Saikosaponin B2 Suppresses Inflammatory Responses Through IKK/IκBα/NF-κB Signaling Inactivation in LPS-Induced RAW 264.7 Macrophages.

Bupleurum falcatum (Umbelliferae) have been widely used to treat inflammatory diseases as traditional medicines in East Asian region. Although saikosaponins are main bioactive molecules of B. falcatum, there is little information on bioactivity of saikosaponin B2 (SSB2). This study was conducted to assess the anti-inflammatory activities and the involved mechanisms of SSB2 in LPS-induced RAW 264.7 macrophages. SSB2 suppressed the releases of nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor α (TNF-α), interleukins (IL)-6, and IL-1ß by suppressing mRNA levels of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, IL-1ß, and IL-6 in LPS-induced macrophages. SSB2 blocked LPS-induced DNA binding and nuclear factor kappa B (NF-κB) transcriptional activity by inhibiting nuclear translocation p65 and p50, inhibitory κBα (IκBα) degradation, and IκB kinase ß (IKKß) phosphorylation and activity. In IKKß-overexpressing cells, SSB2 significantly suppressed IKKß-dependent NF-κB transcriptional activity. Moreover, SSB2 reduced phosphorylation of p38 and extracellular signal-regulated kinase1/2 (ERK1/2). SSB2 effectively inhibits LPS-induced pro-inflammatory mediator releases by interfering with IKKß and IκBα activation, thus preventing NF-κB activation. Our data indicates that SSB2 could be a potential therapeutic application for inflammation-associated diseases.

Preparation and evaluation of enteric-coated delayed-release pellets of duloxetine hydrochloride using a fluidized bed coater.

In this study, the enteric-coated delayed-release pellets of duloxetine hydrochloride (DLX) were formulated using a fluidized bed coater. Three separate layers, the drug layer, the barrier layer, and the enteric layer, were coated onto inert core pellets. Among the three formulations (F1-F3), the dissolution profiles of formulation F2 were most similar to those of the marketed product, with similarity and difference factors of 83.99 and 3.77, respectively. In addition, pharmacokinetic parameters of AUC, C(max), T(max), t(1/2), K(el), and MRT of DLX for the developed formulation (F2) did not differ significantly from those for the marketed product in beagle dogs, suggesting that they were bioequivalent. Our results demonstrated that the in vitro dissolution data resembled the in vivo performance of the drug. Therefore, this study has a positive scope for further scale up and development of the formulation for achievement of the generic product.

Enhanced oral bioavailability of paclitaxel by solid dispersion granulation.

The main objective of this study was to develop novel orally administrable tablets containing solid dispersion granules (SDG) of amorphous paclitaxel (PTX) prepared by fluid bed technology, and to evaluate its in vitro dissolution and in vivo pharmacokinetics (PK) in beagle dogs. The SDG were prepared using optimized composition by fluid bed technology, and characterized for solid-state properties. The release study of SDG tablet (SDG-T) in simulated gastric fluid showed a rapid release of PTX, reaching maximum dissolution within 20 min. Finally, the PK profile of SDG-T and a reference formulation Oraxol™ (oral solution formulation used in Phase I clinical study) at a dose of 60 mg orally with co-administration of P-gp inhibitor HM38101, and Taxol® at a dose of 10 mg intravenously (i.v.) was investigated in beagle dogs. The mean absolute BA% of PTX following SDG-T and Oraxol™ solution was 8.23 and 6.22% in comparison to i.v. administration of Taxol®. The relative BA% of PTX from SDG-T in comparison to Oraxol™ solution was 132.25% at a dose of 60 mg following oral administration. In conclusion, we have successfully prepared PTX tablets with solid dispersion granules (SDG) of amorphous PTX using fluid bed technology that could provide plasma PTX concentration in the range of 10-150 ng/mL for a period of 24 h following oral administration in dogs with a P-gp inhibitor. Hence, this could be a promising formulation for PTX oral delivery and could be used in our intended clinical studies following pre-clinical efficacy studies.

Sildenafil vaginal suppositories: preparation, characterization, in vitro and in vivo evaluation.

AIM: The main objective was to investigate the in vitro release profile/kinetics, and in vivo plasma pharmacokinetics (PK) and organ biodistribution (BD) of the prepared sildenafil vaginal suppositories (SVS). METHODS: Suppositories containing 25 mg of sildenafil were prepared by the cream melting technique using Witepsol H-15 as a suppository base. The suppositories were characterized for weight variation, content uniformity, hardness, disintegration time and crystallinity change. The in vitro dissolution in pH 4.5, and in vivo plasma PK and organ BD of sildenafil from SVS in female Sprague Dawley rats, were also investigated. RESULTS: The mean weight variation, content uniformity, hardness and disintegration time of the prepared SVS were 1.127 ± 0.020 g, 98.25 ± 2.50%, 2.5 ± 0.08 kg and 9 ± 1.0 min, respectively. The release of sildenafil from the SVS was more than 90% at 30 min, with a release kinetic of Hixson--Crowell model and non-Fickian diffusion (n = 0.464). The plasma PK study demonstrated a significantly lower Cmax (∼10 times) and AUC0-24 h (∼13 times) of sildenafil in plasma following intravaginal (IVG) administration of suppositories compared to oral (PO) administration of sildenafil solution. Nevertheless, the organ BD study showed a phenomenally higher Cmax (∼40 times) and AUC0-24 h (∼20 times) of sildenafil in uterus following IVG administration of suppositories than PO administration of sildenafil solution. CONCLUSION: This study demonstrated enhanced sildenafil exposure in the uterus following IVG administration of SVS, which could be used to target the uterus for therapeutic benefits.

Zanamivir oral delivery: enhanced plasma and lung bioavailability in rats.

The objective of this study was to enhance the oral bioavailability (BA) of zanamivir (ZMR) by increasing its intestinal permeability using permeation enhancers (PE). Four different classes of PEs (Labrasol(®), sodium cholate, sodium caprate, hydroxypropyl ß-cyclodextrin) were investigated for their ability to enhance the permeation of ZMR across Caco-2 cell monolayers. The flux and Papp of ZMR in the presence of sodium caprate (SC) was significantly higher than other PEs in comparison to control, and was selected for further investigation. All concentrations of SC (10-200 mM) demonstrated enhanced flux of ZMR in comparison to control. The highest flux (13 folds higher than control) was achieved for the formulation with highest SC concentration (200 mM). The relative BA of ZMR formulation containing SC (PO-SC) in plasma at a dose of 10 mg/kg following oral administration in rats was 317.65% in comparison to control formulation (PO-C). Besides, the AUC0-24 h of ZMR in the lungs following oral administration of PO-SC was 125.22 ± 27.25 ng hr ml(-1) with a Cmax of 156.00 ± 24.00 ng/ml reached at 0.50±0.00 h. But, there was no ZMR detected in the lungs following administration of control formulation (PO-C). The findings of this study indicated that the oral formulation PO-SC containing ZMR and SC was able to enhance the BA of ZMR in plasma to an appropriate amount that would make ZMR available in lungs at a concentration higher (>10 ng/ml) than the IC50 concentration of influenza virus (0.64-7.9 ng/ml) to exert its therapeutic effect.

An optimized analytical method of fluconazole in human plasma by high-performance liquid chromatography with ultraviolet detection and its application to a bioequivalence study.

A sensitive and accurate HPLC-UV method for the quantification of fluconazole (FLA) level in human plasma has been developed. The sample was prepared by one-step liquid-liquid extraction (LLE) of FLA from plasma using dichloromethane. Phenacetin was used as the internal standard. The chromatographic retention times of FLA and phenacetin were 4.6 and 8.3 min, respectively. The lower limit of quantitation (LLOQ) was 0.05 microg/mL, and no interferences were detected in the chromatograms. The devised HPLC-UV method was validated by evaluating its intra- and inter-day precisions and accuracies in a linear concentration range between 0.05 and 10.00 microg/mL. The devised method was successfully applied to a bioequivalence studies involving the oral administration of a single 150 mg FLA tablet and 3 x 50 mg FLA capsules in healthy Korean male volunteers.

Anti-inflammatory effect of buddlejasaponin IV through the inhibition of iNOS and COX-2 expression in RAW 264.7 macrophages via the NF-kappaB inactivation.

Buddlejasaponin IV isolated from Pleurospermum kamtschatidum is an anti-inflammatory compound that inhibits NO, PGE(2) and TNF-alpha production. Here, we studied the mode of action of this compound. Buddlejasaponin IV (2.5-10 microM) reduced lipopolysaccaride (LPS (1 microg ml(-1)))-induced levels of iNOS and COX-2 at the protein levels, and iNOS, COX-2, TNF-alpha, interleukin (IL)-1beta and IL-6 mRNA expression in RAW 264.7 macrophages in a concentration-dependent manner, as determined by Western blotting and RT-PCR, respectively. Buddlejasaponin IV inhibited the LPS-induced activation of nuclear factor-kappaB (NF-kappaB), a transcription factor necessary for proinflammatory mediators, iNOS, COX-2, TNF-alpha, IL-1beta and IL-6 expression. This effect was accompanied by a parallel reduction in IkappaB-alpha degradation and phosphorylation, and by the nuclear translocation of the NF-kappaB p65 subunit. The effects of buddlejasaponin IV on acute phase inflammation were studied on serotonin- and carrageenan-induced paw edema. The antiedematous effect of buddlejasaponin IV was compared with 10 mg kg(-1) of indomethacin p.o. Maximum inhibitions of 26 and 41% were noted at a dose of 20 mg kg(-1) for serotonin- and carrageenan-induced paw edema, respectively. The analgesic effect of buddlejasaponin IV was evaluated using acetic acid-induced writhing and hot-plate tests. Buddlejasaponin IV (10 and 20 mg kg(-1), p.o.) was found to have a marked analgesic effect in both models. These results suggest that the inhibitions of the expressions of iNOS, COX-2, TNF-alpha, IL-1beta and IL-6 by blocking NF-kappaB activation, are responsible for the anti-inflammatory effects of buddlejasaponin IV isolated from P. kamtschatidum.

Rapid and highly sensitive liquid chromatography/electrospray ionization tandem mass spectrometry method for the quantitation of buspirone in human plasma: application to a pharmacokinetic study.

A rapid and sensitive method for the quantitation of buspirone in human plasma by liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was developed. Plasma samples were treated by liquid-liquid extraction with methyl tert-butyl ether (MTBE). The chromatographic separation was performed isocratically on a reversed-phase Shiseido C18 column (50 mm x 2.0 mm, 3 microm) with a mobile phase of acetonitrile/0.1% acetic acid (1:1, v/v). The acquisition was performed in multiple reaction monitoring (MRM) mode, monitoring the transitions m/z 386 --> 122 for buspirone and m/z 409 --> 238 for amlodipine (the internal standard). The method was validated to determine its specificity, recovery, limit of quantitation, accuracy and precision. The lower limit of quantitation was 0.02 ng/mL with a relative standard deviation of less than 10%. The present method provides an accurate, precise and sensitive tool for buspirone and was successfully applied to a pharmacokinetic study in eight subjects.

Simple method for the assay of clindamycin in human plasma by reversed-phase high-performance liquid chromatography with UV detector.

A rapid and simple high-performance liquid chromatography (HPLC) method was developed and validated for the quantification of clindamycin in human plasma. After precipitation with 50% trichloroacetic acid (TCA) containing the internal standard, propranolol, the analysis of the clindamycin level in the plasma samples was carried out using a reverse-phase cyano (CN) column with ultraviolet detection (204 nm). The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile-distilled water-7.6 mm tetramethylammonium chloride (TMA) (60:40:0.075, v/v/v), adjusted to pH 3.2. The proposed method was specific and sensitive with a lower limit of quantitation (LLOQ) of 0.2 microg/mL. This HPLC method was validated by examining the precision and accuracy for inter- and intraday analysis in the concentration range 0.2-20.0 microg/mL. The relative standard deviations (RSD) in the inter- and intraday validation were 6.1-14.9 and 6.0-16.1%, respectively. In the stability test, clindamycin was found to be stable in human plasma during the storage and assay procedure. The present HPLC method was applied to the analysis of samples taken up to 12 h after a single oral administration of clindamycin in healthy volunteers.

Quantification of propiverine by liquid chromatography/electrospray tandem mass spectrometry: application to a bioequivalence study of two formulations in healthy subjects.

Here we report on the development and validation of a sensitive and rapid reversed-phase liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantitative determination of propiverine in human plasma. After adding an internal standard (oxybutynin chloride) to human plasma, samples were extracted using n-hexane/ethylacetate (8:2, v/v). Compounds extracted were analyzed by reversed-phase high-performance liquid chromatography (HPLC) with multiple reaction monitoring (MRM) mode for analyte detection. This method for determination of propiverine proved accurate and reproducible, with a limit of quantitation of 0.5 ng/ml in human plasma. The standard calibration curve for propiverine was linear (r2=0.9988) over the concentration range 0.5-1000.0 ng/ml in human plasma. The intra- and inter-day precision over this concentration range was lower than 8.66% (relative standard deviation, %R.S.D.), and accuracy was between 99.46 and 109.41%, respectively. This method was successfully applied to a bioequivalence study of two propiverine hydrochloride tablet formulations (20 mg) in 24 healthy subjects after a single administration.