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.
Hayashi
Hideki
H
Department of Clinical Pharmacology and Genetics, School of Pharmaceutical Sciences, University of Shizuoka, Shizuoka, Japan.
Fujimaki
Chihiro
C
Tsuboi
Seiji
S
Matsuyama
Taiji
T
Daimon
Takashi
T
Itoh
Kunihiko
K
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