Immunosuppressive Agents

Chapter 59

Immunosuppressive Agents

Over the last half of the twentieth century immunosuppressive agents have evolved from nonspecific, myelotoxic drugs to agents that target specific enzymes catalyzing reactions required for normal immune function. Much of our current understanding of T-cell function has been 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 is unraveled, investigators are devising specific, less toxic, and more efficacious agents for interrupting the immune response. This process, termed rational drug development, 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 with the use of naturally induced and genetically engineered antibodies, soluble receptor fragments, and other biologic methods is available for the treatment of human diseases, most of these therapies are either inapplicable or unavailable for treatment of animal diseases. For the foreseeable future in veterinary medicine, immunosuppression will rely on chemotherapeutic agents. As new immunosuppressive agents become more available and clinicians become familiar with their indications, effects, and adverse effects, immunosuppressive therapy should become more specific, effective, and safe.

Myelotoxic Agents


The major effect of cyclophosphamide results from alkylation of DNA during the S phase of the cell cycle. The alterations in DNA structure can be lethal to the cell or can produce miscoding errors that inhibit cell replication or 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 immune-mediated hemolytic anemia (IMHA), corticosteroid-resistant immune-mediated thrombocytopenia (IMTP), rheumatoid arthritis (RA), and polymyositis (in conjunction with corticosteroids). Cyclophosphamide is administered to cats for the treatment of IMHA and RA. 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 the enzymes required for the biosynthesis of purine nucleotides. The triphosphate analogs that do form become incorporated into DNA and result in ribonucleic acid 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 generally is administered in conjunction with a corticosteroid or cyclophosphamide.

Azathioprine has been used for the treatment of IMTP, IMHA, autoimmune skin diseases, chronic hepatitis, myasthenia gravis, immune-mediated glomerulopathy, chronic atrophic gastritis, systemic lupus erythematosus, and inflammatory bowel disease. In recent studies azathioprine combined with prednisolone was no more effective than prednisolone administered alone in the treatment of IMHA, and azathioprine monotherapy was only moderately effective in the treatment of perianal fistulas. Azathioprine monotherapy was not effective enough to be recommended for general use in the treatment of atopic dermatitis in dogs. Although very 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 renal allografts not matched for major histocompatibility complex (MHC) antigens. However, when administered on an every-other-day schedule (1 to 3 mg/kg PO) with cyclosporine, azathioprine has been used to maintain canine MHC-matched renal allografts successfully. In feline renal transplantation patients with suspected inflammatory bowel disease or following an allograft rejection episode, azathioprine is administered with cyclosporine at a dosage of 0.3 mg/kg every 3 days PO. The dose is decreased if the total white blood cell count falls below 3000 cells/µl or there is biochemical evidence of hepatotoxicity. The primary complication encountered with the administration of azathioprine is bone marrow suppression, which can result in leukopenia, anemia, and thrombocytopenia. Acute pancreatitis and hepatotoxicity may also occur.



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 inhibition 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 redistribution affects primarily T cells. T-cell activation and cytotoxicity also are reduced. Glucocorticoids suppress cytokine activity and alter macrophage function. Prednisolone and prednisone are considered to be the first-line immunosuppressive agents for the treatment of immune-mediated and chronic inflammatory diseases in dogs and cats because of their general efficacy and low cost. IMHA and IMTP, 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 has been used in both dogs and cats to slow allograft rejection; however, when administered as a single agent, prednisolone is not capable of preventing allograft rejection. For the prevention of allograft rejection in cats, prednisolone is administered with cyclosporine at an initial dosage of 2.5 to 5 mg q12h PO. This dosage generally is reduced to 2.5 to 5 mg q24h PO 30 days following transplantation. Over the next several months, with monitoring serum creatinine and blood urea nitrogen concentrations, the prednisolone dosage is reduced to 2.5 mg q24h PO or discontinued.

Although corticosteroids are inexpensive and often effective, the long-term use of these drugs can result in severe complications, usually manifested as signs of iatrogenic hyperadrenocorticism. The potential for 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 the serine-threonine phosphatase activity of calcineurin. 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 (G0 phase of the cell cycle) and prevents synthesis of several cytokines, in particular interleukin-2 (IL-2). Without stimulation by IL-2, further T-cell proliferation is inhibited, and T-cell cytotoxic activity is reduced. Cyclosporine also may exert an immunosuppressive effect because it stimulates mammalian cells to secrete transforming growth factor-β (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 has gained widespread use in veterinary medicine. Immunosuppression with a combination of cyclosporine and prednisolone has maintained normal function of non–MHC-matched feline renal allografts for longer than 12 years. Cyclosporine in combination with azathioprine, prednisolone, and antithymocyte serum has been used to maintain non–MHC-matched canine renal allografts. Bone marrow transplantation has been performed successfully in cats with the use of cyclosporine immunosuppression. Cyclosporine also has been used to control corticosteroid-resistant IMHA and IMTP in dogs. Cyclosporine is available in an ophthalmic preparation (Optimmune) for the control of keratoconjunctivitis sicca in dogs. An ophthalmic preparation of cyclosporine was shown to be an effective treatment for feline eosinophilic keratitis. Lifelong therapy is recommended. Cyclosporine (10 to 20 mg/kg q24h PO) was found to significantly reduce the size and depth of perianal fistulas in dogs (Mathews and Sukhiani, 1997). Most dogs did not require further therapy, either medical or surgical, after 6 to 8 weeks of treatment. In a more recent study (Hardie et al, 2005) cyclosporine resolved or reduced anal furuncular lesions in 25 of 26 dogs. However, residual or recurrent lesions were encountered that required long-term medical therapy or surgical resection. Cyclosporine (5 mg/kg q24h PO induction dose, reduced by 50% to 75% over weeks to months, depending on response) has been shown to be effective in the reduction of skin lesions and pruritus in dogs with atopic dermatitis (Steffan et al, 2006). Cyclosporine also has been shown to be effective in the treatment of atopic dermatitis in cats and is an alternative treatment to prednisolone (Wisselink and Willemse, 2009).

Cyclosporine has been used for the treatment of other dermatologic diseases such as pemphigus foliaceus (Guaguere et al, 2004). Cyclosporine also has been shown to be effective in the treatment of granulomatous meningoencephalitis (Adamo and O’Brien, 2004) and, combined with prednisolone, in the treatment of necrotizing meningoencephalitis. Cyclosporine has been used to treat steroid-refractory inflammatory bowel disease (5 to 10 mg/kg q24h PO for 10 weeks; Allenspach et al, 2006) and recently has been shown to control inflammatory colorectal polyps in miniature dachshunds when combined with prednisolone. Cyclosporine appears to be effective in the management of myasthenia gravis in dogs, especially when anticholinesterase medication fails and other immunosuppressive medications such as corticosteroids lead to deterioration in clinical signs. Very recently, feline idiopathic pure red cell aplasia was shown to be responsive to combination therapy with cyclosporine and glucocorticoids. Most cats required long-term low-dose therapy.

Cyclosporine is available in two oral formulations: Sandimmune and Neoral. Both contain cyclosporine at a concentration of 100 mg/ml, but the two solutions are not biologically equivalent. Sandimmune has an olive oil base, and adsorption of cyclosporine requires emulsification of the agent by bile salts and digestion by pancreatic enzymes. The absorption percentage can be as little as 4%, and there is a tremendous variation in dose-trough whole blood levels among individuals of the same species. Neoral (and now various generics) is a microemulsion preconcentrate of cyclosporine that becomes a microemulsion when in contact with gastrointestinal fluids. The microemulsion is absorbed directly through the gut epithelium, which results in more sustained and consistent blood levels of the drug. When Neoral replaces Sandimmune as treatment, most feline renal transplant recipients have had a reduction in the dosage level necessary to maintain the same trough whole blood levels. In addition, feline renal transplant patients have been administered Sandimmune at a dosage of 10 to 15 mg/kg q24h PO to initiate immunosuppression at the time of surgery. To achieve the same trough whole blood levels of cyclosporine (approximately 500 ng/ml), Neoral is administered at a dose of 1 to 4 mg/kg q24h PO. Neoral appears to be a more effective immunosuppressant than Sandimmune because of its more complete absorption, which results in a more sustained and predictable blood level. In addition, it is more economical to use. The various generic formulations of the microemulsion form of cyclosporine do not appear to be interchangeable; that is, the same dosage does not necessarily result in the same trough blood concentrations in patients. If one microemulsion form is exchanged for another, it is important to check whole blood trough concentrations to ensure both that adequate levels have been achieved and that the concentration of the drug in the blood has not become too high.

To achieve immunosuppression using cyclosporine in dogs, I recommend treating to attain a 12-hour whole blood trough level (measured just before the next oral dose) of at least 500 ng/ml. With Sandimmune, achieving this level requires an oral dosage of 10 to 25 mg/kg q24h divided into two doses. Neoral can be initiated at 5 to 10 mg/kg q24h divided into two doses. With either formulation the presence of gastrointestinal inflammation increases the dosage requirements, and blood levels of the agent must be measured starting 24 to 48 hours after initiation of therapy to ensure that adequate blood levels are achieved. Blood levels of cyclosporine should be measured at periodic intervals during the time of therapy. To reduce the cost of the cyclosporine necessary to treat medium-sized to large dogs, I administer ketoconazole at a dosage of 10 mg/kg q24h PO in addition to the cyclosporine. Ketoconazole interferes with the hepatic metabolism of cyclosporine, and it reduces the dosage requirement of cyclosporine by as much as 60%. I have not encountered toxic effects with the coadministration of these agents, but it has been reported that the long-term administration of ketoconazole to dogs may result in cataract formation.

To achieve immunosuppression with cyclosporine in cats, I recommend attaining a 12-hour whole blood trough level of 250 to 500 ng/ml. With Sandimmune, obtaining this level requires a dose of 4 to 15 mg/kg q24h PO divided into two doses. Neoral can be initiated at 1 to 5 mg/kg q24h PO divided into two doses. Again it is imperative to measure blood levels 24 to 48 hours after initiation of therapy to ensure that adequate blood levels have been achieved. Blood levels must also be measured periodically during the course of therapy. Based on pharmacokinetic studies in the cat, trough whole blood concentrations of cyclosporine may not correlate well with drug exposure (Mehl et al, 2003). The whole blood concentrations 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 is recommended for therapeutic drug monitoring in human renal transplant patients.

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

Unlike the situation in humans, cyclosporine does not appear to be hepatotoxic in dogs and cats unless extremely high blood levels are maintained (>3000 ng/ml). Although nephrotoxicity is not as frequently encountered in humans, cyclosporine can be nephrotoxic in the cat. Nephrotoxicity in the cat does not seem to be related to the level of the drug in whole blood and can occur at relatively low plasma concentrations. Cats with extremely high cyclosporine whole blood concentrations (>4000 ng/ml) often show no toxicity at all. In my years of practice I have seen cyclosporine nephrotoxicity develop in only one dog. This dog had a chylothorax that resulted in trough whole blood concentrations of cyclosporine of more than 3000 ng/ml. Levels higher than 1000 ng/ml can cause inappetence in cats. If levels of 1000 ng/ml are maintained for several weeks or months, opportunistic bacterial and fungal infections can occur. As in humans, cyclosporine can promote the development of neoplasia, particularly lymphomas, in cats and dogs. The administration of high levels of prednisolone (1 to 2 mg/kg q24h PO) with cyclosporine increases the likelihood of tumor formation. As in humans, cyclosporine has resulted in a marked increase in hair growth in several of my feline renal transplant recipients. When administered to dogs, cyclosporine can result in severe gingival hyperplasia, fibropapillomatosis, and severe or fatal pyodermas, especially when combined with azathioprine (Gregory et al, 2006).

Cyclosporine has a distinctly unpleasant taste to both humans and animals, which necessitates administration in gelatin capsules or mixed with other fluids. Novartis supplies capsules (Atopica) containing 10, 25, 50, or 100 mg of cyclosporine. Novartis now has an oral solution specifically marketed for cats that is deemed to be palatable. I still place the oral solution in No. 0 or No. 1 gelatin capsules. Some cats need only a very small dose of cyclosporine (1 to 3 mg per dose). Measuring and administering this small amount (0.01 to 0.03 ml) of a drug is very difficult and imprecise. Sandimmune can be diluted and stored in olive oil; I usually make a 1 : 10 dilution. Neoral can be diluted in any oral solution, but it must be administered immediately after it is diluted because it is a microemulsion concentrate. I dilute Neoral in tap water.

Cyclosporine also is available in an intravenous solution (Sandimmune IV) that must be diluted in 0.9% sodium chloride or 5% dextrose in water. For the treatment of acute renal allograft rejection, I 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, treat an acute hemolytic crisis, or provide therapy during periods when a patient cannot tolerate oral medications.

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Jul 18, 2016 | Posted by in PHARMACOLOGY, TOXICOLOGY & THERAPEUTICS | Comments Off on Immunosuppressive Agents

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