Liver transplantation and Immunosuppression

The early 1970s saw the introduction of a new class of immunosuppressants, the so-called “calcineurin inhibitors” (CNIs). These compounds form the backbone of immunosuppression in liver transplantation recipients. Currently, two CNIs are available: Cyclosporine A and Tacrolimus (TAC).

CyA was discovered by Jean-Francois Borel in 1973 from a soil fungus, Tolypocladium inflatum. Food and Drug Administration approval was obtained in 1983. CyA causes selective suppression of cell-mediated immunity via inhibition of T-cell activation. After forming a complex with its cytoplasmic receptor protein (cyclophilin), CyA binds to and inhibits the calcium and calmodulin-dependent phosphatase calcineurin. It is believed that calcineurin plays a vital role in the transcriptional process by which interleukin (IL) 2 and other cytokines are activated. The production of these substances by T helper cells is a vital component of the immune response and is central to the graft rejection process.

Nephrotoxicity is one of the main side effects of CyA therapy. This can be both an acute and a longterm complication, inducing a post-OLT rate of renal failure up to 20%.Common metabolic abnormalities include hyperkalemia, hypomagnesemia, hyperlipidemia, and, to a lesser extent, hyperglycemia. Hypertension, gingival hyperplasia and hirsutism are also a common occurrence. Between 10% and 28% of patients have neurological manifestations
that range from mild tremor and peripheral neuropathy to psychoses, hallucinations, motor
weakness, and seizures.

In 1984, a soil sample containing the fungus Streptomyces tsukubaensis was discovered from near mount Tsukuba in Japan. Two years after this discovery, TAC was isolated.TAC is 100 times more potent than CyA and exerts its action by binding to FK binding protein (FKBP12).
This complex then inhibits calcineurin, which is responsible for transcription of IL-2, IL-3, IL-4, IL-8, and various chemotactic factors. TAC absorption occurs in the duodenum and jejunum. Unlike CyA, TAC absorption is not influenced by presence of bile, which is advantageous in cholestatic patients or those with biliary diversion or ileus. Food reduces bioavailability, and TAC should be taken on an empty stomach. Metabolism occurs in the liver via the cytochrome P450-3A. Side effects are similar to CyA, including nephrotoxicity, neurotoxicity, posttransplant diabetes mellitus, and hyperkalemia.

Corticosteroids

As mentioned earlier, corticosteroids have been a mainstay since early days of liver transplant. It is by far the most heavily utilized non-calcineurin inhibitor in liver transplant. Corticosteroids exert their most critical immunosuppressive effect by blocking T-cell– derived
and antigen-presenting cell-derived cytokine expression. This includes IL-1, IL-2, IL-3, and IL-6. Corticosteroids continue to be used in reversing acute rejection and in maintenance therapy. Side effects are numerous and include hypertension, hyperglycemia, osteoporosis, hyperlipidemia, increased risk of gastric ulcers, risk of fungal and bacterial infections, and suppression of HPA axis. Raising concern over negative impact of this class of medication in the recurrence of hepatitis C.

Antimetabolites

Azathioprine was the first antimetabolite used in liver transplant but its use has since decreased dramatically over time. Azathioprine is an imidazolyl derivative of mercaptopurine and antagonizes purine metabolism. The result is an inhibition in synthesis of DNA, RNA, and proteins. Current use stands at 5% of U.S. transplant centers. This has been primarily due to the side effect profile, which includes significant myelosuppression and hepatotoxicity.

Mycophenolate mofetil (MMF, CellCept) and mycophenolic acid (MPA, Myfortic) are the most
recent additions to the antimetabolite arena, with MMF approval in 1995 and MPA in 2004. MPA is a delayed-release product in contrast to MMF, which is immediately released. Both formulations inhibit the de novo purine nucleotide synthesis via abrogation of the inosine monophosphate dehydrogenase and the production of guanosine nucleotides. This action leads to a blockage of DNA replication in T and B lymphocytes that are unable to use alternate salvage pathways.

Studies have shown a large variation in MMF pharmacokinetics in liver transplantation related to fluctuations in serum albumin concentrations, changes not seen in the renal transplant population. Liver dysfunction impairs MPA conjugation and prolongs MPA half-life. Furthermore, TAC has been shown to augment the bioavailability of MPA, resulting in higher exposure to MPA (Tables 6A and 6B). The incidence of adverse effects (nausea, gastritis abdominal pain, diarrhea, and neutropenia) requiring dose reduction or withdrawal is high, ranging from 24% to 57%

Antibody Induction

Antibody therapy has been used as a means of delaying the introduction of maintenance therapy and/or helps facilitate the removal of an immunosuppressive agent, particularly corticosteroids. Antibody therapy can be seen as depleting or receptor modulating or both. reiterate, dose reductions are common and in some patients removed completely, especially when combined combined other myelosuppressive medications, such as sirolimus.


Antithymocyte Globulin (ATG)

Polyclonal antilymphocyte antibody preparations are heterologous preparations. Animals (rabbits and horses) are immunized with human T cells and thymocytes. Antisera are then collected. A purified gamma globulin fraction (ATG) is used to reduce the likelihood of serum sickness. The ATG preparations approved by the Food and Drug Administration are ATGAM (of equine origin) and Thymoglobulin (of rabbit origin). These polyclonal preparations are directed at multiple different epitopes on the T cell (CD2, CD3, CD4, CD8, CD28, and the T-cell
receptor) as well as CD16 found on natural killer cells and macrophages. These antibodies cause depletion of T cells by apoptosis, antibody mediated cytolysis and internalization of the cell surface receptors. The biologic effects of the depleting antibodies are profound and last longer than the presence of heterologous antibody. Side effects can include a “first-dose” effect (cytokine release syndrome) and is related to the myriad of cytokines released by these lymphocytes upon their demise. The symptoms typically include fever, chills, tachycardia, gastrointestinal disturbances, bronchospasm, and fluctuations of blood pressure, which all can be ameliorated by pretreatment with corticosteroids, diphenhydramine, and acetaminophen.

Monoclonal Anti–T-Cell Antibodies

Muromonab-CD3 (OKT3) is a murine-derived antibody directed to a specific portion of T cells. It exerts its activity by binding to the CD3 antigen on the surface of T lymphocytes. This binding inactivates the adjacent T-cell receptor, which is critical for activation of T lymphocytes. The end result is a rapid fall in the number of mature lymphocytes. OKT3 was first used in liver transplantation in 1987 for prophylaxis against acute cellular rejection and later to reduce CNI exposure and treatment of steroid-resistant rejection.More recent data has shown a significant drop-off in favor or IL-2 receptor antibodies and polyclonal preparations.
A cytokine-release syndrome is more frequently associated with the first dose as compared to the polyclonal antibody preparations and starts 1-3 hours following administration. Reactions can be quite severe and can range from flulike symptoms (pyrexia, chills, dyspnea, chest pain, and tightness) to severe and lifethreatening shocklike reactions (pulmonary edema).

These symptoms can be mitigated with the administration of a corticosteroid, diphenhydramine, and acetaminophen just prior to dose administration. Reexposure to OKT3 may result in lower efficacy due to antimurine antibodies that may form. In the early transplant period when the incidence of acute cellular rejection was as high as 71% and steroid-resistant rejection was more common, OKT3 was the mainstay in the treatment of steroid-resistant rejection with high salvage rates. Currently, steroid-resistant rejection rates are much lower, thanks to improved immunosuppressive agents and strategies. OKT3 use today is much lower, with many centers preferring treatment regimens for acute cellular rejection to increasing TAC blood levels and then adding corticosteroid boluses if rejection
is still present.

IL-2 Receptor Antibodies

Two products are currently marketed: basiliximab (Simulect) and daclizumab (Zenapax). Daclizumab is a humanized product while basiliximab shows chimeric properties. Both bind to the IL-2R -chain, which is upregulated on the surface of activated T lymphocytes. Immunosuppression is achieved by competitive antagonism of IL-2-induced T-cell proliferation.

Miscellaneous

The following agents are used at different stages in liver transplant, from induction to conversion. Rapamycin Sirolimus (Rapamune, RAP) is a macrocyclic triene antibiotic (structurally related to TAC) with immunosuppressive, antitumor, and antifungal properties, although the latter 2 are not clinically significant. RAP binds to the immunophilin FKBP12 and blocks the response of T- and B-cell activation by cytokines, which prevents cell-cycle progression and proliferation; in contrast, TAC and CyA inhibit the production of cytokines.

Interestingly, although RAP binds to the same immunophilin (FKBP12) as TAC, it has a very different mechanism of action—i.e., blockage of cell-cycle progression at the juncture of G1 and S phase. Because of this, many refer to the binding site for RAP as the “target of RAP.” Multiple serious side effects have limited its use early on in liver transplantation. This includes leukopenia, thrombocytopenia, elevated serum cholesterol and triglyceride levels, anemia, lymphocele, wound dehiscence, and oral ulcerations. The most serious side effect has been the issue of hepatic artery thrombosis and includes the following black box warning in liver transplantation: “The safety and efficacy of Sirolimus (Rapamune) as immunosuppressive therapy have not been established in liver transplant patients, and therefore such use is not recommended.”

Current Therapeutic Strategies

Steroid Avoidance

Interests in corticosteroid abstention stems from the well-know side effects of osteoporosis, hyperglycemia, cushingoid features, hypertension, as well as the deleterious effects on recurrence of hepatitis C. The first randomized study concerning complete steroid avoidance was published by the Eason et al.

Renal Sparing Protocols

The sentinel articles by Ojo et al. and Gonwa et al. brought a stark realization about the true incidence of renal failure after transplant. Up to 21% of patients developed chronic renal failure within 5 years after receiving a nonrenal transplant.Thirteen years’ post–liver transplant, 18.1% of patients were diagnosed severe renal dysfunction, a significantly higher percentage when compared to controls.9 Both articles alluded to a direct role of CNIs.
Different strategies have been developed to help in this ongoing struggle. One approach has examined adding MMF and reducing the dose of the calcineurin inhibitor or eliminating the calcineurin inhibitor altogether. These studies have produced some encouraging results with up to 50% of patients showing at least a 15% improvement in renal function.

Conversion from CNI to Sirolimus

Recent studies with small numbers looked at the impact of switching patients with chronic renal impairment of CNI to RAP. Questions still remain on the optimal time to conversion and whether it can be used in the early posttransplant period. Large randomized trials are ongoing in an effort to answer these questions.

Calcineurin Inhibitor Avoidance

Very few studies have looked at complete calcineurin inhibitor avoidance. The latest data from the scientific registry of transplant recipients database show 99% of patients are discharged on either TAC (majority) or CyA. The concept of using agents without nephrotoxicity in solid-organ-transplant recipients where rejection has less of a negative impact on the long-term survival has sparked some enthusiasm, but to date little has been published to demonstrate success with this strategy.

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Lancet 2000;355:376-377; Liver Transpl Surg 1999;5:502-508; Transplantation 2005;80:18-25.