ARTHRITIS & RHEUMATISM Vol. 48, No. 3, March 2003, pp 757–766 DOI 10.1002/art.10968 © 2003, American College of Rheumatology Attenuation of Autoimmune Disease in Fas-Deficient Mice by Treatment With a Cytotoxic Benzodiazepine Jeffrey J. Bednarski, 1 Roscoe E. Warner, 1 Tharaknath Rao, 1 Francesco Leonetti, 2 Raymond Yung, 1 Bruce C. Richardson, 1 Kent J. Johnson, 1 Jonathan A. Ellman, 2 Anthony W. Opipari, Jr., 1 and Gary D. Glick 1 Objective. Elimination of autoreactive cells relies on Fas-dependent activation-induced cell death mecha- nisms, an important component of peripheral tolerance. Defects in Fas or its cognate ligand lead to inefficient activation-induced cell death and are specific causes of lymphoproliferative and autoimmune diseases. The present study was undertaken to investigate a novel 1,4-benzodiazepine (Bz-423) that induces apoptosis and limits autoimmune disease in NZB/NZW mice, to deter- mine its activity against lupus-like disease associated with defective Fas expression. We investigated the Fas- dependence of its cytotoxic actions, its therapeutic po- tential in mice deficient in Fas, and its therapeutic mechanism of action. Methods. Primary lymphocytes isolated from Fas- deficient MRL/MpJ-Fas lpr (MRL-lpr) mice were tested for sensitivity to Bz-423. Bz-423 was administered to MRL-lpr mice for short (1-week) or long (14-week) periods, and its effects on cell survival were determined along with measures of nephritis, arthritis, antibody titers, and Th subpopulations. BALB/c mice were simi- larly treated to determine if Bz-423 alters normal im- mune functions in vivo. Results. Administration of Bz-423 to MRL-lpr mice significantly reduced autoimmune disease includ- ing glomerulonephritis and arthritis. Treatment was associated with decreases in CD4 T cells and an alteration in the Th1/Th2 balance. At the therapeutic dosage, Bz-423 did not interfere with normal T and B cell responses in BALB/c mice, suggesting that this agent is not globally immunosuppressive. Conclusion. Bz-423 is a novel immunomodulatory agent that is active against disease even in the context of defective Fas signaling. It is a leading compound for further investigation into the development of selective therapies for lupus. Systemic lupus erythematosus (SLE) is an auto- immune disease characterized by a spectrum of antibod- ies that recognize self antigens (1–3). Autoantibody– antigen immune complexes deposit in the tissues of the heart, brain, lungs, and kidney, incite tissue destruction, and impair organ function by activating complement and recruiting inflammatory cells (3). The kidney is particu- larly vulnerable to disease: at least 40% of all SLE patients have renal involvement, and the renal disease associated with lupus has a high mortality rate (4,5). Lymphocytes are primary targets for pharmacologic therapy used to treat organ-threatening SLE, and effec- tive immunosuppressive drugs include corticosteroids, azathioprine, and cyclophosphamide (6–9). Although the mechanisms by which these agents exert their effects on SLE are not fully understood, cytotoxicity against B and T lymphocytes is strongly implicated (10). Advances have been made in devising effective treatment regi- mens; however, use of these agents is still accompanied by serious complications that limit their administration and overall clinical benefit (11). Acquisition of agents that more selectively target lupus-determining lymphoid cells would significantly ad- vance SLE treatment. In pursuit of this goal, we previ- ously identified a novel 1,4-benzodiazepine (designated Supported by NIH grants AI-47450 and GM-42168 (to Dr. Glick) and GM-50353 (to Dr. Ellman), and by the University of Michigan Multipurpose Arthritis Center. 1 Jeffrey J. Bednarski, Roscoe E. Warner, PhD, Tharaknath Rao, MD, Raymond Yung, MD, Bruce C. Richardson, MD, PhD, Kent J. Johnson, MD, Anthony W. Opipari, Jr., MD, PhD, Gary D. Glick, PhD: University of Michigan, Ann Arbor; 2 Francesco Leonetti, PhD, Jonathan A. Ellman, PhD: University of California, Berkeley. Address correspondence and reprint requests to Gary D. Glick, PhD or Anthony W. Opipari, Jr., MD, PhD, University of Michigan, 930 North University, Ann Arbor, MI 48109-1055. E-mail: gglick@umich.edu. Submitted for publication September 19, 2002; accepted in revised form December 13, 2002. 757