Dayana Rubio Gouvea

Low expression of CD39 on regulatory T cells as a biomarker for resistance to methotrexate therapy in rheumatoid arthritis

Significance Methotrexate (MTX) is the first-line therapy for rheumatoid arthritis (RA). However, about 40% of patients are resistant to MTX. Furthermore, MTX resistance is only apparent after a prolonged continuous MTX treatment (>3 mo), by which time the disease of the nonresponders would have aggravated. Thus, there is a considerable unmet need for a biomarker to select MTX-resistant patients and place them immediately on alternative therapy. We found here that the low density of CD39 on peripheral regulatory T cells in RA patients is a rapid, convenient, and reliable (P < 0.01) biomarker for MTX resistance. Our findings also provide previously unrecognized information on aspects of immune regulation in RA and the mechanism of action of MTX. Rheumatoid arthritis (RA) is an inflammatory autoimmune disease characterized by joint destruction and severe morbidity. Methotrexate (MTX) is the standard first-line therapy of RA. However, about 40% of RA patients are unresponsive to MTX treatment. Regulatory T cells (Tregs, CD4+CD25+FoxP3+) are thought to play an important role in attenuating RA. To investigate the role of Tregs in MTX resistance, we recruited 122 RA patients (53 responsive, R-MTX; 69 unresponsive, UR-MTX) and 33 healthy controls. Three months after MTX treatment, R-MTX but not UR-MTX showed higher frequency of peripheral blood CD39+CD4+CD25+FoxP3+ Tregs than the healthy controls. Tregs produce adenosine (ADO) through ATP degradation by sequential actions of two cell surface ectonucleotidases: CD39 and CD73. Tregs from UR-MTX expressed a lower density of CD39, produced less ADO, and had reduced suppressive activity than Tregs from R-MTX. In a prospective study, before MTX treatment, UR-MTX expressed a lower density of CD39 on Tregs than those of R-MTX or control (P < 0.01). In a murine model of arthritis, CD39 blockade reversed the antiarthritic effects of MTX treatment. Our results demonstrate that MTX unresponsiveness in RA is associated with low expression of CD39 on Tregs and the decreased suppressive activity of these cells through reduced ADO production. Our findings thus provide hitherto unrecognized mechanism of immune regulation in RA and on mode of action of MTX. Furthermore, our data suggest that low expression of CD39 on Tregs could be a noninvasive biomarker for identifying MTX-resistant RA patients.

Re-examination of the anion derivatives of isoflavones by radical fragmentation in negative electrospray ionization tandem mass spectrometry: experimental and computational studies

This paper reports theoretical and experimental studies of gas-phase fragmentation reactions of four naturally occurring isoflavones. The samples were analyzed in negative ion mode by direct infusion in ESI-QqQ, ESI-QqTOF and ESI-Orbitrap systems. The MS/MS and MS(n) spectra are in agreement with the fragmentation proposals and high-resolution analyses have confirmed the formulae for each ion observed. As expected, compounds with methoxyl aromatic substitution have showed a radical elimination of •CH(3) as the main fragmentation pathway. A second radical loss (•H) occurs as previously observed for compounds which exhibit a previous homolytic •CH(3) cleavage (radical anion) and involves radical resonance to stabilize the anion formed. However, in this study we suggest another mechanism for the formation of the main ions, on the basis of the enthalpies for each species. Compounds without methoxy substituent dissociate at the highest energies and exhibit the deprotonated molecule as the most intense ion. Finally, energy-resolved experiments were carried out to give more details about the gas-phase dissociation reaction of the isoflavones and the results are in agreement with the theoretical approaches.

In vitro metabolism of the alkaloid piplartine by rat liver microsomes

Because piplartine (PPT) has demonstrated biological activities, such as cytotoxic, anxiolytic, antidepressant, antifungal and antiplatelet activities, this molecule is a relevant drug candidate. The metabolic fate of drug candidates is an essential requirement in assessing their safety and efficacy. Based on this requirement, the biotransformation of PPT by cytochrome P450 enzymes (CYP) was investigated for the first time. To determine the in vitro enzymatic kinetic parameters, an HPLC method was developed and validated to quantify PPT. All samples were separated on a reversed-phase C18 column using a mobile phase of acetonitrile:water (40:60, v/v). The method exhibited a linear range of 2.4-157.7 μmol/L, with the following calibration curve: y=0.0934 (±0.0010)x+0.0027, r=0.9975. The lower limit of quantitation was verified to be 2.4 μmol/L, with an RSD below 7%. The precision and accuracy were assessed for both within-day and between-day determinations; neither relative standard (RSD%) deviations nor relative errors (RER) exceeded a value of 15%. The mean absolute recovery was 85%, with an RSD value below 6%. The enzymatic kinetic parameters revealed a sigmoidal profile, with V(max)=4.7±0.3 μmol/mg mL⁻¹/min, h=2.5±0.4, S₅₀=44.7±0.3 μmol/L and CL(max)=0.054 μL/min/mg protein, indicating cooperativity behavior. Employing a mammalian model, PPT metabolism yielded two unreported monohydroxylated products (m/z 334). The identification and structural elucidation of the metabolites were performed by comparing their mass spectra with those spectra of the parent drug. For the first time, the in vitro metabolism studies employing microsomes were demonstrated to be a suitable tool for data regarding enzymatic kinetics and for the metabolites formed in the PPT mammalian metabolism.

Effects of chlorogenic acid on neutrophil locomotion functions in response to inflammatory stimulus

AIM OF THE STUDY Species of Lychnophora are used in Brazilian folk medicine as analgesic and anti-inflammatory agents. Chlorogenic acid (CGA) and their analogues are important components of polar extracts of these species, as well in several European and Asian medicinal plants. Some of these phenolic compounds display anti-inflammatory effects. In this paper we report the isolation of CGA from Lychnophora salicifolia and its effects on functions involved in neutrophils locomotion. MATERIALS AND METHODS LC-MS(n) data confirmed the presence of CGA in the plant. Actions of CGA were investigated on neutrophils obtained from peritoneal cavity of Wistar rats (4h after 1% oyster glycogen solution injection; 10 ml), and incubated with vehicle or with 50, 100 or 1000 μM CGA in presence of lipopolysaccharide from Escherichia coli (LPS, 5 μg/ml). Nitric oxide (NO; Griess reaction); prostaglandin E(2) (PGE(2)), interleukin-1β (IL-1β) and tumor necrosis factor-α [TNF-α; enzyme-linked immunosorbent assay (EIA)]; protein (flow cytometry) and gene (RT-PCR) expression of L-selectin, β(2)integrin and platelet-endothelial cell adhesion molecule-1 (PECAM-1) were quantified. In vitro neutrophil adhesion to primary culture of microvascular endothelial cell (PMEC) and neutrophil migration in response to formyl-methionil-leucil-phenilalanine (fMLP, 10(-8)M, Boyden chamber) was determined. RESULTS CGA treatment did not modify the secretion of inflammatory mediators, but inhibited L-selectin cleavage and reduced β(2) integrin, independently from its mRNA synthesis, and reduced membrane PECAM-1 expression; inhibited neutrophil adhesion and neutrophil migration induced by fMLP. CONCLUSIONS Based on these findings, we highlight the direct inhibitory actions of CGA on adhesive and locomotion properties of neutrophils, which may contribute to its anti-inflammatory effects and help to explain the use of Lychnophora salicifolia as an anti-inflammatory agent.

A new HPLC-DAD-MS/MS method for the simultaneous determination of major compounds in the crude extract of Lychnophora salicifolia Mart. (Brazilian arnicão) leaves: application to chemical variability evaluation.

Lychnophora salicifolia Mart., which occurs in the Brazilian Cerrado in the states of Bahia and Minas Gerais as well as in the southeast of the state of Goiás, is the most widely distributed and also the most polymorphic species of the genus. This plant is popularly known to have anti-inflammatory and analgesic activities. In this work, we have studied the variation in terms of polar metabolites of ninety-three Lychnophora salicifolia Mart. specimens collected from different regions of the Brazilian Cerrado. Identification of the constituents of this mixture was carried out by analysis of the UV spectra and MS data after chromatographic separation. Twenty substances were identified, including chlorogenic acid derivatives, a flavonoid C-glucoside, and other sesquiterpenes. The analytical method was validated, and the reliability and credibility of the results was ensured for the purposes of this study. The concentration range required for analysis of content variability within the analyzed group of specimens was covered with appropriate values of limits of detection and quantitation, as well as satisfactory precision and recovery. A quantitative variability was observed among specimens collected from the same location, but on average they were similar from a chemical viewpoint. In relation to the study involving specimens from different locations, there were both qualitative and quantitative differences among plants collected from different regions of Brazil. Statistical analysis revealed that there is a correlation between geographical localization and polar metabolites profile for specimens collected from different locations. This is evidence that the pattern of metabolites concentration depends on the geographical distribution of the specimens.

The Protective Role of Lychnophora ericoides Mart. (Brazilian Arnica) in 1,2-Dimethylhydrazine-Induced Experimental Colon Carcinogenesis

Aberrant crypt foci (ACF) and colon rectal mucosal epithelial cell proliferation have been shown to be increased in patients with colon cancer and have been largely used for early detection of factors that influence colorectal carcinogenesis in rats. Fifty male Wistar rats were randomly divided into 5 groups. The groups G1 to G4 were given 4 injections of the carcinogen 1,2-dimethylhydrazine (DMH). The G2 group received Lychnophora ericoides (LE) extracts for 6 wk. The groups G3 and G4 received LE for 4 wk and 2 wk, respectively, at the postinitiation and initiation phases of colonic carcinogenesis. The group G5 was the control. Forty-two days after the first injections of DMH for the neoplasic induction, we observed a statistically significant decrease in the number of aberrant crypt foci (ACF) and an attenuation of the increase in cell proliferation induced by DMH in all the LE-treated groups. Thus, we concluded that Lychnophora ericoides extracts were effective against the development of cancer. These data suggest that LE has a protective influence on the process of colon carcinogenesis, suppressing both the initiation and the promotion of colonic carcinogenesis.

Sensitive method for determination of piplartine, an alkaloid amide from Piper species, in rat plasma samples by liquid chromatography-tandem mass spectrometry

Piplartine (PPTN) is an alkaloid amide found in Piper species that presents different activities. PPTN determination in rat plasma is necessary to better understand its biological effects. The aim of this study was to develop a sensitive LC-MS/MS method for the determination of PPTN in rat plasma. The performance criteria for linearity, sensitivity, precision, accuracy, recovery, and stability have been assessed and were within the recommended guidelines. The validated method proved to be suitable in a pilot study of PPTN kinetic disposition in rat plasma after a single intraperitoneal dose, and represents an appropriate tool to further pharmacokinetic studies.

Gas-phase fragmentation of protonated piplartine and its fungal metabolites using tandem mass spectrometry and computational chemistry

Piplartine, an alkaloid produced by plants in the genus Piper, displays promising anticancer activity. Understanding the gas-phase fragmentation of piplartine by electrospray ionization tandem mass spectrometry can be a useful tool to characterize biotransformed compounds produced by in vitro and in vivo metabolism studies. As part of our efforts to understand natural product fragmentation in electrospray ionization tandem mass spectrometry, the gas-phase fragmentation of piplartine and its two metabolites 3,4-dihydropiplartine and 8,9-dihydropiplartine, produced by the endophytic fungus Penicillium crustosum VR4 biotransformation, were systematically investigated. Proposed fragmentation reactions were supported by ESI-MS/MS data and computational thermochemistry. Cleavage of the C-7 and N-amide bond, followed by the formation of an acylium ion, were characteristic fragmentation reactions of piplartine and its analogs. The production of the acylium ion was followed by three consecutive and competitive reactions that involved methyl and methoxyl radical eliminations and neutral CO elimination, followed by the formation of a four-member ring with a stabilized tertiary carbocation. The absence of a double bond between carbons C-8 and C-9 in 8,9-dihydropiplartine destabilized the acylium ion and resulted in a fragmentation pathway not observed for piplartine and 3,4-dihydropiplartine. These results contribute to the further understanding of alkaloid gas-phase fragmentation and the future identification of piplartine metabolites and analogs using tandem mass spectrometry techniques. Copyright

Pharmacokinetic Evaluation of Avicularin Using a Model-Based Development Approach

The aim of this study was to use the pharmacokinetic information of avicularin in rats to project a dose for humans using allometric scaling. A highly sensitive and specific bioanalytical assay to determine avicularin concentrations in the plasma was developed and validated for UPLC-MS/MS. The plasma protein binding of avicularin in rat plasma determined by the ultrafiltration method was 64%. The pharmacokinetics of avicularin in nine rats was studied following an intravenous bolus administration of 1 mg/kg and was found to be best described by a two-compartment model using a nonlinear mixed effects modeling approach. The pharmacokinetic parameters were allometrically scaled by body weight and centered to the median rat weight of 0.23 kg, with the power coefficient fixed at 0.75 for clearance and 1 for volume parameters. Avicularin was rapidly eliminated from the systemic circulation within 1 h post-dose, and the avicularin pharmacokinetic was linear up to 5 mg/kg based on exposure comparison to literature data for a 5-mg/kg single dose in rats. Using allometric scaling and Monte Carlo simulation approaches, the rat doses of 1 and 5 mg/kg correspond to the human equivalent doses of 30 and 150 mg, respectively, to achieve comparable plasma avicularin concentrations in humans.

Spatial and Temporal Variations in Secondary Metabolites Content of the Brazilian Arnica Leaves (Lychnophora ericoides Mart., Asteraceae)

The genus Lychnophora (Asteraceae) is a medicinal plant traditionally used as anti-inflammatory or analgesic agent. Its biological properties have been widely investigated and correlated with its chemical composition, which could be different between the species and even accordingly to the place and season of collection. In this work, it was investigated the spatial and temporal variations in the secondary metabolites from five populations of Lychnophora ericoides. Among them, a more diversified secondary metabolism was observed for the plants collected at Ibiraci-MG (Brazil), a city placed between two biomes, Cerrado and Atlantic Forest. The highest concentrations of chlorogenic acids were found in the Cerrado dry season (winter), while the flavonoids were higher during the rainy season (summer). Significant reduction was observed in the contents of all the secondary metabolites in old leaves, and also in flowery branches. These variations detected in L. ericoides leaves could highly influence the intensity of the biological activities of this plant.