Polyglutamate Forms Research Articles

Abstract 4730: Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors

Abstract These studies investigate factors that impact the antitumor activity of SHMT2 targeting including drug accumulation, metabolism, expression of one carbon (C1) related enzymes, and tumor hypoxia. Mitochondrial C1 metabolism is upregulated in a variety of cancer types including leukemias and solid tumors. The mitochondrial C1 enzymes serine hydroxymethyltransferase (SHMT) 2 and 5,10-methylene tetrahydrofolate (THF) dehydrogenase 2 (MTHFD2) are among the top 5 overexpressed metabolic enzymes in tumors vs normal tissues in a wide range of tumor types. Thus, SHMT2 and MTHFD2 are potential therapeutic targets for cancer, sparing normal tissues that are less reliant on mitochondrial C1 metabolism. SHMT2 metabolizes serine and THF to produce glycine and 5,10-methylene THF; MTHFD2 metabolizes 5,10-methylene THF to NADH and 10-formyl THF which is converted to formate by MTHFD1L. Thus, mitochondrial C1 metabolism is the principal source of C1 units for cellular biosynthesis and provides the majority of glycine for protein, purine and glutathione synthesis. We previously discovered novel 5-substituted pyrrolo[3,2-d]pyrimidine antifolate compounds which exhibit primary inhibition at mitochondrial SHMT2 along with cytosolic SHMT1 and de novo purine biosynthesis at glycinamide ribonucleotide and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferases. The lead compound AGF347 is taken up into cells by the facilitative transporters reduced folate carrier (SLC19A1) and the proton coupled folate transporter (SLC46A1) and like folate cofactors is metabolized to polyglutamate (PG) forms by folylpolyglutamate synthase (FPGS) which promotes retention in cytosolic and mitochondrial compartments and target enzyme engagement. We investigated the impact of folate transporter expression on metabolism to PGs for SHMT2 targeted antifolates. Folate transporter expression inversely correlated with anti-tumor efficacy of SHMT2 targeted antifolates and overexpression of FPGS resulted in dramatically increased sensitivities. However, for pyrazolopyran inhibitors of SHMT2 (e.g. SHIN1) which enter cells by diffusion and are not metabolized to polyglutamates, increased transporter and FPGS levels dramatically decreased anti-tumor activity. As SHMT2 is a target of HIF1α, we studied the impact of multitargeting SHMT2 and purine biosynthesis by pyrrolo[3,2-d]pyrimidine antifolates under hypoxic conditions. Our results establish that multi-targeting SHMT2 and purine biosynthesis preserves anti-tumor activity under hypoxia. Collectively, our results demonstrate that SHMT2 targeted pyrrolo[3,2-d]pyrimidine antifolates exhibit increased antitumor efficacies in cells expressing low folate transporter expression and is further enhanced by increased FPGS activity. Additionally, we find multitargeting of SHMT2 and de novo purine biosynthesis manifests as antitumor activity even under hypoxic conditions. Citation Format: Mathew Joseph Schneider, Carrie O'Connor, Xun Bao, Md. Junayed Nayeen, Zhanjun Hou, Jing Li, Aleem Gangjee, Larry Matherly. Pharmacodynamic determinants of anti tumor activity of SHMT2 inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4730.

Methotrexate Triglutamate as a Determinant of Clinical Response in Mexican Patients With Rheumatoid Arthritis: Pharmacokinetics and Dose Recommendation.

Methotrexate is the gold standard treatment in rheumatoid arthritis. Once absorbed, it is internalized in cells, where glutamate residues are added to produce polyglutamated forms, which are responsible for the effect of methotrexate. The aim of the current study is to determine the relationship between methotrexate triglutamate concentrations and the clinical evolution in rheumatoid arthritis patients, as well as to characterize the variability in both features to propose strategies for low-dose methotrexate optimization. The quantification of methotrexate triglutamate concentration in red blood cells was performed through ultra-performance liquid chromatography coupled with mass spectrometry. Polymorphisms of genes involved in the formation of polyglutamates were determined by real-time polymerase chain reaction. A multivariate regression was performed to determine the covariates involved in the variability of methotrexate triglutamate concentrations and a population pharmacokinetics model was developed through nonlinear mixed-effects modeling. Disease activity score changed according to methotrexate triglutamate concentrations; patients with good response to treatment had higher concentrations than moderate or nonresponding patients. The methotrexate triglutamate concentrations were related to time under treatment, dose, red blood cells, and body mass index. A 1-compartment open model was selected to estimate the pharmacokinetic parameters; the typical total clearance (L/day) was determined as 1.45 * (body mass index/28 kg/m2 ) * (red blood cells/4.6 × 106 cells/μL) and the volume of distribution was 52.4 L, with an absorption rate of 0.0346/day and a fraction metabolized of 1.03%. Through the application of the model, the initial dose of methotrexate is proposed on the basis of stochastic simulations and considering methotrexate triglutamate concentrations found in responders patients.

Folates: Key Nutrients to Remember

Folic acid or vitamin B9 or pteroylglutamic acid, is a relatively simple molecule with two characteristics; firstly, it must be reduced by 2 or 4 hydrogen atoms to be metabolically active which makes it sensitive to oxidation and must be protected by ascorbic acid, secondly it may include in addition to the constituent residues of the molecule, 1-7 glutamate residue at one of its ends. These polyglutamate forms that make up the largest share of food folate, must be deconjugated by a specific enzyme present in the intestinal lumen before being absorbed in the jejunum. It is in the methylated form after passing through the enterocyte it is transported in the blood, excreted in bile and reabsorbed. It must be demethylated to integrate folic cell cycle and methyl transfer, that allows the synthesis of methionine (only possible in the presence of vitamin B12), purine, serine and especially thymidylic acid, constitutive DNA. As a methyl donor that plays a fundamental role in cerebral and nervous metabolism. Folates are involved in cell division thus; any folic acid deficiency causes a slowdown in rapid multiplication systems which may lead to red blood cell disorders (macrocytic anemia), immunity, and neural tube defects, in addition to physiological disorders (cardiovascular, cancer ...). Folic acid supplementation appears to allow the correction of these disorders.

Using Methotrexate Metabolites to Make Clinical Decisions in JIA

Determining how to optimally treat juvenile idiopathic arthritis (JIA) remains a challenge, although we are better equipped to do so now than ever before. Capitalizing on currently available therapies for rapid and sustained disease control remains incredibly important since long-term outcomes are shown to be impacted by the duration of active disease, and arthritis in childhood can influence growth and development with long-term consequences in adulthood. Although achieving remission remains difficult, we are now better able to achieve clinically inactive disease utilizing the therapeutic armamentarium that has become available over the last several decades. The current challenge is determining which medication to use and when, since significant variability is observed in treatment response and toxicity and there is a wide array of costs associated with currently available treatments. The search for viable biomarkers and prediction tools to assist with these clinical decisions remains underway. Methotrexate is an important therapeutic option either alone or in combination with other medications, and although it has a relatively long history of clinical use, there remains significant variability in clinical response and an incomplete understanding of its mechanism of action. The polyglutamated form of methotrexate (MTXGlun) is a more stable and reliable cellular marker of methotrexate (MTX) activity and has received attention in recent years as a potentially useful biomarker for MTX. Improvements in the sensitivity of MTXGlun quantitation via HPLC-tandem mass spectrometry (LC-MS/MS) and observed associations between MTXGlun and clinical outcomes keep the optimism high for this biomarker. However, utilizing MTXGlun “real time” for clinical decision making is neither a currently mainstream practice in pediatrics, nor is it ready to be. Available data in pediatrics have not identified or validated targetable MTXGlun concentrations associated with clinical improvement in JIA, and the currently available high-performance liquid chromatography (HPLC) clinical assays are not as sensitive as LC-MS/MS methods reported in current research studies in JIA. Additionally, factors such as time to transport for processing, need for sample refrigeration, and protection from light can affect sample stability as improper handling can result in continued action of cellular enzymes within the folate pathway resulting in continued deglutamation and falsely low MTXGlun concentrations. Along these same lines, pharmacogenomic studies searching for genetic factors that impact MTX outcomes have also been challenged with lack of validation. Based on the complex genetic architecture of the folate pathway (discussed below), inherent redundancy in the system likely requires changes in many genes, rather than a single gene variant, to result in a significant impact on drug outcomes. More robust and consistent evidence is needed on the association of MTXGlun concentrations with clinical response in JIA. Furthermore, since MTX dosing in children is higher per body surface area than in adults, meaningful MTXGlun levels in children also need to be identified and validated before utilizing this clinical assay in JIA. Despite the limitations of use in the clinical setting at the current moment, MTXGlun measurement may prove to be additionally useful in exploring the mechanisms of action of MTX and the effect of this drug on the complex endogenous folate pathway—all important in understanding the complete picture of MTX action in JIA.

The membrane transport and polyglutamation of pralatrexate: a new-generation dihydrofolate reductase inhibitor

To characterize, directly and for the first time, the membrane transport and metabolism of pralatrexate, a new-generation dihydrofolate reductase inhibitor approved for the treatment for peripheral T-cell lymphoma. [(3)H]pralatrexate transport was studied in unique HeLa cell lines that express either the reduced folate carrier (RFC) or the proton-coupled folate transporter (PCFT). Metabolism to active polyglutamate derivatives was assessed by liquid chromatography. These properties were compared to those of methotrexate (MTX). The pralatrexate influx K t, mediated by RFC, the major route of folate/antifolate transport at systemic pH, was 0.52μΜ, 1/10th the MTX influx K i. The electrochemical potential of pralatrexate within HeLa cells far exceeded the extracellular level and was greater than for MTX. In contrast, MTX transport mediated by PCFT, the mechanism of folate/antifolate absorption in the small intestine, exceeded that for pralatrexate. After a 6h exposure of HeLa cells to 0.5μM pralatrexate, 80% of intracellular drug was its active polyglutamate forms, predominantly the tetraglutamate, and was suppressed when cells were loaded with natural folates. There was negligible formation of MTX polyglutamates. The difference in pralatrexate and MTX growth inhibition was far greater after transient exposures (375-fold) than continuous exposure (25-fold) to the drugs. Pralatrexate's enhanced activity relative to MTX is due to its much more rapid rate of transport and polyglutamation, the former less important when the carrier is saturated. The low affinity of pralatrexate for PCFT predicts a lower level of enterohepatic circulation and increased fecal excretion of the drug relative to MTX.

Folylpoly-Glutamate Synthetase Expression Is Associated with Tumor Response and Outcome from Pemetrexed-Based Chemotherapy in Malignant Pleural Mesothelioma

Pemetrexed-based chemotherapy represents the standard of care in first-line treatment of advanced malignant pleural mesothelioma (MPM). However, there are no established predictors of clinical benefit. Pemetrexed inhibits multiple enzymes involved in pyrimidine and purine synthesis, but the main target is thymidylate synthase (TS). After cellular uptake pemetrexed is converted into more effective polyglutamated forms by folylpoly-γ-glutamate synthetase (FPGS). We hypothesized that FPGS and TS protein expressions are associated with clinical outcome after pemetrexed-based chemotherapy. Pretreatment tumor samples from 84 patients with histologically confirmed MPM, who received pemetrexed combined with platinum (79 of 84) or single-agent pemetrexed (5 of 84) as first-line treatment, were retrospectively analyzed. FPGS and TS protein expressions were semiquantitatively assessed by using the Hybrid (H)-scoring system (range, 0-300). H-scores were correlated with radiological response according to modified Response Evaluation Criteria in Solid Tumors, progression-free survival (PFS) and overall survival (OS). Median H-score of the entire cohort was 230 for FPGS (range, 100-300), and 210 for TS (range, 100-300). High FPGS protein expression was significantly associated with longer PFS (pCOX = 0.0337), better objective tumor response (partial response versus stable disease + progressive disease; pKW = 0.003), and improved disease-control rate (partial response + stable disease versus progressive disease; pKW = 0.0208), but not with OS. In addition, high TS protein expression was associated with progressive disease under pemetrexed-based therapy (p = 0.0383), and shorter OS (pCOX = 0.0071), but no association with PFS was observed. FPGS and TS expressions were associated with clinical response and outcome to pemetrexed-based first-line chemotherapy in MPM. Prospective evaluation of FPGS and TS expressions and their prognostic/predictive power in MPM patients is warranted.

Understanding the variation in response to methotrexate

ORIGINAL ARTICLE, p 165 Methotrexate (MTX) has been used in the management of psoriasis since the 1960s and despite the increasing use and success of newer biologic agents, it remains a first‐line treatment in patients with moderate to severe psoriasis, due to its efficacy and cost‐effectiveness.1 It is used as a standard comparator in randomized controlled trials of biologic agents in psoriasis. However, a significant number of patients on MTX do not experience benefit and up to 30% of patients are unable to tolerate the drug because of toxicity including nausea, myelosuppression or hepatotoxicity.2 Reasons for this inter‐individual variability in both toxicity and response are partially unknown but may include demographic factors (such as patient age and weight), external or environmental influences (for instance adherence to treatment, drug dosage), and genetic variation in pharmacokinetics (for instance, drug metabolizing enzymes) or pharmacodynamics (drug targets). Pharmacogenetics is the study of those inherited factors that influence the inter‐individual variability in response to drugs and is a rapidly developing field. The pharmacokinetics and pharmacodynamics of MTX, as have recently been unravelled, have proved to be complicated, and early attempts at pharmacogenetic studies in both rheumatology and dermatology using a candidate gene approach have not been definitive. MTX is a pro‐drug which is actively transported into the cell via a transmembrane reduced folate carrier protein (SLC19A1) and actively exported from the cell by ATP‐binding cassette transporters ABCC1 and ABCG2. Polymorphisms in ABCC1 and ABCG2 have been found to correlate with a better response to MTX (with odds ratios of about 2) and polymorphisms in SLC19A1 and ABCC1 have been associated with MTX toxicity. Within the cell, MTX is activated by the sequential addition of between two and seven glutamic acid residues under the action of the enzyme folylpolyglutamate synthetase, thus converting the parent drug to the active and more stable polyglutamated form. MTX polyglutamates (MTXPGs) persist intracellularly, exerting a sustained effect which allows for its once‐weekly administration.3 Some studies of patients with rheumatoid arthritis have shown a correlation between intracellular MTXPG levels and MTX treatment efficacy.4, 5

Abstract 1713: Folylpoly-α-glutamate synthetase and thymidylate synthase are associated with clinical outcome from pemetrexed-based therapy in advanced non-small cell lung cancer (NSCLC)

Abstract Background: The antifolate pemetrexed targets multiple enzymes involved in pyrimidine and purine synthesis including thymidylate synthase (TS). After entry into cells, pemetrexed is converted to polyglutamated forms by folylpoly-α-glutamate synthetase (FPGS), a critical step to achieve full target inhibition. We hypothesized that FPGS and TS protein expression may predict outcome following pemetrexed-treatment of patients with advanced NSCLC, like in malignant pleural mesotheliomas (Christoph et al., J Clin Oncol. 2011: 29 suppl.). This is the largest report on pemetrexed-treatment outcome based on TS and FPGS in Caucasian patients with advanced NSCLC. Methods: Pretreatment tumor samples from 161 patients (pts) with metastatic NSCLC, treated with PMX (82 pts (51%)), a combination of PMX with platinum (74 pts (46%)) or within other combinations (5 pts (3%)), were retrospectively analyzed. FPGS and TS protein expression levels were evaluated by IHC using the H-Scoring system (0-300), which relies on the product of intensity (range 0 to 3) and the percentage of positive tumor cells (0-100%). Radiographic evaluation of response was performed according to RECIST criteria (version 1.1). Results: Median pretreatment H-scores were 180 for FPGS (range: 0-280) and 205 (range: 120-290) for TS. Using the log-rank test and the median H-score as cut-off, we found a significant association between low TS protein expression and improved progression-free survival (PFS) (median PFS of 5.5 months vs 3.4 months; hazard ratio [HR] 0.66, 95% CI, 0.45 to 0.96; P=0.03) or prolonged overall survival (median OS of 33.9 months vs 15.0 months; hazard ratio [HR] 0.52, 95% CI, 0.31 to 0.86; P=0.01). Moreover, high FPGS protein expression was only associated with better PFS (median PFS of 5.5 months vs 3.4 months; hazard ratio [HR] 0.58, 95% CI, 0.37 to 0.89; P=0.03). Considering exclusively patients suffering from adenocarcinomas (110 pts (68%)), TS was associated with objective response to pemetrexed-based treatment (mean H-score 192 for responders vs 210 for non-responders, P=0.03). Conclusions: We have investigated FPGS and TS protein expressions in tumor specimens from the largest series of PMX-treated Caucasian NSCLC patients. Baseline determination of TS and FPGS expression by IHC using the H-score system is associated with clinical outcome from PMX-based therapy in advanced NSCLC. Further prospective validation studies are warranted. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1713. doi:1538-7445.AM2012-1713

Red blood cell folate concentrations and polyglutamate distribution in juvenile arthritis

Methotrexate (MTX) has several enzymatic targets in the folate pathway. To better understand the variability in response to MTX, we characterized the interindividual variability of intracellular folate pools in children with juvenile arthritis (JA) and determined clinical and genetic contributors to this variability. This exploratory single-center cross-sectional study evaluated 93 patients with JA not currently receiving MTX. Whole blood, plasma, and erythrocyte folate concentrations were determined after deconjugation and analyzed through reversed-phase separation and stable isotope dilution tandem mass spectrometry. Folate polyglutamates were measured in red blood cell lysates using an ion-pair reversed phase chromatography tandem mass spectrometry method. Intracellular concentrations of 5-methyl-tetrahydrofolate (5-CH3-THF) and 5,10-methenyl-tetrahydrofolate varied approximately 20-fold and 80-fold, respectively. The polyglutamated forms of 5-CH3-THF as a percentage of total 5-CH3-THF (5-CH3-THFGlun) were also measured. Hierarchical clustering of 5-CH3-THFGlun revealed two groups, each with two distinct clusters. There was an inverse relationship between 5-CH3-THFGlun chain length and plasma 5-CH3-THF concentrations. A subgroup of patients with a historical intolerance to MTX had significantly lower cellular folate concentrations (P<0.0001). In univariate analyses, clinical variables including sex, age, and folate supplementation in addition to variations in MTHFR, MTR, and SLC25A32 were associated with differential intracellular folate redox concentrations. Multivariate analysis further supported the association of single nucleotide polymorphisms in SLC25A32, MTHFR, and MTR with variability in intracellular 5-CH3-THF and 5,10-methenyl-tetrahydrofolate concentrations, respectively. Measurement of intracellular folate isoforms may contribute toward a better understanding of individual MTX effects in JA. Clinical variables in addition to genotypic differences beyond MTHFR may additionally explain differential intracellular folate concentrations and variable responses to MTX.

Abstract B6: Folylpoly-glutamate synthetase and thymidylate synthase are associated with clinical outcome from pemetrexed-based therapy in non-small cell lung cancer (NSCLC)

Abstract Background: The antifolate pemetrexed targets multiple enzymes involved in pyrimidine and purine synthesis including thymidylate synthase (TS). After entry into cells, pemetrexed is converted to polyglutamated forms by folylpoly-γ-glutamate synthetase (FPGS), a critical step to achieve full target inhibition. We hypothesized that FPGS and TS protein expression may predict outcome following pemetrexed-treatment of patients with advanced NSCLC, like in malignant pleural mesotheliomas (Christoph et al., J Clin Oncol. 2011: 29 suppl.). This is the largest report on pemetrexed-treatment outcome based on TS and FPGS in Caucasian patients with advanced NSCLC. Methods: Pretreatment tumor samples from 161 patients (pts) with metastatic NSCLC, treated with pemetrexed combined with platinum (74 pts (46%)) or as single agent (82 pts (51%)) or within other combinations (5 pts (3%)), were retrospectively analyzed. FPGS and TS protein expression levels were evaluated by IHC using the H-Scoring system (0–300), which relies on the product of intensity (range 0 to 3) and the percentage of positive tumor cells (0–100%). Radiographic evaluation of response was performed according to RECIST criteria (version 1.1). Results: Median pretreatment H-scores were 180 for FPGS (range: 0–280) and 205 (range: 120–290) for TS. Using the log-rank test and the median H-score as cut-off, we found a significant association between low TS protein expression and improved progression-free survival (PFS) (median PFS of 5.5 months vs. 3.4 months; hazard ratio [HR] 0.66, 95% CI, 0.45 to 0.96; P=0.03) or prolonged overall survival (median OS of 33.9 months vs. 15.0 months; hazard ratio [HR] 0.52, 95% CI, 0.31 to 0.86; P=0.01). Moreover, high FPGS protein expression was only associated with better PFS (median PFS of 5.5 months vs. 3.4 months; hazard ratio [HR] 0.58, 95% CI, 0.37 to 0.89; P=0.03). Considering exclusively patients suffering from adenocarcinomas (110 pts (68%)), TS was associated with objective response to pemetrexed-based treatment (mean H-score 192 for responders vs. 210 for non-responders, P=0.03). Conclusions: We have investigated FPGS and TS protein expressions in tumor specimens from the largest series of pemetrexed-treated Caucasian NSCLC patients. Baseline determination of TS and FPGS expression by IHC using the H-score system is associated with clinical outcome from pemetrexed-based therapy in advanced NSCLC. Further prospective validation studies are warranted.

Differences in folate–protein interactions result in differing inhibition of native rat liver and recombinant glycine N-methyltransferase by 5-methyltetrahydrofolate

Glycine N-methyltransferase (GNMT) is a key regulatory enzyme in methyl group metabolism. In mammalian liver it reduces S-adenosylmethionine levels by using it to methylate glycine, producing N-methylglycine (sarcosine) and S-adenosylhomocysteine. GNMT is inhibited by binding two molecules of 5-methyltetrahydrofolate (mono- or polyglutamate forms) per tetramer of the active enzyme. Inhibition is sensitive to the status of the N-terminal valine of GNMT and to polyglutamation of the folate inhibitor. It is inhibited by pentaglutamate form more efficiently compared to monoglutamate form. The native rat liver GNMT contains an acetylated N-terminal valine and is inhibited much more efficiently compared to the recombinant protein expressed in E. coli where the N-terminus is not acetylated. In this work we used a protein crystallography approach to evaluate the structural basis for these differences. We show that in the folate–GNMT complexes with the native enzyme, two folate molecules establish three and four hydrogen bonds with the protein. In the folate–recombinant GNMT complex only one hydrogen bond is established. This difference results in more effective inhibition by folate of the native liver GNMT activity compared to the recombinant enzyme.

Predictive value of thymidylate synthase and folylpoly-glutamate synthetase for clinical benefit from pemetrexed in malignant pleural mesothelioma.

7024 Background: The antifolate pemetrexed (PMX) targets multiple enzymes involved in pyrimidine and purine synthesis including thymidylate synthase (TS). After entry into cells, PMX is converted to more potent polyglutamated forms by folylpoly-γ-glutamate synthetase (FPGS), a critical step to achieve full target inhibition. We hypothesized that FPGS and TS protein expression may predict response and outcome following PMX treatment of patients (pts) with malignant pleural mesothelioma (MPM). Methods: Pre-treatment tumor samples from 64 pts with MPM, treated with PMX combined with platinum (58/64) or as single agent (6/64), were retrospectively analyzed. TS and FPGS protein expression levels were evaluated by immunohistochemistry using the H-Scoring system (ranging from 0 to 300), which relies on the product of intensity of specific tumor cell immunoreactivity (range 0 to 3) and the percentage of positive tumor cells. Radiologic evaluation of response was performed according to RECIST. Results: Median pre-treatment H-scores were 220 (range: 50-300) for TS and 240 for FPGS (range: 110-300). Using the log-rank test and the median H-score as cut-off, we found a significant association of improved progression-free survival (PFS) with low TS protein expression (median PFS of 223 vs 181 days; hazard ratio [HR] 0.574, 95% CI, 0.330 to 0.997; P=0.049). High FPGS protein expression was associated with objective response (mean H-score 254 for responders vs 233 for non-responders, P=0.026) as well with absolute clinical benefit meaning objective response and disease control (mean H-score 248 for responders and stable disease pts vs 220 for progressive disease pts, P=0.005). We found an association of high FPGS protein expression with prolonged PFS (median PFS of 199 vs 97 days; HR 0.487, 95% CI, 0.093 to 1.068; P=0.013), additionally. Conclusions: This is the first report of FPGS examination in MPM. Based on our retrospective study, base line determination of TS and FPGS expression may be predictive for clinical benefit of PMX-based therapy in MPM. Confirmation of the prognostic and predictive value of TS and FPGS expression in MPM by prospective studies is warranted.

Abstract IA6-1: The evolution of folate analogs: Drugs targeted to folate transporters, drugs targeted to novel enzyme sites, effects on signaling pathways

Abstract IA6-1: The evolution of folate analogs: Drugs targeted to folate transporters, drugs targeted to novel enzyme sites, effects on signaling pathways

6043 Late adverse effects of preoperative hyperfractionated radiation therapy (RT) for advanced rectal cancer

We have studied the molecular effects of a LFD in a murine model in order to better define the biochemical changes associated with folate deficiency. In addition, we have demonstrated the effect of a LFD on the pharmacokinetic profile and therapeutic activity and toxicity of lometrexol. These studies showed increased density of FR in tumors implanted in LFD mice and a decrease in the affinity of these receptors for folic acid. The results suggest that tumors can compensate for low folate bioavailability by up-regulation of a second FR with slightly lower affinity for folic acid. The higher density of this FR would provide greater capacity for garnering serum folate.FPGS activity increased in several tumors and liver and kidney of LFD mice. The increase in this enzyme activity would result in enhanced polyglutamation of folates and classical antifolates and thus increased cellular retention. Consistent with these changes in liver FPGS, mice injected i.v. with a single dose of lometrexol accumulated significantly more drug in liver and tumors of LFD animals compared to SD mice. Also, higher liver concentrations of lometrexol persisted longer in LFD mice. Polyglutamate analysis showed that longer polyglutamate forms appeared earlier in liver of LFD mice. After 7 days, longer polyglutamyl forms were recovered from liver of LFD mice (octa- and hepta-glutamyl lometrexol) compared to those on SD.A comparison of the efficacy and toxicity of lometrexol in C3H mammary tumor-bearing mice showed that in mice on LFD, lometrexol treatment produced a delayed toxicity with an LD50 of 0.1-0.3 mg/kg, a 3000-fold increase in lethality compared to SD mice. Supplementation of mice with folic acid restored anti-tumor activity and increased the therapeutic dose-range over which efficacy could be assessed.These studies support the use of folic acid supplementation for cancer patients treated with antifolate therapy in order to prevent the biochemical changes in FR and FPGS associated with folate deficiency, prevent delayed toxicity to GARFT inhibitors and enhance the therapeutic potential of this class of drugs.

Forms of Folate in the Monkey.

The predominant forms of folate found in monkey liver three days after parenteral administration of 3H pteroylglutamate contained five and six glutamyl residues and were primarily the 5-methyl, with lesser quantities of 5- and 10-formyl tetrahydro derivatives. Formation of polyglutamate forms occurred more slowly in kidney. A complex mixture of the polyglutamates was also found in intestinal mucosal cells.

Novel insights into intestinal and renal folate transport. Focus on “Apical membrane targeting and trafficking of the human proton-coupled folate transporter in polarized epithelia”

FOLATE IS AN ESSENTIAL MICRONUTRIENT that functions as a coenzyme in DNA and RNA synthesis and in the metabolism of several amino acids, including homocysteine (11). Folate deficiency can occur in humans because of poor dietary intake, malabsorption, metabolic blocks, or increased requirements as seen during pregnancy and lactation (3). Symptoms of deficiency are severe and include megoblastic anemia and neural tube defects in children. Mammals must absorb folate from the diet, because they do not have the capability to synthesize folate. Intestinal absorption occurs by both passive and carriermediated mechanisms, with the second process predominating in the proximal small intestine at normal intake levels (3). Intestinal transport is critical for overall body folate homeostasis, as exemplified by the fact that individuals with hereditary folate malabsorption exhibit signs of folate deficiency (5, 6). Dietary folate exists in the polyglutamate form, which is converted to the monoglutamate form before absorption. Transport across the intestinal epithelium is a two-step process; the putative brush-border membrane transporter is pH dependent (i.e., higher activity at low pH) (12), and movement across the basolateral surface is also a carrier-mediated process that has been previously described (13). Recently, three folate transport systems have been identified in mammals: the reduced folate carrier (RFC) (4, 8), the folate receptor (FR) (7), and the newly described proton-coupled folate transporter (PCFT; also called SLC46A1) (9). The relative role of each of these transport systems in intestinal and renal folate absorption is currently not completely understood (although the FR, which was described in renal cells, has not been identified in the intestine). The recent studies by Subramanian et al. (17) have provided novel evidence that makes a major contribution to our understanding of folate homeostasis. These authors have clearly demonstrated, for the first time, that the human PCFT is a brush-border membrane protein in polarized epithelia; they show that it is functionally expressed on the apical membrane in widely accepted models of the intestinal and renal epithelia. Moreover, in polarized MDCK cells, they noted that the PCFT and the RFC showed differential localization, with the RFC present on the basolateral domain and the PCFT in the apical membrane. Additional experiments were aimed at determining the trafficking motifs of the PCFT and the effect of clinically relevant mutations; results showed that the COOH terminus was not important for apical targeting and that mutation of a region (DXXGR[113]R) associated with hereditary folate malabsorption (the R[113]S mutation has been identified in a human patient) (20) abolished a -turn between membrane-spanning domains 2 and 3 and led to protein retention in the endoplasmic reticulum. Further analyses found that cell surface delivery of the PCFT is dependent on an intact microtubular network. The findings presented in this article reveal new aspects of folate transport that can only be appreciated in the context of previous studies in this field. Prior investigations suggested that the RFC is important in intestinal and renal folate absorption. Some studies found RFC expressed on the intestinal brush-border membrane in mouse (18) and rat (1) intestine, where it is presumably involved in folate absorption, while others described expression at the basolateral surface of renal epithelial cells (2) and at the plasma membrane when expressed in Xenopus oocytes (16). Moreover, the RFC was found to be ontogenically regulated during rat development and was reflective of overall intestinal folate transport (1). Despite these intriguing observations, certain inconsistencies exist between the known properties of intestinal folate transport and those ascribed to the RFC, including the higher activity of the RFC at neutral as opposed to acidic pH (15).

Physicochemical effect of pH and antioxidants on mono- and triglutamate forms of 5-methyltetrahydrofolate, and evaluation of vitamin stability in human gastric juice: Implications for folate bioavailability

This report examines the physico-chemical properties of mono and triglutamate forms of 5-methyltetrahydrofolate n (5CH 3–H 4–PteGlu n ) in human gastric juice and under artificial conditions, typical of the upper gastrointestinal tract. Degradation of 5CH 3–H 4–PteGlu n to 5-methyldihydrofolate n (5CH 3–5,6-H 2PteGlu n ) and on to the C 9–N 10 scission product p-aminobenzoylglutamate (P-ABG), and the salvage of 5CH 3–H 4–PteGlu n from 5CH 3–5,6-H 2PteGlu n was examined with emphasis on the influence of pH and ascorbic acid. At pH 3.5, physiological levels of ascorbic acid (50 μMol/l) salvage both 5CH 3–H 4PteGlu 1 and 5CH 3–H 4PteGlu 3 from acid labile 5CH 3–5,6-H 2PteGlu n and prevent loss of vitamin activity as indicated by P-ABG n formation. This process is less efficient at pH 7.0, although under such conditions 5CH 3–5,6-H 2PteGlu n is rendered relatively stable. Ascorbic acid (50 μMol/l) reduces degradation of 5CH 3–H 4PteGlu 1 and 5CH 3–H 4PteGlu 3 at both pH 3.5 and 7.0. However, irrespective of ascorbic acid, 5CH 3–H 4PteGlu 1 and 5CH 3–H 4PteGlu 3 are both more stable at pH 3.5 than pH 7.0. There is a clear differential between mono- and polyglutamate stability, since, irrespective of pH or the presence of ascorbic acid, 5CH 3–H 4PteGlu 1 is inherently more stable than 5CH 3–H 4PteGlu 3. While ascorbic acid clearly stabilises mono- and triglutamate forms of methylfolate in human gastric juice, a factor in gastric juice removes any differential instability as seen in buffer solution. We speculate that this endogenous factor could be either a binding protein or some other antioxidant. These in vitro findings provide information that may be useful in evaluating the in vivo bioavailability of natural polyglutamate forms of the vitamin.

Folate Metabolism and the Role of Dihydropteridine Reductase (DHPR)

That dihydropteridine reductase (DHPR) has a key role to play in folate metabolism in the CNS is made clear by low concentrations of folate species in the CSF and blood of patients with DHPR deficiency. CSF folate concentrations, for reasons not yet explained, are made even lower by administration of folic acid although peripheral total folate concentrations are normalised. The clinical condition deteriorates markedly with folic acid but in contrast improves with 5-formyltetrahydrofolic acid administration (1). Folate metabolism in the CNS differs from that in the periphery in that CSF total folate concentrations are twice that in peripheral plasma whilst vitamin B12 levels are an order of magnitude lower. DHPR is ubiquitous throughout the body and its function in folate metabolism may be explored using peripheral blood samples without recourse to invasive techniques. In DHPR deficiency there is an increase in 7,8-dihydrobiopterin in the peripheral circulation as there is in classical phenylketonuria, i.e., phenylalanine hydroxylase deficiency where there is also significant perturbation of folate metabolism (2). Presumably the same mechanism prevails but folate speciation in DHPR deficiency appears not to have been explored, therefore the precise role of DHPR in folate metabolism remains unclear. With the elegant methods used by Lucock, vide supra (2) and the existence of patients with active DHPR but absence of pterin carbinolamine dehydratase (PCD) activity (see Fig. 1) there is possibility for this to be resolved For normal salvage of quinonoid dihydrobiopterin (and perhaps folates) both PCD and DHPR are required. With inactive PCD, there is moderate transient hyperphenylalaninaemia which does not require treatment but 7-substituted forms of biopterin and neopterin appear (3). We propose that it would be valuable to look at these rare cases to see if 7-folates are also created as this would elucidate further the role of this salvage pathway in folate metabolism. Folates exist in varying polyglutamated forms, have varying configurations at C5 and C10 positions and as fully oxidised, dihydro and tetrahydro forms. The distribution of postulated 7-folates between states of reduction and glutamate numbers could then be compared with normal values. This would be pertinent to normal CNS function and development and long-term health care of patients with DHPR or PCD deficiency, as well as expanding our limited knowledge of these complex metabolic pathways.

5-Amino-4-Imidazolecarboxamide Riboside Potentiates Both Transport of Reduced Folates and Antifolates by the Human Reduced Folate Carrier and Their Subsequent Metabolism

Transport is required before reduced folates and anticancer antifolates [e.g., methotrexate (MTX)] exert their physiologic functions or cytotoxic effects. The folate/antifolate transporter with the widest tissue distribution and greatest activity is the reduced folate carrier (RFC). There is little evidence that RFC-mediated influx is posttranscriptionally regulated. We show that [(3)H]MTX influx in CCRF-CEM human childhood T-leukemia cells is potentiated up to 6-fold by exogenous 5-amino-4-imidazolecarboxamide riboside (AICAr) in a AICAr and MTX concentration-dependent manner. Metabolism to more biologically active polyglutamate forms is also potentiated for MTX and other antifolates. That potentiation of influx by AICAr is mediated by effects on the RFC is supported by analyses +/-AICAr showing (a) similarity and magnitude of kinetic constants for [(3)H]MTX influx; (b) similarity of inhibitory potency of known RFC substrates; (c) lack of potentiation in a CCRF-CEM subline that does not express the RFC; and (d) similarity of time and temperature dependence. Potentiation occurs rapidly and does not require new protein synthesis. Effects of specific inhibitors of folate metabolism and the time and sequence of AICAr incubation with cells suggest that both dihydrofolate reductase inhibition and metabolism of AICAr are essential for potentiation. Acute folate deficiency or incubation of CCRF-CEM with AICAr-related metabolites (e.g., adenosine) does not initiate potentiation. AICAr increases growth inhibitory potency of MTX and aminopterin against CCRF-CEM cells when both AICAr and antifolate are present for the first 24 hours of a 120-hour growth period. AICAr is the first small molecule that regulates RFC activity.

Quantitation of in vivo human folate metabolism

A quantitative understanding of human folate metabolism is needed. The objective was to quantify and interpret human folate metabolism as it might occur in vivo. Adults (n = 13) received 0.5 nmol [(14)C]pteroylmonoglutamate (100 nCi radioactivity) plus 79.5 nmol pteroylmonoglutamate in water orally. (14)C was measured in plasma, erythrocytes, urine, and feces for >/=40 d. Kinetic modeling was used to analyze and interpret the data. According to the data, the population was healthy and had a mean dietary folate intake of 1046 nmol/d, and the apparent dose absorption of (14)C was 79%. The model predictions showed that only 0.25% of plasma folate was destined for marrow, mean bile folate flux was 5351 nmol/d, and the digestibility of the mix (1046 + 5351 nmol/d) was 92%. About 33% of visceral pteroylmonoglutamate was converted to the polyglutamate form, most of the body folate was visceral (>99%), most of the visceral folate was pteroylpolyglutamate (>98%), total body folate was 225 micromol, and pteroylpolyglutamate synthesis, recycling, and catabolism were 1985, 1429, and 556 nmol/d, respectively. Mean residence times were 0.525 d as visceral pteroylmonoglutamate, 119 d as visceral pteroylpolyglutamate, 0.0086 d as plasma folate, and 0.1 d as gastrointestinal folate. Across subjects, folate absorption, bile folate flux, and body folate stores were larger than prior estimates. Marrow folate uptake and pteroylpolyglutamate synthesis, recycling, and catabolism are saturable processes. Visceral pteroylpolyglutamate was an immediate precursor of plasma p-aminobenzoylglutamate. The model is a working hypothesis with derived features that are explicitly model-dependent. It successfully quantitated folate metabolism, encouraging further rigorous testing.