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.

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

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.

Development of a method to quantify clindamycin in vitreous humor of rabbits' eyes by UPLC-MS/MS: application to a comparative pharmacokinetic study and in vivo ocular biocompatibility evaluation.

Ocular toxoplasmosis may result in uveitis in the posterior segment of the eye, leading to severe visual complications. Clindamycin-loaded poly(lactide-co-glycolide) (PLGA) implants could be applied to treat the ocular toxoplasmosis. In this study, the pharmacokinetic profiles of the drug administrated by PLGA implants and by intravitreal injections in rabbits' eyes were evaluated. The implant released the drug for 6 weeks while the drug administrated by intravitreal injections remained in the vitreous cavity for 2 weeks. Compared to the injected drug, the implants containing clindamycin had higher values of area under the curve (AUC) (39.2 vs 716.7 ng week mL(-1)) and maximum vitreous concentration (Cmax) (8.7 vs 13.83 ng mL(-1)). The implants prolonged the delivery of clindamycin and increased the contact of the drug with the eyes' tissues. Moreover, the in vivo ocular biocompatibility of the clindamycin-loaded PLGA implants was evaluated regarding to the clinical examination of the eyes and the measurement of the intraocular pressure (IOP) during 6 weeks. The implantable devices caused no ocular inflammatory process and induced the increase of the IOP in the fourth week of the study. The IOP augmentation could be related to the maximum concentration of clindamycin released from the implants. In conclusion, the PLGA implants based on clindamycin may be a therapeutic alternative to treat ocular toxoplasmosis.

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.

Evaluation of intestinal permeability of vicenin-2 and lychnopholic acid from Lychnophora salicifolia (Brazilian arnicão) using Caco-2 cells.

Lychnophora salicifolia, commonly known as "arnicão", is used as an anti-inflammatory agent and as a flavoring agent in the Brazilian traditional spirit "cachaça". In this work, the permeation process of vicenin-2 (1) and lychnopholic acid (2) (major secondary metabolites from the hydroalcoholic extract) was investigated using Caco-2 cells. For this investigation, a new HPLC-DAD method was developed and validated for the quantification step. It was observed that 2 crosses the Caco-2 cell monolayer by passive diffusion. On the other hand, 1 was not transported, suggesting no absorption and no efflux of this compound in Caco-2 cells.

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.

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.

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.

A new high-performance liquid chromatography assay for the determination of sesquiterpene lactone 15-deoxygoyazensolide in rat plasma.

A simple, rapid and sensitive high-performance liquid chromatography method was developed for the analysis of the sesquiterpene lactone 15-deoxygoyazensolide (LAC15-D) in rat plasma samples. The chromatographic separation was achieved on a LiChrospher RP18 column using methanol:water (50:50, v/v) containing 0.6% acetic acid as mobile phase, at a flow rate of 0.7 mL min(-1). UV detection was carried out at 270 nm. Phenytoin was used as internal standard. Prior to the analysis, the rat plasma samples were submitted to liquid-liquid extraction with dichloromethane. The mean absolute recoveries were 73% with R.S.D. values lower than 3.5. The method was linear over the 6.0-2000 ng mL(-1) concentration range and the quantification limit was 6.0 ng mL(-1). Within-day and between-day assay precision and accuracy were studied at three concentration levels (15, 300 and 480 ng mL(-1)) and were lower than 15%. The validated method was used to measure the plasmatic concentration of LAC15-D in rats that received a single intraperitoneal dose of 30 mg kg(-1).