Tohoku J. Exp. Med., 2008, 215(1)

Application of Fluorescence Polarization Immunoassay for Determination of Methotrexate-Polyglutamates in Rheumatoid Arthritis Patients

HIDEKI HAYASHI,¹ CHIHIRO FUJIMAKI,¹ SEIJI TSUBOI,² TAIJI MATSUYAMA,³ TAKASHI DAIMON⁴ and KUNIHIKO ITOH¹

¹Department of Clinical Pharmacology and Genetics, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan
²Department of Rheumatology, Shizuoka Kousei Hospital, Shizuoka, Japan
³Department of Pharmacy, Shizuoka Kousei Hospital, Shizuoka, Japan
⁴Department of Drug Evaluation and Informatics, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan

Rheumatoid arthritis (RA) is a chronic disease characterized by the painful joints, inflammation, uncontrolled proliferation of synovial tissue and multisystem comorbidities. Weekly low-dose methotrexate (MTX) has been established as effective treatment in RA patients. MTX is converted to γ-glutamyl polyglutamates, an active form of MTX, through the action of folylpolyglutamate synthetase in the cells. MTX-polyglutamates (MTX-PGs) in red blood cells (RBCs) may be useful as a therapeutic marker of RA. However, the previously reported methods for the quantification of MTX and MTX-PGs in RBCs are impractical for clinical use due to time-consuming, laborious and high cost. We attempted to apply a method with the commercially available fluorescence polarization immunoassay (FPIA) kit. We found that anti-MTX monoclonal antibody showed the reactivity to 4-amino-10-methylpteroylheptaglutamic acid (MTX-PG₇) as equal to MTX. Good agreement was observed in the concentration-response curves between MTX and MTX-PG₇ spiked samples. Accordingly, the anti-MTX monoclonal antibody for FPIA appeared to show the equal reactivity to MTX and MTX-PGs. The recoveries of MTX and MTX-PG₇ from RBCs were 99.0% and 94.1%, respectively. Furthermore, we determined total MTX-PGs concentrations in RBCs of 71 patients with RA treated with weekly pulse MTX. Total MTX-PGs concentrations in 70% of the patients were found to be more than 50 nM that is the lower limit of MTX-PGs concentration in RBCs for expected therapeutic outcome. The routine measurement of total MTX-PGs concentration in RBCs might be useful for prediction about therapeutic outcome of MTX in RA patients.

keywords —— methotrexate; methotrexate-polyglutamate; rheumatoid arthritis; therapeutic drug monitoring; fluorescence polarization immunoassay.

© 2008 Tohoku University Medical Press

===============================

Tohoku J. Exp. Med., 2008, 215, 95-101

Correspondence: Kunihiko Itoh, Department of Clinical Pharmacology and Genetics, School of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku, Shizuoka 422-8526, Japan.

e-mail: itohk@u-shizuoka-ken.ac.jp