Pralatrexate Sustainably Released from Polypeptide Thermogel Is Effective for Chondrogenic Differentiation of Mesenchymal Stem Cells.

Folic acid was reported to significantly improve chondrogenic differentiation of mesenchymal stem cells. In a similar mechanism of action, we investigated clinically approved antifolates by the U.S. Food and Drug Administration as chondrogenic-promoting compounds for tonsil-derived mesenchymal stem cells. A poly(ethylene glycol)-poly(l-alanine) thermogelling system was used as a three-dimensional cell culture matrix, where stem cells and antifolates could be incorporated simultaneously during a heat-induced in situ sol-to-gel transition. The antifolates could be supplied over several days by the sustained release of the drug from the thermogel. Initially, seven antifolates were prescreened based on cell viability and expression of a typical chondrogenic biomarker of type II collagen (COL II) at the mRNA level. Then, dapsone, pralatrexate, and trimethoprim were selected as candidate compounds in the second round screening, and detailed studies were carried out on the mRNA and protein expression of various chondrogenic biomarkers including COL II, SRY box transcription factor 9, and aggrecan. Three-dimensional cultures of stem cells in the thermogel in the absence of a chondrogenic promoter compound and in the presence of kartogenin (KGN) were performed as a negative control and positive control, respectively. The chondrogenic biomarkers were significantly increased in the selected antifolate-incorporating systems compared to the negative control system, without an increase in type I collagen (an osteogenic biomarker) expression. Pralatrexate was the best compound for inducing chondrogenic differentiation of the stem cells, even better than the positive control (KGN). Nuclear translocation of the core-binding factor ß subunit (CBFß) and enhanced nuclear runt-related transcription factor 1 (RUNX1) by antifolate treatment suggested that the chondrogenesis-enhancing mechanism is mediated by CBFß and RUNX1. An in silico modeling study confirmed the mechanism by proving the high binding affinity of pralatrexate to a target protein of filamin A compared with other antifolate candidates. To conclude, pralatrexate was rediscovered as a lead compound, and the polypeptide thermogel incorporating pralatrexate and mesenchymal stem cells can be a very effective system in promoting chondrogenic differentiation of stem cells and might be used in injectable tissue engineering for cartilage repair.

Targeting folate receptor beta on monocytes/macrophages renders rapid inflammation resolution independent of root causes.

Provoked by sterile/nonsterile insults, prolonged monocyte mobilization and uncontrolled monocyte/macrophage activation can pose imminent or impending harm to the affected organs. Curiously, folate receptor beta (FRß), with subnanomolar affinity for the vitamin folic acid (FA), is upregulated during immune activation in hematopoietic cells of the myeloid lineage. This phenomenon has inspired a strong interest in exploring FRß-directed diagnostics/therapeutics. Previously, we have reported that FA-targeted aminopterin (AMT) therapy can modulate macrophage function and effectively treat animal models of inflammation. Our current investigation of a lead compound (EC2319) leads to discovery of a highly FR-specific mechanism of action independent of the root causes against inflammatory monocytes. We further show that EC2319 suppresses interleukin-6/interleukin-1ß release by FRß+ monocytes in a triple co-culture leukemic model of cytokine release syndrome with anti-CD19 chimeric antigen receptor T cells. Because of its chemical stability and metabolically activated linker, EC2319 demonstrates favorable pharmacokinetic characteristics and cross-species translatability to support future pre-clinical and clinical development.

Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance.

BACKGROUND: Pralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance. METHODS: To investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay. RESULTS: Compared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [14C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3ß (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines. CONCLUSIONS: The resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX.

Efficacy and tolerability of folate-aminopterin therapy in a rat focal model of multiple sclerosis.

BACKGROUND: Activated macrophages in the experimental model of multiple sclerosis (MS) express folate receptor-ß (FR-ß), representing a promising target for the treatment of MS. Here, we both evaluated the efficacy of a novel folate-aminopterin construct (EC2319) in a rat focal model of multiple sclerosis (MS) and investigated the utility of 68Ga-labeled 1,4,7-triazacyclononane-1,4,7-triacetic acid-conjugated folate (68Ga-FOL) for assessing inflammatory lesions. In addition, we investigated whether FR-ß is expressed in the brain of patients with MS. METHODS: Focal delayed-type hypersensitivity experimental autoimmune encephalomyelitis (fDTH-EAE) was induced in 40 Lewis rats; 20 healthy Lewis rats were used as controls. Rats were divided into six groups according to the duration of disease (control, acute, or chronic) and intervention (vehicle versus EC2319). 68Ga-FOL analyses, histology, and immunofluorescence of the brain were performed to evaluate the efficacy of subcutaneously administered EC2319 on lesion development. Immunofluorescence was used to assess FR-ß expression in postmortem brain samples from 5 patients with MS and 5 healthy controls. RESULTS: Immunofluorescence and histological analyses revealed significant reductions in FR-ß expression (P < 0.05) and lesion size (P < 0.01), as well as improved inducible nitric oxide synthase/mannose receptor C type 1 ratios (P < 0.01) in macrophages and microglia during the chronic but not acute phase of fDTH-EAE in EC2319-treated rats. The uptake of IV-injected 68Ga-FOL in the brain was low and did not differ between the groups, but the in vitro binding of 68Ga-FOL was significantly lower in EC2319-treated rats (P < 0.01). FR-ß positivity was observed in chronically active lesions and in normal-appearing white matter in MS brain samples. CONCLUSIONS: EC2319 was well tolerated and attenuated inflammation and lesion development in a rat model of a chronic progressive form of MS. Human MS patients have FR-ß-positive cells in chronically active plaques, which suggests that these results may have translational relevance.

A novel virtual screening procedure identifies Pralatrexate as inhibitor of SARS-CoV-2 RdRp and it reduces viral replication in vitro.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus poses serious threats to the global public health and leads to worldwide crisis. No effective drug or vaccine is readily available. The viral RNA-dependent RNA polymerase (RdRp) is a promising therapeutic target. A hybrid drug screening procedure was proposed and applied to identify potential drug candidates targeting RdRp from 1906 approved drugs. Among the four selected market available drug candidates, Pralatrexate and Azithromycin were confirmed to effectively inhibit SARS-CoV-2 replication in vitro with EC50 values of 0.008µM and 9.453 µM, respectively. For the first time, our study discovered that Pralatrexate is able to potently inhibit SARS-CoV-2 replication with a stronger inhibitory activity than Remdesivir within the same experimental conditions. The paper demonstrates the feasibility of fast and accurate anti-viral drug screening for inhibitors of SARS-CoV-2 and provides potential therapeutic agents against COVID-19.

Cost-utility analysis of pralatrexate for relapsed or refractory peripheral T-cell lymphoma based on a case-matched historical control study along with single arm clinical trial.

BACKGROUND: Patients with relapsed or refractory peripheral T-cell lymphoma (R/R PTCL) treated with pralatrexate have previously shown superior overall survival (OS) compared to those who underwent conventional chemotherapy (CC, 15.4 vs. 4.07 months). We conducted an economic evaluation of pralatrexate from a societal perspective in Korea based on data from the PROPEL phase II study. METHODS: Using a Markov model with a weekly cycle, we simulated the experience of patients with R/R PTCL receiving pralatrexate or CC for 15 years. The model consists of five health states; initial treatment, treatment pause, subsequent treatment, stem cell transplantation (SCT) success, and death. Comparative effectiveness was based on PROPEL phase II single-arm study and its matched historical control analysis. Costs included drug, drug administration, monitoring, adverse event management, and SCT costs. RESULTS: The incremental cost-effectiveness ratio of the base case was $39,153 per quality-adjusted life-year (QALY) gained. The results of one-way sensitivity analysis ranged from $33,949 to $51,846 per QALY gained, which remained within an implicit willingness-to-pay (WTP) threshold of anticancer drugs in Korea. CONCLUSIONS: Pralatrexate is a cost-effective intervention with improved OS and incremental costs within the WTP limit. Pralatrexate could function as a new therapeutic option for patients suffering from life-threatening R/R PTCL.

Auxiliary in vitro and in vivo biological evaluation of hydrogen peroxide sensitive prodrugs of methotrexate and aminopterin for the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease that causes severe joints damage and other extra-articular alterations. Despite the efficacy of low-dose methotrexate (LD-MTX) in RA treatment, adverse effects are the predominant reasons for discontinuation of therapy. As a therapeutic targeting strategy, the presence of increased concentrations of reactive oxygen species (ROS) in the inflammatory environment can serve as the stimulus for prodrug activation in site-selective drug delivery systems. Our group has previously reported novel ROS sensitive prodrugs (1-3) of MTX and aminopterin (AMT) for site-selective delivery to inflammatory tissue associated with RA, with the aim of reducing side effects in RA therapy. Herein, we investigate the effect and toxicity of the same prodrugs in a rat CIA (collagen-induced arthritis) model of RA. We find that prodrug 1, an arylboronic acid ROS-sensitive MTX-prodrug, displays similar in vivo efficacy as MTX at an equimolar dose, while avoiding adverse effects known to restrict MTX treatment. To further characterize prodrug 1 and its ROS mediated activation, we synthesized compound 4, a negative control lacking the boronic acid moiety. We then investigated the effect of molecules on cell proliferation and cytotoxicity in the presence of the ROS scavenger pyruvate, as well as their stability in buffer and cell media, demonstrating a direct correlation between ROS concentration and the prodrug activity. Moreover, the in vitro ADME properties were investigated, including permeability, rat plasma and microsomal stability.

Pralatrexate in Combination with Oxaliplatin in Advanced Esophagogastric Cancer: A Phase II Trial with Predictive Molecular Correlates.

The purpose of our study was to evaluate the efficacy of a combination of pralatrexate plus oxaliplatin in advanced esophagogastric cancer (EGC), analyze the impact of polymorphisms in folate metabolism pathway genes on toxicity and efficacy of pralatrexate, and to evaluate microRNA profile of tumor epithelium as a predictive biomarker. This was a two-stage trial with a safety lead in cohort and a primary endpoint of overall response rate (ORR). Patients received biweekly intravenous oxaliplatin (85 mg/m2) and pralatrexate (Dose level 1 [D1], 120 mg/m2; dose level-1 [D-1] 100 mg/m2). Single-nucleotide polymorphisms (SNP) in genes encoding proteins involved in pralatrexate metabolism were evaluated in germline DNA. microRNA profiling of the tumor epithelium was performed. ORR was 26%. Dose-limiting toxicities were observed in 2 of 4 patients at D1 and none at D-1. The T>C polymorphism in DHFR rs11951910 was significantly associated with lower progression-free survival (PFS; P ≤ 0.01), whereas the presence of the SLC19A1 rs2838957 G>A polymorphism was associated with improved PFS (P = 0.02). Presence of the GGH rs3780130 A>T and SLC19A1 rs1051266 G>A polymorphisms were significantly associated with better overall survival (OS; P = 0.01), whereas GGH rs7010484 T>C polymorphism was associated significantly with reduced OS (P = 0.04). There was no correlation between epithelial microRNA expression profile with disease progression or response. We conclude that the combination of oxaliplatin and pralatrexate is safe, is well tolerated, and has modest efficacy in advanced EGC. Pharmacogenomic analysis may be relevant to the use of pralatrexate in combination with platinum agents.

Antifolate SERS-active nanovectors: quantitative drug nanostructuring and selective cell targeting for effective theranostics.

One of the frontiers of nanomedicine is the rational design of theranostic nanovectors. These are nanosized materials combining diagnostic and therapeutic capabilities, i.e. capable of tracking cancer cells and tissues in complex environments, and of selectively acting against them. We herein report on the preparation and application of antifolate plasmonic nanovectors, made of functionalized gold nanoparticles conjugated with the folic acid competitors aminopterin and methotrexate. Due to the overexpression of folate binding proteins on many types of cancer cells, these nanosystems can be exploited for selective cancer cell targeting. The strong surface enhanced Raman scattering (SERS) signature of these nanovectors acts as a diagnostic tool, not only for tracing their presence in biological samples, but also, through a careful spectral analysis, to precisely quantify the amount of drug loaded on a single nanoparticle, and therefore delivered to the cells. Meanwhile, the therapeutic action is implemented based on the strong toxicity of antifolate drugs. Remarkably, supplying the drug in the nanostructured form, rather than as a free molecule, enhances its specific toxicity. The selectivity of the antifolate nanovectors can be optimized by the design of a hybrid folate/antifolate coloaded nanovector for the specific targeting of folate receptor α, which is overexpressed on numerous cancer cell types.

Chromosome Transplantation: Correction of the Chronic Granulomatous Disease Defect in Mouse Induced Pluripotent Stem Cells.

In spite of the progress in gene editing achieved in recent years, a subset of genetic diseases involving structural chromosome abnormalities, including aneuploidies, large deletions and complex rearrangements, cannot be treated with conventional gene therapy approaches. We have previously devised a strategy, dubbed chromosome transplantation (CT), to replace an endogenous mutated chromosome with an exogenous normal one. To establish a proof of principle for our approach, we chose as disease model the chronic granulomatous disease (CGD), an X-linked severe immunodeficiency due to abnormalities in CYBB (GP91) gene, including large genomic deletions. We corrected the gene defect by CT in induced pluripotent stem cells (iPSCs) from a CGD male mouse model. The Hprt gene of the endogenous X chromosome was inactivated by CRISPR/Cas9 technology thus allowing the exploitation of the hypoxanthine-aminopterin-thymidine selection system to introduce a normal donor X chromosome by microcell-mediated chromosome transfer. X-transplanted clones were obtained, and diploid XY clones which spontaneously lost the endogenous X chromosome were isolated. These cells were differentiated toward the myeloid lineage, and functional granulocytes producing GP91 protein were obtained. We propose the CT approach to correct iPSCs from patients affected by other X-linked diseases with large deletions, whose treatment is still unsatisfactory. Stem Cells 2019;37:876-887.

Pralatrexate: a comprehensive update on pharmacology, clinical activity and strategies to optimize use.

It has been nearly 8 years since pralatrexate became the first drug approved by the U.S. Food and Drug Administration for the treatment of relapsed or refractory peripheral T-cell lymphoma (PTCL). Like most drugs approved for a particular clinical indication, as much or more is learned once it enters mainstream use as in the years leading up to regulatory approval. Over the past several years, many diverse lines of research have shed new insight into both the agent, and the diseases it treats. In this review, we will bring the reader up to date on the many new aspects related to pralatrexate's pharmacology, activity across the panoply of T-cell lymphoproliferative malignancies, as well as some new and emerging guidelines that are likely to improve its safety profile. Finally, the review will close with the many new lines of evidence building a rationale for the combination of these novels: novel combination, and the vision for new platforms in PTCL care.

'Rod and ring' formation from IMP dehydrogenase is regulated through the one-carbon metabolic pathway.

'Rods and rings' (RRs) are conserved, non-membrane-bound intracellular polymeric structures composed, in part, of inosine monophosphate dehydrogenase (IMPDH), a key enzyme leading to GMP and GTP biosynthesis. RR formation is induced by IMPDH inhibitors as well as glutamine deprivation. They also form upon treatment of cells with glutamine synthetase inhibitors. We now report that depriving cells of serine and glycine promotes RR formation, and we have traced these effects to dihydrofolate reductase (DHFR) and serine hydroxymethyltransferase-2 (SHMT2), pivotal enzymes in one-carbon metabolism and nucleotide biosynthesis. RR assembly is likewise induced upon DHFR inhibition by methotrexate or aminopterin as well as siRNA-mediated knockdown of DHFR or SHMT2. Because RR assembly occurs when guanine nucleotide biosynthesis is inhibited, and because RRs rapidly disassemble after the addition of guanine nucleotide precursors, RR formation might be an adaptive homeostatic mechanism, allowing IMPDH to sense changes in the one-carbon folate pathway.

Methotrexate and Pralatrexate.

This article reviews methotrexate and the more potent, related compound, pralatrexate, for the treatment of cutaneous T-cell lymphomas, including mycosis fungoides, Sézary syndrome, and CD30+ lymphoproliferative disorders. Although these folate antagonists are traditionally viewed as antiproliferative cell cycle inhibitors, it is recognized that they inhibit DNA methylation, providing a rationale for their use as epigenetic regulators and cell proliferation inhibitors. The underlying mechanisms are outlined, key supporting data presented, followed by brief mention of recent mathematical modeling supporting the general superiority of combination therapy. Several novel examples involving folate antagonists are proposed.

Methylthioadenosine phosphorylase (MTAP)-deficient T-cell ALL xenografts are sensitive to pralatrexate and 6-thioguanine alone and in combination.

PURPOSE: To investigate the effectiveness of a combination of 6-thioguanine (6-TG) and pralatrexate (PDX) in methylthioadenosine phosphorylase (MTAP)-deficient T-cell acute lymphoblastic leukemia (T-cell ALL). METHODS: CCRF-CEM (MTAP(-/-)) and Molt4 (MTAP(+/+)) T-cell ALL cell lines were treated with 6-TG or PDX and evaluated for efficacy 72 h later. NOD/SCID gamma mice bearing CEM or Molt4 xenografts were treated with 6-TG and PDX alone or in combination to evaluate antitumor effects. RESULTS: CEM cells were more sensitive to 6-TG and PDX in vitro than Molt4. In vivo, CEM cells were very sensitive to PDX and 6-TG, whereas Molt4 cells were highly resistant to 6-TG. A well-tolerated combination of PDX and 6-TG achieved significant tumor regression in CEM xenografts. CONCLUSIONS: The loss of MTAP expression may be therapeutically exploited in T-cell ALL. The combination of 6-TG and PDX, with the inclusion of leucovorin rescue, allows for a safe and effective regimen in MTAP-deficient T-cell ALL.

Leucovorin rescue allows effective high-dose pralatrexate treatment and an increase in therapeutic index in mesothelioma xenografts.

PURPOSE: To investigate the ability of leucovorin (LV) to abrogate dose-limiting toxicities of pralatrexate (PDX) while maintaining efficacy, in vivo. METHODS: H2052 mesothelioma cells were treated with the antifolates methotrexate (MTX), PDX and pemetrexed, with and without LV rescue 24 h later. Cell killing was evaluated 48 h later. Female nude mice bearing H2052 xenografts were treated with varying doses and schedules of the antifolate PDX and LV. RESULTS: In vitro, H2052 cells were more sensitive to PDX as compared to MTX and pemetrexed. Administration of LV 24 h after antifolate treatment reduced efficacy of antifolates MTX and pemetrexed, but not PDX. In vivo, LV was found to reduce toxicity of PDX at the maximum tolerated dose without sacrificing efficacy. Lethal doses of PDX were rescued by LV, and mice bearing the H2052 tumor demonstrated prolonged and enhanced tumor regression. CONCLUSIONS: High-dose PDX with subsequent LV rescue may be a viable treatment strategy in mesothelioma and other cancers. The inclusion of LV rescue into new and existing PDX treatment protocols should be explored as a way to expand the tolerability and effectiveness of PDX in the clinic.

LD-aminopterin in the canine homologue of human atopic dermatitis: a randomized, controlled trial reveals dosing factors affecting optimal therapy.

BACKGROUND: Options are limited for patients with atopic dermatitis (AD) who do not respond to topical treatments. Antifolate therapy with systemic methotrexate improves the disease, but is associated with adverse effects. The investigational antifolate LD-aminopterin may offer improved safety. It is not known how antifolate dose and dosing frequency affect efficacy in AD, but a primary mechanism is thought to involve the antifolate-mediated accumulation of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). However, recent in vitro studies indicate that AICAR increases then decreases as a function of antifolate concentration. To address this issue and understand how dosing affects antifolate efficacy in AD, we examined the efficacy and safety of different oral doses and schedules of LD-aminopterin in the canine model of AD. METHODS AND FINDINGS: This was a multi-center, double-blind trial involving 75 subjects with canine AD randomized to receive up to 12 weeks of placebo, once-weekly (0.007, 0.014, 0.021 mg/kg) or twice-weekly (0.007 mg/kg) LD-aminopterin. The primary efficacy outcome was the Global Score (GS), a composite of validated measures of disease severity and itch. GS improved in all once-weekly cohorts, with 0.014 mg/kg being optimal and significant (43%, P<0.01). The majority of improvement was seen by 8 weeks. In contrast, GS in the twice-weekly cohort was similar to placebo and worse than all once-weekly cohorts. Adverse events were similar across all treated cohorts and placebo. CONCLUSIONS: Once-weekly LD-aminopterin was safe and efficacious in canine AD. Twice-weekly dosing negated efficacy despite having the same daily and weekly dose as effective once-weekly regimens. Optimal dosing in this homologue of human AD correlated with the concentration-selective accumulation of AICAR in vitro, consistent with AICAR mediating LD-aminopterin efficacy in AD.

The impact of 5-formyltetrahydrofolate on the anti-tumor activity of pralatrexate, as compared to methotrexate, in HeLa cells in vitro.

PURPOSE: To investigate the impact of 5-formyltetrahydrofolate on the activities of pralatrexate, as compared to methotrexate (MTX), in vitro. METHODS: Cells were exposed to (6S)5-formyltetrahydrofolate (5-formylTHF) for 24 h, before or after a 6-h exposure to antifolates following which the cellular accumulation and activities of the drugs were evaluated in HeLa cells. RESULTS: A 24-h delay between a 6-h exposure to antifolates and a subsequent 24-h exposure to 4 µM 5-formylTHF sustained the full activities of both antifolates. A 72-h interval was required between a single exposure of up to 4 µM 5-formylTHF and subsequent exposure to drugs to sustain activities of the antifolates. When cells were incubated with 4 µM 5-formylTHF for 24 h weekly, for 4 weeks, there was no significant increase in the IC50 for pralatrexate, but the MTX IC50 increased 2.5-fold as compared to cells growing continuously in 25 nM 5-formylTHF. This cyclical exposure to 5-formylTHF increased the cell folate pool by 16 %, had no significant effect on the intracellular pralatrexate level, but decreased intracellular MTX by 15 %. An extracellular concentration of MTX 50-fold higher than that of pralatrexate was required to achieve an intracellular level, and growth inhibition, comparable to that of pralatrexate. CONCLUSIONS: Cyclical exposures to 5-formylTHF at levels in excess of what is achieved in most clinical "rescue" regimens do not affect pralatrexate accumulation nor antitumor activity in HeLa cells, in contrast to MTX. An important element in preserving pralatrexate activity is achieving a sufficient interval between exposure to 5-formylTHF and the next dose of antifolate.

Folate receptor-targeted aminopterin therapy is highly effective and specific in experimental models of autoimmune uveitis and autoimmune encephalomyelitis.

EC0746 is a rationally designed anti-inflammatory drug conjugate consisting of a modified folic acid-based ligand linked to a γ-hydrazide analog of aminopterin. In this report, EC0746's effectiveness was evaluated against experimental retinal S-antigen (PDSAg) induced autoimmune uveitis (EAU) and myelin-basic-protein induced autoimmune encephalomyelitis (EAE). In both models, functional FR-ß was detected on activated macrophages in local (retinal or central-nervous-system, respectively) and systemic (peritoneal cavity) sites of inflammation. In myelin-rich regions of EAE rats, an increased uptake of (99m)Tc-EC20 (etarfolatide; a FR-specific radioimaging agent) was also observed. EC0746 treatment at disease onset suppressed the clinical severity of both EAU and EAE, and it strongly attenuated progressive histopathological changes in the affected organs. In all parameters assessed, EC0746 activity was completely blocked by a benign folate competitor, suggesting that these therapeutic outcomes were specifically FR-ß mediated. EC0746 may emerge as a useful macrophage-modulating agent for treating inflammatory episodes of organ-specific autoimmunity.

Can the L5178Y Tk+/- mouse lymphoma assay detect epigenetic silencing?

The mouse lymphoma L5178Y Tk(+/-) assay is broadly used in toxicology to assess genotoxicity because of its known sensitivity to genotoxicants that act through a variety of mechanisms, which may include epigenetic DNA methylation. This brief article highlights the studies that have contributed to this conjecture and suggests an addition to the experimental design that could identify if the test substance is a potential epimutagen acting via hypermethylation.