Loss of reduced folate carrier function and folate depletion result in enhanced pemetrexed inhibition of purine synthesis.

Pemetrexed is a novel antifolate with polyglutamate derivatives that are potent inhibitors of thymidylate synthase (TS) and to a lesser extent glycinamide ribonucleotide formyltransferase (GARFT). Conditions that might modulate relative suppression of these sites were assessed by the pattern of hypoxanthine and thymidine protection. When grown with 25 nmol/L racemic 5-formyltetrahydrofolate, thymidine alone fully protected wild-type HeLa cells to at least 1 micromol/L pemetrexed, but protection of a reduced folate carrier (RFC)-null subline required both thymidine and hypoxanthine above a concentration of 30 nmol/L pemetrexed. As medium 5-formyltetrahydrofolate was decreased, protection by thymidine alone decreased, and was further diminished when HeLa cells were grown in dialyzed serum. There was little protection by thymidine of RFC-null HeLa cells under the latter conditions. Thymidine alone was not protective, and hypoxanthine alone produced only a small (2-fold) increase in IC(50), in a HeLa-derived line 8-fold resistant to pemetrexed due to a modest increase in TS. Finally, in MCF-7 breast cancer cells there was greater protection with thymidine alone than in HeLa cells when cells were grown in medium containing a low concentration of 5-formyltetrahydrofolate. These observations indicate that as intracellular folates decrease in HeLa cells, due to decreased extracellular reduced folate, or loss of RFC function, pemetrexed inhibition of GARFT increases. These data support the concept that the contribution to pemetrexed activity by inhibition of GARFT, particularly at low folate levels, is a contributing factor to drug activity but relative inhibition of TS and GARFT may vary among human tumors and cell lines.     

Multidrug resistance proteins and folate supplementation: therapeutic implications for antifolates and other classes of drugs in cancer treatment

Over the past decades, numerous reports have covered the crucial role of multidrug resistance (MDR) transporters in the efficacy of various chemotherapeutic drugs. Specific cell membrane-associated transporters mediate drug resistance by effluxing a wide spectrum of toxic agents. Although several excellent reviews have addressed general aspects of drug resistance, this current review aims to highlight implications for the efficacy of folate-based and other types of chemotherapeutic drugs. Folates are vitamins that are daily required for many biosynthetic processes. Folate supplementation in our diet may convey protective effects against several diseases, including cancers, but folate supplementation also makes up an essential part of several current cancer chemotherapeutic regimens. Traditionally, the folate leucovorin, for instance, is used to reduce antifolate toxicity in leukemia or to enhance the effect of the fluoropyrimidine 5-fluorouracil in some solid tumors. More recently, it has also been noted that folic acid has the ability to increase antitumor activity of several structurally unrelated regimens, such as alimta/pemetrexed and cisplatin. Moreover, studies from our laboratory demonstrated that folates could modulate the expression and activity of at least two members of the MDR transporters: MRP1/ABCC1, and the breast cancer resistance protein BCRP/ABCG2. Thus, folate supplementation may have differential effects on chemotherapy: (1) reduction of toxicity, (2) increase of antitumor activity, and (3) induction of MRP1 and BCRP associated cellular drug resistance. In this review the role of MDR proteins is discussed in further detail for each of these three items from the perspective to optimally exploit folate supplementation for enhanced chemotherapeutic efficacy of both antifolate-based chemotherapy and other classes of chemotherapeutic drugs.     

Expression of a folate binding protein in L1210 cells grown in low folate medium

We have isolated variants of L1210 cells (L1210B) expressing, in addition to the "classical" high affinity/low capacity system for reduced folate uptake, high levels of a membrane-associated folate binding protein. This folate binding protein was expressed in L1210 cells grown at low physiological folate levels (less than 0.5 nM), but down-regulated after transfer in standard high folate (2 microM) medium. The binding capacity of L1210B cells for [3H]folic acid and [3H]-methotrexate was identical (5-11 pmol/10(6) cells) but affinities were different. The affinities relative to folic acid were 0.5 for 5-methyltetrahydrofolate, 0.25 for 5-formyltetrahydrofolate, 0.08 for 10-ethyl-10-deazaaminopterin, and 0.05 for methotrexate, respectively. L1210B cells exposed to low extracellular concentrations of [3H]folic acid (25 nM) accumulated 15 pmol [3H]folic acid/10(7) cells over a 5-h period. [3H]Folic acid accumulation by wild-type L1210 cells could not be demonstrated under these conditions. The folate binding protein in L1210B cells could be specifically and covalently labeled at 4 degrees C with a N-hydroxysuccinimide ester of [3H]-methotrexate or [3H]folic acid. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of detergent-solubilized membrane proteins showed a major labeled band with Mr 42,000-44,000.     

Folate and antifolate cell binding and accumulation at physiologic folate concentrations

Cellular transport of folate has been shown to be via either a carrier-mediated or -facilitated diffusion process. The transport constant (Kt) values have ranged from 1 to 20 microM in vitro. These studies have generally been done on cells grown in high concentrations of folic acid (10(-6) to 10(-5) M). When cells were grown in folate-depleted medium for 72 hours, growth was nearly equivalent to that observed in complete medium, but a significant quantity of a high-affinity membrane receptor for folate was detected. The dissociation constants for folic acid, 5-methyltetrahydrofolate and methotrexate were 0.4, 4, and 18 nM, respectively. Incubation of folate-depleted cells for 3 hours at 10(-8) M methyltetrahydrofolate restored the intracellular folate content to values equivalent to those found in cells grown in folate-complete medium and decreased membrane binding by greater than 95%. Binding of 5-methyltetrahydrofolate was inhibited by methotrexate, folic acid, and 5-formyltetrahydrofolate, but not by glutamic acid, pteroic acid, or p-aminobenzoylglutamic acid. Antisera against a folate-binding protein (FBP) blocked cell binding of folate and purified FBP also blocks cell binding of folate. These results suggest that there is a receptor-mediated uptake of folate, functional at physiologic concentrations of plasma folate. This receptor could potentially be a new membrane target for antifol, antineoplastic agents.     

Pharmacology and mechanism of action of pemetrexed

Pemetrexed is a novel multitargeted antifolate that inhibits > or = 3 enzymes involved in folate metabolism and purine and pyrimidine synthesis. These enzymes are thymidylate synthase, dihydrofolate reductase, and glycinamide ribonucleotide formyltransferase. This agent has broad antitumor activity in phase II trials in a wide variety of solid tumors, and is approved in combination with cisplatin for the therapy of malignant mesothelioma. In a recent phase III trial, pemetrexed demonstrated equivalent efficacy to docetaxel, but with significantly less toxicity, in second-line treatment of non-small-cell lung cancer. The most common and serious toxicities of pemetrexed--myelosuppression and mucositis--have been significantly ameliorated by folic acid and vitamin B12 supplementation. More important, vitamin supplementation has not been shown to adversely affect efficacy in some tumor types. Tumors with codeletion of the methylthioadenosine phosphorylase gene, as a consequence of p16 deletions, may be particularly sensitive to pemetrexed. In this review, the biochemistry and mechanism of action of pemetrexed are discussed.     

Extent of the requirement for folate transport by L1210 cells for growth and leukemogenesis in vivo

In these studies the extent of the requirement for 5-methyltetrahydrofolate by L1210 cells for growth and leukemogenesis in vivo was addressed from the aspect of its cellular membrane transport. Growth characteristics and leukemogenesis in vivo were determined for parental and methotrexate-resistant L1210 cell variants with reduced capacity for folate coenzyme transport inward. These variants exhibited 6-, 16-, and 100-fold reductions compared to parental cells in influx Vmax for the high-affinity system transporting 5-substituted reduced folates and methotrexate. They also exhibited reduced saturability for methotrexate influx (3-fold higher Km), but not for influx of 5-formyltetrahydrofolate or 5-methyltetrahydrofolate. The reduced influx capacity in these variants correlated with their increased requirement for reduced folates during growth in vitro and with the ability of the variants to proliferate and develop leukemia in vivo. Lack of growth potential in vivo for one variant appears to reflect the inability for net intracellular accumulation of reduced folate per se, since growth of this variant could be restored by treatment of mice with folic acid, but not with 5-methyltetrahydrofolate or 5-formyltetrahydrofolate, and following reversion to a more transport-proficient phenotype.     

Fluorouracil-based chemotherapy in patients with gastrointestinal malignancies: influence of nutritional folate status on toxicity

This study aimed to prospectively evaluate the potential influence of folate status on the toxicity induced by 5-fluorouracil (5-FU)-based chemotherapy in patients with gastrointestinal tumors. 105 patients with colorectal, pancreatic or gastric cancer were entered into the study. Treatment regimens consisted of bolus 5-FU/leucovorin or infusional 5-FU combined with cisplatin. Baseline homocysteine, vitamin B(12) and folic acid serum levels were determined in all patients. Univariate and multivariate logistic regression models were used to identify predictive factors for toxicity. Univariate analysis showed a significant association between older age, low BSA, gastric/pancreatic cancer and treatment with 5-FU/cisplatin and the incidence of grade 3-4 hematological toxicity, and between female sex, low BSA and gastric/pancreatic cancer and the incidence of severe non-hematological toxicity. Variables that retained independent prognostic value in the multivariate model were tumor type, chemotherapy schedule and BSA for both hematological and non-hematological toxicities. Baseline homocysteine, vitamin B(12) or folate status were not significant predictors of any kind of toxicity either according to univariate or multivariate analysis. This study failed to demonstrate a significant association between a patient s nutritional folate status and the toxicity induced by fluoropyrimidine-based chemotherapy in a cohort of patients with various gastrointestinal malignancies.     

Sensitivity to 5,10-dideazatetrahydrofolate is fully conserved in a murine leukemia cell line highly resistant to methotrexate due to impaired transport mediated by the reduced folate carrier.

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.     

Pemetrexed disodium: a novel antifolate clinically active against multiple solid tumors

Pemetrexed disodium (ALIMTA), "pemetrexed") is a novel, multi-targeted antifolate that has demonstrated promising clinical activity in a wide variety of solid tumors, including non-small cell lung, breast, mesothelioma, colorectal, pancreatic, gastric, bladder, cervix, and head and neck. Pemetrexed inhibits multiple folate-dependent enzymes involved in both purine and pyrimidine synthesis including thymidylate synthase, dihydrofolate reductase, glycinamide ribonucleotide formyltransferase, and aminoimidazole carboxamide ribonucleotide formyltransferase. As a single agent, pemetrexed exhibits a moderate toxicity profile at a dose of 500 mg/m(2) by 10-minute infusion once every 21 days with myelosuppression being the dose-limiting toxicity. Folic acid added to the diet in preclinical studies reduced toxicities while maintaining antitumor activity. Based on this observation and clinical toxicities, folic acid and vitamin B(12) dietary supplementation has been recently introduced into all ongoing trials. Studies combining pemetrexed with other active chemotherapeutic agents demonstrate that these combination therapies may become important treatment regimens in a variety of cancer types. Currently, pemetrexed phase III trials are ongoing in mesothelioma and non-small cell lung cancer with future trials planned to explore this unique multitargeted antifolate.     

Responses of biomarkers of folate and riboflavin status to folate and riboflavin supplementation in healthy and colorectal polyp patients (the FAB2 Study).

Epidemiologic data suggest that increasing folate intake may protect against colorectal cancer. Riboflavin may interact with folate to modulate the effect. A double-blind randomized placebo-controlled intervention study (the FAB2 Study) was carried out in healthy controls and patients with colorectal polyps (adenomatous and hyperplastic) to examine effects of folic acid and riboflavin supplements on biomarkers of nutrient status and on putative biomarkers of colorectal cancer risk (DNA methylation and DNA damage; to be reported elsewhere). Ninety-eight healthy controls and 106 patients with colorectal polyps were stratified for the thermolabile variant of methylene tetrahydrofolate reductase, MTHFR C677T, and were randomized to receive 400 microg of folic acid, 1,200 microg of folic acid, or 400 microg of folic acid plus 5 mg of riboflavin or placebo for 6 to 8 weeks. Blood samples and colon biopsy samples were collected for the measurement of biomarkers of folate and riboflavin status. Supplementation with folic acid elicited a significant increase in mucosal 5-methyl tetrahydrofolate, and a marked increase in RBC and plasma, with a dose-response. Measures of riboflavin status improved in response to riboflavin supplementation. Riboflavin supplement enhanced the response to low-dose folate in people carrying at least one T allele and having polyps. The magnitude of the response in mucosal folate was positively related to the increase in plasma 5-methyl tetrahydrofolate but was not different between the healthy group and polyp patients. Colorectal mucosal folate concentration responds to folic acid supplementation to an extent comparable to that seen in plasma, but with a suggestion of an upper limit.     

Clinical activity of pemetrexed: a multitargeted antifolate anticancer agent

Pemetrexed is a new generation antifolate anticancer agent that inhibits several folate-dependent enzymes required for production of DNA and RNA intermediates. Early studies showed significant hematologic and nonhematologic toxicities with this agent. However it was found that many of these toxicities related to functional folate status and could be markedly reduced through routine supplementation with folic acid and vitamin B(12), without adversely affecting efficacy. Phase III studies with pemetrexed have established a clinical role for this drug as a single agent in the second-line treatment of non-small cell lung cancer and in combination with cisplatin for the frontline treatment of unresectable malignant pleural mesothelioma. Clinical trials of pemetrexed alone or in combination with other chemotherapeutic agents have shown considerable activity in many tumor types including colorectal, pancreatic and breast cancer, and urothelial tumors.     

Farletuzumab, an anti-folate receptor α antibody, does not block binding of folate or anti-folates to receptor nor does it alter the potency of anti-folates in vitro

Purpose

Folate is a cofactor in the synthesis of purines and pyrimidines; folate analogs are potent cytotoxic drugs. Folate receptor alpha (FR??), a protein-mediating cellular accumulation of folate (and anti-folates), has limited expression in normal tissues and is overexpressed by numerous carcinomas. Limited distribution and high affinity for folic acid have resulted in the development of antibodies or the use of folic acid coupled to toxins or radionuclides as therapeutic and imaging agents. Farletuzumab is an anti-FR?? antibody in clinical trials for ovarian and non-small cell lung cancers. Our goal was to evaluate the effect of farletuzumab on binding and uptake of folates and anti-folates and the potency of anti-folates in vitro.

Methods

Direct binding and uptake of radiolabeled folates and anti-folates and the assessments of drug concentration of drug that inhibited cell growth 50?% (IC₅₀) in vitro in the presence or absence of antibody.

Results

Farletuzumab did not block membrane binding of radiolabeled folic acid, 5-methyltetrahydrofolate, pemetrexed, and other anti-folates; folic acid blocked >95?%. Farletuzumab had a minimal effect on the cytoplasmic accumulation of 5-methyltetrahydrofolate or pemetrexed; folic acid had a considerable but variable effect on the different cell lines. As a single agent, farletuzumab did not affect cell viability or the IC₅₀ of pemetrexed and other anti-folates in vitro.

Conclusions

Farletuzumab does not block FR?? binding of folates and anti-folates, minimally retards folate delivery via FR??-mediated transport, and minimally retards the growth of cells in vitro. Concomitant use of farletuzumab and pemetrexed is not contraindicated.     

Effect of folic Acid supplementation on the folate status of buccal mucosa and lymphocytes.

Folate deficiency may be associated with an increased risk of cancer at certain sites. There is a need to measure folate status and putative biomarkers of cancer risk in the same target tissue, or in surrogate tissues. A study was carried out to develop a method for the rapid measurement of folate in human buccal mucosa and lymphocytes and to evaluate the responsiveness of this measurement in both tissues to folic acid supplementation in healthy subjects, relative to conventional markers of folate status. Three hundred and twenty-three adults, ages between 20 and 60 years, were screened for RBC folate concentrations. Sixty-five subjects with red cell folate between 200 and 650 nmol/L participated in a randomized, double blind, placebo-controlled, folic acid (1.2 mg) intervention trial, lasting 12 weeks. As anticipated, a significant baseline correlation (r = 0.36, P < 0.01) was observed between red cell folate and plasma 5-methyltetrahydrofolate (5-MeTHF). Lymphocyte total folate was significantly associated with plasma 5-MeTHF (r = 0.28, P < 0.05) and plasma total homocysteine concentration (r = -0.34, P < 0.05). Buccal mucosa total folate showed no correlation with either red cell folate or 5-MeTHF, but was significantly associated with lymphocyte total folate (r = 0.35, P < 0.01). Supplementation elicited a significant increase in lymphocyte total folate (P < 0.01), and this was strongly associated with the increase in RBC total folate (P < 0.01) and plasma 5-MeTHF (P < 0.01). Buccal mucosa total folate was not influenced by folate supplementation. Methods have been developed for the rapid measurement of lymphocyte and buccal mucosal total folate. Lymphocyte folate is sensitive to folate intake and is reflected by plasma 5-MeTHF.     

Phase I and pharmacokinetic study of pemetrexed with high-dose folic acid supplementation or multivitamin supplementation in patients with locally advanced or metastatic cancer.

PURPOSE: This phase I study evaluated the effect of folate supplementation on the toxicity, tolerability, and pharmacokinetics of pemetrexed in patients with locally advanced or metastatic cancer. It also examined two different types of vitamin supplementation and whether the extent of prior myelosuppressive therapy affected pemetrexed tolerability. Patients and Methods: Patients received a 10-min infusion of 600 to 14,00 mg/m(2) pemetrexed every 3 weeks. Patients were stratified into cohorts by pretreatment status [lightly pretreated (LPT) or heavily pretreated (HPT)] and were supplemented with intermittent high-dose folic acid (HDFA) or with continuous daily multivitamins (MVI) containing nutritional doses of folic acid. Pemetrexed plasma pharmacokinetics were evaluated for cycle 1. RESULTS: Sixty-two HDFA patients (28 HPT and 34 LPT) were treated with 204 cycles of pemetrexed, and 43 MVI patients (20 HPT and 23 LPT) were treated with 182 cycles. Hematologic dose-limiting toxicities included grade 4 neutropenia (5 of 105 patients), grade 4 thrombocytopenia (4 of 105 patients), and febrile neutropenia (3 of 105 patients). Nonhematologic toxicities included fatigue, vomiting, diarrhea, and nausea. Pemetrexed doses of 800 and 1,050 mg/m(2) were well tolerated when administered with vitamin supplementation to HPT and LPT patients, respectively. There were no clinically relevant differences in toxicities or pemetrexed pharmacokinetics for LPT versus HPT patients or for patients receiving HDFA versus daily MVI supplementation. CONCLUSIONS: The pemetrexed doses tolerated in this study with vitamin supplementation were significantly higher than those tolerated in earlier studies without supplementation, and toxicities were independent of the type of vitamin supplementation or prior myelosuppressive treatment. The recommended dose of pemetrexed is 1,050 mg/m(2) in LPT patients and 800 mg/m(2) in HPT patients, irrespective of the type of vitamin supplementation.     

Pemetrexed: a novel antifolate agent enters clinical practice

Pemetrexed (Alimta, Eli Lilly) is a multitargeted antifolate that inhibits at least three enzymes in the nucleic acid synthetic pathways. The US Food and Drug Administration recently approved pemetrexed, in combination with cisplatin, for the first-line treatment of advanced malignant pleural mesothelioma. Moreover, pemetrexed was recently shown to be as efficacious as docetaxel (Taxotere, Aventis) in the second-line treatment of non-small cell lung cancer, and its toxicity profile was preferable. The main toxicity seen with pemetrexed is myelosuppression, which is considerably reduced by coadministration of folic acid and vitamin B12. Multiple Phase II clinical trials have demonstrated that pemetrexed has promising single-agent activity in many other solid tumors, including head and neck, breast and colorectal cancers. Combination regimens consisting of pemetrexed and other chemotherapeutics or novel molecular-targeted agents are currently under investigation. Future studies will better define and likely expand the role of pemetrexed for the treatment of cancer.     

A prominent low-pH methotrexate transport activity in human solid tumors: contribution to the preservation of methotrexate pharmacologic activity in HeLa cells lacking the reduced folate carrier.

Whereas the major folate transporter, the reduced folate carrier (RFC), has a physiological pH optimum, transport activities for folates and antifolates have been detected with low pH optima. Because the interstitial pH in solid tumors is generally acidic, the mechanisms by which antifolates are transported at low pH could be an important determinant of drug activity under these conditions. The current study quantitated the low pH methotrexate (MTX) transport activity in human solid tumor cell lines from the National Cancer Institute tumor panel and other sources. MTX influx at pH 5.5 was equal to, or greater than, influx at pH 7.4 in 29 of 32 cell lines. To assess the role of RFC in transport at low pH in one of these cell lines, a HeLa clonal line (R5) was selected for MTX resistance due to a genomic deletion of the carrier gene. MTX influx was depressed by 70% in R5 versus wild-type HeLa cells at pH 7.4. At pH 6.5, influx in these two lines was similar; as the pH was decreased to 5.5 influx increased in both cell lines. Similarly, whereas net MTX uptake over 1 h was markedly decreased in R5 cells at pH 7.4, net uptake in HeLa and R5 cells was comparable at pH 6.5. Also, as compared with MCF7 breast cancer cells, MTX uptake was markedly decreased at pH 7.4, but only minimally at pH 6.5, in the MDA-MB-231 human breast cancer cell line that lacks RFC expression. When grown with folic acid (2 micro M) at pH 7.4, the IC(50) for MTX was 14-fold higher in R5 as compared with wild-type HeLa cells; the difference was only 4-fold when cells when grown at pH 6.9; the IC(50)s were identical at this pH when the medium folate was 25 nM 5-formyltetrahydrofolate. These data demonstrate that transport activity at low pH is prevalent in human solid tumors, is RFC-independent in R5 cells and MDA-MB-231 breast cancer cells, and can preserve MTX activity in the absence of RFC at an acidic pH relevant to solid tumors in vivo.     

Characteristics of a novel transport system for folate compounds in wild-type and methotrexate-resistant L1210 cells

The growth requirements and transport characteristics of folate compounds in a methotrexate-resistant L1210/R81 cell line were compared with parental cells. Concentrations for half-maximal growth of the resistant cells with folate (350 nM) and 5-formyltetrahydrofolate (20 nM) were found to be higher by 2.7-fold and 20-fold, respectively, relative to the parent, suggesting that changes had occurred in the transport of these folate compounds. Transport measurements revealed that the resistant cells have lost the capacity to transport methotrexate and other folate compounds via the reduced-folate transport system but that a second previously undescribed transport system is present. Uptake of folate via this second route is energy dependent, exhibits saturation kinetics, can be inhibited by substrate analogues, and is activated by a reduction in pH. The pH effect is substantial since uptake at 5.0 microM folate can be increased 10-fold by decreasing the pH from 7.4 to 6.2. The observed Kt for half-maximal influx of folate at pH 6.8 was 5.2 microM, and the Vmax was 0.55 pmol/min/mg of protein. Ki values for methotrexate, 5-methyltetrahydrofolate, and 5-formyltetrahydrofolate were 10.2, 3.2, and 2.7 microM, respectively. Transport activity was not affected by depleting internal stores of folate by growth in folate-free medium, and this same transport system was also present in comparable amounts in wild-type cells. In the latter case, interfering uptake via the reduced-folate transport system was blocked by bromosulfophthalein and thiamine pyrophosphate. Methotrexate and 5-formyltetrahydrofolate were also transported via this alternative, pH-dependent route, whereas little or no uptake of 5-methyltetrahydrofolate could be detected. The results indicate that antifolate resistance in L1210/R81 cells was induced by inactivating the reduced-folate transport system and by utilizating a secondary route which has a low capacity for transporting methotrexate but whose activity is sufficient to permit cell growth in medium supplemented with folate or 5-formyltetrahydrofolate.     

Maternal folate and other vitamin supplementation during pregnancy and risk of acute lymphoblastic leukemia in the offspring

The Australian Study of Causes of Acute Lymphoblastic Leukemia in Children (Aus‐ALL) was designed to test the hypothesis, raised by a previous Western Australian study, that maternal folic acid supplementation during pregnancy might reduce the risk of childhood acute lymphoblastic leukemia (ALL). Aus‐ALL was a national, population‐based, multicenter case–control study that prospectively recruited 416 cases and 1,361 controls between 2003 and 2007. Detailed information was collected about maternal use of folic acid and other vitamin supplements before and during the index pregnancy. Data were analyzed using logistic regression, adjusting for matching factors and potential confounders. A meta‐analysis with the results of previous studies of folic acid supplementation was also conducted. We found weak evidence of a protective effect of maternal folate supplementation before pregnancy against risk of childhood ALL, but no evidence for a protective effect of its use during pregnancy. A meta‐analysis including this and 2 other studies, but not the study that raised the hypothesis, also found little evidence that folate supplementation during pregnancy protects against ALL: the summary odds ratios (ORs) for folate supplementation were 1.06 [95% confidence interval (CI): 0.77–1.48] with reference to no folate supplementation and 1.02 (95% CI: 0.86–1.20) with reference to no vitamin supplementation. For vitamin supplementation in general, the summary OR from a meta‐analysis of 5 studies—including Aus‐ALL—was 0.83 (95% CI: 0.73–0.94). Vitamin supplementation in pregnancy may protect against childhood ALL, but this effect is unlikely to be large or, if real, specifically due to folate.     

99mTc-Tetraethylenepentamine-Folate--a new 99mTc-based folate derivative for the detection of folate receptor positive tumors: synthesis and biological evaluation

A new radiopharmaceutical, 99mTc-Tetraethylenepentamine(TEPA)-Folate has been synthesized introducing TEPA to the gamma-carboxyl group of folic acid. This binds with 99mTc high efficiency at ambient temperature. The resulting 99mTc-N5-Folate is stable under physiological conditions at least for 24 h after radiocomplexation. TEPA is a known open chain pentamine (N5) chelator, its four-nitrogen act as the binding site for 99mTc. The folate membrane receptor binding of the 99mTc-TEPA-Folate by established human tumor cell lines (KB, U-87MG and MDA-MB-468) showed Kd in microM range in normal DMEM (10% serum, 10 microM folic acid). The blood kinetic studies showed more than 70% clearance within five minutes from the circulation. The KB cell line tumors in mice were readily identifiable in the gamma images and revealed major accumulation of radiotracer in liver, kidneys and intestines. High tumor uptake was shown in the tumor bearing nude mice; tumorto-blood ratios reached 2.68 +/- 0.52 and 5.5 +/- 1.47 at 1 and 4 h after post injection respectively. Surviving fractions as obtained in clonogenic assay were 1.02 +/- 0.07 and 1.03 +/- 0.05 in U-87MG and MDA-MB-468 cell lines respectively. The 99mTc-N5-Folate conjugate have promising utility as a receptor specific radiopharmaceutical for imaging neoplastic tissues known to over express folate-binding protein.