Statins and Immunomodulation

1. Statins and Immunomodulation Jon A. Kobashigawa, MD DSL/Thomas D. Gordon Chair in Heart Transplantation Medicine Associate Director, Cedars-Sinai Heart Institute Director, Advanced Heart Disease Section Director, Heart Transplant Program Cedars-Sinai Medical Center, Los Angeles, California  Cedars-Sinai Heart Institute

2. Conflict of Interest/Affiliation Disclosure Statement Ihave the following relationship(s) to disclose: Research grants Name of Company/ies with which relationship exists: Novartis, XDx, TransMedics, Alexion Cedars-Sinai Heart Institute

3. Cardiac Allograft Vasculopathy Histology: Section of Affected Coronary Artery

6. Pravastatin in Heart Transplantation • The original paper1, “Outcomes of pravastatin in heart transplant patients” demonstrated benefit in survival, clinically severe rejection, and cardiac allograft vasculopathy at one year after transplant. • Inhibition of natural killer cells2 in the pravastatin treated patients suggested an added immunosuppressive effect when given concomitantly with cyclosporine. 1 Kobashigawa JA et al. N Engl J Med. 1995:333:621-627. 2 Wang M, Terasaki PI et al. Human Immunology 1993; 37: 264-270 Cedars-Sinai Heart Institute

7. Pravastatin Randomized Trial in Heart Transplantation • 97 heart transplant patients randomized to pravastatin (n=47) or no pravastatin (n=50) • Primary end points at 1 year included cholesterol levels, rejection, survival, and cardiac allograft vasculopathy by angiography and intravascular ultrasound (IVUS) Kobashigawa JA et al. N Engl J Med. 1995:333:621-627. Cedars-Sinai Heart Institute

8. First-year Results of Pravastatin Study NKC = natural killer cell IVUS = intravascular ultrasound Kobashigawa JA et al. N Engl J Med 1995:333:621-627

9. Terasaki Microtest Tray: Use of Calcein AM Vital Dye • Natural killer cells, lymphokine-activated killer cells and cytotoxic T-lymphocytes are readily detectable by this microtest. • The advantages of this microtest (1993) over the standard chromium-release assay are: • no radioactivity • tenfold fewer cells required • measurement time of 1 second per reading, which is about 60 times faster than with chromium release • ease in setting up multiple tests, numbering in the hundreds • results can be confirmed by microscopic examination. Wang M, Terasaki PI et al. Human Immunology 1993; 37: 264-270

10. Statins as Immunomodulators • Suppresses T-cell responses.1 • Reduces expression of class II MHC on APC’s due to suppression of CIITA promoter gene.2 • Reduces chemokine synthesis in PBM cells.3 • Blocks LFA-1 to decrease lymphocyte adhesion to ICAM-1 and impair T-cell costimulation.4 1. Kurakata, et al. Immunopharm 1996;34:51-61 2. Kwak, et al. Nature Med 2000;6:1399-1402 3. Romano, et al. Lab Invest 2000;80:1095-1100 4. Weitz, et al. Nature Med 2001;7:687-692 Cedars-Sinai Heart Institute

11. Cholesterol Synthesis Pathway HMG-CoA HMG-CoA Reductase Inhibitors Mevalonate Farnesyl P-P Farnesylated RAS Cholesterol

12. Farnesyltransferase Inhibition: A Novel Method of Immunomodulation • Farnesyltransferase inhibitors (FTIs) block farnesylation of proteins. • A new potent FTI, A228839, is analyzed for its effects on T cell proliferation, T cell polarized shape, intracellular calcium kinetics, cytokine production and apoptosis. • Si MS, Imagawa DK, et al. .Int Immunopharmacol 2003 Apr;3(4):475-483 Cedars-Sinai Heart Institute

13. A228839 Prevents Lectin-Induced Lymphocyte Proliferation Absorbance Lambda=490 nm A228839 ul PHA - + + + + + + + + • Si MS, Imagawa DK, et al. .Int Immunopharmacol 2003 Apr;3(4):475-483

14. A228839 Disrupts 1ES T cell Polarized Shape • Si MS, Imagawa DK, et al. .Int Immunopharmacol 2003 Apr;3(4):475-483

15. A228839 Promotes Apoptosis in PHA-Activated Lymphocytes A228839 treated lymphocytes (overlay) demonstrated greater FL-1 channel signals reflecting increased staining with annexin V. Approx 83% of A228839 treated lymphocytes were apoptotic as compared to 31% of untreated controls. Apoptotic Region A228839 Control • Si MS, Imagawa DK, et al. .Int Immunopharmacol 2003 Apr;3(4):475-483

16. Farnesyltransferase Inhibition: A Novel Method of Immunomodulation • Prevents lectin-induced lymphocyte proliferation. • Prevents APC-induced T cell proliferation. • Disrupts 1E5 T cell polarized shape. • Alters lymphocyte [Ca ++]i homeostasis. • Potently inhibits PBMC cytokine production. • Promotes apoptosis in lectin-activated lymphocytes. • Si MS, Imagawa DK, et al. .Int Immunopharmacol 2003 Apr;3(4):475-483 Cedars-Sinai Heart Institute

17. Summary • Benefit from statins are derived not only from lipid-lowering but also through the pleiotropic,cholesterol-independent effects which include: • vasculoprotective • anti-inflammatory • immunomodulatory properties • Statin therapy administered to patients after heart transplant on calcineurin inhibitor results in a decrease in rejection, cardiac allograft vasculopathy, and an increase in survival. • Various mechanistic studies of potential benefit of statins have been realized supporting its routine use in heart transplant recipients. Cedars-Sinai Heart Institute