An LC/MS quantitative and microdialysis method for cyclovirobuxine D pharmacokinetics in rat plasma and brain: The pharmacokinetic comparison of three different drug delivery routes.

To explore the brain-targeting of cyclovirobuxine D(CVB-D) after administered intranasally, the pharmacokinetics of CVB-D via three different drug delivery routes: intragastric (i.g.), intranasal (i.n.), and intravenous (i.v.) in rat brain and blood was compared. Firstly, an in vivo microdialysis method for sampling CVB-D in both plasma and brain of the rat was established. Secondly, a liquid chromatography-tandem mass spectrometry method has been developed and validated for determination of CVB-D in microdialysis samples. For plasma and brain microdialysis samples, liquid-liquid extraction was used and donepezil was chosen as internal standard. Both were followed by HPLC separation and positive electrospray ionization tandem mass spectrometry detection (ESI-MS/MS). Chromatographic separation was achieved on a agilent C18 column with a mobile phase of methanol-water (50:50, v/v) (pH 3.2) containing 0.1% formic acid and 5mM ammonium acetate. Mass spectrometric detection in the positive ion mode was carried out by selected reaction monitoring (MRM) of the transitions at m/z 403.4→372.3 for CVB-D and m/z 380.2→243.1 for donepezil (IS). Good linearities were obtained in the range of 10-4000ng/mL in rat microdialysates for CVB-D. The lowest limit of quantitation was 5ng/mL, with an extraction recovery >75%, and no significant matrix effects. Intra- and inter-day precisions were all <15% with accuracies of 97.26-116.20%. All of which proved that the established method was successfully applied to the pharmacokinetic study of CVB-D. Simultaneously, brain uptake and pharmacokinetic studies were performed by determination of CVB-D concentration in blood and brain respectively for CVB-D i.g., i.n. and i.v.. Results showed that the intranasal CVB-D could improve brain targeting and had advantages for direct nose to brain transport of CVB-D when compared with injection and oral delivery routes, which indicates that intranasal administration of CVB-D could be a promising approach for the treatment of cerebrovascular disease.

[Determination of six C-Glycoside flavones and antitumor activity of water-soluble total flavonoids from Isodon lophanthoides var. gerardianus].

This research established an HPLC method for determination of six C-Glycoside flavones of warer-soluble total flavonoids from Isodon lophanthoides var. gerardianus (Benth.) H. Hara, and studied the antitumor activity of the warer-soluble total flavonoids. The HPLC system consisted of Kromasil 100-5 C18 (4.6 mm x 250 mm, 5 microm) column and a solution system of methanol, acetonitrile and 0.5% formic acid gradient elution at a flow rate of 0. 8 mL x min(-1) and the wavelength of detector was at 334 nm. The column temperature was 25 degrees C. The antitumor activity of water-soluble flavonoids was assayed using HepG2 cell as the tested cell. The linear ranges of vicenin II, vicenin III, isoschaftoside, schaftoside, vitexin, 6, 8-di-C-a-L-arabinosylapigenin were 0.25-2.53, 0.12-1.20, 0.37-3.69, 0.16-1.63, 0.19-1.92, 0.14-1.42 microg, respectively. The average recoveries (n = 6) were 99.6% (RSD 0.87%), 100.2% (RSD 2.0%), 99.6% (RSD 1.8%), 97.9% (RSD 1.5%), 98.8% (RSD 1.2%), 98.6% (RSD 1.2%), respectively. After exposure in 24, 48, 72 h, the total flavonoids showed inhibitory effect on the proliferation of HepG2 cells with IC50 as the evaluation index, the IC50 values of 1.89, 1.71, 1.51 g x L(-1), respectively. The method is quick, simple and accurate with good re- producibility, and can be used for determination of vicenin II, vicenin III, isoschaftoside, schaftoside, vitexin, 6, 8-di-C-a-L-arabino- sylapigenin in the warer-soluble total flavonoids from L lophanthoides var. gerardianus. The warer-soluble total flavonoids from L lophanthoides have inhibitory effect on the proliferation of HepG2 cells.

Evaluation of a 13-hexyl-berberine hydrochloride topical gel formulation.

13-hexyl-berberine hydrochloride (HB-13) is a derivative from berberine which finds widespread applications in the treatment of infectious pathogens including fungi, bacteria, parasites and viruses. As our continuing efforts for treatment of herpes simplex virus (HSV), we studied the topical delivery and safety of HB-13 in a gel formulation (0.5%) in a pig model. Our studies demonstrated the maximal HB-13 concentration was 2.51 µg/mL, which was more than the half maximal inhibitory concentration (IC50) as we previously reported. In addition, there was no sign of irritation or histological aberrance for stripped skin continuously applied with 0.5% HB-13 gel for 21 days. In conclusion, 0.5% HB-13 gel can achieve effective anti-HSV concentration in the dermis and it is safe to use.

Effects of tacrolimus on IFN-γ signaling in keratinocytes: possible mechanisms by which tacrolimus affects IFN-γ-dependent skin inflammation.

Interferon-gamma (IFN-γ) signaling in keratinocytes plays an important role in IFN-γ-induced skin inflammation. A novel tacrolimus topical ointment has shown remarkable efficacy in treating skin inflammation. This study explored the mechanism of tacrolimus-modulated IFN-γ signal transduction in HaCaT keratinocytes and the effects of tacrolimus on IFN-γ-associated cytokine production in HaCaT cells. Tacrolimus down-regulated the recombinant human IFN-γ (rhIFN-γ)-induced expression of IFN-γ receptor α (IFN-γRα). The IFN-γ induced expression of phosphorylated Janus kinase 2 (pJAK2) and phosphorylated signal transducer and activator of transcription-1 (pSTAT-1) was also inhibited by tacrolimus. Tacrolimus up-regulated the IFN-γ-induced expression of suppressor of cytokine signaling-1 (SOCS-1). Tacrolimus was also demonstrated to down-regulate IFN-γ-induced the secretion of chemotactic factor CXCL-8 and the expression of intercellular adhesion molecule-1 and human leucocyte antigen HLA-DR. The findings in this work indicate that the direct effects of tacrolimus on IFN-γ signaling in keratinocytes may contribute to its therapeutic efficacy as a topical ointment in the treatment of IFN-γ-dependent skin inflammation.

Inhibition of the nuclear factor-kappaB signaling pathway by leflunomide or triptolide also inhibits the anthralin-induced inflammatory response but does not affect keratinocyte growth inhibition.

We performed this study to determine the relationship between activation of nuclear factor (NF)-kappaB and inhibition of keratinocyte growth by anthralin, which not only might be useful for a better understanding of the role of NF-kappaB in the pathogenesis of psoriasis, but also indicate whether the inflammatory reaction induced by anthralin is inseparable from its antipsoriatic activity. The involvement of NF-kappaB was assessed using the antipsoriatic drugs leflunomide and triptolide (T0) as effectors, since they can inhibit NF-kappaB activation induced by anthralin. The results showed that the inhibition of keratinocyte growth by anthralin was not related to the activation of NF-kappaB. Using sodium salicylate, a known NF-kappaB inhibitor, further confirmed this conclusion. Thus it might be possible to inhibit the inflammatory response induced by anthralin via repression of NF-kappaB activation. We found that leflunomide or T0 could significantly inhibit the mRNA overexpression of interleukin-8 and intercellular adhesion molecule-1 in keratinocytes induced by anthralin. Taken together, our data indicate that the growth inhibition of anthralin is related to the NF-kappaB-independent signaling pathway, and that leflunomide or T0 could control proinflammatory cytokine expression induced by anthralin via inhibiting the activation of NF-kappaB.