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Biomedical subjects

D G Priest

Publications and source records attributed to D G Priest.

At least 37 records · Page 2Linked to original sources
PubMed1994

Disposition of folic acid and its metabolites: a comparison with leucovorin.

A pharmacokinetic study of folic acid and its metabolites was conducted to provide a basis to consider folic acid as a therapeutic alternative to leucovorin. Leucovorin has been used in various folate antagonist rescue regimens and to modulate fluorouracil activity in the treatment of solid tumors. Although leucovorin is typically administered intravenously in fluorouracil modulation therapy, limited oral administration trials have yielded equivalent responses. Because metabolites rather than leucovorin are the predominant circulating species after oral administration, these clinical results indicate that metabolites themselves can be modulating agents. Folic acid could be an attractive alternative to leucovorin provided it effectively elevates the same plasma metabolites. Hence, folic acid at doses of 25 and 125 mg/m2 was administered orally and intravenously to normal volunteers. Plasma folic acid and its reduced folate metabolites were monitored over a 24-hour period by use of a previously developed radioenzymatic method. The metabolites that accumulated--5-methyltetrahydrofolate, 5,10-methylenetetrahydrofolate, tetrahydrofolate, and 10-formyltetrahydrofolate--were the same metabolites that were observed previously after leucovorin administration. Folic acid metabolites accumulated more slowly and persisted longer than leucovorin metabolites, which can be attributed to slower metabolism of the fully oxidized vitamin. Based on these results, it is concluded that folic acid could be an attractive therapeutic alternative to leucovorin for fluorouracil modulation.

Adult↗
PubMed1991

Pharmacokinetics of leucovorin metabolites in human plasma as a function of dose administered orally and intravenously.

Studies have shown that conversion of leucovorin to the metabolite 5,10-methylenetetrahydrofolate (5,10-CH2FH4) is responsible for enhancement of the antitumor effects of fluorouracil given in combination with leucovorin, but the biochemical basis of this conversion in humans is not fully understood. To determine a possible sequence of metabolic steps, we studied the pharmacokinetics of leucovorin and its reduced folate metabolites in plasma in healthy volunteers. Groups of five subjects were given two equal doses of 10, 25, 125, 250, or 500 mg/m2 leucovorin, one orally and one intravenously at a 30-day interval. A sensitive radioenzymatic method that we developed previously was used to measure plasma concentrations of [S]5-formyltetrahydrofolate, 10-formyltetrahydrofolate (10-CHOFH4), 5-methyltetrahydrofolate (5-CH3FH4), and the combined 5,10-CH2FH4 plus tetrahydrofolate (FH4) pools. Intravenous administration of leucovorin resulted in dose-dependent accumulation of 5,10-CH2FH4 + FH4 exceeding 2 microM at peak levels. After oral and intravenous administration, 10-CHOFH4 and 5,10-CH2FH4 + FH4 exhibited peak levels earlier and were eliminated more rapidly than 5-CH3FH4. Accumulation of all metabolites after intravenous administration was linearly dose dependent, while oral administration appeared to result in saturation. We propose that the host activation of leucovorin suggested by these findings could be responsible for elevation of intratumor 5,10-CH2FH4 levels, thus enhancing the antitumor effects of fluorouracil. These results also suggest that 10-CHOFH4, 5,10-CH2FH4, and FH4 are intermediate metabolites and that 5-CH3FH4 is the terminal metabolite. In addition, our results indicate that attainment of high plasma levels of the metabolites active in modulation of the therapeutic effects of fluorouracil is best achieved through intravenous administration of high doses of leucovorin. Our future studies will address the proposed sequential conversion pathway and, thus, the mechanism by which pharmacologically relevant reduced folates accumulate in plasma after leucovorin administration.

Administration, Oral↗
PubMed1991

A sensitive radioenzymatic assay for (S)-5-formyltetrahydrofolate.

A highly sensitive, radioenzymatic method has been developed for the specific and quantitative estimation of (S)-5-formyltetrahydrofolate. This method is based on enzymatic cycling of the 5-formyl derivative to methylenetetrahydrofolate followed by entrapment into a stable ternary complex with thymidylate synthase and tritiated fluorodeoxyuridylate. Determination of bound radiolabeled ligand permits estimation of the original folate. The initial cycling step is catalyzed by the enzyme, methenyltetrahydrofolate synthetase, which is specific for the (S)-diastereomer of 5-formyltetrahydrofolate and generates a product which can be further cycled to tetrahydrofolate using either 10-formyltetrahydrofolate deacylase or glycinamide ribonucleotide transformylase. Tetrahydrofolate is ultimately converted to the entrapable methylene derivative in the presence of excess formaldehyde. Using this assay recovery of reference (S)-5-formyltetrahydrofolate was linear over the range 0.03-1.9 pmol with an average recovery of 83 +/- 2%. The method has been applied to estimation of plasma (S)-5-formyltetrahydrofolate from a volunteer who had been administered (R,S)-5-formyltetrahydrofolate. Where comparison was possible, estimation of plasma (S)-5-formyltetrahydrofolate by this one step ternary complex-based method yielded results that were very similar to those observed by Straw et al. (Cancer Res., 44, 3114, 1984) who used an HPLC-based method for separation of diastereomeric mixtures of reduced folates and microbiological growth dependence to determine (S)-5-formyltetrahydrofolate.

Animals↗
PubMed1991

Human red blood cell-mediated metabolism of leucovorin [(R,S)5-formyltetrahydrofolate].

The ability of human blood in vitro, and partially purified red blood cells, to metabolize leucovorin, or 5-formyltetrahydrofolate, has been examined. A radioenzymatic assay based upon entrapment of 5,10-methylenetetrahydrofolate, and other reduced folates after cycling to this form, into a stable ternary complex with thymidylate synthase and tritiated 5-fluoro-2'-deoxyuridine-5'-monophosphate was used to estimate reduced folate metabolites. Incubation of whole blood samples with (R,S)5-formyltetrahydrofolate resulted in a time- and concentration-dependent extracellular accumulation of the reduced folates, 5-methyltetrahydrofolate, tetrahydrofolate, 10-formyltetrahydrofolate, and 5,10-methylenetetrahydrofolate. While accumulation with time was nonlinear, the tetrahydrofolate pool showed the greatest overall increase in concentration. 5-Methyltetrahydrofolate, which was the only reduced folate detected in plasma prior to introduction of (R,S)5-formyltetrahydrofolate, accumulated more slowly than tetrahydrofolate. 10-Formyltetrahydrofolate and 5,10-methylenetetrahydrofolate accumulated even more slowly but exhibited nonlinear kinetic patterns similar to those of tetrahydrofolate and 5-methyltetrahydrofolate. When blood cells were removed by centrifugation, a complete loss of metabolic activity was observed. Exposure of purified red blood cells to (R,S)5-formyltetrahydrofolate resulted in accumulation of extracellular reduced folates that was similar to that in whole blood samples while partially purified white blood cells exhibited little activity. Metabolism of the (S) diastereomer of 5-formyltetrahydrofolate accounted for essentially all of the observed extracellular accumulation of reduced folates. We propose that red blood cell-mediated metabolism of 5-formyltetrahydrofolate could, in part at least, account for reduced folate accumulation in plasma when leucovorin is administered to humans.

Biotransformation↗
PubMed1991

Role of folylpolyglutamates in biochemical modulation of fluoropyrimidines by leucovorin.

The growth-inhibitory effect of fluoropyrimidines combined with a short-term exposure to leucovorin and the pattern of polyglutamylation of folates were compared between parental CCRF-CEM cells and a cell line with impaired ability to form polyglutamates (CCRF-CEM/P). The combination of leucovorin with 5-fluorouracil or 5-fluorodeoxyuridine increased the growth inhibition of CCRF-CEM cells compared to the fluoropyrimidine alone in the parent cell line but not in CCRF-CEM/P cells. In addition, leucovorin produced a significant increase in the inhibition of intracellular thymidylate synthase activity caused by 5-fluorouracil or 5-fluorodeoxyuridine as compared to these drugs alone in CCRF-CEM cells, but no increase in inhibition over that produced by the single drugs alone was observed in CCRF-CEM/P cells. Although levels of 5,10-methylene tetrahydrofolate after leucovorin administration were similar in both cell lines, polyglutamylation of this coenzyme was decreased in the CCRF-CEM/P cell line. The inability of CCRF-CEM/P cells to form significant levels of polyglutamates of N5,N10-methylenete-trahydrofolate, may be responsible for the lack of enhanced cell kill observed when a short exposure to leucovorin is used with fluoropyrimidines.

Cell Division↗
PubMed1991

Inhibition of mammalian thymidylate synthase by 10-formyltetrahydropteroylpolyglutamate.

Reduced derivatives of 10-formylfolate have been evaluated as inhibitors of mammalian thymidylate synthase (EC 2.1.1.45) from H35 hepatoma cells. With 5,10-methylenetetrahydrofolylheptaglutamate as the substrate, 10-formyltetrahydrofolylmonoglutamate is a competitive inhibitor with a Ki of 2.4 microM, which is reduced to 0.1 microM for the heptaglutamate derivative. 10-Formyldihydrofolylmono- and -heptaglutamate are approximately threefold less inhibitory than the tetrahydro analog. The concentrations of 10-formyltetrahydrofolate and 10-formyldihydrofolate were measured in dividing hepatoma cells by a novel enzymatic assay and were found to be 5 microM and undetectable, respectively. These results suggest that the concentration of 10-formyltetrahydrofolate within the dividing cells has the potential to severely inhibit or modulate thymidylate biosynthesis.

Animals↗
PubMed1990

Regulation of drosophila folylpolyglutamates during development.

The polyglutamate status of reduced folates during the larval, pupal and adult stages of Drosophila melanogaster development was investigated. The chain length distribution is very similar and is predominantly pentaglutamate. Half-life estimates of the hydrolytic degradation to the monoglutamate showed larva less than pupa less than adult. This raises the possibility that polyglutamate hydrolase may have a role in regulating the total intracellular reduced folate content of the different developmental stages.

Animals↗
PubMed1990

Role of substrate depletion in the inhibition of thymidylate biosynthesis by the dihydrofolate reductase inhibitor trimetrexate in cultured hepatoma cells.

The effects of the lipid-soluble dihydrofolate reductase inhibitor, trimetrexate, on the inhibition of thymidylate biosynthesis as a result of perturbation in cellular folate pools in H35 hepatoma cells in vitro has been investigated. Exposure of the cultures to increasing concentrations of trimetrexate between 2 and 20 nM causes a marked reduction in de novo thymidylate biosynthesis and a concomitant decrease in (6R)5,10-methylenetetrahydropteroylpolyglutamate (5,10-CH2H4PteGlun) from 2.0-0.2 microM, respectively. This is accompanied by an increase in H2PteGlun from 1.2 microM in control cultures to 4.7 microM in cultures exposed to 20 nM trimetrexate. The dependency of de novo thymidylate biosynthesis on intracellular 5,10-CH2H4PteGlun in trimetrexate-treated cells is compared with (a) the relationship of thymidylate biosynthesis on intracellular levels of 5,10-CH2H4PteGlun in folate-depleted cells supplemented with increments of folic acid and (b) the substrate (5,10-CH2H4PteGlun) dependence of purified thymidylate synthase from the same source. All three results are nearly identical demonstrating that trimetrexate-dependent inhibition of de novo thymidylate biosynthesis is primarily a result of substrate depletion. These results coupled with the weak inhibitory properties of H2PteGlun for thymidylate synthase Ki = 5.0 microM) suggest that H2PteGlun accumulation is not the major determinant in inhibiting thymidylate synthase following trimetrexate inhibition but under certain conditions has the potential to enhance the inhibition caused by substrate depletion.

Carcinoma, Hepatocellular↗
PubMed1989

Relationship of reduced folate changes to inhibition of DNA synthesis induced by methotrexate in L1210 cells in vivo.

Reduced folate levels and DNA synthesis were examined in L1210 cells in mice after exposure to a wide range of methotrexate doses. A radioenzymatic assay based upon entrapment of tissue 5,10-methylenetetrahydrofolate (CH2FH4), and other reduced folates after cycling to this form, into a stable ternary complex with thymidylate synthase and [3H]-5-fluoro-2'-deoxyuridine 5'-monophosphate was used to estimate reduced folates. DNA synthesis was estimated from incorporation of [3H]-2'-deoxyuridine into DNA. The predominant reduced folate in cells from untreated animals was 10-formyltetrahydrofolate (10-CHOFH4; 3.59 pmol/10(6) cells). The four other reduced folates measured, tetrahydrofolate (FH4), CH2FH4, dihydrofolate (FH2), and 5-methyltetrahydrofolate (5-CH3FH4), were present in nearly equal amounts yielding a total reduced folate level of 6.24 pmol/10(6) cells. When methotrexate was administered s.c. at doses of 1.5, 12, and 400 mg/kg, the level of FH2 increased dramatically and total tetrahydrofolates measured decreased extensively within 1 h. DNA synthesis was completely inhibited during the first 1-2 h after administration of each dose of methotrexate with onset of recovery after 4 and 20 h at 1.5 and 12 mg/kg and not at all after the highest dose. Both FH4 and CH2FH4 were extensively depleted at 12 and 400 mg/kg methotrexate but considerably less depletion of CH2FH4, and none of FH4, was observed at 1.5 mg/kg during a period when DNA synthesis was essentially abolished. The metabolically linked 5-CH3FH4 and 10-CHOFH4 pools were also extensively depleted following treatment with methotrexate. While FH2 levels expanded extensively after drug treatment, the total increase did not account for the extent of depletion of the combined tetrahydrofolate pools. The change in concentration with time of any one folate pool was apparently not sufficient to explain completely the duration of inhibition of DNA synthesis; however, sustained inhibition of DNA synthesis was generally associated with maintenance of an expanded FH2 pool and delay in repletion of the combined tetrahydrofolate pools. Discussion is presented with respect to the impact of these results on changing notions of the mode of action of classical antifolates.

Animals↗
PubMed1989

The role of cellular folates in the enhancement of activity of the thymidylate synthase inhibitor 10-propargyl-5,8-dideazafolate against hepatoma cells in vitro by inhibitors of dihydrofolate reductase.

Exposure of growing cultures of hepatoma cells in vitro to the lipid-soluble dihydrofolate reductase inhibitors metoprine (36 nM) or trimetrexate (2 nM) at subtoxic concentrations causes little change in cell growth rate, colony forming ability, cell cycle distribution, and de novo purine and thymidylate biosynthesis. The reductase inhibitors augment the cytotoxic activity of the thymidylate synthase inhibitor, 10-propargyl-5,8-dideazafolate by nearly 10-fold under optimal conditions. Treatment of the hepatoma cells with the reductase inhibitors for 72 h during growth caused approximately a 75% reduction in total cellular folates and 5,10-methylenetetrahydrofolate (primarily as polyglutamates) the substrate for thymidylate synthase. The reductase inhibitors also cause a doubling in the accumulation of 10-propargyl-5,8-dideazafolate polyglutamates. The combined antifolate treatment (metoprine or trimetrexate plus 10-propargyl-5,8-dideazafolate) expands the dUMP pool by 30-fold, which is more than the sum of either of the antifolates alone. Consequently, it is postulated that the enhanced activity of 10-propargyl-5,8-dideazafolate in combination with low concentrations of dihydrofolate reductase inhibitors is due to an increase in the ratio of inhibitor to substrate for thymidylate synthase of nearly 10-fold and an extensive enhancement of the dUMP pool. These conditions predispose the target enzyme and the cells to more effective metabolic blockade by 10-propargyl-5,8-dideazafolate which is presumably caused by the formation of an inhibited 10-propargyl-5,8-dideazafolate[polyglutamate]-thymidylate synthase-dUMP ternary complex.

Animals↗
PubMed1989

The effect of methionine on methotrexate metabolism in rat hepatocytes in monolayer culture.

The effect of methyl donors on the metabolism of methotrexate has been investigated in rat hepatocytes in monolayer culture. Pulse exposure to low concentrations of methotrexate (1 microM, 3h) in the absence of methionine results in the facile formation of the di- to pentaglutamates with the di- and triglutamate predominating. Further incubation after the removal of methotrexate (MTX) results in a shift to the tetra- and pentaglutamate at the expense of the shorter chain length derivatives. The same measurement in the presence of 1 mM methionine causes approx. an 80% inhibition in the formation of polyglutamates. This effect can be partially achieved when methionine is replaced by choline or betaine. No alteration in the formation of 7-hydroxymethotrexate could be detected by similar changes in methionine concentrations in the medium. The activity of the enzymes which synthesize and degrade methotrexate polyglutamates, folylpolyglutamate synthetase and gamma-glutamyl hydrolase, respectively, were the same in extracts of cells grown in the absence and in the presence of 1 mM methionine. Incubation of the hepatocytes with methionine causes a significant increase in 5,6,7,8-tetrahydrofolate (H4folate), 5,10-methylenehydrofolate and 10-formyltetrahydrofolate and a decrease in 5-methyltetrahydrofolate. These results suggest that the inhibition of glutamylation of methotrexate could be due in part to an elevation in reduced folates which can more effectively compete with methotrexate as a substrate for folylpolyglutamate synthetase. Inhibition in methotrexate glutamylation by methionine, betaine and choline in hepatocytes may contribute to the alleviation of hepatic toxicity by methyl donors.

Animals↗
PubMed1989

Accumulation of tetrahydrofolates in human plasma after leucovorin administration.

Reduced folates in plasma after i.v. and oral leucovorin administration were estimated by a ternary complex assay based on the incorporation of CH2FH4 into a stable complex with Lactobacillus casei thymidylate synthase and [3H]FdUMP. Each of the reduced folates, CH2FH4, FH4, and 5CH3FH4, could be quantitatively recovered from plasma by this approach even in the presence of high concentrations of the parent compound leucovorin. Examination of the accumulation kinetics of these reduced folates showed that after i.v. administration of 20 mg D,L-leucovorin to a healthy volunteer, FH4 and, to a lesser extent, CH2FH4 accumulated to maximal levels very early (less than 15 min), with a subsequent depletion that had a half-life of approximately 30 min. Accumulation of FH4 reached a peak level that was 12% of the maximal level of 5CH3FH4 achieved and more than 3 times greater than the pretreatment level of this common, circulating reduced folate form. Similar accumulation patterns were observed in a female patient with metastatic colonic cancer who was undergoing methotrexate (MTX)/fluorouracil therapy followed by i.v. leucovorin (15 mg). FH4 also accumulated, but to a lesser extent and over a longer period of time, when the same dose of leucovorin given orally. When several similar doses of leucovorin were given prior to the experimental dose, greater accumulation and duration of the FH4 response was observed. We propose that accumulation of FH4 and CH2FH4 could provide a circulating source of the reduced folate thought to be the active form for both high-dose MTX with leucovorin rescue and enhancement of fluorouracil activity.

Administration, Oral↗
PubMed1988

Role of methylenetetrahydrofolate depletion in methotrexate-mediated intracellular thymidylate synthesis inhibition in cultured L1210 cells.

The effect of low methotrexate levels on methylenetetrahydrofolate and four other reduced folate pools in cultured L1210 cells has been examined over a 48-h period. Media folate levels and methotrexate were used to alter intracellular levels of reduced folates, and the distribution among individual reduced folates, so that they could be evaluated in terms of their effects on thymidylate synthesis and cell proliferation. Over the media folate concentration range of 0.25-50 microM, growth rate and thymidylate synthesis remained essentially unchanged while total intracellular reduced folates, determined from the summation of the five individual pools measured, increased approximately 25-fold. The 5-methyltetrahydrofolate and 10-formyltetrahydrofolate pools accounted for over 90% of the total reduced folate at the highest media folate level, while low media folate resulted in a much more equal distribution among the five reduced folates examined. Methotrexate, over the concentration range of 0.25-30 nM, caused extensive growth and intracellular thymidylate synthesis inhibition at media folate levels used in RPMI 1640 media (2.5 microM) and lower. However, growth inhibition was much less at the highest media folate level used, and thymidylate synthesis was not inhibited to a statistically significant extent. Intracellular reduced folates also responded differently to methotrexate depending upon the level of media folate. Depletion of the thymidylate synthase substrate, methylenetetrahydrofolate, could not account for diminished growth or thymidylate synthesis inhibition, since at 0.25 and 2.5 microM media folate no depletion occurred in response to methotrexate and only slight depletion was observed at 50 microM media folate. Dihydrofolate showed a tendency to increase at each of the media folate levels used with the least increase at the highest folate level. However, the ratio of dihydrofolate to total reduced folates was quantitatively most consistent with thymidylate synthesis and growth inhibition results.

Animals↗
PubMed1988

Enhancement of polyacrylamide gel slice dissolution in hydrogen peroxide by cupric sulfate.

An improved method is described for quantitation of radio-labelled protein by scintillation counting after polyacrylamide gel electrophoresis. The method is based upon copper catalyzed dissolution of gel slices in hydrogen peroxide under ambient conditions. Complete dissolution of gel sections was accomplished by incubation at 25 degrees C in 30% H2O2 that contained 0.9 mM CuSO4. Recovery of tritiated protein was greater than 90% under these conditions while in the absence of CuSO4 recovery was less than 50%.

Acrylic Resins↗