Fig. 21.1
Steps in T lymphocyte activation and proliferation. Panel a Multiple signals are required for T cell activation and proliferation in response to alloantigen recognition. Panel b Donor antigens on the surface of antigen presenting cells (APC) are recognized by the T cell receptor (TCR) on the surface of T lymphocytes (signal 1). Panel c A second signal, involving binding of the B7 molecules on the APC to CD28 on the surface of T lymphocytes is required for T lymphocyte activation to occur (signal 2). Signals 1 and 2 trigger an increase in the cytoplasmic levels of calcium, which in turn activates the cytoplasmic protein phosphatase calcineurin. Calcineurin dephosphorylates a transcription factor called nuclear factor of activated T cells (NFAT), allowing it to enter the nucleus, where it promotes the expression of interleukin 2 (IL-2). Panel d Secreted IL-2 binds to the IL-2 receptor (IL-2R) on the surface of activated T lymphocytes (signal 3), providing the stimulus needed for cell growth and proliferation through the mammalian target of rapamycin (mTOR) pathway
Effector Mechanisms Leading to Tissue Injury
Allograft rejection is mediated through both cellular and humoral effector mechanisms (Fig. 21.2). Activated, antigen-specific CD8+ T lymphocytes directly affect donor cell death via releasing a number of cytotoxic proteins that result in cell lysis and that induce apoptosis within the target cell. Similarly, activated CD4+ helper T lymphocytes secrete a variety of cytokines, including interleukin-4 and interleukin-5 that promote the maturation of B-lymphocytes and the production of donor-specific alloantibodies. Alloantibodies bind to their specific MHC targets on the surface of vascular endothelial cells within the allograft, where they cause active damage to the graft by activating the complement cascade and by targeting cells for destruction by natural killer cells and macrophages in a process called antibody-dependent cell-mediated cytotoxicity (ADCC). The later cells have specific receptors on their surfaces that recognize tissue-bound antibody and kill targeted cells through the release of pore-forming proteins and proteolytic enzymes. Additionally, CD4+ T lymphocytes initiate a nonspecific delayed-type hypersensitivity (DTH) response whereby non-antigen-specific cells such as macrophages, natural killer cells, and monocytes are recruited into the graft to enhance the inflammatory response.
Fig. 21.2
Effector mechanisms leading to allograft rejection. Activated T lymphocytes undergo clonal expansion and differentiation into effector cells. CD8+ cytotoxic T lymphocytes directly affect donor cell death by causing cell lysis and inducing apoptosis. In contrast, CD4+ helper T cells secrete cytokines and chemokines that stimulate B lymphocyte maturation and alloantibody production and that help macrophages, natural killer cells, and monocytes to induce a delayed-type hypersensitivity response
Principles of Immunosuppression
The goal of immunosuppression is to blunt the alloimmune response to prevent or treat cardiac allograft rejection while minimizing both drug toxicities as well as the major sequella of immune suppression, namely infection and malignancy. Most clinically used immunosuppressive regimens consist of a combination of several agents used concurrently and follow several general principles. The first principle is that immune reactivity and tendency toward graft rejection are highest early (within the first 3–6 months) after graft implantation and decrease with time. Thus, most regimens employ the highest intensity of immunosuppression immediately after surgery and decrease the intensity over the first year, eventually settling on the lowest maintenance levels of immunosuppression that are compatible with preventing graft rejection and minimizing drug toxicities. The second general principle is to use low doses of several drugs without overlapping toxicities in preference over higher (and more toxic) doses of a single drug whenever feasible. The third principle is that too intense immunosuppression is undesirable because it leads to undesirable effects such as susceptibility to infection and malignancy.
Immunosuppressive regimens can be classified as induction, maintenance, or anti-rejection. Induction regimens provide intense early post-operative immune suppression while maintenance regimens are used throughout the patient’s life to prevent both acute and chronic rejection. This chapter will review the induction and maintenance immunosuppressive regimens used in heart transplantation. The treatment of acute rejection will be discussed in subsequent chapters.
Induction Therapy
Currently, slightly fewer than 50 % of heart transplant programs employ a strategy of augmented immunosuppression, or induction therapy, during the early post-operative period [1]. The goal of induction therapy is to provide intense immunosuppression when the risk of allograft rejection is highest. From a clinical perspective, the main advantages of induction therapy are to allow delayed initiation of nephrotoxic immunosuppressive drugs in patients with compromised renal function prior to or following surgery and to provide some flexibility with respect to early corticosteroid weaning or use of corticosteroid-sparing maintenance immunosuppressive regimens after transplantation [2–4]. Several anti-lymphocyte antibodies that target specific epitopes on the surface of both B and T cells have been used as part of induction therapy. However, the overall strategy of universal induction therapy and the optimal drugs to achieve a state of early intense immunosuppression remain controversial. The decreased early rejection observed with induction therapy may be negated by an increase in late rejection after induction therapy is completed and by the potential for increased rates of infection and malignancy associated with such therapy [5–11]. However, patients at highest risk for fatal rejection, including younger patients, African American patients, patients with high levels of pre-formed antibodies against HLA epitopes, and patients supported on ventricular assist devices may derive a benefit from induction therapy [12].
Muromonab-CD3 (OKT3)
OKT3 is a murine monoclonal antibody that binds to the T cell receptor-CD3 complex on the surface of circulating T cells. It exerts its immunosuppressive effects via a variety of mechanisms, including rapid T cell depletion from the peripheral circulation as a result of opsonization in the liver and spleen, and modulation of the T cell receptor-CD3 antigen recognition complex, thereby blocking the immunologic function of these cells [13, 14].
OKT3 administration is associated with a number of important acute and long-term side effects. The first or second drug dose is typically associated with a cytokine release syndrome characterized by fevers, rigors, nausea, vomiting, diarrhea, hypotension, chest pain, dyspnea or wheezing, arthralgias, and myalgias. This syndrome is caused by initial activation of T cells and release of multiple cytokines. It can be attenuated by pre-medication with intravenous steroids, antihistamines, antipyretics, and H2-blockers. Rare life-threatening complications have included pulmonary edema, aseptic meningitis, and encephalopathy. Long-term adverse reactions include an increased risk of life-threatening opportunistic infections, particularly with cytomegalovirus, and post-transplant lymphoproliferative disorders. Finally, prolonged use of OKT3 can elicit a host anti-mouse antibody response that can blunt future drug efficacy and increase the risk of antibody-mediated rejection [15–17]. Due to these adverse effects and the availability of alternate agents, the use of OKT3 has been abandoned.
Polyclonal Anti-thymocyte Antibodies
Polyclonal antibodies are derived by immunization of horses (ATGAM) or rabbits (Thymoglobulin) with human thymocytes. These preparations contain antibodies directed against a wide variety of human T-cell antigens and cause rapid depletion of T-lymphocytes by inducing complement-mediated cytolysis and cell-mediated opsonization in the spleen and liver. There are no head-to-head comparison trials of ATGAM and Thymoglobulin in heart transplantation, but data from the kidney transplant literature suggests that thymoglobulin may result in a lower incidence of both short and long-term acute rejection compared to ATGAM, possibly because of more profound and durable lymphopenia after Thymoglobulin administration [18, 19]. These agents combined are currently employed in 20 % of heart transplant recipients based upon the most recent international transplant registry data [1].
The major acute side effects associated with this class of drugs include a serum sickness reaction characterized by fevers, chills, tachycardia, hypertension or hypotension, myalgias, and rash. The reaction is typically noticed during the first or second drug infusion and can be treated by temporarily stopping the drug infusion and restarting at a lower infusion rate. Pre-medication with intravenous steroids, antihistamines, antipyretics, and H2 blockers can prevent or reduce the severity of symptoms. Dose-dependent leukopenia (30–50 %) and thrombocytopenia (30–40 %) have also been observed and typically respond to dose reduction or drug discontinuation for severe cases (WBC < 2000 cells/mm3 or platelet count < 50,000 cells/mm3). These agents do not induce a host antibody response to horse or rabbit sera and can be re-used for the treatment of allograft rejection. Long-term side effects include a pre-disposition to opportunistic infections, particularly with cytomegalovirus, and a possible increase in the incidence and aggressiveness of post-transplant malignancies [11, 20, 21].
Interleukin-2 Receptor Antagonists
In recent years, the use of interleukin-2 (IL-2) receptor antagonists for induction therapy has increased, and these drugs are now used in 28 % of patients undergoing heart transplantation [1]. Compared to OKT3 and anti-thymocyte antibodies, this class of drugs has a significantly lower incidence of drug-related adverse reactions [22, 23]. The currently available agent, Basiliximab (Simulect), is an anti-IL-2 receptor monoclonal antibodies that selectively binds to the IL-2 receptor of T-lymphocytes, blocks binding of IL-2 to the receptor complex, and exhibits its immunosuppressive effects by inhibiting IL-2 mediated T-lymphocyte proliferation.
Basiliximab was studied in a pilot multicenter, placebo-controlled randomized study of 56 de novo heart transplant recipients designed to assess the safety, tolerability, and pharmacokinetics of the drug. Patients were randomized to two doses of either basiliximab or to placebo in addition to a background immunosuppressive regimen that included cyclosporine, mycophenolate mofetil, and corticosteroids. There were no significant differences between treatment groups with respect to drug-related adverse events or infections. At 6 months, a non-statistically significant trend toward a decrease in the mean number of days to a first biopsy-proven acute rejection episode of ISHLT Grade ≥ 2R or to a rejection episode with hemodynamic compromise was observed in the basiliximab group compared to placebo (74 versus 41 days) [24].
Alemtuzumab
Alemtuzumab (Campath-1H) is a humanized rat monoclonal antibody that targets the CD52 antigen expressed on both T and B cells. This powerful cytolytic agent produces a profound lymphopenia that lasts for approximately 6 months and that may persist for up to 3 years in some individuals [25]. The agent was originally developed to treat chronic lymphocytic leukemia but has also been used as induction therapy in kidney and heart transplantation, where it has permitted use of lower intensity maintenance immunosuppression [26, 27]. Currently, the use of alemtuzumab as induction therapy is limited to only 2 % of heart transplant recipients [28].
Maintenance Immunosuppressive Regimens
The strategies and drugs used for immune suppression have advanced considerably since the first heart transplant was performed in 1967. Beginning with the introduction of cyclosporine in 1983, significant advances have been made in moving from drugs that provide broad and non-specific immunosuppression to newer agents that provide more targeted immunosuppression through selective inhibition of lymphocyte activation and proliferation. Drug selectivity has resulted in a marked increase in patient survival due to a decrease in the incidence of both life-threatening opportunistic infections and rejection episodes. Most maintenance immunosuppressive protocols employ a three-drug regimen consisting of a calcineurin-inhibitor (cyclosporine or tacrolimus), an antimetabolite agent (mycophenolate mofetil or less commonly azathioprine), and tapering doses of corticosteroids over the first year post-transplantation. The commonly used drugs in heart transplantation and their toxicities are outlined in Table 21.1 [29].
Drug | Dosing | Target levels | Major toxicities |
|---|---|---|---|
Calcineurin inhibitors | |||
Cyclosporine | 4–8 mg/kg/day in 2 divided doses, titrated to keep target 12-h trough levels | 0–6 months: 250–350 ng/mL 6–12 months: 200–250 ng/mL >12 months: 100–200 ng/mL | Renal insufficiency Hypertension Dyslipidemia Hypokalemia and hypomagnesemia Hyperurecemia Neurotoxicity (encephalopathy, seizures, tremors, neuropathy) Gingival hyperplasia Hirsutism |
Tacrolimus | 0.05–0.1 mg/kg/day in 2 divided doses, titrated to keep target 12-hr. trough levels | 0–6 months: 10–15 ng/mL 6–12 months: 5–10 ng/mL >12 months: 5–10 ng/mL | Renal dysfunction Hypertension Hyperglycemia and diabetes mellitus Dyslipidemia Hyperkalemia Hypomagnesemia Neurotoxicity (tremors, headaches) |
Cell cycle agents | |||
Azathioprine | 1.5–3.0 mg/kg/day, titrated to keep WBC ~ 3 K | None | Bone marrow suppression Hepatitis (rare) Pancreatitis Malignancy |
Mycophenolate mofetil | 2000–3000 mg/day in 2 divided doses | Mycophenolic acid (MPA): 2–5 mcg/ml | Gastrointestinal disturbances (nausea, gastritis, and diarrhea) Leukopenia |
Mycophenolic acid | 1440 mg/day in 2 divided doses | None | Less gastrointestinal disturbances compared to mycophenolate mofetil Leukopenia |
Proliferation signal inhibitors | |||
Sirolimus | 1–3 mg/day, titrated to keep therapeutic 24-h trough levels | 5–10 ng/mL | Oral ulcerations Hypercholesterolemia and hypertriglyceridemia Poor wound healing Lower extremity edema Pulmonary toxicities (pneumonitis, alveolar hemorrhage) Leukopenia, anemia, and thrombocytopenia Potentiation of CNI nephrotoxicity Proteinuria |
Everolimus | 1–1.5 mg/day, titrated to keep therapeutic 12-h trough levels | 3–8 ng/mL | Similar to sirolimus |
Corticosteroids | |||
Prednisone | 0.5–1 mg/kg/day in 2 divided doses, tapered to 0.05 mg/kg/day by 6–12 month | None | Weight gain Hypertension Hyperlipidemia Osteopenia Hyperglycemia Poor wound healing Salt and water retention Proximal myopathy Cataracts Peptic ulcer disease Growth retardation |
Calcineurin-Inhibitors
Since the introduction of cyclosporine in the early 1980’s, the calcineurin inhibitors have remained the cornerstone of maintenance immunosuppressive therapy in heart and other solid organ transplantation. These drugs exert their immunosuppressive effects by inhibiting calcineurin, which is normally responsible for the transcription of IL-2 and several other cytokines, including TNF-α, granulocyte-macrophage colony-stimulating factor, and interferon-gamma (Fig. 21.1). The end result is blunting of T-lymphocyte activation and proliferation in response to alloantigens. The two available calcineurin inhibitors, cyclosporine and tacrolimus, form complexes with different intracellular binding proteins, and these drug-protein complexes subsequently bind to and inhibit calcineurin. The drugs differ with respect to both efficacy and side effect profile.
Cyclosporine
Cyclosporine is a peptide derived from the fungus Tolypocladium inflatum that has powerful immunosuppressive properties. It binds to the cytoplasmic protein cyclophilin to inhibit calcineurin. The drug is available in several formulations. The older oil-based formulation, called Sandimmune®, was characterized by variable and incomplete absorption. The newer modified formulations, including Gengraf® and Neoral®, are microemulsion formulations that result in improved and more reproducible drug absorption. Due to their improved pharmacokinetic profile, the microemulsion preparations are generally preferred over the oil-based formulation. The two formulations are not considered bioequivalent, and patients should not be routinely switched from one to the other without close monitoring of drug levels.
Dosing and Therapeutic Drug Monitoring
Cyclosporine is available as oil-based or microemulsion capsules, as an oral microemulsion solution, and as a concentrate for injection. When given intravenously, approximately one third of the daily oral dose should be given as a continuous infusion over 24 h. The drug is typically titrated to achieve therapeutic 12-h trough levels. In general, cyclosporine levels are kept highest in the first year post-transplantation (200–350 ng/mL) and lowered in subsequent periods (100–200 ng/mL). However, target drug levels should be individualized according to a patient’s risk of rejection, renal function, and susceptibility to drug toxicities and infection.
Major Toxicities
The major toxicities of cyclosporine include renal insufficiency, hypertension, dyslipidemia, hypokalemia and hypomagnesemia, and neurotoxicity (see Table 21.1). Gingival hyperplasia and hirsutism are two additional side effects that are unique to cyclosporine.
Tacrolimus
Tacrolimus (Prograf®), previously known as FK-506, is a macrolide compound derived from the fungus Streptomyces tsukubaensis. It binds to a cytoplasmic protein called FK binding protein and inhibits calcineurin via a similar pathway to that of cyclosporine. In recent years, the use of tacrolimus in heart transplantation has increased, and it is currently the most widely used calcineurin-inhibitor.
Multiple single-center and multi-center randomized comparisons between de novo use of tacrolimus and cyclosporine after heart transplantation have been reported [30–37]. As a whole, these trials have shown similar survival between patients treated with the two agents but fewer episodes of biopsy-proven or drug-treated acute rejection among patients treated with tacrolimus. Additionally, tacrolimus is associated with a more favorable side effect profile compared to cyclosporine. Compared to patients treated with cyclosporine, patients on tacrolimus had less hypertension, less hyperlipidemia, but a higher incidence of post-transplant diabetes mellitus.
Dosing and Therapeutic Drug Monitoring
Tacrolimus is available as oral capsules and as an injectable solution. The drug is typically given orally. When intravenous administration is required, approximately one third of the daily oral dose should be given as a continuous infusion over 24 h. Drug dosing is titrated to achieve therapeutic 12-h trough levels. In general, target levels are typically highest in the first 6 months (10–15 ng/mL) and lower thereafter (5–10 ng/mL).
Major Toxicities
Compared to cyclosporine, the use of tacrolimus is associated with less hypertension and dyslipidemia. However, an increased frequency of new-onset diabetes mellitus has been observed in patients on tacrolimus compared with cyclosporine.
Antimetabolites
The antimetabolites, or antiproliferative agents, interfere with the synthesis of nucleic acids and exert their immunosuppressive effects by inhibiting the proliferation of both T and B lymphocytes.
Azathioprine
Azathioprine (Imuran®) is a prodrug that is first rapidly hydrolyzed in the blood to its active form, 6-mercaptopurine, and subsequently converted to a purine analogue, thio-inosine-monophosphate. This antimetabolite is incorporated into DNA and inhibits further nucleotide synthesis, thereby preventing mitosis and proliferation of rapidly dividing cells such as activated T and B lymphocytes. This drug is typically used as an adjunctive immunosuppressive agent with either corticosteroids or more commonly in conjunction with a calcineurin inhibitor. The major side effects include dose-dependent myelosuppression, particularly leukopenia. Azathioprine should be temporarily withheld if the white cell count falls below 3000/mm2 or drops by 50 % compared to the previous value. Other potentially serious side effects include hepatotoxicity and pancreatitis.
Mycophenolate Mofetil
Mycophenolate mofetil (Cellcept®) has replaced azathioprine as the preferred antimetabolite agent in recent years. It is also prodrug that is rapidly hydrolyzed to its active form, mycophenolic acid (MPA). MPA is a reversible inhibitor of inosine monophosphate dehydrogenease, a critical enzyme for the de-novo synthesis of guanine nucleotides. Lymphocytes lack a key enzyme in the guanine salvage pathway and are dependent upon the de novo pathway for the production of purines necessary for RNA and DNA synthesis. Therefore both T- and B-lymphocytes proliferation are selectively inhibited.
In a multi-center, active-controlled, randomized trial, mycophenolate mofetil was compared with azathioprine when used in conjunction with cyclosporine and corticosteroids in 650 de novo heart transplant recipients. Because an intravenous form of the study drug (mycophenolate mofetil) was not available at the time of the trial, 11 % of the patients withdrew before receiving the drug. Survival and rejection were similar in both groups when analyzed in an intention-to-treatment manner. However, among treated patients, mycophenolate mofetil was associated with a significant reduction in both mortality (6 % versus 11 %, p=0.031) and in the incidence of treatable rejection (66 % versus 74 %, p=0.026) at 1 year [38].
Dosing and Therapeutic Drug Monitoring
Mycophenolate mofetil is available as an oral tablet or capsule and as a powder for injection. The intravenous solution is given at the same oral dose as a 2 h infusion every 12 h. The drug is typically administered at a starting dose of 1000–1500 mg twice daily and subsequently decreased as needed in response to leukopenia or gastrointestinal intolerance. While drug monitoring is not routinely performed, some centers target MPA trough levels between 2 and 5 ng/mL.
Major Toxicities
Mycophenolate mofetil is not nephrotoxic and causes less bone marrow suppression compared to azathioprine. The main side effects include dose related leukopenia and gastrointestinal toxicities such as nausea, gastritis, and diarrhea. A possible association between mycophenolate mofetil and Progressive Multifocal Leukoencephalopathy (PML) has been reported [39].
Mycophenolic Acid
Mycophenolate sodium (Myfortic®) is an enteric coated, delayed release salt of mycophenolic acid, developed to improve the upper gastrointestinal tolerability of mycophenolate. Mycophenolic acid is available in 180 mg and 360 mg enteric coated tablets. Because of this coating, the tablet should not be crushed. The following conversions between mycophenolate mofetil (MMF) and mycophenolate sodium should provide equimolar amounts of MPA:
1000 mg MMF = 720 mg mycophenolate sodium
1500 mg MMF = 1080 mg mycophenolate sodium
Single and multi-center studies in de novo heart transplant recipients have shown that EC-MPS is therapeutically similar to MMF with respect to prevention of both biopsy-proven and treated acute rejection episodes, graft loss, or death. However, significantly fewer patients in the EC-MPS group required dose reductions during treatment [40, 41].
Proliferation Signal Inhibitors
In recent years, a new class of drugs called proliferation signal inhibitors, or mammalian target of rapamycin (mTOR) inhibitors, has been used in selected patients with renal insufficiency, cardiac allograft vasculopathy, or malignancies, in an attempt to reverse or slow progression of these conditions. However, the high incidence of drug-related adverse effects, including pericardial effusions, delayed sternal wound healing after transplantation, and the potential for enhanced nephrotoxicity when used with standard-dose cyclosporine, may limit the widespread use of these agents as de-novo therapy following transplantation [36, 42–44]. The two drugs in this class, Sirolimus and Everolimus, have similar mechanisms of action. They are structurally similar to Tacrolimus and also bind to the FK binding protein; however, they exert their immunosuppressive effects via a calcineurin-independent mechanism. The drug-immunophilin complex inhibits a protein kinase in the cytoplasm called mammalian target of rapamycin (mTOR) (Fig. 21.1d). mTOR is involved in the transduction signals from the IL-2 receptor to the nucleus. The consequence of mTOR inhibition is cell cycle arrest at the G1 to S phase, preventing both T- and B-cell proliferation in response to cytokine signals.
Sirolimus
Sirolimus (Rapamune®) is a macrolide antibiotic derived from the fungus Streptomyces hygroscopicus. The efficacy of sirolimus as an alternative to azathioprine was evaluated in a prospective, open-label, randomized trial of 136 de novo heart transplant recipients. Patients were randomized 2:1 to receive one of two sirolimus doses (3 or 5 mg) or to azathioprine. Sirolimus doses were subsequently adjusted in both groups to achieve similar target blood levels. All patients received concurrent immunosuppression with cyclosporine and corticosteroids. Compared with azathioprine, the use of either dose of sirolimus was associated with fewer biopsy-proven acute cellular rejection episodes at 6 months. Additionally, the development of cardiac allograft vasculopathy, as defined by intravascular ultrasound, was significantly reduced in the sirolimus groups at both 6 months and 2 years. Patient survival at 12 months was comparable among groups [43]. The combination of sirolimus with tacrolimus was also compared against mycophenolate mofetil with tacrolimus and mycophenolate mofetil with cyclosporine in a multi-center randomized trial involving 343 de novo heart transplant recipients. In this study, there was no statistically significant difference in the incidence of acute cellular rejection or hemodynamically compromising rejection among the three groups, but patients in both the sirolimus plus tacrolimus group and in the mycophenolate mofetil plus tacrolimus group experienced fewer treated rejection episodes compared to patients in the mycophenolate mofetil plus cyclosporine group. However, patients in the sirolimus plus tacrolimus group experienced an increased incidence of renal dysfunction and wound healing complications compared to the other two groups [36].
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