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Carbon monoxide (CO) interferes with inflammatory and apoptotic processes associated with ischemia–reperfusion injury and graft rejection. Here, the in vitro effects of carbon monoxide releasing molecule-3 (CORM-3), a novel water-soluble carbonyl CO carrier, have been investigated on porcine aortic endothelial cells (PAEC) and primate peripheral blood mononuclear cells (PBMC). Furthermore, the pharmacodynamics and pharmacotolerance of CORM-3 after administration of single and multiple doses in the primate have been assessed in view of its potential application in pig-to-primate xenotransplantation models. Methods: For in vitro studies, PAEC and primate PBMC were exposed for 24, 48 and 72 h to CORM-3 (20 to 1000 μm) and viability was measured using an MTS assay. PAEC and primate PBMC proliferation after exposure to CORM-3 was assessed by CFSE labelling. Proliferation of primate PBMC against irradiated pig lymphocytes was also assessed. Tumor necrosis factor alpha (TNF-α) production and Caspase-3 and -7 activity in Concanavalin A (conA)-stimulated primate PBMC were measured following treatment with CORM-3. In vivo, CORM-3 was administered i.v . to cynomolgus monkeys at 4 mg/kg, as single or multiple doses for up to 30 days. The effect of CORM-3 was evaluated by the assessment of production of TNF-α and interleukin 1β following PBMC stimulation with LPS by species-specific ELISA. Complete hematologic and biochemical analyses were routinely performed in treated primates. Results: At concentrations <500 μm, CORM-3 did not alter the viability of PAEC or primate PBMC cultures in vitro, nor did it induce significant levels of apoptosis or necrosis. Interestingly, at concentrations of 300 and 500 μm, significant PAEC proliferation was observed, whilst concentrations ≥50 μm inhibited conA-activated primate lymphocyte proliferation (IC50 of 345.8 ± 51.9 μm) and the primate xenogeneic response against pig PBMC. Such responses were demonstrated to be CO-dependent. In addition, CORM-3 significantly inhibited caspase-3 and -7 activity at concentrations between 200 and 500 μm and caused a significant reduction in TNF-α production (IC50 332.8 ± 33.9 μm). In vivo, following the administration of multiple doses, TNF-α production was significantly reduced in comparison to pre-treatment responses, with decreased levels maintained throughout the study. Moreover, a slight and transient increase in transaminases and bilirubin was observed in animals exposed to multiple doses of CORM-3. Conclusions: These studies suggest that CORM-3 has anti-inflammatory and immunomodulatory properties in primates that may result in clinical benefit to allo- and xenografted organs. [ABSTRACT FROM AUTHOR]

Background: Cyclophosphamide (CYP) and methotrexate (MTX) have been used as immunosuppressants in induction or maintenance protocols in a large variety of xenotransplantation models. Combining the use of transgenic porcine organs expressing human decay-accelerating factor (hDAF) with immunosuppressive therapy that included the use of CYP or MTX, survival of primate recipients of life-supporting renal xenografts has been prolonged. However, both drugs can cause significant systemic toxicity and, in particular, gastrointestinal (GI) toxicity. To date only limited data have been reported on the histopathological features deriving from the use of such agents in non-human primates. Methods: Cyclophosphamide or MTX was used as part of the immunosuppressive regimen in 15 bilaterally nephrectomized non-human primate ( Macaca fascicularis) recipients of a life-supporting hDAF porcine kidney. At post-mortem, a detailed analysis of the GI tract in animals receiving either CYP or MTX was performed. Paraffin-embedded sections of each portion of the GI tract were prepared and stained with hematoxylin and eosin (H&E). In some animals, additional investigations by immunohistochemistry (CD3, CD5, CD20, CD79 αcy, lambda, and kappa light chains) and by in situ hybridization for EBV encoded RNA (EBER) were undertaken. Results: The xenografted animals from the CYP group had a mean survival of 31 days (range: 0 to 90 days); animals from the MTX group survived a median of 14 days (range: 0 to 39 days). GI complications were the most frequent cause of euthanasia after renal failure. In CYP-treated animals GI-tract lesions were primarily characterized by diffuse, severe lymphoplasmocytic mucosal inflammatory infiltrate. Variable degrees of villi atrophy and fusion, gut-associated lymphoid tissue (GALT) and goblet cell hyperplasia were also observed. In MTX-treated primates, findings were consistent with severe villi atrophy associated with mild-to-moderate disseminated lymphoplasmocytic infiltration. Conclusions: In conclusion, GI tract lesions are an early and consistent finding when CYP or MTX are used as induction agents in this model. The two compounds induce different types of GI tract damage, however, in agreement with their different mechanisms of action. Whilst CYP primarily determines inflammatory lesions, MTX leads to a degenerative type of damage. This study indicates that immunosuppressive drugs can cause severe GI tract damage in primate recipients of renal xenografts and may be responsible for life-threatening lesions. [ABSTRACT FROM AUTHOR]

Cozzi E, Cadrobbi R, Baldan R, Dedja A, Calabrese F, Castagnaro M, Fante F, Boldrin M, Iacopetti I, Ravarotto L, Carraro P, Bronte V, De Santo C, Busetto R, Plebani M, Cancellotti FM, Rigotti P, Thiene G and Ancona E. Methotrexate for immunosuppression in life-supporting pig-to-cynomolgus monkey renal xenotransplantation. Xenotransplantation 2003; 10: 587–595. © Blackwell Munksgaard, 2003 Methotrexate (MTX) has been used successfully as an immunosuppressant in rodent xenotransplantation models, but the data generated so far with MTX in pig-to-baboon cardiac transplantation studies have been disappointing. The potential of this agent was consequently explored in a life-supporting pig-to-primate renal model using the cynomolgus monkey as the recipient species. Introductory in vitro and in vivo pharmacokinetic and pharmacodynamic studies with MTX were conducted in three cynomolgus monkeys. Subsequently,10 cynomolgus monkey recipients of a life-supporting kidney from human decay-accelerating factor transgenic pigs were administered MTX intravenously according to three different regimens. All the animals also received cyclosporine A and steroids. In addition, mycophenolate sodium (MPS) was administered post-operatively in two of the three groups of transplanted animals. At clinically relevant concentrations, MTX is able in vitro to inhibit the mixed lymphocyte reactions (MLR) in cynomolgus monkeys. After intravenous administration, moreover, exposure of cynomolgus monkeys to MTX appeared to be higher than had been previously reported in baboons. Graft function was observed in the transplanted animals, which survived from 0 to 41 days. All but two animals revealed acute humoral rejection in the explanted graft and developed diarrhea. Diarrhea was the cause of euthanasia in five cases. It was unrelated to the administration of MPS and associated with severe histopathological signs of enteritis. This study demonstrates that the pharmacokinetic and pharmacodynamic profiles of MTX vary substantially between non-human primate species. In vitro, MTX has immunosuppressive properties in the cynomolgus monkey at clinically relevant concentrations. In vivo, MTX has a very narrow therapeutic window in cynomolgus monkeys, however, as it does in baboons. We conclude that MTX is scarcely effective as an immunosuppressant, be it for induction or maintenance, in pig-to-cynomolgus monkey renal xenotransplantation. [ABSTRACT FROM AUTHOR]