Monomethyl Fumarate Research Articles

Diroximel fumarate to treat multiple sclerosis.

On October 29, 2019, the Food and Drug Administration (FDA) of the United States approved diroximel fumarate (DRF) as an oral fumarate for the treatment of relapsing forms of multiple sclerosis. Another oral fumarate, dimethyl fumarate (DMF), was approved for the same indication on March 27, 2013. Prior to its approval, DRF did not undergo rigorous testing to determine its efficacy, as its active metabolite, monomethyl fumarate, is the same as that of DMF (bioequivalency). The efficacy, safety and tolerability of DMF have previously been demonstrated in a number of clinical trials and real-world studies. For DRF, one phase III study has been completed, and another is in progress to determine its safety, tolerability and efficacy. In this paper, we review the pharmacology, pharmacokinetics, metabolism, clinical studies and drug safety of DRF.

Dimethyl fumarate transfer into human milk.

Dimethyl fumarate (DMF) is approved for the treatment of relapsing-remitting multiple sclerosis. It is unknown whether DMF or its primary metabolite monomethyl fumarate (MMF) are excreted into human milk. We present two cases of lactating patients who donated milk samples to study the transfer of MMF into human milk following a week of 2 × 240 mg daily oral dose. Samples were analyzed using liquid chromatography mass spectrometry. The calculated relative infant dose was 0.019% and 0.007%. This is the first study to demonstrate that MMF is transferred into human milk, with only limited exposure to an infant.

Nicotinic acid promotes sleep through prostaglandin synthesis in mice

Nicotinic acid has been used for decades for its antiatherogenic properties in humans. Its actions on lipid metabolism intersect with multiple sleep regulatory mechanisms, but its effects on sleep have never been documented. For the first time, we investigated the effects of acute systemic administration of nicotinic acid on sleep in mice. Intraperitoneal and oral gavage administration of nicotinic acid elicited robust increases in non-rapid-eye movement sleep (NREMS) and decreases in body temperature, energy expenditure and food intake. Preventing hypothermia did not affect its sleep-inducing actions suggesting that altered sleep is not secondary to decreased body temperature. Systemic administration of nicotinamide, a conversion product of nicotinic acid, did not affect sleep amounts and body temperature, indicating that it is not nicotinamide that underlies these actions. Systemic administration of monomethyl fumarate, another agonist of the nicotinic acid receptor GPR109A, fully recapitulated the somnogenic and thermoregulatory effects of nicotinic acid suggesting that they are mediated by the GPR109A receptor. The cyclooxygenase inhibitor indomethacin completely abolished the effects of nicotinic acid indicating that prostaglandins play a key role in mediating the sleep and thermoregulatory responses of nicotinic acid.

Diroximel fumarate (DRF) in patients with relapsing–remitting multiple sclerosis: Interim safety and efficacy results from the phase 3 EVOLVE-MS-1 study

Background: Diroximel fumarate (DRF) is a novel oral fumarate for patients with relapsing–remitting multiple sclerosis (RRMS). DRF and the approved drug dimethyl fumarate yield bioequivalent exposure to the active metabolite monomethyl fumarate; thus, efficacy/safety profiles are expected to be similar. However, DRF’s distinct chemical structure may result in a differentiated gastrointestinal (GI) tolerability profile. Objective: To report interim safety/efficacy findings from patients in the ongoing EVOLVE-MS-1 study. Methods: EVOLVE-MS-1 is an ongoing, open-label, 96-week, phase 3 study assessing DRF safety, tolerability, and efficacy in RRMS patients. Primary endpoint is safety and tolerability; efficacy endpoints are exploratory. Results: As of March 2018, 696 patients were enrolled; median exposure was 59.9 (range: 0.1–98.9) weeks. Adverse events (AEs) occurred in 84.6% (589/696) of patients; the majority were mild (31.2%; 217/696) or moderate (46.8%; 326/696) in severity. Overall treatment discontinuation was 14.9%; 6.3% due to AEs and <1% due to GI AEs. At Week 48, mean number of gadolinium-enhancing lesions was significantly reduced from baseline (77%; p < 0.0001) and adjusted annualized relapse rate was low (0.16; 95% confidence interval: 0.13–0.20). Conclusion: Interim data from EVOLVE-MS-1 suggest DRF is a well-tolerated treatment with a favorable safety/efficacy profile for patients with RRMS.

Dimethyl fumarate therapy reduces memory T cells and the CNS migration potential in patients with multiple sclerosis

BackgroundDimethyl fumarate (DMF) is a disease-modifying therapy for patients with relapsing-remitting multiple sclerosis (RRMS). T cells are major contributors to the pathogenesis of RRMS, where they regulate the pathogenic immune response and participate in CNS lesion development. ObjectivesIn this study we evaluate the therapeutic effects of DMF on T cell subpopulations, their CNS migration potential and effector functions. MethodsBlood and CSF from untreated and DMF-treated patients with RRMS and healthy donors were analyzed by flow cytometry. ResultsDMF reduced the prevalence of circulating proinflammatory CD4+ and CD8+ memory T cells, whereas regulatory T cells were unaffected. Furthermore, DMF reduced the frequency of CD4+ T cells expressing CNS-homing markers. In coherence, we found a reduced recruitment of CD4+ but not CD8+ T cells to CSF. We also found that monomethyl fumarate dampened T cell proliferation and reduced the frequency of TNF-α, IL-17 and IFN-γ producing T cells. ConclusionDMF influences the balance between proinflammatory and regulatory T cells, presumably favoring a less proinflammatory environment. DMF also reduces the CNS migratory potential of CD4+ T cells whereas CD8+ T cells are less affected. Altogether, our study suggests an anti-inflammatory effect of DMF mainly on the CD4+ T cell compartment.

Gasdermin D Hypermethylation Inhibits Pyroptosis And LPS-Induced IL-1β Release From NK92 Cells.

IntroductionAlthough natural killer (NK) are major cells used to treat cancer patients, recent clinical trials showed that NK92 cells can be also used for the same purpose due to their high anti-tumor activity. Here, we examined whether these cells might be inflammatory due to the release of interleukin-1β (IL-1β), and whether the anti-inflammatory molecules dimethyl fumarate (DMF), or monomethyl fumarate (MMF) impair this activity.MethodsNK92 cells were examined for the synthesis and release of IL-1β utilizing RT-PCR and ELISA assay, respectively. The expression of hydroxy-carboxylic acid receptors (HCA)1, HCA2 and HCA3 was detected by immunoblotting, flow cytometry, immunofluorescence and RT-PCR assays. The activation of caspase-1 and Gasdermin D (GSDMD) was evaluated by immunoblot assay. Pyroptosis was demonstrated by immunofluorescence imaging. Expression of DNA methyltransferases (DNMTs) mRNA was determined by whole transcriptome and immunoblot analyses.ResultsLPS-induced the release of IL-1β from NK92 cells, whereas DMF or MMF inhibited this induction. The effect of these drugs was due to inhibiting the conversion of procaspase-1 into active caspase-1. NK92 cells highly expressed GSDMD, a pyroptotic-mediated molecule. However, LPS induced the distribution of GSDMD into the cell membranes, corroborated with the presence of pyroptotic bodies, an activity that was inhibited by DMF or MMF. These molecule also inhibited the generation of GSDMD through DNMT-mediated hypermethylation of the promoter region of GSDMD gene. These results were supported by increased expression of DNMTs mRNA as determined by whole transcriptome analysis.DiscussionOur results are the first to show that NK92 cells utilize GSDMD pathway to release IL-1β. Further, DMF and MMF which were previously shown to enhance NK cell cytotoxicity, also inhibit the inflammatory effects of these cells, making them most suitable for treating cancer patients.

Structural Study of Monomethyl Fumarate-Bound Human GAPDH.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a core enzyme of the aerobic glycolytic pathway with versatile functions and is associated with cancer development. Recently, Kornberg et al. published the detailed correlation between GAPDH and di- or monomethyl fumarate (DMF or MMF), which are well-known GAPDH antagonists in the immune system. As an extension, herein, we report the crystal structure of MMF-bound human GAPDH at 2.29 Å. The MMF molecule is covalently linked to the catalytic Cys152 of human GAPDH, and inhibits the catalytic activity of the residue and dramatically reduces the enzymatic activity of GAPDH. Structural comparisons between NAD+-bound GAPDH and MMF-bound GAPDH revealed that the covalently linked MMF can block the binding of the NAD+ co-substrate due to steric hindrance of the nicotinamide portion of the NAD+ molecule, illuminating the specific mechanism by which MMF inhibits GAPDH. Our data provide insights into GAPDH antagonist development for GAPDH-mediated disease treatment.

Dimethyl fumarate and monomethyl fumarate attenuate oxidative stress and mitochondrial alterations leading to oxiapoptophagy in 158N murine oligodendrocytes treated with 7β-hydroxycholesterol

Oxidative stress and mitochondrial dysfunction contribute to the pathogenesis of neurodegenerative diseases and favor lipid peroxidation, leading to increased levels of 7β-hydroxycholesterol (7β-OHC) which induces oxiapoptophagy (OXIdative stress, APOPTOsis, autoPHAGY). The cytoprotective effects of dimethylfumarate (DMF), used in the treatment of relapsing remitting multiple sclerosis and of monomethylfumarate (MMF), its main metabolite, were evaluated on murine oligodendrocytes 158 N exposed to 7β-OHC (50 μM, 24 h) with or without DMF or MMF (25 μM). The activity of 7β-OHC in the presence or absence DMF or MMF was evaluated on several parameters: cell adhesion; plasma membrane integrity measured with propidium iodide (PI), trypan blue and fluoresceine diacetate (FDA) assays; LDH activity; antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)); generation of lipid peroxidation products (malondialdehyde (MDA), conjugated dienes (CDs)) and protein oxidation products (carbonylated proteins (CPs)); reactive oxygen species (ROS) overproduction conducted with DHE and DHR123. The effect on mitochondria was determined with complementary criteria: measurement of succinate dehydrogenase activity, evaluation of mitochondrial potential (ΔΨm) and mitochondrial superoxide anions (O2●−) production using DiOC6(3) and MitoSOX, respectively; quantification of mitochondrial mass with Mitotracker Red, and of cardiolipins and organic acids. The effects on mitochondrial and peroxisomal ultrastructure were determined by transmission electron microscopy. Intracellular sterol and fatty acid profiles were determined. Apoptosis and autophagy were characterized by staining with Hoechst 33,342, Giemsa and acridine orange, and with antibodies raised against caspase-3 and LC3. DMF and MMF attenuate 7β-OHC-induced cytotoxicity: cell growth inhibition; decreased cell viability; mitochondrial dysfunction (decrease of succinate dehydrogenase activity, loss of ΔΨm, increase of mitochondrial O2●− production, alteration of the tricarboxilic acid (TCA) cycle, and cardiolipins content); oxidative stress induction (ROS overproduction, alteration of GPx, CAT, and SOD activities, increased levels of MDA, CDs, and CPs); changes in fatty acid and cholesterol metabolism; and cell death induction (caspase-3 cleavage, activation of LC3-I in LC3-II). Ultrastructural alterations of mitochondria and peroxisomes were prevented. These results demonstrate that DMF and MMF prevent major dysfunctions associated with neurodegenerative diseases: oxidative stress, mitochondrial dysfunction, apoptosis and autophagy.

Reactivity studies on a trihydride niobocene complex towards α,β-Unsaturated carboxylic acids

The reactions of the trihydride niobocene complex [NbCp’2H3] (Cp’ = η5-C5H4SiMe3) (1) with different α,β-unsaturated carboxylic acids have been studied. The reactions with fumaric and maleic acids (A and B) gave a new dinuclear carboxylato-containing niobocene derivative [(NbCp’2)2(μ-κ2O,O-OOC-CH2-CH2-COO-κ2O,O)] (2), due to the occurrence of two processes, dihydrogen elimination and hydrogenation of the CC bond of the α,β-unsaturated carboxylic acid. Likewise, the use of an α,β-unsaturated carboxylic acid derivative such as mono-methyl fumarate (ester) (C) gave the mononuclear [NbCp’2(OOC-CH2-CH2-COOMe-κ2O,O)] (3). When other α,β-unsaturated carboxylic acids with less activated alkenes were used, such as trans-cinnamic acid (F), (E)-2-methyl-3-phenylacrylic acid (G), cyclohex-1-ene-1-carboxylic acid (H), 3-methylbut-2-enoic acid (I), (E)-2-methylbut-2-enoic acid (J) or (E)-pent-2-enoic acid (K), only the complexes [NbCp’2(OOCR-κ2O,O)], (R = trans-CH=CHPh (4), trans-CMe = CHPh (5), 1-cyclohexenyl (6), (CH=CMe2) (7), (cis-CMe = CHMe) (8), (trans-C(H) = CH-Et) (9)), were isolated, respectively, resulting from a process with dihydrogen elimination and the hydrogenation of the CC bond do not takes place.Treatment of 2–9 with CO (3 atm) promote that the coordination mode of the carboxylate ligand changes from (κ2O,O-OOC-R) to (κ1O-OOC-R) and yielding new carbonyl derivatives [(NbCp’2)2(μ-κ1O-OOC-CH2-CH2-COO-κ1O) (CO)2] (10); and [NbCp’2(OOCR-κ1O) (CO)] (R = CH2-CH2-COOMe (11), trans-CH=CHPh (12), trans-CMe = CHPh (13), 1-cyclohexenyl (14), CH=CMe2 (15), cis-CMe = CHMe (16) and R = trans-CHCH-Et) (17)).Reaction of 2 with O2 afford a new diperoxo compound [{NbCp’2(O2-κ2O,O)}2 (μ−κ1O-OOC-CH2-CH2-COO-κ1O)](19), through a monoperoxo derivative [NbCp’2(O2-κ2O,O) (μ-κ1O-OOC-CH2-CH2-COO-κ2O,O)NbCp’2] (18). All the new complexes were characterized by usual IR and NMR spectroscopic methods and the crystal structure of 16 and 18 were determined by X-ray diffraction studies.

Monomethyl fumarate protects cerebral hemorrhage injury in rats via activating microRNA-139/Nrf2 axis.

Monomethyl fumarate (MF) exerts anti-inflammatory and antioxidant capacities. Whether microRNA-139 and nuclear factor (erythroid-derived 2)-like 2 (Nrf2) are involved in the pharmacological activity of MF remain unclear. We aim to elucidate the potential function of MF in intracerebral hemorrhage (ICH), and its possible mechanism. Twenty-four Sprague Dawley (SD) rats were randomly assigned into sham group, ICH group and MF group, with 8 rats in each group. Rats in ICH and MF group were subjected to ICH procedures. Rat brain tissues were harvested at 48 h after ICH procedures. Evans blue extravasation was performed to evaluate ICH-induced rat brain damage. Content of cerebral edema and neurological deficit were examined to reflect the neuronal pathological lesions. Reactive oxygen species (ROS) content in rat brain was examined by immunofluorescence. Activities of oxidative stress indexes in rat brain homogenate were detected using relative commercial kits. MicroRNA-139 expression in rat brain was quantified by quantitative Real-time polymerase chain reaction (qRT-PCR). Finally, protein levels of Nrf2, HO-1, NQO1 and nuclear factor-kappa B (NF-κB) in rat brain tissues were examined by Western blot. Compared with rats in sham group, neurological deficit scores of rats in ICH group were lower. Disruption of blood-brain barrier and brain tissue edema of rats were pronounced in ICH group. However, MF pretreatment markedly alleviated the above mentioned cerebral lesions. In addition, MF pretreatment increased activities of SOD, GSH and CAT, but decreased MDA and ROS contents in rat brain homogenate relative to those in ICH group (p<0.05). Western blot analysis found that expression levels of Nrf2, HO-1 and NQO-1 were markedly upregulated after MF pretreatment, while the expression level of NF-κB was downregulated. At the cellular level, we altered microRNA-139 expression in SH-SY5Y cells by transfection of microRNA-139 mimics or inhibitor. Overexpression of microRNA-139 remarkably increased Nrf2 expression and decreased NF-κB expression. Treatment of high-dose MF upregulated Nrf2, downregulated NF-κB and decreased ROS content in SH-SY5Y cells. MF protects ICH in rats by inhibiting oxidative stress and inflammatory response through activating microRNA-139/Nrf2 axis.

Monomethyl Fumarate Protects the Retina From Light-Induced Retinopathy.

PurposeWe determine if monomethyl fumarate (MMF) can protect the retina in mice subjected to light-induced retinopathy (LIR).MethodsAlbino BALB/c mice were intraperitoneally injected with 50 to 100 mg/kg MMF before or after exposure to bright white light (10,000 lux) for 1 hour. Seven days after light exposure, retinal structure and function were evaluated by optical coherence tomography (OCT) and electroretinography (ERG), respectively. Retinal histology also was performed to evaluate photoreceptor loss. Expression levels of Hcar2 and markers of microglia activation were measured by quantitative PCR (qPCR) in the neural retina with and without microglia depletion. At 24 hours after light exposure, retinal sections and whole mount retinas were stained with Iba1 to evaluate microglia status. The effect of MMF on the nuclear factor kB subunit 1 (NF-kB) and Nrf2 pathways was measured by qPCR and Western blot.ResultsMMF administered before light exposure mediated dose-dependent neuroprotection in a mouse model of LIR. A single dose of 100 mg/kg MMF fully protected retinal structure and function without side effects. Expression of the Hcar2 receptor and the microglia marker Cd14 were upregulated by LIR, but suppressed by MMF. Depleting microglia reduced Hcar2 expression and its upregulation by LIR. Microglial activation, upregulation of proinflammatory genes (Nlrp3, Caspase1, Il-1β, Tnf-α), and upregulation of antioxidative stress genes (Hmox1) associated with LIR were mitigated by MMF treatment.ConclusionsMMF can completely protect the retina from LIR in BALB/c mice. Expression of Hcar2, the receptor of MMF, is microglia-dependent in the neural retina. MMF-mediated neuroprotection was associated with attenuation of microglia activation, inflammation and oxidative stress in the retina.

Neuro-protective effect of monomethyl fumarate on ischemia reperfusion injury in rats: Role of Nrf2/HO1 pathway in peri-infarct region

Post stroke recanalization has been associated with increased risk of oxidative stress. Stimulating endogenous antioxidant pathway by activation of nuclear factor erythroid-2-related factor-2 (Nrf2) plays a key role in neuronal defense against inflammation and oxidative stress in penumbra. Here, we explored whether monomethyl fumarate (MMF) could produce neuro-protection after ischemia/reperfusion (I/R) injury via Nrf2/HO1 activation. In male SD rats, middle cerebral artery was occluded for 90 min and confirmed using Laser Doppler flowmeter. MMF (10, 20 and 40 mg/kg) was administered in two divided doses at 30 min post ischemia and 5–10 min after reperfusion. After 24 h, effect on neurobehavioral parameters, infarct damage by TTC staining and MRI, oxidative stress and inflammatory cytokines were assessed. Expression studies of nuclear Nrf2 and cytoplasmic HO1 were performed in peri-infarct cortex and striatum; followed by dual immunofluorescence study to check the specific cell type. I/R induced neurobehavioral deficits and infarct damage were significantly (p < 0.05) attenuated by MMF (20 and 40 mg/kg). MMF, 20 mg/kg, significantly normalized I/R induced altered redox status and increased levels of TNF-α, IL-1β in the ipsilateral cortex. MRI data showed significantly reduced infarct in cortex but not in striatum after MMF treatment. Expression of nuclear Nrf2 and cytoplasmic HO1 were significantly (p < 0.05) increased in peri-infarct cortex after treatment with MMF. Additionally, dual immunofluorescence showed increased Nrf2 expression in neurons and HO1 expression in neurons as well as astrocytes in peri-infarct cortex after MMF treatment. Our results show the neuro-protective potential of MMF probably by restricting the progression of damage from striatum to cortex through activation of Nrf2/HO1 pathway in peri-infarct cortex.

HCT-116 colorectal cancer cells secrete chemokines which induce chemoattraction and intracellular calcium mobilization in NK92 cells.

We recently reported that pretreatment of IL-2 activated human natural killer (NK) cells with the drugs dimethyl fumarate (DMF) and monomethyl fumarate (MMF) upregulated the expression of surface chemokine receptor CCR10. Ligands for CCR10, namely CCL27 and CCL28, induced the chemotaxis of these cells. Here, we performed a bioinformatics analysis to see which chemokines might be expressed by the human HCT-116 colorectal cancer cells. We observed that, in addition to CCL27 and CCL28, HCT-116 colorectal cancer cells profoundly express CXCL16 which binds CXCR6. Consequently, NK92 cells were treated with DMF and MMF for 24h to investigate in vitro chemotaxis towards CXCL16, CCL27, and CCL28. Furthermore, supernatants collected from HCT-116 cells after 24 or 48h incubation induced the chemotaxis of NK92 cells. Similar to their effects on human IL-2-activated NK cells, MMF and DMF enhanced the expression of CCR10 and CXCR6 in NK92 cells. Neutralizing anti-CXCL16 or anti-CCL28 inhibited the chemotactic effects of 24 and 48 supernatants, whereas anti-CCL27 only inhibited the 48h supernatant activity, suggesting that 24h supernatant contains CXCL16 and CCL28, whereas HCT-116 secretes all three chemokines after 48h in vitro cultures. CXCL16, CCL27, and CCL28, as well as the supernatants collected from HCT-116, induced the mobilization of (Ca)2+ in NK92 cells. Cross-desensitization experiments confirmed the results of the chemotaxis experiments. Finally, incubation of NK92 cells with HCT-116 induced the lysis of the tumor cells. In summary, these results might have important implications in directing the anti-tumor effectors NK cells towards tumor growth sites.

LC-MS Analysis of Dimethyl Fumarate in Rat plasma with Measurement Uncertainty Estimation

Dimethyl fumarate (DMF) is the methyl ester of fumaric acid initially recognized as a very effective hypoxic cell radio sensitizer. Phase III clinical trials found that DMF successfully reduced relapse rate and increased time to progression of disability in multiple sclerosis. Small molecules like dimethyl fumarate pose particular difficulties when analyzing biological samples due to the increased possibility of matrix effects and in this scenario DMF immediately converts to its active metabolite MMF (Monomethyl fumarate) by oral route. It is for this reason that a sensitive liquid chromatography-tandem mass spectrometry method has now been developed for the analysis of DMF and for studying the pharmacokinetic profile in rats. Sample preparation was by rapid protein precipitation with acetonitrile. Analyte separation was achieved on a reversed-phase XTerra MS C18 column (100 x 3.9 mm, 3.5g) with 0. 01M ammonium formate and acetonitrile in gradient mode as the mobile phase at a flow rate of 1.0 mL/min and analyzed by a hybrid triple-quadrupole linear ion trap mass spectrometer in positive electrospray ionization mode for both DMF and MMF. Limits of detection, and quantification were 20 and 50 ng/mL, for DMF and 1 and 10 ng/mL for MMF respectively. Calibration curve showed excellent linearity within the 50–2500 ng/mL range for DMF and 10–500 ng/mL range (r2 > 0.999) for MMF. Intra-and inter-day precision defined by coefficient of variation was <10% and accuracy (bias %) was within 90–110%. Measurement uncertainty estimation was 8.6% for DMF and 11.6% for MMF. The method has been successfully used in the analysis of DMF and MMF in rat plasma following its administration to male wistar rats for pharmacokinetic studies.

Dimethyl fumarate: Regulatory effects on the immune system in the treatment of multiple sclerosis.

Dimethyl fumarate (DMF) is an important oral treatment option for various autoimmune diseases, such as multiple sclerosis (MS) and psoriasis. DMF and its dynamic metabolite, monomethyl fumarate (MMF) are the major compounds that exert therapeutic effects on several pathologic conditions in part, through downregulation of immune responses. The exact mechanism of DMF is yet to be fully understood even though its beneficial effects on the immune system are extensively studied. It has been shown that DMF/MMF can affect various immune cells, which can get involved in both the naive and adaptive immune systems, such as T cells, B cells, dendritic cells, macrophages, neutrophils, and natural killer cells. It is suggested that DMF/MMF may exert their effect on immune cells through inhibition of nuclear factor-κB translocation, upregulation of nuclear factor erythroid-derived 2(E2)-related factor antioxidant pathway, and activation of hydroxyl carboxylic acid receptor 2. In this review, the mechanisms underlying the modulatory functions of DMF or MMF on the main immune cell populations involved in the immunopathogenesis of MS are discussed.

Monomethyl fumarate alleviates sepsis-induced hepatic dysfunction by regulating TLR-4/NF-κB signalling pathway

AimsSepsis is a potentially fatal illness that can lead to impairment of multiple organs such as liver. The condition is deeply associated with oxidative stress and inflammation. Monomethyl fumarate (MMF) has manifested antioxidant and immunomodulatory properties. The aim of current study was to evaluate protective effects of MMF in sepsis-induced hepatic dysfunction. Main methodsSepsis was induced by cecal ligation and puncture (CLP). Wistar rats were assigned to one of sham, CLP, CLP + dexamethasone (as positive control of inflammation) and CLP + MMF groups. Levels of serum IL-1β, IL-6, IL-10, AST, ALT and γ‑GT were quantified. Furthermore, Hepatic levels of GSH and MDA and mRNA expression of TNF and NFKBIA along with hepatic protein level of TLR-4 were assessed. Also, histopathological study of liver was carried out to evaluate hepatic injuries. Key findingsSeptic rats demonstrated risen levels of IL-1β, IL-6, IL-10, AST, ALT and γ‑GT, while treatment with dexamethasone or MMF attenuated these levels. Moreover, enhancements in protein level of TLR-4 and mRNA levels of TNF and NFKBIA were observed in CLP rats. These elevations were mitigated in CLP-induced rats that were treated with either dexamethasone or MMF. Treatment with dexamethasone or MMF also shifted sepsis-induced disturbance in the levels of GSH and MDA towards sham levels. Hepato-protective effects of dexamethasone and MMF were further confirmed by histopathological observations. SignificanceOur findings imply that MMF alleviates sepsis-induced hepatic dysfunction by mitigating the inflammatory and oxidative state and this effect is at least partly mediated by the inhibition of TLR-4/NF-κB signalling pathway.

Visible Light-Initiated Bioorthogonal Photoclick Cycloaddition.

Here we report a visible light-triggered, catalyst free bioorthogonal reaction that proceeds via a distinct pathway from reported bioorthogonal reactions. The prototype of this bioorthogonal reaction was the photocycloaddition of 9,10-phenanthrenequinone with electron-rich alkenes to form fluorogenic [4+2] cycloadducts. The bioorthogonal photoclick cycloaddition was readily initiated using a conventional visible light source such as a hand-held LED lamp. The reaction proceeded rapidly under biocompatible conditions, without observable competition from side reactions such as nucleophilic additions by water or common nucleophilic species. The bioorthogonal functionality in this reaction did not cross react with various alkynes and electron-deficient alkenes such as monomethyl fumarate. We demonstrated orthogonal labeling of two proteins using this reaction together with a strain promoting azide-alkyne click reaction or the UV-triggered reaction of tetrazole with monomethyl fumarate. The application of this reaction in the temporal and spatial labeling of live cells was also demonstrated.

The Immunometabolomic Interface Receptor Hydroxycarboxylic Acid Receptor 2 Mediates the Therapeutic Effects of Dimethyl Fumarate in Autoantibody-Induced Skin Inflammation.

The drug dimethyl fumarate (DMF) is in clinical use for the treatment of psoriasis and multiple sclerosis. In addition, it has recently been demonstrated to ameliorate skin pathology in mouse models of pemphigoid diseases, a group of autoimmune blistering diseases of the skin and mucous membranes. However, the mode of action of DMF in inflammatory skin diseases has remained elusive. Therefore, we have investigated here the mechanisms by which DMF improves skin pathology, using the antibody transfer model of bullous pemphigoid-like epidermolysis bullosa acquisita (EBA). Experimental EBA was induced by transfer of antibodies against collagen VII that triggered the infiltration of immune cells into the skin and led to inflammatory skin lesions. DMF treatment reduced the infiltration of neutrophils and monocytes into the skin explaining the improved disease outcome in DMF-treated animals. Upon ingestion, DMF is converted to monomethyl fumarate that activates the hydroxycarboxylic acid receptor 2 (HCA2). Interestingly, neutrophils and monocytes expressed Hca2. To investigate whether the therapeutic effect of DMF in EBA is mediated by HCA2, we administered oral DMF to Hca2-deficient mice (Hca2−/−) and wild-type littermates (Hca2+/+) and induced EBA. DMF treatment ameliorated skin lesions in Hca2+/+ but not in Hca2−/− animals. These findings demonstrate that HCA2 is a molecular target of DMF treatment in EBA and suggest that HCA2 activation limits skin pathology by inhibiting the infiltration of neutrophils and monocytes into the skin.

Abstract B26: Thermally abused frying oil enhances metastatic progression in vivo and in vitro: A link to elevated fumarate levels

Abstract For the past four decades, dietary fat intake has composed a significant portion of the typical Western diet, at 33-35% of total kilocalories. Approximately 8% of this dietary fat is from consumption of deep fat fried foods. Oil has been demonstrated to be absorbed by food during frying and contributes between 8 to 40% of the food’s final weight. As oils are used and reused for frying, complex chemical reactions take place creating deleterious polar compounds (e.g., acrylamide, sterol derivatives, or other reactive oxidative species.) Although these compounds are associated with health risks, i.e., heart disease, diabetes mellitus, and cancer, there is no regulation in the United States for upper limits of polar material in frying oil. This raises an important question as to whether thermally abused fryer oil (TAFO) can alter metastatic breast cancer progression. We initially conducted an in vivo study in which a modest amount of dietary fat, 25% of kilocalories, came from fresh fryer oil or TAFO. Ovariectomized BALB/C mice fed the diet containing TAFO for 16 weeks and inoculated with luciferase-expressing 4T1 murine mammary cancer cells had significantly more metastasis to lungs than control-fed animals (p &amp;lt; 0.05, n=21). Serum metabolic analysis in these animals revealed significantly elevated levels of fumarate, a known onco-metabolite, in TAFO-fed compared with control-fed animals (p &amp;lt; 0.05, n=9). To confirm that fumarate was mechanistically involved in metastatic progression, we conducted in vitro studies. 4T1 cells exposed to 500 nM to 10 µM monomethyl fumarate (MMF), a cell-permeable form of fumarate, trended toward an increase in the healing rate over 24 hours in a wound healing (scratch) assay compared to control (n =2). This preliminary evidence supports the hypothesis that fumarate accumulation creates a protumorigenic and metastatic environment and needs confirmation in subsequent investigation. This information can help elucidate mechanistic details of the effects of TAFO on breast cancer metastasis. Further work will assess rates of 4T1 cellular proliferation and invasion induced by MMF. In summary, dietary intake of TAFO increased metastasis of tumor cells to lungs, and dysregulation of the metabolite fumarate may be mechanistically involved. Citation Format: Jennifer R. Hughes, Kaitlyn M. Joyce, Anthony Cam, William G. Helferich, Zeynep Madak-Erdogan. Thermally abused frying oil enhances metastatic progression in vivo and in vitro: A link to elevated fumarate levels [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B26.

Dimethyl fumarate therapy suppresses B cell responses and follicular helper T cells in relapsing-remitting multiple sclerosis

Background: Dimethyl fumarate (DMF) is a disease-modifying therapy used for patients with relapsing-remitting multiple sclerosis (RRMS). B cells are important contributors to the pathogenesis of RRMS, where they regulate the inflammatory immune responses and participate in development of lesions in the central nervous system (CNS). The impact of DMF on B cell subpopulations remains incompletely understood. Objectives: In this study, we evaluated the effects of DMF on B cell subpopulations and their effector functions. Methods: Blood from 21 DMF-treated and 18 untreated patients with RRMS was analyzed by flow cytometry. Results: We found that DMF reduces the frequency of circulating antigen–experienced B cells, a reduction likely related to a reduced frequency of follicular helper T (TFH) cells and an increased frequency of follicular regulatory T (TFR) cells. Studying the impact of monomethyl fumarate (MMF), the primary metabolite of DMF, on B cell effector function in vitro showed that MMF increased the frequency of transforming growth factor (TGF)-β-producing B cells and decreased the frequency of B cells secreting lymphotoxin (LT)-α, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and to a lesser extent IL-10. Conclusion: In summary, these data suggest an anti-inflammatory role of DMF and its metabolite MMF on the B cell compartment.