Organic anion transporter 3- and organic anion transporting polypeptides 1B1- and 1B3-mediated transport of catalposide

Abstract

We investigated the in vitro transport characteristics of catalposide in HEK293 cells overexpressing organic anion transporter 1 (OAT1), OAT3, organic anion transporting polypeptide 1B1 (OATP1B1), OATP1B3, organic cation transporter 1 (OCT1), OCT2, P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP). The transport mechanism of catalposide was investigated in HEK293 and LLC-PK1 cells overexpressing the relevant transporters. The uptake of catalposide was 319-, 13.6-, and 9.3-fold greater in HEK293 cells overexpressing OAT3, OATP1B1, and OATP1B3 transporters, respectively, than in HEK293 control cells. The increased uptake of catalposide via the OAT3, OATP1B1, and OATP1B3 transporters was decreased to basal levels in the presence of representative inhibitors such as probenecid, furosemide, and cimetidine (for OAT3) and cyclosporin A, gemfibrozil, and rifampin (for OATP1B1 and OATP1B3). The concentration-dependent OAT3-mediated uptake of catalposide revealed the following kinetic parameters: Michaelis constant (K m) =41.5 μM, maximum uptake rate (V max) =46.2 pmol/minute, and intrinsic clearance (CL int) =1.11 μL/minute. OATP1B1- and OATP1B3-mediated catalposide uptake also showed concentration dependency, with low CL int values of 0.035 and 0.034 μL/minute, respectively. However, the OCT1, OCT2, OAT1, P-gp, and BCRP transporters were apparently not involved in the uptake of catalposide into cells. In addition, catalposide inhibited the transport activities of OAT3, OATP1B1, and OATP1B3 with half-maximal inhibitory concentration values of 83, 200, and 235 μM, respectively. However, catalposide did not significantly inhibit the transport activities of OCT1, OCT2, OAT1, P-gp, or BCRP. In conclusion, OAT3, OATP1B1, and OATP1B3 are major transporters that may regulate the pharmacokinetic properties and may cause herb-drug interactions of catalposide, although their clinical relevance awaits further evaluation.

Keywords: OAT3; OATP1B1; OATP1B3; catalposide; drug transporters; herb–drug interaction.

Figure 1

Chemical structure of catalposide.

Figure 2

Effects of representative inhibitors on the uptake of catalposide in HEK293 cells expressing (A) organic anion transporter 3 (OAT3), (B) organic anion transporting polypeptide 1B1 (OATP1B1), and (C) OATP1B3 transporters. Notes: Concentrations of 1, 10, and 200 μM probenecid and 10, 100, and 1,000 μM cimetidine and furosemide were used as inhibitors of OAT3. In addition, 0.1, 1, and 10 μM cyclosporin A; 10, 100, and 1,000 μM gemfibrozil; and 1, 10, 100 μM rifampin were used as inhibitors of OATP1B1 and OATP1B3 transporters. Bars represent the means ± standard deviation of three independent experiments. *P<0.01, significantly different compared with control group.

Figure 3

Concentration dependence of the uptake of catalposide (2–500 μM) in HEK293 cells expressing (A) organic anion transporter 3 (OAT3), (B) organic anion transporting polypeptide 1B1 (OATP1B1), and (C) OATP1B3 transporters. Notes: V_max represents the maximum uptake rate, K_m represents the concentration of a substrate that is required for 50% of V_max, Cl_int represents intrinsic clearance, and n is the Hill coefficient. Each data point represents the mean ± standard deviation of three independent experiments.

Figure 4

Inhibitory effect of catalposide on the transport activities of (A) organic anion transporter 3 (OAT3), (B) organic anion transporting polypeptide 1B1 (OATP1B1), (C) OATP1B3, (D) OAT1, (E) organic cation transporter 1 (OCT1), (F) OCT2, (G) P-glycoprotein (P-gp), and (H) breast cancer resistant protein (BCRP). Notes: Probe substrates were used as follows: 0.1 μM [³H]estrone-3-sulfate (ES; a substrate for OAT3, OATP1B1, and BCRP), 0.1 μM [³H]estradiol-17b-D-glucuronide (EG; a substrate for OATP1B3), 1 μM [¹⁴C]para-aminohippuric acid (PAH; a substrate for OAT1), 0.1 μM [³H]methyl-4-phenylpyridinium (MPP⁺; a substrate for OCT1 and OCT2), and 0.1 μM [³H]digoxin (a substrate for P-gp). Data represent the means ± standard deviation of three independent experiments. Data were fitted to an inhibitory effect maximal effect model and the half-maximal inhibitory concentration (IC₅₀) value was calculated.