Immunosuppressive drugs: beyond glucocorticoids (Proceedings)

Glucocorticoids are the most commonly used drugs for immunosuppression of dogs and cats with immune-mediated diseases because they induce rapid, non-specific inhibition of the immune system by reducing inflammation-associated gene transcription, inhibiting intracellular signaling pathways, down-regulating cell membrane expression of adhesion proteins, and slowing cell proliferation. Glucocorticoids unfortunately also modulate metabolic pathways in many non-immune system cell populations, which may result in life-threatening side-effects such as hypercoagulability, hypertension, increased susceptibility to opportunistic infections, congestive heart failure, pancreatitis, and insulin resistance and secondary diabetes mellitus. In addition, although weight gain, alopecia, polyuria, polydipsia, and polyphagia are usually only temporary annoyances, some owners may find these signs to be intolerable, leading to frustration or even euthanasia. Newer or alternative immunosuppressive agents may have synergistic immunosuppressive effects and therefore allow clinicians to maintain disease remission with a lower dose of glucocorticoids 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 effectiveness, recommended doses, or prognosis when these drugs are administered in conjunction with or in place of prednisone.

Azathioprine

Azathioprine is a pro-drug that lacks immunosuppressive effects until it is converted by the liver into 6-mercaptopurine (6-MP). 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 insertion of 6-MP into DNA which 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. 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 to adenine and guanine. Although 6-MP is commercially available, administration of the activated drug to people increases the prevalence of adverse affects, and therefore is not recommended in dogs or cats.

Abundant anecdotal experience exists on the benefits of azathioprine for treatment of immune-mediated diseases in dogs, but few controlled studies have been published. Use of azathioprine in dogs with immune-mediated diseases that typically require prolonged treatment with glucocorticoids (including immune-mediated hemolytic anemia [IMHA], immune-mediated thrombocytopenia [ITP], systemic lupus erythematosus, immune-mediated polyarthritis, and pemphigus foliaceus) is common. The primary benefit of azathioprine in these diseases is due to its steroid-sparing affects; 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 who fail to achieve remission with glucocorticoids alone, or who following 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) choose to begin azathioprine administration in cases of severe immune-mediated disease at the time of diagnosis (i.e. at the same time that glucocorticoid therapy is initiated).

Most support for use of azathioprine is as part of retrospective studies of dogs with IMHA. In the largest retrospective study evaluating treatment of dogs with prednisone and azathioprine, most dogs which survived the initial 14-day high-mortality period could be weaned off drugs within 3 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. Other retrospective studies of dogs with IMHA that did not have uniform treatment protocols also support the belief that outcome is improved with azathioprine. Whether the presumptive beneficial effects of this drug on survival are true 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.

Use of azathioprine for adjunctive treatment of other immune-mediated conditions, including ITP, Evan's syndrome, immune-mediated neutropenia, immune-mediated skin diseases, and systemic lupus erythematosus has also been suggested. Azathioprine monotherapy for treatment of newly-diagnosed disease has been reported in dogs with myasthenia gravis, atopy, and perianal fistulae. Although 3 of 4 dogs with myasthenia gravis were successfully managed, time until remission was up to three months, with one dog dying due to myasthenic crisis before therapeutic serum concentrations had been presumptively attained. Likewise, clinical signs were completely controlled in only some dogs with atopy or perianal fistulae.

Azathioprine may result in rare but severe adverse effects in dogs. These include fulminant hepatic necrosis with massive (i.e. >10,000 IU) increases in ALT 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. The prevalence of bone marrow suppression in dogs with IMHA treated with long-term azathioprine was 12.5% in one study; fortunately, suppressed cell lines rapidly resolved to discontinuation of azathioprine. Frequent monitoring for hepatic and bone marrow adverse effects is recommended in people, with complete blood counts and liver enzyme activity re-evaluated on an approximately 3-month basis. Toxicity in people is dependent in large part on tissue concentrations of thiopurine methyltransferase (TPMT), the enzyme responsible for 6-MP degradation. Approximately 10% of dogs may have decreased tissue concentrations of TPMT, with some breeds (i.e. giant schnauzers) possibly being predisposed to toxic effects.

Healthy cats have significantly lower TPMT concentrations than dogs or people, and therefore severe, fatal bone marrow suppression is induced when azathioprine is mistakenly prescribed at the same dose as used in dogs. 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 following failure of other agents, consultation with an internist to discuss appropriate drug dosing and monitoring is highly recommended.

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. This 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, its effects are cell-cycle independent (in contrast to most other immunosuppressive drugs). Although this is an advantage when used for treatment of neoplastic diseases, there may be a higher likelihood of severe immunosuppression and systemic toxicity with this drug. Side-effects in dogs include bone marrow suppression, hemorrhagic cystitis, and gastrointestinal disturbances.

Although the benefits of cyclophosphamide in multiagent chemotherapy therapy of lymphoma are well-establish, 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. Other, non-controlled 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 administration of cyclophosphamide. In this latter study, cyclophosphamide administration was associated with delayed resolution of anemia and longer clinical recovery, possibly due to drug-induced suppression of bone marrow stem cells. 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 of dogs and cats.

Cyclosporine

Cyclosporine (and tacrolimus) prevents activation of T-lymphocytes despite appropriate stimulation of T-cell surface receptors by antigen-derived peptides. These drugs bind to intracellular cyclophilin, producing a drug-protein complex which inactivates the enzyme calcineurin, an inducer of the lymphocyte DNA transcription factor which in turn up-regulates 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-cells, macrophages, and cytotoxic T-cells. Despite the theoretically broad immunosuppressive effects of this drug on much of the adaptive immune response, conflicting evidence as to its benefit in canine IMHA combined with the 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-450 system. Therefore any drug that inhibits, activates, or competes for these enzymes will alter serum concentrations of cyclosporine. For example, concurrent administration of phenobarbital, a cytochrome-450 enzyme inducer, results in lower than expected serum cyclosporine concentrations. Ketoconazole and erythromycin, on the contrary, increase serum cyclosporine concentrations via cytochrome-450 inhibition. This interaction can be exploited in patients with severe cyclosporine-induced gastrointestinal tract signs or where drug cost is prohibitive; ketoconazole, a relatively inexpensive drug, can be administered in order to allow lowering of the cyclosporine dose. However, ketoconazole co-administration is not routinely recommended because this drug may also result in hepatotoxicity or gastrointestinal disturbances.

Cyclosporine pharmacokinetics are strongly influenced by administration of drug in conjunction with a meal, diet composition, and bioavailability of the drug formulation being administered. Most current formulations of cyclosporine are well-absorbed following oral administration, although non-aqueous formulations (i.e. Sandimmune®) 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 the patient can also be appropriately fed a high-fat content diet. Therapeutic serum cyclosporine concentration cannot be predicted purely based on mg/kg dosing, and regular measurement of trough cyclosporine concentration is required. Trough concentration (i.e. immediately prior to the next pill) can be measured as early as 48 hours following change in drug dose, and many commercial laboratories offer this assay. Target trough concentration for most immune-mediated diseases is 400-600 ng/ml.

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

Most published support for the use of systemic cyclosporine exists for dogs with perianal fistulae, IMHA, and immune-mediated dermatologic diseases; however, sporadic reports exist on the use of this drug for many other diseases. Glucocorticoids are rarely used as first-line therapy for treatment of perianal fistulae, as affected dogs often respond completely to cyclosporine, with permanent remission possible. Topical tacrolimus may also be used, although this drug can be toxic if licked and requires gloved application. Patients with perianal fistulae appear to not require as high trough cyclosporine concentrations (100-300 ng/mL) than dogs with other diseases. Also, because larger breeds are more commonly affected, adjunctive therapy with ketoconazole is more commonly recommended. Dogs with IMHA which 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, many internists report that some dogs will in fact achieve disease remission with administration of this drug. If cyclosporine is used for treatment of canine IMHA, simultaneous administration of azathioprine should be avoided, because the cumulative immunosuppression anecdotally increases the prevalence of opportunistic infections.

Mycophenolate mofetil

Mycophenolate mofetil (frequently referred to as 'mycophenolate' or 'MMF') interferes with DNA replication, thus inhibiting lymphocyte proliferation. 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 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 up-regulate an isoform of inosine monophosphate dehydrogenase that is more sensitive to the inhibitory effects of MPA, thus magnifying their susceptibility to this drug.

Mycophenolate is used primarily as a maintenance immunosuppressive agent in people. Although it may be prescribed at the time of first 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 which include MMF have been established for a number of human diseases including rheumatoid arthritis, systemic lupus erythematosus, some immune-mediated glomerulonephritides, and following organ transplantation.

Serum MPA concentrations following oral MMF administration in dogs are highly variable and call into question its suitability as a post-transplant immunosuppressant. 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. 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. Use of prednisone and MMF has been reported in dogs with IMHA, with 7 of 8 dogs having complete resolution of anemia within one month, only one dog of which had transient, mild enteritis. 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. Additional diseases of which this author is aware that MMF may have had a beneficial effect include as an adjunctive agent to cyclosporine in dogs with non-regenerative IMHA (personal communications), and in dogs with pemphigus complex.

Prospective studies are required in order to establish the effectiveness of MMF in these various diseases, and in particular whether use of this drug 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 versus 15 dogs treated with pyridostigmine and MMF failed to demonstrate improved case outcome with MMF. 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.

Leflunomide

Leflunomide is an inactive pro-drug which is hydrolyzed in the intestine and plasma to the active metabolite teriflunomide (formerly known as 'A77 1726'). The mechanism of immunosuppression by teriflunomide is similar to that of mycophenolic acid, 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 via inhibition of tyrosine kinases associated with several cytokine and growth factor receptors, although whether this occurs at clinically relevant drug concentrations is unclear. Finally, although not directly related to its use as an immunosuppressive agent, teriflunomide also inhibits replication of a number of herpesvirus, including feline herpesvirus-1.

Leflunomide is primarily used in people for treatment of immune-mediated arthritides, including rheumatoid arthritis and psoriatric arthritis. Use in other autoimmune diseases or for immunosuppression of transplant recipients has also been reported, including Crohn's disease, systemic lupus erythematosus, immune-mediated vasculitides, and some nephropathies. However, leflunomide is still limited to use as a rescue drug in most diseases because of the high prevalence of side-effects, many of which are quite severe. In addition to gastrointestinal upset and skin rashes, life-threatening reactions may include acute and chronic hepatotoxicity, severe myelosuppression with secondary infections, interstitial lung disease, and toxic epidermal necrolysis have been reported.

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. Two dogs and all 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 approximately two-thirds of animals; however, disease remission in both groups was primarily determined by presence of clinical signs (i.e. lameness and semi-objective 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 non-steroidal anti-inflammatory drug for pain relief during the initial leflunomide treatment period. Additional diseases in dogs and cats which have been treated with leflunomide following failure of conventional therapy include immune-mediated hemolytic anemia, immune-mediated thrombocytopenia, Evan's syndrome, non-suppurative meningoencephalomyelitis, systemic histiocytosis, immune-mediated polyarthritis, pemphigus foliaceus; a majority of cases resulted in disease remission. Larger scale studies comparing efficacy of leflunomide to traditional immunosuppressive drug regimens are still lacking, but the lack of severe side effects in any o reported cases thus far holds promise for more widespread use of this drug in the future.

*Portions of this manuscript have been reproduced from a similar article by the author, published in Veterinary Medicine August 2010 (anticipated).