LC/MS/MS and radioisotope method combined for recognizing the affinity between catalpol and OCT2 transporter

Abstract

More and more herbal medicines are found to be the substrates of drug transporters. In this paper, chromatography/tandem mass spectrometry (LC/MS/MS) combined with radioisotope method was used for the quantification of catalpol, a traditional Chinese medicine, to study the affinity relationship between herbal medicines and transporters. Catalpol uptake experiment was carried out by using several transporters (OAT1, OCT2, OAT3, OATP1B1 and OATP2B1). And samples were precipitated with methanol and quantified with LC/MS/MS. The results show that catalpol has a good affinity with OCT2-transfected S2 cells. After studying drug-drug interaction between catalpol and ¹⁴C-tetraethylammonium (TEA), we found that catalpol is able to facilitate TEA transport mediated by OCT2, suggesting that catalpol could probably be a new promoter of OCT2.

Key words： catalpol; LC/MS/MS; OCT2

Acknowledgments

This study was supported by the National Basic Research Program of China(Grant Nos. 2010CB735602, 2012CB72400-2) and the National Significant New Drug Creation Project Foundation of China (2011ZX09102-009-002, 2012ZX09304-002).

Supporting information

S1 Sample preparation

S1.1 Incubation condition

HEK293 cells were incubated in CO₂ incubator (Thermo Scientific) at 37°C and 5% CO₂. S2 cells were incubated in CO₂ incubator at 33°C and 5% CO₂. Culture solution was DMEM with 10% fetal bovine serum.

S1.2 Difference of 24-well plates

HEK293 and S2 cells were seeded in poly-D-lysine coated 24-well plates (Biocoat, BD) and non-poly-D-lysine coated plates, respectively. The initiation cell concentration was 2 × 10⁵ cells/mL.

S1.3 Post-processing method

After extraction (including cells and methanol), vigorous vortex for 30s and centrifugation at 12000 r∙min^-1 for 5 min, supernatant was separated.

S2 Cell function confirmation

S2.1 Pre-processing method

DMEM solution was disposed and cells were incubated in DPBS buffer (adding MgCl₂ and CaCl₂) at 37°C for 10 min.

During uptake experiment, all dosing volume was 500 µL.

S2.2 Test the function of cell lines

³H-estrone sulfate (50 nmol/L) was investigated for 1 min uptake time mediated by OATP1B1, OATP2B1 and 2 min uptake time mediated by OAT3. For comparison, 30 μmol/L non-radiolabeled estrone sulfate was used as inhibitor for OATP1B1 and 300 μmol/L estrone sulfate was used as inhibitor for OATP2B1 and OAT3. ¹⁴C-PAH (5 μmol/L) was investigated for 2 min uptake time mediated by transporter OAT1. For comparison, 1 mmol/L non-radiolabeled PAH was used as inhibitor. ¹⁴C-TEA (60 μmol/L) was investigated for 2 min uptake time mediated by transporter OCT2. For comparison, 1 mmol/L non radiolabeled TEA was used as inhibitor.

S2.3 Post-processing method

Each well of 24 well plates was added 500 µL 0.1 mol/L NaOH after disposing DPBS. After extraction, each sample was added 3 mL of scintillation solution.

S3 Catalpol uptake experiment

S3.1 Termination and post-process method

All cell samples were washed by 0°C DPBS after uptake experiment and each well of 24 well plates was added 300 µL methanol after disposing DPBS. After extraction (including cells and methanol), votex (30s) and centrifugation (12000 r∙min^-1 for 5 min), supernatant was separated.

S4 Drug-drug interaction

S4.1¹⁴C-TEA uptake in the presence of catalpol or non-radiolabeled TEA

Thirty μmol/L, 100 μmol/L and 300 μmol/L catalpol were added respectively to investigate if there would be any influence to ¹⁴C-TEA uptake. In comparison, 30 μmol/L, 100 μmol/L and 300 μmol/L non-radiolabeled TEA were also added respectively to investigate the influence to ¹⁴C-TEA uptake.

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