The effect of breast cancer resistance protein (Bcrp/Abcg2) on the disposition of the phytoestrogens daidzein, genistein, and coumestrol was investigated using Bcrp Ϫ/Ϫ mice. Expression of the genes for either mouse Bcrp or human BCRP in MDCK II cells induced apically directed transport of the three phytoestrogens, whereas their transcellular transport was identical in mock and LLC-PK1 cells expressing mouse Mdr1a. After oral administration, the plasma levels of daidzein and genistein were increased in Bcrp Ϫ/Ϫ mice, but only a minimal change was observed for coumestrol. At steady state, tissue-to-plasma concentration ratios of the three phytoestrogens in the brain and testis of wild-type mice were very small and similar to those of [ 14 C]inulin, whereas those were significantly increased in the brain and testis of Bcrp Ϫ/Ϫ mice. The largest increases were observed with genistein (9.2-and 5.8-fold in the brain and testis, respectively). The distributions of genistein in the epididymis and fetus, but not the ovary, were also increased in Bcrp Ϫ/Ϫ mice. The Bcrp protein was localized in the luminal membrane of the endothelial cells in the testis and the body of the epididymis and in both the luminal and abluminal side of ducts in the head of the epididymis. These results suggest that Bcrp limits the oral availability and distribution into the brain and testis, epididymis, and fetus of phytoestrogens.

ABSTRACT:The role of breast cancer resistance protein (BCRP/ABCG2) in limiting the brain and testis penetration of xenobiotic compounds in the blood-brain and -testis barriers was investigated using Bcrp The brain and testis are highly protected from the invasion of xenobiotic compounds by the blood-brain barrier (BBB) and bloodtestis barrier (BTB). The BBB is formed by brain microvascular endothelial cells, whereas myoid and Sertoli cells are involved in the BTB in addition to the endothelial cells (Bart et al., 2002;Kusuhara and Sugiyama, 2005;Scherrmann, 2005). In addition to the highly developed tight junctions between adjacent cells, the BBB and BTB express multiple xenobiotic transporters characterized by broad substrate specificities. They actively extrude xenobiotic compounds into the circulating blood and, thereby, limit tissue penetration from the circulating blood. On the luminal side of the BBB, ABC transporters, such as P-glycoprotein (P-gp/MDR1/ABCB1), multidrug resistanceassociated protein 1 (MRP1/ABCC1) and -4 (MRP4/ABCC4), and breast cancer resistance protein (BCRP/ABCG2), are expressed (Schinkel, 1999;Leggas et al., 2004;Lee et al., 2005;Leslie et al., 2005;Scherrmann, 2005). Among them, P-gp is the best characterized transporter providing a barrier function for the BBB. Cumulative studies have shown that it plays a key role in limiting the penetration of a variety of drugs and toxins (Schinkel, 1999;Scherrmann, 2005). Immunohistochemical analysis has demonstrated the expression of P-gp, MRP1, and BCRP in the BTB (Bart et al., 2004;Augustine et al., 2005). In vivo studies using Mdr1a Ϫ/Ϫ or Mrp1 Ϫ/Ϫ mice have shown their roles in protecting the testis from invasion by their substrates (Wijnholds et al., 1998;Uhr et al., 2000).The present study is focused on BCRP in the BBB and BTB. BCRP is a member of the ABC G-transporter family . Cumulative in vivo studies, particularly using Bcrp Ϫ/Ϫ mice, have shown the importance of BCRP in limiting oral absorption and mediating the biliary and urinary excretion of xenobiotic compounds (Jonker et al., 2002;van Herwaarden et al., 2003;Mizuno et al., 2004;Adachi et al., 2005;Kondo et al., 2005;Ando et al., 2007). The functional role of BCRP in the BBB was first demonstrated by Breedveld et al. (2005), who showed that the brain distribution of imatinib was significantly increased in Bcrp Ϫ/Ϫ mice, although subsequent analysis showed that P-gp plays a significant role in limiting

ABSTRACT:Breast cancer resistance protein (Bcrp/Abcg2) is a member of the ATP-binding cassette transporter family with the ability to transport a variety of sulfate conjugates. In the present study, the regional expression and activity of Bcrp and sulfotransferases (SULTs/Sults) were investigated in mouse intestine. Western blotting analysis revealed the highest expression of Bcrp in the ileum over the duodenum, jejunum, and colon. Functional analysis of Bcrp was performed in everted intestinal sacs using 4-methylumbelliferone (4MU). The mucosal secretion clearance of 4MU sulfate formed in the enterocytes was markedly reduced in the jejunum, ileum, and colon of Bcrp (؊/؊) mice in comparison with wild-type mice, whereas a slight and nonsignificant reduction was observed in the duodenum. The reduction in the mucosal secretion clearance was most marked in the ileum followed by the colon and jejunum. In addition, the mucosal secretion clearance of minoxidil sulfate, an active metabolite of minoxidil, was also significantly reduced in the intestine of Bcrp (؊/؊) mice. The sulfation activity of 4MU was higher in the colon than in the small intestine where glucuronidation activity was somewhat higher than the sulfation activity. Real-time polymerase chain reaction analysis showed that the expression of sulfotransferases, such as Sult1a1/2, Sult1b1, and Sult1d1, was also highest in the colon. These results suggest that Bcrp activity is higher in the mid to lower intestine and that the cooperation of Bcrp and SULT provides an important detoxification pathway, particularly in the colon.

Engineered cysteine residues are particularly convenient for site-specific conjugation of antibody-drug conjugates (ADC), because no cell engineering and additives are required. Usually, unpaired cysteine residues form mixed disulfides during fermentation in Chinese hamster ovarian (CHO) cells; therefore, additional reduction and oxidization steps are required prior to conjugation. In this study, we prepared light chain (Lc)-Q124C variants in IgG and examined the conjugation efficiency. Intriguingly, Lc-Q124C exhibited high thiol reactivity and directly generated site-specific ADC without any pretreatment (named active thiol antibody: Actibody). Most of the cysteine-maleimide conjugates including Lc-Q124C showed retro-Michael reaction with cysteine 34 in albumin and were decomposed over time. In order to acquire resistance to a maleimide exchange reaction, the facile procedure for succinimide hydrolysis on anion exchange resin was employed. Hydrolyzed Lc-Q124C conjugate prepared with anion exchange procedure retained high stability in plasma. Recently, various stable linkage schemes for cysteine conjugation have been reported. The combination with direct conjugation by the use of Actibody and stable linker technology could enable the generation of stable site-specific ADC through a simple method. Actibody technology with Lc-Q124C at a less exposed position opens a new path for cysteine-based conjugation, and contributes to reducing entry barriers to the preparation and evaluation of ADC.

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