Test can help plan therapy against rheumatoid arthritis
Karina Toledo/Agência FAPESP/DICYT A test developed and patented by researchers in Ribeirão Preto, São Paulo State, Brazil, makes it possible to identify patients with rheumatoid arthritis who will not respond to the drug methotrexate (MTX) even before treatment has begun. MTX is considered the gold standard for combating the disease.
Epidemiological data show that the drug is not effective in approximately 40% of cases. Currently, it is necessary to wait three to six months after the start of treatment before learning whether the patient is responding satisfactorily. This delay can reduce the likelihood of a cure, according to physicians.
The research that enabled the development of the test kit was conducted as part of Raphael Sanches Peres’s work for his master’s degree and PhD at the Center for Research on Inflammatory Diseases (CRID), one of the Research, Innovation and Dissemination Centers (RIDCs) supported by FAPESP.
The findings were recently published in Proceedings of the National Academy of Sciences (PNAS).
“This is a good example of translational research, and CRID permitted its crystallization. We’re now negotiating with biopharmaceutical companies to license the methodology,” said Fernando de Queiroz Cunha, full professor at the University of São Paulo’s Ribeirão Preto School of Medicine (FMRP-USP) and a member of CRID’s executive committee.
The kit uses monoclonal antibodies coupled to fluorescent molecules to quantify the expression of CD39, an enzyme essential to the anti-inflammatory action of methotrexate.
“CD39 is one of the ectonucleotidases, enzymes expressed on the membranes of some immune system cells, as well as on endothelial cells, that are responsible for breaking down molecules of ATP [adenosine triphosphate] to create adonesine. Previous studies had already shown that the main mechanism of methotrexate’s action on rheumatoid arthritis is mediated by adenosine,” Peres said.
During his master’s research, Peres analyzed blood samples from a group of patients with rheumatoid arthritis treated at the teaching hospital of FMRP-USP and observed that patients who did not respond to MTX had deficient CD39 expression.
During his PhD research, Peres monitored a group of patients with the disease who had never been treated to determine whether they arrived at the clinic with reduced expression of the enzyme.
Blood samples were collected and analyzed before the start of treatment with methotrexate and again three months later. The results showed that in patients who improved with the therapy, there was a greater increase in the number of regulatory T cells (Tregs), a type of lymphocyte that is responsible for CD39 expression and inflammation control.
Furthermore, the Tregs of patients who did not respond to methotrexate expressed proportionally less CD39 than the lymphocytes of either those who responded or healthy individuals. “This difference was observed even before patients were treated with MTX,” Peres said.
To confirm these findings, the scientists at CRID standardized a protocol for treatment with MTX in an animal model of rheumatoid arthritis.
“We induced the disease in mice and treated the animals for five weeks. The results proved the importance of an increase in Tregs to mediate the effects of MTX. When we administered a molecule capable of inhibiting CD39, MTX lost its anti-inflammatory effect,” Peres said.
After completing his PhD, Peres plans to conduct new experiments to investigate why CD39 expression is lower in patients who do not respond to the drug.
Window of opportunity
According to Peres, the cost of the test to measure CD39 expression is about US$10. Only a small amount of blood is needed, and the test can be performed easily and quickly in any laboratory equipped with a flow cytometer. The results are available in approximately two days.
The method can help physicians plan therapy. “The idea is to perform the test as soon as a patient is diagnosed with rheumatoid arthritis. If the result shows low CD39 expression, the physician should prescribe an alternative treatment,” Peres said.
The best alternative to methotrexate available today is treatment with immunobiological drugs such as tumor necrosis factor-alpha (TNF-α) inhibitors. The disadvantages are higher cost and a lack of efficacy for some patients.
According to rheumatologist Paulo Louzada Junior, one of Peres’s PhD supervisors and a co-author of the paper published in PNAS, the test developed by the group at CRID enables physicians to skip stages and take advantage of the “window of opportunity” for the early treatment of rheumatoid arthritis.
“Studies show that if the inflammation is controlled within a year there’s a greater likelihood of achieving total remission of the disease, meaning suspension of the drug and discharge of the patient,” Louzada Junior said.
When the window of opportunity is missed, a drug that could work if administered early in the inflammatory process becomes less effective. Moreover, Louzada Junior said, the longer it takes to control the inflammatory process, the greater the likelihood of joint injury.
Rheumatoid arthritis is an autoimmune condition in which the body's immune system mistakenly attacks normal cells. Synovial joints are the main targets. RA is estimated to affect between 1% and 2% of the world’s population. Fifty thousand new cases are reported each year in Brazil.
The CRID group also plans to investigate whether low expression of the enzyme CD39 may interfere with the treatment of other rheumatic diseases, such as psoriasis, systemic lupus erythematosus and systemic sclerosis.