Modulation of the de novo purine nucleotide pathway as a therapeutic strategy in mitochondrial myopathy.

Mitochondrial myopathy (MM) is characterised by muscle weakness, exercise intolerance and various histopathological changes. Recently, a subset of MM has also been associated with aberrant activation of mammalian target of rapamycin complex 1 (mTORC1) in skeletal muscle. This aberrant mTORC1 activation promotes increased de novo nucleotide synthesis, which contributes to abnormal expansion and imbalance of skeletal muscle deoxyribonucleoside triphosphates (dNTP) pools. However, the exact mechanism via which mTORC1-stimulated de novo nucleotide biosynthesis ultimately disturbs muscle dNTP pools remains unclear. In this article, it is proposed that mTORC1-stimulated de novo nucleotide synthesis in skeletal muscle cells with respiratory chain dysfunction promotes an asymmetric increase of purine nucleotides, probably due to NAD+ deficiency. This in turn could disrupt purine nucleotide-dependent allosteric feedback regulatory mechanisms, ultimately leading to dNTP pools aberration. Pharmacological down-modulation of aminoimidazole carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC) activity is also proposed as a potential therapeutic strategy in MM exhibiting mTORC1-driven abnormal metabolic reprogramming, including aberrant dNTPs pools.

Characterization of AICAR transformylase/IMP cyclohydrolase (ATIC) bifunctional enzyme from Candidatus Liberibacer asiaticus.

The bifunctional enzyme, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) transformylase/inosine monophosphate (IMP) cyclohydrolase (ATIC) is involved in catalyzing penultimate and final steps of purine de novo biosynthetic pathway crucial for the survival of organisms. The present study reports the characterization of ATIC from Candidatus Liberibacer asiaticus (CLasATIC) along with the identification of potential inhibitor molecules and evaluation of cell proliferative activity. CLasATIC showed both the AICAR Transformylase (AICAR TFase) activity for substrates, 10-f-THF (Km, 146.6 µM and Vmax, 0.95 µmol/min/mg) and AICAR (Km, 34.81 µM and Vmax, 0.56 µmol/min/mg) and IMP cyclohydrolase (IMPCHase) activitiy (Km, 1.81 µM and Vmax, 2.87 µmol/min/mg). The optimum pH and temperature were also identified for the enzyme activity. In-silico study has been conducted to identify potential inhibitor molecules through virtual screening and MD simulations. Out of many compounds, HNBSA, diosbulbin A and lepidine D emerged as lead compounds, exhibiting higher binding energy and stability for CLasATIC than AICAR. ITC study reports higher binding affinities for HNBSA and diosbulbin A (Kd, 12.3 µM and 34.2 µM, respectively) compared to AICAR (Kd, 83.4 µM). Likewise, DSC studies showed enhanced thermal stability for CLasATIC in the presence of inhibitors. CD and Fluorescence studies revealed significant conformational changes in CLasATIC upon binding of the inhibitors. CLasATIC demonstrated potent cell proliferative, wound healing and ROS scavenging properties evaluated by cell-based bioassays using CHO cells. This study highlights CLasATIC as a promising drug target with potential inhibitors for managing CLas and its unique cell protective, wound-healing properties for future biotechnological applications.

The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL.

Anaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)-ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

Identification of ATIC as a Novel Target for Chemoradiosensitization.

PURPOSE: Mutations in the gene encoding 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (ATIC), a bifunctional enzyme that catalyzes the final 2 steps of the purine de novo biosynthetic pathway, were identified in a subject referred for radiation sensitivity testing. Functional studies were performed to determine whether ATIC inhibition was radiosensitizing and, if so, to elucidate the mechanism of this effect and determine whether small molecule inhibitors of ATIC could act as effective radiosensitizing agents. METHODS AND MATERIALS: Both small interfering RNA knockdown and small molecule inhibitors were used to inactivate ATIC in cell culture. Clonogenic survival assays, the neutral comet assay, and γH2AX staining were used to assess the effects of ATIC inhibition or depletion on cellular DNA damage responses. RESULTS: Depletion of ATIC or inhibition of its transformylase activity significantly reduced the surviving fraction of cells in clonogenic survival assays in multiple cancer cell lines. In the absence of ionizing radiation exposure, ATIC knockdown or chemical inhibition activated cell cycle checkpoints, shifting cells to the more radiosensitive G2/M phase of the cell cycle, and depleted cellular adenosine triphosphate but did not result in detectable DNA damage. Cells in which ATIC was knocked down or inhibited and then treated with ionizing radiation displayed increased numbers of DNA double-strand breaks and a delay in the repair of those breaks relative to irradiated, but otherwise untreated, controls. Supplementation of culture media with exogenous adenosine triphosphate ameliorated the DNA repair phenotypes. CONCLUSIONS: These findings implicate ATIC as an effective, and previously unrecognized, target for chemoradiosensitization and, more broadly, suggest that purine levels in cells might have an underappreciated role in modulating the efficiency of DNA damage responses that could be exploited in radiosensitizing strategies.

Characterization of a novel AICARFT inhibitor which potently elevates ZMP and has anti-tumor activity in murine models.

AICARFT is a folate dependent catalytic site within the ATIC gene, part of the purine biosynthetic pathway, a pathway frequently upregulated in cancers. LSN3213128 is a potent (16 nM) anti-folate inhibitor of AICARFT and selective relative to TS, SHMT1, MTHFD1, MTHFD2 and MTHFD2L. Increases in ZMP, accompanied by activation of AMPK and cell growth inhibition, were observed with treatment of LY3213128. These effects on ZMP and proliferation were dependent on folate levels. In human breast MDA-MB-231met2 and lung NCI-H460 cell lines, growth inhibition was rescued by hypoxanthine, but not in the A9 murine cell line which is deficient in purine salvage. In athymic nude mice, LSN3213128 robustly elevates ZMP in MDA-MB-231met2, NCI-H460 and A9 tumors in a time and dose dependent manner. Significant tumor growth inhibition in human breast MDA-MB231met2 and lung NCI-H460 xenografts and in the syngeneic A9 tumor model were observed with oral administration of LSN3213128. Strikingly, AMPK appeared activated within the tumors and did not change even at high levels of intratumoral ZMP after weeks of dosing. These results support the evaluation of LSN3213128 as an antineoplastic agent.

Antifolates targeting purine synthesis allow entry of tumor cells into S phase regardless of p53 function.

The class of folate antimetabolites typified by (6R)-dideazatetrahydrofolate (lometrexol, DDATHF) are specific inhibitors of de novo purine synthesis because of potent inhibition of glycinamide ribonucleotide formyltransferase (GART) but do not induce detectable levels of DNA strand breaks. As such, they are a test case of the concept that ribonucleotide depletion can be sensed by p53, resulting in a G(1) cell cycle block. The GART inhibitors have been proposed previously to be cytotoxic in tumor cells lacking p53 function but only cytostatic in p53 wild-type tumor cells. We have investigated this concept. Cell cycle progression into and through S phase was slowed by DDATHF, but both p53 +/+ and -/- human colon carcinoma cells entered and completed one S phase in the presence of drug. This inability of p53 to initiate a G(1) arrest after DDATHF treatment was mirrored by an independence of the cytotoxicity of DDATHF on p53 function. We conclude that carcinoma cells are killed equally well by DDATHF and related compounds whether or not the p53 pathway is intact and that the utility of GART inhibitors would not be limited to p53-negative tumors.

A sensitive and specific radioimmunoassay for LY309887, a potent inhibitor of glycinamide ribonucleotide formyltransferase.

LY309887, a reduced analogue of folic acid, is a potent inhibitor of glycinamide ribonucleotide formyltransferase and possesses a broad spectrum of antitumor activity. During preclinical studies using supplementation with oral folic acid, this second-generation inhibitor displayed both the desired safety profile and the pharmacology to warrant clinical investigation. A sensitive analytical method was needed to assess the pharmacokinetics of LY309887 due to the low doses planned for Phase I studies and the potential for low concentrations in plasma long after i.v. administration. We therefore undertook the development of a competitive RIA. A highly specific antiserum was raised in rabbits following immunization with LY309887 coupled to BSA. A RIA tracer was prepared by radioiodination of compound 389753, the adduct of LY309887 with p-tyramine. We developed a competitive-binding RIA procedure and used superparamagnetic particles coated with goat antirabbit IgG as a method for separating the bound and free forms of LY309887. The RIA is sensitive (0.5 ng/ml in serum and 25 ng/ml in urine), specific (negligible interference from endogenous folates), and reproducible (interassay coefficients of variation ranging from 8.1 to 15.4% and 7.6 to 8.3% for serum and urine controls, respectively). We used the RIA to assess the i.v. pharmacokinetics of LY309887 in both patients with metastatic cancer and dogs. The sensitivity of the RIA permitted the demonstration that serum concentrations of LY309887 decline in a multiexponential manner with a prolonged terminal elimination phase. We conclude that the RIA is a valid method for quantifying LY309887 in biological fluids.

Cellular ATP depletion by LY309887 as a predictor of growth inhibition in human tumor cell lines.

The antifolate LY309887 is a specific glycinamide ribonucleotide formyltransferase inhibitor that blocks de novo purine synthesis and produces a depletion of purine nucleotides. The activity of LY309887 in six human tumor cell lines has been examined by growth inhibition and clonogenic assay after continuous exposure for three cell doubling times and by ATP depletion at 24 h. Three cell lines (CCRF-CEM, MCF7, and GC3) were sensitive to LY309887-induced growth inhibition (IC50: 5.6-8.1 nM), whereas the other cell lines (COR-L23, T-47D, and A549) were comparatively resistant (IC50: 36-55 nM). Sensitivity to LY309887 cytotoxicity was consistent with sensitivity to growth inhibition in four of five cell lines tested (MCF7/GC3: 0.01% survival and COR-L23/T-47D: 1-5% survival at 100 nM LY309887). LY309887-induced ATP depletion was measured by luciferase-based ATP assay and confirmed by high performance liquid chromatography measurements. There was a linear relationship between ATP depletion and growth inhibition when data were analyzed for all six cell lines (r2 = 0.93; P < 0.0001). Depletion of 24-h cellular ATP concentrations to < 1 mM was associated with both cell growth inhibition and cytotoxicity in all cell lines studied. In conclusion, cellular ATP depletion induced by LY309887 can be used to predict growth inhibition and cytotoxicity in human tumor cells.

Pharmacokinetic and pharmacodynamic evaluation of the glycinamide ribonucleotide formyltransferase inhibitor AG2034.

Glycinamide ribonucleotide formyltransferase (GARFT) is a component of the de novo purine synthesis pathway. AG2034 is a specific inhibitor of GARFT that was designed based on the GARFT crystal structure. In conjunction with Phase I studies at four clinical centers in the United States and United Kingdom, AG2034 pharmacology was evaluated in 54 patients receiving 1-11 mg/m2 AG2034 as a 2-5 min injection. Blood samples were obtained just prior to and 5, 15, 30, and 45 min, and 1, 1.5, 2, 4, 6, 8, 12, 24, 48, 72, and 96 h after bolus injection during course 1. Limited sampling was also performed on course 3. Plasma AG2034 was measured using a sensitive and reproducible ELISA assay. AG2034 demonstrated a trimodal elimination pattern over 24 h, with median half-life (t(1/2))alpha = 8.7 min, t(1/2)beta = 72.6 min, and t(1/2)gamma = 364.2 min. AG2034 systemic clearance ranged from 9.4-144.5 ml/min/m2, and volume of distribution was 1.2-7.6 liters/m2. Course 1 AG2034 area under the concentration versus time curve (AUC) had a linear relationship with dose (r(s) = 0.86). Accumulation of AG2034 was evident, because course 3 AUC was higher than course 1 in 23 of 23 evaluable patients, but was not associated with an increase in erythrocyte AG2034. AG2034 systemic exposure had an impact on toxicity, because course 1 and course 3 AG2034 AUCs were significantly higher for patients with grade III/IV toxicity than patients with less than grade II toxicity (P < 0.001 and P = 0.001 for course 1 and course 3, respectively). This study demonstrates rapid systemic clearance of AG2034 and suggests pharmacokinetic approaches that may minimize patient toxicity and aid the development of this interesting class of anticancer agents.

AMPK Activation via Modulation of De Novo Purine Biosynthesis with an Inhibitor of ATIC Homodimerization.

5-Aminoimidazole-4-carboxamide ribonucleotide (known as ZMP) is a metabolite produced in de novo purine biosynthesis and histidine biosynthesis, but only utilized in the cell by a homodimeric bifunctional enzyme (called ATIC) that catalyzes the last two steps of de novo purine biosynthesis. ZMP is known to act as an allosteric activator of the cellular energy sensor adenosine monophosphate-activated protein kinase (AMPK), when exogenously administered as the corresponding cell-permeable ribonucleoside. Here, we demonstrate that endogenous ZMP, produced by the aforementioned metabolic pathways, is also capable of activating AMPK. Using an inhibitor of ATIC homodimerization to block the ninth step of de novo purine biosynthesis, we demonstrate that the subsequent increase in endogenous ZMP activates AMPK and its downstream signaling pathways. We go on to illustrate the viability of using this approach to AMPK activation as a therapeutic strategy with an in vivo mouse model for metabolic disorders.

A new nonlinear mixture response surface paradigm for the study of synergism: a three drug example.

A flexible approach to response surface modeling for the study of the joint action of three active anticancer agents is used to model a complex pattern of synergism, additivity and antagonism in an in vitro cell growth assay. The method for determining a useful nonlinear response surface model depends upon a series of steps using appropriate scaling of drug concentrations and effects, raw data modeling, and hierarchical parameter modeling. The method is applied to a very large in vitro study of the combined effect of Trimetrexate (TMQ), LY309887 (LY), and Tomudex (TDX) on inhibition of cancer cell growth. The base model employed for modeling dose-response effect is the four parameter Hill equation [1]. In the hierarchical aspect of the final model, the base Hill model is treated as a function of the total amount of the three drug mixture and the Hill parameters, background B, dose for 50% effect D50, and slope m, are understood as functions of the three drug fractions. The parameters are modeled using the canonical mixture polynomials from the mixture experiment methodologies introduced by Scheff [2]. We label the model generated a Nonlinear Mixture Amount model with control observations, or zero amounts, an "NLMAZ" model. This modeling paradigm provides for the first time an effective statistical approach to modeling complex patterns of local synergism, additivity, and antagonism in the same data set, the possibility of including additional experimental components beyond those in the mixture, and the capability of modeling three or more drugs.

Methionine in and out of proteins: targets for drug design.

The increasing need for new antibiotics to overcome rapidly developing resistance mechanisms observed in clinical isolates of Gram-positive and Gram-negative eubacteria has placed critical emphasis on the search for new antibacterial enzyme targets and the structural and mechanistic investigation of such targets. Among these potential targets, the enzymes responsible for integrating the amino acid methionine into proteins, along with its subsequent post-translational modification and repair, have emerged as promising candidates for the development of novel antibiotics. As well, there is increasing evidence for the importance of several of these enzymes in the development of anti-cancer, anti-parasitic, and anti-atherosclerotic drugs. Within the last three years, the crystal structures of all of these enzymes have been determined, which offers an unprecedented source of structural information for inhibitor design. The development of combinatorial chemistry and high throughput screening procedures has quickly provided several potent, specific inhibitors for a number of these enzymes, particularly the peptide deformylase, methionine aminopeptidase, and methionyl-tRNA synthetase enzymes. This review critically analyzes the future potential for inhibition of enzymes in this pathway, allowing for a pragmatic view of the success of inhibitor developments and highlighting areas in which further investigations are warranted.

Deaza analogs of folic acid as antitumor agents.

Derivatives of the vitamin folic acid function in the body for the synthesis of thymidylate, purines and amino acids and are necessary for normal metabolism and growth. Methotrexate (MTX), an inhibitor of dihydrofolate reductase (DHFR) is the outstanding example of an antitumor antifolate. MTX is clinically useful in the treatment of childhood leukemia, choriocarcinoma and psoriasis, where it corrects abnormal growth, and in rheumatoid arthritis and other autoimmune diseases where it corrects abnormal immune function. Since 1949, when the chemical synthesis of MTX was reported by workers at the Lederle Laboratories of the American Cyanamid Company, much has been learned about the basis of antifolate cytotoxicity and selectivity. This review will focus on deaza antifolates which are: 1). presently under clinical development and 2). less developed compounds which represent novel approaches. Compounds will be grouped according to their enzyme targets; DHFR, thymidylate synthase (TS) and glycinamide ribonucleotide formyltransferase (GARFT). In addition to inhibition of target enzymes, antifolate membrane transport into cells and conversion to poly-L-gamma-glutamate forms are important considerations in drug design along with the reverse processes, cellular hydrolysis of antifolate poly-L-gamma-glutamates to monoglutamates and the extrusion of the monoglutamates through the cell membrane. These processes can be modulated by competition with folates.

Folate status and the safety profile of antifolates.

Throughout the history of cancer chemotherapy, the control of drug-related toxicity has been a major concern. Antifolates such as methotrexate also have a reputation for sporadic and unpredictable toxicity. Pretreatment levels of plasma or red cell folate have not been found to be useful in predicting which patients will develop toxicity. During the phase II development of pemetrexed, the plasma levels of homocysteine and methylmalonic acid were studied as sensitive surrogate markers for folate and vitamin B(12) status, respectively. These were found to be strongly correlated with the subsequent development of serious drug-related toxicities (myelosuppression, diarrhea, mucosal toxicity, and infection), suggesting that toxicity was related to relative folate deficiency in some cancer patients. A policy of nutritional supplementation was introduced and led to a marked reduction in toxicity and the abolition of treatment-related deaths with apparent preservation of anticancer activity.

Update on antifolate drugs targets.

Antifolate drugs are molecules directed to interfere with the folate metabolic pathway at some level. They can be recognized among the first rationally designed compounds applying the principle of structural analogy with the substrate developing the antimetabolite strategy. This strategy has taken advantage of the basic different features of the microbial and human folate metabolism and therefore allows targeting the pathway at different steps generating a specificity tools for Medicinal Chemists. Two main problems are giving renewed importance to such targets and therefore improving the efforts to discover new targets in the folate metabolism area. The first one is the increasing resistance to the present drugs due to different mechanisms such as the enzyme modification and the increased production of enzymes with not well recognized importance. The second one is the development of techniques directed to highlight the interference at genetic level of molecular probes as antifolate drug to develop new enzymes previously unknown. This approach is defined as genetic approach to drug discovery, from gene to drugs. The present article describes the importance in drug design and discovery of some antifolate targets among the best known at the present status of research such as thymidylate synthase (TS), dhydrofolate reductases, (DHFR) serine hydroxymethyltransferase (SHMT), folyilpolyglutamyl synthetase (FPGS), gamma-glutamyl hydrolase (gamma-GH), glycinamide-ribonucleotide transformylase (GARTfase), amino-imidazole-carboxamide-ribonucleotide transformylase (AICARTfase) and Folate transporters. Discovery, known functions, structure/function studies and inhibition will be described.

Virtual screening of human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase against the NCI diversity set by use of AutoDock to identify novel nonfolate inhibitors.

AICAR transformylase (5-aminoimidazole-4-carboxamide ribonucleotide transformylase) is a folate-dependent activity of the bifunctional protein ATIC (AICAR transformylase and IMP cyclohydrolase) and is responsible for catalyzing the penultimate step of the de novo purine biosynthetic pathway. As such, AICAR transformylase has been proposed as a potential target for antineoplastic drug design. Virtual screening of the human AICAR transformylase active site by use of AutoDock against the NCI diversity set, a library of compounds with nonredundant pharmacophore profiles, has revealed 44 potential inhibitor candidates. In vitro inhibition assay of 16 soluble compounds from this list revealed that eight compounds with novel scaffolds, relative to the general folate template, had micromolar inhibition. Subsequent extension of docking trials on compounds with similar scaffolds from the entire NCI-3D database has unveiled 11 additional inhibitors that were confirmed by the in vitro inhibition assay. In particular, one compound, NSC30171, had nanomolar inhibition (K(i) = 154 nM, IC(50) = 600 nM) against AICAR transformylase. These 19 inhibitors serve as novel templates/scaffolds for development of more potent and specific non-folate-based AICAR transformylase inhibitors.

Folic acid-enhanced synergy for the combination of trimetrexate plus the glycinamide ribonucleotide formyltransferase inhibitor 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin -6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034): comparison across sensitive and resistant human tumor cell lines.

Folic acid (PteGlu)-enhanced intense synergy has been observed between nonpolyglutamylatable dihydrofolate reductase (DHFR) inhibitors and polyglutamylatable inhibitors of other folate-requiring enzymes, such as glycinamide ribonucleotide formyltransferase (GARFT) and thymidylate synthase. Since this phenomenon is potentially therapeutically useful, we explored its universality by examining the combined action of a DHFR inhibitor, trimetrexate (TMQ), with a GARFT inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]++ +thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034), in eight human cultured cell lines. Using a 96-well plate cell growth inhibition assay, four ileocecal adenocarcinoma cell lines [HCT-8, HCT-8/DW2 (Tomudex-resistant), HCT-8/DF2 (Tomudex-/FdUrd-resistant), and HCT-8/50 (adapted to 50 nM PteGlu)], three head and neck carcinoma cell lines [A253, FaDu, and Hep-2/500 (FdUrd-resistant)], and a non-small cell lung carcinoma cell line [H460] were treated for 96 hr with TMQ + AG2034 in the presence of 23 or 40 microM PteGlu. Cell growth was measured with the sulforhodamine B assay at the end of this period. Drug interactions were assessed by fitting a 7-parameter model including a synergism parameter, alpha, to data with weighted nonlinear regression. Isobologram analysis was also applied. At 23 microM PteGlu, cells exhibited similar intensities of Loewe synergy for the combination of TMQ + AG2034. Loewe synergy was abolished in HCT-8/50 cells cultured and studied in 50 nM PteGlu. At 40 microM PteGlu, the intensity of the combined action in all cell lines was increased However, the most intense Loewe synergy was seen with HCT-8, HCT-8/DF2, H460, FaDu, A253, and Hep-2/500 cells, whereas the HCT-8/50 subculture showed less of the phenomenon, and PteGlu enhancement was the least with HCT-8/DW2, a subline deficient in folylpolyglutamate synthetase (FPGS). The universality of the PteGlu-enhanced intense synergy phenomenon is suggested. Impaired FPGS activity and low-folate adaptation prior to treatment significantly lessen the degree of PteGlu enhancement.

Thymidine and hypoxanthine protection patterns of the folic acid-enhanced synergies for combinations of trimetrexate plus a polyglutamylatable inhibitor of purine or thymidylate synthesis against human ileocecal HCT-8 cells.

In order to examine the intracellular locus of the folic acid (PteGlu)-enhanced synergies of trimetrexate (TMQ) plus the thymidylate synthase (TS) inhibitor, raltitrexed (RTX), and TMQ plus the glycinamide ribonucleotide formyltransferase (GARFT) inhibitor, AG2034, comprehensive protection studies with thymidine (dThd) and hypoxanthine (HX) were conducted in a 96-well plate cell growth inhibition (sulforhodamine B) assay. Current modeling techniques were extended to characterize these protection patterns involving multiple-agent interaction. Wild-type human ileocecal HCT-8 cells and DW2, a subline deficient in folylpoly-gamma-glutamate synthetase (FPGS) were individually treated for 96 h with TMQ, AG2034 and a 1:1 mixture of TMQ:AG2034 or with TMQ, RTX, and a 1:1 mixture of TMQ:RTX in the presence of PteGlu (2.3 or 40 micro M) and the protection agents (10 micro M dThd and/or 100 micro M HX). Drug treatments were randomly assigned to wells. Both isobols and 3-dimensional concentration-effect surfaces were used to assess the nature and the intensity of drug interactions. The structural Hill model was fitted to data with weighted non-linear regression for most cases. A so-called 'double Hill' model was sometimes more appropriate when a plateau in the middle of the concentration-effect curve was found. In HCT-8 and DW2 cells at 2.3 and 40 micro M PteGlu, inhibition of DHFR by TMQ induced antithymidylate and antipurine effects; AG2034 and RTX selectively inhibited de novo purine or thymidine synthesis, respectively. dThd protection increased the PteGlu-enhancement of the TMQ + AG2034 synergy, whereas HX protection increased the PteGlu-enhancement of the TMQ + RTX synergy. The PteGlu-enhanced synergies of TMQ + AG2034 and TMQ + RTX occur primarily through inhibition of purine synthesis and inhibition of thymidylate synthesis, respectively. These results further substantiate the hypothesis that the nonpolyglutamylatable DHFR inhibitor, TMQ, acts as a modulator by decreasing the protection by PteGlu of cells against the polyglutamylatable AG2034 and RTX.

Whole-body disposition and polyglutamate distribution of the GAR formyltransferase inhibitors LY309887 and lometrexol in mice: effect of low-folate diet.

PURPOSE: The whole-body autoradiographic distribution of two radiolabeled antifolate inhibitors of GAR formyltransferase, lometrexol and LY309887, were compared in tumor-bearing mice maintained on standard diet (SD) and a low-folate diet (LFD) in order to determine the total amounts of drug that accumulated in blood, tumor, liver and kidney. The time-dependent changes in tissue distribution were evaluated over a 7-day period in order to compare the pharmacokinetic properties of both inhibitors and to assess the influence of dietary folate on this distribution. In addition, the effect of dietary folate on polyglutamation of compound accumulating in the liver was measured. The results have bearing on the potential of these two clinical agents to produce delayed toxicity in cancer patients and the use of dietary folate to modulate or prevent the development of this toxicity. METHODS: Single equimolar i.v. doses of [14C]LY309887 and [14C]lometrexol were administered to C3H mammary tumor bearing mice on SD or LFD, and the disposition of these compounds was quantitated using whole-body autoradiography. Livers were also harvested and extracted for determination of polyglutamate distribution. Animals were sacrificed both early and late (7 days) after dosing to determine the long-term retention of these compounds. RESULTS: Whole-body autoradiography revealed that the highest concentrations of both compounds were in liver and kidney. Concentrations of both compounds were two-fold higher in livers from LFD mice than in livers from SD mice. Lometrexol concentrations in liver averaged 2.8- and 2.2-fold higher than LY309887 in SD and LFD livers, respectively. In SD livers, the polyglutamate profiles of both compounds were similar, with hexaglutamates being the longest chain species detected. In LFD livers, hexaglutamates of LY309887 were observed, while hepta- and octaglutamates of lometrexol were detected after 168 h. CONCLUSIONS: The reduced hepatic retention and biochemical profile of LY309887 compared to lometrexol suggest that it may be less likely to produce delayed cumulative toxicity while still retaining antitumor activity. However, the increased hepatic accumulation observed in LFD mice emphasizes the importance of assessing and supplementing folate in cancer patients treated with this class of compounds.

Phase I study of AG2034, a targeted GARFT inhibitor, administered once every 3 weeks.

PURPOSE: To identify a recommended phase II dose for the second generation glycinamide ribonucleotide transformylase (GARFT) inhibitor, AG2034, administered by intravenous bolus every 3 weeks without folate supplementation and to describe AG2034 pharmacokinetics. METHODS: Adults with advanced malignancies were enrolled in cohorts of three per dose level with expansion to six upon observation of dose-limiting toxicity (DLT). The maximum tolerated dose (MTD) was defined as the dose at which two of up to six patients experienced DLT. Upon identification of an MTD and evidence of cumulative toxicity, a lower intermediate dose was explored as a candidate phase II dose. AG2034 plasma concentrations were measured using an ELISA assay. RESULTS AND CONCLUSIONS: The recommended phase II dose is 5.0 mg/m2. DLTs were anemia, thrombocytopenia, mucositis, diarrhea, hyperbilirubinemia, fatigue, and insomnia. Toxicities were modestly cumulative over three courses. Pharmacokinetic analysis showed a dose-AUC0-24 relationship and a progressive increase in AG2034 AUC0-24 over three courses. Both pharmacokinetic and pharmacodynamic factors may contribute to the modest cumulative toxicity observed with AG2034.