Peijin Zhang

CHARACTERIZATION OF TRANSPORT PROTEIN EXPRESSION IN MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN (MRP) 2-DEFICIENT RATS

Multidrug resistance-associated protein (Mrp) 2-deficient transport-deficient (TR–) rats, together with their transport-competent Wistar counterparts (wild type), have been used to examine the contribution of Mrp2 to drug disposition. However, little is known about potential variation in expression of other transport proteins between TR– and wild-type rats or whether these differences are tissue-specific. Sections of liver, kidney, brain, duodenum, jejunum, ileum, and colon were obtained from male TR– and wild-type Wistar rats. Samples were homogenized in protease inhibitor cocktail and ultracentrifuged at 100,000g for 30 min to obtain membrane fractions. Mrp2, Mrp3, Mrp4, P-glycoprotein, sodium-dependent taurocholate cotransporting polypeptide, organic anion transporting polypeptides 1a1 and 1a4, bile salt export pump, breast cancer resistance protein, ileal bile acid transporter, UDP-glucuronosyl transferase (UGT1a), glyceraldehyde-3-phosphate dehydrogenase, and β-actin protein expression were determined by Western blot. Mrp3 was significantly up-regulated in the liver (∼6-fold) and kidney (∼3.5-fold) of TR– rats compared with wild-type controls. Likewise, the expression of UGT1a enzymes was increased in the liver and kidney of TR– rats by ∼3.5- and ∼5.5-fold, respectively. Interestingly, Mrp3 expression was down-regulated in the small intestine of TR– rats, but expression was similar to wild type in the colon. Mrp4 was expressed to varying extents along the intestine. Expression of some transport proteins and UGT1a enzymes differ significantly between TR– and wild-type rats. Therefore, altered drug disposition in TR– rats must be interpreted cautiously because up- or down-regulation of other transport proteins may play compensatory roles in the presence of Mrp2 deficiency.

Roles of P-glycoprotein, Bcrp, and Mrp2 in biliary excretion of spiramycin in mice

ABSTRACT The multidrug resistance proteins P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein 2 (Mrp2) are the three major canalicular transport proteins responsible for the biliary excretion of most drugs and metabolites. Previous in vitro studies demonstrated that P-gp transported macrolide antibiotics, including spiramycin, which is eliminated primarily by biliary excretion. Bcrp was proposed to be the primary pathway for spiramycin secretion into breast milk. In the present study, the contributions of P-gp, Bcrp, and Mrp2 to the biliary excretion of spiramycin were examined in single-pass perfused livers of male C57BL/6 wild-type, Bcrp-knockout, and Mrp2-knockout mice in the presence or absence of GF120918 (GW918), a P-gp and Bcrp inhibitor. Spiramycin was infused to achieve steady-state conditions, followed by a washout period, and parameters governing spiramycin hepatobiliary disposition were recovered by using pharmacokinetic modeling. In the absence of GW918, the rate constant governing spiramycin biliary excretion was decreased in Mrp2− knockout mice (0.0013 ± 0.0009 min−1) relative to wild-type mice (0.0124 ± 0.0096 min−1). These data are consistent with the ∼8-fold decrease in the recovery of spiramycin in the bile of Mrp2-knockout mice and suggest that Mrp2 is the major canalicular transport protein responsible for spiramycin biliary excretion. Interestingly, biliary recovery of spiramycin in Bcrp-knockout mice was increased in both the absence and presence of GW918 compared to wild-type mice. GW918 significantly decreased the rate constant for spiramycin biliary excretion and the rate constant for basolateral efflux of spiramycin. In conclusion, the biliary excretion of spiramycin in mice is mediated primarily by Mrp2 with a modest P-gp component.

KNOCKING DOWN TRANSPORT: APPLICATIONS OF RNA INTERFERENCE IN THE STUDY OF DRUG TRANSPORT PROTEINS

RNA interference (RNAi) is a gene silencing process mediated by double-stranded RNA (dsRNA). The silencing process is comprised of an initiation step, in which small interfering RNA (siRNA) is introduced to the cell, and an effector step, which involves degrading mRNA molecules of the target gene. RNA interference has been observed in most organisms from plants to vertebrates. As a gene silencing approach, RNAi has proven to be extremely useful in characterizing gene function and developing new tools in cancer therapy and drug delivery. The development of RNAi-related technologies is an emerging area in biomedical research. In this review, recent progress in the application of RNAi to the study of transport proteins is summarized and evaluated; the advantages, disadvantages and future directions of RNAi technology are discussed.