Topical bioequivalence of acyclovir creams using dermal microdialysis in pigs: a new model to evaluate bioequivalence for topical formulations.

The aim was to evaluate the bioequivalence of topically applied Acyclovir (ACV) creams using dermal microdialysis (DMD) in a pig model. Three ACV creams (3%), ACV1, ACV2 and ACV3, were topically administrated on the dorsum of pigs, and the DMD sampling technique was used to continuously collect microdialysate. The concentration of ACV in microdialysate was measured by HPLC and the concentration-time profiles were used to calculate pharmacokinetic parameters. The results showed that 90% confidence interval (CI) of the ratio of AUC(0-4 h) of ACV2 and ACV3 was between 88.2 and 105.7%, which was within the acceptance range (80-125%). Ninety percent CI of the ratio of C(max) of ACV2 and ACV3 was between 87.4 and 124.4%, which was within the acceptance range (80-125%). These data indicate that ACV2 and ACV3 used in this study were bioequivalent. This study demonstrates that the pig model coupled with DMD sampling can potentially provide a cost-effective strategy to evaluate topical drug delivery and its associated pharmacokinetic studies.

Determination of clobetasol propionate, tazarotene and tazarotenic acid in Bama mini-pig skin by UPLC-MS/MS: Application to pharmacokinetic and drug-drug interaction studies.

Tazarotene and clobetasol propionate are efficacious for the treatment of psoriasis. The plasma pharmacokinetic assessments of tazarotene or clobetasol propionate have been reported. However, the pharmacokinetic characteristics of tazarotene and clobetasol propionate in skin when used together have not been studied. In the present study, sensitive and rapid methods were developed for the determination of clobetasol propionate, tazarotene and its active metabolite tazarotenic acid in Bama mini-pig skin by UPLC-MS/MS. After homogenization and pretreatment of skin samples, the separation was performed on a WondaSiL C18 column (4.6 × 150 mm, 5 µm) for tazarotene and clobetasol propionate. The separation of tazarotenic acid was achieved on a BDS HYPERSIL C18 column (4.6 × 100 mm, 2.4 µm). All the analytes were quantified with positive electrospray ionization and multiple reactions monitoring mode. The assay was validated in the range of 22-1111 ng/g for tazarotene and clobetasol propionate, 2-111 ng/g for tazarotenic acid in skin samples. The methods were fully validated to meet the requirements for bioassay in accuracy, precision, recovery, reproducibility, stabilities and matrix effects, and successfully applied to evaluate the novel combination ointment of tazarotene and clobetasol propionate. The obtained intradermal content-time curves characterized the dermal absorption and metabolism features of the combination ointment. It was also found that there was no significant drug-drug interaction trend between tazarotene and clobetasol propionate. The obtained results would be essential for the development and clinical applications of this novel combination ointment.

[Effects of triptolide on the production of interferon-gamma in human peripheral blood mononuclear cell and phosphorylation of signal transducer and activator of transcription-1 and production of interleukin-8].

OBJECTIVE: To investigate the effects of triptolide on the production of interferon-gamma (IFN-gamma) in human peripheral blood mononuclear cell (PBMC) and interleukin-8 (IL-8) in HaCaT keratinocytes and phosphorylation of signal transducer and activator of transcription-1 (STAT1) of IFN-gamma signal transduction pathways in HaCaT cells. METHODS: Human PBMC was induced by phytohaemagglutinin (PHA-L) and HaCaT cells were stimulated by recombinant human IFN-gamma (rhIFN-gamma). The productions of IFN-gamma and IL-8 in cells were detected by ELISA. The expression of STAT1 and its phosphorylation were analyzed by Western blot. RESULTS: Triptolide inhibited the production of IFN-gamma in human PBMC induced by PHA-L in a dose-dependent manner (P < 0.05, P < 0.01, P < 0.001) and the 50% inhibitory concentration (IC50) value was 5.96 x 10(-11) mol/L. IL-8 production in HaCaT cells induced by rhIFN-gamma in vitro was also inhibited by triptolide (P < 0.001) and the IC50 value was about 1.15 x 10(-13) mol/L. The expressions of phosphorylated STAT1 in HaCaT cells stimulated by rhIFN-gamma was inhibited by triptolide (P < 0.01) and the IC50 value was about 9.45 x 10(-11) mol/L. CONCLUSION: Triptolide can inhibit the production of IFN-gamma in human PBMC and downregulate IL-8 level in HaCaT keratinocytes induced by rhIFN-gamma. Triptolide can inhibit the phosphorylations of STAT1 of IFN-gamma signal pathway in HaCaT keratinocytes stimulated by IFN-gamma.

Percutaneous penetration kinetics of lidocaine and prilocaine in two local anesthetic formulations assessed by in vivo microdialysis in pigs.

The aim of this study was to characterize and compare the percutaneous penetration kinetics of lidocaine (L) and prilocaine (P) in two local anesthetic formulations by in vivo microdialysis coupled with HPLC. The microdialysis system for studying lidocaine and prilocaine was calibrated by a no-net-flux method in vitro and retrodialysis method in vivo, respectively. A dosage of 0.2 g/cm2 of an in-house P-L formulation (2.5% lidocaine and 2.5% prilocaine, methylcellulose-based) and commercially available Eutectic Mixture of Local Anesthesia (EMLA, 2.5% lidocaine and 2.5% prilocaine, carbopol-based) was separately but symmetrically applied in the dorsal region of pigs. Saline (0.9%, w/v) was perfused into the linear microdialysis probe at a flow rate of 1.5 microl/min. Dialysate was collected upon topical application up to 6 h at 20-min intervals and assessed by HPLC. The results demonstrated the area under the concentration-time curve (AUC(0-6 h)) of lidocaine and prilocaine in EMLA was 71.95+/-23.36 microg h/ml and 38.01+/-14.8 microg h/ml, respectively, in comparison to 167.11+/-56.12 microg h/ml and 87.02+/-30.38 microg h/ml in the P-L formulation. The maximal concentrations (Cmax) of lidocaine and prilocaine in the dermis were 29.2+/-9.08 microg/ml and 16.54+/-5.31 microg/ml in EMLA and 80.93+/-17.98 microg/ml and 43.69+/-12.87 microg/ml in the P-L formulation, respectively. This study indicates a well-calibrated microdialysis system can provide vital real-time information on percutaneous drug delivery and specifically a methylcellulose-based P-L formulation can increase percutaneous absorption of both lidocaine and prilocaine in pigs compared to carbopol-based EMLA.

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.