Organ Transplantation - Immunosuppressive Agents

Modification of the Rejection Response [1,2]

Transplanted organs are rejected by a process that occurs when the host's immune system recognizes the grafted tissue as foreign and mounts a response. Modification of this rejection response or altering the immune system's ability to respond to the grafted tissue can be produced in a number of ways including reducing the immune system's exposure to antigens, depleting the number of lymphocytes, suppressing antibody formation, and altering cellular function, such as blocking activation of lymphocytes. The most direct from of immunomodulation is to reduce the exposure of the host immune system to alloantigens. In organ transplantation, this is accomplished by matching the donor and recipient for MHC antigens. Transplantation antigens can also be hidden from allosensitive T cells. Prior to implantation into the peritoneal cavity, pancreatic islet cells can be encapsulated in fenestrated plastic spheres that prohibit T cells from coming into contact with the islet cells.

Lymphocyte populations can be depleted by the administration of cytotoxic drugs, antilymphocyte serum, lymphocytopheresis, or by irradiation of lymphoid organs. Organs of the immune system, in particular the spleen and thymus, can be surgically removed. Splenectomy results in impairment of phagocytic functions and a reduced production of antibodies.

Antibody formation may be suppressed by cytotoxic drugs or lymphoid irradiation of B cells and plasma cells. Destruction of T cells indirectly decreases antibody production by decreasing the effects of T-cell-derived cytokines on B cells. Antibodies and cytokines can be directly eliminated from the host by plasmapheresis.

Alteration of cellular function, directly or indirectly, without generalized cytotoxicity has been in the forefront of immunologic research for the past two decades. Cyclosporine was the first antirejection agent that specifically altered T-cell function. A number of antibodies have been developed that inhibit cell surface molecules involved in T-cell activation and/or T-cell-mediated cytotoxicity. OKT3 is a murine monoclonal antibody directed against the human CD3/T-cell receptor complex on the cell surface of T cells. Within minutes of administration, a marked decrease occurs in the number of circulating T cells. Gradually, these cells reappear but have internalized the CD3/T-cell receptor complex from the cell surface. Without this receptor complex, T cells cannot respond to the class I or class II antigen complex on the surface of allogeneic or antigen-presenting cells. Other antibodies have been directed against cytokines and cytokine receptors. Anti-IL-2 receptor antibodies are now available for human transplant patients and have been safely used to reduce the incidence and severity of acute rejection episodes. Anti-tumor necrosis factor, anti-IFN-γ antibodies and others have been shown to increase allograft survival in animal models.

Antibodies have also been directed against the accessory cell-adhesion molecules, resulting in reduction of T cell binding and activation. Interference with the binding of LFA-1 on T cells and with ICAM-1 on allogeneic or antigen-presenting cells has prolonged allograft survival in rodent and primate models.

The antibodies used against T-cell receptors, cytokines, and adhesion molecules are usually murine in origin. Mice are challenged with the foreign glycoproteins and produce antibody that is collected and administered to another animal or human being facing a rejection response. In most cases, the organ recipient will form antibodies against the murine antibody that eventually neutralize its effect. To reduce this problem in human transplantation, the new anti-IL-2 receptor antibodies have taken the active portion of the mouse antibody (antigen binding or Fab portion) and combined it in various ways to human antibody (Fc and variable regions) to produce antibodies of lower or nil antigenicity (Zenapax, Roche, Nutley, NJ; Simulect, Novartis, East Hanover, NJ).

Other schemes of specific immunosuppression are under investigation; for example, the generation of T-cell-specific antibody/toxin complexes. As T-cell activation and the factors involved in signal transduction are better understood, agents are being directed against the molecular events that occur in the cell membrane and in the cytosol following T-cell antigen recognition.

Immunosuppressive Agents [1,2]

Over the last half of the 20th century, immunosuppressive agents evolved from nonspecific cytotoxic drugs to agents that target specific enzyme pathways that catalyze reactions required for normal immune function. Much of our current knowledge of T-cell function was provided by research performed to understand the mechanism of action of cyclosporine. As each element of antigen recognition, T-cell activation, cytokine synthesis, and T-cell-dependent cytolysis are unraveled, investigators are devising more specific, less toxic and more efficacious agents for interrupting the immune response. This is termed rational drug development and replaces the selection of potential immunosuppressive agents based on their ability to lyse or inhibit the activation of T and B cells in vitro. Although specific immunosuppression using naturally induced and genetically engineered antibodies, soluble receptor fragments, and other biological methods are available for therapy of human diseases, most are not applicable or available for use in the treatment of animal diseases. For the foreseeable future, in clinical veterinary medicine, immunosuppression will continue to be accomplished using chemotherapies. As the new immunosuppressive agents become more readily available and clinicians become familiar with their indications, effects, and side effects, immunosuppression should become more specific, effective, and safe.

Myelotoxic Agents

The major effect of cyclophosphamide results from alkylation of deoxyribonucleic acid (DNA) during the S phase of the cell cycle. The alterations in DNA structure can be lethal to the cell or may produce miscoding errors that inhibit cell replication of DNA transcription. Cyclophosphamide produces T- and B-cell lymphopenia and suppresses both T-cell activity and antibody production. Cyclophosphamide is administered to dogs for the treatment of corticosteroid-resistant autoimmune hemolytic anemia, corticosteroid-resistant thrombocytopenia, rheumatoid arthritis, and polymyositis (in conjunction with corticosteroids). Cyclophosphamide is administered to cats for the treatment of autoimmune hemolytic anemia and rheumatoid arthritis. Myelosuppression, gastroenteritis, alopecia, and hemorrhagic cystitis are the major complications associated with cyclophosphamide therapy.

Azathioprine is a purine analog that is metabolized to ribonucleotide monophosphates. Poor conversion to diphosphates and triphosphates leads to an intracellular accumulation of monophosphates that produces a feedback inhibition of enzymes required for the biosynthesis of purine nucleotides. The triphosphate analogs that do form become incorporated into DNA and result in ribonucleic acid (RNA) miscoding and faulty transcription. Azathioprine has a greater effect on humoral than on cell-mediated immunity. For the treatment of immune-mediated diseases in dogs, azathioprine is generally administered in conjunction with corticosteroids and/or cyclophosphamide. Azathioprine has been used for the treatment of autoimmune thrombocytopenia, autoimmune hemolytic anemia, autoimmune skin diseases, chronic hepatitis, myasthenia gravis, immune-mediated glomerulopathy, chronic atrophic gastritis, systemic lupus erythematosus, and inflammatory bowel disease. Although myelotoxic in cats, azathioprine has been used for the treatment of feline autoimmune skin diseases. Azathioprine and prednisolone, when administered at maximally tolerated levels, do not effectively suppress the rejection response against canine MHC-nonmatched renal allografts. However, when administered on an every other day schedule (1 to 5 mg/kg) with cyclosporine, azathioprine has been used to successfully maintain both canine MHC-matched and MHC-mismatched renal allografts. When administered at a dose of 0.3 mg/kg every third day, adjusting the dosage to maintain a white blood cell count of at least 3000 cells/ µl, azathioprine has been used in combination with cyclosporine to reverse or control renal allograft rejection in cats. The primary complication encountered with the administration of azathioprine is bone marrow suppression that can result in leukopenia, anemia, and thrombocytopenia. Acute pancreatitis and hepatotoxicity may also occur.

Methotrexate competitively inhibits folic acid reductase necessary for the reduction of dihydrofolate to tetrahydrofolate and affects the production of both purines and pyrimidines. The effects of methotrexate occur during the S-phase of the cell cycle. Methotrexate is used primarily as an antineoplastic agent in dogs and cats for lymphomas, carcinomas, and sarcomas. In human medicine, methotrexate is administered for the treatment of rheumatoid arthritis and psoriasis. Gastrointestinal toxicity is the most common complication encountered with the administration of methotrexate.

Glucocorticoids

Glucocorticoids, and in particular, prednisolone, have both direct and indirect effects on the immune response. Glucocorticoids stabilize the cell membrane of endothelial cells and inhibit the production of local chemotactic factors, thus decreasing infiltration of neutrophils, monocytes, and lymphocytes. In allogeneic tissues, the secretion of destructive proteolytic enzymes such as collagenase, elastase, and plasminogen activator is inhibited. Glucocorticoids also inhibit the release of arachidonic acid from membrane phospholipids. This prevents the synthesis of prostaglandins, thromboxanes, and leukotrienes, which are major mediators of inflammation. Glucocorticoids redistribute monocytes and lymphocytes from the peripheral circulation to the lymphatics and bone marrow. This affects primarily T cells. T-cell activation and cytotoxicity are also reduced. Glucocorticoids suppress cytokine activity and alter macrophage function. Owing to their general efficacy and low cost, prednisolone or prednisone are considered to be the first line immunosuppressive agents for the treatment of immune-mediated and chronic inflammatory diseases in dogs and cats. Autoimmune hemolytic anemia and thrombocytopenia, autoimmune and allergic skin diseases, myasthenia gravis, allergic pneumonitis and bronchitis, immune-mediated arthritis, and systemic lupus erythematosus are just some of the indications for corticosteroid therapy in animals. Prednisolone at a dose of 0.25 to 2 mg/kg/day has been used in both dogs and cats to slow allograft rejection; administered as a single agent, however, prednisolone is not capable of preventing allograft rejection. Although inexpensive and often effective, the chronic use of corticosteroids in both human beings and animals can result in severe complications, usually manifested as signs of hyperadrenocorticism. This complication, in addition to the fact that corticosteroids suppress multiple elements of the immune response, has led to the search for steroid-sparing immunosuppressive protocols.

Calcineurin Inhibitors

Cyclosporine is bound in the cytosol of lymphocytes by cyclophilins (cyclosporine-binding proteins). The cyclosporine-cyclophilin complexes associate with calcium-dependent calcineurin/calmodulin complexes to impede calcium-dependent signal transduction. Transcription factors that promote cytokine gene activation are either direct or indirect substrates of calcineurin's serine/threonine phosphatase activity. This enzymatic activity is reduced by association of the cyclosporine-cyclophilin bimolecular complex with calcineurin. Via this mechanism of action, cyclosporine inhibits early T-cell activation (Go phase of the cell cycle) and prevents synthesis of several cytokines, in particular, IL-2. Without stimulation by IL-2, further T-cell proliferation is inhibited, and T-cell cytotoxic activity is reduced. Cyclosporine may also exert an immunosuppressive effect as it stimulates mammalian cells to secrete transforming growth factor beta (TGF-β) protein. TGF-β is a potent inhibitor of IL-2-stimulated T-cell proliferation and generation of antigen-specific cytotoxic lymphocytes. Cyclosporine is not cytotoxic or myelotoxic, and is specific for lymphocytes. This specificity spares other rapidly dividing cells, and allows nonspecific host defense mechanisms to continue to function.

Cyclosporine is gaining wide use in veterinary medicine. Combination cyclosporine and prednisolone immunosuppression has maintained normal function of MHC-nonmatched feline renal allografts for over 13 years. Cyclosporine in combination with azathioprine and prednisolone, or with azathioprine, prednisolone, and antithymocyte serum has been used to maintain MHC-nonmatched canine renal allografts. Bone marrow transplantation has been successfully performed in cats using cyclosporine immunosuppression.

Cyclosporine has also been used to control corticosteroid-resistant autoimmune hemolytic anemia and thrombocytopenia in dogs. Cyclosporine is available in an ophthalmic preparation (Optimmune, Schering-Plough, Kenilworth, NJ) for the control of keratoconjunctivitis sicca in dogs. Recently, cyclosporine was found to significantly reduce the size and depth of perianal fistulas in dogs. Most dogs did not require further therapy, either medical or surgical, after 6 to 8 weeks of therapy.

Cyclosporine is available in two oral formulations: Sandimmune and Neoral (Sandoz, East Hanover, NJ). Both contain cyclosporine in a concentration of 100 mg/ml, but the two solutions are not biologically equivalent. Sandimmune consists of an olive oil base and absorption of cyclosporine requires emulsification of the agent by bile salts and digestion by pancreatic enzymes. Absorption percentage can be as little as 4% and tremendous variation exists in dose/trough whole blood concentrations among individuals of the same species. Neoral is a microemulsion preconcentrate of cyclosporine that becomes a microemulsion when in contact with gastrointestinal fluids. The microemulsion is directly absorbed through the gut epithelium, resulting in more sustained and consistent blood concentrations of the drug. When converting to Neoral from Sandimmune, most feline renal transplant recipients have had a reduction in dose level necessary to maintain the same trough whole blood concentrations. In addition, feline renal transplant patients were administered Sandimmune at 10 to 15 mg/kg/24 hours to initiate immunosuppression at the time of surgery. To achieve the same trough whole blood concentrations of cyclosporine (approximately 500 ng/ml), Neoral is administered at 4 to 6 mg/kg/24 hours. Owing to a more complete absorption that results in a more sustained and predictable blood concentration, Neoral appears be a more effective immunosuppressant than Sandimmune. In addition, it is more economical to use.

To achieve immunosuppression with cyclosporine in dogs, the authors recommend attaining a 12-hour whole blood trough (measured just before the next oral dose) concentration of at least 500 ng/ml. Using Sandimmune, this will require an oral dose of 10 to 25 mg/kg/24 hours divided into 2 doses. Neoral can be initiated at 6 to 10 mg/kg/24 hours divided into 2 doses. With either formulation, gastrointestinal inflammation will increase the dose requirements, and blood concentrations of the agent must be measured starting 24 to 48 hours after initiation of therapy to assure that adequate blood concentrations are achieved. Blood concentrations of cyclosporine should be measured at periodic intervals during the time of therapy.

To reduce the cost of cyclosporine necessary to treat medium- to large-size dogs, ketoconazole can be administered at 10 mg/kg/24 hours in addition to the cyclosporine. Ketoconazole interferes with the hepatic metabolism of cyclosporine, and will reduce the dose requirement of cyclosporine by as much as 60%. Possible toxic effects with the coadministration of these agents include hepatitis and cataract formation.

To achieve immunosuppression with cyclosporine in cats, the authors recommend attaining a 12-hour whole blood trough concentration of 250 to 500 ng/ml. Using Sandimmune, this will require an oral dose of 4 to 15 mg/kg/24 hours divided into 2 doses. Neoral can be initiated at 1 to 5 mg/kg/24 hours divided into 2 doses. Again, it is imperative to measure blood concentrations 24 to 48 hours after initiation of therapy to ensure that adequate blood concentrations have been achieved. Blood concentrations must also be measured periodically during the time of therapy.

Whole blood or plasma concentrations of cyclosporine can be determined by high-pressure liquid chromatography, fluorescence polarization immunoassay, and specific monoclonal antibody radioimmunoassay. Most medical centers that serve human patients perform cyclosporine assays and will serve veterinary needs.

Based on pharmacokinetic studies in the cat, trough whole blood concentrations of cyclosporine may not correlate well with drug exposure [3]. The whole blood concentration measured at 2 hours after administration of the drug may correlate better with drug exposure and give a better index for drug dosage and change in dose. The blood concentration of cyclosporine measured 2 hours after administration, or C2, is recommended for therapeutic drug monitoring in human renal transplant patients [3].

Unlike the situation in human beings, cyclosporine does not appear to be hepatotoxic in dogs and cats unless extremely high blood levels are maintained (> 3000 ng/ml). Although not as frequently encountered in human beings, cyclosporine can be nephrotoxic in the cat. Nephrotoxicity in the cat does not seem to be related to the concentration of the drug in the whole blood; it can occur at relatively low drug concentrations. Cats with extremely high concentrations in whole blood may show no nephrotoxicity at all. Whole blood concentrations of cyclosporine greater than 1000 ng/ml can cause inappetance in cats. If concentrations of 1000 ng/ml are maintained for several weeks or months, opportunistic bacterial and fungal infections can occur. As in human beings, cyclosporine can promote the development of neoplasia, particularly lymphomas, in cats and dogs. The administration of high levels (1 to 2 mg/kg/24 hours) of prednisolone with cyclosporine increases the likelihood of tumor formation. As in humans, cyclosporine has resulted in a marked increase in hair growth in several of our feline renal transplant recipients.

Cyclosporine has a distinctly unpleasant taste to both humans and animals. It is necessary to administer the drug in gelatin capsules. Novartis supplies capsules containing 25 mg or 100 mg of cyclosporine. For most cats, these capsules contain far too much drug. The authors place the oral solution in #0 or #1 gelatin capsules. Some cats require only a small dose of cyclosporine: 1 to 3 mg/dose. Measuring and administering this small amount (0.10 to 0.03 ml) of drug is difficult and imprecise. Sandimmune can be diluted and stored in olive oil. Neoral can be diluted in any oral solution, including tap water, but because it is a microemulsion concentrate, it must be administered immediately after it is diluted.

Cyclosporine is also available in an intravenous solution (Sandimmune IV) that must be diluted in 0.9% sodium chloride or 5% dextrose in water. The authors administer a dose of 6 mg/kg over 4 hours in the calculated maintenance fluid requirement. Intravenous cyclosporine is administered to control organ rejection episodes, an acute hemolytic crisis, or during periods when a patient cannot tolerate oral medications.

Tacrolimus (FK506, Prograf, Fujisawa USA, Dearfield, IL), although structurally different from cyclosporine, shares a similar mechanism of action. Tacrolimus binds in the cytosol of lymphocytes with an immunophilin, FK-binding protein (FKBP). As with the cyclosporine-cyclophilin complex, the tacrolimus-FKBP complex binds to calcineurin and inhibits its phosphatase activity. This directly and indirectly inhibits de novo expression of nuclear regulatory proteins and T-cell activation genes. The transcription of cytokines (IL-2, -3,-4, -5, IFN-γ, TNF-α, and GM-CSF) responsible for lymphocyte activation is suppressed as is the expression of IL-2 and IL-7 receptors. Tacrolimus, in vitro, is 50 to 100 times more potent an inhibitor of lymphocyte activation than cyclosporine. Tacrolimus also inhibits B-cell proliferation and production of antibody by unknown mechanisms. Tacrolimus decreases hepatic injury associated with ischemia/reperfusion injury, perhaps by inhibiting production of TNF and IL-6 by hepatocytes, and stimulates hepatic regeneration following liver injury.

Experimentally, allograft recipients from many species have been treated successfully with tacrolimus with doses several times less than for cyclosporine. Tacrolimus has prolonged the survival of renal, liver, pancreas, heart, lung, and vascularized limb grafts in rodents, dogs, and nonhuman primates. In human organ recipients, tacrolimus is superior to cyclosporine for the reversal of ongoing rejection. Tacrolimus also seems to have a greater steroid-sparing effect over Sandimmune but may not be superior to Neoral. The toxicity of tacrolimus is similar to that of cyclosporine in human beings.

Little, if any, use of tacrolimus has been applied clinically to veterinary patients. Experimentally, tacrolimus was shown to significantly prolong MHC-mismatched renal allograft survival in cats without serious side-effects [4]. Based on its effectiveness in other experimental animal trials, tacrolimus could be effective in controlling a wide range of immune-mediated conditions. Owing to its inhibition of antibody synthesis, in addition to T-cell proliferation, tacrolimus may be particularly effective in controlling immune-mediated anemia, thrombocytopenia, and arthritis.

Despite the potential benefits of tacrolimus for treating diseases in dogs, a major concern is the possible toxicity of the drug. A dose of 0.16 mg/kg/intramuscularly/day and 1.0 mg/kg/orally/day has been reported to be effective in prolonging renal allograft survival in beagle dogs. Side effects included anorexia, vasculitis, and intestinal intussusception. In a study using mongrel dogs, the same doses were not effective in prolonging renal allograft survival and most of the dogs developed severe vasculitis leading to fatal myocardial infarction, hepatic failure, and intussusception. Combination therapy with cyclosporine appears to have an additive effect with less toxicity. Blood concentrations of tacrolimus are assayed at human medical centers using monoclonal immunoassays. The effective serum trough concentration of tacrolimus in dogs is approximately 0.1 to 0.4 ng/ml; about 100 times lower than that of cyclosporine. Trough levels of 2.0 ng/ml or greater can result in death.

Inhibitors of Cytokine and Growth Factor Action

Sirolimus (rapamycin, Rapamune, Wyeth-Ayerst, Philadelphia, PA) is a macrocyclic antibiotic with a structure similar to tacrolimus that also binds in the cell cytosol to FKBP. However, sirolimus and tacrolimus affect different and distinct sites in the signal transduction pathway. The immunosuppressive activity of sirolimus appears to be a consequence in part of the sirolimus-FKBP complex blocking the activation of the mammalian target of rapamycin, (mTOR) [5]. mTOR is a serine/threonine protein kinase and is involved in the regulation of cell proliferation through the initiation of gene translation in response to amino acids, growth factors, cytokines, and mitogens. The kinase activity of additional cell cycle regulatory proteins, cyclin-dependent kinase-2 and -4, is also inhibited by sirolimus. Sirolimus blocks IL-2 and other growth factor-mediated signal transduction (signal 3 of the allograft rejection response) and the calcium-independent CD28/B7 (CD80/CD86) costimulatory pathway. Whereas cyclosporine and tacrolimus block T-cell cycle progression at the G0 to G1 stage, sirolimus prevents cells from progressing from G1 to the S phase. Sirolimus blocks T-cell activation by IL-2, -4, and -6 and stimulation of B-cell proliferation by lipopolysaccaride. Sirolimus directly inhibits B cell immunoglobulin synthesis caused by interleukins. Sirolimus has been shown to prevent acute, accelerated, and chronic rejection of skin, heart, renal, islet, and small bowel allografts in rodent, rabbit, dog, pig, and nonhuman primate graft recipients. It has also been shown to be efficacious in models of autoimmunity; insulin-dependent diabetes, and systemic lupus erythematosus.

Sirolimus' antagonism of cytokine and growth factor action is not limited to cells of the immune system. Growth factor (PDGF, FGF)-induced proliferation of fibroblasts, endothelial cells, hepatocytes, and smooth muscle cells is inhibited by sirolimus. Sirolimus has been very effective in preventing intimal smooth muscle proliferation (arteriosclerosis) following mechanical or immune-mediated arterial injury. In human clinical trials, supplementation of cyclosporine-based protocols is associated with a reduction in acute renal allograft rejection; however, the combination of the two drugs increases the risk of nephrotoxicity, hemolytic-uremic syndrome, and hypertension [5]. Other reported side effects include hyperlipidemia, thrombocytopenia, delayed wound healing, delayed graft function, mouth ulcers, pneumonitis, and interstitial lung disease. Everolimus, another inhibitor of mTOR, is a derivative of sirolimus.

Mycophenolate mofetil (RS-61443, mycophenolic acid, Cellcept, Roche Laboratories, Palo Alto, CA) is a prodrug hydrolyzed by liver esterases to mycophenolic acid. Mycophenolic acid is cytostatic for lymphocytes owing to its inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme necessary for de novo purine biosynthesis. Mycophenolic acid is a relatively selective inhibitor of T- and B-cell proliferation during the S phase of the cell cycle via its ability to prevent guanosine and deoxyguanosine biosynthesis. Mycophenolic acid has been shown to reduce allograft rejection in multiple animal models, being most effective when combined with cyclosporine, tacrolimus, and/or sirolimus. Mycophenolic acid was developed, in part, as a non myelotoxic replacement for azathioprine in human allograft patients. Early clinical trials in human renal allograft recipients showed a decrease in biopsy-proven acute rejection episodes in patients receiving mycophenolic acid in place of azathioprine. At therapeutic doses, mycophenolic acid can be toxic to animals. The primary dose-limiting effects are anemia and weight loss in rats; leukopenia, diarrhea, and anorexia in monkeys; and gastrointestinal hemorrhage, anorexia, and diarrhea in dogs. To reduce the toxic effects, the dose can be lowered or mycophenolic acid can be given in combination with other immunosuppressive agents. Mycophenolic acid can also inhibit growth factor-induced smooth muscle and fibroblast proliferation. Sirolimus and mycophenolic acid, in combination, are extremely effective in preventing arterial intimal smooth muscle proliferation following mechanical injury. This has marked implications for the control of chronic allograft rejection.

Leflunomide (Hoechst AG, Wiesbaden, Germany) is a synthetic organic isoxazole that the intestinal mucosa metabolizes to the active form, A77 1726. Leflunomide mediates at least part of its antiproliferative activity during the S phase of the cell cycle by inhibiting the de novo pathway of pyrimidine biosynthesis. The target of A77 1726 in this pathway is the enzyme dihydroorotate dehydrogenase. At higher concentrations, leflunomide is also an inhibitor of tyrosine kinases associated with growth factor receptors. In addition to T and B lymphocytes, leflunomide also has an antiproliferative effect on smooth muscle cells and fibroblasts, which is also owing to inhibition of the pyrimidine biosynthetic pathway in these cells. Leflunomide is currently approved for the treatment of rheumatoid arthritis in human beings. It has been shown to be an effective disease-modifying antirheumatic drug free from the side effects commonly associated with currently approved immunosuppressants. In addition to its efficacy in humans and animal models with autoimmune diseases, leflunomide has been found to control acute, ongoing, and chronic allograft rejection of the kidney, skin, heart, vessels, lung, and composite grafts in small and large animal models. Leflunomide has been used to successfully treat steroid-resistant autoimmune hemolytic anemia and systemic histiocytosis in dogs. In combination with cyclosporine, leflunomide has completely prevented the rejection of canine MHC-nonmatched renal allografts in both experimental and clinical studies.

At doses used in humans, leflunomide causes gastrointestinal toxicity in dogs because of the accumulation of a metabolite, trimethylfluoroanaline (TMFA). Fortunately, the canine lymphocyte is far more sensitive than the human lymphocyte to the effects of the active agent, A77 1726, and much lower oral doses are equally effective in achieving immunosuppression. The authors currently recommend a dose of 4 mg/kg/24 hours orally, and adjust the dose as needed to obtain a 24-hour serum trough concentration of 20 µ/ml. Early studies in the cat suggest that TMFA does not present the toxicity problem encountered in dogs; however, cats metabolize the drug much slower and require approximately half the oral dose to achieve effective blood concentrations. Both cats and dogs with diminished renal function may be subject to TMFA toxicity, as it is excreted by the kidneys. Leflunomide is marketed under the trade name, Arava. Owing to the short half-life of the drug in dogs, as compared with that in human beings, the use of leflunomide in dogs is very expensive.

Leflunomide analogs are currently being developed for transplantation applications. A combination of cyclosporine and FK778, a leflunomide analog, significantly prolonged MHC-mismatched canine renal allograft survival [5].

Experimental Compounds

FTY 720 is derived from myriocin, a fungus-derived sphingosine analogue [6]. After phosphorylation, FTY 720 engages lymphocyte sphingosine-1-phosphate receptors and profoundly alters lymphocyte traffic, acting as a functional sphingosine-1-phosphate antagonist. FTY 720 sequesters naïve and activated CD4+ and CD8+ T cells and B cells from the blood into lymph nodes and Peyer's patches, without affecting their functional properties. Importantly, FTY 720 does not impair cellular or humoral immunity to systemic viral infection, and it does not affect T- cell activation, expansion/proliferation, or immunologic memory.

FTY 720 synergizes effectively with inhibitors of T-cell activation and proliferation to prevent allograft rejection in a wide range of animal models. In combination with subtherapeutic concentrations of cyclosporine, FTY 720 has been shown to delay or prevent the rejection of skin, heart, small bowel, liver, and kidney allografts in rats, dogs, and nonhuman primates [7]. Similar results have been seen when FTY 720 is combined with rapamycin and tacrolimus. FTY 720 is extensively metabolized in the liver via cytochrome enzymes that are not involved in the metabolism of cyclosporine, rapamycin, or tacrolimus and, therefore, variations in drug concentrations when these agents are co-administered are unlikely to occur. In Phase I and II clinical trials in human renal transplant patients, FTY 720 was well tolerated and did not cause any significant toxicity, allograft loss, or increase in infection rates or other complications such as diabetes. The pharmacokinetic profile of FTY 720 was characterized by linear dose-proportional exposure over a wide range of doses, only moderate inter-patient variability, and a prolonged elimination half-life (89 - 150 hours). These factors suggest that FTY 720 will be administered once daily, without the need for monitoring blood concentrations or dose titration [7]. Human renal transplant patients experienced a significant reduction in peripheral blood lymphocyte counts by up to 85%. It is hoped that FTY 720 may be useful in the future design of more effective and less toxic immunosuppressive regimens for prevention of allograft rejection.

Combination Therapy

Most of the currently used or soon to be available immunosuppressant agents have differing mechanisms of action and are effective at different stages of the cell cycle. Experimentally and clinically, combining agents often results in more effective immunosuppression with fewer drug-induced side effects. In human transplant patients, cyclosporine and tacrolimus are currently considered to be the first line immunosuppressive agents. To increase their effectiveness and decrease toxicity, azathioprine, sirolimus, prednisolone, and/or mycophenolic acid are added to antirejection protocols. Few of the new non-myelotoxic agents have been used in veterinary patients, but many published experimental animal trials investigating autoimmune disease and organ transplantation provide indications and insight into their use. Based on experimental and clinical experience in canine MHC-non matched organ transplantation, the combination of cyclosporine and leflunomide or cyclosporine with azathioprine are extremely effective in preventing renal allograft rejection.