TS proportional to the estimated log kill/dose of 5-FUra; in contrast, the resistant tumors showed no such decrease from pretreatment levels. Assay of FdUMP showed that the free nucleotide was formed rapidly in all tumors in excess over available TS-binding sites. However, tumor sensitivity did not correlate with peak or residual FdUMP levels or with deoxyuridylate levels, which were low and remained so in all tumors. Tumor sensitivity to 5-FUra also could not be explained by the small differences among the tumors in total perchloric acid-soluble metabolites of 5-FUra or drug incorporation into RNA. We conclude from these data that levels of free TS in the tumor after 5-FUra treatment are predictive of chemotherapeutic response in these murine models of human colonic adenocarcinoma.