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Azathioprine and Mycophenolates
Introduction
Whereas calcineurin inhibitors (cyclosporine and tacrolimus) block early signaling events and cytokine production, both azathioprine and mycophenolates, often termed antiproliferatives, exert effects downstream in the cell cycle, interfering with cytokine-dependent signals and lymphocyte proliferation. Azathioprine, usually in combination with corticosteroids, was the backbone of immunosuppression in renal transplantation from the early 1960s to the early 1980s, after which cyclosporine (CsA) was added to the mix. “Triple” therapy, with CsA, azathioprine, and steroids then became standard in most centers through the mid-1990s. Mycophenolate mofetil (MMF) was developed in the early 1990s as an alternative to azathioprine, supplanting it as standard therapy (in combination with calcineurin inhibitors and steroids) by the end of the decade in most developed countries. At the time of this writing, MMF is the most widely prescribed transplant immunosuppressant in the world, although some would maintain its superiority compared with azathioprine is modest. In addition, because azathioprine is less expensive, it will continue to have a role in transplantation, in particular, in developing countries where cost is a greater determinant of immunosuppressive protocols. Thus both remain relevant to clinical practice in transplantation.
History
Azathioprine
6-Mercaptopurine was developed by Elion and Hitchings at Burroughs Wellcome as an anticancer agent in the 1950s. Subsequently, 6-mercaptopurine was shown to be immunosuppressive by Schwartz and Dameshek ; it attenuated the humoral response to a foreign protein and prolonged survival of skin allografts in rabbits, termed drug-induced immunologic tolerance. This work was noted by Calne in the UK and Hume in the US; independently, these investigators found that 6-mercaptopurine delayed or prevented rejection of renal allografts in dogs. In Calne’s laboratory, although only two dogs survived the kidney transplant operation, at death a little more than a month later, there was no histologic evidence of rejection, a unique finding at the time. Almost simultaneously, similar results in a much larger series of canine transplants emerged from Hume’s unit. Soon thereafter, Elion and colleagues produced azathioprine, an imidazolyl derivative of 6-mercaptopurine that appeared somewhat less toxic than 6-mercaptopurine. Azathioprine was first used in humans in Boston at the Peter Bent Brigham Hospital in 1961 and then subsequently utilized (at doses of 2.5–3 mg/kg/day) in a rapidly increasing number of kidney transplant units globally.
In those early days, rejection seemed inevitable, and almost every patient experienced at least one episode. At first, corticosteroids were used to treat rejection in patients on azathioprine; later they were added to azathioprine-based prophylaxis by Starzl at the time of transplantation. In this so-called azathioprine era, arbitrarily large doses of steroids were administered from the time of transplantation, with a gradual reduction over 6 to 12 months to maintenance levels. These high doses of steroids may have been primarily responsible for significant morbidity (discussed in Chapter 16 ); only in the 1970s, after a series of randomized trials and clinical observations documenting the efficacy of lower doses in preventing rejection, was a major reduction in steroid-related complications noted (see Chapter 16 ). By the late 1970s, azathioprine and low-dose steroids, sometimes used together with an antilymphocyte serum or globulin for induction (particularly in North America), had become standard immunosuppressive therapy.
After CsA emerged in the early 1980s, azathioprine was deemed obsolete. However, it quickly reemerged, utilizing lower doses (1.5–2 mg/kg/day) as adjunct therapy to enable administration of lower, presumably less-toxic, doses of CsA. For more than a decade, triple therapy with CsA (4–8 mg/kg/day), azathioprine, and low-dose corticosteroids was considered standard immunosuppressive therapy for kidney recipients in most units in the developed world. During this era, acute rejection rates fell from 80% or greater to around 50%, with some improvement in graft survival and patient survival, and substantially less morbidity than in preceding decades. It was the search for even better immunosuppressants in the late 1980s that led to the introduction of mycophenolates.
Mycophenolates (Mycophenolic Acid, Mpa)
MPA is a fermentation product of Penicillium brevicompactum and related fungi. It was first isolated by Gosio in 1896, named in 1913 by Alsberg and Black, and subsequently found to have weak antibacterial and antifungal activity. Raistrick and colleagues published the structure of MPA in 1952. In the 1960s, it was found to have a strong antimitotic effect in mammalian cells and was regarded as a potential antitumor agent. Subsequent work by Franklin and Cook documented its major mechanism of action (inhibition of the do novo pathway of purine synthesis). At about the same time, a Japanese group documented MPA’s immunosuppressive properties, which were initially viewed as an impediment to clinical utility.
As noted earlier, discovery and development of CsA changed the landscape for transplant immunosuppression. In the search for new agents targeting other pathways, Allison and Eugui, among others, working first in the UK and subsequently at Syntex Pharmaceuticals, began examining in vitro the effect of MPA on lymphocyte function, documenting inhibition of cytotoxic T cell generation, antibody formation, lymphocyte adhesion, and the mixed lymphocyte reaction. Scientists at Syntex, via morpholinoethyl esterification of MPA, produced a prodrug (RS61443, mycophenolate mofetil, or MMF) with substantially improved oral bioavailability in humans and other mammals. Initial animal studies demonstrated the agent to be difficult to administer to dogs and primates, with substantial gastrointestinal toxicity, especially at higher doses (>20 mg/kg). Optimal kidney graft outcomes in dogs occurred when RS61443 was combined with low-dose CsA and prednisone. MMF monotherapy, even at substantially higher doses, demonstrated only limited efficacy. The initial human experience was in rheumatoid arthritis, where a dose of 2 g/day was associated with “significant clinical improvement” among refractory patients. Side effects were minimal and primarily of gastrointestinal origin (nausea, vomiting, abdominal pain, diarrhea). No other significant toxicities (including bone marrow suppression) were noted.
In 1988, it was elected to proceed with clinical trials in kidney transplantation. The initial study was a phase I/II trial involving 48 kidney recipients at two centers. All the patients received polyclonal antibody induction, CsA, and prednisone. Eight dosing groups of six subjects each received MMF in daily doses ranging from 100 to 3500 mg. The greatest efficacy in preventing acute rejection with an acceptable adverse event profile was seen in the 2000 and 3000 mg dosing groups, providing the basis for the design of three large registration trials.
The phase III MMF trials, commencing globally in 1992, were the first of their kind: rigorously conducted studies involving large numbers of patients in solid-organ transplantation. Each study included approximately 500 subjects assigned to one of three treatment arms. Two of the groups in each study received MMF, either 2 or 3 g/day, in combination with CsA and corticosteroids. In the US trial, all the patients underwent polyclonal antibody induction, with the comparator group receiving azathioprine instead of MMF. In the Tricontinental trial, treatment arms were identical, with an azathioprine control group, but without antibody induction. In the European trial, there was no antibody induction, and comparator patients received placebo instead of azathioprine. Findings of all three trials were remarkably similar: a 50% reduction in rates of acute rejection with MMF (to less than 20% overall) within the first 6 months after transplantation, with identical graft and patient survival among treatment arms at 1 and 3 years ( Fig. 15.1 ) For most patients, the 2-g dose appeared to offer optimal efficacy with fewer adverse effects. Based on these findings, on May 3, 1995, the US Food and Drug Administration (FDA) approved MMF (CellCept) 2 to 3 g/day for use in combination with CsA and corticosteroids, with global availability soon to follow. A starting dose of 2 g/day became widely accepted in adults, and, by 2002, MMF was the most widely prescribed immunosuppressant in the US.
In ensuing years, there have been at least three other landmark developments affecting the use of mycophenolates in transplantation. First, to address the gastrointestinal adverse events that plague some patients, Novartis developed an enteric-coated formulation, mycophenolate sodium (EC-MPS, Myfortic). Designed to delay MPA release and absorption in the gut (with the assumption that gastric and early small-bowel exposure were major determinants of gastrointestinal toxicity), clinical trials documented EC-MPS efficacy equivalent to MMF, with trends to fewer adverse gastrointestinal events. EC-MPS became available in 2004. Second, in 2008, patent protection for the innovator formulation of MMF (CellCept) began expiring in the US and globally. The clinical effect of the availability of multiple MMF preparations on patients and the marketplace remains controversial. Finally, in 2009, Tacrolimus/MMF became a FDA-approved combination in kidney transplantation. Previously, use of MMF or EC-MPS with tacrolimus, the most common immunosuppressant combination in the world, had been “off-label,” limiting its use as a comparator protocol in drug development. This label change altered the landscape for new drug development and provided guidance for MPA dosing in tacrolimus-based protocols.
Mechanism of Action
Although both azathioprine and mycophenolate are considered antiproliferative agents, their effects result from differing mechanisms of action. Azathioprine is a thiopurine, an imidazolyl derivative of 6-mercaptopurine. It is transformed to 6-mercaptopurine in the liver, in turn, converted to thioinosinic acid and 6-thioguanine. These latter metabolites are incorporated into DNA and RNA, inhibiting lymphocyte proliferation by suppressing de novo purine synthesis.
Given the documented immunosuppressive effect of azathioprine, a relatively nonspecific inhibitor of nucleotide synthesis, MPA use in transplantation was based on the observation that inherited deletions in nucleic acid synthesis resulted in immunodeficiencies: children lacking adenosine deaminase show combined T cell and B cell deficits. In contrast, subjects with absence of hypoxanthine-guanine phosphoribosyl transferase display essentially normal immune function, indicating the purine salvage pathway as relatively unimportant in lymphocytes ( Fig. 15.2 ). MPA acts as a reversible, noncompetitive inhibitor of inosine 5′-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo synthesis of guanine nucleotides. This effect arrests new DNA synthesis in proliferating cells at the G 1 /S interface, with guanosine triphosphate (GTP) decreasing to 10% of levels measured in unstimulated T cells. Addition of guanosine or deoxyguanosine reverses the inhibition, confirming the IMPDH target. T and B lymphocytes are highly dependent on this pathway for proliferation in response to stimulation. There are two isoforms of IMPDH, with type I expressed in many cell lines, but type II the predominant isoform in activated T and B lymphocytes; IMPDH type II is four times more sensitive to MPA. These two factors impart some degree of lymphocyte-specific selectivity to the antiproliferative activity of MPA. Although monocyte and dendritic cell replication may also be affected by MPA, there is relatively little effect on neutrophils.
The action of MPA at the G 1 /S interface is also selective. Neither the production of interleukin-2 nor the expression of its receptor is affected, showing a lack of influence on signal 1 of lymphocyte activation. The primary antiproliferative effect also retards induction of cytotoxic T cells. Similarly, MPA mitigates primary and ongoing B cell responses, presumably as a result of blockade of cell division, with subjects receiving MMF in the US pivotal trial demonstrating far less production of xenoantibody toward equine antithymocyte globulin (ATG) than those on azathioprine. MPA treatment also blunts the synthesis of natural xenoantibodies after plasma exchange and splenectomy in rats and inhibits the humoral response to influenza vaccine in humans.
Another distinct mechanism of MPA activity may relate to the requirement for GTP to activate fucose and mannose transfer and glycoprotein synthesis, thus diminishing expression of adhesion molecules. When GTP is depleted as a consequence of MPA, adhesion of lymphocytes and monocytes to activated endothelial cells is decreased in a dose-dependent fashion.
Cell types other than lymphocytes may also be sensitive to MPA inhibition, with some models indicating vascular smooth muscle cells, mesangial cells, and myofibroblasts to display dampened proliferation. In a subhuman primate model of orthotopic allograft vasculopathy, intravascular ultrasound documented dose-dependent inhibition of the progression of intimal volume changes. The efficacy of MMF was confirmed using aortic allograft in another subhuman primate model and in a rodent chronic rejection system. The effects were potentiated when MMF was administered with sirolimus, a combination that also reduced transforming growth factor-β 1 and its effects on extracellular matrix synthesis and degradation. There may also be a more direct antifibrotic effect of MPA mediated by upregulation of neutral endopeptidase in the kidney.
Clinical Pharmacology and Blood Monitoring
Azathioprine
Dosing, Metabolism, and Monitoring
Azathioprine is given as a single daily dose. If used with steroids alone, a suitable starting dose is 2.5 mg/kg/day, with careful monitoring of leukocyte counts. The dose of azathioprine may be reduced somewhat over time, particularly in the face of evidence of marrow suppression. Analysis of data from the Collaborative Transplant Study suggested that, for patients receiving azathioprine and steroids only, long-term doses greater than 1.5 mg/kg/day were associated with superior graft survival. When azathioprine is administered with a calcineurin inhibitor (CNI) and steroids (triple therapy), lower doses are appropriate. A fairly standard dose of azathioprine in a triple protocol is 1.5 mg/kg, 100 mg/day. At this level, hematologic toxicity is uncommon.
Xanthine oxidase has an important role in the catabolism of 6-mercaptopurine, and, therefore, azathioprine. Concomitant use of xanthine oxidase inhibitors (e.g., allopurinol) requires significant azathioprine dose reduction to avoid excessive toxicities, particularly in the bone marrow. Although the metabolites are excreted in the urine, these are inactive, and no reduction in dosage is required in acute kidney failure.
Regular monitoring of hematologic parameters is an important aspect of azathioprine therapy; common practice is to reduce the dose when the leukocyte count decreases to less than 3 × 10 9 /L. As already mentioned, if xanthine oxidase inhibitors are used, the azathioprine dose should be reduced by at least 25% or consideration given to conversion to MPA. Blood levels of azathioprine or its metabolites are not routinely monitored in clinical practice. It has been noted, however, that the development of leukopenia can also result from viral infection, leading to the suggestion that erythrocyte 6-thioguanine nucleotide levels may be a better indicator of azathioprine activity in transplant patients.
A number of genetic variations in the thiopurine S-methyltransferase (TMPT) gene have been identified, which have been related to azathioprine-induced toxicities. Polymorphisms in the TMPT enzyme, which catalyzes the S-methylation of 6-mercaptopurine and azathioprine, may be associated with an increased likelihood of myelotoxicity and leukopenia and require dose modification. Genotyping for this polymorphism before commencing azathioprine might allow the appropriate azathioprine dose for an individual to be determined, however, the cost/benefit ratio of this approach remains controversial.
Side Effects
The major complication of azathioprine therapy is bone marrow suppression, most commonly manifest as leukopenia, although, in more severe settings, anemia and thrombocytopenia may also occur. Megaloblastic anemia has been described in association with the use of azathioprine. Although hepatotoxicity has been attributed to azathioprine for many years; it is probably rarer than thought, with many cases likely reflective of unrecognized viral hepatitis. Hair loss is an uncommon complication. Increased risk of nonmelanoma skin cancers and other malignancies in transplant recipients has been attributed to azathioprine. Evolving experience, with both skin and colon cancer, does indeed indicate azathioprine-treated patients to be at greater risk, with a recent meta-analysis indicating increased skin cancers like squamous cell, but not basal cell carcinoma. Mechanistically, Hofbauer and colleagues have shown that mercaptopurine metabolites intercalated into DNA of skin increase sensitivity to ultraviolet irradiation, providing a plausible mechanistic explanation for the clinical observations.
Mycophenolates
Dosing
In adults with kidney transplants, MMF is most commonly administered orally at a dose of 1 g twice daily. In pediatric patients, the recommended dose of MMF oral suspension is 600 mg/m 2 at 12-hour intervals up to a maximum of 2 g daily. Intravenous MMF is administered at the same dose and frequency as the oral preparation, infused over 2 hours. EC-MPS is administered orally at a dose of 720 mg twice daily in adults. This suggested dose of EC-MPS in a stable pediatric patient is 400 mg/m 2 twice daily, but its use is not recommended in patients less than 5 years old.
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