Erythrocyte Folyl Polyglutamate Synthetase Activity Profiling as a Potential Tool for the Prediction of Methotrexate Efficacy and Toxicity in Rheumatoid Arthritis
Methotrexate (MTX) is the cornerstone of therapy in the treatment of rheumatoid arthritis (RA). However, its efficacy and toxicity are variable and remain unpredictable. Interindividual variation in the metabolism of MTX by the enzyme folyl polyglutamate synthetase (FPGS) has been associated with re...
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MDPI AG
2023-11-01
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| Series: | Future Pharmacology |
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| Online Access: | https://www.mdpi.com/2673-9879/3/4/49 |
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| author | Amar Kumar Mudassar Iqbal Arain Pooja Bhadbhade Ryan Funk |
| author_facet | Amar Kumar Mudassar Iqbal Arain Pooja Bhadbhade Ryan Funk |
| author_sort | Amar Kumar |
| collection | DOAJ |
| description | Methotrexate (MTX) is the cornerstone of therapy in the treatment of rheumatoid arthritis (RA). However, its efficacy and toxicity are variable and remain unpredictable. Interindividual variation in the metabolism of MTX by the enzyme folyl polyglutamate synthetase (FPGS) has been associated with response variability in RA. In this work, we propose the development of a FPGS phenotyping assay that can be evaluated as a tool for the prediction of efficacy and toxicity in patients with RA prior to initiating MTX therapy. FPGS activity was measured in erythrocyte lysate by monitoring methotrexate polyglutamate (MTX + Glu<sub>n</sub>) formation using ultra-performance liquid chromatography tandem–mass spectrometry (UPLC/MS/MS). Erythrocyte FPGS activity was measured in newly diagnosed RA (n = 35) and osteoarthritis (n = 7) patients. The enzymatic assay was optimized for measuring FPGS activity in 25 µL of packed erythrocytes over two hours. The coefficient of variation for intra- and inter-day analysis was found to be 5% and 12%, respectively. The method was used to measure FPGS enzyme kinetics, resulting in a mean (SD) K<sub>m</sub> of 30.3 (4.8) µM and a V<sub>max</sub> of 612 (193) pmol MTX + Glu<sub>2</sub>/h/mL of packed erythrocytes. Mean (SD) erythrocyte FPGS activity in patients with RA was found to be 445.93 (344.50) pmol MTX + Glu<sub>2</sub>/h/mL and with a 26-fold difference in the range (range: 83–2179 pmol MTX + Glu<sub>2</sub>/h/mL) whereas for patients with OA, it was found to be 409.80 (157.66) pmol MTX + Glu<sub>2</sub>/h/mL with a 3.5-fold difference in the range (range: 200.95–683.93 pmol MTX + Glu<sub>2</sub>/h/mL). Monitoring erythrocyte FPGS activity may be a feasible strategy of phenotyping for methotrexate efficacy and toxicity in patients with RA. |
| first_indexed | 2024-03-08T20:45:06Z |
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| issn | 2673-9879 |
| language | English |
| last_indexed | 2024-03-08T20:45:06Z |
| publishDate | 2023-11-01 |
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| spelling | doaj.art-cd7edce8167043e5932148d7264c98992023-12-22T14:10:16ZengMDPI AGFuture Pharmacology2673-98792023-11-013481983310.3390/futurepharmacol3040049Erythrocyte Folyl Polyglutamate Synthetase Activity Profiling as a Potential Tool for the Prediction of Methotrexate Efficacy and Toxicity in Rheumatoid ArthritisAmar Kumar0Mudassar Iqbal Arain1Pooja Bhadbhade2Ryan Funk3Center for Computational Biology, University of Kansas, Kansas City, KS 66047, USADepartment of Pharmacy Practice, University of Kansas, Kansas City, KS 66047-1620, USADivision of Allergy, Clinical Immunology, and Rheumatology, University of Kansas Medical Center, Kansas City, KS 66160, USACenter for Computational Biology, University of Kansas, Kansas City, KS 66047, USAMethotrexate (MTX) is the cornerstone of therapy in the treatment of rheumatoid arthritis (RA). However, its efficacy and toxicity are variable and remain unpredictable. Interindividual variation in the metabolism of MTX by the enzyme folyl polyglutamate synthetase (FPGS) has been associated with response variability in RA. In this work, we propose the development of a FPGS phenotyping assay that can be evaluated as a tool for the prediction of efficacy and toxicity in patients with RA prior to initiating MTX therapy. FPGS activity was measured in erythrocyte lysate by monitoring methotrexate polyglutamate (MTX + Glu<sub>n</sub>) formation using ultra-performance liquid chromatography tandem–mass spectrometry (UPLC/MS/MS). Erythrocyte FPGS activity was measured in newly diagnosed RA (n = 35) and osteoarthritis (n = 7) patients. The enzymatic assay was optimized for measuring FPGS activity in 25 µL of packed erythrocytes over two hours. The coefficient of variation for intra- and inter-day analysis was found to be 5% and 12%, respectively. The method was used to measure FPGS enzyme kinetics, resulting in a mean (SD) K<sub>m</sub> of 30.3 (4.8) µM and a V<sub>max</sub> of 612 (193) pmol MTX + Glu<sub>2</sub>/h/mL of packed erythrocytes. Mean (SD) erythrocyte FPGS activity in patients with RA was found to be 445.93 (344.50) pmol MTX + Glu<sub>2</sub>/h/mL and with a 26-fold difference in the range (range: 83–2179 pmol MTX + Glu<sub>2</sub>/h/mL) whereas for patients with OA, it was found to be 409.80 (157.66) pmol MTX + Glu<sub>2</sub>/h/mL with a 3.5-fold difference in the range (range: 200.95–683.93 pmol MTX + Glu<sub>2</sub>/h/mL). Monitoring erythrocyte FPGS activity may be a feasible strategy of phenotyping for methotrexate efficacy and toxicity in patients with RA.https://www.mdpi.com/2673-9879/3/4/49methotrexateFPGSrheumatoid arthritisenzyme kineticstherapeutic drug monitoringbiomarker |
| spellingShingle | Amar Kumar Mudassar Iqbal Arain Pooja Bhadbhade Ryan Funk Erythrocyte Folyl Polyglutamate Synthetase Activity Profiling as a Potential Tool for the Prediction of Methotrexate Efficacy and Toxicity in Rheumatoid Arthritis Future Pharmacology methotrexate FPGS rheumatoid arthritis enzyme kinetics therapeutic drug monitoring biomarker |
| title | Erythrocyte Folyl Polyglutamate Synthetase Activity Profiling as a Potential Tool for the Prediction of Methotrexate Efficacy and Toxicity in Rheumatoid Arthritis |
| title_full | Erythrocyte Folyl Polyglutamate Synthetase Activity Profiling as a Potential Tool for the Prediction of Methotrexate Efficacy and Toxicity in Rheumatoid Arthritis |
| title_fullStr | Erythrocyte Folyl Polyglutamate Synthetase Activity Profiling as a Potential Tool for the Prediction of Methotrexate Efficacy and Toxicity in Rheumatoid Arthritis |
| title_full_unstemmed | Erythrocyte Folyl Polyglutamate Synthetase Activity Profiling as a Potential Tool for the Prediction of Methotrexate Efficacy and Toxicity in Rheumatoid Arthritis |
| title_short | Erythrocyte Folyl Polyglutamate Synthetase Activity Profiling as a Potential Tool for the Prediction of Methotrexate Efficacy and Toxicity in Rheumatoid Arthritis |
| title_sort | erythrocyte folyl polyglutamate synthetase activity profiling as a potential tool for the prediction of methotrexate efficacy and toxicity in rheumatoid arthritis |
| topic | methotrexate FPGS rheumatoid arthritis enzyme kinetics therapeutic drug monitoring biomarker |
| url | https://www.mdpi.com/2673-9879/3/4/49 |
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