Immunosuppression in Pediatric Kidney Transplantation

Abstract

Because it is currently not possible to induce specific tolerance, transplantation requires immunosuppressive therapies. The goal is to use immunosuppressive agents that are potent, selective, and reversible, with reliable delivery and long-term safety. A careful balance is required to find the dose that prevents rejection of the graft, while minimizing the risks of over-immunosuppression leading to infection and cancer. The common immunosuppressive agents used in pediatric renal transplantation include the glucocorticoids, azathioprine, mycophenolate mofetil, the calcineurin inhibitors tacrolimus and ciclosporin, the mammalian target of rapamycin inhibitors sirolimus (SRL) and everolimus, and antibodies to cell surface antigens on lymphocytes (antithymocyte globulin), anti-CD25 antibodies (anti-interleukin-2 receptor antibodies), alemtuzumab (a humanized anti-CD52 pan-lymphocytic monoclonal antibody) and belatacept, a costimulation blocker of T cells. This chapter focuses on these agents and how they inhibit the immune response. Although data from adult renal transplantation trials are used to help guide management decisions in pediatric patients, immunosuppression often must be modified because of the unique clinical effects of some of these agents in children, including their impact on growth and development.

Introduction

Ideally, a recipient (host) would accept a kidney transplant without induction of an antigen-specific response. However, it is not currently possible to induce specific immunologic tolerance, and transplantation requires immunosuppressive therapies. The goal is to use immunosuppressive agents that are not only potent and selective, but allow reversibility of their action, can be reliably delivered and display long-term safety. Most therapies alter immune response mechanisms but are not immunologically specific, and a careful balance is required to find the dose that prevents rejection of the graft, while minimizing the risks of over-immunosuppression leading to infection and cancer.

Current immunosuppressive agents reduce acute rejection, but do not induce tolerance. A few patients with organ transplants can successfully withdraw their immunosuppressive therapy without rejecting their grafts for long periods of time. However, these are rare exceptions, and such patients may eventually reject, even after years as this operational state of prope tolerance is unstable and poorly understood. The hunt for biomarkers to harness this “operational” tolerance state has remained elusive [1]. Even though antigen-specific T cells with reactivity to the foreign antigen persist in the host indefinitely, some graft and host adaptation must occur, since the level of immunosuppression required long-term is very low compared to the levels required within the first weeks post-transplant. This adaptation makes long-term immunosuppression possible; however, the long-term risk of cancer in the immunosuppressed patient remains increased. Thus, the distinction between immunosuppression and tolerance induction is partly artificial: any immunosuppression involves some apparent antigen-specific adaptation, i.e. down-regulation of the host response to the graft; and many tolerance protocols involve some non-specific immunosuppressive therapies.

The goal of immunosuppressive therapy is to prevent acute rejection while minimizing drug side effects. In children who undergo renal transplantation, immunosuppression is divided into 3 categories: (i) Induction therapy, i.e. intense immunosuppression administered during the perioperative period to prevent acute rejection, (ii) maintenance therapy, i.e. immunosuppressive treatment to prevent acute rejection after the perioperative period, and (iii) immunosuppressive therapy to treat graft rejection. In general, immunosuppression should be most intense during the first 3 months after transplantation when the risk of acute rejection and allograft loss is greatest. Immunosuppression is tapered slowly to a maintenance level by 6–12 months post-transplant. The goal remains to find the best combination of immunosuppressive agents that optimizes allograft survival by preventing rejection episodes while limiting drug toxicities. Although data from adult renal transplantation trials are used to help guide management decisions in pediatric patients, immunosuppression must often be modified because of the unique clinical effects of some of these agents in children, including their impact on longitudinal growth and development.

Allograft survival rates vary among the various immunosuppressive agents due to patient-specific clinical characteristics, such as age, ethnicity, obesity, hyperlipidemia, hypertension, proteinuria, delayed allograft function and some donor-related factors such as living-related versus deceased kidney donation. Immunosuppressive agents should therefore be chosen in part based on patient characteristics. Other issues to be considered are related to the immunologic history of the patient, such as ABO compatibility and the degree of HLA matching, pre-sensitization, re-transplantation, history of acute rejection episodes and the risk of recurrent disease.

The common immunosuppressive agents used in pediatric renal transplantation include glucocorticoids (steroids), azathioprine (AZA), mycophenolate mofetil (MMF), the calcineurin inhibitors tacrolimus (TAC) and ciclosporin (CSA), the mammalian target of rapamycin (mTOR) inhibitors sirolimus (SRL) and everolimus (EVR), antibodies to cell surface antigens on lymphocytes, including anti-thymocyte globulin (ATG), anti-CD25 antibodies (anti-interleukin-2 [IL-2] receptor antibodies), alemtuzumab (a humanized anti-CD52 pan-lymphocytic monoclonal antibody) and belatacept, a co-stimulation blocker of T cells. The structures of some immunosuppressives are shown in Fig. 67.1 [2]. Our discussion will focus on these agents and how they inhibit the immune response.

Fig. 67.1

Structure of mycophenolic acid, ciclosporin, azathioprine, tacrolimus, and sirolimus. These are all small molecules with molecular weights of 320, 1203, 277, 804, and 914, respectively. (Used with permission from Johnson RJ, Feehally J (eds): Comprehensive Clinical Nephrology, 2nd ed. Philadelphia: Elsevier; 2003) [2]

The Immune Response

By the time transplant surgery is completed, the graft has undergone acute injury leading to an increased expression of major histocompatibility complex (MHC) molecules by cells within the graft that are either constitutively expressed (class I, HLA-A and HLA-B) or inducible (class II, HLA-DR) antigens. Injury recruits lymphocytes and antigen-presenting cells (APCs), typically monocytes, macrophages, and dendritic cells from the host. These injury-related events may influence the probability of rejection and thereby contribute to the superior outcome in transplants from live donors (with less injury) vs. those from deceased donors.

Allorecognition of donor MHC molecules may occur either by the direct route (host T cells recognize donor MHC on donor cells) or indirectly (host T cells recognize donor MHC as peptides in the MHC groove of host APCs). T cell receptors (TCR) engaging MHC–peptide complexes provide signal 1. Co-stimulatory signals from the APC engaging receptors on the T cells provide signal 2 (Fig. 67.2) [3]. The major co-stimulatory molecules of the APC are B7–1/B7–2, which bind CD28 on the T cells. Activated T cells express CD40L that can activate the APC by engaging CD40 on the APC, and Fas ligand (FasL), which binds Fas on other lymphocytes or other cells to induce apoptosis in the Fas-bearing cell. Activation of signals 1 and 2 is followed by T cell activation with production of many cytokines. Cytokines, such as IL–2, engage specific receptors on the T cells to provide signal 3, the signal for cell division and clonal expansion. The engagement of CD40 by CD40L and the cytokines and growth factors from T cells regulate the T cell response, recruit and activate inflammatory cells, and alter adhesion molecules to cause mononuclear cells to accumulate in the graft. Depending on the type and degree of signaling, full activation of the T cell may occur, or T cells may undergo partial activation, apoptosis, anergy, or neglect (ignoring the antigen). T cells also bind via CD40L to CD40 on B cells, thereby directing the switch from IgM to IgG production by B cells and promoting the maturation of IgG-producing B cells.

Fig. 67.2

The 3 events in T cell activation. Engagement of the T cell receptor with the antigenic peptide in the context of self–MHC class II molecule leads to the activation of the calcineurin pathway and results in the induction of cytokine genes (e.g., IL–2) (signal 1). Signal 2, the co-stimulatory signal, involves the engagement of CD28 with members of the B7 family. This synergizes with signal 1 to induce cytokine production. Interaction between cytokine production and its corresponding receptor leads to induction of cell division, probably through the target of rapamycin (TOR) pathway. This constitutes signal 3. (Used with permission from Feehally J, Floege J, Johnson RJ (eds.): Comprehensive Clinical Nephrology, 3rd ed. Philadelphia: Mosby, 2007) [3]

Chemotactic factors (chemokines) and expression of adhesion proteins and foreign (MHC) antigens mediate localization (homing) of CD4 and CD8 T cells to the graft endothelium. Lymphocyte recirculation depends on the ability to enter and leave lymphoid tissue. CD8 T cells that recognize peptide in the groove of class I MHC become cytotoxic T cells (CTL). Graft rejection is associated with infiltration by cytotoxic CD8 lymphocytes. Delayed type hypersensitivity may also be involved in T cell-mediated damage. Antibody-mediated injury may also occur and causes damage to endothelium. A summary of the effects of immunosuppressive drugs is presented in Fig. 67.3 [3].

Fig. 67.3

Individual immunosuppressive drugs and sites of action in the 3-signal model. Anti-CD154 antibody has been withdrawn from clinical trials but remains of interest. FTY720 engagement of sphingosine-1-phosphate (S-1-P) receptors triggers and internalizes the receptors and alters lymphocyte recirculation, causing lymphopenia. Antagonists of chemokine receptors (not shown) are also being developed in preclinical models. MPA denotes mycophenolic acid. (Reproduced with permission from [4])

Classification of Immunosuppressive Agents

Immunosuppressive and immunomodulatory drugs can be pharmacologically categorized on the basis of their mechanism of action. The 3-signal model of T cell activation and proliferation is helpful in understanding the molecular mechanisms and site of action of various immunosuppressive drugs. Figure 67.3 depicts a schematic representation of the 3-signal model along with the site of action of common immunosuppressive drugs [4]. Signal 1 features APCs (macrophages and dendritic cells) presenting the foreign antigen to the T lymphocyte, activating the TCR, which further relays the signal through the transduction apparatus known as the CD3 complex. Signal 2 is a non-antigen-specific co-stimulatory signal which occurs as a result of binding of the B7 molecule on the APC to CD28 on the T cell. Both signal 1 and signal 2 activate signal transduction pathways: the calcium–calcineurin pathway, mitogen-activated protein (MAP) pathway, and the nuclear factor-κB (NF-κB) pathway. This in turn leads to increased expression of IL-2, which through its receptor (IL-2R) activates the cell cycle (signal 3). Signal 3 activation requires the enzyme mTOR for translation of mRNA and cell proliferation. Thus, various drugs act on different cellular signals and achieve immunosuppression by a number of mechanisms: depleting lymphocytes, diverting lymphocyte traffic, or blocking lymphocyte response pathways.

Induction Immunosuppressive Therapy

Induction refers to the administration of an intensive immunosuppressive regimen during the perioperative period. The rationale behind this approach is that the risk of acute rejection is greatest in the first weeks and months after transplantation. Induction therapies often involve the use of polyclonal or monoclonal antibodies to achieve rapid and profound early immunosuppression. Polyclonal antibodies used for this purpose include those against thymocytes (e.g., commercially available rabbit or equine preparations); monoclonal antibodies include basiliximab (a chimeric human–murine anti-CD25 or anti–IL-2R antibody) and alemtuzumab (an anti-CD52 antibody targeting both B and T cells), which is not always readily available.

A number of trials have been and are being conducted in adult and pediatric renal transplant recipients to look into the effects of prophylactic antibody induction therapies. Evaluation of any induction protocol requires consideration of the following factors: (i) Incidence and severity of delayed allograft function or primary non-function, including post-transplant dialysis requirements; (ii) incidence of acute rejection; (iii) incidence, type, and severity of associated infections; (iv) long-term allograft survival and function; (v) mortality and morbidity, including length of hospitalization, (vi) cost, and (vii) incidence and type of malignancy during long-term follow-up. Several studies from single centers and registries, as well as meta-analyses, have found that induction with antibodies may be superior to non-antibody-based regimens, especially in high-risk groups [5]. Unfortunately, most if not all published studies have addressed only some of the above issues. As a result, although each protocol may have specific advantages and disadvantages in a particular patient population, none is yet proven to be superior when all the above factors are considered. The optimal prophylactic induction immunosuppressive therapy to prevent renal transplant rejection remains therefore controversial. Figure 67.4 presents the induction antibody use from 2007–2018, as reported in the Scientific Registry of Renal Transplant Recipients (SRTR) 2018 annual report, indicating that administration of ATG has increased while that of anti-IL-2R and no induction have decreased over time. This is in contrast to reports from the Cooperative European Paediatric Renal Transplant Initiative (CERTAIN) Registry showing that 60% of pediatric kidney transplant recipients in Europe do not receive any induction therapy, 35% receive basiliximab and 5% ATG [6]. Hence, the frequency and type of the chosen immunosuppressive anti-lymphocyte regimens for induction therapy vary markedly between North America and Europe and probably reflect differences in patient characteristics and estimated immunological risk, but is unfortunately not based on comprehensive clinical trials.

Fig. 67.4

Induction agent use in pediatric kidney transplant recipients in the United States of America. Immunosuppression at transplant reported to the Organ Procurement and Transplantation Network (OPTN) (https://pubmed.ncbi.nlm.nih.gov/31898417). IL2-RA, interleukin-2 receptor antagonist

In fact, induction therapy produces the greatest benefits in groups at high risk of allograft rejection. These high-risk groups include African-Americans, recipients of kidneys with prolonged cold ischemia time, and those at high immunologic risk, particularly individuals who are pre-sensitized. The sequential induction regimen of thymoglobulin followed by a combination of TAC and MMF with or without steroids is recommended in these high-risk groups.

Lymphocyte Depleting Antibodies

Polyclonal Antibodies

Because of the redundancy of the immune system, polyclonal antibodies, which have a broad specificity, should theoretically be more effective in induction therapy than monoclonal anti-lymphocyte agents. Anti-thymocyte globulin (ATGAM) is a purified gamma globulin solution obtained by immunization of horses with human thymocytes. It contains antibodies to a wide variety of human T cell surface antigens, including the major MHC antigens. Thymoglobulin is a rabbit-derived polyclonal antibody preparation approved for the treatment of rejection and induction therapy by the US Federal Drug Administration (FDA). As for ATGAM, thymoglobulin contains antibodies to a wide variety of T cell antigens and MHC antigens.

Polyclonal antibodies act in 3 ways: by activating or altering the function of lymphocytes, by lysing lymphoid cells, and by altering the traffic of lymphoid cells and sequestering them. These antibodies are potently immunosuppressive, but often produce side effects. By triggering T cells, they generate significant first-dose effects, with the release of tumor necrosis factor alpha (TNFα), interferon γ (IFN-γ), and other cytokines, causing a first-dose reaction (flu-like syndrome, fever, and chills).

Dosage of Thymoglobulin

Thymoglobulin induction is usually dosed from 1 to 6 mg/kg per dose, and the duration may range from 1 to 10 days, although a more typical regimen is 1.5 mg/kg per dose for 3–5 days [7,8,9,10]. In animal models, higher initial doses of shorter duration approximating a human-equivalent dose of 6 mg/kg were associated with more peripheral and central lymphocyte depletion and better allograft survival [11]. Based on these models, the optimal induction dose is felt to total 6 mg/kg [9, 12]. Total doses of 5.7 mg/kg, on average given as 1.5 mg/kg per day, have been shown to produce similar outcomes as higher doses in high-risk recipients who received an average of 10.3 mg/kg [10]. Higher doses and prolonged duration of induction agents are thought to be associated with an increased risk of infection and the potential development of lymphoma, whereas cumulative doses of less than 3 mg/kg may not effectively prevent acute rejection [13].

Efficacy and Safety

Few studies have compared the relative efficacy of thymoglobulin and ATGAM for induction therapy. In one study in adult renal transplant recipients, 72 patients were randomly assigned in a double-blind 2:1 fashion to receive intravenous doses of thymoglobulin at 1.5 mg/kg or ATGAM at 15 mg/kg intra-operatively, then daily for at least 6 days [14]. The delayed graft function rate was only 1 percent for both groups. At 1 year, the thymoglobulin group had a significantly lower acute rejection rate (4% vs. 25%, respectively) and higher allograft survival (98% vs. 83%). The lower rejection rate was thought to be due in part to a more sustained lymphopenia with thymoglobulin, while the exceptionally low delayed graft function rate seen in both groups may have been due to the intra-operative use of the ATGs. Both antibodies have the ability to block a number of adhesion molecules, cytokines, chemokines, and their receptors, which may contribute to ischemia reperfusion injury and delayed graft function. At 5 years, allograft survival was significantly better in the thymoglobulin arm (77% vs. 57%, respectively) [15]. Two cases of post-transplant lymphoproliferative disorder (PTLD) developed with ATGAM, while none were observed with thymoglobulin. The mean 5-year serum creatinine concentration was similar in both groups.

In pediatric renal transplant recipients, a historical cohort study compared the rates of survival, rejection, and infection in patients who received induction therapy with ATGAM (n = 127) or thymoglobulin (n = 71) [16]. Maintenance immunosuppression included CSA, AZA or MMF, and prednisone. Mean follow-up was 90 ± 25 months for ATGAM recipients and 32 ± 15 months for thymoglobulin recipients. Overall, the incidence of acute rejection was lower in thymoglobulin recipients vs. ATGAM recipients (33% vs. 50%, P = 0.02). Epstein-Barr virus (EBV) infection was higher in thymoglobulin recipients versus ATGAM recipients (8% vs. 3%, P = 0.002). But the two groups did not significantly differ in patient and graft survival rates, incidence of chronic rejection, EBV lymphoma, or other infections. The authors concluded that thymoglobulin induction was associated with a reduced incidence of acute rejection and an increased incidence of EBV infection in pediatric renal transplant recipients.

IL-2 Receptor Antibodies

Full T cell activation leads to the calcineurin-mediated stimulation of the transcription, translation, and secretion of IL-2, a key autocrine growth factor that induces T cell proliferation. Thus, an attractive therapeutic option is abrogation of IL-2 activity via the administration of anti-IL-2R antibodies. Currently, only basiliximab, a chimeric monoclonal antibody, is commercially available and has been approved by the FDA for use in renal transplantation in adults and pediatric patients (Fig. 67.5). The IL-2R consists of 3 transmembrane protein chains: CD25, CD122, and CD132. CD25 is present on nearly all activated T cells, but not on resting T cells. IL-2 induces clonal expansion of activated T cells. Although CD25 does not transduce the signal, it is responsible for the association of IL-2 with the β- and γ-chains, which triggers the activated T cell to undergo rapid proliferation. This antibody binds to activated T cells and render them resistant to IL-2 by blocking, shedding or internalizing the receptor; it may also deplete and sequester some activated T cells. However, IL-2R functions are partially redundant because other cytokine receptors have overlapping functions, e.g., IL-15R. Therefore, saturating IL-2R produces stable, but relatively mild immunosuppression, and is only effective in combination with other immunosuppressants.

Fig. 67.5

Chimeric vs. humanized monoclonal antibodies. (Used with permission from Feehally J, Floege J, Johnson RJ (eds.): Comprehensive Clinical Nephrology, 3rd ed. Philadelphia: Mosby, 2007) [3]

Dosage of Basiliximab

The dosing schedule for basiliximab is the following: intravenous administration of two 10 mg doses to children <35 kg in weight and two 20 mg doses to children ≥35 kg in weight, with the first dose given within 2 h prior to surgery, and the second on post-transplant day 4. In the 14 patients who were evaluated for the pharmacokinetics and pharmacodynamics of basiliximab and received concomitant immunosuppression with CSA and AZA, the mean duration of IL-2R saturation was 42 ± 16 days [17]. In a larger study of 82 patients who received basiliximab in combination with MMF, MMF reduced basiliximab clearance and thereby prolonged CD25 saturation from 5 to 10 weeks [18].

Efficacy and Safety

The effectiveness of IL-2R antibody therapy was best reported in a meta-analysis involving 38 trials that enrolled nearly 5000 patients that assessed the impact of therapy on allograft loss and rejection [19]. Data were derived from published trials and abstracts of completed and ongoing trials. From these 38 trials, 14 trials enrolling 2410 patients compared IL-2R antagonists with placebo for at least one outcome. Compared with placebo, IL-2R antagonists reduced acute rejection rates at 6 months (relative risk [RR 0.66], CI 0.59–0.74) and 1 year (RR 0.67, CI 0.60–0.75), but the incidence of graft loss was the same.

In pediatric renal transplant recipients, two large prospective randomized controlled trials showed that induction therapy with basiliximab in patients with low to medium immunological risk on maintenance therapy (TAC in conjunction with AZA, or CSA in conjunction with MMF) did not lead to a statistically significant reduction in the incidence of acute rejection episodes [20, 21]. As a result, there is presently no consensus amongst pediatric renal transplantation centers regarding the use of and regimen for immunosuppressive induction therapy. Considerations in choosing the appropriate agent include the efficacy in the patient population (e.g., recipients with high or low risk of graft loss), the side effect profile, and the concomitant immunosuppressive therapy (steroid avoidance, early steroid withdrawal, or conventional steroid therapy).

Comparison of Basiliximab with Thymoglobulin

Few studies have compared the use of different induction immunosuppressive regimens. In adult patients at increased risk of acute rejection, thymoglobulin is more effective than basiliximab in preventing rejection [22,23,24,25]. A multicenter, international, randomized, prospective study of 278 first deceased-donor kidney transplant recipients compared the safety and efficacy of a 5-day course of thymoglobulin (n = 141) or 2 doses of basiliximab (n = 137) [26]. Recipients and donors were chosen based upon characteristics that would predict an increased risk of rejection or delayed graft function. Patients in both arms were administered CSA, MMF, and prednisone for maintenance immunosuppression, and received antiviral prophylaxis with ganciclovir. The primary endpoint was a composite of acute rejection, delayed allograft function, transplant loss, and death. At 1 year, there was no difference between thymoglobulin and basiliximab in the incidence of the composite endpoint. However, thymoglobulin was associated with a significantly lower acute rejection rate (16% vs. 26%), and incidence of acute rejection that required antibody treatment (1.4% vs. 8%). Although overall adverse event and serious adverse event rates were similar, thymoglobulin was associated with a higher incidence of infection (86% vs. 75%), but lower incidence of cytomegalovirus (CMV) disease (8% vs. 18%).

At 5-year follow-up, the incidence of acute rejection and need for antibody treatment of acute rejection remained lower among those treated with thymoglobulin, compared with basiliximab (16% vs. 30% and 3% vs. 12%, respectively) [22]. Patients treated with thymoglobulin also had a significantly lower composite endpoint of acute rejection, graft loss, and death at 5 years (39% vs. 52%) and incidence of treated CMV infection (7% vs. 17%); however, the incidence of malignancy did not differ. Hence, the relative benefits of thymoglobulin were sustained over a 5-year period.

Alemtuzumab

Alemtuzumab (Campath-1H, MabCampath) is a humanized IgG1 monoclonal antibody directed against CD52, a glycoprotein expressed on mononuclear cells, including T and B lymphocytes, monocytes, and natural killer cells [23]. Its efficacy for induction and maintenance immunosuppression with low-dose calcineurin inhibitors (CNIs) was first introduced by Calne et al. [24] and later supported by the Pittsburgh group [25] in adults and children. Alemtuzumab induction has been associated with lower rates of acute rejection than basiliximab and daclizumab in low immunological risk patients and was associated with similar efficacy as compared with rabbit anti-thymocyte globulin in high-risk patients [26]. Although most of these studies have involved adult kidney transplant recipients, there is also pediatric experience that supports this efficacy claim, especially in highly sensitized, high-risk children [27, 28]. A significant reduction of white blood cell and absolute lymphocyte counts, up to 1 year post-transplant, has been observed in children receiving alemtuzumab treatment [29].There is no currently recommended dose for children, but 0.3 mg/kg per dose has been most frequently used in pediatric studies [30]. The most common number of doses administered was 2 doses, with a range from 1 to 4 doses during the first week post-transplant. Due to infusion-related reactions, pre-medication with methylprednisolone, acetaminophen, and diphenhydramine is recommended in addition to administration of anti-emetics to avoid nausea and vomiting. The major side effect that limits the use of alemtuzumab is profound lymphopenia, which may contribute to significant adverse events, including infections, autoimmune complications, and malignancies [30]. The Cooperative Clinical Trials in Pediatric Transplantation program of the National Institute of Allergy and Infectious Disease has completed a multicenter pilot trial of alemtuzumab induction in pediatric kidney transplant recipients with initial maintenance immunosuppression of TAC and MMF, but the results have yet to be published.

Recommendations

There is currently no consensus for immunosuppressive induction therapy following kidney transplantation in children. At the present time, no consistent evidence exists that induction therapy is beneficial or cost-effective in low-risk patients on triple therapy with CNIs in conventional doses, MMF and steroids. According to 2 prospective, randomized, controlled trials, induction therapy with basiliximab in pediatric patients with low or normal immunological risk on maintenance therapy with either TAC in conjunction with AZA or CSA in combination with MMF did not lead to a statistically significant reduction in the incidence of acute rejection episodes [20, 21]. In contrast, the Kidney Disease: Improving Global Outcomes clinical practice guidelines recommend induction therapy for all adult patients, with an IL-2R blocker as first line for those not at high immunological risk [31]. There is some evidence favoring the use of IL-2R blockers over no induction in adult renal transplantation [32]. However, it has been pointed out in the more recent literature that these studies mainly used outdated maintenance regimens [33]. No large randomized trial has examined the effect of IL-2R antibodies or rabbit ATG induction vs. no induction in patients receiving TAC, mycophenolic acid (MPA) and steroids. With this triple maintenance therapy, the addition of induction may achieve an absolute risk reduction for acute rejection of only 1–4% in standard-risk patients without improving renal allograft or patient survival. In contrast, rabbit ATG induction lowers the relative risk of acute rejection by almost 50% vs. IL-2R antibodies in patients with high immunological risk. These recent data raise questions about the need for IL-2R antibodies in kidney transplantation, as it may no longer be beneficial in standard-risk transplantation. Although augmentation of immunosuppression by IL-2R antibody induction may allow steroid minimization, it may be inferior to rabbit ATG in high-risk situations. Updated evidence-based guidelines are necessary to support clinicians deciding whether and what induction therapy is required for their transplant patients today [33]. Studies in the US and Europe have investigated the potential of IL-2R antibodies or thymoglobulin in replacement of steroids with promising results (see section in this chapter on “Steroid Withdrawal or Avoidance”). Another potential application is delayed graft function when the use of CNIs should be avoided.

Maintenance Immunosuppressive Therapy

Maintenance immunosuppressive therapy is administered to kidney transplant recipients for prevention of acute rejection. Although an adequate level of immunosuppression is required to dampen the immune response to the allograft, the level of chronic immunosuppression is slowly decreased over time to help lower the overall risk of infection and malignancy. The type of immunosuppression may also be modified to decrease the risk of developing chronic antibody-mediated rejection, the most common underlying long-term cause of allograft loss.

Conventional maintenance regimens consist of a combination of immunosuppressive agents that differ in their mechanism of action. This strategy minimizes morbidity and mortality associated with each class of agent while maximizing overall effectiveness. Such regimens vary by transplant center and geographic area. There are a number of important issues to consider when deciding upon the immunosuppressive protocol to administer in a particular patient. First, the risk of acute rejection and allograft loss is highest in the first 3 months post-transplant. As a result, immunosuppression should be at its highest during this period (see section in this chapter on “Induction Immunosuppressive Therapy”). Second, the occurrence of the most serious side effects of immunosuppressive therapy, infections and malignancy, correlate with the total amount of immunosuppression. It is therefore essential that immunosuppression is gradually tapered to a maintenance level by 6–12 months post-transplant.

Allograft survival rates vary among the various immunosuppressive agents due to patient-specific clinical characteristics and co-morbidities, such as age, ethnicity, obesity, hyperlipidemia, hypertension, proteinuria, and/or delayed allograft function. The choice of immunosuppression should consider these factors, but also take into account the “immunologic” history of the patient. Transplant physicians must reflect the following questions: Is the patient sensitized? Is this the first kidney transplant or a re-transplant? How many acute rejection episodes has the patient had? What is the degree of HLA matching?

The optimal maintenance immunosuppressive therapy in pediatric kidney transplantation is not established. The major immunosuppressive agents currently used in various combination regimens are TAC, CSA (in standard form or microemulsion), MMF, EVR, SRL, AZA and steroids (primarily oral prednisone or methylprednisolone). We and most transplant centers currently utilize a maintenance regimen consisting of triple immunosuppression therapy with a CNI (TAC or CSA), an anti-metabolite (MMF or AZA), and methylprednisolone. EVR or SRL are also used by some transplant centers in triple therapy regimens, often in place of the CNI or the antimetabolite. Within the North American Pediatric Renal Trials and Collaborative Studies (NAPRTCS) registry, marked changes in the type of maintenance immunosuppression and dosing strategies have been observed over time [34]. These are substantially caused by the introduction of newer drugs such as MMF and TAC. In the transplant era 2008–2017, the most popular immunosuppressive regimen at post-transplant day 30, utilized in 48.9% of patients, was triple therapy with TAC, MMF and prednisone (Table 67.1).

Table 67.1 Observed drug utilization rates in North American pediatric renal transplant recipients among transplanted grafts with ≥30 days function that have occurred since 1996. (Data are from [34])

Glucocorticoids

Glucocorticoids, developed in the early 1950s, represent one of the principal agents used for both maintenance immunosuppression and treatment of acute rejection.

Mechanism of Action

Steroids have both anti-inflammatory and immunosuppressive actions [35]. Lymphopenia and monocytopenia occur with the inhibition of lymphocyte proliferation, survival, activation, homing, and effector functions. Steroids suppress production of MHC molecules and numerous cytokines and vasoactive substances, including IL-1, TNFα, IL-2, chemokines, prostaglandins (via inhibition of phospholipase A2), and proteases. Steroids also cause neutrophilia (often with a left shift), but neutrophil chemotaxis and adhesion are inhibited. They also affect non-hematopoietic cells.

Steroids exert their effect by binding to glucocorticoid receptors (GR), which belong to a family of ligand-regulated transcription factors called nuclear receptors. GR are normally present in the cytoplasm in an inactive complex with heat shock proteins (hsp90, hsp70, and hsp56). The binding of steroids to the GR dissociates hsp from the GR and forms the active steroid–GR complex, which migrates to the nucleus and dimerizes on palindromic DNA sequences, called the glucocorticoid response element (GRE), in many genes. The binding of GR in the promoter region of the target genes can lead to either induction or suppression of gene transcription (e.g., of cytokines). GR also exert effects by interacting directly with other transcription factors independent of DNA binding. One principal effect of steroids on immune and inflammatory responses may be attributable to their ability to affect gene transcription by regulating key transcription factors involved in immune regulation: activator protein-1 (AP-1) and NF-κB. The regulation of NF-κB by steroids may be via induction of IkB, the inhibitor of NF-κB. Other effects of steroids are mediated through the release of a regulatory protein, lipocortin, which inhibits phospholipase A2, thereby inhibiting the production of leukotrienes and prostaglandins. The total immunosuppressive effect of steroids is complex, reflecting effects on cytokines, adhesion molecules, apoptosis, and activation of inflammatory cells.

Pharmacokinetics and Drug Interactions

The major steroids used are prednisone or prednisolone (given orally with comparable efficacy) and methylprednisolone (given orally or intravenously with 25% more potency). These agents are rapidly absorbed and have short plasma half-lives (60–180 min), but long biological half-lives (18–36 h). The effect of prednisone (dose per body weight) is greater in the setting of renal failure or hypalbuminemia, in women, and in the elderly, but less prednisone effect is observed in children. Certain drugs can decrease steroid efficacy by increasing metabolism: rifampicin, phenytoin, phenobarbital, and carbamazepine. In contrast, increased steroid effects may be observed in patients receiving oral contraceptives, estrogens, ketoconazole, and erythromycin.

Administration

In many transplant centers, the initial dose of steroids is usually administered during surgery as intravenous methylprednisolone, at doses between 2 and 10 mg/kg body weight. The oral dose of steroids used for maintenance therapy varies between 15 and 60 mg/m² per day (0.5–2 mg/kg body weight per day), which is gradually tapered over time to approximately 3–5 mg prednisone per m² body surface area, usually taken as a single morning dose. Alternate-day dosing is often administered 6–12 months post-transplant to minimize the effect of steroids on growth.

Side Effects

Steroids have multiple side effects in children, including growth impairment, susceptibility to infections, cushingoid appearance, body disfigurement, acne, cardiovascular complications, hypertension, hyperglycemia, aseptic bone necrosis, osteopenia, cataracts, poor wound healing, and psychological effects (Table 67.2). The negative impact that steroids have on appearance may play a role for poor adherence, especially in the body image conscious adolescent. The risk for infection is excessive if high-dose pulse therapy is prolonged (typically >3 g per 1.73 m²). Steroids dosage should therefore be decreased gradually during rejection treatment even if serum creatinine fails to improve. Interestingly, steroids are not associated with an increased risk of malignancy. One of the most important reasons for stopping steroids or switching to alternate-day therapy is statural growth impairment, which is frequently observed in those on continuous treatment.

Table 67.2 Semi-quantitative comparison of safety profiles of current primary immunosuppressive compounds

Steroid Withdrawal or Avoidance

Because of the multiple adverse effects of maintenance steroid therapy, attempts have been made to withdraw or minimize steroid therapy in children with a renal allograft [36,37,38,39,40]. There are two major strategies in steroid minimization in pediatric kidney transplantation: (i) Late steroid withdrawal (>1 year post-transplantation) and (ii) either complete steroid avoidance or early steroid withdrawal (<7 days post-transplantation). In the late steroid withdrawal approach, the patients suitable for minimization are identified by a stable post-transplant clinical course and renal function. In late steroid withdrawal, there is no need for an antibody induction in the perioperative period [41]. In early withdrawal or complete avoidance protocols, the criteria of suitability are predefined before transplantation (e.g., low immunological risk), and antibody induction is used in all patients [42,43,44,45,46,47]. There is also an intermediate approach, combining elements from early and late withdrawal protocols, in which antibody induction is used; however, the decision of steroid withdrawal is delayed until 6–9 months post-transplant, when stable renal graft function (sometimes combined with a normal protocol biopsy) allows identification of suitable candidates (as in the late withdrawal approach) [48]. Steroid withdrawal has the advantage over steroid avoidance that immunologically high-risk patients and those with unstable graft function can easily be identified beforehand and be excluded from steroid-free immunosuppression.

Late steroid withdrawal without induction therapy was investigated in a trial of 42 patients with low immunologic risk who were maintained on CSA, MMF, and steroids. At 1 year post-transplant, patients were randomly assigned to either steroid continuation or withdrawal over a 3-month period [40]. At 1 year follow-up, the steroid withdrawal group had increased catch-up growth, lower arterial blood pressure, and a better carbohydrate and lipid profile than those on continuous steroid therapy. In a subsequent follow-up report, longitudinal growth in the steroid withdrawal group continued to be superior to controls; catch-up growth was especially pronounced in pre-pubertal patients off steroids (Fig. 67.6) [41]. Although the relative height gain after steroid withdrawal was less pronounced in pubertal patients, they still benefited from cessation of steroid treatment. Also, the prevalence of the metabolic syndrome declined in the withdrawal group from 39% at baseline to 6% 2 years after discontinuing steroid therapy [41]. Allograft function remained stable in the withdrawal group compared with controls, and the incidence of acute rejections was similar in the steroid withdrawal and control groups (4% vs. 11%, respectively). An earlier retrospective case–control study had reported a beneficial effect of late steroid withdrawal (mean time 1.5 ± 1.3 years post-transplant) on growth in similarly treated (pre-pubertal) children. The patients in this study who had steroids withdrawn also had better blood pressure control with a lower requirement for antihypertensive therapy [49].

Fig. 67.6

Randomized controlled trial on late steroid withdrawal: Change of height SDS (mean ± SEM) in the steroid withdrawal group (filled circles) and the control group (open triangles) during the 27-month study period. Panel A, all patients; panel B, pre-pubertal patients; panel C, pubertal patients. ∗P < 0.05 vs. control; ∗∗P < 0.01 vs. control; ∗∗∗P < 0.001 vs. control; #P < 0.05 vs. baseline; ##P < 0.01 vs. baseline. (Used with permission of Oxford University Press from Höcker B, Weber L, Feneberg R, Drube J, John U, Fehrenbach H, et al. Improved growth and cardiovascular risk after late steroid withdrawal: 2-year results of a prospective, randomized trial in paediatric renal transplantation. Nephrol Dial Transplant 2010; 25:617–624) [41]

More recently, a single-center study has reported on efficacy and safety of a different regimen combining the intermediate withdrawal of steroids (>6 months and <1 year) with minimization of exposure to CSA. The protocol initially included a 2-dose basiliximab induction, standard exposure to CSA (C₀ 150–200 μg/L) and prednisolone. This was followed by adding EVR at 2 weeks (C₀ 4–6 μg/L), reducing CSA exposure by half (C₀ 75–100 μg/L), and then steroid withdrawal at 9 months in patients with a normal protocol biopsy. Results of the open-label uncontrolled trial have been promising, without acute rejection and a 100% 3-year graft survival [48, 50,51,52], while another study on intermediate steroid withdrawal (2 doses of basiliximab and sequential replacement of tapered steroids with MMF at 6 months post-transplant under protocol biopsy) reported an acute rejection rate of 13% [53]. Another intermediate steroid withdrawal protocol was used in a US multicenter trial, during which patients received 2 doses of basiliximab, combined with SRL, TAC or CSA, and steroids. After 6 months and a protocol biopsy without signs of rejection, patients were randomized to withdraw or maintain steroids [37]. It should be noted that during the first phase of the trial (prior to randomization) 6.9% of the patients developed PTLD. This was mainly seen in young EBV-naïve children, receiving an EBV-seropositive renal allograft; however, this complication should not be regarded as directly related to steroid minimization but rather to initial over-immunosuppression [53].

Early steroid withdrawal or steroid avoidance may eventually be found to provide the best overall risk-to-benefit ratio with maintenance immunosuppressive therapy in renal transplantation. Early steroid withdrawal or steroid avoidance protocols have been used successfully and have undergone extensive evaluation both in the US and in Europe. However, many of these protocols have chosen low-risk individuals and utilized intensive induction therapy with extended daclizumab induction therapy or thymoglobulin, TAC, and MMF [42]. A randomized, controlled study in 196 pediatric kidney transplant recipients investigated the efficacy and safety of early steroid withdrawal. Two doses of daclizumab in patients treated with a regimen of TAC and MMF allowed early steroid withdrawal on day 5 post-transplant [45]. There was a comparable rate of biopsy-proven acute rejection after 6 months in patients off steroids compared with controls (10.2% vs. 7.1%). In addition, pre-pubertal patients with early steroid withdrawal showed better growth and lipid and glucose metabolism profiles compared to controls, without increases in graft loss. These favorable effects were confirmed in a follow-up study over a 2-year observation period [54]. The steroid avoidance strategy was examined in a North American, randomized, controlled, multicenter study [55]. One hundred thirty children receiving primary kidney transplants were randomized to steroid-free or steroid-based immunosuppression, with concomitant TAC, MMF and standard dose daclizumab (steroid-based group) or extended dose daclizumab (steroid-free group). Follow-up was 3 years post-transplant. Recipients under 5 years of age showed improved linear growth under a steroid-free regimen compared to controls on steroids. No differences in the rates of biopsy-proven acute rejection were observed at 3 years post-transplant (16.7% in steroid-free vs.17.1% in steroid-based; P = 0.94). Patient survival was 100% in both arms; graft survival was 95% in the steroid-free and 90% in the steroid-based arms (P = 0.30) at 3 years follow-up. Over the 3-year follow-up period, the steroid-free group had lower systolic blood pressure (P = 0.017) and cholesterol levels (P = 0.034) (Fig. 67.7). The authors concluded that complete steroid avoidance is safe and effective in unsensitized children receiving primary kidney transplants [55].

Fig. 67.7

North American randomized controlled multicenter study on steroid avoidance: Estimated group mean standardized change in growth (Z-score) among infants and young children (a), mean eGFR level (by Schwartz method) (b), mean diastolic (c) and systolic (d) blood pressure levels and serum cholesterol (e) and triglyceride (f) levels from transplantation up to 3 years. (Used with permission of John Wiley and Sons from Sarwal MM, Ettenger RB, Dharnidharka V, Benfield M, Mathias R, Portale A, et al. Complete corticosteroid avoidance is effective and safe in children with renal transplants: a multicentre randomized trial with three years follow-up. Am J Transpl 2012; 12: 2719–29) [55]

Despite these encouraging results, steroid withdrawal or avoidance following kidney transplantation remains a controversial issue. Although the benefits of using steroid-free protocols in pediatric patients are obvious, they may increase risk in patients with certain immunological constellations. Further studies need to identify new monitoring tools to assess the immunologic safety to allow successful and safe conversion of patients to steroid-free immunosuppression at later times after transplantation, even after having initially started on a steroid-based maintenance immunosuppression protocol.

Recurrent Kidney Disease

Because recurrent disease is the fourth most common cause of graft loss in children undergoing kidney transplantation, there have been concerns that steroid withdrawal may be associated with an increased risk of graft loss. Although information is limited, a study demonstrated no difference in graft survival due to recurrent disease in children (4–18 years of age) treated with a rapid prednisone discontinuation protocol compared with historical controls who received steroid therapy [56]. However, more data are needed to ensure there is not an untoward increased risk of recurrent disease, for example lupus erythematosus-associated nephritis, following steroid withdrawal.

Calcineurin Inhibitors

CSA, a lipophilic cyclic peptide of 11 amino acid residues, and TAC, a macrolide antibiotic, are drugs with similar mechanisms of action that have become major maintenance immunosuppressive agents used in transplantation.

Mechanism of Action

CSA and TAC act by inhibiting the calcium-dependent serine phosphatase calcineurin, which normally is rate-limiting in T cell activation (Fig. 67.8). Calcineurin is activated by the engagement of the T cell receptor, followed by activation of tyrosine kinases and phospholipase C-γ1, release of inositol triphosphate, release of calcium stored in the endoplasmic reticulum, and opening of membrane calcium channels. Calcineurin provides an essential step for transducing signal 1 to permit cytokine and CD40L transcription. A high cytoplasmic calcium concentration activates calcineurin, which then dephosphorylates regulatory sites in key transcription factors, the ‘nuclear factors of activated T lymphocytes’ (NFAT_p and NFAT_c). This causes the NFAT proteins to translocate (with calcineurin) into the nucleus and bind to their DNA target sequences in the promoters of cytokine genes. Calcineurin has been implicated in the dephosphorylation of transcription factor Elk-1, and indirectly in the activation of Jun/AP-1 and NF-κB.

Fig. 67.8

Calcineurin inhibition. During normal T cell activation, calcium release activates calcineurin’s phosphatase activity, causing dephosphorylation of the transcription factor nuclear factor of activated T cells (NF-AT) and subsequent translocation to the nucleus. Ciclosporin and tacrolimus form a complex with immunophilins (cyclophilin or FK-binding protein 12, respectively), which bind calcineurin and sterically inhibit the phosphatase activity, preventing dephosphorylation and nuclear translocation of NF-AT. (Used with permission from Floege J, Johnson RJ, Feehally J, (eds.): Comprehensive Clinical Nephrology, 4th ed. St. Louis: Elsevier, 2010)

CSA and TAC cross cell membranes freely and bind to immunophilins (cyclophilin and FK-binding protein 12 [FKBP12], respectively), which are ubiquitous and abundant intracellular proteins with isomerase activity. The active complex then binds to a site on calcineurin and blocks its interactions with key substrates. The inactivation of calcineurin bound to CSA–cyclophilin or TAC–FKBP12 is the key to the immunosuppressive effect and some of the toxic effects of these drugs. While inhibition of calcineurin has many effects on the T cell, the best studied is the blocking of the translocation of the NFAT proteins from the cytoplasm into the nucleus.

CSA and TAC partially inhibit the calcineurin pathway at therapeutic blood levels (e.g., trough levels of 200 μg/L CSA or 5–20 μg/L TAC) [57]. However, even partial inhibition of calcineurin reduces the transcription of many genes associated with T cell activation (e.g., IL-2, IFN-γ, granulocyte–macrophage colony-stimulating factor (GM-CSF), TNF-α, IL-4, CD40L). Therefore, the functional consequence of partial calcineurin inhibition is probably a quantitative limitation in cytokine production, CD40L expression, and lymphocyte proliferation. The effect of CSA and TAC on calcineurin in vivo is rapidly reversible, emphasizing the importance of patient compliance, drug monitoring, and reliable formulations for delivery. The effects on non-T cells could also be clinically significant.

Pharmacokinetics and Drug Interactions

CSA and TAC are both variably absorbed and are metabolized extensively by the liver via the cytochrome P450 system. CSA is excreted primarily by the biliary system. The absorption of some CSA preparations may be bile-dependent, and therefore may be reduced in the presence of cholestatic liver disease. The absorption of the microemulsion formulations of CSA or TAC is bile-independent. Neither CSA nor TAC pharmacokinetics are affected by alterations in renal function. Both CSA and TAC bind to cells and to plasma components (primarily lipoproteins for CSA and albumin for TAC) in the blood; consequently, they must be assayed in whole-blood. Many drugs and agents can affect CSA and TAC levels through effects on their absorption or metabolism (see Table 67.3).

Table 67.3 Examples of common drug interactions of immunosuppressants used in solid-organ transplantation: ciclosporin, tacrolimus, sirolimus, and everolimus

Since the absorption of CSA is decreased and its metabolism increased in children compared to adults, relatively higher dosages are required in pediatric patients. CSA is usually administered initially as 8–15 mg/kg daily in divided doses (or intravenously using one third the oral dose over a 24-h period) during the induction phase, with target trough blood levels of 150–300 μg/L for the first 3–6 months post-transplant. Doses are reduced after 3–6 months (typically 4–6 mg/kg daily); long-term target trough blood levels of 75–125 μg/L appear to provide comparable patient and graft survival as higher blood levels, but with less risk of malignancy [58]. A microemulsion form of CSA (Neoral™, Novartis, East Hanover, NJ) gives more reliable and slightly higher absorption, and may allow a slightly lower dose. Generic forms of CSA are available, but oral formulations of CSA may not be equivalent and readily interchangeable. Knowledge of the characteristics of the oral formulations is necessary before switching between them.

TAC is 20- to 30-fold more potent than CSA, and is therefore administered at a 20-fold lower dose. Initial dosing is usually 0.2–0.3 mg/kg daily in two divided doses orally (or 0.05–0.1 mg/kg daily intravenously over 24 h), and target trough levels are 5–15 μg/L. Since TAC is more water-soluble than CSA, it is not as dependent upon bile salts for absorption. However, food intake can reduce the absorption of TAC by up to 40 percent; thus, it is recommended that this agent be taken on an empty stomach [59]. In addition, TAC is best absorbed in the morning. There is also an extended release formulation of TAC available that is designed to be given once per day with similar efficacy and safety.

Genetic polymorphisms in genes encoding TAC-metabolizing enzymes partly explain the inter-patient variability in TAC pharmacokinetics [60]. The key enzymes involved in the metabolism of TAC are CYP3A4 and CYP3A5 [61]. Individuals are considered expressers of CYP3A5 if they carry at least one CYP3A5*1 allele, whereas individuals homozygous for the CYP3A5*3 allele are classified as CYP3A5 non-expressers. In addition to CYP3A5*3, the CYP3A5*6 and CYP3A5*7 variant alleles can also lead to non-functional CYP3A5 protein [62]. There are ethnic distribution differences of CYP3A5 variant alleles, with expressers (carriers of the CYP3A5*1 variant allele) being more frequently found among non-Caucasian populations. Approximately 10–40% of Caucasians are CYP3A5 expressers, 33% of Asians and 55% of African-Americans [63]. CYP3A5 expressers require a TAC dose that is approximately 1.5–2-fold higher than non-expressers to reach the same exposure [64, 65]. This implies that, following a standard, bodyweight-based TAC dose, CYP3A5 expressers are prone to have sub-therapeutic TAC concentrations whereas non-expressers are expected to have supra-therapeutic TAC concentrations [66].

Efficacy: Comparison of Ciclosporin and Tacrolimus

To help assess the relative efficacy of TAC and CSA, a 2005 meta-analysis and meta-regression was performed based upon 30 trials consisting of 4102 adult patients [67]. Despite a certain variability between studies, the overall conclusion from data in adults is that TAC-based immunosuppression is associated with decreased acute rejection rates, superior long-term renal function and more favorable cardiovascular risk profile than CSA-based immunosuppression, which translates into improved long-term renal allograft survival. This statement is supported by the results of the SYMPHONY trial, which compared standard immunosuppression vs. 3 regimens with low-dose or no CNI in de novo single-organ renal transplant patients over 1 year [68]. In this prospective, randomized, open-label study with 4 parallel arms, 1645 adult patients in 15 countries were randomized to standard immunosuppression with normal-dose CSA (target trough level 150-300 μg/L for 3 months, 100-200 μg/L thereafter), MMF (1 g bid) and steroids, or to one of three regimens consisting of daclizumab induction, MMF (1 g bid) and steroids potentiated by a low-dose of either CSA (50–100 μg/L), TAC (3-7 μg/L) or SRL (4-8 μg/L). The low-dose TAC group was significantly superior to all other groups with respect to glomerular filtration rate (GFR) and biopsy-proven acute rejection (p < 0.01) and to normal-dose CSA and low-dose SRL for graft survival (pair-wise p < 0.05). The authors concluded that immunosuppression consisting of daclizumab induction, MMF, low-dose TAC and corticosteroids provides the most optimal balance between efficacy (control of acute rejection) and toxicity (preserving graft function and graft survival) [68].

In pediatric patients, the efficacy and safety of TAC and CSA were compared in one multicenter trial in 196 patients, who were randomly assigned to receive either TAC or CSA microemulsion administered concomitantly with AZA and steroids [69]. TAC therapy resulted in a significantly lower incidence of acute rejection (36.9% vs. 5.91% with CSA therapy (59.1%); P = 0.003) and of steroid-resistant rejection (7.8% vs. 25.8%, P = 0.001) compared with the CSA group. The difference was also significant for biopsy-confirmed acute rejection (16.5% vs. 39.8%, P < 0.001). At 1 year post-transplant, patient survival was similar (96.1% vs. 96.6%); ten grafts were lost in the TAC group compared with 17 in the CSA group (P = 0.06). The TAC group had a significantly better eGFR. A follow-up study at 4 years showed that patient survival was similar (94% vs. 92%, P = 0.86), but graft survival significantly favored TAC (86% vs. 69%; P = 0.025) [70]. The mean eGFR was superior in TAC-treated patients vs. those on CSA (Fig. 67.9). Cholesterol remained significantly higher with CSA throughout follow-up. Three patients in each arm developed PTLD. Incidence of insulin-dependent diabetes mellitus did not differ. From these studies, the authors concluded that TAC was significantly more effective than CSA in preventing acute rejection and preserving renal function in pediatric renal recipients.

Fig. 67.9

Mean glomerular filtration rate (±1 SD) over 4 years post-transplant. Multicenter trial in 196 pediatric patients, who were randomly assigned to receive either tacrolimus or ciclosporin administered concomitantly with azathioprine and corticosteroids. (Used with permission of John Wiley and Sons from Filler G, Webb NJ, Milford DV, Watson AR, Gellermann J, Tyden G, et al. Four-year data after pediatric renal transplantation: a randomized trial of tacrolimus vs. cyclosporin microemulsion. Pediatr Transplant. 2005; 9:498–503) [70]

A retrospective study of the NAPRTCS database of 986 pediatric renal transplant recipients who were treated either with CSA, MMF and steroids (n = 766) or TAC, MMF and steroids (n = 220) revealed that TAC and CSA, in combination with MMF and steroids, produce similar rejection rates and graft survival in pediatric renal transplant recipients [71]. However, TAC was associated with improved graft function at 1 and 2 years post-transplant (Fig. 67.10). There was no difference in time to first rejection, as well as in risk of rejection or graft failure at 1 or 2 years post-transplant. TAC-treated patients were significantly less likely to require antihypertensive medication at 1 and 2 years post-transplant. TAC-treated patients had a higher mean eGFR at both 1 year (99 vs. 78 mL/min/1.73 m², P = 0.0003) and 2 years post-transplant (97 vs. 73 mL/min/1.73 m², P < 0.0001). Hence, there is evidence that TAC is superior to CSA (conventional or microemulsion form) in preventing acute rejection after kidney transplantation in adult and pediatric populations. It also seems more effective in improving long-term graft survival in adults.

Fig. 67.10

(a) Kaplan–Meier estimates of the percentage of patients experiencing a first acute rejection in the first 2 years post-transplant, by treatment group: tacrolimus (TAC), mycophenolate mofetil (MMF) and steroids vs. ciclosporin (CSA), MMF and steroids and donor source. Patients were included in this analysis if they were transplanted between 1997 and 1999 and had a 2-year follow-up in the database. (b) Mean eGFR as calculated by the Schwartz formula at 1 and 2 year post-transplant in patients treated with TAC, MMF and steroids or CSA, MMF and prednisone. (Used with permission of John Wiley and Sons from Neu AM, Ho PL, Fine RN, Furth SL, Fivush BA. Tacrolimus vs. cyclosporine A as primary immunosuppression in pediatric renal transplantation: a NAPRTCS Study. Pediatr Transplant. 2003; 7:217–22) [71]

Side Effects

CSA and TAC have similarities and differences in their toxicity profiles (Table 67.2). Both can cause nephrotoxicity, hyperkalemia, hyperuricemia with occasional gouty attacks, hypomagnesemia secondary to urinary loss, hypertension, diabetes mellitus, and neurotoxicity, especially tremor. In the European pediatric study, the incidence of hypomagnesaemia was significantly higher in the TAC-treated group (34%) compared with the CSA-treated group (12.9%) [69]. Similarly, diarrhea was more frequent in TAC-treated patients (13.6% vs. 3.2%). Hypertrichosis, gum hyperplasia and flu syndrome were reported only in CSA-treated patients, and tremor was reported only in TAC-treated patients [69]. These results are similar to that found in adults, where tremor is consistently more common with TAC, and hirsutism and gum disease more common with CSA [72]. Also, hypertension and hyperlipidemia are more commonly observed with CSA. Interestingly, higher CSA doses are more likely to induce higher blood pressure in older girls compared to boys [73]. In the NAPRTCS study, during which CNIs were used in combination with MMF and steroids, TAC-treated patients were significantly less likely to require antihypertensive medications at 1 and 2 years post-transplant [71]. This is similar to adults, where a lower systemic blood pressure was reported in TAC-treated patients in several studies [72]. In the European pediatric study, the mean total cholesterol levels were reported to decline in the TAC group and increase in the CSA group at the end of 6 months [69]. In the multicenter analysis from the European CERTAIN Registry, the prevalence of dyslipidemia was 95% before transplantation, and 88% at 1 year post-transplant [74]; the use of TAC and MMF was associated with significantly lower concentrations of all lipid parameters compared to regimens containing CSA and mTOR inhibitors. Regimens consisting of CSA, MPA, and corticosteroids as well as of CSA, mTOR inhibitors, and steroids were associated with a 3- and 25-fold increased risk of having more than one pathologic lipid parameter as compared to the use of TAC, MMF, and steroids [74]. Similarly, several studies in adults have shown remarkably lower lipid levels in TAC-treated patients than in those receiving CSA [72]. The improved lipid profiles on TAC may contribute to a better long-term outcome with less cardiovascular morbidity in adult patients.

On the other hand, tremor and glucose intolerance are more common with TAC. In the pediatric multicenter European study, there was no difference in the incidence of new onset insulin-dependent diabetes mellitus between TAC- (3%) and CSA-treated patients (2.2%) [69, 70]. Although in early clinical trials of TAC, a significantly higher incidence of diabetes mellitus was reported in TAC-treated adult patients than in recipients on CSA, the incidence of diabetes mellitus under TAC immunosuppression has become less frequent in recent randomized trials comparing these two CNIs [75]. Post-transplant diabetes regresses after dose reduction in some but not all patients. Both the reduction of steroid dosage and low target trough TAC concentrations contribute to this marked reduction of the incidence of diabetes mellitus under TAC immunosuppression in both adults and children [69, 72]. CSA may also be associated with coarsening of facial features, especially in children. Bone pain that is responsive to calcium-channel blockers may also occur with CSA use and sometimes may require changing to TAC.

The most common serious problem with CNIs is nephrotoxicity, with both a reversible vasomotor component and an irreversible component. Both CSA and TAC can cause acute elevations in serum creatinine that reverse with reduction of the dose, apparently caused by renal vasoconstriction, which may be mediated by calcineurin inhibition. Chronically, CSA and TAC can induce interstitial fibrosis and tubular atrophy with characteristic hyalinosis of the afferent arteriole [76]. This lesion appears to result from long-standing renal vasoconstriction, perhaps mediated in part by an increase in local vasoconstrictor tone (increased angiotensin II, endothelin-1, thromboxane, and sympathetic nerve transmitters) and an inadequate vasodilatory response (impaired nitric oxide formation). The importance of this lesion is apparent from studies in cardiac and liver transplant recipients, in whom CSA or TAC use is associated with chronic kidney disease progressing to end-stage kidney disease (ESKD) in a significant fraction of patients [77]. This problem was more relevant at a time when higher doses of CSA were administered for longer periods. Fortunately, currently, CSA and TAC toxicity is associated with only mild to moderate declines in renal function. However, as the number of patients with long-standing non-renal transplants rises, there is increasing concern about future ESKD in this population. In these cases, it is important to establish the diagnosis of CNI toxicity by renal biopsy and reduce or stop calcineurin inhibition whenever possible [75, 78, 79]. Experimentally, CSA nephropathy is exacerbated in the presence of salt restriction/volume depletion and is lessened by treatment with angiotensin-converting enzyme (ACE) inhibitors, calcium-channel blockers, vasodilators (hydralazine), and steroids.

CSA and TAC treatment can cause hemolytic uremic syndrome, probably through endothelial dysfunction. This complication, which is usually associated with elevated drug levels, may respond to temporary withdrawal of CSA or TAC, plasma exchange, switching to another CNI, or conversion to another immunosuppressive drug class.

There is no difference in the incidence of PTLD between TAC- and CSA-treated recipients when used in combination with AZA/steroids [1% (1/103) vs. 2.1% (2/93)] [69] or when given in conjunction with MMF/steroids (1.4% vs. 2%) [71]. This is similar to adults, in whom recent large, randomized studies showed no differences in the incidence of malignancy between patients treated with TAC or CSA [72].

Therapeutic Drug Monitoring

CSA is a drug with a narrow therapeutic index and broad intra-individual and inter-individual pharmacokinetic variability. Serious clinical consequences may occur because of underdosing or overdosing. Hence, individualization of CSA dosage by therapeutic drug monitoring is required. The traditional monitoring strategy for CSA is based on pre-dose trough level measurements (C₀). However, C₀ shows a relatively poor correlation with CSA exposure (area under the concentration-time curve [AUC]) and with clinical outcome [80]. Studies on the pharmacokinetic and pharmacodynamic relationship of CSA have shown that CSA induces a partial inhibition of calcineurin activity, the rate-limiting step in the activation of primary human T lymphocytes and the target of the CSA/cyclophilin complex [81]. The greatest calcineurin inhibition and the maximum inhibition of IL-2 production occur in the first 1–2 h after dosing. Calcineurin is only partially inhibited in patients, which can result in rejection even when CSA blood concentrations are in the putative therapeutic range. From these observations, it was hypothesized that the CSA AUC_0–4(absorption profile) or the C₂concentration (sample 2 h after oral intake) may be a better predictor of immunosuppressive efficacy than the CSA AUC_0–12. However, a prospective, randomized study in adult renal transplant recipients did not show any advantage of C₂ monitoring strategy in the early post-transplant period compared to a C₀ monitoring strategy, but led to significantly higher CSA doses and blood levels than C₀ monitoring [82]. In a large study in pediatric renal transplant recipients, CSA absorption profiles predicted the risk of acute rejection, while the single pharmacokinetic parameters C₀ or C₂ did not [83]. A disadvantage of C₂ monitoring is the fact that it requires a timed blood sample within a narrow time window (±15 min) that necessitates further organizational requirements for physicians and nursing staff, which may be judged differently between transplant centers. In our center, we routinely monitor CSA therapy by 12-h pre-dose trough concentrations. We aim for the following trough levels in combination with MMF therapy and prednisone: 120–200 μg/L during months 0–3 post-transplant and 80–160 μg/L thereafter. We feel that a monitoring strategy based on CSA C₂ concentrations in the stable period post-transplant is an additional tool in preventing chronic CSA-induced nephrotoxicity. In patients with low or normal immunological risk, who are on additional maintenance therapy with MMF, we aim for CSA C₂ concentrations between 300–600 μg/L beyond the first year post-transplant; C₂ concentrations are monitored every 3–6 months.

When TAC is utilized, a monitoring strategy based on trough levels is in general sufficient because trough levels are good indicators of systemic exposure. In most transplant centers, doses are adjusted to attain target whole-blood trough concentrations of 8–12 μg/L during the first 3 months post-transplant, between 5 and 10 μg/L during months 4–12, and 4–8 μg/L thereafter [84]. It must be emphasized that these target ranges are dependent on the concomitant immunosuppressive therapy. In the SYMPHONY trial, for example, low tacrolimus exposure (trough levels between 3 and 7 μg/L) in the first year post-transplant, in conjunction with MMF, prednisone and daclizumab induction, was associated with excellent efficacy and little TAC-associated toxicity [68]. However, although TAC trough level goals in the low-dose TAC group of the Symphony study were protocol specified at 3–7 μg/L, the achieved levels were 6.4 and 6.5 μg/L at 12 and 36 months post-transplant, respectively. Hence, a more appropriate interpretation of the SYMPHONY trial is that, in combination with MMF, TAC trough level goals of 5–8 μg/L should be considered as standard of care in adult patients. In addition, recent studies in adult kidney allograft recipients lend support to maintain TAC trough levels above 5 μg/L in order to reduce the risk of de novo donor-specific antibodies [85].

Recent registry data indicate that that a more consistent and less variable exposure to the main immunosuppressant TAC later than 1 year post-transplant is associated with a better 5-year graft survival, especially in adolescents and young adults (Fig. 67.11) [86]. In adolescent and young adult patients, the risk of premature graft loss associated with a low 1-year TAC trough level <4.0 ng/mL was 2.38-fold higher compared to a trough level of 4.0–10.9 ng/mL, whereas the risk was not significantly increased in recipients aged 0–11 years. In 24–34-year-old adult patients, the risk of premature graft loss due to a low 1-year TAC trough level <4.0 ng/mL was 1.94-fold increased, but still lower than in adolescent and young adult patients. Importantly, trough levels in the range of 4.0–10.9 ng/mL resulted in a good 5-year graft survival of 85% in the group of 12–23-year-old recipients, comparable to the 88% survival rate observed in 24–34-year-old adult patients (Fig. 67.11). Hence, it appears that optimal TAC exposure can at least partially counteract the enhanced immunoreactivity in this high-risk age group.

Fig. 67.11

Association of 1-year tacrolimus trough level (ng/mL) with graft survival during post-transplant years 2–5 for the age groups (a) 0–11, (b) 12–23, and (c) 24–34 years at the time of transplantation. Log rank P values of Kaplan–Meier analysis are shown. (Reproduced from [86], with permission)

Antimetabolic Agents: Azathioprine

AZA, developed by Nobel Prize laureates Elion and Hitchings in the1950s, has been widely used in renal transplantation for 4 decades. AZA is a purine analog derived from 6-mercaptopurine (6-MP).

Mechanism of Action

AZA is metabolized in the liver to 6-MP and further converted to the active metabolite thioinosinic acid (TIMP) by the enzyme hypoxanthine–guanine phosphoribosyltransferase. Some but not all of the immunosuppressive activity of AZA is attributable to 6-MP. AZA acts mainly as an anti-proliferative agent by interfering with normal purine pathways, by inhibiting DNA synthesis, and by being incorporated into DNA, thereby affecting the synthesis of DNA and RNA [64]. By inhibiting the synthesis of DNA and RNA, AZA suppresses the proliferation of activated B and T lymphocytes. In addition, AZA has been shown to reduce the number of circulating monocytes by arresting the cell cycle of promyelocytes in the bone marrow. The anti-proliferative action of AZA probably explains much of its observed effects on the immune system and its toxicity. AZA shows some selectivity in its effects with certain cell types and different kinds of immune reactions [87]. For instance, it has been shown that primary immune responses are more susceptible to AZA than secondary responses despite the fact that there is a more rapid proliferation of lymphocytes during a secondary response.

Pharmacokinetics and Drug Interactions

AZA is administered orally at 1.5 mg/kg per day in conjunction with CNIs and 2.5 mg/kg per day when used without CNIs. Higher initial doses (5 mg/kg per day) combined with monitoring of 6-thioguanine nucleotide levels in red blood cells are associated with an approximately 20 percent reduction in the acute rejection rate as compared to lower doses [88]. It is metabolized in the liver to 6-MP and further converted to the active metabolite TIMP. Because 6-MP is degraded by xanthine oxidase, allopurinol, a xanthine oxidase inhibitor, will increase the levels of TIMP. Severe leukopenia can occur if allopurinol, used for the treatment of hyperuricemia and gout, is given with AZA. Thus, allopurinol should generally be avoided in patients treated with AZA. If, however, the patient has severe gout and allopurinol must be used, AZA doses must be reduced by about two-thirds, and the white blood cell count must be carefully monitored. AZA eventually has to be discontinued in many such patients. A possible alternative is switching from AZA to MMF, which does not interact with allopurinol.

Side Effects

The major side effect of AZA is bone marrow suppression. All 3 hematopoietic cell lines can be affected, leading to leukopenia, thrombocytopenia, and anemia. The hematologic side effects are dose-related and can occur late in the course of therapy. They are usually reversible upon dose reduction or temporary discontinuation of the drug. AZA should be temporarily withheld if the white cell count falls below 3000/mm³ or if the count drops by 50 percent between blood draws. Recovery usually occurs within 1–2 weeks. The drug can then be restarted at a lower dose and increased gradually to the usual maintenance dose while monitoring the white cell count. Occasionally, AZA has to be discontinued because of recurrent or persistent leukopenia. The mean cell volume is commonly increased in patients on full-dose AZA, and red cell aplasia can eventually result. Interactions between AZA and ACE inhibitors have been reported, causing anemia and leukopenia.

Another potentially serious side effect of AZA, which requires decreasing the dose or even stopping the drug, is hepatotoxicity. This complication is manifested by abnormal liver function tests, usually showing a cholestatic picture. The diagnosis of AZA-induced liver disease is one of exclusion, and the patient should be evaluated for other more serious causes of hepatic dysfunction. AZA has also been linked to the development of skin cancer, the most common malignancy in renal transplant patients. As a result, patients taking AZA for a prolonged period should be instructed to avoid direct exposure to sunlight or to use heavy sun screens when exposed. Other side effects include increased susceptibility to infection and hair loss.

Antimetabolic Agents: Mycophenolate Mofetil

MMF impairs lymphocyte function by blocking purine biosynthesis via inhibition of the enzyme inosine monophosphate dehydrogenase (IMPDH). MMF was developed as a replacement for AZA for maintenance immunosuppression. It is not nephrotoxic and has less bone marrow toxicity than AZA. However, gastrointestinal toxicity can occur, usually manifested by gastritis and diarrhea. MMF is currently available in intravenous, capsule, and liquid formulations.

Mechanism of Action

MPA, the active ingredient of the prodrug MMF, acts by blocking de novo purine synthesis in lymphocytes. Purines can be generated either by de novo synthesis or by recycling (salvage pathway). Lymphocytes preferentially use de novo purine synthesis, whereas other tissues such as brain use the salvage pathway. MPA uncompetitively inhibits IMPDH, which is the rate-limiting enzyme in the de novo synthesis of guanosine monophosphate (GMP) (Fig. 67.12). Inhibition of IMPDH creates a relative deficiency of GMP and a relative excess of adenosine monophosphate (AMP). GMP and AMP levels act as a control on de novo purine biosynthesis, which is essential for T and B lymphocyte proliferation, but not for division in other cells. Therefore, MMF, by blocking IMPDH, creates a block in de novo purine synthesis that selectively interferes with proliferative responses of T and B lymphocytes, inhibiting clonal expansion, and thus inhibiting antibody production, the generation of cytotoxic T cells, and the development of delayed type hypersensitivity. Furthermore, MPA impairs the ability of dendritic cells to present antigen, suppresses the recruitment of monocyte lineage cells, suppresses the glycosylation of adhesion molecules, inhibits vascular smooth muscle proliferation, improves endothelial function, and inhibits mononuclear cell recruitment into allografts and nephritic kidneys [89]. MPA also decreases cytokine-induced nitric oxide synthesis and prevents the formation of reactive species such as peroxynitrite. Furthermore, MPA exhibits antioxidant effects in experimental nephropathies. These properties of MPA likely augment its immunosuppressive properties by limiting fibrosis and vascular sclerosis after immunological injury [90].

Fig. 67.12

Immunosuppressive mechanism of mycophenolic acid. By inhibiting inosine monophosphate dehydrogenase (IMPDH), mycophenolic acid (MPA) antagonizes the de novo pathway of purine synthesis on which lymphocytes particularly depend. Accumulation of adenosine monophosphate inhibits 5-phosphoribosyl-1-pyrophosphate (PRPP) activity thereby diminishing the substrate of IMPDH. Depletion of guanosine phosphates inhibits DNA and RNA synthesis. Lymphocytes lack the salvage pathway of purine synthesis, which depends on the activity of the enzyme hypoxanthine guanosine phosphoribosyl transferase (HGPRTase)

Dosage and Pharmacokinetics

MMF, a semi-synthetic ethyl ester of MPA, is rapidly and completely absorbed and hydrolyzed by esterases to yield the active drug MPA. The recommended dose in pediatric patients in conjunction with CSA is 1200 mg/m² per day in two divided doses, the recommended MMF dose in conjunction with TAC is 800 mg/m² per day in two divided doses. However, recent data from a large, prospective, randomized study in both pediatric and adult renal transplant recipients on fixed dose MMF vs. a concentration-controlled regimen, the FDCC study, indicate that a higher initial MMF dose, for example 1800 mg MMF/m² per day in conjunction with CSA and 1200 mg MMF/m² per day in conjunction with TAC for the first 2–4 weeks post-transplant, is required to achieve adequate MPA exposure in the majority of patients [91, 92]. The MMF dose should be reduced with active CMV infection. When MMF is associated with diarrhea (a side effect of MMF, see below), dividing the daily dosing to 3–4 doses per day may be effective in controlling the diarrhea.

The difference in MMF dosing depending on the concomitant CNI is explained by a pharmacokinetic interaction of CSA with the main MPA metabolite 7-O-MPA glucuronide (7-O-MPAG). CSA inhibits the multidrug resistance protein 2-mediated transport of 7-O-MPAG into the bile. MPAG is subject to enzymatic and non-enzymatic hydrolysis in bile and more importantly in the intestine, thereby liberating the unconjugated drug MPA, which is then reabsorbed into the systemic circulation. This enterohepatic circulation is responsible for a secondary MPA peak occurring 6–12 h after administration. The impact of the enterohepatic cycle on the MPA plasma concentration varies within and between individuals due to factors such as meal times or co-medication of drugs that interrupt the enterohepatic circulation (e.g., bile acid sequestrants, antibiotics). These factors should be considered when evaluating MPA concentrations (particularly pre-dose concentrations) in clinical practice. Furthermore, genetic differences and disease can affect enterohepatic cycling and thus the bioavailability of MPA [93]. If CSA doses are tapered, the pre-dose concentrations of MPA significantly increase, and after complete discontinuation of CSA they can reach about twice the values seen in patients still on CSA co-therapy. When using MMF in combination with TAC or SRL, lower MMF doses can be used to achieve comparable MPA exposure, guided by therapeutic drug monitoring, to that seen with CSA [93]. However, an uncritical approach used by some centers to reduce the MMF dose generally by 50% when co-administered with TAC or SRL is not advisable.

The metabolism of MPA due to glucuronidation can also be affected by drug induction. Steroids are known inducers of UDP-glucuronosyltransferases in vitro, and there is evidence that this may hold true in vivo. In one study, for example, the effect of steroid withdrawal on MPA bioavailability was studied in 26 kidney transplant recipients [94]. When steroids were completely withdrawn 12 months post-transplant, a 33% increase in the mean dose-normalized MPA pre-dose concentrations and MPA-AUCs was observed compared with concentrations at 6 months, when the patients were still receiving maintenance doses of steroids. The relevant drug-drug interactions are summarized in Table 67.4.

Table 67.4 Drug-Drug-Interactions between mycophenolate mofetil and frequently used co-medications

An important pharmacokinetic property of MPA is its extensive and tight protein binding particularly to serum albumin. The free MPA fraction in individuals with conserved renal function ranges from 1% to 3%. Based on in vitro studies, the free MPA fraction is responsible for the pharmacological activity of the drug. Furthermore, it is an important determinant of the MPA clearance. Of the factors evaluated for their effect on MPA protein binding, serum albumin was the most important. In patients with delayed graft function or renal impairment, there are many factors which can affect MPA protein binding. These may lead to substantially elevated free MPA concentrations despite total MPA levels similar to those found in patients with relatively preserved renal function [95, 96].

Efficacy

Following the success of the early MMF studies in adults, MMF was investigated in pediatric renal transplant recipients in open-label studies with historical controls, since randomized, controlled trials were quite difficult to carry out due to the relatively small numbers of pediatric kidney transplants performed each year. Since studies in adult renal transplant recipients had previously established the superiority of MMF over AZA or placebo in reducing the risk of acute rejection, it was important for the pediatric transplant community to have prompt access to open-label studies. Data from 3 large multicenter studies [97,98,99,100,101] and one smaller study [102] provided support for the safety and efficacy of MMF in the pediatric renal transplant population when used with CSA and prednisone. Induction therapy was optional in one study [97] and not used in the other two studies [99, 102]. The incidence of acute rejection within the first 6 months to 1 year for patients receiving MMF in these studies ranged from 28% to 37% [97, 99, 101]. Those studies comparing MMF patient groups to historical controls reported significant reductions in the incidence of acute rejection with MMF vs. AZA [99, 101]. There was also a significant improvement in the incidence of acute rejection between patients receiving MMF and those receiving AZA at 3 years in a follow-up report to one study [100]. In one large study [97], no differences in the incidence of acute rejection were observed when results were stratified by age. Long-term (3-year) graft and patient survival were excellent, with a 30% incidence of acute rejection [98]. MMF has a role in the prevention and/or treatment of chronic rejection. Among children with chronic rejection, some evidence suggests that substituting MMF for AZA may improve renal function [103, 104].

Side Effects

The major toxicity of MMF is gastrointestinal, mainly diarrhea, possibly as a result of the high concentrations of acyl-MPAG in the gut. MMF is devoid of intrinsic renal, cardiovascular or metabolic toxicities, but can increase the risk for CMV infections, leukopenia, and perhaps, mild anemia (Table 67.2). MPA has been associated with protection from Pneumocystis jirovecii pneumonia (PJP) and may actually have some anti-PJP activity because Pneumocystis jirovecii has IMPDH activity. MMF should not be used in pregnant transplant patients since its safety in pregnancy has not yet been established.

In the MMF suspension trial in pediatric renal transplant recipients, MMF safety was evaluated based on the occurrence of adverse events, including the development of opportunistic infections and malignancies. The most frequently noted adverse events were hematological problems such as leukopenia and gastrointestinal disorders like diarrhea, which occurred in 25% and 16% of all patients, respectively, and were observed more often in the youngest age group. In general, the risk of developing side effects declined with increasing age.

Therapeutic Drug Monitoring

Patients on standard-dose MMF therapy show considerable between-patient variability in pharmacokinetic parameters. This variability is attributable to factors that influence exposure to MMF, such as renal function, serum albumin levels, concomitant medications such as CSA that inhibit enterohepatic recirculation of the active metabolite of MMF, MPA, (Table 67.4) and genetic polymorphisms of MPA-metabolizing enzymes. This variability is clinically relevant, as higher plasma concentrations of MPA are correlated with reduced risk of acute rejection after kidney transplantation [96, 105]. These findings have suggested that individualizing the dose regimen of MMF may further improve clinical outcomes compared with a standard-dose regimen.

There has been considerable debate regarding the utility of measuring MPA levels. Advocation for MPA monitoring is based on the premise that monitoring will result in avoiding both underdosing, which prevents rejection, and overdosing, which increases the risk of adverse reactions [105]. One study in adults, for example, showed significantly fewer treatment failures and acute rejection episodes in the monitoring arm compared with the fixed dose arm with no significant difference in side effects [106]. Within this study, MPA exposure and MMF dosing were higher in the monitoring arm based on 3 levels measured over the first 3 h post-dose. Awareness of the potential for a more personalized dosing has led to development of methods to estimate MPA AUC based on the measurement of drug concentrations in only a few samples. This approach is feasible clinically, and has proven successful in terms of correlation with outcome [107]. An MPA-AUC > 40 mg x L/12 h has been recommended for sufficient MPA exposure for the prevention of acute rejection episodes [92]. In general, monitoring of MPA exposure by MPA pre-dose plasma levels is more popular in clinical practice than monitoring of the MPA-AUC by a limited sampling strategy, but less precise. Therefore, some transplant centers monitor MPA pre-dose and target levels between 1.5 and 4 mg/L. In addition, they use levels as a measure of adherence.

Target of Rapamycin (TOR) Inhibitors

SRL (sirolimus, rapamycin) is a macrocyclic triene antibiotic that is produced by the actinomycete Streptomyces hygroscopicus. SRL was approved in September 1999 by the US FDA and in December 2000 by the European Medicines Agency for use in adult renal transplant recipients. EVR is an analog of SRL that has similar effectiveness and side effect profile.

Mechanism of Action

SRL displays a novel mechanism of immunosuppressive action. Interaction with at least two intracellular proteins is required to elicit its anti-proliferative activity. SRL first binds to the cytosolic immunophilin FK-binding protein 12 (FKBP12). In contrast to the TAC-FKBP12 complex, the complex of SRL with FKBP12 does not inhibit calcineurin activity. Instead, this complex binds to and inhibits the activation of mTOR, a key regulatory kinase. This inhibition suppresses cytokine-mediated T-cell proliferation, inhibiting the progression from the G1 to the S-phase of the cell cycle. Thus, SRL acts at a later stage in the cell cycle than do the CNIs CSA and TAC. SRL can, therefore, be used in combination with the CNIs to produce a synergistic effect [108].

Dosage and Pharmacokinetics

EVR is available as tablets and dispersible tablets for administration in water. The current evidence from pediatric renal transplantation suggests that EVR should be administered at an initial dose of 0.8 mg/m² body surface area twice daily when given in combination with CSA therapy, adjusted to target a trough concentration of 3–8 μg/L [109, 110]. There is a well-documented drug-drug interaction between mTOR inhibitors and CSA [111], arising from a shared metabolic pathway via the cytochrome P450 CYP3A4 isoenzyme system, and the fact that both are substrates for the drug transporter P-glycoprotein. EVR exposure is increased by up to three-fold in patients receiving concomitant CSA [112, 113], while TAC exerts only a minimal effect [113, 114]. In patients receiving EVR with concomitant TAC, a dose of 2 mg/m² body surface area twice daily is therefore appropriate [112]. Similarly, SRL bioavailability is higher in the presence of CSA than TAC [115, 116].

SRL is available as tablets form and an oral solution. Data on target doses and blood concentrations for SRL in pediatric transplant recipients are more limited than for EVR. The half-life of SRL increases with age in children [117]. Twice-daily dosing and daily dosing is therefore recommended in young children and older recipients, respectively. However, there is no clear guidance regarding the age or body weight when the switch should be considered. One trial used 13 years of age as a cutoff point [118]. In a pharmacokinetic study of 13 children receiving SRL in a CNI-free regimen, with a median age of 15.5 years, the authors concluded that twice-daily dosing was required in this setting due to more rapid metabolism of SRL in the absence of concomitant CNI therapy [119]. High SRL trough concentrations (>10 μg/L) either with or without concomitant CNI appear inadvisable in children in view of the high risk of toxicity and discontinuation. One small prospective study (n = 19) converted pediatric kidney transplant patients to a CNI-free regimen of SRL with MMF using a single SRL loading dose of 5–7 mg/m² body surface area, then a daily dose of 2–4 mg/m² body surface area adjusted to target a SRL trough concentration of 5–10 μg/L, and achieved a low rate of rejection with a good renal response [118].

EVR and SRL are both macrolide derivatives and share many pharmacokinetic features, including a close correlation between total exposure and trough concentration, low absorption that varies between and within patients, and differences in absorption between adults and children [111, 120]. However, EVR is the 40-O-(2-hydroxyethyl) derivative of SRL, a modification that results in some important pharmacokinetic differences between the two drugs. EVR is more hydrophilic than SRL and is absorbed more rapidly from the gut with more systemic clearance than SRL [121]. As a result, the elimination half-life of EVR is shorter than for SRL (mean 28 h vs. 62 h) [122, 123]. The clinical effect is that no loading dose is required for EVR whereas a loading dose of 3 times the maintenance dose has been recommended for starting SRL in adults to accelerate achievement of steady-state concentration [124]. EVR is administered twice a day in pediatric and adult patients whereas once daily dosing is appropriate for SRL in older children and adult patients (see above for considerations regarding SRL dosing in younger children).

Efficacy

The most frequent reason to include an mTOR inhibitor in the immunosuppressive regimen is to facilitate a reduction in CNI exposure, or to eliminate CNI therapy entirely. The current evidence suggests that de novo administration of EVR with low-exposure CNI therapy in children undergoing renal transplantation is efficacious and safe. The recently published 36-month, multicenter, open-label randomized study investigated EVR with reduced-dose TAC and steroid elimination from month 5 post-transplant compared with a standard-dose TAC regimen with MMF and steroids (control) [125]. The incidence of composite efficacy failure (biopsy-proven acute rejection [BPAR], renal allograft loss, or death) at month 36 was 9.8% vs. 9.6% for EVR with reduced-dose TAC and MMF with standard-dose TAC, respectively, which was driven by BPARs. Kidney allograft loss was low (2.1% vs. 3.8%) with no deaths. Mean eGFR rate at 3 years post-transplant was comparable between groups. Growth in pre-pubertal patients on EVR with reduced-dose TAC without steroids was better (P = 0.05) vs. MMF with standard-dose TAC and steroids. The overall incidence of adverse events and serious adverse events was comparable between groups. Rejection was the leading adverse event for study drug discontinuation in the EVR with reduced-dose TAC group. The authors concluded that, although adverse events-related study drug discontinuation was higher, an EVR with reduced-dose TAC regimen represents an alternative treatment option that enables steroid withdrawal as well as CNI reduction in pediatric kidney transplant recipients [125].

Use of de novo EVR with complete CNI avoidance has not been explored in large trials in pediatric transplant recipients, but is unlikely to be preferable to concomitant reduced-exposure CNI. Primarily mTOR-based, CNI-free immunosuppression is associated with a significantly increased risk of the development of DSA [126]. Switching maintenance patients to an mTOR inhibitor to facilitate CNI minimization can improve renal allograft function or avoid further functional deterioration, particularly when undertaken before irreversible damage has developed. Late switch below an eGFR of 40 mL/min/1.73 m², however, may be associated with an increase in pre-existing proteinuria, favoring early conversion. It remains unresolved whether CNI therapy should be reduced or, indeed, eliminated in maintenance patients regardless of whether renal dysfunction is believed to be due to CNI-related nephrotoxicity. Currently, many transplant centers use mTOR inhibitors as part of a maintenance immunosuppressive regimen only in the following patient subsets in which this drug class may have particular utility: (i) Patients, who have histologically proven CNI nephrotoxicity despite low levels and doses of the CNI; (ii) patients with malignancy (e.g., skin cancers and Kaposi sarcoma), either in remission or being actively treated; (iii) patients after treatment of B cell PTLD; (iv) patients with recurrent CMV viremia, because EVR has anti-CMV activity in vitro and is associated with less CMV replication and disease in vivo compared to MMF [127, 128]. Notably, the incidence of EBV or BK polyomavirus infection is not lower in EVR- compared to MMF-treated patients [125].

Side Effects

Clinically relevant adverse effects of SRL and EVR that require a specific therapeutic response or can potentially influence short- and long-term patient morbidity and mortality as well as graft survival include hypercholesterolemia, hypertriglyceridemia, infectious and non-infectious pneumonia, anemia, lymphocele formation and impaired wound healing (Table 67.2). These drug-related adverse effects are important determinants in the choice of a tailor-made immunosuppressive drug regimen that matches the individual patient risk profile. Equally important in the latter decision is the lack of severe intrinsic nephrotoxicity associated with SRL and EVR and its advantageous effects on hypertension, post-transplantation diabetes mellitus and esthetic changes induced by CNIs. Mild and transient thrombocytopenia, leukopenia, gastrointestinal adverse effects and mucosal ulcerations are all minor complications of SRL and EVR therapy that have less impact on the decision for choosing this drug as the basis for tailor-made immunosuppressive therapy.

An additional side effect in the setting of CNI withdrawal and mTOR inhibitor introduction is aggravation of proteinuria in patients with pre-existing proteinuria by a still incompletely defined mechanism. Available data are consistent with the hypothesis that the increase of proteinuria is causally related to CNI withdrawal and not because of initiation of an mTOR inhibitor [129]. On the other hand, it cannot be excluded that SRL and EVR might also affect glomerular permeability in some patients. The potential complication of increased proteinuria, which is an independent risk factor for decreased long-term kidney allograft function, should therefore be considered when converting from a CNI-based to an mTOR-containing maintenance therapy. Preliminary results show that mTOR inhibitor treatment may impair gonadal function after kidney transplantation, but the clinical significance of these effects is unknown [129, 130].

Generic Immunosuppressive Drugs

The number of immunosuppressive drugs prescribed to prevent rejection is relatively small. Not more than 10 different compounds have been used over a period of 50 years. For most of these drugs, the patents have expired (AZA, CSA, TAC, MMF, SRL), or will expire within the next few years (EVR, mycophenolate sodium) [131]. Policy makers consider generic drugs an attractive option to enable savings on medication cost, allowing the savings to be used for funding high-cost medicines. Generic immunosuppressive drugs are available in Europe, Canada, and the US. Between countries, there are large differences in the market penetration of generic drugs in general, and for immunosuppressive drugs in particular. To allow for safe substitution, a number of criteria need to be fulfilled. Generic substitution should not be taken out of the hands of the treating physicians. Generic substitution can only be done safely if initiated by the prescriber, and in well-informed and prepared patients. Payers should refrain from forcing pharmacists to dispense generic drugs in patients on maintenance treatment with brand drugs. Instead, together with transplant societies, they should design guidelines on how to implement generic immunosuppressive drugs into clinical practice. Substitutions must be followed by control visits to check if the patient is taking the medication correctly and if drug exposure remains stable. Inadvertent, uncontrolled substitutions from one generic to another, initiated outside the scope of the prescriber, must be avoided as they are unsafe. Repetitive subsequent generic substitutions result in minimal additional cost savings and have an inherent risk of medication errors [131].

Adherence to Immunosuppressive Medication

Adherence to medications is defined as the process by which patients take their medication as prescribed. Adherence is of critical importance and it is often lacking, especially in teenagers. Adolescent age at transplant is an independent and significant risk factor for worse long-term renal allograft survival in all major pediatric solid organ transplant types [132]. Adolescents have also been identified to have a multitude of psychiatric and socioeconomic risk factors that deserve greater attention and customized care delivery programs [133]. Immunosuppression obviously only works if it is taken reliably, and there is ample evidence in the literature that this reliability is lacking far too often, leading to adverse outcomes. Moreover, immunosuppressive regimens may need to be adjusted to support adherence, e.g. by combination products to reduce the pill burden and/or by once daily dosing. The latter is realized by a prolonged-release TAC formulation available for older children and adolescents (Advagraf™ in Europe, Astagraf™ in the US). Comparative pharmacokinetic studies have shown that stable pediatric transplant recipients can be converted from immediate-release to prolonged-release TAC at the same total daily dose, using the same therapeutic drug monitoring method [134, 135].

Conclusions

Transplantation in children carries unique challenges. While issues such as controlling rejection and minimizing side effects are similar between adults and children, maintenance immunosuppressant regimens that affect developmental processes have a disproportionate impact on children. This is particularly true for steroids, which have many side effects, including some that can be quite devastating in pediatric patients. Steroid avoidance has been very successful when MMF is combined with TAC and either basiliximab or anti-thymocyte globulin for induction therapy is added; long-term follow-up on these patients with regards to improvements in kidney allograft survival and transplant function will be very informative, as steroid-free patients also have reduction of co-morbidities that drive chronic allograft dysfunction. With the goal of eliminating steroids, the combination of MMF and TAC may strike the correct balance between adequate and excessive immunosuppression. Recently, the use of EVR has been advocated for minimizing CNI exposure after kidney transplantation, but its use is limited by mTOR-related side effects. There is at present no consensus for immunosuppressive therapy following renal transplantation in children.

Newer drugs such as belatacept have not been systematically studied in the pediatric transplant patient population. Since the approval of belatacept in 2011 for use in de novo adult renal transplantation, this CD80/86-CD28 co-stimulation blocker has been shown to be a valuable treatment option for maintenance immunosuppression [136]. Belatacept in adults has been associated with a superior GFR as compared to CNI-based treatments because of the absence of nephrotoxicity. Additionally, belatacept avoids cardiovascular side effects (e.g. hypertension and dyslipidemia) caused by a CNI-based-regimen [136]. However, its use is limited to EBV-seropositive patients, since EBV-naïve patients on belatacept have an increased PTLD risk. Nevertheless, there is great interest in belatacept as possible “depot” immunosuppression in adolescent pediatric recipients. The latter population has the highest rates of kidney allograft loss due to immunosuppression non-adherence and will most likely have prior exposure to EBV. Belatacept has been shown to be safe in adolescents, but not yet approved for children or EBV-naïve patients given the additional risk for developing PTLD [137]. Studies are underway to explore safe protocols for the use of belatacept in adolescent children.

Currently, there is a paucity of novel maintenance immunosuppressive drugs in the pipeline. Iscalimab, a non-B cell depleting anti-CD40 monoclonal antibody, has been investigated in a phase 2 trial in de novo adult kidney allograft recipients with a CNI-free regimen, and other agents targeting co-stimulation blockade are in pre-clinical development [85].

Clearly, much additional work is needed to define optimal immunosuppressive regimens in pediatric renal transplant patients, particularly with respect to newer and evolving regimens. The safety and efficacy of these protocols, with special emphasis on long-term renal allograft survival, PTLD and other malignancies, but also co-morbidities need to be established. The “one size fits all” strategy needs to evolve into tailored strategies based upon a child’s medical history and circumstance (Fig. 67.13) [138].

Fig. 67.13

Modern era immunosuppressive strategies in children. The role of a low-dose CNI regimen is illustrated within the context of other maintenance therapies. CNI calcineurin inhibitor, EBV Epstein-Barr virus, MMF mycophenolate mofetil, mTORi mammalian target of rapamycin inhibitors. (Reproduced from [138], with permission)