In vitro and in vivo effects of the carbon monoxide-releasing molecule, CORM-3, in the xenogeneic pig-to-primate context.
BACKGROUND
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 microm) 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-alpha) 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-alpha and interleukin 1beta following PBMC stimulation with LPS by species-specific ELISA. Complete hematologic and biochemical analyses were routinely performed in treated primates.


RESULTS
At concentrations <500 microm, 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 microm, significant PAEC proliferation was observed, whilst concentrations > or =50 microm inhibited conA-activated primate lymphocyte proliferation (IC(50) of 345.8 +/- 51.9 microm) 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 microm and caused a significant reduction in TNF-alpha production (IC(50) 332.8 +/- 33.9 microm). In vivo, following the administration of multiple doses, TNF-alpha 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.
Vadori
Marta
M
Consorzio per la Ricerca sul Trapianto d'Organi, University of Padua, Via dei Giustiniani 2, Padua, Italy.
Seveso
Michela
M
Besenzon
Federica
F
Bosio
Erika
E
Tognato
Elena
E
Fante
Fabio
F
Boldrin
Massimo
M
Gavasso
Sabrina
S
Ravarotto
Licia
L
Mann
Brian E
BE
Simioni
Paolo
P
Ancona
Ermanno
E
Motterlini
Roberto
R
Cozzi
Emanuele
E
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects; D002166:Camptothecin / Q000494:pharmacology; D002248:Carbon Monoxide / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D003208:Concanavalin A / Q000494:pharmacology; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects*; D006801:Humans; D053583:Interleukin-1beta / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000502:physiology; D008252:Macaca fascicularis; D008934:Mitogens / Q000494:pharmacology; D009942:Organometallic Compounds / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D034421:Sus scrofa; D014183:Transplantation, Heterologous; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D053583:Interleukin-1beta; D008934:Mitogens; D009942:Organometallic Compounds; D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha; C476530:tricarbonylchloro(glycinato)ruthenium(II); D003208:Concanavalin A; D002248:Carbon Monoxide; D020169:Caspases; D002166:Camptothecin