Chunshan Gui

Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family

1. The organic anion transporting polypeptides (humans OATP; other species Oatp) belong to the SLCO gene superfamily of transporters and are twelve transmembrane domain glycoproteins expressed in various epithelial cells. Some OATPs/Oatps are expressed in a single organ, while others are expressed ubiquitously. 2. The functionally characterized members mediate sodium-independent transport of a variety of structurally independent, mainly amphipathic organic compounds, including bile salts, hormones and their conjugates, toxins, and various drugs. 3. This review summarizes the general features and the substrates of the eleven human OATPs. Furthermore, it reviews what is known about the mechanism of their multispecificity, their predicted structure, their role in drug–food interactions, and their role in cancer. 4. Finally, some open questions are raised that need to be addressed to advance OATP research in the near future.

Effect of pregnane X receptor ligands on transport mediated by human OATP1B1 and OATP1B3

The pregnane X receptor is a ligand-activated transcription factor that is abundantly expressed in hepatocytes. Numerous drugs are pregnane X receptor ligands. To bind to their receptor they must cross the sinusoidal membrane. Organic anion transporting polypeptides 1B1 and 1B3 (OATP1B1 and OATP1B3) are polyspecific transporters expressed at the sinusoidal membrane of human hepatocytes. They mediate transport of a variety of drugs including the pregnane X receptor ligands rifampicin and dexamethasone. To test whether additional pregnane X receptor ligands interact with OATP1B1- and 1B3-mediated transport, we developed Chinese Hamster Ovary (CHO) cell lines stably expressing OATP1B1 or 1B3 at high levels. OATP1B1- and 1B3-mediated estradiol-17beta-glucuronide uptake was inhibited by several pregnane X receptor ligands in a concentration dependent way. IC(50) values for rifampicin, paclitaxel, mifepristone, and troglitazone were within their respective pharmacological free plasma concentrations. Kinetic analysis revealed that clotrimazole inhibits OATP1B1-mediated estradiol-17beta-glucuronide transport with a K(i) of 7.7+/-0.3 microM in a competitive way. However, uptake of OATP1B3-mediated estradiol-17beta-glucuronide was stimulated and this stimulation was due to an increased apparent affinity. Transport of estrone-3-sulfate was hardly affected while all other substrates tested were inhibited. Additional azoles like fluconazole, ketoconazole and miconazole did not stimulate OATP1B3-mediated estradiol-17beta-glucuronide transport. In summary, these results demonstrate that pregnane X receptor ligands, by inhibiting or stimulating OATP-mediated uptake, can lead to drug-drug interactions at the transporter level.

Development of a Cell-Based High-Throughput Assay to Screen for Inhibitors of Organic Anion Transporting Polypeptides 1B1 and 1B3

The two organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are expressed at the sinusoidal membrane of hepatocytes. They have a broad and overlapping substrate specificity and transport many endobiotics and drugs. Specific inhibitors are required to determine the contribution of each OATP to the hepatocellular uptake of common substrates. We have developed a cell-based high-throughput assay to screen chemical libraries in order to identify such inhibitors for OATP1B1 and OATP1B3. We have used OATP1B1- or OATP1B3-expressing Chinese Hamster Ovary cells on 96-well plates and determined uptake of fluorescein-methotrexate (FMTX). We validated the assay with known inhibitors and screened the well characterized Prestwick library of 1120 drugs. Along with several known OATP inhibitors including rifampicin, cyclosporine A and mifepristone we identified some new inhibitors. For inhibitors that seemed to be able to distinguish between OATP1B1- and OATP1B3-mediated FMTX uptake IC50 values were determined. Estropipate (estrone-3-sulfate stabilized with piperazine) was the most selective OATP1B1 inhibitor (IC50 = 0.06 μM vs. 19.3 μM for OATP1B3). Ursolic acid was the most selective OATP1B3 inhibitor (IC50 = 2.3 μM vs. 12.5 μM for OATP1B1). In conclusion, this cell-based assay should allow us to identify even more specific inhibitors by screening larger chemical libraries.

Amino acid residues in transmembrane domain 10 of organic anion transporting polypeptide 1B3 are critical for cholecystokinin octapeptide transport.

Human organic anion transporting polypeptides (OATP) 1B1 and 1B3 are multispecific transporters that mediate uptake of amphipathic organic compounds into hepatocytes. The two OATPs contain 12 transmembrane domains (TMs) and share 80% amino acid sequence identity. Besides common substrates with OATP1B1, OATP1B3 specifically transports cholecystokinin octapeptide (CCK-8). To determine which structural domains and/or residues are important for the substrate selectivity of OATP1B3, we constructed a series of chimeric proteins between OATP1B3 and 1B1, expressed them in HEK293 cells, and determined rates of uptake of CCK-8 along with surface expression of the proteins. Replacing TM10 in OATP1B3 with TM10 of OATP1B1 resulted in a dramatically reduced degree of CCK-8 transport, indicating that TM10 is crucial for recognition and/or translocation of CCK-8. Using site-directed mutagenesis, we identified three key residues within TM10, namely, Y537, S545, and T550. When we replaced these residues with the corresponding amino acid residues found in OATP1B1, the level of CCK-8 transport was similarly low as for the replacement of the whole TM10. Kinetic experiments showed that the K m values for CCK-8 transport in the TM10 replacement and triple mutant were only 1.3 and 1.1 microM, respectively, as compared to 16.3 microM for wild-type OATP1B3. Similarly, the V max values dropped from 495.5 pmol (normalized mg) (-1) min (-1) for wild-type OATP1B3 to 13.3 and 19.0 pmol (normalized mg) (-1) min (-1) for the TM10 replacement and triple mutant, respectively. Molecular modeling indicated that two of the three identified residues might form hydrogen bonds with CCK-8. In conclusion, we have identified three amino acid residues (Y537, S545, and T550) in TM10 of OATP1B3 that are important for CCK-8 transport.

Role of transmembrane domain 10 for the function of organic anion transporting polypeptide 1B1

The liver‐specific organic anion transporting polypeptides OATP1B1 and OATP1B3 are highly homologous and share numerous substrates. However, at low concentrations OATP1B1 shows substrate selectivity for estrone‐3‐sulfate. In this study, we investigated the molecular mechanism for this substrate selectivity of OATP1B1 by constructing OATP1B1/1B3 chimeric transporters and by site‐directed mutagenesis. Functional studies of chimeras showed that transmembrane domain 10 is critical for the function of OATP1B1. We further identified four amino acid residues, namely L545, F546, L550, and S554 in TM10, whose simultaneous mutation caused almost complete loss of OATP1B1‐mediated estrone‐3‐sulfate transport. Comparison of the kinetics of estrone‐3‐sulfate transport confirmed a biphasic pattern for OATP1B1, but showed a monophasic pattern for the quadruple mutant L545S/F546L/L550T/S554T. This mutant also showed reduced transport for other OATP1B1 substrates such as bromosulfophthalein and [D‐penicillamine2,5]enkephalin. Helical wheel analysis and molecular modeling suggest that L545 is facing the substrate translocation pathway, whereas F546, L550, and S554 are located inside the protein. These results indicate that L545 might contribute to OATP1B1 function by interacting with substrates, whereas F546, L550, and S554 seem important for protein structure. In conclusion, our results show that TM10 is critical for the function of OATP1B1.

Identification, Ki determination and CoMFA analysis of nuclear receptor ligands as competitive inhibitors of OATP1B1-mediated estradiol-17β-glucuronide transport

Evidence shows that drug-drug interactions can occur at the level of drug transporters such as the organic anion transporting polypeptides (OATPs), a group of membrane solute carriers that mediate the sodium-independent transport of a wide range of amphipathic organic compounds. The polyspecific OATP1B1 is exclusively expressed at the basolateral membrane of hepatocytes and mediates uptake of amphipathic organic compounds from blood into hepatocytes. Nuclear receptors are ligand-activated transcription factors that play an important role in xenobiotic disposition and human diseases. Quite a few nuclear receptor ligands interact with transport proteins. A high-resolution three-dimensional structure is critical to understand the polyspecificity of OATP1B1 to predict and prevent adverse drug-drug interactions. Unfortunately there are no crystal structures of OATPs/Oatps available to date. Therefore, in this study we attempted to elucidate the characteristics of the substrate binding site of OATP1B1 based on small molecules interacting with it. First, we identified inhibitors of the OATP1B1 model substrate estradiol-17beta-glucuronide from about 40 nuclear receptor ligands. Among them, GW1929, paclitaxel and troglitazone were strong inhibitors, while 5 alpha-androstane, 5 alpha-androstane-3beta, 17beta-diol-17-hexahydrobenzoate and estradiol-3-benzoate were weak inhibitors. Then, we selected 25 compounds and performed inhibition kinetic studies to identify competitive inhibitors and determine their K(i) values which ranged from submicromolar to submillimolar. Finally, we performed CoMFA analysis on the identified competitive inhibitors. The CoMFA results indicate that the substrate binding site of OATP1B1 consists of a large hydrophobic middle part with basic residues at both ends that could be very important for substrate binding.

Cloning/characterization of the canine organic anion transporting polypeptide 1b4 (Oatp1b4) and classification of the canine OATP/SLCO members.

The human liver-specific organic anion transporting polypeptides (OATPs) 1B1 and 1B3 are involved in the elimination of numerous xenobiotics and drugs. Although dogs are frequently used for toxicologic and pharmacokinetic characterization of novel drugs, nothing is known about their OATP1B1/1B3 ortholog. Therefore, we cloned and characterized the first canine organic anion transporting polypeptide from dog liver, termed Oatp1b4. The isolated Oatp1b4 cDNA comprises 3661 base pairs (bp) with an open reading frame of 2076bp, encoding a 692-amino acid protein with a molecular mass of approximately 85kDa. The Oatp1b4 gene is approximately 61kb long and has a similar organization as the human OATP1B1 and OATP1B3 with 13 exons identical in length. Northern blot analysis shows that Oatp1b4 is predominantly expressed in the liver. Oatp1b4 mediates sodium-independent transport of typical organic anions including bromosulfophthalein (BSP), [D-penicillamine(2,5)]enkephalin (DPDPE), estradiol-17beta-glucuronide (E17betaG), estrone-3-sulfate and taurocholate. In addition, Oatp1b4 transports the OATP1B3-specific substrate cholecystokinin octapeptide (CCK-8). Kinetic studies showed that Oatp1b4-mediated E17betaG and estrone-3-sulfate transports were monophasic with K(m) values of 5+/-1microM and 33+/-4microM, respectively. In conclusion, the cloned canine Oatp1b4 will provide additional molecular basis to further characterize the species difference of the OATP1B family members.

Influence of Drug Formulation on OATP1B-Mediated Transport of Paclitaxel

Taxane antineoplastic agents are extensively taken up into hepatocytes by OATP1B-type transporters before metabolism and excretion. Because the biodistributional properties imposed upon these agents by different solubilizers drive clinically important pharmacodynamic endpoints, we tested the hypothesis that the in vitro and in vivo interaction of taxanes with OATP1B transporters is affected by the choice of drug delivery system. Transport of paclitaxel, docetaxel, and cabazitaxel was studied in vitro using various cell lines transfected with OATP1B1, OATP1B3, or the rodent equivalent OATP1B2. Pharmacokinetic studies were done in wild-type and OATP1B2-knockout mice in the presence or absence of polysorbate 80 (PS80) or Kolliphor EL (formerly Cremophor EL; CrEL). Paclitaxel and docetaxel, but not cabazitaxel, were transported substrates of OATP1B1, OATP1B3, and OATP1B2, and these in vitro transport processes were strongly reduced in the presence of clinically relevant concentrations of PS80 and CrEL. When paclitaxel was administered without any solubilizers, deficiency of OATP1B2 in mice was associated with a significantly decreased systemic clearance because of a liver distribution defect (P=0.000484). However, this genotype dependence of paclitaxel clearance was masked in the presence of PS80 or CrEL because of significant inhibition of OATP1B2-mediated hepatocellular uptake of the drug (P<0.05). Our findings confirm the importance of OATP1B-type transporters in the hepatic elimination of taxanes and indicate that this process can be inhibited by PS80 and CrEL. These results suggest that the likelihood of drug-drug interactions mediated by these transporters is strongly dependent on the selected taxane solubilizer.