CORTICOSTEROIDS

Glucocorticoids exert their action via binding to intracellular receptors and translocating to the nucleus. They bind receptor sites that regulate action of nuclear factors (such as NFkB) and control expression of glucocorticoid-responsive genes (for example, expression of cytokines, and immune function). Other functions also are responsible for their anti-inflammatory and immunosuppressive action. In clinical veterinary medicine, the action of glucocorticoids is dose-dependent. The lowest doses (using prednisolone as an example) produce physiological effects (0.25 mg/kg/day), higher doses produce anti-inflammatory effects (1.0 mg/kg/day), and still higher doses are considered immunosuppressive (2-4 mg/kg/day). (Some experts contend that immunosuppressive effects can be observed even at low doses.)

For several immune-mediated disorders (for example, autoimmune skin disease, immune-mediated hemolytic anemia and thrombocytopenia, and SLE) glucocorticoids suppress the immune system response to alleviate clinical signs in many patients. Initial (induction) dosage regimens employed are in the range of daily doses of 2.2 to 6.6 mg/kg per day (prednisolone or prednisone). Reported optimal induction doses for skin diseases are 4.4 mg/kg/day for dogs and 6.6 mg/kg/day for cats, but optimal effective doses have not been evaluated for other diseases. A commonly cited immunosuppressive dose for dogs is 2 mg/kg of prednisolone or prednisone administered twice daily orally. During the initial induction period, the daily dose can be divided into a twice-daily dose to lessen (but not eliminate) some of the acute effects such as gastrointestinal problems or behavioral changes. After the induction treatment phase, (at least 10-14 days, or until the animal responds), the dose interval can be lengthened to once daily for another period of time until it is determined that the animal's disease is stable. If this is possible, maintenance therapy is in the range of 1-2 mg/kg every other day.

ALKYLATING AGENTS

If a patient does not show an initial response to corticosteroids, or if the disease is refractory to corticosteroids, an alkylating agent such as cyclophosphamide (Cytoxan) has been used for some diseases. Cyclophosphamide is one of the nitrogen mustards and is one of the most potent immunosuppressive drugs available. The immunosuppressive effects of the nitrogen mustards can be attributed to the metabolites phosphoramide mustard and acrolein. The cytotoxic effect on lymphocytes is caused by damage to DNA. Because cyclophosphamide is directly cytotoxic to lymphocytes, it can suppress B-cell activity and antibody formation. B-cells are reported to be affected more than T-cells because their rate of recovery from an alkylating agent is slower.

Cyclophosphamide is a potent immunosuppressive drug, but the adverse effects can be serious. In people, long-term therapy is discouraged because of the risk that cyclophosphamide will induce secondary malignancies. In dogs the most serious toxic effect is bone marrow suppression, which can lead to secondary infections. Another adverse effect in dogs is sterile hemorrhagic cystitis, which is a severe toxic reaction involving the urinary bladder mucosa. The metabolites cause toxic injury to the bladder epithelium (especially acrolein), because they are concentrated and excreted in the urine. Various strategies have been used to decrease the injury to the bladder epithelium.

When used clinically, the dose administered to dogs is 50 mg/m2, which is approximately 1.5 mg/kg for large dogs and 2.5 mg/kg for small dogs. It is available in 25 and 50 mg tablets. This dose has been administered on an every-other-day (EOD) basis, with corticosteroids administered on the alternate days. Cats are more resistant to the toxic effects on the bladder. They have received a total dose of 6.25-12.5 mg, once daily, 4 days per week. In dogs, cyclophosphamide also has been administered as a "pulse dose" as high as 10 mg/kg q21days.

THIOPURINES: AZATHIOPRINE

As first-line therapy, or as an alternative to nitrogen mustard alkylating agents for treatment of immune-mediated disease, thiopurines have been administered. In veterinary medicine, azathioprine has been used for immune-mediated anemia, colitis, immune-mediated skin disease, and acquired myasthenia gravis. Azathioprine is available as 50 mg tablets. It is dosed at 2 mg/kg, orally, q24h. Long-term therapy is administered at a dose of 0.5 to 1.0 mg/kg every other day, with prednisone administered on the alternate days. In people the lag-period before successful treatment is recognized with azathioprine may be as long as 2-8 months. In veterinary medicine this lag-period is probably shorter and therapeutic benefits have been observed after only 3-5 weeks.

Bone marrow suppression is a concern in all animals treated with azathioprine. Leukopenia and thrombocytopenia are reported adverse effects. Gastrointestinal toxicity and hepatotoxicity also are possible. Gastrointestinal effects such as nausea and diarrhea may be only temporary and subside after several days of therapy. Sterile hemorrhagic cystitis, the complication cited for cyclophosphamide has not been seen with azathioprine. There has also been association (but not well documented) between the administration of azathioprine plus prednisolone and the development of acute pancreatitis in dogs. It has been suggested that this effect is caused by azathioprine's effect on decreasing pancreatic secretion in animals.

Doses of 2.2 mg/kg every other day produced profound neutropenia in cats (Beale et al, 1992). Some veterinarians have administered 1.1 mg/kg every day, or every other day, but other references have discouraged its use in cats because of the bone-marrow suppressing effects (Helton-Rhodes, 1995). Differences in metabolism may explain the susceptibility in cats (discussed below under metabolism). Because cats may be at a risk of bone marrow suppression from administration of azathioprine, currently recommended doses are as low as 0.3 mg/kg once daily or every other day. Careful monitoring of CBC is recommended during this treatment and doses lowered if necessary.

After azathioprine is converted to 6-MP, it is further metabolized by three routes to other metabolites. One metabolic route is via thiopurine methyltransferase (TPMT), which is responsible for conversion to non-toxic 6-MP nucleotides. In people there is genetic polymorphism that determines high or low levels of TPMT (Lennard et al, 1989). Most of the human population has high TPMT activity, but about 11% have low levels and are more prone to toxicity. In people with low TPMT activity, doses must be lowered. It appears that in dogs, this proportion may be similar with 90% of dogs showing normal TPMT activity and 10% with low activity (White et al 1998). Cats, as expected, have low TPMT activity (Foster et al 1999). Animals that are treated with azathioprine should have their bone marrow function monitored during initial therapy to identify those that may have low TPMT activity so doses can be adjusted accordingly.

CYCLOSPORINE

Cyclosporine (also called cyclosporin A, and CsA) is a fat-soluble, cyclic polypeptide fungal product with potent immunosuppressive activity. It has been an important drug used in humans, primarily to produce immunosuppression in organ transplant patients. This drug binds to a specific cellular receptor on calcineurin and inhibits the T-cell receptor-activated signal transduction pathway. Particularly important are its effects to suppresses interleukin-2 (IL-2) and other cytokines, and blocks proliferation of activated T-lymphocytes. The action of cyclosporine is more specific for T-cells as compared to B-cells. One important advantage in comparison to other immunosuppressive drugs is that it does not cause significant myelosuppression or suppress nonspecific immunity.

In veterinary medicine, cyclosporine has been used for various immune-mediated conditions, including perianal fistulas, sebaceous adenitis, and idiopathic sterile nodular panniculitis. However, in small pilot studies, results for treating pemphigus foliaceous in dogs have been disappointing.

Because of the efficacy of cyclosporine for treating atopic dermatitis in people, the use of cyclosporine for treating canine atopic dermatitis has been investigated in dogs as summarized in the review by Marsella & Olivry (2001). The dose used in these studies was 5 mg/kg/day and was as effective as prednisolone (Olivry et al, 2000). Cyclosporine is well tolerated in cats and has been used for immunosuppression in cats undergoing kidney transplantation. It also has been investigated for treating skin diseases. A good response to a dose of 25 mg/cat was seen in 6 cases of eosinophilic plaque and 3 cases of oral eosinophilic granuloma (Guaguere & Prelaud, 2000). In three cases of indolent lip ulcers, the response was less impressive.

Because low doses are used for treating perianal fistulas and atopic dermatitis, usually 5 mg/kg/day or less, adverse effects in dogs are not serious. The most common problem is nausea and loss of appetite. Because cyclosporine undergoes complicated metabolism in the intestine and liver (Wu et al 1995), metabolizing enzyme systems can be turned on (induced) or turned off (inhibited) by other drugs to alter the amount of cyclosporine absorbed. Inducing drugs include rifampin and may decrease concentrations of cyclosporine. Inhibiting drugs include ketoconazole, erythromycin, and in people, flavonoids from grapefruit juice (bergamottin). Cimetidine does not affect metabolism in dogs. Because ketoconazole will reduce cyclosporine clearance in dogs by as much as 85%, (Myre et al 1991) cyclosporine doses can be drastically reduced if a patient is also receiving ketoconazole.

Monitoring of blood concentrations is not necessary during routine therapy, especially if response is favorable and there is an absence of adverse effects. However, in refractory patients, or when there are drug interactions to be monitored, blood concentrations can be measured by clinical laboratories. In the cited studies for perianal fistula treatment, (Mathews and Sukhiani, 1997) 2.5 to 6 mg/kg/day (3 mg/kg q12h) was given to achieve an effective blood concentration of 100-300 ng/ml. For organ transplant in dogs, 10 mg/kg q12h to achieve concentrations of 500-600 ng/mL has been used (Mathews et al, 2000). For treating dermatitis in animals, doses of 3-6 mg/kg/day (dogs and cats) have been administered to achieve concentrations of 200-300 ng/mL. Concentrations above 600 ng/mL may increase risk of toxicity. One must be cognizant of the assay used when monitoring cyclosporine because some immune-mediated assays will overestimate concentrations (e.g., TDx FPIA) (Steimer 1999). Our laboratory has documented that the fluorescence polarization immunoassay using the popular TDx technology, will overestimate the concentration of cyclosporine in cats by 2x compared to a specific HPLC assay, even when the monoclonal assay is used. The TDx assay will overestimate the concentration of cyclosporine in dogs by a factor of 1.6-1.7.

REFERENCES

1.  Beale KM, Altman D, Clemmons RR et al: Systemic toxicosis associated with azathioprine administration in domestic cats. Am J Vet Res 53: 1236-1240, 1992.

2.  Foster AP, Shaw SE, Duley JA, and Harbour DA. (1999) An evaluation of thiopurine methyltransferase in the cat. ESVD/ECVD Congress, pp. 133.

3.  Guaguère E, Prélaud P. Efficacy of cyclosporin in the treatment of 12 cases of eosinophilic granuloma complex. Vet Dermatol 2000; 11 (Supplement 1): 31.

4.  Helton-Rhodes K. Feline immunomodulators. In Bonagura JD (ed) Current Veterinary Therapy XII, 1995. pp. 581-584.

5.  Lennard L, Van Loon JA, Weinshilboum RM: Pharmacogenetics of acute azathioprine toxicity. Relationship to thiopurine methyltransferase genetic polymorphism. Clin Pharmacol Ther 46: 149-154, 1989.

6.  Marsella R, Olivry T. The ACVD task force on canine atopic dermatitis (XXII): nonsteroidal anti-inflammatory pharmacotherapy. Vet Immunol Immunopathol 2001; 81(3-4):331-45.

7.  Mathews KA, Sukhiani HR: Randomized controlled trial of cyclosporine for treatment of perianal fistulas in dogs. J Am Vet Med Assoc 211: 1249-1253, 1997.

8.  Mathews KG, Gregory CR. Renal transplants in cats: 66 cases (1987-1996). J Am Vet Med Assoc 211: 1432-1436.

9.  Myre SA, Schoeder TJ, Grund VR, et al. Critical ketoconazole dosage range for cyclosporin clearance inhibition in the dog. Pharmacology 43: 233-241, 1991.

10. Olivry T, Rivierre C, Jackson HA, Murphy KM, Sousa CA: Cyclosporin-A decreases skin lesions and pruritus in dogs with atopic dermatitis: a prednisolone-controlled blinded trial. Veterinary Dermatology 11(Suppl.1): 47 (abstract P-19) , 2000.

11. Steimer W. Performance and specificity of monoclonal immunoassays for cyclosporine monitoring: How specific is specific? Clin Chem 45: 371-381.

12. White SD, Rosychuk RAW, Scott KV. Investigation into the role of thiopurine methyltransferase in the use of azathioprine in dogs: Phase one. 14th Proceedings of AAVD/ACVD meeting, pp. 111-112.

13. Wu, C-Y, Benet LZ, Hebert MF, et al. (1995) Differentiation of absorption, first pass gut and hepatic metabolism in man. Studies with cyclosporine. Clin Pharmacol Therap 58: 492-497.