Qualitative and quantitative analysis of Arnebiae Radix and Dictamni Cortex and efficacy study of herbal extracts on allergic contact dermatitis using 3D human reconstructed epidermis.

Objective: To evaluate the quality of Arnebiae Radix (AR) and Dictamni Cortex (DC) and study the efficacy of herbal extracts of these two herbs on the treatment of allergic contact dermatitis (ACD). Methods: Qualitative and quantitative analysis of effective components was performed using High Performance Thin Layer Chromatography (HPTLC), High Performance Liquid Chromatography (HPLC), and HPLC-Quadrupole Time of Flight-Mass Spectrometry (HPLC-QTOF-MS). In vitro allergic ACD 3D model was established by incubating 3D reconstructed human epidermis (RHE) with skin sensitizer, potassium dichromate. A total of 65 gene expression that were associated with ACD, which included 24 antioxidant responsive element (ARE) and 41 SENS-IS genes were quantified by qRT-PCR. More than or equal to 10 ARE genes and 18 SENN-IS genes were induced by 1.3-fold, demonstrating the successful establishment of in vitro ACD model. Oil extracts of AR and DC were applied on the in vitro ACD model to study the efficacy. Results: Batch 3 of AR and batch 2 of DC showed presence of all active ingredients with the highest concentrations. Active ingredients of the herbs were extracted using a special oil and formulated into herbal oil extracts. The herbal oil extracts were able to down regulate the induced genes in the in-vitro ACD skin model, bringing the tissue back to homeostatic status. Conclusion: The oil extracts showed the potent efficacy of using AR and DC in ACD treatment. The combination study will be done to optimize the formulation ratio which will be developed into a topical cream.

Directional trans-epithelial transport of organic anions in porcine LLC-PK1 cells that co-express human OATP1B1 (OATP-C) and MRP2.

The transcellular transport of many compounds, which cannot readily cross the lipid bilayer, is mediated by drug uptake and efflux transporters. Human OATP1B1 and MRP2 have been implicated in the hepato-biliary transport of many endogenous and exogenous compounds. Here, we have established epithelial porcine kidney LLC-PK1 derived cell lines, that express both transporters in a polarized fashion, as a model to predict hepato-biliary transport. Immunological identification of OATP1B1 in the recombinant cell lines was greatly facilitated by its C-terminal tagging with a peptide sequence derived from hemagglutinin (HA) avoiding the generation of OATP1B1 specific antibodies. Importantly, the tag did not interfere with the functionality of the transporter. Compared to LLC-PK1 cells and cells which expressed only OATP1B1, the cell line that co-expressed MRP2 and OATP1B1 displayed high directional basolateral-to-apical transport of 17 beta-estradiol-17 beta-glucuronide and estrone-3-sulfate. Dehydroepiandrosterone sulfate already displayed a significant basolateral-to-apical transport in the parental cell line, which was further stimulated upon expression of both transporters. Transcellular flux of all steroid conjugates in the opposite direction (apical-to-basolateral) was much lower. By employing this cellular model we were able to demonstrate for the first time that OATP1B1 together with MRP2 mediates the trans-cellular transport of rifampicin. It is anticipated that the models established herein will greatly facilitate the identification of transporters involved in the disposition of novel drug candidates.

Endogenous drug transporters in in vitro and in vivo models for the prediction of drug disposition in man.

The epithelial canine and porcine kidney cell lines MDCK, MDCKII and LLC-PK1, respectively are employed to establish recombinant models of drug transport. Endogenous drug carriers in these cells may contribute to the activities of recombinant drug transporters, thus making it difficult to assess their properties. We analysed the expression of endogenous transporters in these cell lines by RT-PCR and by determining drug transporter activities. Concerning drug efflux, multidrug resistance protein 1 (MDR1) and MRP1 mRNAs were found in all lines. MRP2 mRNA was expressed in all cell lines except MDCK. Transepithelial transport of vinblastine and its modulation by a MDR1-specific inhibitor or by the MDR1- and MRP-inhibitor verapamil, indicated that MDCKII cells have, in comparisons to the other cell lines, relatively high levels of functional MDR1 while vinblastine transport in MDCK cells is likely to be mediated more by MRP1. Notably, LLC-PK1 cells displayed little activity attributable to either MDR1 and MRP1, thus making them suitable for the expression of these efflux pumps. Of the drug uptake carriers, OATP-A mRNA was only expressed in MDCK cells. OATP-C mRNA was barely detectable in MDCK cells and absent in MDCKII and LLC-PK1 cells. In agreement with transcriptional profiling, the OATP-mediated uptake of either estradiol-glucuronide or estrone-sulfate was either absent or barely detectable in all cell lines thus implying that they are suitable to establish recombinant models for human OATP's. Transcriptional profiling was also performed on porcine and canine tissues and revealed that MRP1 was expressed in canine but not in human or porcine liver, whereas surprisingly OATP-C was expressed in canine kidney but only in human and porcine liver. The findings presented are relevant to the use of porcine and canine models for drug disposition.