Search

Introduction: The folate receptor (FR) is frequently overexpressed in tumors and can be targeted with folate-based (radio)pharmaceuticals. However, significant accumulation of radiofolates in FR-positive kidneys represents a drawback. We have shown that preadministration of the antifolate pemetrexed (PMX) significantly improved the tumor-to-kidney ratio of radiofolates in mice. The aim of this study was to investigate the dose dependence of these effects and whether the same results could be achieved with folic acid (FA) or 5-methyl-tetrahydrofolate (5-Me-THF)., Methods: Biodistribution was assessed 4 h postinjection of the organometallic (99m)Tc-picolylamine monoacetic acid folate in nude mice bearing FR-positive KB tumor xenografts. PMX (50-400 microg/mouse) was injected 1 h previous to radioactivity. The effects of FA and 5-Me-THF (0.5-50 microg/mouse) were investigated likewise. Tissues and organs were collected and counted for radioactivity and the values tabulated as percentage of injected dose per gram tissue (% ID/g)., Results: PMX administration reduced renal retention (<1.6% ID/g vs. control: >10% ID/g), while the tumor uptake (average 1.35%+/-0.40% ID/g vs. control: 1.79%+/-0.49% ID/g) was only slightly affected independent of the PMX dose. Replacement of PMX by FA or 5-Me-THF (50 microg/mouse) resulted in a significant renal blockade (<0.1% ID/g) but at the same time in an undesired reduction of tumor uptake (<0.2% ID/g)., Conclusions: Selective reduction of radiofolate uptake in kidneys under retention of high tumor accumulation could be achieved in combination with PMX over a broad dose range but not with FA or 5-Me-THF.

Pemetrexed is a novel antifolate recently approved for the treatment of pleural mesothelioma and non-small cell lung cancer. In clinical regimens, pemetrexed is administered in conjunction with folic acid to minimize toxicity. However, excessive folate supplementation may also diminish the activity of this agent. The current study demonstrates, in several human solid tumor cell lines, that when extracellular 5-formyltetrahydrofolate levels are increased in vitro, within the range of normal human blood levels, there is a substantial decrease in pemetrexed activity upon continuous exposure to the drug. This was accompanied by a comparable lower level of trimetrexate activity consistent with an expansion of tumor cell folate pools. Likewise, when cells were exposed to pemetrexed with a schedule that simulates in vivo pharmacokinetics, there was markedly less cell killing with higher extracellular folate levels. Data are provided to indicate that 5-formyltetrahydrofolate is an acceptable surrogate for 5-methyltetrahydrofolate, the major blood folate, for this type of in vitro study. These observations and other reports suggest that, in view of the rise in serum folate and fall in serum homocysteine that has accompanied folic acid supplementation of food in the U.S., the addition of folic acid to regimens with pemetrexed should be limited to the lowest recommended level that provides optimal protection from pemetrexed toxicity.

Purpose: In this phase I trial in humans the safety and pharmacology of LY309887 on a weekly schedule combined with daily oral 5-mg doses of folic acid were evaluated., Background: LY309887 is an inhibitor of folate-dependent enzymes involved in de novo purine biosynthesis and has a broad preclinical antitumor activity. In murine systems, combining this agent with exogenous folic acid results in an enhanced therapeutic index., Methods: This study was a single-institution, open-label, clinical trial of dose escalation with toxicity and pharmacokinetic parameters determined. The dose range studied was 0.5-4 mg/m2 per week x6 and then a modified schedule weekly x3 every 6 weeks., Results: Dose-limiting toxicities were of delayed onset and associated with hematologic, neurologic, and mucosal effects. Pharmacokinetic parameters revealed dose linearity for AUC and Cmax. Low circulating levels of drug persisted for over 200 h. Urinary excretion accounted for approximately 50% of the parent drug but was highly variable. The urinary excretion was near maximal within 24 h of dosing., Conclusions: The modified dosing schedule allowed repetitive dosing in patients. Further evaluation of the 2 mg/m2 per week x3 every 6 weeks with daily oral folate supplement as a potential phase II dose may be warranted.

A murine leukemia cell line was identified that is highly resistant to methotrexate (MTX), due to impaired transport, but fully sensitive to 5,10-dideazatetrahydrofolate (DDATHF). A valine-to-methionine substitution at amino acid 104 in the reduced folate carrier (RFC1) explains this disparity in drug resistance. Transfection of the V104M cDNA into an RFC1-deficient cell line markedly increased DDATHF influx (32x) but only modestly increased influx of MTX and 5-formyltetrahydrofolate (4- and 6-fold, respectively). The growth inhibition or growth requirements for these folates fell by factors of 18, 2, and 4, respectively, in the transfectant. Preservation of DDATHF influx in cells with V104M RFC1 resulted in even greater preservation (60%) of the exchangeable drug level. Another major element in the preservation of DDATHF activity was the impact of the mutated carrier on cellular folate pools. For folic acid, folate pools were essentially unchanged but DDATHF polyglutamate levels decreased in lines that express the V104M carrier. However, with 5-formyltetrahydrofolate as the growth source, there was a marked decrease in folate pools in the lines carrying the mutated carrier, and DDATHF polyglutamate levels were unchanged. Hence, DDATHF activity was preserved in cells with V104M RFC1 due to (a) relative conservation of DDATHF transport, and (b) depletion of cellular THF cofactors with diminishing folate cofactor competition at folylpolyglutamate synthetase and possibly glycinamide ribonucleotide formyltransferase. Hence, resistance to one antifolate, in this case MTX, because of a loss of RFC1 transport activity need not exclude the subsequent utility of another antifolate that uses the same carrier.

Purpose: Lometrexol [(6R)-5,10-dideaza-5,6,7,8-tetrahydrofolate] is the prototype folate antimetabolite that targets the de novo purine synthesis pathway. Early phase I trials were confounded by cumulative myelosuppression that prevented repetitive administration. Subsequent preclinical and clinical studies suggested that coadministration of folic acid might favorably modulate lometrexol toxicity without eliminating potential antitumor activity. We set out to determine if concurrent folic acid would allow administration of lometrexol on a weekly schedule, and, if so, to identify an appropriate dose combination for phase II trials. Pharmacokinetic and metabolism studies were undertaken in an attempt to improve our understanding of lometrexol pharmacodynamics., Methods: Patients with advanced cancer received daily oral folic acid beginning 7 days before lometrexol and continuing for 7 days beyond the last lometrexol dose. Lometrexol was administered by short i.v. infusion weekly for 8 weeks. Scheduled lometrexol doses were omitted for toxicity of more than grade 2 present on the day of treatment, and dose-limiting toxicity was prospectively defined in terms of frequency of dose omission as well as the occurrence of severe toxic events. Plasma and whole blood total lometrexol contents (lometrexol plus lometrexol polyglutamates) were measured in samples taken just prior to each lometrexol dose., Results: A total of 18 patients were treated at five lometrexol dose levels. The maximum tolerated dose was identified by frequent dose omission due to thrombocytopenia and mucositis. The recommended phase II dose combination is lometrexol 10.4 mg/m(2) per week i.v. with folic acid 3 mg/m(2) per day orally. One patient with melanoma experienced a partial response, and three patients, two with melanoma and one with renal cell carcinoma, experienced stable disease. Lometrexol was not detectable in any predose plasma sample tested. The total red blood cell content of lometrexol increased over several weeks and then appeared to plateau., Conclusions: Weekly administration of lometrexol is feasible and well-tolerated when coadministered with daily oral folic acid. The nature of the interaction between natural folates and lometrexol that renders this schedule feasible remains unclear. A definition of dose-limiting toxicity that incorporated attention to dose omissions allowed efficient identification of a recommended phase II dose that reflects the maximum feasible dose intensity for a weekly schedule. Lometrexol is a promising, anticancer agent.

Antifolate drugs, as a class, have broad-spectrum activity against both hematologic and solid human malignancies. The pharmacokinetics of the classical antifolate methotrexate have been well-defined and pharmacokinetic data can be exploited to reduce the toxicity and enhance the activity of the drug. Methotrexate remains the only anticancer drug for which plasma drug level monitoring is used in routine clinical practice. Recently, novel classical and nonclassical antifolates have been developed that target either specific folate-dependent enzymes (e.g., thymidylate synthase [CB3717, raltitrexed, ZD9331, 1843U89, nolatrexed, AG331], glycinamide ribonucleotide transformylase [lometrexol, LY309887, AG2034] or multiple folate-dependent enzymes (e.g., MTA/LY231514). In the early clinical trials of these agents, a number of pharmacokinetic-pharmacodynamic relationships were identified and it is highly likely that the full therapeutic potential of these new drugs will also require the exploitation of pharmacokinetic data.

Lometrexol inhibits the first folate-dependent enzyme in de novo purine biosynthesis and is avidly polyglutamated and retained in tissues expressing folylpolyglutamate synthetase. Although clinical studies have been limited by cumulative toxicity, preclinical studies show that pretreatment with folic acid can protect normal tissue while maintaining tumor cytotoxicity. Therefore, a Phase I study of lometrexol every 21 days preceded by i.v. folic acid was initiated. Lometrexol was studied in six patients at 15 mg/m2, in six patients at 20 mg/m2, in three patients at 25 mg/m2, and in nine patients at 30 mg/m2. Patients received either 5 mg of folic acid 1 h before or 25 mg/m2 3 h before lometrexol. Blood samples were obtained around the first course and weekly thereafter for determination of plasma and erythrocyte (RBC) lometrexol concentrations. Bioactive folates in plasma and RBCs were determined in a subset of patients. Lometrexol pharmacokinetics were best described by a three-compartment model. Mean clearance and volume of distribution were 1.6 +/- 0.6 liters/h/m2 and 8.9 +/- 4.1 liters/m2. Mean half-lives were 0.23 +/- 0.1, 2.9 +/- 1.4, and 25.0 +/- 48.7 h. Pharmacokinetics were independent of either lometrexol or folic acid dose. In the weekly blood samples, RBC lometrexol levels rose, long after plasma lometrexol was undetectable. RBC lometrexol levels were independent of folic acid or lometrexol dose. Bioactive folates measured in plasma and RBCs during this same time period did not accumulate. Rising RBC levels were correlated with a fall in hematocrit, hemoglobin, and platelet count. This study indicates that the cumulative toxicity of lometrexol is related to tissue concentration and not plasma pharmacokinetics. RBC lometrexol may be an indicator of cumulative drug exposure and effect.

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.

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.

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.

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.

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.

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.