Electronegative LDL induction of apoptosis in macrophages: Involvement of Nrf2

The aim of this study was to determine the apoptotic pathways and mechanisms involved in electronegative LDL [LDL(-)]-induced apoptosis in RAW 264.7 macrophages and the role of Nrf2 in this process. Incubation of RAW 264 7 macrophages with LDL(-) for 24 11 resulted in dose-dependent cell death. Activated caspases were shown to be involved in the apoptosis induced by LDL(-): incubation with the broad caspase inhibitor z-VAD prevented apoptosis in LDL(-)-treated cells CD95 (Fas), CD95 ligand (FasL). CD36 and the tumor necrosis factor (TNF) ligand Tnfsf10 were overexpressed in LDL(-)-treated cells However, Bax, Bcl-2 and Mcl-1 protein levels remained unchanged after LDL(-) treatment. LDL(-) promoted hyperpolarization of the mitochondrial membrane, elevated reactive oxygen species (ROS) production and translocation of Nrf2 to the nucleus, a process absent in cells treated with native LDL Elicited peritoneal macrophages from Nrf2-deficient mice exhibited an elevated apoptotic response after challenge with LDL(-), together with an increase in the production of ROS in the absence of alterations in CD36 expression These results provide evidence that CD36 expression induced by LDL(-) is Nrf2-dependent. Also, it was demonstrated that Nrf2 acts as a compensatory mechanism of LDL(-)-induced apoptosis in macrophages. (C) 2009 Elsevier B V. All rights reserved

Binary and ternary inclusion complexes of finasteride in HP beta CD and polymers: Preparation and characterization

The aim of this study was to determine whether inclusion complexes between 2-hydroxypropyl-beta-cyclodextrin (HP beta CD) and finasteride (FIN) are formed, and to characterize these. Equimolar FIN/HP beta CD solid systems in the presence or absence of 0.1% (w/v) of polyvinylpyrrolidone K30 (PVP K30) or 0.3% of chitosan were prepared by coevaporation and freeze-drying methods. The systems were characterized by phase solubility, NMR, DSC, and XRD analysis. The results suggest that true binary and ternary inclusion complexes were formed. (c) 2009 Elsevier Ltd. All rights reserved.

HPLC-UV determination of metformin in human plasma for application in pharmacokinetics and bioequivalence studies

In this study, a simple, rapid and sensitive HPLC method with UV detection is described for determination of metformin in plasma samples from bioequivalence assays. Sample preparation was accomplished through protein precipitation with acetonitrile and chromatographic separation was performed on a reversed-phase phenyl column at 40 degrees C. Mobile phase consisted of a mixture of phosphate buffer and acetonitrile at flow rate of 1.0 ml/min. Wavelength was set at 236 nm. The method was applied to a bioequivalence study of two drug products containing metformin, and allowed determination of metformin at low concentrations with a higher throughput than previously described methods. (c) 2007 Elsevier B.V. All rights reserved.

Bioequivalence Evaluation of Two Different Tablet Formulations of Tinidazole in Healthy Volunteers

The bioequivalence of two different tablet formulations of tirtidazole (CAS 19387-91-8) was determined in healthy volunteers after a single dose in a randomized crossover study, with a 1-week washout period between the doses. Reference and test products were administered to 24 volunteers with 240 mL water after overnight fasting. Plasma concentrations of tinidazole were monitored by a high-performance liquid chromatographic method (HPLC) over a period of 72 h after the administration. The pharmacokinetic parameters AUC(0-t), AUC(0-infinity), C(max), T(max), T((1/2)el) and beta were determined from plasma concentration time profile of both formulations and found to be in good agreement with previously reported values. The calculated pharmacokinetic parameters were compared statistically to evaluate bioequivalence between the two brands. The analysis of variance (ANOVA) did not show any significant difference between the two formulations and 90% confidence intervals for the ratio of C(max) (93.9 - 102.6%), AUC(0-t), (94.9-101.1%) and AUC(0-infinity) (94.6-100.8%) values for the test and reference products were within the 80 - 125% interval, satisfying bioequivalence criteria of the European Committee for Proprietary Medicinal Products and the US Food and Drug Administration Guidelines. These results indicate that the test and the reference products of tinidazole are bioequivalent and, thus, may be prescribed interchangeably.

Growth and acidification performance of probiotics in pure culture and co-culture with Streptococcus thermophilus: The effect of inulin

Inulin was used as a prebiotic to improve the quality and consistency of skim milk fermented by co-cultures and pure Cultures of Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus bulgaricus and Bifidobacterium lactis with Streptococcus thermophilus. We compared, either in the presence or absence of 4 g inulin/100 g, the results of the main kinetic parameters, specifically the generation time (t(g)), the maximum acidification rate (V(max)). and the times to reach V(max) (t(max)), to attain pH 5.0 (t(pH5.0)) and to complete the fermentation (t(pH4.5)). Post-acidification, lactic acid formation and cell counts were also determined and compared, either 1 day after the fermentation was complete or after 7 day storage at 4 degrees C. In general, inulin addition to the milk increased in co-cultures V(max), decreased t(max), t(g) and t(pH4.5), favored post-acidification, exerted a bifidogenic effect, and preserved almost intact cell viability during storage. In addition, S. thermophilus was shown to stimulate the metabolism of the other lactic bacteria. Contrary to co-cultures, most of the effects in pure Cultures were not statistically significant. The most important aspect of this paper is the use of the generation time as a toot to investigate the microbial response to inulin addition. (c) 2009 Elsevier Ltd. All rights reserved.

Contribution of peripheral vanilloid receptor to the nociception induced by injection of spermine in mice

Polyamines (putrescine, spermidine and spermine) are important endogenous regulators of ion channels, such as vanilloid (TRPV1), glutamatergic (NMDA or AMPA/kainate) and acid-sensitive (ASIC) receptors. In the present study, we have investigated the possible nociceptive effect induced by polyamines and the mechanisms involved in this nociception in vivo. The subcutaneous (s.c.) injection of capsaicin (as positive control), spermine, spermidine or putrescine produced nociception with ED(50) of 0.16 (0.07-0.39) nmol/paw, 0.4 (0.2-0.7) mu mol/paw, 0.3 (0.1-0.9) mu mol/paw and 3.2 (0.9-11.5) mu mol/paw, respectively. The antagonists of NMDA (MK801, 1 nmol/paw), AMPA/kainate (DNQX, 1 nmol/paw) or ASIC receptors (amiloride, 100 nmol/paw) failed to reduce the spermine-trigged nociception. However, the TRPV1 antagonists capsazepine or SB366791 (1 nmol/paw) reduced spermine-induced nociception, with inhibition of 81 +/- 10 and 68 +/- 9%, respectively. The previous desensitization with resiniferatoxin (RTX) largely reduced the spermine-induced nociception and TRPV1 expression in the sciatic nerve, with reductions of 82 +/- 9% and 67 +/- 11%, respectively. Furthermore, the combination of spermine (100 nmol/paw) and RTX (0.005 fmol/paw), in doses which alone were not capable of inducing nociception, produced nociceptive behaviors. Moreover, different concentrations of spermine (3-300 mu M) enhanced the specific binding of [(3)H](center dot)-RTX to TRPV1 receptor. Altogether, polyamines produce spontaneous nociceptive effect through the stimulation of TRPV1, but not of ionotropic glutamate or ASIC receptors. (C) 2011 Elsevier Inc. All rights reserved.

Pharmacokinetics of tetracycline in plasma, synovial fluid and milk using single intravenous and single intravenous regional doses in dairy cattle with papillomatous digital dermatitis

The purpose of this study was to compare the pharmacokinetics of tetracycline in plasma, synovial fluid, and milk following either a single systemic intravenous (i.v.) injection or a single i.v. regional antibiosis (IVRA) administration of tetracycline hydrochloride to dairy cattle with papillomatous digital dermatitis (PDD). To this end, plasma and synovial fluid tetracycline concentrations were compared with the minimal inhibitory concentration (MIC) values of the major bacteria, which are known to cause digital diseases and thus assess its efficacy in PDD. Residual tetracycline concentrations in milk from cows treated by both methods were also determined. Twelve Holstein cows with various stages of PDD were randomly assigned to two groups of six animals. Group 1 received a single systemic i.v. injection of 10 mg/kg of tetracycline hydrochloride. Group 2 received 1000 mg of tetracycline hydrochloride by IVRA of the affected limb. Blood, synovial fluid and milk samples were taken prior to tetracycline administration (time 0 control), and then at 22, 45 and 82 min, and 2, 3, 4, 6, 8, 12, 24, 48, 72, 96, and 120 h following drug administration. Tetracycline concentrations were determined by high-performance liquid chromatography. Mean tetracycline plasma and milk concentrations in Group 1 were higher than Group 2. The opposite was observed for synovial fluid concentrations. Group 2 synovial fluid concentrations were higher than the MIC value over 24 h for the bacteria most frequently responsible for claw disease. Compared with i.v. administration, IVRA administration of tetracycline produced very high synovial fluid and low plasma and milk concentrations.

Determination of chlorpheniramine in human plasma by HPLC-ESI-MS/MS: application to a dexchlorpheniramine comparative bioavailability study

In the present study a fast, sensitive and robust validated method to quantify chlorpheniramine in human plasma using brompheniramine as internal standard (IS) is described. The analyte and the IS were extracted from plasma by LLE (diethyl ether-dichloromethane, 80:20, v/v) and analyzed by HPLC-ESI-MS/MS. Chromatographic separation was performed using a gradient of methanol from 35 to 90% with 2.5 mm NH(4)OH on a Gemini Phenomenex C(8) 5 mu m column (50 x 4.6 mm i.d.) in 5.0 min/run. The method fitted to a linear calibration curve (0.05-10 ng/mL, R > 0.9991). The precision (%CV) and accuracy ranged, respectively: intra-batch from 1.5 to 6.8% and 99.1 to 106.6%, and inter-batch from 2.4 to 9.0%, and 99.9 to 103.1%. The validated bioanalytical procedure was used to assess the comparative bioavailability in healthy volunteers of two dexchlorpheniramine 2.0 mg tablet formulations (test dexchlorpheniramine, Eurofarma, and reference Celestamine (R), Schering-Plough). The study was conducted using an open, randomized, two-period crossover design with a 2 week washout interval. Since the 90% confidence interval for C(max) and AUC ratios were all within the 80-125% interval proposed by ANVISA and FDA, it was concluded that test and reference formulations are bioequivalent concerning the rate and the extent of absorption. Copyright (C) 2009 John Wiley & Sons, Ltd.

New Rapid Derivative Spectrophotometric and Chromatographic Methods for Assay of Loratadine in Tablets and Syrups

New rapid first-derivative spectrophotometric (UVDS) and a stability-indicating high performance liquid chromatographic (HPLC) methods were developed, validated and successfully applied in the analysis of loratadine (LT) in tablets and syrups. In the UVDS method, 0.1 M HCl was used as solvent. The measurements were made at 312.4 nm in the first order derivative spectra. The HPLC method was carried out on a RP-18 column with a mobile phase composed of methanol-water-tetrahydrofuran (50:30:20, v/v/v). UV detection was made at 247 nm. For HPLC methods the total analysis time was <3min, adequate for routine quality control of tablets and syrups containing loratadine.

UV-Derivative Spectrophotometric and Stability-Indicating High-Performance Liquid Chromatographic Methods for Determination of Simvastatin in Tablets

A stability-indicating high-performance liquid chromatographic (HPLC) and a second-order derivative spectrophotometric (UVDS) analytical methods were validated and compared for determination of simvastatin in tablets. The HPLC method was performed with isocratic elution using a C18 column and a mobile phase composed of methanol:acetonitrile:water (60:20:20, v/v/v) at a flow rate of 1.0 ml/min. The detection was made at 239 nm. In UVDS method, methanol and water were used in first dilution and distilled water was used in consecutive dilutions and as background. The second-order derivative signal measurement was taken at 255 nm. Analytical curves showed correlation coefficients > 0.999 for both methods. The quantitation limits (QL) were 2.41 mu g/ml for HPLC and 0.45 mu g/ml for UVDS, respectively. Intra and inter-day relative standard deviations were < 2.0 %. Statistical analysis with t- and F-tests are not exceeding their critical values demonstrating that there is no significant difference between the two methods at 95 % confidence level.

Bioequivalence study of two oral formulations of cefadroxil in healthy volunteers

Two different cefadroxil (CAS 50370-12-2) formulations were evaluated for their relative bioavailability in 24 healthy volunteers who received a single 500 mg oral dose of each preparation. An open, randomized clinical trial designed as a two-period crossover study with a 7-day washout period between doses was employed. Plasma samples for assessments of their cefadroxil concentration by HPLC-UV were obtained over 8 h after administration. Values of 48.94 +/- 10.18 mu g . h/ml for test, and 48.51 +/- 9.02 mu g . h/ml for the reference preparation AUC(0-t) demonstrate a nearly identical extend of drug absorption. Maximum plasma concentration C-max of 16.04 +/- 4.94 mu g/ml and 16.01 +/- 4.02 mu g/ml achieved for the test and reference preparations did not differ significantly. The parametric 90% confidence intervals (CI) of the mean of the difference (test-reference) between log-transformed values of the two formulations were 96.80% to 104.51% and 92.01% to 107.00% for AUC(0-t) and C-max, respectively. Since for both AUC(0-t) or C-max the 90% CI values are within the interval proposed by the Food and Drug Administration, the test product is bioequivalent to the reference product for both the rate and extent of absorption after single dose administration.

Sero-epidemiology of toxocariasis in a rural settlement in Sao Paulo state, Brazil

The seroprevalence of Toxocara canis and risk factors for infection with this parasite were explored in a rural settlement in Sao Paulo state, Brazil. Total IgA and IgE levels in 79 subjects were determined by turbidimetry and chemiluminescence, respectively. Total counts of leucocytes and erythrocytes and differential counts of leucocytes were made by flow cytometry. ELISA for the detection of anti-Toxocara IgG, IgA and IgE were standardized using Toxocara excretory-secretory antigens (TES) obtained from the cultured second-stage larvae of T. canis. Seventeen (21.5%) of the subjects were found positive for anti-Toxocara IgG, with no significant differences in such seropositivity with age or gender. Thirty (38%) of the subjects showed eosinophilia and 70 (89%) had elevated levels of total IgE. Among the 17 subjects found seropositive for anti-Toxocara IgG, the percentage of leucocytes represented by eosinophils (P=0.0069) and total levels of IgE (P=0.0452) were positively correlated with the levels of anti-TES IgE. Although anti-TES IgA was detected in 10 (59%) of the subjects, there was no significant correlation between the levels of total IgA and those of Toxocara-specific IgA. Only one of the 17 subjects found positive for anti-Toxocara IgG had attended a secondary school and all but two belonged to households with monthly incomes of < U.S.$100. In the study community at least, seropositivity may be related to poor living standards and lack of basic sanitary conditions. The presence of anti-Toxocara IgE and IgA may facilitate the diagnosis of toxocariasis and may well be useful for monitoring the success of treatment.

Negative ion `chip-based` nanospray tandem mass spectrometry for the analysis of flavonoids in glandular trichomes of Lychnophora ericoides Mart. (Asteraceae)

This paper reports a method for the analysis of secondary metabolites stored in glandular trichomes, employing negative ion `chip-based` nanospray tandem mass spectrometry. The analyses of glandular trichomes from Lychnophora ericoides, a plant endemic to the Brazilian `cerrado` and used in traditional medicine as an anti-inflammatory and analgesic agent, led to the identification of five flavonoids (chrysin, pinocembrin, pinostrobin, pinobanksin and 3-O-acetylpinobanksin) by direct infusion of the extracts of glandular trichomes into the nanospray ionisation source. All the flavonoids have no oxidation at ring B, which resulted in a modification of the fragmentation pathways compared with that of the oxidised 3,4-dihydroflavonoids already described in the literature. The absence of the anti-inflammatory and antioxidant di-C-glucosylflavone vicenin-2, or any other flavonoid glycosides, in the glandular trichomes was also demonstrated. The use of the,`chip-based` nanospray QqTOF apparatus is a new fast and useful tool for the identification of secondary metabolites stored in the glandular trichomes, which can be useful for chemotaxonomic studies based on metabolites from glandular trichomes. Copyright (C) 2008 John Wiley & Sons, Ltd.

Antioxidant activity of flavonoids in isolated mitochondria

Mitochondria are important intracellular sources and targets of reactive oxygen species (ROS), while flavonoids, a large group of secondary plant metabolites, are important antioxidants. Following our previous study on the energetics of mitochondria exposed to the flavonoids quercetin, taxifolin, catechin and galangin, the present work addressed the antioxidant activity of these compounds (1-50 mu mol/L) on Fe2+/citrate-mediated membrane lipid peroxidation (LPO) in isolated rat liver mitochondria, running in parallel studies of their antioxidant activity in non-organelle systems. Only quercetin inhibited the respiratory chain of mitochondria and only galangin caused uncoupling. Quercetin and galangin were far more potent than taxifolin and catechin in affording protection against LPO (IC50 = 1.23 +/- 0.27 and 2.39 +/- 0.79 mu mol/L, respectively), although only quercetin was an effective scavenger of both 2,2-diphenyl-1-picrylhydrazyl (DPPH) and superoxide radicals. These results, together with the previous study, suggest that the 2,3-double bond in conjugation with the 4-oxo function in the flavonoid structure are major determinants of the antioxidant activity of flavonoids in mitochondria, the presence of an o-di-OH structure on the B-ring, as occurs in quercetin, favours this activity via superoxide scavenging, while the absence of this structural feature in galangin, favours it via a decrease in membrane fluidity and/or mitochondrial uncoupling. Copyright (c) 2008 John Wiley & Sons, Ltd.