Teriflunomide shifts the astrocytic bioenergetic profile from oxidative metabolism to glycolysis and attenuates TNFα-induced inflammatory responses.

Astrocytes utilize both glycolytic and mitochondrial pathways to power cellular processes that are vital to maintaining normal CNS functions. These cells also mount inflammatory and acute phase reactive programs in response to diverse stimuli. While the metabolic functions of astrocytes under homeostatic conditions are well-studied, the role of cellular bioenergetics in astrocyte reactivity is poorly understood. Teriflunomide exerts immunomodulatory effects in diseases such as multiple sclerosis by metabolically reprogramming lymphocytes and myeloid cells. We hypothesized that teriflunomide would constrain astrocytic inflammatory responses. Purified murine astrocytes were grown under serum-free conditions to prevent acquisition of a spontaneous reactive state. Stimulation with TNFα activated NFκB and increased secretion of Lcn2. TNFα stimulation increased basal respiration, maximal respiration, and ATP production in astrocytes, as assessed by oxygen consumption rate. TNFα also increased glycolytic reserve and glycolytic capacity of astrocytes but did not change the basal glycolytic rate, as assessed by measuring the extracellular acidification rate. TNFα specifically increased mitochondrial ATP production and secretion of Lcn2 required ATP generated by oxidative phosphorylation. Inhibition of dihydroorotate dehydrogenase via teriflunomide transiently increased both oxidative phosphorylation and glycolysis in quiescent astrocytes, but only the increased glycolytic ATP production was sustained over time, resulting in a bias away from mitochondrial ATP production even at doses down to 1 µM. Preconditioning with teriflunomide prevented the TNFα-induced skew toward oxidative phosphorylation, reduced mitochondrial ATP production, and reduced astrocytic inflammatory responses, suggesting that this drug may limit neuroinflammation by acting as a metabolomodulator.

Design, Synthesis, and Biological Evaluation of a Novel Series of Teriflunomide Derivatives as Potent Human Dihydroorotate Dehydrogenase Inhibitors for Malignancy Treatment.

Human dihydroorotate dehydrogenase (hDHODH), as the fourth and rate-limiting enzyme of the de novo pyrimidine synthesis pathway, is regarded as an attractive target for malignancy therapy. In the present study, a novel series of teriflunomide derivatives were designed, synthesized, and evaluated as hDHODH inhibitors. 13t was the optimal compound with promising enzymatic activity (IC50 = 16.0 nM), potent antiproliferative activity against human lymphoma Raji cells (IC50 = 7.7 nM), and excellent aqueous solubility (20.1 mg/mL). Mechanistically, 13t directly inhibited hDHODH and induced cell cycle S-phase arrest in Raji cells. The acute toxicity assay indicated a favorable safety profile of 13t. Notably, 13t displayed significant tumor growth inhibition activity with a tumor growth inhibition (TGI) rate of 81.4% at 30 mg/kg in a Raji xenograft model. Together, 13t is a promising inhibitor of hDHODH and a preclinical candidate for antitumor therapy, especially for lymphoma.

Teriflunomide Inhibits JCPyV Infection and Spread in Glial Cells and Choroid Plexus Epithelial Cells.

Several classes of immunomodulators are used for treating relapsing-remitting multiple sclerosis (RRMS). Most of these disease-modifying therapies, except teriflunomide, carry the risk of progressive multifocal leukoencephalopathy (PML), a severely debilitating, often fatal virus-induced demyelinating disease. Because teriflunomide has been shown to have antiviral activity against DNA viruses, we investigated whether treatment of cells with teriflunomide inhibits infection and spread of JC polyomavirus (JCPyV), the causative agent of PML. Treatment of choroid plexus epithelial cells and astrocytes with teriflunomide reduced JCPyV infection and spread. We also used droplet digital PCR to quantify JCPyV DNA associated with extracellular vesicles isolated from RRMS patients. We detected JCPyV DNA in all patients with confirmed PML diagnosis (n = 2), and in six natalizumab-treated (n = 12), two teriflunomide-treated (n = 7), and two nonimmunomodulated (n = 2) patients. Of the 21 patients, 12 (57%) had detectable JCPyV in either plasma or serum. CSF was uniformly negative for JCPyV. Isolation of extracellular vesicles did not increase the level of detection of JCPyV DNA versus bulk unprocessed biofluid. Overall, our study demonstrated an effect of teriflunomide inhibiting JCPyV infection and spread in glial and choroid plexus epithelial cells. Larger studies using patient samples are needed to correlate these in vitro findings with patient data.

Effect of Teriflunomide on Cells From Patients With Human T-cell Lymphotropic Virus Type 1-Associated Neurologic Disease.

OBJECTIVE: To test the hypothesis that teriflunomide can reduce ex vivo spontaneous proliferation of peripheral blood mononuclear cells (PBMCs) from patients with human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). METHODS: PBMCs from patients with HAM/TSP were cultured in the presence and absence of teriflunomide and assessed for cell viability, lymphocyte proliferation, activation markers, HTLV-1 tax and HTLV-1 hbz messenger ribonucleic acid (mRNA) expression, and HTLV-1 Tax protein expression. RESULTS: In culture, teriflunomide did not affect cell viability. A concentration-dependent reduction in spontaneous proliferation of PBMCs was observed with 25 µM (38.3% inhibition), 50 µM (65.8% inhibition), and 100 µM (90.7% inhibition) teriflunomide. The inhibitory effects of teriflunomide were detected in both CD8+ and CD4+ T-cell subsets, which are involved in the immune response to HTLV-1 infection and the pathogenesis of HAM/TSP. There was no significant change in HTLV-1 proviral load (PVL) or tax mRNA/Tax protein expression in these short-term cultures, but there was a significant reduction of HTLV-1 PVL due to inhibition of proliferation of CD4+ T cells obtained from a subset of patients with HAM/TSP. CONCLUSIONS: These results suggest that teriflunomide inhibits abnormal T-cell proliferation associated with HTLV-1 infection and may have potential as a therapeutic option in patients with HAM/TSP.

New bioassay cage methodology for in vitro studies on Varroa destructor and Apis mellifera.

Varroa destructor Anderson and Trueman, is an ectoparasitic mite of honey bees, Apis mellifera L., that has been considered a major cause of colony losses. Synthetic miticides have been developed and registered to manage this ectoparasite, however, resistance to registered pyrethroid and organophosphate Varroacides have already been reported in Canada. To test toxicity of miticides, current contact-based bioassay methods are designed to evaluate mites and bees separately, however, these methods are unlikely to give an accurate depiction of how miticides interact at the colony level. Therefore, the objective of this study was to develop a bioassay cage for testing the toxicity of miticides on honey bees and Varroa mites simultaneously using amitraz as a reference chemical. A 800 mL polypropylene plastic cage holding 100-150 bees was designed and officially named "Apiarium". A comparison of the effects of three subsequent dilutions of amitraz was conducted on: Varroa mites placed in glass vials, honey bees in glass Mason jars, and Varroa-infested bees in Apiariums. Our results indicated cumulative Varroa mortality was dose-dependent in the Apiarium after 4 h and 24 h assessments. Apiarium and glass vial treatments at 24 h also had high mite mortality and a positive polynomial regression between Varroa mortality and amitraz dose rates. Moreover, chemical application in the Apiarium was less toxic for bees compared to the Mason jar method. Considering these results, the Apiarium bioassay provides a simple, cheap and reliable method for simultaneous chemical screening on V. destructor and A. mellifera. Furthermore, as mites and bees are tested together, the Apiarium simulates a colony-like environment that provides a necessary bridge between laboratory bioassay testing and full field experimentation. The versatility of the Apiarium allows researchers to test a multitude of different honey bee bioassay experiments including miticide screening, delivery methods for chemical products, or development of new mite resistance-testing methodology.

Ponesimod Compared With Teriflunomide in Patients With Relapsing Multiple Sclerosis in the Active-Comparator Phase 3 OPTIMUM Study: A Randomized Clinical Trial.

Importance: To our knowledge, the Oral Ponesimod Versus Teriflunomide In Relapsing Multiple Sclerosis (OPTIMUM) trial is the first phase 3 study comparing 2 oral disease-modifying therapies for relapsing multiple sclerosis (RMS). Objective: To compare the efficacy of ponesimod, a selective sphingosine-1-phosphate receptor 1 (S1P1) modulator with teriflunomide, a pyrimidine synthesis inhibitor, approved for the treatment of patients with RMS. Design, Setting, and Participants: This multicenter, double-blind, active-comparator, superiority randomized clinical trial enrolled patients from April 27, 2015, to May 16, 2019, who were aged 18 to 55 years and had been diagnosed with multiple sclerosis per 2010 McDonald criteria, with a relapsing course from the onset, Expanded Disability Status Scale (EDSS) scores of 0 to 5.5, and recent clinical or magnetic resonance imaging disease activity. Interventions: Patients were randomized (1:1) to 20 mg of ponesimod or 14 mg of teriflunomide once daily and the placebo for 108 weeks, with a 14-day gradual up-titration of ponesimod starting at 2 mg to mitigate first-dose cardiac effects of S1P1 modulators and a follow-up period of 30 days. Main Outcomes and Measures: The primary end point was the annualized relapse rate. The secondary end points were the changes in symptom domain of Fatigue Symptom and Impact Questionnaire-Relapsing Multiple Sclerosis (FSIQ-RMS) at week 108, the number of combined unique active lesions per year on magnetic resonance imaging, and time to 12-week and 24-week confirmed disability accumulation. Safety and tolerability were assessed. Exploratory end points included the percentage change in brain volume and no evidence of disease activity (NEDA-3 and NEDA-4) status. Results: For 1133 patients (567 receiving ponesimod and 566 receiving teriflunomide; median [range], 37.0 [18-55] years; 735 women [64.9%]), the relative rate reduction for ponesimod vs teriflunomide in the annualized relapse rate was 30.5% (0.202 vs 0.290; P < .001); the mean difference in FSIQ-RMS, -3.57 (-0.01 vs 3.56; P < .001); the relative risk reduction in combined unique active lesions per year, 56% (1.405 vs 3.164; P < .001); and the reduction in time to 12-week and 24-week confirmed disability accumulation risk estimates, 17% (10.1% vs 12.4%; P = .29) and 16% (8.1% vs 9.9; P = .37), respectively. Brain volume loss at week 108 was lower by 0.34% (-0.91% vs -1.25%; P < .001); the odds ratio for NEDA-3 achievement was 1.70 (25.0% vs 16.4%; P < .001). Incidence of treatment-emergent adverse events (502 of 565 [88.8%] vs 499 of 566 [88.2%]) and serious treatment-emergent adverse events (49 [8.7%] vs 46 [8.1%]) was similar for both groups. Treatment discontinuations because of adverse events was more common in the ponesimod group (49 of 565 [8.7%] vs 34 of 566 [6.0%]). Conclusions and Relevance: In this study, ponesimod was superior to teriflunomide on annualized relapse rate reduction, fatigue, magnetic resonance imaging activity, brain volume loss, and no evidence of disease activity status, but not confirmed disability accumulation. The safety profile was in line with the previous safety observations with ponesimod and the known profile of other S1P receptor modulators. Trial Registration: ClinicalTrials.gov Identifier: NCT02425644.

Restriction of intracellular Salmonella typhimurium growth by the small-molecule autophagy inducer A77 1726 through the activation of the AMPK-ULK1 axis.

Autophagy plays an important role in restricting the growth of invading intracellular microbes. Salmonella (S) Typhimurium, an intracellular pathogen that causes gastroenteritis and food poisoning in humans, evades autophagic detection by multiple mechanisms. There has been growing interest in developing autophagy inducers as novel antimicrobial agents for treating intracellular bacterial infections. We recently reported that A77 1726, the active metabolite of the anti-inflammatory drug leflunomide, induces autophagy by activating AMP-activated protein kinase (AMPK) and Unc-51 like autophagy activating kinase 1 (ULK1). Our present study aims to determine if A77 1726 was able to restrict intracellular Salmonella growth by inducing autophagy. We first confirmed the ability of A77 1726 to induce autophagy by activating the AMPK-ULK1 axis in uninfected RAW264.7 (a murine macrophage cell line) and HeLa cells (a human cervical carcinoma cell line). A77 1726 enhanced autophagy in S. Typhimurium-infected cells, as evidenced by increased levels of LC3 lipidation and increased numbers of autophagosomes and autolysosomes. Confocal microscopy revealed that A77 1726 induced xenophagy in macrophages, as evidenced by an increased number of LC3-coated bacteria in the cytoplasm. A77 1726 significantly decreased the number of intracellular S. Typhimurium in macrophages. Taken together, our study has demonstrated the ability of A77 1726 to restrict intracellular S. Typhimurium growth in vitro by enhancing xenophagy.

Effect of the Intramolecular Hydrogen Bond on the Active Metabolite Analogs of Leflunomide for Blocking the Plasmodium falciparum Dihydroorotate Dehydrogenase Enzyme: QTAIM, NBO, and Docking Study.

BACKGROUND: Leflunomide (LFM) and its active metabolite, teriflunomide (TFM), have drawn a lot of attention for their anticancer activities, treatment of rheumatoid arthritis and malaria due to their capability to inhibit dihydroorotate dehydrogenase (DHODH) and Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) enzyme. In this investigation, the strength of intramolecular hydrogen bond (IHB) in five analogs of TFM (ATFM) was analyzed employing density functional theory (DFT) using B3LYP/6-311++G (d, p) level and molecular orbital analysis in the gas phase and water solution. A detailed electronic structure study was performed using the quantum theory of atoms in molecules (QTAIM) and the hydrogen bond energies (EHB) of stable conformer obtained in the range of 76-97 kJ/mol, as a medium hydrogen bond. The effect of substitution on the IHB nature was studied by natural bond orbital analysis (NBO). 1H NMR calculations showed an upward trend in the proton chemical shift of the enolic proton in the chelated ring (14.5 to 15.7ppm) by increasing the IHB strength. All the calculations confirmed the strongest IHB in 5-F-ATFM and the weakest IHB in 2-FATFM. Molecular orbital analysis, including the HOMO-LUMO gap and chemical hardness, was performed to compare the reactivity of inhibitors. Finally, molecular docking analysis was carried out to identify the potency of inhibition of these compounds against PfDHODH enzyme. TFM acts as an inhibitor of dihydroorotate dehydrogenase (DHODH) and Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) enzyme. Leflunomide and its active metabolite teriflunomide have been identified as drugs for treatment of some diseases, such as multiple sclerosis (MS), rheumatoid arthritis (RA), malaria, and cancer. Hydrogen bonds play a key role in the interaction between drugs and enzymes. OBJECTIVES: The aim of the present work is to investigate the effect of the strength of intramolecular hydrogen bonds (IHBs) in the active metabolite analogs of leflunomide or analogs of teriflunomide (ATFMs) and study the interaction of these inhibitors against the PfDHODH enzyme using quantum mechanical methods. METHODS: At first, intramolecular hydrogen bonds in five ATFMs were evaluated by the DFT method, quantum theory of atoms in molecules (QTAIM), nuclear magnetic resonance (NMR), natural bond orbital (NBO), and molecular orbital (MO) analyses. Then, the interaction of these inhibitors against the PfDHODH enzyme were compared using molecular docking study. RESULTS: All the computed results confirm the following trend in the intramolecular hydrogen bond strength in five mono-halo-substituted 2-cyano-3-hydroxy-N-phenylbut-2-enamide (ATFM): 5-FATFM> 4-Br-ATFM ≈ 3-Br-ATFM>3-Cl-ATFM>TFM-Z>2-F-ATFM which is in agreement with QTAIM, NMR, and NBO results. Docking results show that 5-F-ATFM (EHB=97kJ/mol) has the minimum MolDock score due to its considerable IHB strength. CONCLUSION: For strong IHBs (EHB>100kJ/mol), C=O and O-H group are involved in the intramolecular interactions and do not contribute to the external interactions. Also, the docking study revealed maximum binding energy between TFM-Z and PfDHODH enzyme.

Long non-coding RNAs as targets for immunosuppressive drug teriflunomide in anti-cancer potential for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the most common form of liver cancer. Because of the relatively chemotherapy-refractory nature of HCC and significant potential poor hepatic reserve, chemotherapy has not been used consistently in the treatment of HCC. Effective new drugs for HCC are urgently needed. Teriflunomide, which was approved for the treatment of relapsing forms of multiple sclerosis (MS), has been identified as a potential antineoplastic drug. Long noncoding RNAs (lncRNAs) are a novel class of RNA molecules defined as transcripts longer than 200 nucleotides that lack protein coding potential. In this study, we investigated the ability of teriflunomide to act as an antineoplastic drug by examining the effects of teriflunomide treatment on HCC cells. Teriflunomide strongly inhibited the proliferation of HCC cells, induced cell apoptosis and induced cell accumulation in S phases of the cell cycle. LncRNA and mRNA expression profiles of HCC cells treated with teriflunomide compared with controls were performed by using microarray analysis. For comparison, the differentially expressed mRNAs were annotated by using gene ontology (GO) and pathway analyses. The microarray revealed that 2085 lncRNAs and 1561 mRNAs differed in the cells treated with teriflunomide compared with controls. Several GO terms including protein folding, mitochondrial outer membrane, transmembrane receptor protein phosphatase activity, negative regulation of cellular biosynthetic process, DNA packaging complex, and receptor signaling protein activity were enriched in gene lists, suggesting a potential correlation with the action mechanism of teriflunomide. Pathway analysis then demonstrated that JAK-STAT signaling pathway may play important roles in the cell apoptosis induced by teriflunomide. Co-expression network analysis indicated that a number of lncRNAs and mRNAs were included in the co-expression network, and p34710_v4 is the lncRNA with highest degree. Then the mRNAs associated with those differentially expressed lncRNAs were also annotated by using gene ontology (GO) and pathway analyses. The pathway analyses shows that teriflunomide significantly inhibited cell proliferation and promoted cell apoptosis partly by participating in Wnt signaling pathways. These findings suggest that teriflunomide could be a potential drug for chemotherapy and molecularly targeted therapies of HCC.

Facile functionalization of Teriflunomide-loaded nanoliposomes with Chondroitin sulphate for the treatment of Rheumatoid arthritis.

This research aims to coat Teriflunomide (TEF) loaded conventional nanoliposomes (CON-TEF-LIPO) with Chondroitin sulphate (CS) to produce CS-TEF-LIPO for the effective treatment of Rheumatoid arthritis (RA). Both CON-TEF-LIPO and CS-TEF-LIPO were produced, characterized and evaluated for their active targeting potential towards CD44 receptors. Cell cytotoxicity, cell viability and intracellular uptake study on differentiated U937 and MG-63 cells demonstrated the active targeting of CS-TEF-LIPO towards CD44 receptors. Furthermore, in vivo pharmacodynamic, biochemical, radiological and histopathological studies performed in adjuvant induced arthritic (AIA) rat model showed a significant (P < 0.05) reduction in inflammation in arthritic rat paw in CS-TEF-LIPO group compared to TEF and CON-TEF-LIPO groups. Moreover, liver toxicity study revealed that CS-TEF-LIPO showed no signs of toxicity and biodistribution study revealed the accumulation of CS-TEF-LIPO in synovial region of arthritic rat. Taken together, results suggest that CS-TEF-LIPO could provide a new insight for an effective treatment of RA.

The in vitro effect of antirheumatic drugs on platelet function.

Several antirheumatic drugs lower the cardiovascular risk among rheumatoid arthritis patients. It is, however, unknown whether inhibition of platelet function contributes to this risk reduction. Only few studies have investigated the potential role of platelets as a target of antirheumatic drugs. In this study, platelet function was tested in vitro in samples from 24 healthy individuals spiked with antirheumatic drugs in clinically relevant concentrations or vehicle. Platelet aggregation was tested with 96-well light transmission aggregometry (LTA), and when an effect ≥20% compared to vehicle was observed, flow cytometric platelet aggregation and activation were evaluated and closure time was measured by Platelet Function Analyzer (PFA-200). When evaluated by LTA, teriflunomide (the active metabolite of leflunomide), tocilizumab, and prednisolone reduced ADP- and collagen-induced platelet aggregation ≥20%, while adalimumab increased TRAP-induced platelet aggregation ≥20%. Using flow cytometry, agonist-induced platelet aggregation with teriflunomide or vehicle was mean ± standard deviation (SD); 30.7% ± 5.8 vs. 41.7% ± 6.5, p = 0.02 using ADP, and 34.7% ± 13.9 vs. 55.8% ± 3.9, p = 0.01 using collagen. Results indicate that teriflunomide, prednisolone, and tocilizumab inhibit, and adalimumab increases platelet aggregation. The study suggests that the majority of antirheumatic drugs mainly reduced cardiovascular risk through indirect effects (e.g., reducing inflammation).

Targeting pyrimidine synthesis accentuates molecular therapy response in glioblastoma stem cells.

Glioblastoma stem cells (GSCs) reprogram glucose metabolism by hijacking high-affinity glucose uptake to survive in a nutritionally dynamic microenvironment. Here, we trace metabolic aberrations in GSCs to link core genetic mutations in glioblastoma to dependency on de novo pyrimidine synthesis. Targeting the pyrimidine synthetic rate-limiting step enzyme carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase (CAD) or the critical downstream enzyme dihydroorotate dehydrogenase (DHODH) inhibited GSC survival, self-renewal, and in vivo tumor initiation through the depletion of the pyrimidine nucleotide supply in rodent models. Mutations in EGFR or PTEN generated distinct CAD phosphorylation patterns to activate carbon influx through pyrimidine synthesis. Simultaneous abrogation of tumor-specific driver mutations and DHODH activity with clinically approved inhibitors demonstrated sustained inhibition of metabolic activity of pyrimidine synthesis and GSC tumorigenic capacity in vitro. Higher expression of pyrimidine synthesis genes portends poor prognosis of patients with glioblastoma. Collectively, our results demonstrate a therapeutic approach of precision medicine through targeting the nexus between driver mutations and metabolic reprogramming in cancer stem cells.

Development and Evaluation of a Water-in-oil Microemulsion Formulation for the Transdermal Drug Delivery of Teriflunomide (A771726).

Teriflunomide (TEF, A771726) is the active metabolite of leflunomide (LEF), a disease-modifying anti-rheumatic drug. The main purpose of this study was to develop and evaluate water-in-oil (W/O) microemulsion formulation of TEF. The W/O microemulsion was optimized formula is the physical and chemical stability of lecithin, ethanol, isopropyl myristate (IPM) and water (20.65/20.78/41.52/17.05 w/w) by using the pseudo-ternary phase diagram and the average droplet size is about 40 nm. The permeability of TEF microemulsion is about 6 times higher than control group in vitro penetration test. The results of anti-inflammatory effect showed that compared with the control group, the external TEF microemulsion group could significantly inhibit swelling of paw in rats, and no significant difference compared with oral LEF group. The results of hepatotoxicity test show that there were normal content of alanine aminotransferase (ALT)/aspartate aminotransferase (AST) and no obvious inflammatory infiltration of TEF microemulsion group compared with LEF group. The plasma concentration curve showed that compared with LEF group, the peak concentration of TEF microemulsion group was decreased, the half-life (t1/2) was prolonged, and the relative bioavailability of TEF microemulsion was 75.35%. These results suggest that TEF W/O microemulsion can be used as a promising preparation to play an anti-inflammatory role while significantly reducing hepatotoxicity.

Tryptophan-kynurenine pathway attenuates ß-catenin-dependent pro-parasitic role of STING-TICAM2-IRF3-IDO1 signalosome in Toxoplasma gondii infection.

Recent studies have documented the diverse role of host immunity in infection by the protozoan parasite, Toxoplasma gondii. However, the contribution of the ß-catenin pathway in this process has not been explored. Here, we show that AKT-mediated phosphorylated ß-catenin supports T. gondii multiplication which is arrested in the deficiency of its phosphorylation domain at S552 position. The ß-catenin-TCF4 protein complex binds to the promoter region of IRF3 gene and initiates its transcription, which was also abrogated in ß-catenin knockout cells. TBK-independent phosphorylation of STING(S366) and its adaptor molecule TICAM2 by phospho-AKT(T308S473) augmented downstream IRF3-dependent IDO1 transcription, which was also dependent on ß-catenin. But, proteasomal degradation of IDO1 by its tyrosine phosphorylation (at Y115 and Y253) favoured parasite replication. In absence of IDO1, tryptophan was catabolized into melatonin, which supressed cellular reactive oxygen species (ROS) and boosted parasite growth. Conversely, when tyrosine phosphorylation was abolished by phosphosite mutations, IDO1 escaped its ubiquitin-mediated proteasomal degradation system (UPS) and the stable IDO1 prevented parasite replication by kynurenine synthesis. We propose that T. gondii selectively utilizes tryptophan to produce the antioxidant, melatonin, thus prolonging the survival of infected cells through functional AKT and ß-catenin activity for better parasite replication. Stable IDO1 in the presence of IFN-γ catabolized tryptophan into kynurenine, promoting cell death by suppressing phospho-AKT and phospho-ß-catenin levels, and circumvented parasite replication. Treatment of infected cells with kynurenine or its analogue, teriflunomide suppressed kinase activity of AKT, and phosphorylation of ß-catenin triggering caspase-3 dependent apoptosis of infected cells to inhibit parasite growth. Our results demonstrate that ß-catenin regulate phosphorylated STING-TICAM2-IRF3-IDO1 signalosome for a cell-intrinsic pro-parasitic role. We propose that the downstream IRF3-IDO1-reliant tryptophan catabolites and their analogues can act as effective immunotherapeutic molecules to control T. gondii replication by impairing the AKT and ß-catenin axis.

Dimethyl fumarate and teriflunomide for multiple sclerosis in a real-life setting: a French retrospective cohort study.

BACKGROUND AND PURPOSE: Dimethyl fumarate (DMF) and teriflunomide are approved oral disease-modifying treatments for relapsing-remitting multiple sclerosis (MS). Phase 3 trials established these agents to be effective and generally well tolerated, although comparative efficacy and discontinuation rates are still unknown. The aim of this study was to assess real-world efficacy and discontinuation of DMF and teriflunomide in patients with relapsing-remitting MS. METHODS: This retrospective observational cohort study was carried out in a French administrative region between March 2014 and July 2017. Patients who were followed by private or hospital neurologists were included. Efficacy and tolerance of the two treatments were assessed and compared by multivariate analysis, considering the duration of MS, annualized relapse rate and Expanded Disability Status Scale score at treatment initiation, treatment duration, type of prescriber and tobacco use. RESULTS: We identified 189 DMF- and 157 teriflunomide-treated patients who had been treated for 22 ± 10 months. After correction for confounders, DMF more efficiently reduced the annualized relapse rate after 2 years than teriflunomide (0.06 vs. 0.21; P = 0.03). DMF-treated patients had more clinical and biological adverse events, resulting in a higher rate of treatment discontinuation (28% vs. 12%, P = 0.03). CONCLUSION: In this retrospective cohort study, DMF demonstrated significantly better efficacy over 2 years than teriflunomide, but tolerance to teriflunomide was better.

Correlation Analysis of Potential Breast Cancer Resistance Protein Probes in Different Monolayer Systems.

Breast cancer resistance protein (BCRP) is a point of interest in drug-drug interaction safety testing. Therefore, a consensus probe that can be applied as victim in multiple experimental settings is of great benefit. Identification of candidates has been driven by the amount and quality of available clinical data, and as a result, drugs such as sulfasalazine and rosuvastatin have been suggested. In this article, the in vitro performance of 5 possible alternatives was evaluated: atorvastatin, chlorothiazide, dantrolene, topotecan, and teriflunomide, and benchmarked against sulfasalazine and rosuvastatin in reference in vitro assays for BCRP drug-drug interaction testing. Based on the results, teriflunomide is proposed as an alternate in vitro BCRP probe.

Anti-inflammatory drug, leflunomide and its metabolite teriflunomide inhibit NSCLC proliferation in vivo and in vitro.

Lung cancer causes more than 150000 deaths annually in the United States alone, of which non-small cell lung cancer (NSCLC) accounts for 80%. Our studies demonstrated that NSCLC cells were sensitive to leflunomide and its metabolite teriflunomide, a FDA approved drug, which was a well-known immunomodulatory drug for relapsing multiple sclerosis (MS). In the present studies, we found first time that they displayed anti-tumor activity of NSCLC in vitro and in vivo. Potent anti-cancer effects in NSCLC in vitro, including inhibiting NSCLC cells viability, arresting cell cycle at the G0/G1 phase, inducing cell apoptosis, delaying and suppressing NSCLC cells colony-forming ability and cell motility, could be achieved with this agent. Meanwhile, we provided evidence that these effects were applicable in vivo by using H460 cells xenograft model in nude mice. In addition, to comprehensively clarify the mechanisms of teriflunomide in NSCLC, we explored a genome-wide transcriptomic analysis, and found that teriflunomide was involved in multiple signaling pathways and cellular processes, such as cell cycle, apoptosis, MAPK and p53 signaling pathway. Taken together, the results of our studies provided insights into a novel anti-cancer effect of leflunomide and teriflunomide on NSCLC and might open new therapeutic avenues for the treatment of NSCLC.

Leflunomide/teriflunomide inhibit Epstein-Barr virus (EBV)- induced lymphoproliferative disease and lytic viral replication.

EBV infection causes mononucleosis and is associated with specific subsets of B cell lymphomas. Immunosuppressed patients such as organ transplant recipients are particularly susceptible to EBV-induced lymphoproliferative disease (LPD), which can be fatal. Leflunomide (a drug used to treat rheumatoid arthritis) and its active metabolite teriflunomide (used to treat multiple sclerosis) inhibit de novo pyrimidine synthesis by targeting the cellular dihydroorotate dehydrogenase, thereby decreasing T cell proliferation. Leflunomide also inhibits the replication of cytomegalovirus and BK virus via both "on target" and "off target" mechanisms and is increasingly used to treat these viruses in organ transplant recipients. However, whether leflunomide/teriflunomide block EBV replication or inhibit EBV-mediated B cell transformation is currently unknown. We show that teriflunomide inhibits cellular proliferation, and promotes apoptosis, in EBV-transformed B cells in vitro at a clinically relevant dose. In addition, teriflunomide prevents the development of EBV-induced lymphomas in both a humanized mouse model and a xenograft model. Furthermore, teriflunomide inhibits lytic EBV infection in vitro both by preventing the initial steps of lytic viral reactivation, and by blocking lytic viral DNA replication. Leflunomide/teriflunomide might therefore be clinically useful for preventing EBV-induced LPD in patients who have high EBV loads yet require continued immunosuppression.

Amitraz changes NE, DA and 5-HT biosynthesis and metabolism mediated by alterations in estradiol content in CNS of male rats.

Amitraz is a formamidine insecticide/acaricide that alters different neurotransmitters levels, among other neurotoxic effects. Oral amitraz exposure (20, 50 and 80 mg/kg bw, 5 days) has been reported to increase serotonin (5-HT), norepinephrine (NE) and dopamine (DA) content and to decrease their metabolites and turnover rates in the male rat brain, particularly in the striatum, prefrontal cortex, and hippocampus. However, the mechanisms by which these alterations are produced are not completely understood. One possibility is that amitraz monoamine oxidase (MAO) inhibition could mediate these effects. Alternatively, it alters serum concentrations of sex steroids that regulate the enzymes responsible for these neurotransmitters synthesis and metabolism. Thus, alterations in sex steroids in the brain could also mediate the observed effects. To test these hypothesis regarding possible mechanisms, we treated male rats with 20, 50 and 80 mg/kg bw for 5 days and then isolated tissue from striatum, prefrontal cortex, and hippocampus. We then measured tissue levels of expression and/or activity of MAO, catechol-O-metyltransferase (COMT), dopamine-ß-hydroxylase (DBH), tyrosine hydroxylase (TH) and tryptophan hydroxylase (TRH) as well as estradiol levels in these regions. Our results show that amitraz did not inhibit MAO activity at these doses, but altered MAO, COMT, DBH, TH and TRH gene expression, as well as TH and TRH activity and estradiol levels. The alteration of these enzymes was partially mediated by dysregulation of estradiol levels. Our present results provide new understanding of the mechanisms contributing to the harmful effects of amitraz.

Inhibition by Teriflunomide of Erythrocyte Cell Membrane Scrambling Following Energy Depletion, Oxidative Stress and Ionomycin.

BACKGROUND/AIMS: Teriflunomide, an inhibitor of pyrimidine synthesis and thus proliferation of activated T and B lymphocytes, is successfully used for treatment of inflammatory disease. Teriflunomide has further been shown to trigger apoptosis of tumor cells and has thus been considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Triggers of cell membrane scrambling include energy depletion, oxidative stress and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored whether teriflunomide modifies eryptosis. METHODS: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, and [Ca2+]i from Fluo3 fluorescence. RESULTS: Oxidative stress (60 min exposure to 0.3 mM tert-butylhydroperoxide), energy depletion (removal of glucose for 48 hours), and exposure to the Ca2+ ionophore ionomycin (1 µM, 60 min) all increased annexin-V-binding, decreased forward scatter and enhanced Fluo3 fluorescence. Teriflunomide (5 µg/ml) did not significantly influence Fluo3 fluorescence, forward scatter and annexin-V-binding under control conditions but significantly blunted the increase of annexin-V-binding following oxidative stress, energy depletion and ionomycin exposure. Teriflunomide further blunted the increase of Fluo3 fluorescence following energy depletion, but did not significantly interfere with increase of Fluo3 fluorescence following oxidative stress and ionomycin exposure. CONCLUSION: Teriflunomide is a novel inhibitor of suicidal erythrocyte death.