Abstract
Benzodiazepine (Bz)-423 is a benzodiazepine derivative that targets activated lymphocytes through the mitochondrial F1F0-ATPase, causing loss of mitochondrial membrane potential and apoptosis without affecting resting lymphocytes. We tested Bz-423 in a non-irradiated B6-Ly5.2 → B6D2F1 model of graft-versus-host disease (GVHD) where donor cells were labeled with CFSE to discriminate 3 days after injection between activated (CFSElo) and unactivated (CFSEhi) Ly5.2+ donor or host Ly5.1+ lymphocytes. Compared to controls, Bz-423 caused loss of mitochondrial membrane polarization within 6h of delivery as measured by 3,3’-dihexyloxacarbocyanine iodide (DiOC6(3)) staining in both activated donor CD4+ (12.6% vs 3.3%, p=0.002) and activated CD8+ (12.9% vs 3.0%, p<0.001) T cells but not in unactivated donor or host cells. Loss of mitochondrial membrane potential was followed by selective apoptosis (Annexin-V+) of donor CD4+ (34.9% vs 16.5%, p=0.04) and CD8+ (29.8% vs 12.2%, p=0.03) T cells. Intraperitoneal injection of 60mg/kg Bz-423 3 times weekly beginning 7d after GVHD induction significantly reduced mortality (50% vs 100%, p<0.02). We next used Bz-423 in a miHA-disparate, CD8+ T cell-mediated model of GVHD (C3H.SW → B6) in which B6 hosts received 9 Gy of TBI followed by injection of 5x106 C3H.SW BM cells and 4x106 T cells. We initiated Bz-423 injections 7d after BMT, when GVHD was already established. The drug significantly reduced GVHD clinical scores and improved survival compared to controls (74% vs 29%, p≤0.02). Bz-423 also significantly reduced quantitative GVHD histologic damage indices in the liver (3.6 vs 11.2, p<0.03) and the GI tract (7.0 vs 15.8, p<0.02). Complete donor engraftment was observed in all animals. Bz-423 reduced IFN-γ, a known mediator of GVHD, in the serum (8.4 vs 21.7 pg/ml, p<0.03) and decreased IFN-γ+CD8+ effector spleen T cells (0.64x105 vs 2.2x105, p=0.008), but did not impair the lysis of tumor targets by CD8+ T cells ex vivo. We tested Bz-423 next in a graft-versus-leukemia (GVL) model where EL-4 lymphoma cells (4x103) that are syngeneic to B6 recipients were injected on the day of BMT. No recipients of syngeneic BMT survived EL-4 challenge (0/12) and no untreated allogeneic BMT survived GVHD (0/12) but 9/14 (64%) of Bz-423 treated allogeneic recipients were alive on day 50 without evidence of lymphoma (p=0.003, Fig 1a). We confirmed the effectiveness of Bz-423 in a third model of GVHD to MHC differences (Balb/c → B6) where again Bz-423 significantly reduced all clinical, biochemical and histologic GVHD parameters and improved day 60 survival (58% vs 8%, p<0.002). Bz-423 also preserved GVL effects in this model where 50% of recipients survived without evidence of EL-4 lymphoma compared to 0% of controls (p<0.04, Fig 1b). We conclude that Bz-423, a first-in-class compound that selectively inhibits mitochondrial respiration and causes apoptosis of activated lymphocytes, can reverse experimental GVHD while preserving beneficial GVL effects.
Figure Figure
Germinated Brown Rice Protects H9c2 Cardiomyocyte against Simulated Ischemic/Reperfusion Injury via Mediated Mitochondria Function
