Immunosuppressive drugs: Beyond glucocorticoids

Glucocorticoids are the most commonly used drugs for immunosuppression in dogs and cats with immune-mediated diseases. Prednisone in particular induces rapid, nonspecific inhibition of the immune system by reducing inflammation-associated gene transcription, inhibiting intracellular signaling pathways, downregulating cell membrane expression of adhesion proteins, and slowing cell proliferation. The inflammatory responses of most leukocytes—including neutrophils, macrophages, lymphocytes, and antigen-presenting cells—are blunted by glucocorticoids, so immunosuppression with this class of drugs downregulates both the innate and acquired immune systems.

Systemic immunosuppression is required to treat most small-animal autoimmune diseases; however, glucocorticoids, unfortunately, modulate metabolic pathways in many nonimmune-system cell populations as well, possibly resulting in life-threatening side effects. For example, prednisone has been associated with hypercoagulability, hypertension, increased susceptibility to opportunistic infections, congestive heart failure, pancreatitis, and insulin resistance and secondary diabetes mellitus. In addition, although the expected glucocorticoid-associated clinical signs of weight gain, alopecia, polyuria, polydipsia, and polyphagia are usually only temporary annoyances in veterinary patients, some owners may find the inappropriate urination, continuous begging for food, or pica to be intolerable, leading to frustration or even euthanasia.

Photos by Greg Kindred

The widespread use of organ transplants in people has motivated pharmaceutical companies to develop newer immunosuppressive agents that more specifically target the immune system and, thus, decrease the likelihood of adverse effects. When these agents are used in dogs and cats, the synergistic immunosuppressive effects may allow veterinarians to maintain disease remission with a lower glucocorticoid dose than would be possible otherwise. Because use of these alternative immunosuppressive drugs is increasing, veterinarians must be aware of those few studies that have evaluated the effectiveness, recommended doses, or prognosis when these drugs are administered in conjunction with or in place of prednisone. This article reviews the mechanisms of action and evidence-based rationale for using the most commonly recommended nonglucocorticoid immunosuppressive drugs and introduces medications that may eventually become standard-of-care for treatment of some immune-mediated diseases in veterinary patients.

AZATHIOPRINE

Azathioprine is a prodrug that lacks immunosuppressive effects until the liver converts it into 6-mercaptopurine (6-MP).1,2 This active metabolite highly resembles adenine and guanine, which are the purine bases that make up much of RNA and DNA. The structural similarity between these molecules results in 6-MP insertion into DNA that is being synthesized (i.e. replication) immediately preceding cell division. Random insertion of 6-MP into DNA results in nonsense mutations and eventual cell death due to disruption of a critical gene or due to apoptosis triggered by a high mutation load. The incorporation of 6-MP into DNA is also promoted by azathioprine-induced interference with purine biosynthesis, thus increasing the relative concentration of 6-MP as compared with adenine and guanine concentrations. Although 6-MP is commercially available, administration of the activated drug to people increases the prevalence of adverse affects, so is not recommended in dogs or cats.

Uses

Abundant anecdotal experience exists on the benefits of azathioprine for treating immune-mediated diseases in dogs, but few controlled studies have been published. Azathioprine is commonly used in dogs with immune-mediated diseases that typically require prolonged glucocorticoid treatment, including immune-mediated hemolytic anemia (IMHA), immune-mediated thrombocytopenia (ITP), systemic lupus erythematosus, immune-mediated polyarthritis, and pemphigus foliaceus (Table 1).

Table 1 Published Initial Doses, Common Uses, and Suggested or Reported Concurrent Immunosuppressive Drugs for Selected Nonglucocorticoid Immunosuppressive Drugs*

The primary benefit of azathioprine in these diseases is its steroid-sparing effects; simultaneous use of this drug may allow lower maintenance doses of glucocorticoids or more rapid tapering with reduced risk of disease recurrence. Azathioprine is frequently first prescribed to dogs that fail to achieve remission with glucocorticoids alone or that, after an appropriate tapering protocol, still require maintenance doses associated with severe side effects. Alternatively, because azathioprine requires at least one to two weeks to reach therapeutic serum concentrations, some veterinary internists (including myself) start azathioprine administration in patients with severe immune-mediated disease at the time of diagnosis (i.e. at the same time that glucocorticoid therapy is initiated).

Most support for azathioprine therapy has been gathered from retrospective studies of dogs with IMHA. In the largest retrospective study evaluating treatment of IMHA in dogs with prednisone and azathioprine, most dogs that survived the initial 14-day high-mortality period could be weaned off drugs within three months, with a 72.6% six-month survival rate for all patients and a 92.5% six-month survival rate for dogs surviving beyond 14 days.3 Other retrospective studies of dogs with IMHA that did not have uniform treatment protocols also suggest that azathioprine improves outcome.4,5 Determining whether azathioprine truly increases the survival rate in dogs with IMHA will require additional studies. For example, azathioprine may be preferentially administered to dogs expected to survive long enough for therapeutic serum concentrations to be reached.

Using azathioprine for adjunctive treatment of other immune-mediated conditions, including ITP, Evans' syndrome (concurrent IMHA and ITP), immune-mediated neutropenia, immune-mediated skin diseases, and systemic lupus erythematosus, has also been suggested.6-10 Azathioprine monotherapy has been used to treat newly diagnosed disease in dogs with myasthenia gravis, atopy, and perianal fistulae.11-13 Although three of four dogs with myasthenia gravis were successfully managed, the time until remission was up to three months, with one dog dying because of myasthenic crisis before therapeutic serum concentrations had been presumptively attained.13 Likewise, clinical signs were completely controlled in only some dogs with atopy or perianal fistulae.11,12

Adverse effects

Azathioprine may result in rare but severe adverse effects in dogs. These effects include fulminant hepatic necrosis with massive (i.e. > 10,000 IU) increases in alanine transaminase (ALT) activity that should be treated with immediate cessation of azathioprine and aggressive supportive care. Bone marrow suppression may also occur, presumptively because dividing bone marrow stem cells will also incorrectly incorporate 6-MP into newly synthesized DNA.14 The prevalence of bone marrow suppression in dogs with IMHA treated long-term with azathioprine was 12.5% in one study; fortunately, cell numbers rapidly returned to normal after azathioprine was discontinued.3 Frequent monitoring for hepatic and bone marrow adverse effects is recommended in people, with complete blood counts and liver enzyme activity reevaluated about every three months. Toxicosis in people depends in large part on tissue concentrations of thiopurine methyltransferase (TPMT), the enzyme responsible for 6-MP degradation.15 About 10% of dogs may have decreased tissue concentrations of TPMT, with some breeds (e.g. giant schnauzers) possibly being predisposed to toxic effects.16

Healthy cats have significantly lower TPMT concentrations than dogs or people, so severe, fatal bone marrow suppression is induced when azathioprine is mistakenly prescribed at the same dose as that used in dogs.17 Because of the severe risk involved, azathioprine should not be routinely used as an immunosuppressant in cats. If therapy with this drug must be considered after failure of other agents, consultation with an internist to discuss appropriate drug dosing and monitoring is highly recommended.

CYCLOSPORINE

Cyclosporine (and tacrolimus) prevents activation of T lymphocytes despite appropriate stimulation of T lymphocyte surface receptors by antigen-derived peptides.1,2 These drugs bind to intracellular cyclophilin, producing a drug-protein complex that inactivates the enzyme calcineurin, an inducer of the lymphocyte DNA transcription factor that, in turn, upregulates interleukin-2 (IL-2) production, the main inducer of T cell proliferation. Calcineurin inhibition, therefore, prevents or slows clonal expansion of activated T cells and blocks the downstream activation of B lymphocytes, macrophages, and cytotoxic T cells. Despite the theoretically broad immunosuppressive effects of cyclosporine on much of the adaptive immune response, conflicting evidence as to its benefit in canine IMHA combined with a much higher cost than that of prednisone has limited its use in most diseases to an adjunctive treatment option rather than as a first-line agent.

Cyclosporine is metabolized primarily by enzymes of the cytochrome P-450 system. So any drug that inhibits, activates, or competes for these enzymes will alter serum cyclosporine concentrations. For example, concurrent administration of phenobarbital, a cytochrome P-450 enzyme inducer, results in lower than expected serum cyclosporine concentrations. On the other hand, ketoconazole and erythromycin increase serum cyclosporine concentrations via cytochrome P-450 inhibition. This interaction can be exploited in patients with severe cyclosporine-induced gastrointestinal tract signs or in cases in which drug cost is prohibitive; for example, concurrently administering ketoconazole, a relatively inexpensive drug, allows a reduction in the cyclosporine dose. However, ketoconazole coadministration is not routinely recommended because this drug may also result in hepatotoxicosis or gastrointestinal disturbances.

Cyclosporine pharmacokinetics is strongly influenced by drug administration in conjunction with a meal, diet composition, and bioavailability of the drug formulation being administered.18 Most current formulations of cyclosporine are well-absorbed after oral administration, although nonaqueous formulations (i.e. Sandimmune—Novartis) result in lower and less predictable serum concentrations when administered in an equivalent mg/kg dose. Cyclosporine is fat-soluble, so having owners administer the drug with a meal may improve absorption, particularly if a patient can also be appropriately fed a high-fat content diet. The therapeutic serum cyclosporine concentration cannot be predicted purely based on mg/kg dosing, and regular measurement of the trough cyclosporine concentration is required. The trough concentration (i.e. immediately before the next pill) can be measured as early as 48 hours after a change in drug dose, and many commercial laboratories offer this assay. The target trough cyclosporine concentration for most immune-mediated diseases is 400 to 600 ng/ml.

Uses

Most published support for the use of systemic cyclosporine exists for dogs with perianal fistulae, IMHA, and immune-mediated dermatologic diseases (Table 1); however, sporadic reports exist on the use of this drug for many other diseases. Glucocorticoids are rarely used as first-line therapy for perianal fistulae since affected dogs often respond completely to cyclosporine, with permanent remission possible.19-21 Topical tacrolimus may also be used, although this drug can be toxic if licked and requires gloved application.22 Patients with perianal fistulae appear to require lower trough cyclosporine concentrations (100 to 300 ng/ml) than do dogs with other diseases. Also, because larger breeds are more commonly affected, adjunctive therapy with ketoconazole is more commonly recommended.23

Dogs with IMHA that fail to respond to prednisone (with or without azathioprine) may benefit from cyclosporine as well. Although a definitive benefit to cyclosporine has not been demonstrated in this disease,24 many internists report that some dogs will achieve disease remission with cyclosporine administration. If cyclosporine is used to treat canine IMHA, avoid simultaneous administration of azathioprine because the cumulative immunosuppression anecdotally increases the prevalence of opportunistic infections.

Cyclosporine is rarely used in cats with immune-mediated diseases despite the successful long-term immunosuppression regularly achieved with this drug in feline renal transplant recipients. Use of cyclosporine has been sporadically reported in cats with presumptively allergic or hypersensitivity-induced skin diseases (such as eosinophilic plaques or indolent ulcers) or as a rescue agent in patients with severe, uncontrolled feline asthma complex, but whether this drug offers any advantages over traditionally used glucocorticoids or alternative immunosuppressive agents is unknown.25,26 Absorption and systemic distribution of cyclosporine in cats does not appear to differ from that in dogs, with similar individual animal variability.18 Unlike dogs, however, whole blood drug concentrations two hours after pill administration may be more accurate than trough drug concentrations.27 Because of this uncertainty in the optimal timing for sample collection, in many cases changes in cyclosporine dose in cats are dictated primarily by the patient's response to therapy.

Adverse effects

The most common cyclosporine-associated adverse effects in dogs and cats are gastrointestinal tract disturbances, including vomiting and diarrhea. These effects are dose-related in most patients and often do not recur after temporary dose decreases. Other less commonly to rarely reported adverse effects include alopecia, gingival hyperplasia, hypertrichosis, and increased prevalence of secondary infections.18 Lymphoproliferative neoplasms occur more commonly after long-term administration of cyclosporine to cats after renal transplantation.28 Renal toxicosis, a common adverse effect in people, has not been reliably reported in small animals.18

MYCOPHENOLATE MOFETIL

Mycophenolate mofetil, frequently referred to as mycophenolate or MMF, interferes with DNA replication, thus inhibiting lymphocyte proliferation.29,30 Mycophenolate is absorbed from the gastrointestinal tract and converted during absorption or in circulation to mycophenolic acid (MPA), the active drug metabolite. MPA is a noncompetitive inhibitor of inosine monophosphate dehydrogenase, the rate-limiting enzyme in de novo synthesis of guanine and other purines. For most cells, this inhibition does not interfere with DNA synthesis because purines can be salvaged from other sources. Lymphocytes, however, not only lack most purine salvage capability, but at times of cell division upregulate an isoform of inosine monophosphate dehydrogenase that is more sensitive to the inhibitory effects of MPA, thus magnifying their susceptibility to this drug.29,31

Uses

Mycophenolate is used primarily as a maintenance immunosuppressive agent in people. Although it may be prescribed at the time of diagnosis, it provides insufficient systemic immunosuppression to induce disease remission when administered alone. Once remission is achieved in combination with other drugs, then MMF administration can be continued for long-term maintenance. Effective immunosuppressive protocols that include MMF have been established for a number of human diseases including rheumatoid arthritis, systemic lupus erythematosus, and some immune-mediated glomerulonephritides as well as for use after organ transplantation.30

Use of prednisolone and MMF has been reported in dogs with IMHA (Table 1), with seven of eight dogs having complete resolution of anemia within one month, only one dog of which had transient, mild enteritis.32 Single case reports include successful treatment of dogs with myasthenia gravis or aplastic anemia that had failed to respond to standard therapy and resolution of suspected glomerulonephritis.33-35 Dogs with pemphigus complex may also be treated with MMF on occasion.10

Prospective studies are required to establish the effectiveness of MMF in treating these various diseases and, in particular, whether its use improves outcome beyond standard therapy. For example, despite the single case of MMF-responsive myasthenia gravis in a dog mentioned above, retrospective analysis of 12 dogs treated with pyridostigmine vs. 15 dogs treated with pyridostigmine and MMF failed to demonstrate improved case outcome with MMF.36 Therefore, MMF should likely be reserved for those dogs in which established immunosuppressive therapies have failed for treatment of a given disease or in which side effects from other drugs are intolerable.

Adverse effects

Serum MPA concentrations after oral MMF administration in dogs are highly variable and call into question its suitability as a post-transplant immunosuppressant.37 In addition, the dose predicted for immunosuppression by pharmacokinetic studies and extrapolation from experience in people results in severe, intractable diarrhea and weight loss in laboratory dogs.38 Nevertheless, the few published reports in dogs with naturally occurring immune-mediated diseases have suggested that, in some patients, MMF is effective at lower doses, with limited side effects.

LEFLUNOMIDE

Leflunomide is an inactive prodrug hydrolyzed in the intestine and plasma to the active metabolite teriflunomide (formerly known as A77 1726).39,40 Teriflunomide's mechanism of immunosuppression is similar to that of MPA, but rather than interfering with purine biosynthesis, inhibition of the enzyme dihydroorotate dehydrogenase selectively prevents lymphocyte de novo pyrimidine production. Teriflunomide may further interfere with lymphocyte proliferation and function by inhibiting tyrosine kinases associated with several cytokine and growth factor receptors, although whether this occurs at clinically relevant drug concentrations is unclear.41 Finally, although not directly related to its use as an immunosuppressive agent, teriflunomide also inhibits replication of a number of herpesviruses, including feline herpesvirus-1.42

Uses

Leflunomide is primarily used in people to treat immune-mediated arthritides, including rheumatoid arthritis and psoriatic arthritis.39,40 Use in other autoimmune diseases or for immunosuppression of transplant recipients has also been reported, including Crohn disease, systemic lupus erythematosus, immune-mediated vasculitides, and some nephropathies.39 However, leflunomide is still limited to use as a rescue agent in people for most diseases in which efficacy has been demonstrated because of the high prevalence of side effects, many of which are quite severe (see Above).

Use of leflunomide has been reported for monotherapy treatment of 14 dogs with immune-mediated polyarthritis and in conjunction with methotrexate in 12 cats with rheumatoid arthritis (Table 1).43,44 Two of the dogs and all of the cats had been previously treated with prednisone with minimal to no improvement noted, or intolerable side effects of prednisone had led to alternative therapy. Complete resolution of clinical signs in both groups of animals occurred in about two-thirds of animals; however, disease remission in both groups was primarily determined by the presence of clinical signs (i.e. lameness and semiobjective pain scoring systems) rather than repeat arthrocentesis, joint radiography, and rheumatoid factor titer measurement. In addition, most dogs with immune-mediated polyarthritis required concurrent administration of a nonsteroidal anti-inflammatory drug (NSAID) for pain relief during the initial leflunomide treatment period.

Additional diseases in dogs and cats that have been treated with leflunomide after failure of conventional therapy include IMHA, ITP, Evans' syndrome, nonsuppurative meningoencephalomyelitis, systemic histiocytosis, immune-mediated polyarthritis, and pemphigus foliaceus; most reported cases have resulted in disease remission.45,46 However, studies comparing the efficacy of leflunomide with traditional immunosuppressive drug regimens are still lacking, and whether this drug should be incorporated into first-line therapy protocols or used as a rescue agent is still unknown.

Adverse effects

Leflunomide may occasionally be associated with vomiting or diarrhea, particularly at the upper end of recommended dose ranges. When these clinical signs do occur, temporary drug dose reductions usually lead to rapid resolution of gastrointestinal upset; following one to two weeks of this decreased dose, patients will oftentimes tolerate a return to the initial higher dose without recurrence of side effects. In people, in addition to gastrointestinal upset and skin rashes, leflunomide has also been associated with several life-threatening idiosyncratic reactions, including acute and chronic hepatotoxicosis, severe myelosuppression with secondary infections, interstitial lung disease, and toxic epidermal necrolysis.39,40 Thus far, none of these severe side effects have been reported in dogs or cats, so leflunomide holds promise for more widespread use in the future.

CYCLOPHOSPHAMIDE

Cyclophosphamide is an alkylating agent; two chloride moieties at opposite ends of the molecule are each capable of covalently bonding with DNA guanine bases.1,2 This bonding results in attachment of cyclophosphamide to the DNA strand at each reaction site and inhibition of DNA strand disassociation that must occur during cell division. Because cyclophosphamide-induced DNA cross-linking occurs regardless of whether a cell is actively undergoing mitosis, cyclophosphamide's effects are cell-cycle independent (in contrast to most other immunosuppressive drugs). Although this is an advantage when used to treat neoplastic diseases, there may be a higher likelihood of severe immunosuppression and systemic toxicosis with this drug.

Uses

Although the benefits of cyclophosphamide in multiagent chemotherapy of lymphoma are well-established in veterinary patients, few studies have evaluated its use in canine or feline immune-mediated diseases. A prospective study of dogs with IMHA treated with cyclophosphamide demonstrated no benefit over prednisone therapy alone.47 Other, noncontrolled retrospective case series have suggested that two different cyclophosphamide dosing regimens do not differ in effectiveness or that prognosis may perhaps even be worsened with cyclophosphamide administration.24,48 In the study which suggested a worsened prognosis, cyclophosphamide administration was associated with delayed resolution of anemia and longer clinical recovery, possibly because of drug-induced suppression of bone marrow stem cells.24 Because of the poor results in adjunctive treatment of IMHA as well as the availability of safer alternatives, cyclophosphamide has fallen out of favor as an adjunctive treatment option for immune-mediated diseases in dogs and cats.

Adverse effects

The most common side effects of cyclophosphamide in dogs include bone marrow suppression, hemorrhagic cystitis, and gastrointestinal disturbances. Specific recommendations on how to minimize or treat these drug-associated complications are beyond the scope of this article, but most veterinary oncology textbooks or chemotherapy references provide this information.

CONCLUSION

At this time, prednisone remains the mainstay drug for immunosuppression in dogs and cats with immune-mediated diseases. However, alternative lymphocyte-specific immunosuppressive medications are widely used in people with autoimmune diseases and could, in theory, be effective in veterinary patients as well. These drugs have been successfully used in small numbers of animals, but prospective studies comparing outcome of patients treated with traditional drug combinations vs. newer immunosuppressants are lacking. Published or suggested doses that have proven effective in limited numbers of animals are summarized in Table 1. Use of these nonglucocorticoid medications for long-term maintenance of immunosuppression may be reasonable if patients cannot tolerate or do not respond to currently recommended drugs. However, informed owner consent is strongly recommended, particularly in regard to the prevalence and severity of drug-associated adverse effects that have been reported in people.

Barrak Pressler, DVM, PhD, DACVIM

Department of Veterinary Clinical Sciences

School of Veterinary Medicine

Purdue University

West Lafayette, IN 47907

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