Characterization of four unknown impurities in azithromycin and erythromycin imino ether using two-dimensional liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry and nuclear magnetic resonance.

RATIONALE: A simple and sensitive method was developed for the separation and characterization of four unknown impurities in azithromycin and erythromycin imino ether using two-dimensional liquid chromatography coupled to high-resolution quadrupole time-of-flight mass spectrometry (2D LC/QTOFMS) with positive and negative electrospray ionization. METHODS: The chromatographic separation in the first dimension was performed with a Waters Xbridge RP18 column in gradient mode using binary mobile phase: (A) phosphate buffer (pH 8.2)-acetonitrile (47:53, v/v) and (B) water-acetonitrile (90:10, v/v). In the second dimension, the chromatographic separation was performed using a Shimadzu Shim-pack GISS C18 column with volatile mobile phases: (A) ammonium formate solution (10 mM) and (B) methanol. RESULTS: The molecular formulae and structures of the four impurities were deduced based on the LC/MS/MS data, and further confirmed using 1 H NMR, 13 C NMR, 1 H-1 H COSY, HSQC and HMBC NMR spectra after semi-preparative isolation of impurities. In addition, the mechanism for the formation of the impurities was also proposed. CONCLUSIONS: The contradiction between the non-volatile salt mobile phase and mass spectrometry was solved by means of a multiple heart-cutting 2D LC approach and on-line desalination technology. Four impurities were separated and characterized. These results could further improve the method of official monographs in pharmacopoeias and guides to improve the process of reducing impurity content.

NF-κB dependent up-regulation of TRPC6 by Aß in BV-2 microglia cells increases COX-2 expression and contributes to hippocampus neuron damage.

The deposition of amyloid ß-protein (Aß) has been involved in neurodegeneration of Alzheimer's disease (AD). Besides Aß plaques and neuronal loss, microglia activation is also common in AD patient brains, suggesting its important role in the pathogenesis of AD. Although activation of microglia by Aß plaques has been demonstrated, the mechanism underlying it is still largely unclear. Here, we found that TRPC6 has a crucial role in microglia activation by Aß. Aß up-regulates the level of TRPC6 via NF-κB in BV-2 microglia and increases the expression of pro-inflammatory factors and oxidative enzyme, COX-2. Knock-down of TRPC6 reduces the Aß-induced expression of pro-inflammatory factors and COX-2 and the damage of hippocampus neurons. Furthermore, inhibition of COX-2 also protects hippocampus neurons from Aß-induced inflammatory damage. Collectively, our studies suggest that Aß increase the expression of TRPC6 via NF-κB in BV-2 microglia and promotes the production of COX-2, which induces hippocampus neuron damage.

Anti-Inflammatory Effects of Schisandrin B on LPS-Stimulated BV2 Microglia via Activating PPAR-γ.

Schisandrin B (Sch B), a dibenzocyclooctadiene lignan isolated from Schisandra chinensis (Turcz.) Baill, has been shown to have anti-inflammatory effect. The purpose of this study was to evaluate the effect of Sch B on LPS-induced inflammation in microglia and to investigate the molecular targets of Sch B. BV2 cells were stimulated by LPS in the presence or absence of Sch B. The results showed that the levels of TNF-α, IL-6, IL-1ß, and PGE2 upregulated by LPS were significantly suppressed by Sch B. LPS-induced NF-κB activation was also inhibited by Sch B. Furthermore, Sch B was found to upregulate the expression of PPAR-γ in a concentration-dependent manner. In addition, the inhibition of Sch B on TNF-α, IL-6, IL-1ß, and PGE2 production were reversed by PPAR-γ antagonist GW9662. In conclusion, these results suggested that Sch B inhibited LPS-induced inflammatory response by activating PPAR-γ.

Flunarizine blocks voltage-gated Na(+) and Ca(2+) currents in cultured rat cortical neurons: A possible locus of action in the prevention of migraine.

Although flunarizine (FLN) has been widely used for migraine prophylaxis with clear success, the mechanisms of its actions in migraine prophylaxis are not completely understood. It has been hypothesized that migraine is a channelopathy, and abnormal activities of voltage-gated Na(+) and Ca(2+) channels might represent a potential mechanism of cortical hyperexcitability predisposing to migraine. The aim of the present study was to investigate the effects of FLN on Na(+) and Ca(2+) channels of cultured rat cortical neurons. Sodium currents (I(Na)) and calcium currents (I(Ca)) in cultured rat cortical neurons were monitored using whole-cell patch-clamp recordings. Both I(Na) and I(Ca) were blocked by FLN in a concentration-dependent manner with IC(50) values of 0.94µM and 1.77µM, respectively. The blockade of I(Na) was more powerful at more depolarizing holding potentials. The steady-state inactivation curve of I(Na) was shifted towards more hyperpolarizing potentials by FLN. FLN significantly delayed the recovery from fast inactivation of I(Na). Furthermore, the action of FLN in blocking I(Na) was enhanced at higher rates of channel activation. Blockades of these currents might help explain the mechanism underlying the preventive effect of FLN on migraine attacks.