LONDON - A drug in clinical trials as an anti-HIV agent may turn out to belong to a new family of immunosuppressants. Researchers in Belgium have shown that azodicarbonamide can delay the rejection of skin grafts in mice.
Michel Goldman, director of the Laboratory of Experimental Immunology at the Universite Libre de Bruxelles in Brussels, told BioWorld International: "Azodicarbonamide appears to work as an immunosuppressant by a new mode of action, and our experiments suggest that it interferes with the influx of calcium into the cell."
The researchers reported their results in a paper in the August edition of Nature Medicine titled, "Azodicarbonamide inhibits T-cell responses in vitro and in vivo." They plan further studies to determine the effect of the drug in mouse models of T cell-mediated inflammatory disorders including diabetes, rheumatoid arthritis and multiple sclerosis. They are seeking development partners.
Goldman said, "The story of this drug is in some ways amazing. It was discovered by a small pharmaceutical company in Brussels called Hubriphar. They found that it interferes with HIV activity in CD4 cells, but that this was not linked with any classical anti-HIV pathway. Since then, it has been shown that azodicarbonamide modifies the zinc finger domains of HIV-1's nucleocapsid protein. However, Hubriphar had some evidence that something more was going on than direct antiviral action. Their scientists visited me and I suggested that the drug might be acting on the target cells of the virus."
Goldman and colleagues in the same department and in the Department of Molecular Biology, together with a collaborator from Hubriphar, embarked on a series of experiments to investigate the drug's action further.
Normally, when CD4+ T cells are activated by antigen-presenting cells, they proliferate and begin to secrete a variety of cytokines, including interleukin-2, interferon-gamma and interleukin-5. This reaction can be mimicked in the laboratory. But when the researchers added azodicarbonamide, it inhibited both the proliferation response and the secretion of cytokines. Further tests showed it was not because the drug was killing the cells.
When a T cell is activated, calcium ions normally rush into the cell as part of the process of signal transduction. This calcium influx can be simulated in the laboratory by stimulating T cells with monoclonal antibodies against CD3 and CD28. However, addition of azodicarbonamide to the cell culture inhibited this movement of calcium ions. The drug also inhibited the calcium influx, which is normally induced when the compound thapsigargin is added, and that normally is induced by the chemokine RANTES.
Goldman and his colleagues wrote in Nature Medicine: "These data strongly indicate that the T cell-suppressive properties of azodicarbonamide are related to a direct inhibition of the calcium mobilization machinery involving depletion-activated calcium channels."
They went on to look at what effect azodicarbonamide had in live animals. Normally, when mice are injected with a monoclonal antibody against the murine CD3 receptor, a widespread activation of T cells occurs, with secretion of many different cytokines, most of them secreted by T cells.
Treating the mice with a dose of azodicarbonamide equivalent to that being used in the human clinical trials (100 mg/kg), they found the drug caused a "large and significant" decrease in the release of both interleukin-2 and interleukin-4.
Most excitingly, they also found that giving mice a dose of azodicarbonamide of 50 mg/kg per day resulted in a significant delay in the animals' rejection of skin allografts, compared with animals receiving a control substance. This confirms, they write, that "azodicarbonamide also exerts suppressive effects on T cells in vivo."
Goldman concluded: "It will be important to look really carefully at the CD4 cell counts and the function of CD4 cells in patients receiving this drug. We will also be examining the possibility of developing azodicarbonamide as an immunosuppressant. The results are very promising because the model used is a tough one - it is usually very difficult to interfere with skin graft rejection in mice."
Future work by the team will include experiments to determine how the drug interferes with calcium metabolism, and to characterize in detail its safety, toxicity and pharmacokinetics in humans. The group also will examine other possible therapeutic applications, such as treatment of autoimmune diseases and allergic disorders.