Monosaccharide Analysis After One-Pot Derivatization Followed by Reverse-Phase Liquid Chromatography Separation and UV/Vis Detection.

Derivatization of monosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP) introduces two chromophores per sugar molecule. Their separation on a superficially porous C18 reverse-phase column, using common liquid chromatography equipment, results in short analysis times (under 20 min) and high sensitivity (limit of quantitation 1 nmol). This method allows for complex monosaccharide mixtures to be separated and quantified using a reasonably simple and safe derivatization procedure.

Discovery of Novel Hybrids of Edaravone and 6-Phenyl-4,5-dihydropyridazin-3(2H)-one with Antiplatelet Aggregation and Neuroprotection for Ischemic Stroke Treatment.

Drugs with anti-platelet aggregation and neuroprotection are of great significance for the treatment of ischemic stroke. A series of edaravone and 6-phenyl-4,5-dihydropyridazin-3(2H)-one hybrids were designed and synthesized. Among them, 6g showed the most effective cytoprotective effect against oxygen-glucose deprivation/reoxygenation-induced damage in BV2 cells and an excellent inhibitory effect on platelet aggregation induced by adenosine diphosphate and arachidonic acid. Additionally, 6g could prevent thrombosis caused by ferric chloride in rats and pose a lower risk of causing bleeding compared with aspirin. It provides better protection against ischemia/reperfusion injury in rats compared with edaravone and alleviates the oxidative stress related to cerebral ischemia/reperfusion by increasing the GSH and SOD levels and decreasing the MDA concentration. Finally, molecular docking results showed that 6g probably acts on PDE3 A and plays an anti-platelet aggregation effect. Overall, 6g could be a potential candidate compound for the treatment of ischemic stroke.

In silico studies reveal structural deviations of mutant profilin-1 and interaction with riluzole and edaravone in amyotrophic lateral sclerosis.

This study aimed to investigate four of the eight PFN-1 mutations that are located near the actin-binding domain and determine the structural changes due to each mutant and unravel how these mutations alter protein structural behavior. Swapaa's command in UCSF chimera for generating mutations, FTMAP were employed and the data was analyzed by RMSD, RMSF graphs, Rg, hydrogen bonding analysis, and RRdisMaps utilizing Autodock4 and GROMACS. The functional changes and virtual screening, structural dynamics, and chemical bonding behavior changes, molecular docking simulation with two current FDA-approved drugs for ALS were investigated. The highest reduction and increase in Rg were found to exist in the G117V and M113T mutants, respectively. The RMSF data consistently shows changes nearby to this site. The in silico data described indicate that each of the mutations is capable of altering the structure of PFN-1 in vivo. The potential effect of riluzole and edaravone two FDA approved drugs for ALS, impacting the structural deviations and stabilization of the mutant PFN-1 is evaluated using in silico tools. Overall, the analysis of data collected reveals structural changes of mutant PFN-1 protein that may explain the neurotoxicity and the reason(s) for possible loss and gain of function of PFN-1 in the neurotoxic model of ALS.

Design, synthesis and biological evaluation of edaravone derivatives bearing the N-benzyl pyridinium moiety as multifunctional anti-Alzheimer's agents.

A series of multi-target directed edaravone derivatives bearing N-benzyl pyridinium moieties were designed and synthesised. Edaravone is a potent antioxidant with significant neuroprotective effects and N-benzyl pyridinium has previously exhibited positive results as part of a dual-site binding, peripheral anionic site (PAS) and catalytic anionic site (CAS), acetylcholinesterase (AChE) inhibitor. The designed edaravone-N-benzyl pyridinium hybrid compounds were docked within the AChE active site. The results indicated interactions with conserved amino acids (Trp279 in PAS and Trp84 in CAS), suggesting good dual-site inhibitory activity. Significant in vitro AChE inhibitory activities were observed for selected compounds (IC50: 1.2-4.6 µM) with limited butyrylcholinesterase inhibitory activity (IC50's >160 µM), indicating excellent selectivity towards AChE (SI: 46 - >278). The compounds also showed considerable antioxidant ability, similar to edaravone. In silico studies indicated that these compounds should cross the blood-brain barrier, making them promising lead molecules in the development of anti-Alzheimer's agents.

Edaravone attenuates myocyte apoptosis through the JAK2/STAT3 pathway in acute myocardial infarction.

Basic and clinical studies have demonstrated that the free radical scavenger edaravone has cytoprotective effects on acute myocardial infarction (AMI) but the underlying mechanism is not fully understood. The aim of this research is to explore the effect of edaravone on the apoptotic process involving the JAK2/STAT3 signaling pathway. AMI in rats was established by left anterior descending coronary artery ligation. Two hours after AMI model established rats were treated with edaravone, edaravone plus AG490, physiological saline, respectively. We detected antioxidant effects by reduced glutathione (GSH), glutathione S-transferase (GST), and Glutathione Peroxidase (GSHPx) Activity. The expressions of t-JAK2, p-JAK2, t-STAT3, p-STAT3 and cleaved caspase-3 were examined by western blot. The mRNA levels for Bcl-2, Bax, Fas, and FasL were measured by RT-PCR and apoptosis was assessed by TUNEL. Edaravone significantly improved hemodynamics after AMI (p < 0.05) and reduced the total infarct volumes (p < 0.05). Compared with Sham rats, the mRNA of Bax, Fas, and FasL increased in different degrees in the AMI group, however, the mRNA of Bcl-2 and the ratio of Bcl-2/Bax decreased, especially the myocardial apoptosis index significantly increased in AMI hearts (all p < 0.05). After treatment with edaravone, the mRNA levels of Bcl-2 and the ratio of Bcl-2/Bax significantly upregulated whereas Bax, Fas, FasL apparently decreased, and the protein expressions of p-JAK2 and p-STAT3 dramatically increased (p < 0.05). In addition, cotreatment with JAK2 inhibitor AG490 abolished the effects of edaravone. We conclude that edaravone attenuated myocardial apoptosis induced by AMI via JAK2/STAT3 signaling pathway.

Chemical reactivity and uses of 1-phenyl-3-methyl-5-pyrazolone (PMP), also known as edaravone.

1-Phenyl-3-methyl-5-pyrazolone is a reagent, known as PMP, used to derivatize monosaccharides for the study of polysaccharides composition and structure, and for the dosage of carbohydrates in complex media. The same molecule is also known as edaravone, a drug approved for the treatment of stroke and amyotrophic lateral sclerosis. It is also a reactive molecule susceptible to form stable adducts with aromatic aldehydes, such as formylpterin and vanillin. In addition, the molecule serves as a scaffold to design of edaravone analogs and drug conjugates, with various pharmacological properties (antioxidant, anticancer, antiviral). We have analyzed the multiple usages of PMP/edaravone to highlight the reactivity of the molecule and its wide range of applications. This phenyl-pyrazolone compound, considered by many as a biochemical reagent and by other as a clinically useful drug, has not yet revealed the full extent of its capacities and benefits.

Development of a RP-HPLC method for determination of glucose in Shewanella oneidensis cultures utilizing 1-phenyl-3-methyl-5-pyrazolone derivatization.

A method was developed and validated for low-level detection of glucose. The method involves quantitation of glucose though derivitization with 1-phenyl-3-methyl-5-pyrazolone (PMP) and HPLC-DAD analysis. The developed method was found to be accurate and robust achieving detection limits as low as 0.09 nM. The applicability of the method was tested against microbial samples with glucose acting as a carbon fuel source. The method was shown to be able to accurately discriminate and quantify PMP-glucose derivatives within Shewanella oneidensis MR-1 samples. The method proved capable at examining glucose usage during the early hours of microbial growth, with detectable usage occurring as early as two hours. S. oneidensis cultures were found to grow more effectively in the presence of oxygen which coincided with more efficient glucose usage. Glucose usage further increased in the presence of competing electron acceptors. The rate at which S. oneidensis reached exponential growth was affected by the presence of ferric iron under microaerobic conditions. Such samples reached exponential growth approximately two hours sooner than aerobic samples.

Edaravone inhibits the conformational transition of amyloid-ß42: insights from molecular dynamics simulations.

Previous work has shown that edaravone inhibits fibrillogenesis of amyloid-ß protein (Aß). However, the detailed mechanism by which edaravone inhibits the conformational transition of the Aß42 monomer is not known at the molecular level. Here, explicit-solvent molecular dynamics (MD) simulations were coupled with molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method to address the issue. MD simulations confirmed that edaravone inhibits the conformational transition of the Aß42 monomer in a dose-dependent manner. It was found that the direct interactions between edaravone and Aß42 are responsible for its inhibiting effects. The analysis of binding free energy using the MM-PBSA method demonstrated that the nonpolar interactions provide favourable contributions (about -71.7 kcal/mol). Conversely, the polar interactions are unfavourable for the binding process. A total of 14 residues were identified as greatly contributing to the binding free energy between edaravone and the Aß42 monomer. In addition, the intra-peptide hydrophobic interactions were weakened and the salt bridge D23-K28 was interrupted by edaravone. Therefore, the conformational transition was inhibited. Our studies provide molecular-level insights into how edaravone molecules inhibit the conformational transition of the Aß42 monomer, which may be useful for designing amyloid inhibitors.Communicated by Ramaswamy H. Sarma.

Optimization of synthesis of carbohydrates and 1-phenyl-3-methyl-5-pyrazolone (PMP) by response surface methodology (RSM) for improved carbohydrate detection.

Reducing sugars can react with 1-phenyl-3-methyl-5-pyrazolone (PMP) to form sugar-PMP derivatives, which can be detected by HPLC-UV or HPLC-DAD due to their high UV absorbance at 248 nm. Six different sugars were synthesized with PMP with aid of response surface methodology (RSM), by which the parameters of the synthesis were designed within temperature ranged between 60 °C and 90 °C, and time from 60 to 180 min, respectively. Consequently, optimal conditions of the glucose (Glu)-, glucosamine (GluN)-, galactose (Gal)-, glucuronic acid (GluA), galacturonic acid (GalA) and glucose-6-phosphate (G6P-PMP) reactions were determined at 71 °C for 129 min, 73 °C for 96 min, 70 °C for 117 min, 75 °C for 151 min, 76 °C for 144 min, and 70 °C for 154 min, respectively. Experiments demonstrated that unique functional groups and delicate differences of carbohydrates' inner pH environment could significantly influence the sugar-PMP reactions. However, sugar stereoisomers did not have remarkable impacts on the reactions.

Development of an Extensive Linkage Library for Characterization of Carbohydrates.

The extensive characterization of glycosidic linkages in carbohydrates remains a challenge because of the lack of known standards and limitations in current analytical techniques. This study encompasses the construction of an extensive glycosidic linkage library built from synthesized standards. It includes an improved liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitation of glycosidic linkages derived from disaccharides, oligosaccharides, and polysaccharides present in complicated matrices. We present a method capable of the simultaneous identification of over 90 unique glycosidic linkages using ultrahigh-performance liquid chromatography coupled with triple quadrupole mass spectrometry (UHPLC/QqQ MS) operated in dynamic multiple reaction monitoring (dMRM) mode. To build the library, known monosaccharides commonly found in plants were subjected to partial methylation to yield partially derivatized species representing trisecting, bisecting, linear, and terminal structures. The library includes glycosidic linkage information for three hexoses (glucose, galactose, and mannose), three pentoses (xylose, arabinose, and ribose), two deoxyhexoses (fucose and rhamnose), and two hexuronic acids (glucuronic acid and galacturonic acid). The resulting partially methylated monosaccharides were then labeled with 1-phenyl-3-methyl-5-pyrazolone (PMP) followed by separation and analysis by UHPLC/dMRM MS. Validation of the synthesized standards was performed using disaccharide, oligosaccharide, and polysaccharide standards. Accuracy, reproducibility, and robustness of the method was demonstrated by analysis of xyloglucan (tamarind) and whole carrot root. The synthesized standards represent the most comprehensive group of carbohydrate linkages to date.

Myeloperoxidase-mediated oxidation of edaravone produces an apparent non-toxic free radical metabolite and modulates hydrogen peroxide-mediated cytotoxicity in HL-60 cells.

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H2O2) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 µM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV-Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 µM H2O2 or a flux of H2O2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H2O2-induced toxicity was ameliorated at high edaravone concentrations (100-200 µM). In contrast, low concentrations of edaravone (1-10 µM) exacerbated the H2O2-induced toxicity. However, the effect of edaravone at low concentration (0-10 µM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug.

Self-nanomicellizing solid dispersion of edaravone: part I - oral bioavailability improvement.

BACKGROUND: Edaravone (EDR) is known for its free radical scavenging, antiapoptotic, antinecrotic, and anticytokine effects in neurological and non-neurological diseases. It is currently available clinically as Radicava® and Radicut®, intravenous medications, recently approved for the treatment of amyotrophic lateral sclerosis and cerebral infarction. However, the oral use of EDR is still restricted by its poor oral bioavailability (BA) due to poor aqueous solubility, stability, rapid metabolism, and low permeability. The present study reports the development of novel EDR formulation (NEF) using self-nanomicellizing solid dispersion (SNMSD) strategy with the aim to enable its oral use. MATERIALS AND METHODS: The selection of a suitable carrier for the development of NEF was performed based on the miscibility study. The optimization of EDR-to-carrier ratio was conducted via kinetic solubility study after preparing SNMSDs using solvent evaporation technique. The drug-polymer carrier interaction and self-nanomicellizing properties of NEF were investigated with advanced characterization studies. In vitro permeation, metabolism, and dissolution study was carried out to examine the effect of the presence of a carrier on physico-chemical properties of EDR. Additionally, the dose-dependent pharmacokinetic study of NEF was conducted and compared with the EDR suspension. RESULTS: Soluplus® (SOL) as a carrier was selected based on the potential for improving aqueous solubility. The NEF containing EDR and SOL (1:5) resulted in the highest enhancement in aqueous solubility (17.53-fold) due to amorphization, hydrogen bonding interaction, and micellization. Moreover, the NEF demonstrated significant improvement in metabolism, permeability, and dissolution profile of EDR. Furthermore, the oral BA of NEF showed 10.2-, 16.1-, and 14.8-fold enhancement compared to EDR suspension at 46, 138, and 414 µmol/kg doses. CONCLUSION: The results demonstrated that SNMSD strategy could serve as a promising way to enhance EDR oral BA and NEF could be a potential candidate for the treatment of diseases in which oxidative stress plays a key role in their pathogenesis.

Self-nanomicellizing solid dispersion of edaravone: part II: in vivo assessment of efficacy against behavior deficits and safety in Alzheimer's disease model.

BACKGROUND: Alzheimer's disease (AD) is a devastating neurodegenerative disorder that lacks any disease-modifying drug for the prevention and treatment. Edaravone (EDR), an approved free radical scavenger, has proven to have potential against AD by targeting multiple key pathologies including amyloid-beta (Aß), tau phosphorylation, oxidative stress, and neuroinflammation. To enable its oral use, novel edaravone formulation (NEF) was previously developed. The aim of the present investigation was to evaluate safety and efficacy of NEF by using in vitro/in vivo disease model. MATERIALS AND METHODS: In vitro therapeutic potential of NEF over EDR was studied against the cytotoxicity induced by copper metal ion, H2O2 and Aß42 oligomer, and cellular uptake on SH-SY5Y695 amyloid-ß precursor protein (APP) human neuroblastoma cell line. For in vivo safety and efficacy assessment, totally seven groups of APP/PS1 (five treatment groups, one each as a basal and sham control) and one group of C57BL/6 mice as a positive control for behavior tests were used. Three groups were orally treated for 3 months with NEF at an equivalent dose of EDR 46, 138, and 414 µmol/kg, whereas one group was supplied with each Donepezil (5.27 µM/kg) and Soluplus (amount present in NEF of 414 µmol/kg dose of EDR). Behavior tests were conducted to assess motor function (open-field), anxiety-related behavior (open-field), and cognitive function (novel objective recognition test, Y-maze, and Morris water maze). For the safety assessment, general behavior, adverse effects, and mortality were recorded during the treatment period. Moreover, biochemical, hematological, and morphological parameters were determined. RESULTS: Compared to EDR, NEF showed superior cellular uptake and neuroprotective effect in SH-SY5Y695 APP cell line. Furthermore, it showed nontoxicity of NEF up to 414 µM/kg dose of EDR and its potential to reverse AD-like behavior deficits of APP/PS1 mice in a dose-dependent manner. CONCLUSION: Our results indicate that oral delivery of NEF holds a promise as a safe and effective therapeutic agent for AD.

Identification of the primary determining factor(s) governing the oral absorption of edaravone in rats.

This study was performed to determine the primary factor(s) governing the oral absorption of edaravone, a novel anti-oxidant for the treatment of amyotrophic lateral sclerosis, in rats. While the aqueous solubility of edaravone widely varied depending on the vehicle used, the oral bioavailability of the drug was not low when it was adequately solubilized, as evidenced by the fact that the oral exposure was high (in terms of the absolute bioavailability of 50-90%) at all dose ranges (i.e., 0.5-27 mg/kg) under solubilized conditions in rats. The sum of the in vitro clearance values for edaravone, 12.7 mL/(min × kg), obtained from metabolic stability studies with tissue-homogenates from the rat liver, kidney, intestine, and with the rat plasma, was found to be virtually identical to the systemic clearance of the drug in rats. It was noted that the liver represented over 83.9% of the total elimination with a hepatic extraction ratio of approximately 0.137, indicative of the minor role of hepatic first pass metabolism in the systemic absorption of edaravone after its oral administration. In studies with Ussing chamber with rat intestinal segments and Madin-Darby canine kidney (MDCKII) cells, edaravone was found to be highly permeable (i.e., Papp over 10 × 10-6 cm/s), and appeared to be a substrate for rat P-glycoprotein (P-gp; estimated Km of 421 µM). In contrast, however, the drug did not appear to be a substrate for human P-gp in transport studies with MDCKII-hMDR1 cells. Collectively, these observations suggest that the primary determining factor for the intestinal absorption of edaravone is its solubilization in vehicle/intestinal fluids, rather than permeability, pre-systemic first-pass metabolism, or efflux transport. Considering the fact that the newly approved indication of the drug would require prolonged administration, probably via oral administration, the findings reported herein provide relevant information regarding its use.

Simultaneous Release and Labeling of O- and N-Glycans Allowing for Rapid Glycomic Analysis by Online LC-UV-ESI-MS/MS.

Most glycoproteins and biological protein samples undergo both O- and N-glycosylation, making characterization of their structures very complicated and time-consuming. Nevertheless, to fully understand the biological functions of glycosylation, both the glycosylation forms need to be analyzed. Herein we report a versatile, convenient one-pot method in which O- and N-glycans are simultaneously released from glycoproteins and chromogenically labeled in situ and thus available for further characterization. In this procedure, glycoproteins are incubated with 1-phenyl-3-methyl-5-pyrazolone (PMP) in aqueous ammonium hydroxide, making O-glycans released from protein backbones by ß-elimination and N-glycans liberated by alkaline hydrolysis. The released glycans are promptly derivatized with PMP in situ by Knoevenagel condensation and Michael addition, with peeling degradation almost completely prevented. The recovered mixture of O- and N-glycans as bis-PMP derivatives features strong ultraviolet (UV) absorbing ability and hydrophobicity, allowing for high-resolution chromatographic separation and high-sensitivity spectrometric detection. Using this technique, O- and N-glycans were simultaneously prepared from some model glycoproteins and complex biological samples, without significant peeling, desialylation, deacetylation, desulfation or other side-reactions, and then comprehensively analyzed by online HILIC-UV-ESI-MS/MS and RP-HPLC-UV-ESI-MS/MS, with which some novel O- and N-glycan structures were first found. This method provides a simple, versatile strategy for high-throughput glycomics analysis.