Chemogenetic activation of mammalian brain neurons expressing insect Ionotropic Receptors by systemic ligand precursor administration.

Chemogenetic approaches employing ligand-gated ion channels are advantageous regarding manipulation of target neuronal population functions independently of endogenous second messenger pathways. Among them, Ionotropic Receptor (IR)-mediated neuronal activation (IRNA) allows stimulation of mammalian neurons that heterologously express members of the insect chemosensory IR repertoire in response to their cognate ligands. In the original protocol, phenylacetic acid, a ligand of the IR84a/IR8a complex, was locally injected into a brain region due to its low permeability of the blood-brain barrier. To circumvent this invasive injection, we sought to develop a strategy of peripheral administration with a precursor of phenylacetic acid, phenylacetic acid methyl ester, which is efficiently transferred into the brain and converted to the mature ligand by endogenous esterase activities. This strategy was validated by electrophysiological, biochemical, brain-imaging, and behavioral analyses, demonstrating high utility of systemic IRNA technology in the remote activation of target neurons in the brain.

Phenylacetic acid, an anti-vaginitis metabolite produced by the vaginal symbiotic bacterium Chryseobacterium gleum.

The human microbiome contains genetic information that regulates metabolic processes in response to host health and disease. While acidic vaginal pH is maintained in normal conditions, the pH level increases in infectious vaginitis. We propose that this change in the vaginal environment triggers the biosynthesis of anti-vaginitis metabolites. Gene expression levels of Chryseobacterium gleum, a vaginal symbiotic bacterium, were found to be affected by pH changes. The distinctive difference in the metabolic profiles between two C. gleum cultures incubated under acidic and neutral pH conditions was suggested to be an anti-vaginitis molecule, which was identified as phenylacetic acid (PAA) by spectroscopic data analysis. The antimicrobial activity of PAA was evaluated in vitro, showing greater toxicity toward Gardnerella vaginalis and Candida albicans, two major vaginal pathogens, relative to commensal Lactobacillus spp. The activation of myeloperoxidase, prostaglandin E2, and nuclear factor-κB, and the expression of cyclooxygenase-2 were reduced by an intravaginal administration of PAA in the vaginitis mouse model. In addition, PAA displayed the downregulation of mast cell activation. Therefore, PAA was suggested to be a messenger molecule that mediates interactions between the human microbiome and vaginal health.

Genome analysis and hyphal movement characterization of the hitchhiker endohyphal Enterobacter sp. from Rhizoctonia solani.

Bacterial-fungal interactions are pervasive in the rhizosphere. While an increasing number of endohyphal bacteria have been identified, little is known about their ecology and impact on the associated fungal hosts and the surrounding environment. In this study, we characterized the genome of an Enterobacter sp. Crenshaw (En-Cren), which was isolated from the generalist fungal pathogen Rhizoctonia solani, and examined the genetic potential of the bacterium with regard to the phenotypic traits associated with the fungus. Overall, the En-Cren genome size was typical for members of the genus and was capable of free-living growth. The genome was 4.6 MB in size, and no plasmids were detected. Several prophage regions and genomic islands were identified that harbor unique genes in comparison with phylogenetically closely related Enterobacter spp. Type VI secretion system and cyanate assimilation genes were identified from the bacterium, while some common heavy metal resistance genes were absent. En-Cren contains the key genes for indole-3-acetic acid (IAA) and phenylacetic acid (PAA) biosynthesis, and produces IAA and PAA in vitro, which may impact the ecology or pathogenicity of the fungal pathogen in vivo. En-Cren was observed to move along hyphae of R. solani and on other basidiomycetes and ascomycetes in culture. The bacterial flagellum is essential for hyphal movement, while other pathways and genes may also be involved.IMPORTANCEThe genome characterization and comparative genomics analysis of Enterobacter sp. Crenshaw provided the foundation and resources for a better understanding of the ecology and evolution of this endohyphal bacteria in the rhizosphere. The ability to produce indole-3-acetic acid and phenylacetic acid may provide new angles to study the impact of phytohormones during the plant-pathogen interactions. The hitchhiking behavior of the bacterium on a diverse group of fungi, while inhibiting the growth of some others, revealed new areas of bacterial-fungal signaling and interaction, which have yet to be explored.

Advances in 4-Hydroxyphenylacetate-3-hydroxylase Monooxygenase.

Catechols have important applications in the pharmaceutical, food, cosmetic, and functional material industries. 4-hydroxyphenylacetate-3-hydroxylase (4HPA3H), a two-component enzyme system comprising HpaB (monooxygenase) and HpaC (FAD oxidoreductase), demonstrates significant potential for catechol production because it can be easily expressed, is highly active, and exhibits ortho-hydroxylation activity toward a broad spectrum of phenol substrates. HpaB determines the ortho-hydroxylation efficiency and substrate spectrum of the enzyme; therefore, studying its structure-activity relationship, improving its properties, and developing a robust HpaB-conducting system are of significance and value; indeed, considerable efforts have been made in these areas in recent decades. Here, we review the classification, molecular structure, catalytic mechanism, primary efforts in protein engineering, and industrial applications of HpaB in catechol synthesis. Current trends in the further investigation of HpaB are also discussed.

Pharmacokinetics, safety, and tolerability of sodium phenylacetate and sodium benzoate in healthy Japanese volunteers: A phase I, single-center, open-label study.

TAK-123, a combination of sodium phenylacetate (NaPA) and sodium benzoate (NaBZ), is an intravenously administered drug developed for the treatment of acute hyperammonemia in infants, children, and adults with urea cycle enzyme deficiencies. The aim of the current study was to evaluate the pharmacokinetics, safety, and tolerability after intravenous infusion of TAK-123 in Japanese healthy adult volunteers. Ten volunteers received a 3.75 g/m2 loading dose of TAK-123 over a period of 1.5 h followed by a maintenance infusion of the same dose over 24 h. Phenylacetate (PA) and benzoate (BZ) and their respective metabolites, phenylacetylglutamine (PAG) and hippurate (HIP) were measured over a 24-h period using a high-performance liquid chromatography/tandem mass spectrometry method. Non-compartmental analysis was performed using WinNonlin® Professional. During the loading dose, plasma levels of both PA and BZ peaked at 1.5 h. Plasma PA levels plateaued and were maintained up to 6.5 h, whereas plasma BZ levels declined rapidly after switching to maintenance infusion. Urinary excretion ratios of PAG and HIP at 48 h after the administration were 99.3% and 104%, respectively, suggesting that almost all NaPA and NaBZ were metabolized and excreted into urine. Overall, TAK-123 was well-tolerated in healthy Japanese adults.

Biodegradation of MC-LR and its key bioactive moiety Adda by Sphingopyxis sp. YF1: Comprehensive elucidation of the mechanisms and pathways.

Microcystins (MCs) are harmful to the ecology and public health. Some bacteria can degrade MCs into Adda, but few can destroy Adda. Adda is the key bioactive moiety of MCs and mainly contributes to hepatotoxicity. We had previously isolated an indigenous novel bacterial strain named Sphingopyxis sp. YF1 that can efficiently degrade MCs and its key bioactive moiety Adda, but the mechanisms remained unknown. Here, the biodegradation mechanisms and pathways of Adda were systematically investigated using multi-omics analysis, mass spectrometry and heterologous expression. The transcriptomic and metabolomic profiles of strain YF1 during Adda degradation were revealed for the first time. Multi-omics analyses suggested that the fatty acid degradation pathway was enriched. Specifically, the expression of genes encoding aminotransferase, beta oxidation (ß-oxidation) enzymes and phenylacetic acid (PAA) degradation enzymes were significantly up-regulated during Adda degradation. These enzymes were further proven to play important roles in the biodegradation of Adda. Simultaneously, some novel potential degradation products of Adda were identified successfully, including 7­methoxy-4,6-dimethyl-8-phenyloca-2,4-dienoic acid (C17H22O3), 2-methyl-3­methoxy-4-phenylbutyric acid (C12H16O3) and phenylacetic acid (PAA, C8H8O2). In summary, the Adda was converted into PAA through aminotransferase and ß-oxidation enzymes, then the PAA was further degraded by PAA degradation enzymes, and finally to CO2 via the tricarboxylic acid cycle. This study comprehensively elucidated the novel MC-LR biodegradation mechanisms, especially the new enzymatic pathway of Adda degradation. These findings provide a new perspective on the applications of microbes in the MCs polluted environment.

The antibacterial mechanism of phenylacetic acid isolated from Bacillus megaterium L2 against Agrobacterium tumefaciens.

Background: Agrobacterium tumefaciens T-37 can infect grapes and other fruit trees and cause root cancer. Given the pollution and damage of chemical agents to the environment, the use of biological control has become an important area of focus. Bacillus megaterium L2 is a beneficial biocontrol strain isolated and identified in the laboratory, which has a good antibacterial effect on a variety of plant pathogens. The antibacterial metabolites of L2 were separated and purified to obtain a bioactive compound phenylacetic acid (PAA). Methods: The potential antibacterial mechanism of PAA against A. tumefaciens T-37 strain was determined by relative conductivity, leakage of nucleic acids, proteins, and soluble total sugars, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and reactive oxygen species (ROS). Results: PAA showed good antibacterial activity against strain A. tumefaciens T-37 with IC50 of 0.8038 mg/mL. Our data suggested that after treatment with PAA, the relative conductivity, nucleic acid, protein, and total soluble sugar of T-37 were increased significantly compared with the chloramphenicol treatment group and the negative treatment group. The total protein synthesis of T-37 cells was inhibited, the consumption of phosphorus decreased with the increase of incubation time, and the content of ROS was significantly higher than that in the negative treatment group. Meanwhile, the activity of two key enzymes (MDH and SDH) involved in the tricarboxylic acid cycle (TCA cycle) decreased. In addition, T-37 cells were found to be damaged by scanning electron microscopy observation. Our results showed that PAA can destroy cell membrane integrity, damage cell structures, affect cell metabolism, and inhibit protein synthesis to exert an antibacterial effect. Conclusions: We concluded that the mechanism of action of the PAA against strain T-37 might be described as PAA exerting antibacterial activity by affecting cell metabolism, inhibiting protein synthesis, and destroying cell membrane integrity and cell ultrastructure. Therefore, PAA has a promising application prospect in the prevention and treatment of root cancer disease caused by A. tumefaciens.

Non-target metabolomics reveals the changes of small molecular substances in duck breast meat under different preservation time.

Poultry products are an essential animal source of protein for humans. Many factors could destroy the balance of the poultry production chain and cause an overstock of products, which need to be stored in the frozen storage warehouse for a long time. The long-term frozen storage may affect the quality of meat products. In this study, the changes of small molecular substances were revealed in duck meat during long-term storage using non-targeted metabolomics. The results showed that compared with fresh meat, even if the meat is stored under frozen storage conditions, the number of differential metabolites of frozen storage meat continues to increase with the prolongation of storage time, indicating that the meat composition has changed significantly with the storage time increased. With the increase in storage time, the nitrogen-containing small molecular compounds in duck meat increased (carnosine and anserine, aspartic acid, and tyrosine, 1H-indole-3-acetamide, 2-Hydroxyphenethylamine, 2-Naphylamine, allocystathionine, and O-phosphoethanolamine), the nucleotides decomposition process strengthened (IMP and AMP, GMP and UMP), and the content of organic acid increased (5-hydroxy indole acetic acid, 5-hydroxypentanoic acid and phenylacetate, taurine) and carbohydrate (1-O-sinapoyl-beta-d-glucose, 4-O-beta-d-glucopyranosyl-d-mannose, and alpha-d-glucose). These small molecular substances can be used as biomarkers to detect long-term stored duck meat deterioration. KEGG enrichment analysis showed that protein catabolism, nucleotide catabolism, fat decomposition and oxidation, and carbohydrate decomposition were the main metabolic processes of meat deterioration during the long-term storage of duck meat. In addition, Non-target metabolome technology is a powerful tool to reveal the meat deterioration process during long-term storage systematically. This study provided a reference for optimizing domestic poultry meat storage methods and ensuring food safety.

Isophthalate:coenzyme A ligase initiates anaerobic degradation of xenobiotic isophthalate.

BACKGROUND: Environmental contamination from synthetic plastics and their additives is a widespread problem. Phthalate esters are a class of refractory synthetic organic compounds which are widely used in plastics, coatings, and for several industrial applications such as packaging, pharmaceuticals, and/or paints. They are released into the environment during production, use and disposal, and some of them are potential mutagens and carcinogens. Isophthalate (1,3-benzenedicarboxylic acid) is a synthetic chemical that is globally produced at a million-ton scale for industrial applications and is considered a priority pollutant. Here we describe the biochemical characterization of an enzyme involved in anaerobic degradation of isophthalate by the syntrophically fermenting bacterium Syntrophorhabdus aromaticivorans strain UI that activate isophthalate to isophthalyl-CoA followed by its decarboxylation to benzoyl-CoA. RESULTS: Isophthalate:Coenzyme A ligase (IPCL, AMP-forming) that activates isophthalate to isophthalyl-CoA was heterologously expressed in E. coli (49.6 kDa) for biochemical characterization. IPCL is homologous to phenylacetate-CoA ligase that belongs to the family of ligases that form carbon-sulfur bonds. In the presence of coenzyme A, Mg2+ and ATP, IPCL converts isophthalate to isophthalyl-CoA, AMP and pyrophosphate (PPi). The enzyme was specifically induced after anaerobic growth of S. aromaticivorans in a medium containing isophthalate as the sole carbon source. Therefore, IPCL exhibited high substrate specificity and affinity towards isophthalate. Only substrates that are structurally related to isophthalate, such as glutarate and 3-hydroxybenzoate, could be partially converted to the respective coenzyme A esters. Notably, no activity could be measured with substrates such as phthalate, terephthalate and benzoate. Acetyl-CoA or succinyl-CoA did not serve as CoA donors. The enzyme has a theoretical pI of 6.8 and exhibited optimal activity between pH 7.0 to 7.5. The optimal temperature was between 25 °C and 37 °C. Denaturation temperature (Tm) of IPCL was found to be at about 63 °C. The apparent KM values for isophthalate, CoA, and ATP were 409 µM, 642 µM, and 3580 µM, respectively. Although S. aromaticivorans is a strictly anaerobic bacterium, the enzyme was found to be oxygen-insensitive and catalysed isophthalyl-CoA formation under both anoxic and oxic conditions. CONCLUSION: We have successfully cloned the ipcl gene, expressed and characterized the corresponding IPCL enzyme, which plays a key role in isophthalate activation that initiates its activation and further degradation by S. aromaticivorans. Its biochemical characterization represents an important step in the elucidation of the complete degradation pathway of isophthalate.

Design, synthesis, and evaluation of phenylpiperazine-phenylacetate derivatives as rapid recovery hypnotic agents.

In this paper, we designed and synthesized a series of novel phenylpiperazine-phenylacetate derivatives as rapid recovery hypnotic agents. The best compound 10 had relatively high affinity for the GABAA receptor and low affinity for thirteen other off-target receptors. In three animal models (mice, rats, and rabbits), compound 10 exerted potent hypnotic effects (HD50 = 5.2 mg/kg in rabbits), comparable duration of the loss of righting reflex (LORR), and significant shorter recovery time (time to walk) than propanidid. Furthermore, compound 10 (TI = 18.1) showed higher safety profile than propanidid (TI = 14.7) in rabbits. Above results suggested that compound 10 may have predictable and rapid recovery profile in anesthesia.

Ottowia caeni sp. nov., a novel phenylacetic acid degrading bacterium isolated from sludge.

A novel bacterium, designated BD-1T, was isolated from a sludge sample. Cells of the novel Gram-stain-negative strain were identified to be facultative anaerobic, non-motile and short rod-shaped. Growth occurred at 15-37 °C (optimum, 30 °C), pH 5.0-10.0 (pH 7.0) and in 0-4.0  % NaCl (2.0 %, w/v). The 16S rRNA gene sequence of strain BD-1T showed the highest sequence similarity to Ottowia thiooxydans DSM 14619T (97.0 %), followed by Ottowia pentelensis DSM 21699T (96.3 %) and less than 96 % to other related strains. The phylogenetic trees revealed that strain BD-1T clustered within the genus Ottowia. Summed feature 3 (C16 : 1 ω7c and/or C16 : 1 ω6c, 48.2 %), C16 : 0 (23.2 %) and summed feature 8 (C18 : 1 ω7c and/or C18 : 1 ω6c, 8.6 %) were the major fatty acids (>5 %), and ubiquinone-8 was the respiratory quinone. Phosphatidylethanolamine, phosphatidylmethylethanolamine and phosphatidylglycerol were identified as the major polar lipids. Meanwhile, the G+C content of the DNA was 63.6 mol% based on the draft genome analysis. The average nucleotide identity and digital DNA-DNA hybridization values between strain BD-1T and DSM 14619T were 74.5 and 21.4  %, respectively. In addition, the novel strain completely degraded 500 mg l-1 phenylacetic acid within 72 h under the condition of 3 % NaCl. Given the results of genomic, phylogenetic, phenotypic and chemotaxonomic analyses, strain BD-1T was considered to represent a novel species of the genus Ottowia, for which the name Ottowia caeni sp. nov. is proposed. The strain is a potential resource for the bioremediation of phenylacetic acid contaminated water. The type strain is BD-1T (=CGMCC 1.18541T=KCTC 82183T).

Production of p-cresol by Decarboxylation of p-HPA by All Five Lineages of Clostridioides difficile Provides a Growth Advantage.

Clostridioides difficile is the leading cause of antibiotic-associated diarrhea and is capable of causing severe symptoms, such as pseudomembranous colitis and toxic megacolon. An unusual feature of C. difficile is the distinctive production of high levels of the antimicrobial compound para-cresol. p-Cresol production provides C. difficile with a competitive colonization advantage over gut commensal species, in particular, Gram-negative species. p-Cresol is produced by the conversion of para-hydroxyphenylacetic acid (p-HPA) via the actions of HpdBCA decarboxylase coded by the hpdBCA operon. Host cells and certain bacterial species produce p-HPA; however, the effects of p-HPA on the viability of C. difficile and other gut microbiota are unknown. Here we show that representative strains from all five C. difficile clades are able to produce p-cresol by two distinct mechanisms: (i) via fermentation of p-tyrosine and (ii) via uptake and turnover of exogenous p-HPA. We observed strain-specific differences in p-cresol production, resulting from differential efficiency of p-tyrosine fermentation; representatives of clade 3 (CD305) and clade 5 (M120) produced the highest levels of p-cresol via tyrosine metabolism, whereas the toxin A-/B+ isolate from clade 4 (M68) produced the lowest level of p-cresol. All five lineages share at least 97.3% homology across the hpdBCA operon, responsible for decarboxylation of p-HPA to p-cresol, suggesting that the limiting step in p-cresol production may result from tyrosine to p-HPA conversion. We identified that elevated intracellular p-HPA, modulated indirectly via CodY, controls p-cresol production via inducing the expression of HpdBCA decarboxylase ubiquitously in C. difficile populations. Efficient turnover of p-HPA is advantageous to C. difficile as p-HPA has a deleterious effect on the growth of C. difficile and other representative Gram-negative gut bacteria, transduced potentially by the disruption of membrane permeability and release of intracellular phosphate. This study provides insights into the importance of HpdBCA decarboxylase in C. difficile pathogenesis, both in terms of p-cresol production and detoxification of p-HPA, highlighting its importance to cell survival and as a highly specific therapeutic target for the inhibition of p-cresol production across C. difficile species.

The regulation of BbLaeA on the production of beauvericin and bassiatin in Beauveria bassiana.

Beauvericin and bassiatin are two valuable compounds with various bioactivities biosynthesized by the supposedly same nonribosomal peptide synthetase BbBEAS in entomopathogenic fungus Beauveria bassiana. To evaluate the regulatory effect of global regulator LaeA on their production, we constructed BbLaeA gene deletion and overexpression mutants, respectively. Deletion of BbLaeA resulted in a decrease of the beauvericin titer, while overexpression of BbLaeA increased its production by 1-2.26 times. No bassiatin could be detected in ΔBbLaeA and wild type strain of B. bassiana, but 4.26-5.10 µg/mL bassiatin was produced in OE::BbLaeA. Furthermore, additional metabolites with increased production in OE::BbLaeA were isolated and identified as primary metabolites. Among them, 4-hydroxyphenylacetic acid showed antibacterial bioactivity against Ralstonia solanacearum. These results indicated that BbLaeA positively regulates the production of beauvericin, bassiatin and various bioactive primary metabolites.

Vulpinic acid as a natural compound inhibits the proliferation of metastatic prostate cancer cells by inducing apoptosis.

BACKGROUND: Lichen secondary metabolites have drawn considerable attention in recent years due to the limitations of current treatment options. Vulpinic acid (VA) obtained from Letharia vulpina lichen species exerts a remarkable cytotoxic effect on different cancer types. However, the therapeutic efficacy of VA in metastatic prostate cancer (mPC) cells has not been investigated. In the present study, we aimed to identify VA-mediated cytotoxicity in PC-3 mPC cells compared with control cells. METHODS AND RESULTS: After identifying the cytotoxic concentrations of VA, VA induced apoptosis was analyzed by Annexin V, cell cycle, acridine orange and propidium iodide staining and RT-PCR analysis. Our findings showed that VA significantly decreased the viability of PC-3 cells (p < 0.01) and caused a considerable early apoptotic effects through G0/G1 arrest, nuclear blebbing and the activation of particularly initiator caspases. CONCLUSIONS: Therefore, VA may be a potential treatment option for mPC patients. However, the underlying molecular mechanisms of VA-induced apoptosis with advanced analysis should be further investigated.

A surgical method for continuous intraportal infusion of gut microbial metabolites in mice.

Gut microbe-derived metabolites influence human physiology and disease. However, establishing mechanistic links between gut microbial metabolites and disease pathogenesis in animal models remains challenging. The major route of absorption for microbe-derived small molecules is venous drainage via the portal vein to the liver. In the event of presystemic hepatic metabolism, the route of metabolite administration becomes critical. To our knowledge, we describe here a novel portal vein cannulation technique using a s.c. implanted osmotic pump to achieve continuous portal vein infusion in mice. We first administered the microbial metabolite trimethylamine (TMA) over 4 weeks, during which increased peripheral plasma levels of TMA and its host liver-derived cometabolite, trimethylamine-N-oxide, were observed when compared with a vehicle control. Next, 4-hydroxyphenylacetic acid (4-HPAA), a microbial metabolite that undergoes extensive presystemic hepatic metabolism, was administered intraportally to examine effects on hepatic gene expression. As expected, hepatic levels of 4-HPAA were elevated when compared with the control group while peripheral plasma 4-HPAA levels remained the same. Moreover, significant changes in the hepatic transcriptome were revealed by an unbiased RNA-Seq approach. Collectively, to our knowledge this work describes a novel method for administering gut microbe-derived metabolites via the portal vein, mimicking their physiologic delivery in vivo.

In vitro gastrointestinal digestion and colonic fermentation of tomato (Solanum lycopersicum L.) and husk tomato (Physalis ixocarpa Brot.): Phenolic compounds released and bioconverted by gut microbiota.

Two of the most important Mexican plant-foods are tomato (Solanum lycopersicum L.) and husk tomato (Physalis ixocarpa Brot.). In this study three objectives were followed: i) to evaluate the bioaccessible phenolic compounds (PC) in T and HT during upper gastrointestinal digestion, ii) to in vitro ferment the indigestible fractions of the samples to evaluate the short-chain fatty acids (SCFA) production, iii) the microbial metabolites, bioconverted PC and volatile organic compounds (VOCs) generated during the fermentation. Vanillic acid was the most bioaccessible PC and after 48 h, 3-hydroxyphenylacetic acid was the most abundant microbial metabolite identified in both samples. The identification of VOCs belonging to terpenes (and derivatives) group in T and HT can be product of the microbial metabolism of carotenoids. The study shows new knowledge of the in vitro intestinal digestion and fermentation of T and HT final compounds with biological potential which should be evaluated in further studies.

An electrochemical method for detecting the biomarker 4-HPA by allosteric activation of Acinetobacterbaumannii reductase C1 subunit.

The C1 (reductase) subunit of 4-hydroxy-phenylacetate (4-HPA) 3-hydroxylase (HPAH) from the soil-based bacterium Acinetobacterbaumannii catalyzes NADH oxidation by molecular oxygen, with hydrogen peroxide as a by-product. 4-HPA is a potent allosteric modulator of C1, but also a known urinary biomarker for intestinal bacterial imbalance and for some cancers and brain defects. We thus envisioned that C1 could be used to facilitate 4-HPA detection. The proposed test protocol is simple and in situ and involves addition of NADH to C1 in solution, with or without 4-HPA, and direct acquisition of the H2O2 current with an immersed Prussian Blue-coated screen-printed electrode (PB-SPE) assembly. We confirmed that cathodic H2O2 amperometry at PB-SPEs is a reliable electrochemical assay for intrinsic and allosterically modulated redox enzyme activity. We further validated this approach for quantitative NADH electroanalysis and used it to evaluate the activation of NADH oxidation of C1 by 4-HPA and four other phenols. Using 4-HPA, the most potent effector, allosteric activation of C1 was related to effector concentration by a simple saturation function. The use of C1 for cathodic biosensor analysis of 4-HPA is the basis of the development of a simple and affordable clinical routine for assaying 4-HPA in the urine of patients with a related disease risk. Extension of this principle to work with other allosteric redox enzymes and their effectors is feasible.

Pharmacokinetics, mass balance, and metabolism of [14C]vicagrel, a novel irreversible P2Y12 inhibitor in humans.

Vicagrel, a novel irreversible P2Y12 receptor inhibitor, is undergoing phase III trials for the treatment of acute coronary syndromes in China. In this study, we evaluated the pharmacokinetics, mass balance, and metabolism of vicagrel in six healthy male Chinese subjects after a single oral dose of 20 mg [14C]vicagrel (120 µCi). Vicagrel absorption was fast (Tmax = 0.625 h), and the mean t1/2 of vicagrel-related components was ~38.0 h in both plasma and blood. The blood-to-plasma radioactivity AUCinf ratio was 0.55, suggesting preferential distribution of drug-related material in plasma. At 168 h after oral administration, the mean cumulative excreted radioactivity was 96.71% of the dose, including 68.03% in urine and 28.67% in feces. A total of 22 metabolites were identified, and the parent vicagrel was not detected in plasma, urine, or feces. The most important metabolic spot of vicagrel was on the thiophene ring. In plasma pretreated with the derivatization reagent, M9-2, which is a methylated metabolite after thiophene ring opening, was the predominant drug-related component, accounting for 39.43% of the radioactivity in pooled AUC0-8 h plasma. M4, a mono-oxidation metabolite upon ring-opening, was the most abundant metabolite in urine, accounting for 16.25% of the dose, followed by M3-1, accounting for 12.59% of the dose. By comparison, M21 was the major metabolite in feces, accounting for 6.81% of the dose. Overall, renal elimination plays a crucial role in vicagrel disposition, and the thiophene ring is the predominant metabolic site.

Assessment of NR4A Ligands That Directly Bind and Modulate the Orphan Nuclear Receptor Nurr1.

Nurr1/NR4A2 is an orphan nuclear receptor transcription factor implicated as a drug target for neurological disorders including Alzheimer's and Parkinson's diseases. Previous studies identified small-molecule NR4A nuclear receptor modulators, but it remains unclear if these ligands affect transcription via direct binding to Nurr1. We assessed 12 ligands reported to affect NR4A activity for Nurr1-dependent and Nurr1-independent transcriptional effects and the ability to bind the Nurr1 ligand-binding domain (LBD). Protein NMR structural footprinting data show that amodiaquine, chloroquine, and cytosporone B bind the Nurr1 LBD; ligands that do not bind include C-DIM12, celastrol, camptothecin, IP7e, isoalantolactone, ethyl 2-[2,3,4-trimethoxy-6-(1-octanoyl)phenyl]acetate (TMPA), and three high-throughput screening hit derivatives. Importantly, ligands that modulate Nurr1 transcription also show Nurr1-independent effects on transcription in a cell type-specific manner, indicating that care should be taken when interpreting the functional response of these ligands in transcriptional assays. These findings should help focus medicinal chemistry efforts that desire to optimize Nurr1-binding ligands.

[Pharmaceutical Properties of Anti-inflammatory Analgesic Patches Using Acrylic Polymer].

The mock patches were prepared with novel acrylic polymers as adhesive layer where biphenyl-4-ylacetic acid (BAA) or 2-(2-fluorobiphenyl-4-yl) propanoic acid (FPA) was used as model active pharmaceutical ingredients (APIs). In addition, the mock patches were formulated with typical ester ingredients for transdermal dosage forms. The molecular state of the model APIs in the adhesive layer was observed by polarized microscope and microscopic Raman spectroscopy, which contains both conventional and low frequency (LF) region. Crystallization behavior would be depended on the interaction between API and polymers in the adhesive layer. In particular, LF Raman measurement was useful to discriminate API polymorphs. The pharmaceutical properties including dissolution and skin permeation of APIs were also evaluated for mock patches. The drug release and transdermal permeation were enhanced with the ester ingredients such as isopropyl myristate and diethyl sebacate due to their diffusion to the test solution or the skin stratum corneum as well as reducing the interaction between API and polymers. Further, the tack strength was not changed, but the peel strength was weakened by the additives. Thus, the adhesive properties were controllable by formulation with the additives. These findings could enable to evaluate the interaction between API and the polymers for adhesive layer and select the appropriate polymer and additives for used APIs when designing the drug products.