Aspects of one-carbon folate cycling related to fluoropyrimidine and antifolate therapy.
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Animals↗
Biomedical subjects
B G Gustavsson
Publications and source records attributed to B G Gustavsson.
Sequential 5-fluorouracil and leucovorin in patients with advanced symptomatic gastrointestinal cancer.
50 patients with advanced symptomatic gastrointestinal cancer were treated with sequential 5-fluorouracil (5-FU)/leucovorin. Patients received an intravenous bolus injection of 5-FU (500 or 600 mg/m2) and leucovorin 30-40 min later, either 50 mg (41 patients) or 200 mg (9 patients). Treatment was given in repeated courses either once weekly or on 2 consecutive days every other week until progression. Toxicity was mild with the lower leucovorin dose, although grade 2 toxicity, particularly diarrhoea, occurred in 27 (66%) patients. All patients receiving the higher leucovorin dose had grade 2-4 toxicity. Toxicity was less with the lower 5-FU dose. Out of 40 patients with colorectal cancer, 34 received leucovorin 50 mg and 6 received 200 mg. Partial response occurred in 10 (29%) and 1 of these patients, respectively. This sequential 5-FU and intermediate-dose leucovorin regimen has acceptable toxicity and a definite anti-tumour activity.
Adult↗
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
Deoxyuracil Nucleotides↗
Folinic acid modulation of fluorouracil: tissue kinetics of bolus administration.
Thymidylate synthase (TS) is the enzyme target of 5-fluorouracil (FUra) that recent laboratory and clinical studies with folinic acid (calcium leucovorin) suggest may mediate important antitumor cytotoxicity. Measurement in carcinoma tissue of parameters related to TS inhibition by 5-fluorodeoxyuridylate (FdUMP), by analogy to hormone receptor analysis, should be useful to determine which patients should receive fluoropyrimidine drug therapy and to evaluate folinic acid requirements. Folinic acid is metabolized to 5,10-methylenetetrahydropteroylglutamine (CH2FH4), which must be present in large excess to effect desired levels of maximal inhibition of TS, by promoting formation and stabilization of TS-FdUMP-CH2FH4 ternary complexes. In patients with metastatic disease, serial biopsies of tumor and normal tissues for studies of pharmacodynamic responses to test-dose FUra or folinic acid are shown to be easily added to routine intraoperative management. A suitable methodologic approach is described and examples given of assays of free TS, FdUMP, dUMP, and CH2FH4 levels after FUra or folinic acid, that may be useful in future studies aimed at improving the cost-effectiveness of FUra-folinic acid combinations.
Aged↗
"Half-way" venous catheters: developmental work on insertion.
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Adolescent↗
Mechanisms of innate resistance to thymidylate synthase inhibition after 5-fluorouracil.
Fifty-four patients with metastatic adenocarcinoma received i.v. bolus 5-fluorouracil, 500 mg/m2, prior to surgical biopsy of tumor at 20-400 min, for analysis of biochemical parameters of resistance to thymidylate synthase (TS) inhibition. The majority of patients, 37, had colon or rectal adenocarcinoma, five had breast cancer, five had gastric primary disease, four had pancreatic adenocarcinoma, and three had hepatocellular adenocarcinoma. Fluorodeoxyuridylate (FdUMP) was assayed by isotope dilution of [3H]FdUMP binding to bacterial TS; free and total TS was determined by [3H]FdUMP binding; and deoxyuridylate (dUMP) was assayed by conversion to [14C]thymidylate. Free levels of TS were lower in breast cancers, 0.08 +/- 0.06 pmol/g, than in other histologies (overall average, 1.41 +/- 2.25), associated with significantly greater percentages of TS inhibition (88.6% versus 62.0% overall). Colorectal tumors showed significantly greater FdUMP levels than other gastrointestinal malignancies, associated with somewhat lower free TS values. Plots of FdUMP levels, or (FdUMP/dUMP) x 100 values versus percentages of TS inhibition suggested minima of 75 pmol/g and 0.10, respectively, for achieving maximal enzyme inhibition. Analyses of normal tissues showed: poor TS inhibition in liver and normal colonic mucosa, related to low FdUMP levels; and very high dUMP levels in bone marrow leukocytes suggestive of reactive increases in dUMP as an important mechanism of recovery in this tissue. Among the 30 of the 37 colorectal tumors that showed suboptimal (less than 85%) inhibition of TS, 16 (53%) showed FdUMP levels less than 75 pmol/g, 8 (27%) showed relatively high dUMP levels (over 35 nmol/g), and 16 (53%) showed poor efficiency of inhibition of TS, with the major overlap between these mechanisms of resistance being high dUMP and poor binding in 6 (20%). These data provide a strong rationale for administration of leucovorin to the majority of patients receiving 5-fluorouracil, since increased intratumoral reduced folates potentially can overcome multiple mechanisms of resistance including low FdUMP, high dUMP, and high total TS levels, in addition to that caused by isolated folate deficiency.
Adenocarcinoma↗
Pharmacodynamics of 5-fluorouracil and leucovorin.
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Adenocarcinoma↗
Methods for thymidylate synthase pharmacodynamics: serial biopsy, free and total TS, FdUMP and dUMP, and H4PteGlu and CH2-H4PteGlu assays.
This report details our methods for performance of the major parameters related to quantitation of TS inhibition resulting from fluoropyrimidine administration to patients, methods equally applicable to preclinical studies. Sampling of tumors before and after drug treatment is done by 4 mm disposable punch biopsy or forceps biopsy via subcutaneous tunneling. Homogenates are prepared using N2 or polytron-mincing. Cytosolic free TS is measured by either the tritium-release method for small biopsies or by [3H]FdUMP ligand-binding. FdUMP and dUMP are separated by DEAE-cellulose column and measured by competitive binding and [14C]dTMP synthesis by the Moran methods. Total, post-FUra TS is measured by pre-incubation dissociation of FdUMP-bound TS after neutral charcoal removal of cytosolic ligands. H4PteGlu and CH2-H4PteGlu are measured by the Priest method using L. Casei TS. The materials and methods are described in sufficient detail to permit wide application of this approach.
Biopsy, Needle↗
Sequential methotrexate/5-FU: FdUMP formation and TS inhibition in a transplantable rodent colon adenocarcinoma.
Parameters for inhibition of thymidylate synthetase were studied after sequential methotrexate/5-fluorouracil (5-FU) administration in a dimethylhydrazine (DMH)-induced transplantable rat colon carcinoma. Tumor-bearing rats were treated with methotrexate (MTX) 40 mg/kg IP Bolus 5-FU, 100 mg/kg IP, was injected after 24 h. Micromethods for assay of 5-fluoro-2'-deoxyuridylate (FdUMP) and thymidylate synthetase (TS) were used to study the in vivo intracellular pharmacokinetics of 5-FU. Formation of FdUMP was equally rapid in tumors regardless of MTX pretreatment, with peak values found at 30 min. Although MTX pretreatment did not increase peak FdUMP levels, it appeared to result in increased persistence of FdUMP, well in excess of available TS-binding sites, at 24 and 48 h. The combination therapy was less effective in terms of TS inhibition over the first 8 h after 5-FU administration, but may have been associated with improved TS inhibition at later time points. Total levels of TS (TStot) steadily increased from a pre-5-FU treatment level of 18.8 pmol to more than 40 pmol/g at 24 h. MTX per se had no apparent effect on baseline TStot levels or on the 5-FU-mediated increases in TStot. We conclude that MTX and 5-FU were antagonistic in terms of TS inhibition over the first 8 h after 5-FU in this DMH-induced rat colon carcinoma, but were possibly synergistic in increasing persistent levels of FdUMP and TS inhibition at later time points. The observation that 5-FU treatment can result in progressive increases in TS levels in some tumors suggests that this may be an important mechanism of 5-FU resistance.
Adenocarcinoma↗
Exchange of occluded, tunnelled, subclavian central venous catheters. A technique reusing the existing tunnel.
A technique for the exchange of occluded, tunnelled, subclavian venous catheters reusing the existing tunnel is described. It was successfully used 14 times in 5 patients needing permanent parenteral nutrition at home, in whom insertion of a new catheter by another route would have been cumbersome and hazardous. The tunnel was opened below the clavicle, and the catheter clamped. The catheter was then cut close to the tunnel exist, and its distal part removed centrally through the opening at the clavicle. A guide wire was simultaneously pulled through. An introducer cannula was placed over the central part of the catheter into the subclavian vein, and the occluded catheter was withdrawn and a new one inserted. The latter was then guided through the tunnel by the guide wire. The catheter was then fixed, and the wound was closed and dressed. Prophylactic cloxacillin was given i.v. for 3 days. No bleeding, pulmonary embolism, local infection or sepsis were observed.
Adult↗
Thymidylate synthetase inhibition in malignant tumors and normal liver of patients given intravenous 5-fluorouracil.
Single surgical biopsies of solid tumor were obtained at 20 to 240 min after drug administration in 21 patients given first-dose bolus i.v. 5-fluorouracil (5-FUra), 500 mg/sq m, and assayed for 5-fluorodeoxyuridylate (FdUMP), deoxyuridylate (dUMP), total thymidylate synthetase (TS), and non-FdUMP-bound, free enzyme. Nineteen patients had cancer of gastrointestinal origin, 13 of these colorectal, and 2 patients had breast adenocarcinoma. In 9 patients, synchronous biopsies of surgically normal liver were obtained along with samples of hepatic tumors metastatic from gastrointestinal sites. Total TS averaged 4.18 pmol/g in the malignant tissues and 2.23 pmol/g in liver. FdUMP levels in the gastrointestinal tumors were higher than in normal liver, were highest at the earliest time interval studied, 20 to 30 min, and appeared to decrease exponentially through 120 min. TS inhibition averaged 70 to 80% in gastrointestinal tumor biopsies and less than 50% in normal liver. Levels of dUMP were low and varied little with time. Those gastrointestinal tumors with higher FdUMP:dUMP ratios showed significantly greater TS inhibition. Tumors of 3 patients who benefited from 5-FUra therapy (1 patient with colonic adenocarcinoma and the 2 patients with breast adenocarcinoma) showed greater TS inhibition than did tumors of remaining patients. It is concluded that the apparent time course changes observed in FdUMP, dUMP, and TS in the grouped data are qualitatively similar to findings of murine studies in vivo and that the relationship between FdUMP:dUMP ratios and TS inhibition are consistent with established in vitro enzymic kinetics. Thus, biopsies of tumors at short time periods after 5-FUra administration may be usefully studied for biochemical parameters of TS inhibition, with the objectives of correlation of sensitivity to subsequent 5-FUra therapy and clarification of mechanisms of drug resistance.
Adult↗
Pharmacokinetic studies of 5-fluorouracil after oral and intravenous administration in man.
The pharmacokinetic conditions for oral administration of 5-fluorouracil (5-FU) were investigated in 16 patients with malignant liver tumours. The concentration of 5-FU in portal and systemic blood was determined by a microbiologic method every 10 min. during 2 hours after oral or intravenous administration of a standard dose of 250 mg 5-FU ( approximately 4 mg/kg b. wt.) or 15 mg 5-FU/kg b. wt. The drug was rapidly absorbed after oral administration with peak values within 10-30 min. 25% of the lower and 40% of the higher oral dose reached the systemic circulation. The reduction of systemic bio-availability was partly accomplished by a loss in the gastrointestinal tract and partly by extraction by the liver. The hepatic extraction ratio was calculated to 0.56 and 0.26 after the lower and the higher dose respectively indicating a saturable process. The availability of 5-FU was significantly higher in portal blood than in systemic blood after oral administration. The opposite conditions were found after intravenous administration. Thus, oral administration of 5-FU to patients with malignant liver tumours seems rational.
Administration, Oral↗
Regional and systemic serum concentrations of 5-fluorouracil after portal and intravenous infusion: an experimental study in dogs.
The serum and urinary concentrations of 5-FU after continuous portal and jugular infusion have been followed by means of a highly sensitive microbiological assay method. Our data indicate that more than 90% of 5-FU was eliminated in the liver after continuous portal infusion of 0.625 mg x kg-1 x hr-1, corresponding to a dose of 15 mg x kg-1 x 24 hr-1. Negligible amounts of intact 5-FU were excreted into the bile, and the urinary excretion was only a few percent of the amount infused. The arterial concentration was on average tenfold higher during the continuous jugular infusion than after the continuous portal infusion, indicating that the route of administration has a pronounced effect on the disposition of 5-FU. Twenty-three percent of the jugular dose reached the liver; 77% was degraded by extrahepatic metabolism. Of these, degradation in the prehepatic splanchnic area accounted for 15%.
Animals↗
5-Fluorouracil plus leucovorin as adjuvant treatment of an experimental liver tumor in rats.
Recent experimental and clinical work has shown that leucovorin potentiates the cytotoxic effect of 5-fluorouracil (5-FU). To investigate the adjuvant role for this combination, 5-FU, 30 mg/kg, combined with leucovorin, 15 mg/kg, was administered intraperitoneally on 3 consecutive days beginning on the same day as tumor cell inoculation in the liver. Tumor take and tumor volume were registered on day 14, followed by recording of survival time. The results of the combined treatment were compared with treatment with 5-FU alone. Leucovorin in combination with 5-FU, but not 5-FU alone, significantly reduced the tumor take compared with untreated animals (p less than 0.01). The combination also resulted in smaller tumors compared with untreated animals (p less than 0.001) or with animals given 5-FU alone (p less than 0.01). The present study supports the use of leucovorin in combination with 5-FU as adjuvant treatment of patients with primary colorectal cancer.
Adenocarcinoma↗