Dear Editor,
Pigs are currently used in models of organ and cell transplantation. Some of the immunosuppressive agents that are used clinically are either not commercially available for use in pigs or must be prepared in the laboratory, e.g., anti-pig anti-thymocyte globulin [1] and anti-pig CD3 monoclonal antibody [2,3], limiting the use of this experimental animal. Except in inbred miniature swine, relatively intense immunosuppressive therapy is required to prevent graft rejection; the dosages of the agents used in humans may be different in pigs [2].
We have been investigating islet allotransplantation (Tx) in pigs, and have documented the limitations of a regimen consisting of tacrolimus and mycophenolate mofetil (MMF) in this model [4]. This regimen did not include induction therapy, which may be important for the success of islet Tx [5]. We therefore explored the effect of cyclophosphamide (CPP) in pigs. CPP is a nitrogen mustard alkylating agent. It is a prodrug, being converted in the liver to active forms that have chemotherapeutic activity [6]. CPP is combined with other chemotherapeutic agents in the treatment of lymphomas, some forms of leukemia, and some solid tumors. It has its effect by inhibiting cell growth. It decreases the immune system's response to various diseases [6], and has therefore been used as an immunosuppressant.
In the field of organ and cell Tx, CPP has been used relatively extensively and successfully in humans and nonhuman primates. In these species, dosages of approximately 1-40 mg/kg and 20-40 mg/kg respectively generally result in a significant reduction in WBC [6-9].
In Landrace-large white cross-breed pigs (weighing approximately 10-15kg), single intravenous (i.v.) doses of CPP (Cytoxan, Bristol-Myers Squibb, Princeton, NJ) at 5, 10, or 20 mg/kg had no effect on the white blood count (WBC) or lymphocyte count. In view of this lack of effect, we searched the literature for experience of CPP in pigs. Very few reports were identified.
In 1981, Mackie et al [10] administered a dose of 30 mg/kg by intraperitoneal injection every 6 or 12 hours for two days. The lymphocyte count fell by 60% after day 4, for a maximum of 18 days, with a preferential effect on B cells, although T cells were significantly reduced. CPP was associated with suppression of the antibody response.
In 1983, Derbyshire [11], in studying the immunological response of pigs to infection with enterovirus, used a single intraperitoneal infusion of 100 mg/kg of CPP. There was no documentation of its effect on T and B cell counts, but the combination of CPP and enterovirus was associated with severe uncontrolled diarrhea and, in one pig, with severe encephalomyelitis.
In 1988, Hoshino et al [12] reported pig kidney Tx using CPP at daily doses of 7 mg/kg/day i.v. for 7 days or 25 or 50 mg/kg/day i.v. for 2 days (together with maintenance cyclosporine). With low-dose CPP, the WBC decreased over the first 7 days to a mean of 1.8/mm[sup]3[/sup], but then, when CPP was discontinued, began to recover. Kidney allograft survival was slightly prolonged compared to cyclosporine alone, but this was not significant (days 10-11). A dose of 25 mg/kg/day for 2 days was associated with a more rapid fall in WBC to a mean of 1.0 mm3 by day 5 and kidney graft survival was prolonged (to day 14). All pigs that received 2 doses of 50 mg/kg/day died from sepsis by day 8.
In 1993, Smith et al. [13] reported that 50-60 mg/kg i.v. of CPP combined with a single high-dose (1,100 cGy) of total body irradiation 24 hours pre-transplantation did not allow the engraftment of a pig allogenic bone marrow transplant, but did result in 100% mortality. CPP combined with fractionated doses of total body irradiation, however, resulted in stable engraftment, which was not achieved when CPP was omitted from the regimen. CPP clearly made a difference to the outcome.
In 2008, Han et al. [14] administered 100mg/kg of CPP intraperitoneally (divided into two doses one week apart), which within 7 days had reduced the total WBC, lymphocyte, monocyte, and neutrophil counts by >50%.
As there is little in the literature on the administration of CPP to pigs, and the data do not provide conclusive evidence of the efficacy of the agent in this species, except at high doses, and as pigs are increasingly being used in studies relating to Tx, we draw our own small experience and that of others to the attention of those working with this species.
References:
1. Mellert J, Hering BJ, Liu X et al: Successful islet auto and allotransplantation in diabetic pigs. Transplantation, 1998; 66: 200-4
2. Dean S, Horley D, Tuch B: The immunosuppression of pigs: a review. Transplantation Rev, 2003; 17: 45-59
3. Pescovitz MD, Book BK, Aasted B et al: Analyses of monoclonal antibodies reacting with porcine CD3: results from the Second International Swine CD Workshop. Vet Immunol Immunopathol, 1998; 60: 261-68
4. Echeverri GJ, McGrath K, Bottino R et al: Endoscopic Gastric Submucosal Transplantation of Islets (ENDO-STI): Technique and initial results in diabetic pigs. Am J Transplant, 2009; 9: 2485-96
5. Van Belle T, von Herrath M: Immunosuppression in islet transplantation. J Clin Invest, 2008; 118: 1625-28
6. FDA: http: //www.accessdata.fda.gov/scripts/cder/onctools/labels.cfm?GN=cyclophosphamide
7. Waterworth PD, Cozzi E, Tolan G et al: Pig to primate cardiac xenotransplantation and cyclophosphamide therapy, Transplantation Proceedings, 1997; 29: 899-900
8. Schuurman HJ, Smith H, Cozzi E: Tolerability of cyclophosphamide and methrotrexate induction immunosuppression in non human primates. Toxicology, 2005; 213: 1-12
9. NIH: http: //ntp.niehs.nih.gov/ntp/roc/eleventh/profiles/s052cycl.pdf
10. Mackie E: Immunosuppressive effects of cyclophosphamide in pigs. Am J Vet Res, 1981; 42: 189-94
11. Derbyshire JB: The effect of immunosuppression with cyclophosphamide on an experimental porcine enterovirus infection in piglets. Can J Comp Med, 1983; 47: 235-37
12. Hoshino T, Tuttle TM, Maley WR et al: Attempts to induce tolerance with cyclophosphamide after renal transplantation in swine. Transplant Proc, 1988; 1(Suppl.1): 144-48
13. Smith C, Suzuki T, Guzzetta P et al: Bone marrow transplantation in miniature swine: IV. Development of myeloablative regimens that allow engraftment across major histocompatiblity barriers. Transplantation, 1993; 56: 541-49
14. Han J, Liu YL, Fan W et al: Dietary L-arginine supplementation alleviates immunosuppression induced by cyclophosphamide in weaned pigs. Amino Acids, 2008; [Epub ahead of print]

Keywords: pigs, Cyclophosphamide, Immunosuppression