The Department of Surgery at the University of Texas Medical School at Houston
Department of Surgery
Basic Science

Basic Science Research

 


Development of new agents to improve the outcomes and reduce the toxicities of immunosuppressive selectin antagonists

The interaction of recipient leukocytes with allograft endothelial cells initiates the process of allograft rejection. During the initial cascade of events, leukocytes roll, attach, spread, and display transendothelial migration. The selectin family of molecules (P-, E-, and L-selectins) slow leukocytes. Rolling is initiated by the interaction of selectins with sialyl Lewisx (sLex) ligand. Two selectins are expressed on endothelial cells with P-selectins stored in the granules and rapidly translocated to the cell surface, and E-selectins induced by inflammatory cytokines. Within minutes of graft reperfusion, endothelial cells express P-selectins that affect leukocyte rolling over the vascular lining. In addition to sLex, P-selectin binds to P-selectin glycoprotein ligand-1 (PSGL-1) expressed on leukocytes, platelets, and other cells. Engagement of PSGL-1 by P-selectins not only slows the movement of leukocytes but also sparks a cascade of signaling pathways leading to the expression of multiple adhesion molecules, including integrins. All of these processes contribute to firm adhesion and migration of leukocytes through the vessel wall and into the inflammatory site. A new potent selectin antagonist, Bimosiamose (BIMO; C46H54O16·0.25 H2O; [867.4 molecular weight]), produced by Encysive Pharmaceuticals Inc. (Bellaire, TX), is a synthetic sLex glycomimetic with potent inhibitory effects on all three selectins.
Previously, we examined whether BIMO inhibits the rejection of kidney allografts as well as the ischemia/reperfusion injury following harvesting of kidney allografts. Although untreated rat recipients acutely reject kidney allografts at a mean survival time (MST) of 8.8±0.75 days, an intravenous 7-day infusion by osmotic pump of 2.5 mg/kg BIMO extended kidney allograft survival to 11.5±2.2 days (p<0.03), 5 mg/kg BIMO to 25.4±11.4 days (p<0.006), 10 mg/kg BIMO to 37.4±13.6 days (p<0.001), and 20 mg/kg BIMO to 39.8±34.5 days (p<0.01). Interestingly, a combination of BIMO and cyclosporine (CsA) produced synergistic interactions, as documented by the combination index (CI) values of 0.34-0.43 (CI<1 is synergistic; CI=1 is additive; and CI>1 is antagonistic). In similar fashion, BIMO interacted synergistically with sirolimus (SRL; CI=0.64), and FTY720 (CI=0.22), documenting a beneficial effect in combination with drugs having different mechanisms of action. To analyze its mechanism of immunosuppression, we showed that administration of BIMO decreased infiltration of kidney allografts with CD4+, CD8+, and macrophages on day 7 post-grafting. At the same time, BIMO reduced expression at the graft site of multiple cytokines on days 3, 5, and 7 post-grafting, including interleukin (IL)-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-12, IL-18, tumor necrosis factor-alpha (TNF-alpha), and interferon-gamma (IFN-gamma). In addition, at similar time points, BIMO inhibited intragraft expression of P-selectin glycoprotein ligand-1, fractalkine (CX3CL1), MIP-3beta/ELC (CCL19), MIP-3alpha/LARC (CCL20), and MCP-1 (CCL2). Thus, BIMO blocks allograft rejection by reduction of intragraft expression of cytokines and chemokines.
We also evaluated the in vivo effects of BIMO on ischemia/reperfusion (I/R) injury. Using a rat model, we perfused kidneys ex vivo with 2 or 4 mg of BIMO suspended in 2 mL of saline immediately after harvesting kidneys prior to transplantation to syngeneic recipients. Within 24 hours after transplantation, the glomerular filtration rate (GFR) was increased, documenting that the 4 mg dose of BIMO produced protection against I/R injury. All these results suggest that the selectin inhibitor has multiple beneficial effects on kidney allografts by mitigating I/R injury and blocking allograft rejection. Our results also indicate that BIMO may be safely used in patients treated with different immunosuppressive drugs to produce even more potent (synergistic) protection from rejection. Since BIMO lacks toxicity, we expect that BIMO may proffer a unique and versatile agent to improve standard immunosuppressive protocols.
We believe that the major advantage of treatment with BIMO is the lack of toxic effects. Our preclinical studies showed that BIMO is well tolerated in rodents and dogs. A single dose as high as 1250 mg/kg BIMO injected i.v. to mice caused no harm. In addition, continuous 14-day i.v. daily injections of 60 mg/kg BIMO to rats caused no observable effects on hematology and clinical chemistry parameters. We propose that BIMO may provide significant benefits to patients of organ allografts without producing toxic effects.

Janus tyrosine kinase 3 (Jak3) inhibitor

Despite improved allograft survivals, current immunosuppressive protocols are marred by side effects. The chief reason for these toxicities is the ubiquitous expression of the target molecules. Immunosuppressants that target calcineurin (CaN)–CsA and tacrolimus (TRL)–cause toxicities in renal, neural, and hepatic tissues. Similarly, sirolimus (SRL) blocks mammalian target of rapamycin (mTOR), which controls nutrient and growth factor-induced cell growth and differentiation. To avoid the toxicities of these inhibitors of critical processes in multiple tissues, we have proposed to target molecules that are exclusively expressed in lymphocytes. One molecule meeting this criterion is Janus tyrosine kinase 3 (Jak3), a cytoplasmic tyrosine kinase with expression limited to T cells, B cells, natural killer (NK) cells, mast cells, and macrophages. Since patients lacking Jak3 are immune compromised, Jak3 seems to represent an excellent therapeutic target for immunosuppression. Over the last 4 years, we have developed a Mannich base compound, NC1153, that inhibits cytokine-induced activation of Jak3 and its downstream substrates signal transducer and activator of transcription (Stat)5a/b 40-fold more effectively than it affects the closely related prolactin-induced Jak2 activity.
Additionally, we have shown that NC1153 did not inhibit several other enzymes, such as growth factor receptor tyrosine kinases, src family members, and serine threonine protein kinases. In vivo studies revealed that NC1153 prolonged the survival of rat kidney allografts. Untreated ACI (RT1a) recipients rejected Lewis (LEW; RT1l) kidney allografts at an MST of 8.8±0.5 days. In contrast, a 7-day therapy by oral gavage of 20-160 mg/kg NC1153 produced similar results as i.v. infusion of the 8-fold-lower NC1153 doses of 2.5-20 mg/kg, documenting approximately 12% oral bioavailability.
An extended 14-day oral course of 240 mg/kg NC1153 produced survivals of 50.6±14.3 days. However, when a 14-day course with 160 mg/kg NC1153 was combined with thrice-weekly delivery up to 90 days, 75% of animals displayed graft survivals beyond 200 days. Since long-surviving recipients again accepted donor-type LEW heart allografts (>100 days; n=3) and rejected third-party Buffalo (RT1b) heart allografts, they likely developed a state of transplantation tolerance.
To examine the mechanism of inhibition by NC1153, we utilized a spleen allograft model to collect a large number of graft infiltrating cells (GICs). Untreated ACI rats acutely reject irradiated LEW spleen allografts within 10 days (n=3). This model allows harvesting on day 7 post-grafting of 50´106 GICs, including anti-donor T cells. Treatment with 160 mg/kg NC1153 prevented rejection and reduced the number of GICs to 20´106. RPA analysis revealed that GICs showed markedly reduced mRNA levels for IFN-gamma, slightly reduced amounts of IL-10 and IL-6, and intact mRNA levels for IL-1alpha, IL-1beta, TNF-beta, and IL-5. When stimulated with IL-2, GICs from untreated recipients showed translocation of Stat5 to the nucleus. In contrast, GICs from recipients treated with NC1153 displayed potent inhibition of Stat5 translocation. In conclusion, inhibition by NC1153 of Jak3/Stat5 signaling in T cells significantly reduced infiltration of grafts with lymphocytes, eventually resulting in diminished production of cytokines.
We also examined the quality of interaction between NC1153 and CsA. When recipients of kidney allografts were treated for 3 days with CsA (2.5-20 mg/kg) and for 7 days with NC1153 (20-160 mg/kg), the two-drug combinations showed synergistic interactions, as measured by CI values of 0.3-0.5. More detailed analysis revealed that the most potent synergism occurred at NC1153/CsA dose ratios of 2:1 with the CI value of 0.3 and the least synergism at dose ratios of 16:1 with the CI value of 0.51. Thus, these in vivo results demonstrate that NC115 blocks allograft rejection, induces transplantation tolerance, and is synergistic with CsA to prolong allograft survival.
We also have documented that NC1153 does not display nephrotoxicity or myelosuppression, nor does it affect cholesterol metabolism. Our previous work in salt-depleted rats revealed that CsA-induced nephrotoxicity was worsened by the addition of SRL. In the same model, SRL alone caused myelosuppression and an increase in cholesterol level. Present results documented that a 28-day course of oral administration of NC1153 (160 mg/kg) did not produce renal dysfunction and did not affect cholesterol metabolism. Although monotherapy with NC1153 or SRL caused no changes in the kidneys, monotherapy with CsA increased glomerular cellularity, enhanced the thickening of vessels (<25%), produced focal tubular dilation, and caused interstitial fibrosis. A combination of SRL and CsA caused more pronounced damage with severe tubular damage and thickening of the walls of small arterioles (>75%). Addition of NC1153 to a CsA regimen did not increase the changes caused by CsA alone. Serum creatinine and creatinine clearance measurements indicated that SRL, but not NC1153, increased CsA-induced nephrotoxicity. Histological examinations confirmed these observations.
Whereas NC1153 alone did not affect lipid metabolism, SRL alone increased levels of total cholesterol, serum LDL-cholesterol, and serum HDL-cholesterol. Although NC1153 alone did not alter femoral bone marrow cellularity, SRL alone produced mild myelosuppression. More importantly, the CsA/SRL combination significantly worsened the cellularity, whereas the NC1153/CsA combination showed no changes.
Based on these experiments, targeting of Jak3 by NC1153 displays none of the traditional side effects produced by current immunosuppressants and does not potentiate toxicities associated with CsA when used in combination. Most importantly, NC1153 produces no effects on hematopoiesis, documenting its selective effect on Jak3, but not on Jak2.

Induction of transplantation tolerance

The best way to protect the allograft from rejection is to induce a state of long-term survival without continuous immunosuppression, namely, transplantation tolerance. One of the experimental methods of tolerance induction is induced by signal 2 blockade with combination of anti-CD40 ligand monoclonal antibody (MAb; MR1) and/or CTLA-4Ig to reduce donor-reactive T cell clones through antigen-induced cell death (AICD). However, long-term lasting tolerance may be induced by MR1 MAb in IL-2-producing Th1-deficient Stat4-/- mice but not in IL-4producing Th2-deficient Stat6-/- mice. Our results and the work of others postulated that IL-4/Stat6-driven regulatory Th2-type (Th2reg) cells are necessary for maintenance of stable tolerance.
Newly discovered cytokine-driven repressors and other regulatory elements seem to be involved in the regulation of differentiation into Th2 cells. We proposed that Stat6, C-maf, and GATA-3 may promote the generation of Th2reg cells. Since Jaks and Stats are regulated by a series of negative feedback loops by a family of suppressors of cytokine signaling (SOCS)1-7, we investigated the expression of these regulatory molecules in tolerant recipients. Therefore, we have examined the pattern of SOCS1, SOCS2, SOCS3, GATA-3, and C-maf expression in the stable state of the Th2reg-driven tolerance. The majority of Balb/c recipients became tolerant to C57BL/6 heart allografts (>200 days; n=5) after treatment with a combination of anti-CD40 ligand (MR-1) MAb and CTLA4-Ig; untreated controls had an MST of 7.8±0.8 days. The same protocol induced tolerance in Stat4-/- recipients but failed in Th2reg-deficient Stat6-/- recipients (20.2±7.0 days; n=5). The real-time PCR showed that levels of SOCS3, GATA-3, and C-maf mRNAs were significantly higher (p<0.01) in purified T cells from tolerant recipients bearing fully functional grafts (>100 days) than from tolerant rejectors that were treated with tolerogenic protocol but rejected heart allografts (30-60 days) or from untreated rejectors (<10 days).
In the in vitro model, purified Stat4-/- T cells cultured in Th2 conditions (concavalin A [ConA]/IL-4/anti-IL-12 Ab) for 35 days had elevated mRNA levels for SOCS3 by 60-fold, GATA-3 by 30-fold, and C-maf by 10-fold in comparison to purified Stat6-/- T cells cultured in Th1 conditions (ConA/IL-12/anti-IL-4 mAb). Based on these results, we propose that SOCS3, GATA-3, and C-maf are involved in regulation of Th2reg cells in transplantation tolerance. Our future plans focus on the development of therapeutic agents that lack side effects and yet induce stable transplantation tolerance.