Immunosuppressives in solid organ transplantation

teaser

Lionel Rostaing
MD PhD
Professor

David Ribes
MD

Nassim Kamar
MD

Federico Sallusto
MD
Multiorgan Transplant Unit
University Hospital
CHU Rangueil
Toulouse
France
E:[email protected]

Solid organ transplantation is a well-established therapy for endstage organ failure, such as kidney, heart, liver, pancreas or lung failure. The first kidney transplant was performed in 1954, and the first liver and heart transplantations were conducted in the 1960s. Other types of organ transplantation are less common and are performed in fewer patients. These include lung, pancreas (whole or islets) and small bowel transplants. Until the early 1980s, solid organ transplantation was associated with a high rate of transplant failure, which, at that time, was mainly due to uncontrolled rejection caused by the use of underpowered immunosuppressants (ie, steroids and azathioprine, as well as a high rate of infectious complications).

In 1983, ciclosporin A (Sandimmun(®) until 1995, Neoral(®) thereafter) was launched: its use was associated with a dramatic increase in allograft survival rate, both in the short and long term. In vital organs, this was associated with a dramatic increase in patient survival. Following this major advance, other immunosuppressants became available and helped improve post-transplant survival. These included tacrolimus in 1995, mycophenolate mofetil (MMF) in 1996 and sirolimus in 1999. To maximise their immunosuppressive effects, these drugs are used in combination to block the T-lymphocyte activation cascade, which gives maximum efficiency but can increase side-effects such as infections or cancers. To minimise the latter, we have to use these drugs at a daily dosage that is average to low. It is also possible to use induction agents during the peritransplant period. These bioreagents include polyclonal antibodies such as Thymoglobulins(®) (which have been in use since the late 1960s), ATG-Fresenius S(®) (in use since 1981) and monoclonal antibodies such as anti-CD3 antibodies (OKT3, Orthoclone(®) [used since 1986]) or anti-CD25 antibodies (daclizumab and basiliximab, used since 1997 and 1998, respectively). None of these immunosuppressants induces tolerance. Therefore, immunosuppression is mandatory in the long term to allow graft survival. In any given patient, there is still no tool to measure the level of immunosuppression, and this can only be assessed by surrogate markers such as rejection (caused by underimmunosuppression) or infections/cancers (caused by overimmunosuppression). Immunosuppressant blood levels are monitored to avoid drug toxicity and achieve blood targets that are recognised as immunosuppressive.

Induction agents
Induction agents are thus termed because it was thought that they could induce tolerance; however, this is not the case. These immunosuppressants are used in the perioperative period and are associated with improved short-term survival at posttransplant because they significantly decrease the rate of acute rejection. Polyclonal antilymphocyte (Thymoglobulins and ATG-Fresenius S) are obtained by immunising rabbits against human thymocytes and Jurkat cells, respectively.(1,2) Thus, the animals produce antibodies directed against numerous T-lymphocyte membrane determinants (CD). The antibodies are then purified and used in ­clinics at 1–1.5mg/kg (Thymoglobulins) or at 2–4mg/kg (ATG-Fresenius S) per infusion, for an average of three to seven injections. These antibodies are associated with rapid and dramatic lymphopenia, which can be monitored on the basis of CD2 or CD3 T-lymphocyte counts. In contrast to Thymoglobulins, ATG-Fresenius S induces weaker lymphopenia. The main clinical indications for the use of these agents are prevention and treatment of acute rejection.(3) At best, to prevent acute rejection, these agents are started before the completion of vascular anastomosis and are continued until post-transplant days 4–10; they are usually given on an alternate-day basis. The other indication for the use of these polyclonal antibodies is the treatment of steroid- resistant acute rejection. In these cases, they are used on a daily basis for five to eight days. As they are xenoantibodies, they may induce immunisation, which could result in:

• Loss of efficacy.
• Serum-sickness disease by days 7–10, or earlier if the patient has been previously exposed to these agents.

This can result in fever, polyarthralgias, increase in serum creatinine, thrombopenia and fall in serum complement and its C4 subfraction. However, the number of patients presenting with serum-sickness disease is low because patients usually receive other coimmunosuppressant medications that limit xenoimmunisation (eg, steroids, MMF). However, when xenoimmunisation occurs, it is mandatory to withdraw xenoantibodies and increase daily steroid dosage. Other side-effects of polyclonal antibodies include:

• Bacterial infections.
• Cytomegalovirus (CMV) infection.
• Thrombopenia.
• Leucopenia.

Polyclonal antibodies that react against many CD markers on the T-lymphocyte membrane can be replaced with monoclonal antibodies that target a specific structure such as CD3 (ie, OKT3 [Orthoclone(®)](4)) or CD52 (ie, alemtuzumab [Campath1(®)](5)). OKT3 was the first to be licensed in the early 1980s. It is a very powerful drug; it is associated with cytokine-release syndrome with the first doses and can promote post-transplant lympho‑proliferative disorders (PTLD) when the cumulative dose is too high. Therefore, this drug is now only used to treat severe steroid-resistant rejections. Alemtuzumab has been licensed for a few years and is used in the perioperative transplant period to prevent acute rejection. There was some debate as to whether it could promote tolerance; however, there are still no data to support this. Basiliximab and dacluzimab are two monoclonal antibodies that target the α chain of the IL-2 receptor (ie, CD25). Basiliximab(6) is used at a fixed dose (20mg) delivered at pretransplant and on postop day 4, whereas daclizumab is administered at 1mg/kg at pretransplant and then every two weeks up to day 60.(7) These two monoclonal antibodies statistically decrease the rate of acute rejection, but they do not promote tolerance and do not improve long-term allograft survival. Basiliximab and daclizumab are easy to use (infusion is via a peripheral vein) and are very well tolerated.

Rituximab is a chimeric monoclonal antibody that binds the B-cell surface antigen CD20; expression of CD20 is restricted to the B-lymphocyte lineage. CD20 appears at the late pre-B stage of development and is lost during terminal differentiation into plasma cells. Rituximab infusion results in rapid B-cell depletion within peripheral blood, which persists for more than six months in patients; however, bone marrow B-cells are within the normal range at six months post­infusion, although they are reduced with respect to baseline. In organ transplantation, rituximab is used as an adjuvant therapy to treat patients with acute vascular or refractory rejections,(8) those receiving ABO-incompatible transplants,(9) those presenting with post-transplant lymphoproliferative disease(10) or in those with cryoglobulinaemia-related glomerulonephritis on the kidney graft.

Belatacept (LEA29Y; Bristol-Myers Squibb) inhibits signal 2 on T-lymphocytes by competing with CD80/86 to bind to CD28 on T-cells. Phase II trials have shown that the long-term infusion of belatacept in kidney-transplant patients is safe, is as efficient as calcineurin inhibitor (CNI)-based therapy and might replace the latter to avoid the long-term nephrotoxicity of CNIs.(11)

Calcineurin inhibitors
Ciclosporin A (CsA) and tacrolimus are the two currently licensed calcineurin-inhibitor agents. In 90–95% of solid organ transplant patients, these drugs are the cornerstone of immunosuppression when used alone or in combination with other immunosuppressants. CsA binds to cyclophiline, whereas tacrolimus binds to the FK-binding protein 12 (FKBP12). This results in the inhibition of calcineurin, an enzyme required for the nuclear translocation of the nuclear factor of activated T-cells (NFAT). The latter activates the IL-2 promotor gene. As calcineurin is an ubiquitous enzyme and is found, for example, within the kidney and central nervous system, its inhibition will lead to numerous side-effects. However, because the inhibition of calcineurin by CsA or tacrolimus is reversible, if their use is discontinued for any reason, then there is a high likelihood of rejection. CsA has been available since 1983 and has allowed a great improvement in graft-survival rates. It is used in regimens of 6–10mg/kg/day, divided into two doses. The therapeutic range varies according to the time elapsed since transplantation and the type of organ transplantation. Because CsA trough levels are not correlated with the area under the curve (AUC), CsA monitoring is based on the C(2) (the value obtained two hours ±10 minutes after drug uptake).(12) The target value varies from 1,000–1,600ng/ml before six months to 600–1,000ng/ml thereafter. CsA is used to prevent acute rejection in organ transplantation and to treat autoimmune diseases such as uveitis, or steroid- resistant idiopathic nephrotic syndrome. Tacrolimus is more potent than CsA; it has been licensed since 1995 for the prevention and treatment of acute rejection, and also for the treatment of autoimmune diseases such as psoriasis. It is administered twice daily (0.1–0.2mg/kg/day) to reach trough levels of 10–20ng/ml before six months, and of 5–12ng/ml thereafter.(13) The major side-effects of CsA and tacrolimus are nephrotoxicity, hypertension and tremors. Nephrotoxicity is dose-related and is initially reversible, but it can become permanent with longer-term exposure to the drugs and ultimately result in end-stage renal failure. Other side-effects vary for the two drugs: CsA can induce hirsutism, gum hyper‑plasia and dyslipidaemia, whereas tacrolimus can induce de-novo diabetes. These side-effects of the drugs can be reduced by achieving blood targets within the low range, provided that patients also receive other immunosuppressants that do not have these side-effects.

Azathioprine
The observation that mercaptopurine (MP), an antimetabolite, prolonged skin-graft survival in rabbits led to its use in human transplant recipients in 1962. This was replaced in 1969 by azathioprine (AZA), the prodrug of MP. The immunosuppressive properties of AZA/6-MP are mediated by the ­intracellular metabolism of 6-MP into its active metabolites, 6-thioguanine nucleotides (6TGN) and 6-methyl‑mercaptopurine (6-MMP).(14) Some studies have suggested that the red blood cell concentration of 6TGN was a potential guide to therapy. Until ­ciclosporin became available, maintenance immuno­suppression in solid organ transplantation relied on AZA (1–3mg/kg/day), in addition to steroids. Because this combination was not immunosuppressive enough, it led to poor allograft survival. The main side-effect of AZA is cytopenia (ie, anaemia, leucopenia and thrombopenia). AZA should never been used with allopurinol, an antigout drug, because its carries the risk of bone marrow aplasia. In 1996, AZA was replaced by MMF.

MMF and EC-MPS
MMF is the ester of mycophenolic acid (MPA), and enteric-coated mycophenolate sodium (EC-MPS) has been developed as an alternative to deliver MPA with the goal of reducing adverse gastro‑intestinal (GI) events caused by MMF. MPA acts by inhibiting inosine monophosphate dehydrogenase (IMPDH), a key enzyme in the de-novo pathway of purine synthesis, which is required for the proliferation and function of T- and B-lymphocytes.(15) Since T- and B-lymphocytes rely solely on this pathway for the production of guanosine nucleotides, the proliferation of these cells is specifically inhibited. GI side-effects might be explained by the fact that enterocytes partly (approximately 50%) rely on the de-novo pathway. The bioavailability of MMF is excellent (90% is found as MPA). MPA glucuronide, which is the glucuronidated (and pharmacologically inactive) metabolite of MPA, undergoes enterohepatic recirculation, allowing sustained plasma concentrations of the drug. In renal transplant patients, the AUC of MPA is generally proportional to dosage over a range of 100–3,500mg/day. The AUC and peak plasma concentration (C(max)) of MPA are approximately 50% lower in the early post-transplant period (<40 days post-transplantation) than in stable renal transplant recipients. The coadministration of anti‑acids and MMF inhibits the absorption of MMF. There is also strong evidence that MPA trough concentrations are influenced by the type of CNIs used. Thus, coadministration of CsA and MMF decreases the exposure to MPA, whereas coadministration of tacrolimus and MMF does not. Typically, in cases of tacrolimus-based therapy, MMF is given on a basis of 1g/day (720mg/day in cases of EC-MPS), whereas, in cases of CsA-based therapy, MMF is given at a daily dose of 2g (1,440mg in cases of EC-MPS). In cases of mammalian target of rapamycin (mTOR)-based therapy, MMF is given at 1.5g/day. MPAs are given to prevent acute rejection (and possibly chronic rejection in heart transplant patients), in association with CNIs or mTOR inhibitors. In some cases, particularly in organ transplant patients who develop CNI-related nephrotoxicity, it is possible to give MMF-based immunosuppression with or without steroids.(16) Side-effects of MPAs include haematological problems (ie, anaemia – particularly when these drugs are used with mTOR inhibitors – leucopenia and thrombopenia) and GI symptoms such as diarrhoea and abdominal discomfort.(17) These GI side-effects are more pronounced with MMF than with EC-MPS.(18)

Glucocorticoids
Glucocorticoids (GCs) have anti-inflammatory properties that result from the pleiotropic effects of the glucocorticoid receptor on multiple signalling pathways. However, their adverse effects are also pleiotropic. GCs may exert their anti-inflammatory properties by either direct or indirect effects on gene expression.(19) Direct effects on gene expression lead to the binding of GC receptors to GC-responsive elements, which results in the inhibition of prostaglandin production (through induction and activation of annexin I, the induction of MAPK phosphatase 1, and the repression of transcription of ­cyclo-oxygenase 2) and of cytosolic phospholipase A2α by MAPK phosphatase 1. Indirect effects on gene expression act through the interaction of GC receptors with the transcriptional activity of nuclear factor (NF)-κB, and thereby the transcription of cyclo-oxygenase 2. In addition, GCs have anti-inflammatory properties through GC receptor-mediated effects on second messenger cascades (ie, the phosphatidylinositol-3- hydroxykinase [PI3K]-Akt-endothelial nitric oxide synthetase [eNOS] pathway). Another mechanism of GC-induced inhibition of inflammation involves decreased stability of mRNA for genes producing inflammatory proteins such as cyclo-oxygenase 2 and vascular endothelial growth factor. GCs also have antiapoptotic properties, thereby efficiently killing lymphoid cells.(20) GCs may induce cell death via different pathways, resulting in apoptotic or necrotic morphologies, depending on the availability/responsiveness of the apoptotic machinery. The former might result from the regulation of typical apoptosis genes, such as members of the Bcl2 family, the latter from detrimental GC effects on essential cellular functions, possibly perpetuated by GC-receptor autoinduction. The pleiotropic side-effects of GCs include Cushing syndrome, adrenal atrophy, hypertension, dys‑lipidaemia, GI bleeding, peptic ulcer, delayed wound healing, hypertrichosis, petechiae, bone necrosis, muscle atrophy, osteoporosis, cataract, hyperglycaemia, hypogonadism and increased sodium retention. GCs were used during the early stages of kidney transplantation as the only immunosuppressant until AZA became available in the 1960s. Since that time, GCs have been used in solid organ transplantation in association with other immunosuppressants. However, the use of powerful immunosuppressive drugs has allowed the dosage of GCs to be minimised to limit their side-effects. For the last few years, both in renal(21) and liver(22) transplantations, it has been shown that effective immunosuppression was achievable without the use of GCs. Conversely, high doses of GCs (eg, intravenous methyl‑prednisolone at 10mg/kg/ day for three consecutive days) is the first-line therapy to treat acute rejection in solid organ transplants and has a nearly 90% success rate. When acute rejection is resistant to GCs, second-line treatment relies on antilymphocyte preparations (eg, Thymoglobulins, ATG, OKT3).

Sirolimus and everolimus
Sirolimus and everolimus inhibit mTOR, a key enzyme in the activation of the kinase cascade, which occurs after ligation of IL-2 on its receptor on the surface of T-lymphocytes. This prevents the cell from leaving the G phase to enter the S phase. mTOR inhibitors also inhibit Bcl2, thus promoting apoptosis. These agents are termed antiproliferative because their use will lead to the inhibition of cell division in many organs. Sirolimus was discovered in 1974. The drug has poor bioavailability and a long half-life.(23) It requires a loading dose, followed by a once-daily uptake thereafter. Therapeutic blood levels range between 8 and 20ng/ml. The drug does not induce nephrotoxicity, except when it is used in combination with anticalcineurin agents. Clinical trials in renal transplant patients have shown that sirolimus-based immunosuppression without CNIs achieved very good long-term results regarding renal function.(24) Everolimus has a molecular structure similar to that of sirolimus. Its half-life is shorter and, therefore, it is administered on a twice-daily basis. Therapeutic trough levels range between 8 and 12ng/ml. In contrast to sirolimus, everolimus can be used with very low doses of CsA to achieve good immunosuppression, with very low nephrotoxicity.(25,26) mTOR inhibitors, in addition to other immunosuppressants, are indicated to prevent acute rejection. In particular, they might prevent chronic rejection in heart and lung transplant patients.(27) However, due to their antiproliferative properties, when these drugs are used at the time of transplantation, they can be associated with delayed wound healing and lymphocele (in renal-transplant patients). These problems occur particularly in patients who are obese or who have hypoalbuminaemia. Conversely, the antiproliferative properties of these drugs might aid in the prevention of post-transplant neoplasia. Typically, in maintenance patients, mTOR inhibitors are of value in organ transplant patients who develop CNI-related nephrotoxicity, or in those who develop neoplasia. In these cases, CNIs can be safely withdrawn under the umbrella of mTOR inhibitors, with or without MPA.(28) Side-effects of mTOR inhibitors include haematological problems (ie, microcytic anaemia – particularly when they are used with MMF – leucopenia and thrombo­cytopenia) dyslipidemia (ie, hypercholesterolaemia and hypertriglyceridaemia), metabolic disorders (eg, hypokalaemia and hypophosphataemia), mouth ulcers, interstitial pneumopathy and proteinuria.

New immunosuppressants

Malononitrilamide FK778
FK778 (a leflunomide derivative) is a synthetic malono­nitrilamide (MNA) that has been demonstrated to have both immunosuppressive and antiproliferative activities.(29) The MNAs inhibit both T- and B-cell function by blocking de-novo pyrimidine synthesis through the blockade of the pivotal mitochondrial enzyme dihydro-orotate dehydrogenase (DHODH). FK778 has been demonstrated to prevent acute allograft rejection in multiple experimental transplant models in rodents, dogs and primates, and to be effective in the rat model of chronic renal allograft rejection. In addition, FK778 has been shown to prevent vascular remodelling after mechanical intimal injury via a mechanism that may be related to tyrosine kinase inhibitory activity in vascular smooth muscle cells. Another intriguing activity of the MNA family is the ability to block replication of members of the Herpes virus family, with in-vitro evidence of efficacy against cytomegalo­virus (CMV) and polyoma virus, which are important pathogens in the transplant recipients. FK778 is currently being explored in a number of trials in solid organ transplant recipients and has been shown to be pharmacologically active, well tolerated and safe in phase II trials.

Fingolimod (FTY720)
Fingolimod (FTY720) is an orally active immunosuppressant that affects lymphocyte recirculation and has the potential to prevent transplant rejection and treat autoimmune diseases, including multiple sclerosis.(30) Fingolimod is a synthetic sphingosine analogue that becomes phosphorylated in vivo and acts as a sphingosine-1- phosphate receptor agonist. Fingolimod induces rapid and reversible sequestration of lymphocytes into secondary lymphoid organs, thereby preventing their migration to sites of inflammation. As prerequisite for its function, phosphorylation of FTY720 to yield a potent agonist of the S1P1 receptor is required in vivo and is catalysed by an (as yet) unknown kinase. In clinical phase II trials, fingolimod at a dose of 2.5mg/day was found to be as effective as MMF in combination with ­ciclosporin for the prevention of acute rejection after renal transplantation. Fingolimod was well tolerated and was not associated with the side-effects commonly observed with immunosuppressant therapies. Phase III trials are ongoing.

Combinations of immunosuppressants
Due to the large number of immunosuppressive agents that are currently available, it is now possible to give immunosuppression à la carte. Current immuno­suppression relies on combined drugs that are able to block the cascade of lymphocyte activation, which ultimately prevents (although imperfectly) acute and chronic rejection. Thus, physicians use regimens based on CNIs, MPAs or mTOR inhibitors, with or without induction therapy and with or without steroids. For example, patients who are at greatest risk of acute rejection (ie, patients undergoing a second transplant, patients highly sensitised against HLA antigens, African-Americans, ­paediatric patients, patients with a history of and/or current positive cross-match and those who are recipients of an ABO-incompatible donor) should receive induction therapy. For patients with low immunological risk, induction therapy significantly decreases the rate of acute rejection but does not improve long-term graft survival.

In renal transplantation, when the kidney is from a marginal donor (ie, an older donor or a donor with a history of hypertension or cardiovascular disease) or when the cold ischaemia time is greater than 36 hours, it is better to use CNI-free immunosuppression. In recipients who are at high risk of developing post-transplant diabetes mellitus (ie, patients older than 45 years, with a family history of diabetes, obese or of Hispanic or African-American ethnicity), both steroids and tacrolimus should be avoided.

For heart and liver transplant patients who develop CNI-related renal insufficiency in the long-term,(31) the progressive withdrawal of CNI and its replacement by MPAs and/or mTOR inhibitors should be contemplated.

It is possible that renal transplant patients who exhibit long-term renal dysfunction (with evidence from the kidney biopsy of CNI-related nephrotoxicity) should be withdrawn from CNI therapy, which should be replaced by mTOR inhibitors with or without MPAs.

When solid organ transplant patients present with a cancer (nonmelanomatous skin cancer, PTLD or organ cancer), it is preferable that CNIs are withdrawn and replaced by mTOR inhibitors, as the latter may also have some antitumour properties.

Conclusion
Current immunosuppressants achieve very good long-term results in solid organ transplantations, when they are used in combination. However, because we still do not know how to measure the degree of immunosuppression, their long-term use is associated with side-effects.

Acknowledgments
The authors acknowledge the skillful secretarial assistance of Ms Danièle Mencia and the editing work of Ms Susanna Lyle

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