Application of fluorescence polarization immunoassay for determination of methotrexate-polyglutamates in rheumatoid arthritis patients.
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 gamma-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-PG(7)) as equal to MTX. Good agreement was observed in the concentration-response curves between MTX and MTX-PG(7) 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-PG(7) 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.
Department of Clinical Pharmacology and Genetics, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
Hideki
H
Hayashi
Chihiro
C
Fujimaki
Seiji
S
Tsuboi
Taiji
T
Matsuyama
Takashi
T
Daimon
Kunihiko
K
Itoh
D000284:Administration, Oral; D000911:Antibodies, Monoclonal; D018501:Antirheumatic Agents / Q000032:analysis* / Q000378:metabolism* / Q000627:therapeutic use; D001172:Arthritis, Rheumatoid / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology; D016231:Fluorescence Polarization Immunoassay; D006801:Humans; D018836:Inflammation Mediators / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use; D011099:Polyglutamic Acid / Q000031:analogs & derivatives* / Q000276:immunology / Q000378:metabolism / Q000627:therapeutic use
D000911:Antibodies, Monoclonal; D018501:Antirheumatic Agents; D018836:Inflammation Mediators; C511775:poly(gamma-glutamic acid); D011099:Polyglutamic Acid; C014085:methotrexate polyglutamate; D008727:Methotrexate