sq-23377 and teriflunomide

Teriflunomide, an inhibitor of pyrimidine synthesis and thus proliferation of activated T and B lymphocytes, is successfully used for treatment of inflammatory disease. Teriflunomide has further been shown to trigger apoptosis of tumor cells and has thus been considered for the treatment of malignancy. In analogy to apoptosis of nucleated cells, erythrocytes may enter suicidal death or eryptosis, which is characterized by cell shrinkage and phospholipid scrambling of the cell membrane with translocation of phosphatidylserine to the erythrocyte surface. Triggers of cell membrane scrambling include energy depletion, oxidative stress and increase of cytosolic Ca2+ activity ([Ca2+]i). The present study explored whether teriflunomide modifies eryptosis.. Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, and [Ca2+]i from Fluo3 fluorescence.. Oxidative stress (60 min exposure to 0.3 mM tert-butylhydroperoxide), energy depletion (removal of glucose for 48 hours), and exposure to the Ca2+ ionophore ionomycin (1 µM, 60 min) all increased annexin-V-binding, decreased forward scatter and enhanced Fluo3 fluorescence. Teriflunomide (5 µg/ml) did not significantly influence Fluo3 fluorescence, forward scatter and annexin-V-binding under control conditions but significantly blunted the increase of annexin-V-binding following oxidative stress, energy depletion and ionomycin exposure. Teriflunomide further blunted the increase of Fluo3 fluorescence following energy depletion, but did not significantly interfere with increase of Fluo3 fluorescence following oxidative stress and ionomycin exposure.. Teriflunomide is a novel inhibitor of suicidal erythrocyte death.

Topics: Aniline Compounds; Annexin A5; Calcium; Crotonates; Eryptosis; Erythrocyte Membrane; Erythrocytes; Flow Cytometry; Fluorescent Dyes; Glucose; Humans; Hydroxybutyrates; Ionomycin; Nitriles; Oxidative Stress; Phosphatidylserines; Primary Cell Culture; tert-Butylhydroperoxide; Toluidines; Xanthenes

We have optimized assays to measure mitogen-stimulated rat lymphocyte activation in whole blood and have used these assays to quantitate the potencies of immunosuppressive drugs with different mechanisms of action. To define the optimal conditions for measuring T cell functions in whole blood, the effects of different concentrations of mitogens that activate T cells through calcium-dependent and -independent pathways were measured over time. Proliferation was measured by tritium-labeled thymidine ([3H]-TdR) incorporation and by flow cytometric analysis of proliferating cell nuclear antigen (PCNA)/DNA content. Furthermore, we detected the increases in percent expression of cell-surface activation antigens (CD25, CD134, CD71, CD11a and CD54). Concanavalin A (Con A) stimulated maximum lymphocyte proliferation and expression of T cell surface activations by 72-96 h, which was 48 h later than stimulation by phorbol 12-myristate 13-acetate (PMA) plus anti-CD28 monoclonal antibody (mAb) or PMA plus ionomycin (IONO). Addition of sirolimus, tacrolimus, cyclosporine or the active metabolite of leflunomide, A77 1726, to mitogen-stimulated whole blood produced drug concentration-dependent inhibitions of lymphocyte proliferation and expression of cell surface activation antigen expression. From these data, we determined drug potencies (inhibitory concentration of 50%, IC(50)) and drug concentrations causing maximum inhibition of T cell functions (I(max)). We developed simple and reproducible assays to measure different lymphocyte functions in whole blood cultures. These assays were used to investigate the mechanisms of different immunosuppressive drugs. These methods can be exploited to measure T cell functions in blood collected from subjects treated with immunosuppressants in vivo.

Topics: Aniline Compounds; Animals; Blood; Calcium; CD28 Antigens; Concanavalin A; Crotonates; Cyclosporine; Flow Cytometry; Hydroxybutyrates; Immunosuppressive Agents; Ionomycin; Kinetics; Lymphocyte Activation; Male; Mitogens; Nitriles; Rats; Rats, Inbred Lew; Sirolimus; T-Lymphocytes; Tacrolimus; Tetradecanoylphorbol Acetate; Toluidines