Immunosuppressive drugs: Beyond glucocorticoids (Proceedings)

There is no question that glucocorticoids (GCs) remain the mainstay of immunosuppressive therapy in small animal medicine. However other drugs are available that can be used in conjunction with GCs in order to provide synergistic immunosuppression and thus allow lower GC dosage, more specifically target certain arms of the immune system, and provide proven superior immunosuppression to GCs in a few diseases. Unfortunately only a few controlled studies have been performed to evaluate these alternative drugs despite anecdotal evidence that they may be of benefit.

Azathioprine

Azathioprine is a purine analog; its biologic structure is similar to the basic building blocks of DNA, but when incorporated into DNA during cell replication it results in slowed cell growth and death via mutations or apoptosis. Although activated lymphocytes are the primary target of this drug, other rapidly dividing cells, particularly bone marrow stem cells, are affected as well. Azathioprine is metabolized in the liver to the active metabolite 6-mercaptopurine; although 6-MP is available in an oral form it is more toxic than azathioprine in people and possibly dogs as well.

Azathioprine is the non-GC immunosuppressant with which we as a profession have the most anecdotal experience, although few controlled studies exist on its use in veterinary patients. For example, use of azathioprine in immune-mediated diseases that require long-term immunosuppressive treatment (such as IMHA, ITP, myasthenia gravis, or SLE) is considered routine. The primary benefit of azathioprine is that because it has a different mechanism of action than GCs, simultaneous use may allow lower doses of GCs or more rapid tapering. However azathioprine requires several weeks to reach therapeutic serum concentrations; therefore many clinicians (including myself) routinely start azathioprine in cases of severe immune-mediated disease at the time of diagnosis. The strongest published evidence for use of azathioprine is a retrospective which examined short and long-term survival of dogs with IMHA. Dogs were treated with prednisone with or without azathioprine (with subgroups receiving other adjunctive therapies). Regardless of the adjunctive therapies, dogs which received azathioprine had better outcomes than those which received prednisone alone in previous reports.

Azathioprine causes hepatic necrosis or bone marrow suppression in some dogs. Frequent monitoring is recommended in people; ideally a full CBC, serum chemistry panel, and urinalysis should be performed every 3 months while receiving azathioprine. Hepatotoxicity in dogs is due to widespread necrosis, with massive increases in ALT activity noted. Withdrawal of the drug and aggressive supportive care is required if this occurs. Bone marrow suppression usually first affects the neutrophil line, although all cell lines may eventually be decreased. Toxicity is partially related to tissue concentrations of thiopurine methyltransferase, an enzyme responsible for degrading 6-MP. 10% of dogs may have decreased concentrations of TPMT, with some breeds (Giant Schnauzers) possibly having lower concentrations.

Cats routinely develop severe bone marrow suppression and/or toxicity with azathioprine, and thus should not routinely receive this drug. I have used this drug at a very low dose as a last-ditch effort in cats with IMHA; if this is being considered I recommend consulting with an internist to discuss appropriate dosing and monitoring.

Cyclophosphamide

Cyclophosphamide is an alkylating agent: it cross-links DNA strands, preventing the disassociation that must occur for cell division to progress. Most immunosuppressive drugs preferentially exert their mechanism of action on actively dividing cells; however, because cyclophosphamide-induced DNA cross-linking occurs regardless of whether a cell is actively dividing, it is cell-cycle independent. Unfortunately this means that there is the potential for more severe immunosuppression and toxicity. Side-effects include bone marrow suppression, gastrointestinal tract signs, and severe hemorrhagic cystitis.

Unfortunately, although cyclophosphamide has a clear benefit for the treatment of neoplasia (particularly lymphoproliferative neoplasms), it may worsen prognosis in dogs with IMHA. Because this drug affects all cells, bone marrow stem cells are also suppressed, and thus erythrocyte regeneration was slowed and recovery was delayed in onestudy. Because of these discouraging results, cyclophosphamide has fallen out of favor as a treatment option for IMHA, and its use in other immune-mediated diseases has likewise diminished.

Cyclosporine and Tacrolimus

Cyclosporine and tacrolimus inhibit activation of T-lymphocytes by inactivating the enzyme calcineurin. Calcineurin is an activator a DNA transcription factor necessary for T-cell proliferation. Therefore inhibition slows T-cell responses, activation of B-cells and macrophages, and T-cell cytotoxicity. Cyclosporine is primarily used as an adjunct to other immunosuppressive drugs rather than as a sole therapy. Sirolimus (Rapamycin®) has a slightly different mechanism of action as the other calcineurin inhibitors, but has not been studied in veterinary patients.

The pharmacokinetics of cyclosporine vary from dog-to-dog, are influenced by diet, and bioavailability differs amongst formulations. Neoral®, Atopica®, and (maybe) generic cyclosporine are better absorbed, whereas Sandimmune® results in lower serum concentrations when administered in an equivalent milligram per kilogram dose. Because of this variability, measurement of serum cyclosporine concentration is required to ensure adequate dosing. Trough cyclosporine concentration can be measured (i.e. immediately prior to the next pill) as soon as 48 hours after a change in drug dose. Gastrointestinal tract signs are most common and often correlate with drug dose; alopecia, gingival hyperplasia, hypertrichosis, and secondary infections are less common to rare in dogs. In people renal toxicity is routine with cyclosporine administration, but this has not been reliably reported in small animals.

Cyclosporine is metabolized primarily by enzymes of the cytochrome-450 system. Therefore any drug that inhibits, activates, or competes for these enzymes will alter the serum concentrations of cyclosporine. Ketoconazole and erythromycin result in increased serum cyclosporine concentration; ketoconazole is occasionally administered to those patients that have severe gastrointestinal tract signs or where drug cost is prohibitive in order to allow lowering of the cyclosporine dose. Despite these advantages, ketoconazole co-administration is not done routinely because this antifungal agent is not a benign drug (hepatotoxicity and gastrointestinal effects are common), and because the change in serum cyclosporine concentration in response to a given ketoconazole dose is variable.

Published support for the use of cyclosporine exists in veterinary patients with perianal fistulae, IMHA, immune-mediated dermatologic diseases, and inflammatory bowel disease. Treatment of perianal fistulae with cyclosporine is considered standard-of-care; affected dogs often respond completely, and many can be tapered off the drug with time. Topical tacrolimus may also be useful, although this drug can be toxic if licked by the patient, and requires gloved application by the owner. Patients with fistulae require lower trough cyclosporine concentrations (100-300 ng/mL) than those with more severe systemic autoimmune diseases, where recommended trough concentrations are 400-600 ng/mL. Also, because dogs with perianal fistulae are usually large breeds, I often offer ketoconazole early on because of cost concerns. For IMHA, prednisone alone or in conjunction with azathioprine is still considered first-line therapy. However non-responding patients (immunosuppressive doses of glucocorticoids for 14-21 days) may benefit from cyclosporine; published reports do not provide definitive support for this drug, but many internists anecdotally feel there is a benefit. A recent publication reported that dogs with histologically-confirmed IBD that first failed a stringent diet trial and then failed immunosuppressive doses of prednisone may benefit from cyclosporine. Finally, the only disease group where cyclosporine appears to be contraindicated is glomerulonephritis, regardless of histologic subtype. A prospective study reported that dogs who received this drug tended to do worse than those who received placebo.

Mycophenolate mofetil

As with azathioprine, mycophenolate mofetil (MMF) interferes with purine synthesis. However rather than being incorporated as an anti-metabolite, MMF is converted to the active metabolite mycophenolic acid which inhibits an enzyme required for purine synthesis. Most cells have the ability to 'salvage' purines from the monophosphate/biphosphate forms (i.e. ADP, GDP, cAMP, etc.), so MMF has little effect. Lymphocytes, on the other hand, rely heavily on synthesis, and thus MMF targets B- and T-cells with fewer side effects than azathioprine.

Whether MMF should be incorporated into standard immunosuppressive protocols in human diseases is still being determined. As with azathioprine, MMF is predominantly used as a maintenance agent, and was designed to be a long-term drug which could be used during periods of remission with few side-effects. It is now an established drug in treatment of rheumatoid arthritis, SLE, some immune-mediated glomerulonephritides, and following transplantation, both as a maintenance agent or for rescue after failing initial immunosupressive regimens. Use as an adjunctive agent during initial induction therapy is still being explored, but results are mixed. Larger-scale and longer-term studies are producing evidence that MMF may be associated with a higher frequency of side-effects that azathioprine without more effective immunosuppression.

No controlled studies using MMF have been performed in dogs or cats, although anecdotes, abstracts, and a few case reports are available. MMF has been used in dogs with naturally occurring immune-mediated hemolytic anemia in conjunction with prednisone; 6 of 7 dogs responded partially to completely, and one dog developed mild enteritis. Reports include successful treatment of dogs with myasthenia gravis, IMHA, aplastic anemia, or in dogs with suspect glomerulonephritis. Hopefully publications will follow evaluating efficacy and short- and long-term side effects. Most available information has been derived from experimental studies in dogs receiving renal or bone marrow transplants, in which case MMF may be effective in preventing some cases of rejection, and may be synergistic with cyclosporine. Toxicity is usually gastrointestinal, with mild to severe enteritis and colitis secondary to epithelial cell inflammation and apoptosis; in people GI signs respond to dose reduction.

As of now I strongly recommend against routine use of MMF in patients with any disease other than immune-mediated cytopenic diseases (i.e. IMHA, ITP, etc.) or myasthenia gravis, and even in this population I only recommend it as a rescue therapy in patients that have failed all other recommended treatment modalities. It is possible that this drug will become part of our arsenal in the future, but evidence-based medicine dictate that caution is still warranted. In some cases that apparently showed a response to MMF, serum mycophenolic acid concentrations were undetectable, arguing that some other mechanism, and therefore other side-effects, may eventually be found. The standard human dose routinely causes unacceptable GI toxicity in dogs; 10-12.5 mg/kg BID (IV or PO) is suggested.