Pharmaceutics, Preformulation and Drug Delivery
6-Mercaptopurine Transport by Equilibrative Nucleoside Transporters in Conditionally Immortalized Rat Syncytiotrophoblast Cell Lines TR-TBTs

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Abstract

Recently, more women were provided with 6-mercaptopurine (6-MP) during pregnancy. Therefore, we attempted to clarify the transport mechanisms of 6-MP through blood–placenta barrier using rat conditionally immortalized syncytiotrophoblast cell lines (TR-TBTs). The uptake of 6-MP was time- and ATP dependent, but sodium independent in TR-TBTs. 6-MP was eliminated over 50% from the cells within 30 min. The uptake of 6-MP was saturable with Michaelis–Menten constant values of 198 μM and 250 μM in TR-TBT 18d-1 and TR-TBT 18d-2, respectively. 6-Thioguanine, azathioprine, and hypoxantine, structural analogues of 6-MP, strongly inhibited [14C]6-MP uptake. Equilibrative nucleoside transporter (ENT) inhibitors, adenosine and uridine, significantly inhibited [14C]6-MP uptake. However, several organic anions and cations had no effect on [14C]6-MP uptake in TR-TBTs. These results suggest that sodium-independent transporters, ENTs, may be involved in 6-MP uptake at the placenta. In addition, multidrug resistance protein (MRP) inhibitors, methotrexate, probenecid, cefmetazole, and sulfinpyrazone, significantly increased the accumulation of [14C]6-MP in the cells. It is indicated that 6-MP may be eliminated across the blood–placental barrier via MRPs. TR-TBTs expressed mRNA of ENT1, ENT2, MRP4, and MRP5. These findings are important for the therapy of acute lymphoblastic leukemia and autoimmune diseases of pregnant women, and should be useful data in elucidating teratogenicity of 6-MP during pregnancy. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:3773–3782, 2011

Section snippets

INTRODUCTION

Recently pregnancy rate has risen in women who are 35 years old or more. This late pregnancy is considered a high-risk pregnancy because of increasing occurrence of malignant diseases such as cancer in women of this age.1,2 In addition, it is known that an attack of chronic inflammatory disease such as inflammatory bowel disease (IBD) in women patients begins at their childbearing age.3 6-Mercaptopurine (6-MP) is one of the antineoplastic and immunosuppressive drugs used as maintenance

Materials

[14C]6-Mercaptopurine (54 mCi/mmol) was obtained from Moravek Biochemicals Inc. (Brea, California, USA). All other chemicals and reagents were purchased from Sigma Chemical (St. Louis, Missouri, USA).

Cell Culture

The TR-TBTs were cultured according to the previous report.15 For the functional study, the cells were seeded on rat tail collagen type I-coated 24-well culture plates (IWAKI, Tokyo, Japan) at a density of 1 × 105 cells/well. After incubation for 3 days at 33°C, the cells were cultured confluent.

Time Course of [14C]6-MP Uptake and Efflux by TR-TBTs

We first experimented the uptake of [14C]6-MP for 60 min in TR-TBTs. [14C]6-MP was taken up time dependently and it was linear for 5 min (Fig. 1). [14C]6-MP uptake was significantly decreased by 0.5 mm unlabeled 6-MP (Fig. 1). To investigate the adenosine triphosphate (ATP)-dependent transporter responsible for [14C]6-MP uptake, we measured the accumulation of 6-MP in the presence of sodium azide, an ATPase inhibitor. The accumulation of 6-MP by TR-TBTs enhanced significantly in the presence of 10 

DISCUSSION

It has been reported that 6-MP blood concentration in fetus merely represents by 1%–2% of its respective maternal blood level, although 6-MP is supposed to be transported to the human placenta.7 The limited 6-MP blood distribution in fetus may allow the embryo and the fetus will not be exposed to the cytotoxicity of 6-MP. Therefore, the study about the BPB transport mechanism of 6-MP is important to explain the teratogenicity of 6-MP during pregnancy. Our results show that the uptake as well as

CONCLUSION

In present study, we clarify that MRPs, including MRP4 and MRP5, and ENTs expressed at the BPB are involved in the transport of 6-MP. Our findings are important for therapy of ALL and autoimmune diseases of pregnant women, and should provide useful data in elucidating teratogenicity of 6-MP during pregnancy.

Acknowledgements

The authors thank Dr. Terasaki for the valuable discussion. This study was supported in grant from the SRC Research Center for Women's Diseases of Sookmyung Women's University.

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