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Purpose: To determine the identity of the transport process that is responsible for the uptake of N5-methyltetrahydrofolate, the predominant form of folate in blood, into cultured human retinal pigment epithelial cells., Methods: Human retinal pigment epithelial cells were cultured on an impermeable plastic support, and the characteristics of the uptake of radiolabeled N5-methyltetrahydrofolate into the cells were investigated. The expression of the folate receptor and the reduced-folate transporter in these cells was evaluated by functional assays and by Northern blot analysis. In addition, the characteristics of N5-methyltetrahydrofolate uptake in these cells were compared with those in folate transport-defective human breast cancer cells that were manipulated to express functionally by transfection the cloned human folate receptor and the human reduced-folate transporter., Results: Transport of N5-methyltetrahydrofolate into these cells occurred by a single saturable process with a Michaelis-Menten constant of 0.13 +/- 0.01 microM. The process was specific for such reduced folates as N5-methyltetrahydrofolate and N5-formyltetrahydrofolate. Nonreduced folate interacted with this transport process only weakly. The transport process was inhibited by anion transport inhibitors. Results of Northern blot analysis indicated the presence of transcripts specific for the reduced-folate transporter in these cells. These cells expressed very small amounts of the folate receptor evidenced from the binding of folate and from the detectable presence of folate receptor-specific transcript, but there was no evidence for participation of the receptor in the observed transport of N5-methyltetrahydrofolate. There was also no evidence in these cells for expression of the folate transporter that prefers nonreduced folate as a substrate., Conclusions: Transport of N5-methyltetrahydrofolate in human retinal pigment epithelial cells occurs exclusively through the reduced-folate transporter. The folate receptor is expressed at negligible levels in these cells and does not participate in the observed transport. Because the cells were cultured on impermeable supports, making the basolateral membrane inaccessible for transport measurements, it is speculated that the observed findings are related to the transport function of the apical membrane of these polarized cells.

Although reabsorption across the apical (AP) membrane of the renal proximal tubule cell plays a vital role in the conservation of plasma 5-methyltetrahydrofolate, basolateral (BL) membrane-directed secretory pathways may also be important in regulating the urinary excretion of folate. Folate transport proteins, folate receptor and the reduced folate carrier have been implicated in the renal conservation of folate across the AP membrane, but their role in BL membrane-directed folate transport has not been studied. 5-Methyltetrahydrofolate transport across the AP and BL membranes of human proximal tubule cells was studied in cells grown on membrane inserts to allow optimum differentiation of AP and BL domains. Colchicine, an inhibitor of vesicular-mediated endocytosis, inhibited AP binding and AP-directed transport without affecting BL transport. Probenecid, an inhibitor of anion exchange, did not affect binding, but inhibited both AP and BL-directed transport with a greater effect on BL transport. Folic acid abolished AP binding of 5-methyltetrahydrofolate, but diminished AP-mediated transport by only 50%. These data suggest that both the folate receptor and the reduced folate carrier participate in AP uptake of folates by human kidney cells, but that BL-mediated uptake occurs primarily by the reduced folate carrier. Folate transport from the secretory direction occurred as readily as that from the reabsorptive direction, indicating that altered secretion could mediate excess urinary folate excretion.

Decreases in plasma folate levels leading to folate deficiency can result from increased urinary loss of folate, due to changes in either the renal reabsorption or secretion of folate. Hence, human proximal tubule (HPT) cells were cultured on microporous membranes to separate apical (AP) and basolateral (BL) domains and to assess the transport of 5-methyltetrahydrofolate from AP-to-BL (i.e., reabsorptive) and BL-to-AP (secretory) directions. Cellular uptake of alpha-methylglucoside occurred specifically from the AP direction, and transport of p-aminohippurate occurred more readily from the BL direction, demonstrating cell polarity similar to that in vivo. Under tight monolayer conditions, binding of folate to the AP membrane occurred more readily from the AP direction, although AP binding also occurred from the BL chamber. Intracellular transport occurred equally from both AP and BL directions. When loaded from either direction, folate was effluxed from HPT cells into both AP and BL chambers. About 20-30% of the internalized substrate was converted to nonfolate catabolites. Thus HPT cells readily take up folate via both the AP and BL membranes, metabolize it intracellularly and secrete the products across both membranes. These studies suggest that renal folate homeostasis is regulated bidirectionally.

Lometrexol (5,10-dideazatetrahydrofolic acid) is a new antifolate that is highly selective in inhibiting the key enzyme of purine synthesis glycinamide ribonucleotide formyltransferase. The most promising preclinical features of lometrexol in animal models were its significant activity against a broad panel of solid tumors, the schedule dependency of its antitumor activity, and the availability of a rescue regimen with folinic or folic acid. In the present study, lometrexol was first given daily for 3 consecutive days, repeated every 4 weeks (part I). The occurrence of delayed myelotoxicity prompted the development of a rescue regimen with lometrexol given in a single dose on day 1, followed by oral folinic acid, 15 mg four times a day, from day 3 to day 5 (part II). Longer time intervals between administration of lometrexol and start of rescue were then evaluated (part III), and in the last part of the study (part IV), the maximum tolerated dose of single intermittent doses of lometrexol with folinic acid given from day 7 to day 9 was established. Sixty adult patients entered the study. In part I, the highest daily dose that could be safely given was 4 mg/m2, for a total dose of 12 mg/m2. Cumulative early stomatitis and delayed thrombocytopenia were dose limiting. The use of oral folinic acid made it possible to escalate the dose up to 60 mg/m2, and the maximum tolerated dose was reached at this dose when folinic was given from day 7 to day 9, with anemia being the dose-limiting toxicity. A shorter time interval between lometrexol and folinic acid administrations (from day 5 to day 7) is recommended for Phase II evaluations to optimize the antitumor effect. Anemia was normochromic and macrocytic, possibly due to a deficiency of folic acid. One partial response of 8 months' duration was reported in a patient with epithelial cancer of the ovary, relapsing after cisplatin and alkylating agents. The use of folic acid as rescue, proposed on the basis of experimental data and pharmacological considerations, has also allowed the repeated administration of lometrexol at doses higher than in the previous studies. The advantages of rescue with folinic acid over supplementation with folic acid, however, are difficult to define.

Previously we showed that the potocytosis of 5-methyltetrahydrofolate is dependent on the acidification of plasmalemmal vesicles created each time a caveolae, closes off from the cell surface. We now report that bafilomycin A1, which is a specific inhibitor of the V-type proton ATPase, inhibits 5-methyltetrahydrofolate uptake into MA104 cells (ED50 = 150 nM). The inhibitory effect was reversed within 30 min after removal of the drug from the cells. Bafilomycin A1 had no effect on the binding of folic acid to its receptor. A concentration of up to 200 nM bafilomycin A1 did not affect sequestration of folate receptors. Immunoblotting showed that the 70-kDa subunit of the V-type proton pump was localized to caveolae-rich fractions isolated from the plasma membrane of these cells. These results suggest that a V-type proton pump acidifies the lumen of plasmalemmal vesicles during potocytosis.

A phase I and pharmacokinetics study of oral uracil, ftorafur, and leucovorin was performed in patients with advanced cancer. Uracil plus ftorafur (UFT) was given in a 4:1 molar ratio in three divided doses for 28 consecutive days. Patient cohorts were treated at 200, 250, 300, and 350 mg/m2 of UFT daily. For all patients, 150 mg of leucovorin was given daily in three oral doses. A 1-week rest period followed each 28-day treatment course. Gastrointestinal toxicity, characterized by diarrhea, nausea, and vomiting, was dose-limiting at 350 mg/m2 UFT in patients who had received prior chemotherapy. Mild fatigue and transient hyperbilirubinemia were also common. In previously untreated patients, UFT at 350 mg/m2 was well-tolerated, suggesting this as an acceptable phase II dose in this schedule with leucovorin. Two of eight previously untreated patients with advanced colorectal cancer had partial responses with UFT (350 mg/m2) plus leucovorin. Pharmacokinetic parameters [ftorafur, uracil, 5-fluorouracil (5-FU), 5-methyltetrahydrofolate] showed wide interpatient variations. Plasma levels of 5-FU (Cmax 1.4 +/- 1.9 microM) were comparable to those achieved with protracted venous infusions, and folate levels (Cmax 6.1 +/- 3.6 microM) were sufficient for biochemical modulation. Ongoing study will determine if this convenient oral regimen will compare favorably in terms of efficacy, toxicity, and cost with intravenous fluoropyrimidine programs.

Lometrexol is an antifolate which inhibits glycinamide ribonucleotide formyltransferase (GARFT), an enzyme essential for de novo purine synthesis. Extensive experimental and limited clinical data have shown that lometrexol has activity against tumours which are refractory to other drugs, notably methotrexate. However, the initial clinical development of lometrexol was curtailed because of severe and cumulative antiproliferative toxicities. Preclinical murine studies demonstrated that the toxicity of lometrexol can be prevented by low dose folic acid administration, i.e. for 7 days prior to and 7 days following a single bolus dose. This observation prompted a Phase I clinical study of lometrexol given with folic acid supplementation which has confirmed that the toxicity of lometrexol can be markedly reduced by folic acid supplementation. Thrombocytopenia and mucositis were the major toxicities. There was no clear relationship between clinical toxicity and the extent of plasma folate elevation. Associated studies demonstrated that lometrexol plasma pharmacokinetics were not altered by folic acid administration indicating that supplementation is unlikely to reduce toxicity by enhancing lometrexol plasma clearance. The work described in this report has identified for the first time a clinically acceptable schedule for the administration of a GARFT inhibitor. This information will facilitate the future evaluation of this class of compounds in cancer therapy.

(6R)-5,10-Dideaza-5,6,7,8-tetrahydrofolic acid (lometrexol) is an antipurine antifolate which selectively inhibits glycinamide ribonucleotide formyltransferase. Lometrexol pharmacokinetics were evaluated in 17 patients (32 courses) as part of a Phase I study in which folic acid supplementation was used to improve tolerance to the drug, its clinical utility being previously limited by severe cumulative toxicity. Lometrexol was administered as an i.v. bolus every 4 weeks at a starting dose of 12 mg/m2, with subsequent interpatient dose escalation to 16, 30, and 45 mg/m2. p.o. folic acid (5 mg/day) was given for 7 days before and 7 days after lometrexol administration. The disposition of total lometrexol in plasma was best described by a biexponential model for data acquired up to 12 h after drug administration, although triexponential plasma pharmacokinetics were often found to give a more adequate description when data were available at later time intervals (24 h and greater). Mean plasma half-lives (+ SD) for model-dependent analysis were t1/2alpha 19 +/- 7 min, t1/2beta 256 +/- 96 min, and t1/2gamma (where measurable) 1170 +/- 435 min. Lometrexol area under plasma concentration versus time curve was proportional to the dose administered. Moderate plasma protein binding of lometrexol was evident (78 +/- 3%) with an inverse linear relationship between fraction of unbound lometrexol and the concentration of serum albumin. The volume of distribution of lometrexol at steady state was between 4.7 and 15.8 l/m2. Renal elimination of lometrexol, studied in 19 patients (21 courses), was considerable, accounting for 56 +/- 17% of the total dose administered within 6 h of treatment, and 85 +/- 16% within 24 h of treatment. These recoveries of unchanged lometrexol indicate that the drug does not appear to undergo appreciable systemic metabolism at the range of concentrations studied. Lometrexol pharmacokinetics were also examined in seven patients who received 45 or 60 mg/m2 lometrexol as part of a separate study of the drug given with folinic acid rescue 5-7 days after treatment. No marked differences were evident in lometrexol plasma half-lives, plasma clearance, or the extent of plasma protein binding, indicating that there is not a pronounced pharmacokinetic interaction between lometrexol and folic acid.

A column-switching chiral HPLC assay was developed that allows the separation and quantitation of the diastereomers of leucovorin (LV, 5-formyltetrahydrofolic acid) and its metabolite 5-methyltetrahydrofolate (METHF) in serum and urine by means of fluorescence detection. The analysis procedure consists of an on-line concentration of the folates in the HPLC system which is followed by the elution and separation of folates on an achiral 3-microns Microbore C18 column in (6R,S)-LV and (6R,S)-METHF. (6R,S)-LV and (6R,S)-METHF are subsequently transferred on-line onto a chiral 7-microns bovine serum albumin column through a Rheodyne valve system and are separated into their diastereometers. Time of analysis is 70 min. Detection limit is 5 ng/ml for each diastereometer. The within-day variation ranges between 3.2 and 15.8% in relation to the measured concentration. Between-day variation is 4.4-12.1% for a concentration of 100 ng/ml for each diastereometer. (6R,S)-LV and (6S)-LV pharmacokinetics were assessed by analyzing serum and urine samples of four-healthy volunteers.

Purpose: To analyze clinical and pharmacokinetic data of cisplatin (CP)/fluorouracil (FU)/l folinic acid (l FA) chemotherapy administered as first-line treatment to locally advanced head and neck cancer patients., Patients and Methods: Thirty-nine patients (35 men and four women; median age, 60 years; six stage III and 33 stage IV) received CP on day 1 (100 mg/m2) followed by l FA (200 mg/m2/d x 5) plus FU (500 mg/m2/d x 5) administered by continuous venous infusion (three cycles planned). Mean plasma concentrations of FU, l FA, and 5-methyltetrahydrofolate (5MTHF) over the cycle were computed., Results: Clinical response was assessable for 33 patients. Response rates on the primary tumor site (n = 33) were 63.7% complete responses (CRs), 24.2% partial responses (PRs), and 12.1% treatment failures. Response rates on lymph nodes (n = 27) were 40.7% CRs, 37.1% PRs, and 22.2% treatment failures. The most frequent toxicity was mucositis (36.2% of cycles grade 3 to 4). Grade 3 to 4 nausea-vomiting, diarrhea, neutropenia, and thrombocytopenia occurred in 6.7%, 1.9%, 13.3%, and 1% of cycles, respectively. Pharmacokinetic analysis showed a wide interpatient variability for both FU (mean, 1.01 mumol/L; range, 0.16 to 2.09), l FA (mean, 1.89, mumol/L; range, 0.52 to 7.88) and 5MTHF plasma concentrations (mean, 3.85 mumol/L; range, 1.30 to 8.11). A significant correlation was demonstrated between FU concentration and hematologic toxicity grade, mucositis grade, and nausea-vomiting/diarrhea grade. Regarding tumor response, patients who failed to respond significantly exhibited lower FU and total folate concentrations than patients with a CR or PR., Conclusion: This study highlights the efficacy of CP/FU/l FA in head and neck carcinoma and establishes the clinical importance of coupled FU/FA pharmacokinetics to predict pharmacodynamic variability.

We investigated the oxidative degradation pathway of 5CH3-H4PteGlu, the main extracellular folate and the predominant form of the vitamin found in food and blood. 5CH3-H4PteGlu is oxidized to 5CH3-5,6-H2PteGlu which subsequently undergoes C9-N10 bond cleavage yielding a pteridine residue and P-ABG, the latter step resulting in irreversible loss of vitamin activity. Under moderately acid conditions typical of the postprandial gut (pH 3.5) 5CH3-H4PteGlu is fairly stable (t1/2 = 273.6 min), while 5CH3-5,6-H2PteGlu is rapidly degraded (t1/2 = 16.9 min). In a neutral environment (pH 6.4) stability is reversed; 5CH3-H4PteGlu t1/2 = 12.0 mins, 5CH3-5,6-H2PteGlu t1/2 = 1504.6 min. Ascorbic acid was efficacious in the facile salvage of 5CH3-H4PteGlu from 5CH3-5,6-H2PteGlu which occurred rapidly and with significant efficiency (100% conversion) under acid (pH 3.5) conditions, t1/2 = 1.3 min (1 mmol/liter ascorbate), but was less efficient under neutral (pH 6.4) conditions t1/2 = 273.6 min (36% conversion). The presence of zinc and iron broadly maintains the pattern of effect, but increases all reaction rates. PteGlu was stable under all conditions studied. These results obtained in an artificial environment were supported by findings in human gastric juice: at a gastric pH of 1.47 with low endogenous ascorbate (7.0 mumol/liter), 5CH3-5,6-H2PteGlu and 5CH3-H4PteGlu both degrade instantly via C9-N10 bond cleavage to yield an equimolar amount of P-ABG. If the same gastric juice is spiked at 58.0 mumol/liter ascorbate (moderate endogenous concentration), 5CH3-H4PteGlu is stable (t1/2 = 334.7 min), while 5CH3-5,6-H2PteGlu is instantly salvaged to 5CH3-H4PteGlu with 43.3% efficiency, and the remaining 5CH3-5,6-H2PteGlu is degraded to P-ABG. In gastric juice with an elevated pH of 7.0 and no endogenous ascorbate, 5CH3-5,6-H2PteGlu and 5CH3-H4PteGlu are both stable, with no C9-N10 bond cleavage. This, for 5CH3-H4PteGlu, is in apparent contrast to findings at pH 6.4 in an artificial environment. The same gastric juice spiked to 50 mumol/liter ascorbate did not result in 5CH3-H4PteGlu salvage from 5CH3-5,6-H2PteGlu.(ABSTRACT TRUNCATED AT 400 WORDS)

We examined the antitumor effects of two antifolate inhibitors of thymidylate synthesis, N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino ]-2-theno yl-L-glutamic acid (D1694; Tomudex) and 1843U89 as well as a folate-based inhibitor of purine synthesis, 5, 10-dideazatetrahydrofolic acid (DDATHF) on human soft tissue sarcoma cell lines having intrinsic resistance to methotrexate (MTX) due to impaired accumulation of polyglutamates of MTX (HS-16 and HS-42 cells) and to increased levels of dihydrofolate reductase and thymidylate synthase activity (HS-18 cells). Growth inhibition studies showed that ED50 values for D1694 and 1843U89 after a 24-h exposure were 11-19-fold and 22-222-fold lower, respectively, than those for MTX in HT-1080, a MTX-sensitive cell line, and the three MTX-resistant cell lines. In contrast, DDATHF was less cytotoxic than MTX in both the MTX-sensitive and the three resistant sarcoma cell lines. Uptake of D1694, 1843U89, or DDATHF was 2.5-4.5-fold higher than MTX in these sarcoma cell lines. However, D1694 and 1843U89, unlike MTX, accumulate in HS-16 and HS-42 cells as polyglutamate forms, reaching 70% of the total intracellular drug level after 24 h. DDATHF polyglutamates (9.4-24%) were less in the same cell lines. Much lower Km values for D1694 and 1843U89 as compared to MTX for folylpolyglutamate synthase were measured in the sarcoma cell lines, with Vmax values equal to or slightly higher than those obtained with MTX. D1694 and 1843U89 are significantly more cytotoxic than MTX in intrinsically MTX-resistant sarcoma cell lines as a result of extensive formation of polyglutamates. These two thymidylate synthase inhibitors should be evaluated in patients with soft tissue sarcomas.

Three high-performance liquid chromatographic methods are described for the detection of the novel antifolate anticancer drug (6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate (lometrexol): one with fluorometric detection and two with detection by UV absorbance. An assay for plasma lometrexol using UV detection (288 nm) and reversed-phase chromatography was developed, with a quantitation limit of 0.2 microgram/ml and linearity up to 10 micrograms/ml. This assay was modified for measurement of lometrexol in urine, with a quantitation limit of 2 micrograms/ml and linearity up to 25 micrograms/ml. An alternative assay for plasma lometrexol using derivatization and fluorescence detection (excitation at 325 nm, emission at 450 nm) was also developed, which proved twenty-fold more sensitive (quantitation limit of 10 ng/ml) than the UV assay, and which was linear up to 250 ng/ml. The fluorometric method requires sample oxidation with manganese dioxide prior to analysis, and uses ion-pair chromatography with tetramethylammonium hydrogensulphate as an ion-pair reagent. All assays use a similar preliminary solid-phase extraction method (recovery as assessed by UV absorption > 73%), with C10-desmethylene lometrexol added for internal standardisation. Each assay is highly reproducible (inter-assay precision in each assay is < 10%). Applicability of the fluorescence-based assay to lometrexol in plasma and the UV-based assay to lometrexol in urine is demonstrated by pharmacokinetic studies in patients treated as part of a Phase I clinical evaluation of the drug.

The effect of folic acid depletion on the tissue distribution and plasma pharmacokinetics of the oncolytic agent 5,10-dideazatetrahydrofolic acid (DDATHF) was evaluated in mice fed either folic acid-deficient or regular diets. Mice were maintained on diets for 2 weeks prior to receiving a single i.v. 30 mg/kg dose of [14C]DDATHF (tissue distribution) or DDATHF (plasma pharmacokinetics). Whole-body autoradiographic evaluation and plasma analysis for DDATHF were conducted in mice at 5 min and 6, 24, 48, 96, 120, and 168 h postdose. Radiocarbon associated with [14C]DDATHF was readily distributed to all tissues in both diet groups at the early time points and was rapidly cleared from most tissues at 24 h postdose. At the later time points, substantial amounts of radioactivity remained in liver from mice fed either diet. However, levels of radiocarbon in liver from mice fed the folic acid-deficient diet were approximately 2.5-4.2-fold the radiocarbon levels in liver from mice fed the regular diet. Similarly, plasma pharmacokinetics indicated that mice fed the folic acid-deficient diet had sustained plasma concentrations of DDATHF compared to plasma concentrations in mice fed the regular diet. These data indicated that a deficiency in dietary folic acid in mice caused increased hepatic retention of radioactivity and sustained plasma concentrations of DDATHF which are probably responsible for the observed toxicity of DDATHF in mice.

(6R)5,10-Dideazatetrahydrofolic acid (DDATHF, Lometrexol), a potent antitumor drug in vivo and in vitro, is an inhibitor of the two folate-dependent enzymes in the de novo purine biosynthesis pathway: glycinamide ribonucleotide (GAR) and amino imidazole carboxamide (AICAR) transformylases. A single dose of DDATHF (50 mg/kg, i.p.) in C57/BL6 mice caused a prolonged depletion of purine nucleotides (ATP and GTP) in colon 38 tumor and only a temporary effect in liver. GAR transformylase activity was higher in colon 38 tumor than in liver, but a kinetic analysis on the purified enzyme showed no differences in Ki values for DDATHF or Km values for the folate substrate. As a consequence of de novo purine synthesis inhibition, there was a 2- to 3-fold elevation of 5-phosphoribosyl-1-pyrophosphate pools in colon 38 tumor between 4 and 12 hr after DDATHF administration. When DDATHF (50 mg/kg) was administered 4 or 8 hr prior to 5-fluorouracil (5-FU; 85 mg/kg, i.p., weekly), these biochemical effects significantly increased the antitumor activity of 5-FU, with a modest increase in toxicity. Lower doses of DDATHF (25 and 37.5 mg/kg) when combined with 5-FU also resulted in an improved antitumor activity without additional toxicity. The two different schedules of administration for DDATHF, 4 and 8 hr prior to 5-FU, showed no differences in antitumor activity or toxicity.

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.

The ethanol-induced increase in urinary folate excretion has been shown to decrease plasma folate levels and may contribute to the development of folate deficiency associated with alcoholism. The mechanism for this effect remains to be elucidated. The present studies were designed to examine the direct effect of ethanol on the renal handling of 5-methyltetrahydrofolate (5-CH3-H4PteGlu), the physiological folate. Rats were given four consecutive hourly doses of ethanol (1 g/kg) or isocaloric doses of glucose solution orally. This treatment generated an average plasma ethanol level of 305 mg/dl. Kidneys from male Sprague-Dawley rats were removed 5 hr after initial treatment and perfused in vitro to eliminate any extrarenal effects that could confound interpretation of results. Ethanol was not added to the perfusate. These treatments had no effect on 5-CH3-H4PteGlu conservation by the isolated perfused rat kidney in comparison to experiments in which the animal received no treatment. Ethanol was then added directly to the perfusate to generate average concentrations of 293 mg/dl. The in vitro addition of ethanol significantly decreased the percentage reabsorption and increased the fractional excretion of 5-CH3-H4PteGlu in comparison to controls (kidneys perfused with or without an isocaloric dose of glucose). This effect did not become significant until the renal tissue was exposed to these levels of ethanol for 1 hr. These results indicate that ethanol directly impairs the renal conservation of 5-CH3-H4PteGlu.

The pharmacokinetic profiles of folinic acid (FA) and its active metabolite, 5-methyltetrahydrofolic acid, were studied after oral administration of decreasing doses of calcium folinate during 37 courses of high and intermediate dose methotrexate treatment in 25 lymphoma patients. FA was administered at a dose of 6 x 50 mg in 15 courses, 6 x 25 mg in seven courses, 6 x 15 mg in 10 courses and 6 x 7.5 mg in 5 courses. FA, 5-methyltetrahydrofolic acid, methotrexate and 70H-methotrexate were assayed simultaneously by high performance liquid chromatography. When FA was administered at doses between 50 and 15 mg, maximum concentrations of both the drug and its metabolite were always obtained after 1 to 2 h and remained stable. The same was true for the equilibrium concentration of the two products at doses over 15 mg. These findings suggest saturation of absorption and metabolism of folinic acid at doses over 15 mg.

A subline of H35 hepatoma cells has been developed which exhibits 80-fold resistance to 5,10-dideazatetrahydrofolate, an antifolate which inhibits glycinamideribonucleotide transformylase. The cells are cross-resistant to methotrexate, an inhibitor of dihydrofolate reductase and 10-propargyl-5,8-dideazafolate and its 2-desamino-2-methyl derivative, both inhibitors of thymidylate synthase. The resistant cells are characterized by an impaired activity of the reduced folate transport system which affects cellular import of methotrexate, 5,10-dideazatetrahydrofolate, and 2-desamino-2-methyl-10-propargyl-5,8-dideazafolate but not 10-propargyl-5,8-dideazafolate. In addition, the resistant cells exhibit a severalfold increased activity of gamma-glutamyl hydrolase, the enzyme which cleaves the intracellular polyglutamate derivatives of the antifolates. Evidence for the involvement of gamma-glutamyl hydrolase in resistance is derived from the observation that polyglutamate derivatives of 10-propargyl-5,8-dideazafolate in resistant cells are maintained at one-third the amount of that in parental cells in the presence of the same extracellular concentration. This is the first observation that an increase in gamma-glutamyl hydrolase contributes to acquired resistance to antifolates.

The pharmacokinetic values of d,l-leucovorin and l-leucovorin were compared in eight healthy volunteers following oral administration of 25 mg d,l-leucovorin and 12.5 mg l-leucovorin. Serum levels of l-5-formyltetrahydrofolate, l-5-methyltetrahydrofolate, and total reduced folates were measured by an established microbiological method. Pharmacokinetic data for both preparations were consistent with those previously reported for d,l-leucovorin, with essentially complete first pass metabolism to l-5-methyltetrahydrofolate, the active metabolite. No differences were found between the two preparations in serum concentrations of active folate fractions, AUC, or Cmax, or in clearance and volume of distribution estimates. These data suggest that after administration of 25 mg of d,l-leucovorin, the d-diastereoisomer has no significant effect on the standard pharmacokinetic measurements of the active l-folates.

Because mechanisms for the renal regulation of folate excretion are poorly understood, a cell culture system representative of the human proximal tubule (HPT) was used for studies of renal folate transport. After confluent cultures of HPT cells were incubated with 3H-labeled folic acid (PteGlu), binding to the apical membrane was determined by an acid removal procedure, and transport was subsequently measured in solubilized cells. Although PteGlu binding was almost all specific (suppressed by excess unlabeled PteGlu), HPT cells transported PteGlu by specific and nonspecific processes. Specific PteGlu binding and transport were both saturable processes, reaching maxima of 0.5 and 0.1 pmol/mg protein, respectively, with half-maximal constants of 12 and 50 nM, respectively. The PteGlu analogues methotrexate and 5-methyltetrahydrofolic acid (5-CH3-H4PteGlu) inhibited both the binding and transport of PteGlu, with 5-CH3-H4PteGlu being more potent (lower half-maximal inhibitory concentration). In contrast, 5-formyltetrahydrofolic acid significantly reduced PteGlu transport at concentrations (100-250 nM) that had no effect on binding. These data suggest that the HPT cells will serve as a good model for studies of renal folate reabsorption. Initial characterization of the transport of folate by HPT cells suggests two distinct processes, binding to a high-affinity membrane folate-binding protein followed by a structurally specific transfer into the cell.

We have characterized the uptake of 5-formyltetrahydrofolate into isolated rat liver mitochondria. Uptake of 5-formyltetrahydrofolate, 5-methyltetrahydrofolate and folic acid was linear for the first 2 min of incubation. 5-Methyltetrahydrofolate and 5-formyltetrahydrofolate were accumulated in mitochondria to 3.7- and 4.3-fold, respectively. 5-Formyltetrahydrofolate transport showed a pH optimum at 5.5 and was saturable with an apparent Km = 2.80 mumol.L-1 and Vmax = 3.49 pmol.min-1.mg protein-1. Uptake of 5-formyltetrahydrofolate was inhibited by the structural analog 5-methyltetrahydrofolate and, to a much lesser extent, folic acid and methotrexate. Uptake was not inhibited by various substrates of known mitochondrial transporters, e.g., succinate, malate, citrate, glutamate and phosphate, nor was uptake inhibited by the sulfhydryl reagent, N-ethylmaleimide. Uptake was not stimulated by preloading of mitochondria with malate or aspartate. Uptake of 5-formyltetrahydrofolate was not affected by the proton ionophore carbonyl cyanide 4-trifluoromethoxyphenyl-hydrazone nor by collapsing the electrical gradient with valinomycin. These results indicate that 5-formyltetrahydrofolate uptake into mitochondria via a carrier-mediated system specific for the reduced, naturally occurring folates. The folates, once in the mitochondria, may be converted to the polyglutamylated forms, which will not readily diffuse out of the mitochondria.

The intestinal absorption and in vivo kinetics of (6S)-[3H]-5-methyl-tetrahydrofolate (5-methyl-H4folate), (6S)-[3H]-5-formyl-H4folate and [3H]folic acid were investigated to determine whether inherent differences exist in the overall bioavailability of these folates in rats. Adult rats (n = 9 per group) were given an intragastric dose of the appropriate folate (50 pmol/100 g body wt) in 50 mmol/L ascorbate (pH 7). Each compound underwent nearly complete absorption within 8 h, and there was no significant difference in the excretion kinetics in relation to the form of folate administered. A biphasic pattern of excretion was observed over the following 8 d. Both urine and feces were important excretory routes. The rapid phase of total isotopic excretion (urinary and fecal) exhibited a half time (t1/2) of 0.11-0.12 d, whereas the t1/2 of the slower phase was 13.4-15.9 d. Isotopic distributions and the pattern of labeled folates in urine and tissues were similar regardless of the form administered. These results indicate that the bioavailability of orally administered folic acid, 5-methyl-H4folate and 5-formyl-H4folate is equivalent in rats under the conditions of this study.

The bioavailability of orally administered monoglutamyl folic acid and various (6S)-tetrahydrofolates was examined in humans with stable-isotope methods. Folic acid (PteGlu), tetrahydrofolate (H4folate), 5-formyl-H4folate, 10-formyl-H4folate, and 5-methyl-H4folate were prepared for oral administration in 3',5'-2H2 labeled (d2) form, and [glu-2H4]folic acid (d4-PteGlu) was prepared for intravenous injection. In each of five trials, fasting adult males (n = 7) on a folate saturation regimen (2 mg/d) were given a single oral dose of one of the d2-folates in apple juice, as well as an intravenous injection of d4-PteGlu as a control. Urine was collected for 48 h and the isotope labeling of urinary folates determined by mass spectrometry. Isotope excretion ratios of urinary folates were used as criteria of bioavailability (pooled SE = 0.10): PteGlu (1.53, least squares mean), 10-formyl-H4folate (1.02), 5-methyl-H4folate (0.99), 5-formyl-H4folate (0.1.13), and H4folate (0.71). These results indicate that differences exist in the bioavailability of monoglutamyl folates under these experimental conditions. This variation, whether due to differences in absorption or postabsorptive events, must be considered in quantitative studies of folate utilization with this type of protocol.

This study compares the antitumor activity and metabolism of the purine de novo biosynthesis inhibitor 5-deazaacyclotetrahydrofolate and a series of analogues. All compounds have similar IC50 values for inhibition of MCF-7 cell growth, activity of glycineamide ribonucleotide transformylase, and methotrexate uptake by MOLT-4 cells, the latter a measure of cellular uptake potential. Only 5-deazaacyclotetrahydrofolate and the 2'-fluoro and 3'-fluoro analogues demonstrated significant inhibition of colon 38 adenocarcinoma or HCT-116 colon carcinoma growth in vivo. This correlated with the Km of these compounds for folylpolyglutamate synthetase. 5-Deazaacyclotetrahydrofolate and 2'-fluoro-5-deazaacyclotetrahydrofolate which displayed the strongest antitumor activity were detectable in colon 38 tumor tissue 24 hr after dosing and were present nearly exclusively as the polyglutamated species. These results indicate that polyglutamation represents a critical step in the in vivo antitumor activity of these compounds.

An analytical method for measurement of tetrahydrofolate (THF) and N5-methyltetrahydrofolate (5MF) in pig plasma by the high performance liquid chromatography and electrochemical detection is described. The plasma sample was deproteinized by perchloric acid and the supernatant was analyzed using the following conditions; (a) phenyl bonded phase column as an analytical column; (b) mobile phase consisting of 20 mM acetate buffer (pH 3.6) containing 0.1 mM EDTA and acetonitrile (96.5:3.5, v/v); (c) an applied potential of +300 mV. Under the above condition, both peaks of THF and 5MF in the plasma were well separated. The detection limits of THF and 5MF were 0.15 and 0.13 ng/ml, respectively, at S/N = 3. The recoveries of THF and 5MF from the plasma spiked with standard THF and 5MF were 77.6 +/- 2.1% and 83.0 +/- 1.7% (mean +/- S.D.), respectively.

We examined 5-methyltetrahydrofolate transport across the basolateral membrane (BLM) of rat liver using isolated membrane vesicles. Uptake of 5-methyltetrahydrofolate by BLM vesicles (BLMV) was found by osmolarity and initial uptake studies to be mostly the result of transport of the substrate into an active, intravesicular space with some binding (approximately 25%) to membrane surfaces. Uptake was similar in the presence of an inwardly directed Na+ or K+ gradient, indicating that transport is not dependent on Na+. Uptake of 5-methyltetrahydrofolate was linear for the first 30 s and was more rapid when an initial pH gradient was imposed across the vesicular membrane [extravesicular pH (pHo) = 5.0, intravesicular pH (pHi) = 7.5]. Under pH gradient conditions, uptake at 3-5 min was about twofold higher than at equilibrium (60 min). The initial rate of uptake at pHo = pHi = 5.0 was more rapid than at pHo = pHi = 7.5, but in neither case was uptake above equilibrium observed. Uptake under pH gradient conditions and at pH 5.0 (no pH gradient) was saturable (apparent Michaelis constants of 0.58 and 0.36 microM, respectively; maximum uptake rates of 1.25 and 0.79 pmol.10 s-1.mg protein-1, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

5,10-Dideazatetrahydrofolic acid (DDATHF) is a potent inhibitor of glycinamide ribonucleotide transformylase, one of the folate-dependent key enzymes in de novo purine biosynthesis. The present report demonstrates that multiple membrane-transport routes may be involved in the cellular uptake of DDATHF. These routes include the classic reduced folate carrier and a membrane-associated folate-binding protein (mFBP). The role of an mFBP in the uptake of DDATHF was suggested from observations that (a) the mFBP showed a very high binding affinity for DDATHF, (b) murine and human leukemia cells expressing an mFBP were highly sensitive to growth inhibition by DDATHF, and (c) protection against this growth inhibition could be achieved using folic acid rather than reduced folate compounds.

5-Deazaacyclotetrahydrofolate is a cytotoxic tetrahydrofolate analogue which inhibits glycinamide ribonucleotide transformylase (Kelley et al., J. Med. Chem., 33: 561-567, 1990). Cultured mouse L-cells and human MCF-7 and MOLT-4 cells concentrated the drug several hundred-fold after 24 h of continuous exposure to a cytotoxic level (100-200 nM) of radiolabeled drug. High performance liquid chromatography analysis revealed that each cell type metabolized greater than or equal to 80% of the internalized drug to polyglutamated forms, which are more potent glycinamide ribonucleotide transformylase inhibitors. In L-cells, 45% of the polyglutamated metabolites were also N-formylated. The pharmacokinetics and distribution of [14C]-deazaacyclotetrahydrofolate were studied in C57BL/6 male mice. Its plasma half-life was 2.15 h. Radiolabel was concentrated to well above plasma level in the kidney, pancreas, and liver. Metabolism was examined in tumor-bearing and in normal mice. Twenty-four h after a single i.p. injection (50 mg/kg), drug equivalents were 0.6 nmol/g (83% polyglutamated) in colon-38 adenocarcinoma carried s.c., 2.4 nmol/g (100% polyglutamated) in ascitic P388 cells, and 3.7 nmol/g (76% polyglutamated and approximately 20% formylated) in mouse liver. Elimination was mostly in the urine as unmetabolized drug. Feces contained 5-deazaacyclotetrahydropteroate (parent compound less glutamate). In conclusion, 5-deazaacyclotetrahydrofolate was shown to be concentrated to well above the extracellular level and metabolized to more active polyglutamated forms by transformed cells grown in culture and in mice.

After the use of d,1-folinic acid (d,1-CHO-THF), pharmacokinetic measurements should take into account 1-CHO-THF and its metabolite 1-methyltetrahydrofolic acid (1-CH3-THF) as well as d-CHO-THF. For this purpose, we developed a simple and rapid assay by combining reversed-phase HPLC to determine total levels of d,1-CHO-THF and CH3-THF and chiral HPLC to separate the biologically active 1-CHO-THF from the inactive d-CHO-THF. We investigated the pharmacokinetics after short-term infusion of 300 mg d,1-CHO-THF in ten healthy volunteers. With a mean of 56.5 min, 1-CHO-THF exhibits a rapid body clearance of 222 ml/min, about 60% of which is caused by metabolism to CH3-THF and 40%, by renal excretion. CH3-THF has a terminal half-life of 208 min and a total body clearance of 88.9 ml/min, which is essentially the same as the renal clearance. Due to the lower clearance of CH3-THF, its AUC (2,132 microM x min) exceeds that of 1-CHO-THF (1445 microM x min) by approximately 50%. In contrast to that of the reduced 1-folates, the total body and renal clearance of d-CHO-THF is very low, with values of 13.2 and 12.9 ml/min, respectively. This results in a very high AUC of 24, 269 microM x min, which is higher by factors of 17 and 11 than those of 1-CHO-THF and CH3-THF, respectively. The implications of the distinct kinetics of the reduced 1-folates and d-CHO-THF for the efficacy of folinic acid/5-fluorouracil therapy and adequate protocols for the treatment of advanced colorectal cancer are discussed.

1. The influence of goat's-milk folate-binding protein (FBP) on the uptake of 5-methyltetrahydrofolate (MTHF) by brush-border-membrane vesicles prepared from the small intestine of the 6-d-old goat was investigated using a rapid-filtration assay. 2. Uptake of MTHF by the membrane vesicles was strongly enhanced by FBP within the pH range 4.5-6.5, with an optimum at pH 5-5.5. 3. Both the initial rate of MTHF uptake and uptake of MTHF at equilibrium were markedly increased in the presence of FBP. 4. Uptake of MTHF by brush-border-membrane vesicles was maximal when the molar ratio FBP:MTHF was 1.0-2.5. 5. The relation between pH and 125I-labelled FBP binding to the membranes was similar to that for uptake of MTHF, with an optimum at pH 5. 6. In experiments in which the osmotic pressure of the incubation medium was progressively increased with cellobiose, 125I-labelled FBP was found to be taken up primarily by binding to the brush-border-membrane surface. 7. Uptake of 125I-labelled FBP was time-dependent and saturable, with a Km of 0.39 (SE 0.07) microM and Vmax of 6.73 (SE 0.92) micrograms/mg protein. 8. Experiments in which various milk proteins (cow FBP, goat FBP, alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin and lactoferrin) were allowed to compete in turn with 125I-labelled FBP for uptake by brush-border-membrane vesicles indicated that high-affinity binding was probably specific to FBP, although lactoferrin reduced uptake possibly by non-specific coating of the mucosal surface. 9. It was concluded that a folate transport mechanism mediated by the FBP in milk exists at the intestinal brush border of neonatal goats. It is suggested that this may reinforce the developing endogenous transport system.

The 90-min-uptake (37 degrees C) of 3H-methotrexate (3H-MTX) by uninduced HL60 cells was inhibited by 2,4-dinitrophenol, iodoacetate, ouabain, p-chloromercuriphenylsulphate and p-chloromercuribenzoate indicating its dependence on intracellular ATP, the Na+ and K+-activated ATPase of the cell membrane and sulphydryl groups on the cell surface. There was a marked reduction in the 90-min-uptake (37 degrees C) of 3H-MTX and 14C-methyltetrahydrofolate (14C-mTHF) when HL60 cells were induced to differentiate into neutrophil myelocytes and metamyelocytes by incubation with DMSO; a smaller reduction was seen when HL60 cells were induced to differentiate into mononuclear phagocytes by incubation with 1 alpha,25-dihydroxyvitamin D3 ([OH]2D3). Although it has been reported that the transport system mediating folate uptake has a higher Km value for mTHF and MTX in normal than in malignant cells, the Km values for these substrates were lower in blood-monocyte-derived macrophages than in uninduced HL60 cells or [OH]2D3-induced-macrophages. Values for the 90-min-uptake (37 degrees C) of 3H-MTX and 14C-mTHF in human blood-monocyte-derived macrophages were higher than the corresponding values in uninduced HL60 cells and [OH]2D3-induced macrophages.

Previously reported work from this laboratory has shown that, in man, the formation of plasma 5-methyltetrahydrofolic acid (5MeTHF) from orally administered pteroylmonoglutamic acid (PGA) obeys Michaelis-Menten kinetics. The regression equation obtained from a plot of the reciprocal amount of 5MeTHF formed (micrograms per litre plasma/min) as a function of reciprocal amount of PGA administered (micrograms/kg body weight) provides a useful means of calculating the improved efficiency of 5MeTHF formation by dividing the total dose of PGA into smaller doses.

The pharmacokinetics of the active and inactive diastereoisomers of leucovorin and its active metabolite, 5-methyltetrahydrofolate (5-CH3-THF) were studied after iv injection of leucovorin in normal human subjects at a dose of 28 mg/m2, and in patients given 500 mg/m2 daily by constant iv infusion for a 5.5 day period. In both studies the plasma half-life (t1/2) of the active isomer, L-formyltetrahydrofolate (CHO-THF), was only 32 to 35 minutes, whereas the inactive isomer, D-CHO-THF had a plasma t 1/2 of 352 to 485 minutes. During constant infusion, the plasma levels reached plateaus of 2.33 and 37.5 microM for L-CHO-THF and D-CHO-THF, respectively. The inactive isomer was cleared from plasma only by urinary excretion of the unchanged drug. The active isomer was also excreted unchanged in the urine but in addition was extensively metabolized to the active metabolite L-5-CH3-THF. The active metabolite achieved a plasma level of 4.85 microM during constant infusion and appeared to have a longer t 1/2 after constant infusion than was observed after iv injection. Furthermore a larger apparent volume of distribution (Vd) of 5-CH3-THF was obtained in the constant infusion study. These findings suggest that constant iv infusion of large doses of leucovorin can considerably expand the intracellular pools of active folate. The consequence of the extensive accumulation of the inactive isomer, D-CHO-THF, is not known. However, the small Vd of D-CHO-THF suggests that it does not extensively accumulate in tissues.

1. The uptake of 5-methyltetrahydrofolate (MTHF) and folic acid (pteroylmonoglutamate) by enterocytes and brush-border-membrane vesicles prepared from the small intestine of the 6-d-old male goat was determined using a rapid-filtration assay. 2. Both MTHF and folic acid were taken up by membrane vesicles at 25 degrees and enterocytes at 37 degrees by a pH-dependent mechanism with maximum uptake when the pH of the incubation medium was 5.0. 3. Experiments in which the osmotic pressure of the medium was raised in successive increments with the non-absorbable sugar cellobiose indicated that transport rather than membrane binding was the main component of uptake. 4. Experiments at pH 5.0 showed that uptake of MTHF and folic acid was saturable and that the characteristics of folate transport were similar in both tissue preparations: (a) transport rates of both MTHF and folic acid were constant during the first 1-2 min for a given folate concentration, then declined to reach a steady-state in 10-30 min; (b) initial velocities of transport of MTHF and folic acid increased in proportion to their concentrations up to 7-10 microM, but the rate of increase slowed thereafter until saturation was reached at 20-25 microM (Km for brush-border-membrane vesicles 40.8 and 62.9 microM, Km for enterocytes 50.9 and 55.2 microM for MTHF and folic acid respectively). Values of Vmax for membrane vesicles (pmol/mg protein per min) were 46.5 MTHF or 40.3 folic acid; enterocytes Vmax (pmol/10(7) cells per min) 15.9 MTHF or 30.6 folic acid; (c) uptake of MTHF and folic acid by brush-border-membrane vesicles and enterocytes measured under steady-state conditions approached saturation at 50 microM for each analogue (Km for membrane vesicles 58.1 and 55.2 microM, Km for enterocytes 43.1 and 49.4 microM for MTHF and folic acid respectively; Vmax for membrane vesicles 49.7 and 77.8 pmol/mg protein, for enterocytes 52.8 and 54.7 pmol/10(7) cells for MTHF and folic acid respectively). 5. It was concluded that transport of MTHF and folic acid was by a similar pH-dependent mechanism both in brush-border-membrane vesicles and in intact enterocytes. At pH 5.0, transport involved a saturable carrier-mediated process located in the brush-border membrane.