ARTHRITIS & RHEUMATISM Vol. 52, No. 2, February 2005, pp 670–678 © 2005, American College of Rheumatology LETTERS DOI 10.1002/art.20770 Drug efflux transporters in rheumatoid arthritis: comment on the article by Kremer To the Editor: We read with interest the article by Kremer (1) review- ing the mechanisms of action of methotrexate (MTX) and potential mechanisms of MTX resistance. One facet that Kremer highlighted is the role of drug efflux transporters in mediating resistance to MTX. Although he highlights the role of multidrug resistance–associated proteins (MRPs) in medi- ating MTX transport, he also emphasizes a study examining permeable glycoprotein (P-gp) expression and response to MTX (2,3). Although it is now established that MRP-1 trans- ports MTX (4), whether P-gp transports MTX is less clear. In order to clarify this issue, we undertook a drug accumulation study using radiolabeled MTX in an in vitro T lymphoblastoid cell line (CEM cells). Parent CEM cells do not express any efflux transporters, but CEM cells pretreated with vinblastine (VBL) constitutively express the efflux transporter P-gp. CEM cells pretreated with epirubicin (E1000) constitu- tively express the efflux transporter MRP-1. This in vitro system is well characterized and has been used to determine the impact of the multidrug resistant transporters on the cellular accumulation of other compounds (5). We incubated 1  10 6 cells/ml with radiolabeled MTX for 18 hours at 37°C across a range of drug concentrations. The excess supernatant was discarded and the cells were extracted in methanol. The intracellular drug concentrations were calculated for the 3 cell lines and results compared using the Kruskal-Wallis test for multiple comparisons. The extent of accumulation of MTX in the MRP-1–expressing cells was significantly reduced (P  0.001), compared with the CEM and VBL cells (Figure 1). These data support the balance of published evidence suggesting that MTX is a substrate for MRP-1 (4) but is not a substrate for P-gp (6). This is also consistent with the obser- vation that P-gp preferentially transports neutral hydrophobic compounds, whereas MRP-1 preferentially transports anionic compounds such as MTX (6). Instead of P-gp it is likely that the transporters MRPs 1–4 (4,6,7), together with breast cancer resistance protein (8), are more important in mediating MTX resistance. Studies suggest that breast cancer resistance protein is also capable of transporting di- and triglutamate forms of MTX (9). This is important, as Kremer (1) highlights, since the polyglutamated forms of MTX are critical to its intracellular activity and efficacy. Further studies are required to clarify the relative importance of the different cellular efflux proteins in mediating an individual’s response to MTX in rheumatoid arthritis. Supported in part by the UK Arthritis Research Campaign. Samantha L. Hider, MRCP, MSc, BM, BS University of Manchester Manchester, UK Patrick Hoggard, PhD Saye Khoo, MD, MRCP David Back, PhD University of Liverpool Liverpool, UK Ian N. Bruce, MD, FRCP University of Manchester Manchester, UK 1. Kremer JM. Toward a better understanding of methotrexate [re- view]. Arthritis Rheum 2004;50:1370–82. 2. Llorente L, Richaud-Patin Y, Diaz-Borjon A, Alvarado dlB, Jakez- Ocampo J, De La FH, et al. Multidrug resistance-1 (MDR-1) in rheumatic autoimmune disorders. Part I: Increased P-glycoprotein activity in lymphocytes from rheumatoid arthritis patients might influence disease outcome. Joint Bone Spine 2000;67:30–9. 3. Ranganathan P, Eisen S, Yokoyama WM, McLeod HL. Will pharmacogenetics allow better prediction of methotrexate toxicity and efficacy in patients with rheumatoid arthritis? Ann Rheum Dis 2003;62:4–9. 4. Hooijberg JH, Broxterman HJ, Kool M, Assaraf YG, Peters GJ, Noordhuis P, et al. Antifolate resistance mediated by the multidrug resistance proteins MRP1 and MRP2. Cancer Res 1999;59:2532–5. 5. Davey RA, Longhurst TJ, Davey MW, Belov L, Harvie RM, Hancox D, et al. Drug resistance mechanisms and MRP expression in response to epirubicin treatment in a human leukaemia cell line. Leuk Res 1995;19:275–82. 6. Jansen G, Scheper RJ, Dijkmans BA. Multidrug resistance proteins in rheumatoid arthritis, role in disease-modifying antirheumatic drug efficacy and inflammatory processes: an overview. Scand J Rheumatol 2003;32:325–36. 7. Zeng H, Chen ZS, Belinsky MG, Rea PA, Kruh GD. Transport of methotrexate (MTX) and folates by multidrug resistance protein (MRP) 3 and MRP1: effect of polyglutamylation on MTX trans- port. Cancer Research 2001;61:7225–32. 8. Volk EL, Farley KM, Wu Y, Li F, Robey RW, Schneider E. Overexpression of wild-type breast cancer resistance protein medi- ates methotrexate resistance. Cancer Res 2002;62:5035–40. 9. Chen ZS, Robey RW, Belinsky MG, Shchaveleva I, Ren XQ, Sugimoto Y, et al. Transport of methotrexate, methotrexate poly- glutamates, and 17-estradiol 17-(-D-glucuronide) by ABCG2: effects of acquired mutations at R482 on methotrexate transport. Cancer Res 2003;63:4048–54. DOI 10.1002/art.20744 Methotrexate and long-term treatment of rheumatic disease: comment on the article by Kremer To the Editor: We read with interest the excellent review on metho- trexate (MTX) by Kremer (1). The author discusses drug Figure 1. Intracellular accumulation of methotrexate (MTX). Values are the mean and SD (n = 4).  = P  0.001 in multidrug resistance–associated protein-1–expressing cells (E1000) versus CEM and vinblastine (VBL) cells. 670