Endotoxin-Induced Sepsis on Ceftriaxone-Treated Rats' Ventilatory Mechanics and Pharmacokinetics.

Sepsis can trigger acute respiratory distress syndrome (ARDS), which can lead to a series of physiological changes, modifying the effectiveness of therapy and culminating in death. For all experiments, male Wistar rats (200-250 g) were split into the following groups: control and sepsis-induced by endotoxin lipopolysaccharide (LPS); the control group received only intraperitoneal saline or saline + CEF while the treated groups received ceftriaxone (CEF) (100 mg/kg) IP; previously or not with sepsis induction by LPS (1 mg/kg) IP. We evaluated respiratory mechanics, and alveolar bronchial lavage was collected for nitrite and vascular endothelial growth factor (VEGF) quantification and cell evaluation. For pharmacokinetic evaluation, two groups received ceftriaxone, one already exposed to LPS. Respiratory mechanics shows a decrease in total airway resistance, dissipation of viscous energy, and elastance of lung tissues in all sepsis-induced groups compared to the control group. VEGF and NOx values were higher in sepsis animals compared to the control group, and ceftriaxone was able to reduce both parameters. The pharmacokinetic parameters for ceftriaxone, such as bioavailability, absorption, and terminal half-life, were smaller in the sepsis-induced group than in the control group since clearance was higher in septic animals. Despite the pharmacokinetic changes, ceftriaxone showed a reduction in resistance in the airways. In addition, CEF lowers nitrite levels in the lungs and acts on their adverse effects, reflecting pharmacological therapy of the disease.

Preclinical Evaluation of Polymeric Nanocomposite Containing Pregabalin for Sustained Release as Potential Therapy for Neuropathic Pain.

This study offers a novel oral pregabalin (PG)-loaded drug delivery system based on chitosan and hypromellose phthalate-based polymeric nanocomposite in order to treat neuropathic pain (PG-PN). PG-PN has a particle size of 432 ± 20 nm, a polydispersity index of 0.238 ± 0.001, a zeta potential of +19.0 ± 0.9 mV, a pH of 5.7 ± 0.06, and a spherical shape. Thermal and infrared spectroscopy confirmed nanocomposite generation. PG-PN pharmacokinetics was studied after a single oral dose in male Wistar rats. PG-PN showed greater distribution and clearance than free PG. The antinociceptive effect of PG-PN in neuropathic pain rats was tested by using the chronic constriction injury model. The parameter investigated was the mechanical nociceptive threshold measured by the von Frey filaments test; PG-PN showed a longer antinociceptive effect than free PG. The rota-rod and barbiturate sleep induction procedures were used to determine adverse effects; the criteria included motor deficit and sedative effects. PG-PN and free PG had plenty of motors. PG-PN exhibited a less sedative effect than free PG. By prolonging the antinociceptive effect and decreasing the unfavorable effects, polymeric nanocomposites with pregabalin have shown promise in treating neuropathic pain.

The liquid crystalline phase behaviour of a nasal formulation modifies the brain disposition of donepezil in rats in the treatment of Alzheimer's disease.

Although nanoparticles, polymeric micelles, liposomes, nanoemulsions, and microemulsions were extensively evaluated as formulations for nasal administration of drugs, lyotropic liquid crystal (LLC) mesophases have been few studied. The phase transition from a low-viscosity microemulsion to a more viscous LLC may improve the mucoadhesion of the formulation. Donepezil is a drug administered orally in the treatment of Alzheimer's disease, and with gastrointestinal side effects that are typical of acetylcholinesterase inhibitors. Based on this, donepezil administration by nasal pathway using a mucoadhesive LLC may be a feasible alternative. A colloidal formulation was selected from a ternary diagram, combining CETETH-10, oleic acid, and water (40:45:15, w/w). Donepezil was incorporated into the formulation, and the characterisation included in vitro studies, such as mucoadhesion and drug release. Pharmacokinetics in Wistar rats included evaluations by the nasal pathway with donepezil incorporated into microemulsion. A phase transition from an isotropic to an anisotropic system was observed after the swelling of the microemulsion with artificial nasal fluid (12-20 %). The release of donepezil in vitro occurred in a sustained manner. Significant levels of donepezil were achieved in the brain after nasal administration of the microemulsion, as a promising strategy for the treatment of Alzheimer's disease.

In vivo schistosomicidal activity of 7-epiclusianone and its quantification in the plasma of healthy and Schistosoma mansoni infected mice using UPLC-MS/MS.

BACKGROUND: The tetraprenylated benzophenone 7-epiclusianone (7-epi) is a substance isolated from the fruits of Garcinia brasiliensis and in vitro studies have demonstrated that 7-epi is effective against Schistosoma mansoni adult worms. HYPOTHESIS/PURPOSE: Here we report the in vivo evaluation of 7-epi and its pharmacokinetic in healthy and Schistosoma mansoni infected mice. STUDY DESIGN AND METHODS: In this work, we assayed the schistosomicidal activity of 7-epi at the dose of 100 mg/kg and 300 mg/kg body weight/day in S. mansoni experimentally infected mice. Besides, two groups of animals were treated and a detailed analysis of plasma samples was performed by liquid chromatography coupled to mass spectrometry (LC-MS/MS). RESULTS: The worm burden showed a reduction in the infected mice after treatment with 300 mg/kg for five days (p < .05). And we found an increase of AUC0-∞ (20846 vs. 32438 ng.h/ml) and a decrease of total apparent clearance (0.006 vs. 0.004 l/h/kg) of 7- epi in the infected group compared to the healthy group. Consequently, the half-life increased (1.73 vs. 6.11 h) and Cmax was reduced (5427.5 vs. 3321.0 ng/ml) in the infected group compared to the healthy group. In addition, histopathological investigations were performed analysing liver samples from healthy and infected mice. CONCLUSION: The results showed significant schistosomicidal in vivo activity at 300 mg/kg. In addition, livers from S. mansoni infected mice showed a greater number of egg granulomas and the changes in the pharmacokinetic parameters in this group could be associated with the pathology of the murine experimental schistosomiasis.

On-Line Restricted Access Molecularly Imprinted Solid-Phase Extraction of Selective Serotonin Reuptake Inhibitors Directly from Untreated Human Plasma Samples Followed by HPLC-UV Analysis.

In this article, we proposed a new restricted access molecularly imprinted polymer coated with bovine serum albumin (RAMIP-BSA) to be used in a multidimensional chromatographic system applied for the analysis of six selective serotonin reuptake inhibitors (SSRIs) directly from untreated human plasma samples. Fluoxetine, methacrylic acid and ethylene glycol dimethacrylate were used as the template, functional monomer and cross-linker, respectively. The imprinted polymer was covered with a bovine serum albumin (BSA) layer via the interconnections between the amine groups of the BSA using glutaraldehyde as a cross-linker. The obtained RAMIP-BSA was able to extract the SSRIs directly from the human plasma, while ∼100% of the proteins were excluded from the sample. Selectivity coefficients were calculated for fluoxetine (template) in comparison with venlafaxine, duloxetine, citalopram, fluvoxamine, paroxetine and sertraline, and the values were >1 in all cases, attesting to the presence of binding sites in the imprinted polymer. The method presented analytical ranges from 20 to 500 µg/L and correlation coefficients >0.99 for all of the SSRIs (fluoxetine, venlafaxine, duloxetine, citalopram, fluvoxamine and sertraline). Precision and accuracy presented variation coefficients and relative errors <14.5% and within the range of -19.18 to 3.8%, respectively. In all cases, the apparent recoveries were >85%. The proposed method was able to analyze three samples per hour, and each column was used at least 50 times without any significant changes in its performance.

On-line restricted access molecularly imprinted solid phase extraction of ivermectin in meat samples followed by HPLC-UV analysis.

A new restricted access molecularly imprinted polymer coated with bovine serum albumin (RAMIP-BSA) was synthesized, characterized and used for direct analysis of ivermectin from bovine meat samples, in a two-dimensional liquid chromatography system with UV detection. Ivermectin, 4-vynilpiridine and ethylene glycol dimethacrylate were employed as template, functional monomer and cross-linker, respectively. A BSA layer was cross-linked around the polymer, resulting in a biocompatible chemical barrier able to eliminate about 100% of protein from the samples. Ivermectin was extracted from the minced meat samples through a solvent extraction using methanol:water (70:30, v:v), and the extracts were directly injected into the two-dimensional liquid chromatography system, without any other treatment. Samples, fortified with ivermectin from 50 to 500 µg kg(-1), were used to build the analytical calibration curve (r=0.996). The limit of quantification was 50 µg kg(-1). Precision and accuracy presented variation coefficients, as well as relative errors lower than 17.0% and within -18.5% and 22.0%, respectively.

Dialkyl phosphates determination by gas chromatography: Evaluation of a microwave-assisted derivatization†.

Dialkyl phosphates are organophosphate insecticide metabolites and their urinary analysis is useful for assessing human exposure to these compounds. This study presents a sample preparation method with microwave-assisted derivatization for two dialkyl phosphates to make the process faster before gas chromatographic analysis. The optimized conditions for derivatization procedure were: 250 µL of 2,3,4,5,6-pentafluorobenzyl bromide 3% in acetonitrile for derivative; microwave for 5 min with intensity of 160 W. The electron ionization mass spectrometric analysis was performed using a gas chromatography with mass spectrometry QP-2010 from the Shimadzu(®) equipped with RTx(®) -5MS capillary column. Ions were monitored at selected-ion monitoring mode at m/z 350 for diethyl thiophosphate and m/z 366 for diethyl dithiophosphate. The developed method was linear for both metabolites. The intra-assay precision was the values ranged between 1.1 and 9.1%, for diethyl thiophosphate, and between 4.06 and 6.9%, for diethyl dithiophosphate. The interassay precision showed relative standard deviation between 10.3 and 15.1%, for diethyl thiophosphate and between 4.9 and 11.9%, for diethyl dithiophosphate. The results obtained suggests that derivatization assisted by microwave, before gas chromatography with mass spectrometry analysis, can be applied to monitoring of exposure to organophosphates once is fast, sensible, and precise method to determinate dialkyl phosphates.

Direct doping analysis of beta-blocker drugs from urinary samples by on-line molecularly imprinted solid-phase extraction coupled to liquid chromatography/mass spectrometry.

The use of beta-blockers to enhance performance in some sports is forbidden. Based on this regulation, there is a demand for dynamic analytical procedures for analyzing these compounds quickly and without manual sample preparation. Therefore, the use of a molecularly imprinted polymer (MIP) in a multidimensional liquid chromatographic system coupled to a mass spectrometer provides a good alternative for improving the selectivity and practicality of the beta-blocker analyses, as described in this paper. A water-compatible MIP for oxprenolol was synthesized by the precipitation method, using methacrylic acid as a functional monomer and 2-hydroxyethyl methacrylate and glycerol dimethacrylate as hydrophilic monomers. A column filled with MIP was coupled to an LC-MS/MS instrument under the multidimensional configuration, with 10.0 mmol L(-1) ammonium formate buffer (pH 5.0) as the loading and reconditioning mobile phase and a 0.01% formic acid aqueous solution-methanol (30 : 70 v : v) as the elution mobile phase. The system was used for on-line extraction and quantization of oxprenolol (from 1.0 to 75.0 µg L(-1)), atenolol, propranolol, nadolol, pindolol, labetalol and metoprolol (all from 3.0 to 50 µg L(-1)) simultaneously, from urine samples. The correlation coefficient was higher than 0.99 for all the analytes. Suitable precision and accuracy were obtained.

Quick and simple LC-MS/MS method for the determination of simvastatin in human plasma: application to pharmacokinetics and bioequivalence studies

A simple, rapid, and sensitive method based on liquid chromatography-tandem mass spectrometry for the quantitative determination of simvastatin in human plasma was developed and validated. After a simple extraction with methyl tert-butyl ether, the analyte and internal standard (lovastatin) were analyzed using reverse-phase liquid chromatography, on a Kinetex C18 column (100 × 4.6 mm, 2.6 μm) using acetonitrile: ammonium acetate (2 mM + 0.025 % formic acid) (70: 30, v/v) as a mobile phase in a run time of 3.5 min. Detection was carried out using electrospray positive ionization mass spectrometry in the multiple-reaction monitoring mode. The method was linear over 0.04-40.0 ng/mL concentration range. The mean extraction recovery of simvastatin was 82% (RSD within 15%). Intraday and interday precisions (as relative standard deviation) were all ≤8,7% with accuracy (as relative error) of ±8%. This rapid and reliable method was successfully applied for a bioequivalence study of 40 mg of simvastatin orally disintegrating tablets in 44 healthy volunteers, showing that this method is suitable for the quantification of simvastatin in human plasma samples for pharmacokinetics and bioequivalence studies.

Desenvolveu-e e validou-se um método simples, rápido e sensível baseado na cromatografia líquida acoplada à espectrometria de massas em tandem para a quantificação de sinvastatina em plasma humano. Após um simples preparo de amostras utilizando extração com éter metil-terc-butílico, o analito e seu padrão interno (lovastatina) foram analisados por cromatografia líquida de fase reversa, em uma coluna Kinetex C18 (100 mm x 4,6 mm x 2,6 μm), utilizando uma fase móvel composta de acetonitrila:acetato de amônio (2 mM + 0,025% ácido fórmico) (70:30, v/v) em tempo total de corrida de 3,5 min. A detecção foi realizada por espectrometria de massas utilizando a ionização por electrospray no modo positivo e monitorando os íons pelo sistema de monitoramento de reação múltipla. O método apresentou linearidade na faixa de 0,4 - 40,0 ng/mL. A recuperação média obtida para sinvastatina foi de 82% (DPR menor que 15%). A precisão intradia e interdias (como desvio padrão relativo) foi ≤8,7% com exatidão (como erro relativo) de ± 8%. Este método rápido e confiável foi aplicado com sucesso em um estudo de bioequivalência de comprimidos de desintegração oral de sinvastatina 40 mg em voluntários sadios, mostrando que este método é adequado para a quantificação de sinvastatina em plasma humano para estudos farmacocinéticos e bioequivalência.

Influence of quinidine, cimetidine, and ketoconazole on the enantioselective pharmacokinetics and metabolism of metoprolol in rats.

Metoprolol is a beta-blocker and its racemic mixture is used for the treatment of hypertension. In the present study we investigated the influence of CYP2D and CYP3A on the stereoselective metabolism of metoprolol in rats. Male Wistar rats (n = 6 per group) received racemic metoprolol (15 mg/kg) orally, with or without pretreatment with the CYP inhibitor ketoconazole (50 mg/kg), cimetidine (150 mg/kg), or quinidine (80 mg/kg). Blood samples were collected up to 48 h after metoprolol administration. The plasma concentrations of the stereoisomers of metoprolol, O-demethylmetoprolol (ODM), alpha-hydroxymetoprolol (OHM) (Chiralpak AD column), and metoprolol acidic metabolite (AODM) (Chiralcel OD-R column) were determined by HPLC using fluorescence detection (lambda(exc) = 229 nm; lambda(em) = 298 nm). CYP3A inhibition by ketoconazole reduced the plasma concentrations of ODM and AODM and favored the formation of OHM. CYP2D and CYP3A inhibition by cimetidine reduced the plasma concentrations of OHM and AODM and favored the formation of ODM. The inhibition of CYP2D by quinidine reduced the plasma concentrations of OHM and favored the formation of ODM. In conclusion, the results suggest that CYP3A is involved in the formation of ODM and CYP2D is involved in the formation of AODM.

Enantioselectivity in the pharmacokinetic interaction between fluvastatin and lercanidipine in healthy volunteers.

Hypertension and dyslipidemia are independent risk factors for cardiovascular mortality and are frequently present in the same patient. Fluvastatin (FV), used to reduce cholesterol levels, and lercanidipine (LER), used to control blood pressure, are marketed as racemic mixtures. Therapeutic activities are 30-fold higher for (+)-3R, 5S-FV and 100- to 200-fold higher for S-LER compared with their respective antipodes. The present study describes the enantioselective pharmacokinetic interaction between LER and FV in healthy volunteers. A crossover randomized study was conducted in 3 phases on 8 volunteers treated with a single oral racemic dose of LER (20 mg) or FV (40 mg) or LER plus FV. Serial blood samples were collected from 0 to 24 hours. Plasma concentrations of the LER and FV enantiomers were determined by liquid chromatography/tandem mass spectrometry, and pharmacokinetic parameters were evaluated using the WinNonlin software. The Wilcoxon and Mann-Whitney tests (P < .05) were used to analyze enantiomer ratios and the pharmacokinetic drug interaction. Data are expressed as medians. In monotherapy, the kinetic disposition of both FV and LER was enantioselective. AUC values were significantly higher for (-)-3S,5R-FV than for (+)-3R,5S-FV (358.20 vs 279.68 ng.h/mL) and for S-LER compared with R-LER (13.90 vs 11.88 ng.h/mL). The pharmacokinetic parameters of FV were not enantioselective when combined with LER (AUC: (-)-3S,5R-FV: 325.21; (+)-3R,5S-FV: 316.44 ng.h/mL). There was a significant reduction in S-LER (8.06 vs 13.90 ng.h/mL) and R-LER (6.76 vs 11.88 ng.h/mL) AUC values when FV was coadministered. In conclusion, the interaction between FV-LER might be clinically relevant because AUC values of (+)-3R,5S-FV were increased when LER was coadministered, and AUC values of the 2 LER enantiomers were reduced when FV was coadministered.

Simultaneous analysis of the enantiomers of verapamil and norverapamil in rat plasma by liquid chromatography-tandem mass spectrometry.

An enantioselective micromethod for the simultaneous analysis of verapamil (VER) and norverapamil (NOR) in plasma was developed, validated and applied to the study of the kinetic disposition of VER and NOR after the administration of a single oral dose of racemic-VER to rats. VER, NOR and the internal standard (paroxetine) were extracted from only 100-microL plasma samples using n-hexane and the enantiomers were resolved on a Chiralpak AD column using n-hexane:isopropanol:ethanol:diethylamine (88:6:6:0.1) as the mobile phase. The analyses were performed in the selected reaction monitoring mode. Transitions 456>166 for VER enantiomers, 441>166 for NOR enantiomers and 330>193 for the internal standard were monitored and the method had a total chromatographic run time of 12 min. The method allows the determination of VER and NOR enantiomers at plasma levels as low as 1.0 ng/mL. Racemic VER hydrochloride (10mg/kg) was given to male Wistar rats by gavage and blood samples were collected from 0 to 6.0 h (n=6 at each time point). The concentration of (-)-(S)-VER was three folds higher than (+)-(R)-VER, with an AUC ratio (-)/(+) of 2.66. Oral clearance values were 12.17 and 28.77 L/h/kg for (-)-(S)-VER and (+)-(R)-VER, respectively. The pharmacokinetic parameters of NOR were not shown to be enantioselective.

Stereoselective analysis of metoprolol and its metabolites in rat plasma with application to oxidative metabolism.

We investigated the stereoselective kinetic disposition and metabolism of metoprolol (MET) in rats. The racemic MET (15 mg/kg) was given by oral gavage and blood samples were collected from 0 to 10h (n=6 at each time point). The enantiomeric concentrations of MET and its metabolites alpha-hydroxymetoprolol (alpha-OHM) and O-demethylmetoprolol (ODM) were determined by HPLC using a Chiralpak AD chiral column and fluorescence detection. The pharmacokinetic parameters of unchanged MET and the formation of ODM did not show to be stereoselective. In contrast, the AUC (ng h/mL) of alpha-hydroxymetoprolol isomers were higher to I'R [638.2(525.2-706.2) for 1'R2R and 659.6(580.4-698.1) for 1'R,2S, mean, (95%CI)] than to I'S products [58.3(47.4-66.1) for 1'S,2R and 57.1(44.7-67.9) for 1'S,2S, mean, (95%CI)]. We conclude that the kinetic disposition of unchanged MET and the formation of ODM are not enantioselective in rats but the metabolism of alpha-OHM yields predominantly the 1'R-product.

Enantioselective determination of metoprolol acidic metabolite in plasma and urine using liquid chromatography chiral columns: applications to pharmacokinetics.

Enantioselective separations on chiral stationary phases with or without derivatization were developed and compared for the HPLC analysis of (+)-(R)- and (-)-(S)-metoprolol acidic metabolite in human plasma and urine. The enantiomers were analysed in plasma and urine without derivatization on a Chiralcel OD-R column, and in urine after derivatization using methanol in acidic medium on a Chiralcel OD-H column. The quantitation limits were 17 ng of each enantiomer/ml plasma and 0.5 microgram of each enantiomer/ml urine using both methods. The confident limits show that the methods are compatible with pharmacokinetic investigations of the enantioselective metabolism of metoprolol. The methods were employed in a metabolism study of racemic metoprolol administered to a patient phenotyped as an extensive metabolizer of debrisoquine. The enantiomeric ratio (+)-(R)/(-)-(S)-acid metabolite was 1.1 for plasma and 1.2 for urine. Clearances were 0.41 and 0.25 l/h/kg, respectively, for the (+)-(R)- and (-)-(S)-enantiomers. The correlation coefficients between the urine concentrations of the acid metabolite enantiomers obtained by the two methods were >0.99. The two methods demonstrated interchangeable application to pharmacokinetics.